A Hybrid Bi-layered Scaffold from Silk Fibroin-Gelatin nanofibers and Egg yolk oil-loaded Tragacanth Hydrogel as a promising wound dressing

A Hybrid Bi-layered Scaffold from Silk Fibroin-Gelatin nanofibers and Egg yolk oil-loaded Tragacanth Hydrogel as a promising wound dressing | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A Hybrid Bi-layered Scaffold from Silk Fibroin-Gelatin nanofibers and Egg yolk oil-loaded Tragacanth Hydrogel as a promising wound dressing Parisa Malekian-Nouri, Adeleh Gholipour-Kanani This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7734190/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Hybrid wound dressings such as nanofibers-hydrogel structures have been developed to prevent infection and accelerate wound healing. The main objective of this study was to fabricate a hybrid scaffold composed of silk fibroin (SF)–gelatin (Ge) nanofibers and tragacanth(TG) hydrogel loaded with egg yolk oil(EYO) for burn wound applications. The TG hydrogel, serving as a flexible drug carrier, was loaded with egg yolk oil, which is well known for its burn-healing efficacy. Silk fibroin was employed for its regenerative properties and low scarring potential. FESEM results revealed that SF-Ge-PVA nanofibers, prepared at a volume ratio of 1:1:2 and electrospun under 18kV with a 12cm nozzle-to-collector distance, exhibited bead-free and uniform morphology with an average diameter of 201 ± 18 nm. Contact angle and swelling studies demonstrated that both the hydrogel layer and the hybrid nanofiber–hydrogel structure were hydrophilic, with swelling ratios of 1106% and 902%, respectively. MTT assay results confirmed the absence of cytotoxicity in both EYO-loaded and unloaded hybrid structures. Furthermore, the SF-Ge-PVA nanofibers combined with EYO-loaded hydrogel exhibited excellent antibacterial activity against Gram-positive bacteria, achieving 99.97% elimination of bacterial colonies. Overall, these findings suggest that the developed hybrid scaffold is a highly promising material for burn wound healing applications. Biological sciences/Biotechnology Physical sciences/Chemistry Physical sciences/Materials science Physical sciences/Nanoscience and technology Silk fibroin nanofibers Tragacanth hydrogels Egg yolk oil Hybrid bi-layered scaffolds Burn Dressing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction The clinical priority for tissue regeneration of different kinds of skin defects like major traumatic wounds, burns, pressure ulcers, and chronic diabetic wounds has increased substantially in recent times. Although, cellular sheets and cultured scaffolds represent current medical approaches, but they have not successfully rebuilt the entire natural structure and functional aspects of skin tissue [ 1 ]. Over the past decades, tissue engineering approaches have focused on mimicking the extracellular matrix (ECM) environment to guide cellular repair [ 2 ]. In particular, biomaterial scaffolds that replicate the composition and three-dimensional fibrous network of the dermal ECM can direct cell adhesion, migration, and differentiation in wound sites [ 2 ], [ 3 ]. Thus, there is growing interest in designing materials that reproduce the nano-scale structure and biochemical cues of native skin to improve healing outcomes. Electrospun nanofibrous membranes possess high porosity and large specific surface area, providing an optimal environment for the complex and dynamic wound healing process and numerous sites for carrying wound healing agents. Such nanofibrous mats have an extremely large surface area and pore volume, creating a microenvironment that can support complex healing processes and accommodate high loadings of bioactive molecules [ 3 ]. Furthermore, the fine fibrous structure closely resembles the dermal ECM, which has been shown to enhance hemostasis, reduce inflammation, and accelerate tissue repair [ 3 ]. Polymer biomaterials demonstrate excellent biocompatibility and biodegradability, together with native ECM-like morphology and suitable physicochemical mechanical and biological controls, which help establish an optimal wound-healing microenvironment [ 4 ]. Burn healing and skin tissue repairing face significant challenges in medical science such as severe infection, surface dehydration and scar formation. Modern wound dressings are thus designed to fulfill key functions – they should maintain a moist, oxygenated environment, absorb exudate, allow gas exchange, and provide a barrier against infection [ 5 ], [ 6 ]. Combinations of fiber mats and hydrogels have gained attention because they can incorporate multiple therapeutic functions (e.g. antimicrobial agents, growth factors, antioxidants) and thus accelerate healing while minimizing scar formation [ 5 ], [ 7 ]. Natural biopolymers in particular are attractive for skin repair because they inherently resemble ECM components and support cell function [ 8 ]. Examples include protein-based materials (e.g. collagen [ 4 ], keratin [ 9 ], gelatin [ 10 ], and silk fibroin [ 11 ]) and polysaccharides (e.g. chitosan [ 12 ], alginate [ 10 ], tragacanth gum [ 13 ]). Gelatin as a collagen-derivative possesses most of collagen properties such as its biocompatibility and gel-forming ability [ 14 ]. Therefore, as a low-cost, non-immunogenic material, gelatin promotes hemostasis and absorbs wound exudate in the inflammatory phase [ 6 ]. It made of composition of amino acids such as proline, glycine and hydroxyproline which can mimic the ECM fibril structure similar to collagen [ 14 ]. So, it can stimulate the fibroblast migration to enhance the formation of granulation tissue in proliferation phase of healing process [ 14 ]. In chronic wound models, gelatin-based matrices have been shown to enhance platelet aggregation and support vascular and epithelial cell regeneration through its hydrophilic and bioactive properties as well as non-antigenicity [ 6 ], [ 15 ]. Silk-fibroin as a protein derived from natural silk has been considered in wound healing application due to its highly anti-inflammatory and pro-angiogenic properties followed by sufficient biocompatibility and biodegradability [ 16 ]. Numerous studies have shown that SF accelerates wound closure by stimulating angiogenesis and modulating inflammation; compared to many conventional dressings, SF-based materials significantly speed skin regeneration [ 16 ]. Moreover, bilayer skin-like scaffolds incorporating silk fibroin have demonstrated scar-inhibition effects in animal models. For example, a recent study reported by Zhou et al. [ 8 ] showed that the SF–hyaluronic acid bilayer scaffold promoted full-thickness skin regeneration while reducing wound contraction and abnormal collagen deposition, thereby suppressing hypertrophic scar formation [ 8 ]. A research by Yerra et al. [ 11 ] developed a nanofibrous scaffold based on silk fibroin loaded with antibiotics for burn wound treatment by showing suitable cell adherence properties and a proper morphology for wound dressing. These data highlight SF’s potential both to rebuild dermal structure and to improve healing quality. Polysaccharide-based hydrogels also play a crucial role in advanced wound dressings. One promising example is Tragacanth gum (TG), a natural hydrocolloid derived from Astragalus plants. TG is a highly branched, anionic polysaccharide containing galacturonic acid, xylose, arabinose, galactose, and so on [ 17 ]. It shows inherent properties in wound healing process due to its biocompatibility, biodegradability, ability to create a moist environment and promote cell growth [ 13 ]. Recent studies have demonstrated that TG-based hydrogels can naturally accelerate wound closure and improve tissue regeneration in vivo, while also lowering infection risk (likely due to TG’s ability to incorporate antimicrobial agents) [ 17 ]. In addition, as a natural hydrogel, tragacanth has a great potential to be an excellent carrier matrix. It can encapsulate drugs, growth factors, or bioactive extracts for sustained release at the wound site [ 17 ]. Bioactive lipids and natural extracts are often incorporated into dressings to further enhance healing. Egg yolk oil (EYO) as a traditional remedy for third degree burns has been considered due to its anti-inflammatory, anti-microbial and tissue regenerative properties [ 18 ]. It contains saturated and unsaturated fatty acids, fat miscible vitamins, phospholipids, immunoglobulin, antioxidants and cholesterol [ 18 ]. Shadman-manesh et al. [ 19 ] developed a nanofibrous scaffold based on PCL which was loaded with EYO for third degree burns in rat models. From in vivo results, they concluded that the rate of wound closure, re-epithelialization and collagen deposition after 21-day treatment with EYO-loaded-scaffold were improved significantly. This study has developed a new bi-layered scaffold based on gelatin-silk fibroin nanofibers combined with egg yolk oil loaded-tragacanth hydrogel layered as a promising structure for burn wound treatment. The inner layer is an electrospun nanofibrous mat composed of gelatin and silk fibroin, chosen to mimic the dermal ECM structure and to leverage the inherent wound-healing activity of these proteins. The fibrous mat provides mechanical strength, cellular attachment sites, and hemostatic capability. The outer layer is a TG hydrogel loaded with egg yolk oil, forming a moist, protective covering. This hydrogel maintains wound hydration and slowly releases EYO’s antibacterial and regenerative factors. In this design, the two layers work in concert: the top hydrogel blocks dehydration and infection, while the bottom fibrous layer promotes cell adhesion and tissue regeneration. The hybrid scaffolds have been characterized using different physicochemical studies such as FESEM, FTIR, contact angle, swelling and release study as well as in vitro studies including cytocompatibility, fibroblast adhesion and proliferation, and antibacterial activity to investigate their efficiency for skin repairing and wound healing applications. In summary, this study presents a fully biomimetic, multifunctional bilayer dressing for burn wounds, combining gelatin/SF nanofibers with an EYO-loaded TG hydrogel. By uniting the favorable properties of these natural materials, the scaffold is expected to provide a moist, antimicrobial, and ECM-like environment that supports rapid, scar-minimizing skin repair. 2. Experimental 2.1. Materials Tragacanth (TG) and Silk ( Bombyx mori ) cocoons were purchased from Attarak Company (Iran). Egg yolk oil was purchased from Giah Taghdis Company (Iran). Gelatin (Ge) (type B, CAS No.: 9000-70-8) and glutaraldehyde (GA) were purchased from Sigma-Aldrich (Germany). Polyvinyl alcohol (PVA) (M w =72000 g.mol − 1 ), calcium chloride dehydrate, lithium bromide (LiBr) and sodium carbonate (Na 2 CO 3 ) were supplied by Merck (USA). 2.2. Extraction of silk fibroin Extraction of silk fibroin from silk cocoons was performed according to the guidelines reported by Cao et al. [ 20 ]. Briefly, 5 g of crushed, dried B. mori cocoons were boiled in 0.02 M Na₂CO₃ solution for 20 min to remove sericin. The fibers were rinsed three times with cold distilled water and dried at ambient temperature for 24 h. The degummed silk was dissolved in 9.3 M LiBr at 60°C for 4 h with stirring. The resulting solution was dialyzed (molecular weight cutoff: 12–14 kDa) against deionized water for 3 days, with water changes every 6 h, to remove residual LiBr. The dialyzed solution was centrifuged at 9000 rpm for 20 min at 4°C to yield a concentrated SF solution, which was stored at 4°C until use. The process is shown in Fig. 1 . The extracted fibroin was analyzed in our previous study [ 21 ]. 2.3. Preparation of SF-Ge-PVA nanofibers Aqueous solutions of 10% (w/v) Ge and 10% (w/v) PVA were prepared at 40°C and blended at Ge:PVA ratios of 1:1 and 1:2 (v/v). SF solution was subsequently added to achieve SF:Ge:PVA ratios of 1:1:1 and 1:1:2 (v/v/v). The mixtures were stirred at 25°C for 2 h to obtain clear, homogeneous solutions, which were loaded into syringes for electrospinning. Electrospinning was performed using a conventional setup (FNM Duos Electroris, HV35P OV, Fanavaran Nanomeghyas Co., Iran) under voltages of 15 kV and 18 kV, nozzle-to-collector distances of 12 cm and 15 cm, and a flow rate of 1 mLh⁻¹. To improve structural stability in aqueous media, fibers were crosslinked via glutaraldehyde (GA) vapor treatment. Following a previous report [ 21 ], nanofibrous mats were exposed to the vapor of 30 mL GA in a sealed desiccator for 18 h at room temperature. Crosslinked mats were washed three times with phosphate-buffered saline (PBS) and dried in a vacuum oven at 40°C for 2 h. 2.4. Preparation of SF-Ge-PVA nanofibers/EYO-loaded TG hydrogel hybrid A 4 wt% TG solution was prepared by dissolving TG in distilled water at 40°C with stirring for 3 h. EYO (15 wt% of the dried polymer weight) was incorporated and stirred for 20 min. Subsequently, CaCl₂ (1.5 wt%) was added as a crosslinker, and the solution was stirred for an additional 30 min. The mixture was cast into 80 mm Petri dishes (4 mm thickness) and dried at 50°C for 1 h in a vacuum oven. Finally, the crosslinked SF-Ge-PVA nanofibrous mat was placed on the surface of the hydrogel and left to dry overnight at room temperature to form the hybrid bilayer scaffold. Figure 2 shows the preparation of both EYO loaded and unloaded hybrids. 2.5. Physico-chemical and morphological characterization ATR-FTIR (Attenuated Total Reflection Fourier Transform Infrared) spectroscopy was performed using EQUINOX 55 spectrophotometer (Bruker, Germany) to study the chemical bonds of the extracted SF and the hybrid scaffolds. Briefly, the analyses was used to investigate the interactions between components in the scaffolds using KBr tablets in the range of 4000–400 cm − 1 . Morphological studies were performed using field emission scanning electron microscopy (FESEM, XL30 PHILIPS SEM, Scanning electron microscope) to determine the optimum conditions of the electro spun nanofibers and hybrid hydrogels. Digimizer software (v. 6.3.0) was used to measure the average fiber diameters. Contact angle measurements were performed according to ASTM D7334-08 to investigate the hydrophilic properties of the produced scaffolds using a static optical contact angle measurement setup (CAG-10, Jikan, Iran) by the sessile drop method. A 1cm×1cm piece of each scaffolds was cut and placed on the instrument’s sample holder and exposure to a single droplet of distilled water (volume∼2µl). The shape of the drops on the scaffold surfaces was recorded using the camera attached to the drop shape analyzer after 3 s. The test was repeated three times and the average value was reported. Swelling studies were carried on both TG hydrogel and the hybrid scaffold to measure the water uptake capacity of the scaffolds. Regards, the samples were cut into squares with an area of 1 cm 2 and dried at 60°C until constant weight ( W d ) was obtained. The specimens were placed in 10 ml water (7.4 pH and 37°C) during 24 h and removed in two time points of 12 and 24 h and after drying their surface they were weighted ( W s ). The swelling ratio (SR) was calculated using the following Eq. 1 , where, W d and W s were the weights of dried and swollen samples, respectively. 2.6. Release study EYO release from hydrogels was evaluated using UV–Vis spectroscopy (UNICO, USA). Pre-weighed EYO-loaded hydrogels were immersed in 2 mL PBS (pH 7.4) at 37°C. At predetermined intervals (0.5, 1, 2, 4, 6, 12, 24, 48, and 72 h), 1 mL of medium was withdrawn and replaced with fresh PBS. Absorbance was recorded between 270–480 nm. Each experiment was performed in triplicate. 2.7. Antibacterial activity The antibacterial activities of both EYO-loaded and unloaded bi-layered scaffolds were evaluated against Escherichia coli ( E. coli , ATCC 25922) as a gram-negative bacteria and Staphylococcus aureus ( S. aureus , ATCC 6538) as a gram-positive bacteria which were purchased from Pasteur Institute of Iran (Tehran, Iran). In this study, the logarithmic count reduction test was performed by the microbial count method according to ASTM E2180 to examine the antibacterial activities of the scaffolds. In order to perform the analysis, the bacterial strains were grown on Mueller Hinton Agar medium plates (Merck, Germany) at 37°C for 18 h. Using distilled salinized water (0.9% NaCl), suspensions from E. coli and S. aureus at a final concentration of 2.2 × 10⁸ CFU/mL and 3.9 × 10⁸ CFU/mL were prepared respectively. Each hybrid scaffold was cut into a 10 mm in diameter discs and exposing to the prepared bacteria suspensions in petri dishes. The petri dishes were incubated for 24 h at 37˚C. Each experiment was performed in triplicate. 2.8. Cytotoxicity and cell attachment Cytotoxicity was evaluated via MTT assay using human foreskin fibroblast cells (HFF-2; purchased from Pasteur Cell Bank (Pasteur Institute, Tehran, Iran) https://en.pasteur.ac.ir/Department-of-Cell-Bank ). Cells (1 × 10⁴ per well) were seeded in 96-well plates in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 50 U·mL⁻¹ penicillin, and 50 U·mL⁻¹ streptomycin, and incubated at 37°C for 24 h. Extracts from scaffolds (90 µL) and 10 µL FBS were added and incubated for another 24 h. MTT reagent (0.5 mg·mL⁻¹, 100 µL) was then added for 4 h. The formazan crystals were dissolved in isopropanol, and absorbance was measured at 570 nm using a microplate reader (BioTek ELx808, USA). Cell viability (%) was calculated relative to control wells by using Eq. 2. For cell attachment, scaffolds seeded with HFF-2 were fixed after 3 days with 3.5% GA for 2 h at 4°C, dehydrated in graded ethanol (50–96%), air-dried, and imaged by SEM. 2.9. Statistical analysis All data are expressed as mean ± standard deviation (SD). Statistical analyses were performed using SPSS v16.0. One-way ANOVA with Tukey’s post hoc test was applied, and differences were considered significant at p < 0.05. 3. Results and Discussion 3.1. Morphological Analysis of SF-Ge-PVA Nanofibers Due to the negative electrostatic charge within silk fibroin chains in aqueous solutions, and the repulsion of like charges in an electric field, the possibility of electrospinning it purely is very poor [22]. Blending SF with a carrier polymer such as polyvinyl alcohol (PVA) is a common strategy to increase chain entanglement and improve spinnability. Fig. 3 presents SEM micrographs of SF:Ge:PVA nanofibers prepared at blend ratios of 1:1:1 and 1:1:2 (v/v/v) electrospun at an applied voltage of 15 kV and a nozzle-to-collector distance of 12 cm. Increasing the PVA fraction yielded smoother fibers with greater morphological uniformity [23]. Concomitantly, the mean fiber diameter decreased from 402 ± 54 nm (Fig. 3a) to 294 ± 37 nm (Fig. 3b). This trend is consistent with modest reductions in effective solution viscosity and enhanced jet stretching in the electric field (within a regime that avoids bead formation), in line with general electrospinning principles and prior reports on SF-blend systems [24]. The blend ratio of 1:1:2 (SF:Ge:PVA) was selected for further optimization of electrospinning conditions. In order to evaluate the influence of voltage and nozzle-to-collector distance, fibers were electrospun at applied voltages of 15 kV and 18 kV and distances of 12 cm and 15 cm and the SEM results are shown in figure 4. At a constant distance of 12 cm, increasing the applied voltage from 15 kV to 18 kV significantly reduced the mean fiber diameter from 294 ± 37 nm to 201 ± 18 nm (Figs. 4a and 4b). A similar trend was observed at 15 cm, where fiber diameter decreased under higher voltage (Figs. 4c and 4d). This behavior can be explained by the dominant effect of increased electric field strength, which enhances jet acceleration and stretching, overriding the tendency for a thicker jet at higher voltages. Stronger fields promote more intense whipping instabilities, resulting in finer and more uniform fibers [25]. Under the condition of 18 kV and 12 cm, highly uniform fibers with smooth morphology were obtained, representing the optimal electrospinning condition. Comparison of Figs. 4a vs. 4c and 4b vs. 4d illustrates the influence of increasing the nozzle-to-collector distance at constant voltage. At 18 kV, increasing the distance from 12 cm to 15 cm led to an increase in fiber diameter from 201 ± 18 nm to 298 ± 30 nm. This is attributed to the reduction in effective field strength at greater distances, leading to less jet stretching during flight and consequently thicker fibers [25]. These findings confirm that an applied voltage of 18 kV and a collection distance of 12 cm represent the most favorable electrospinning conditions for producing uniform SF:Ge:PVA fibers. 3.2. Morphological study of TG hydrogel and the hybrid structure Figure 5 depicts the SEM micrographs of TG hydrogel and the hybrid scaffold comprising SF-Ge-PVA nanofibers on an EYO-TG hydrogel in both surface and cross-sectional views. The TG hydrogel exhibited a relatively smooth surface morphology, while the hybrid sample displayed increased surface roughness due to the nanofibrous layer. According to Figs. 5b and 5c, confirmed strong adhesion between the two layers of hybrid scaffold. It can be seen that the TG hydrogel penetrated into the pores of the SF-Ge-PVA nanofibrous mat prior to complete crosslinking, thereby forming a coherent bilayer structure. These results are consistent with the findings of Irantash et al., [21] who reported similar interpenetration of nanofibers into polysaccharide hydrogel matrices 3.3. FTIR results of SF-Ge-PVA Nanofibers Fig. 6A shows the FTIR spectra of SF, PVA, gelatin and the SF-Ge-PVA nanofibrous mat. For silk fibroin, characteristic peaks at 1642 cm⁻¹ (C=O stretching, amide I), 1530 cm⁻¹ (N–H bending, amide II), and 1233 cm⁻¹ (C–N stretching/C=O bending, amide III) confirm a random coil conformation [26]. Additional peaks at 1069 cm⁻¹ and 1441 cm⁻¹ correspond to C–O–C stretching and C–H bending vibrations, respectively. A broad band at 3000–3600 cm⁻¹ is assigned to N–H and O–H stretching [21]. The PVA spectrum exhibited a broad hydroxyl band centered at 3412 cm⁻¹, asymmetric and symmetric C–H stretching at 2920 cm⁻¹ and 2840 cm⁻¹, and a carbonyl band at 1730 cm⁻¹, attributable to residual acetate groups [21]. Gelatin displayed a strong N–H stretching band at 3284 cm⁻¹, indicating hydrogen bonding, and amide I, II, and III bands at 1636 cm⁻¹, 1539 cm⁻¹, and 1237 cm⁻¹, respectively [10, 27]. The FTIR spectrum of the SF-Ge-PVA nanofibrous mat (Fig. 6A) showed characteristic peaks of all three components with slight shifts, indicating the formation of physical interactions, particularly hydrogen bonding, between functional groups. A broad absorption band centered at 3429 cm⁻¹ corresponds to the overlapping O–H and N–H stretching vibrations of hydroxyl and amine groups. As a result of hydrogen bond formation, the ester C=O stretching of PVA, the amide I (C=O stretching) of gelatin and SF, and the amide II (N–H bending) of both gelatin and SF shifted to 1720, 1647, and 1533 cm⁻¹, respectively. A peak observed at 1244 cm⁻¹ can be assigned to C–O stretching in esters or alcohols and may also correspond to amide III (C–N stretching) vibrations. Together, these spectra confirm the coexistence of SF, PVA, and gelatin within the hybrid scaffold. Fig. 6B presents the spectra of TG, EYO, and the EYO-loaded TG hydrogel. TG displayed characteristic absorptions near 3312 cm⁻¹ (O–H stretching) and 1621 cm⁻¹ (carboxylate stretching and H–O–H bending), which shifted to 3296 and 1632 cm⁻¹, respectively, in the EYO-loaded hydrogel. This shift confirms intermolecular interactions between TG hydroxyl/carboxylate groups and the N–H/C=O groups of EYO, in agreement with earlier findings [19]. TG also exhibited peaks associated with C–H stretching in methylene groups, asymmetric and symmetric stretching of the carboxylate anion, and a pyranose ring vibration at 632 cm⁻¹, which shifted to 627 cm⁻¹ in the EYO-loaded hydrogel [21]. The FTIR spectrum of EYO showed features typical of lipids and proteins: a broad O–H/N–H stretching band at 3294 cm⁻¹, strong symmetric and asymmetric C–H stretching at 2850 and 2919 cm⁻¹, and an ester C=O stretching peak at 1742 cm⁻¹, slightly shifted to 1744 cm⁻¹ in the hydrogel. These observations confirm the successful incorporation of EYO into the TG hydrogel, consistent with previous reports [19]. 3.4. Surface hydrophilicity and swelling behavior Surface wettability as an important property that actively affects biological properties such as cell adhesion and compatibility was evaluated by contact angle test and the results can be seen in Figure 7A and B. Both hydrogel-only and hybrid scaffolds exhibited significant hydrophilicity, with contact angles below 60°. The hybrid scaffold showed lower contact angles than the hydrogel alone, likely due to the increased surface roughness imparted by the nanofibrous layer (SEM images, Fig. 4). Such hydrophilic surfaces are beneficial for wound dressings, especially in burn care where moisture balance is crucial. These results are consistent with the findings of Mohammadi et al. [28]. Swelling studies (Fig. 7C) revealed water uptake capacities of 1106% for TG hydrogel and 902% for the hybrid scaffold after 24 h. These high swelling values indicate that both systems can absorb substantial wound exudate, maintaining a moist healing environment and minimizing desiccation-related complications. The slightly lower swelling of the hybrid reflects the structural contribution of the nanofibrous layer, which restricts excessive hydrogel expansion while maintaining fluid absorption—an advantage for handling and stability. Similar results have been reported for other polysaccharide-based hydrogels with nanofibrous reinforcements [29]. 3.5. Drug Release Results The release of EYO from the TG hydrogel was monitored by UV–Vis spectroscopy over 72 h (Fig. 7D). Based on the reports, crosslinked polysaccharide based hydrogels can provide a proper substrate for encapsulating oily extracts [30]. The release curve exhibited a biphasic pattern. An initial slow diffusion phase released ~22% of the encapsulated oil within the first 18 h. This was followed by a more pronounced release, reaching 56% at 24 h, before gradually plateauing at ~88% by 72 h. This behavior reflects a combination of diffusion through the swollen hydrogel matrix and polymer relaxation/degradation, typical of polysaccharide hydrogels [29]. Such sustained release is advantageous in wound management, providing prolonged antibacterial and regenerative activity at the wound site without repeated dressing changes [31]. 3.5. Antibacterial Results Antibacterial performance was assessed against E. coli (Gram-negative) and S. aureus (Gram-positive) using colony reduction assays (Table 1). Even the unloaded hybrid scaffold showed notable antibacterial effects, particularly against S. aureus , likely attributable to the inherent antibacterial activity of SF [16]. Upon EYO loading, antibacterial efficacy was substantially enhanced: 96.1% reduction in E. coli colonies and 99.8% reduction in S. aureus . These findings highlight the synergistic antibacterial action of EYO with the SF-based matrix, in line with previous studies demonstrating the antimicrobial potency of egg yolk oil in burn wound models [31]. Table 1. Antibacterial activities of EYO-loaded and unloaded hybrid structures. Bacteria Type Material Type T₀ (CFU/mL) T 24 h (CFU/mL) Colony Reduction (%) E. coli Control 2.2 × 10⁸ 1.15 × 10⁸ 47.7% Unloaded hybrid scaffold 2.2 × 10⁸ 3.9 × 10⁷ 82.2% EYO-loaded hybrid scaffold 2.2 × 10⁸ 8.5 × 10⁶ 96.1% S. aureus Control 3.9 × 10⁸ 3.1 × 10⁸ 20.5% Unloaded hybrid scaffold 3.9 × 10⁸ 4.7 × 10⁶ 98.76% EYO-loaded hybrid scaffold 3.9 × 10⁸ 7.9 × 10⁵ 99.79% 3.6. Cell viability and attachment Biocompatibility was evaluated using MTT assays with HFF-2 fibroblasts (Fig. 8A). Both unloaded and EYO-loaded hybrid scaffolds exhibited excellent cell viability, with no cytotoxic effects compared to controls. Although at concentrations of 1 to 5 μg/mL, cell viability in the unloaded sample was significantly higher than that in the oil-containing sample, with increasing concentration from 5 to 100 μg/mL, cell viability on the EYO-loaded scaffold showed a further increase. This suggests that EYO incorporation does not impair, and may even promote, fibroblast growth at therapeutically relevant concentrations. These observations are consistent with previous reports of EYO-enhanced wound healing [19, 32]. SEM images (Fig. 8B,C) further confirmed favorable cell–material interactions. Fibroblasts adhered well, flattened, and spread extensively on the nanofibrous surface of the hybrid scaffold, resembling behavior on natural ECM. Enhanced attachment and spreading, together with high viability, demonstrate that the hybrid construct provides a supportive environment for dermal regeneration. 4. Conclusion A bilayer hybrid scaffold composed of SF–Ge–PVA nanofibers and an EYO-loaded TG hydrogel was successfully fabricated and characterized as a candidate wound dressing. Optimal electrospinning conditions (18 kV, 12 cm) produced bead-free, uniform nanofibers. FTIR confirmed the coexistence of all polymeric and bioactive components with hydrogen bonding interactions. Contact angle and swelling analyses demonstrated favorable hydrophilicity and high water uptake, ensuring a moist healing environment. Controlled EYO release provided sustained delivery, while antibacterial assays revealed nearly complete elimination of S. aureus and strong inhibition of E. coli . Cytocompatibility and SEM analyses confirmed excellent fibroblast viability and attachment. Taken together, these findings establish the SF-Ge-PVA/EYO-TG bilayer scaffold as a promising material for burn wound healing, combining structural support, moisture regulation, antibacterial protection, and cytocompatibility. Declarations Author Contributions Parisa Malekian-Nouri performed experiments and acquisition of data and contributed to write the draft and final approval. Adeleh Gholipour-Kanani contributed to supervision, conception and design of the study, preformed interpretation of data and writing the manuscript as well as revising the draft and final approval. Additional information The authors declare no competing interests. Funding Declaration There is no Funding to declare. Data Availability All data generated or analyzed during this study are included in this published article. References Hama, R., Reinhardt, J. W., Ulziibayar, A., Watanabe, T., Kelly, J., Shinoka, T. 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PH-sensitive starch-based hydrogels: Synthesis and effect of molecular components on drug release behavior, Polymers, 12(9) , 1974 (2020). https://doi.org/10.3390/polym12091974 [31] Rastegar, F., Azarpira, N., Amiri, M., Azarpira, A. The effect of Egg Yolk Oil in the Healing of Third degree burns in Rat, Iranian Red Crescent Medical Journal, 13(10), 739 -743 (2011). Yenilmez E, Başaran E, Arslan R, Berkman MS, Güven UM, Bayçu C, Yazan Y. Chitosan gel formulations containing egg yolk oil and epidermal growth factor for dermal burn treatment. Pharmazie . 70(2) , 67-73 (2015). Additional Declarations No competing interests reported. Supplementary Files GraphicalAbstract.tif Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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10:48:04","extension":"html","order_by":54,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":119052,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7734190/v1/da512c827ac47f7a93b31e7f.html"},{"id":93926875,"identity":"0d507011-72e4-4175-8b65-000705a4cd1c","added_by":"auto","created_at":"2025-10-20 10:48:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2141473,"visible":true,"origin":"","legend":"\u003cp\u003eThe extraction process of silk fibroin (SF) from silk cocoons.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7734190/v1/f59f95fedc2c57b65a14509e.png"},{"id":93926876,"identity":"095def96-a5c1-42ff-a09d-e77cf3c6f4cb","added_by":"auto","created_at":"2025-10-20 10:48:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1283021,"visible":true,"origin":"","legend":"\u003cp\u003ePreparation of the hybrid structure: a) SF-Ge-PVA nanofibers/TG hydrogel and b) SF-Ge-PVA nanofibers/EYO-loaded TG hydrogel.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7734190/v1/34dee80dedb672364b3400ec.png"},{"id":93926880,"identity":"f15ffdd8-781d-46cc-916c-21f6c5702408","added_by":"auto","created_at":"2025-10-20 10:48:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6346191,"visible":true,"origin":"","legend":"\u003cp\u003eThe SEM images of SF-Ge-PVA nanofibers with different volume ratios of: a) 1:1:1 and b) 1:1:2 (SF:Ge:PVA) under constant electrospinning conditions (voltage of 15 kV and distance of 12 cm); 5000x, 10000 x.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7734190/v1/1befdfdf4b4113313ad721ea.png"},{"id":93927663,"identity":"2abaacbe-bd2b-41dc-a334-bdd272c21052","added_by":"auto","created_at":"2025-10-20 10:56:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5814929,"visible":true,"origin":"","legend":"\u003cp\u003eThe SEM images of SF-Ge-PVA (1:1:2) nanofibers electrospun at 15 and 18 kV and 12 and 15 cm nozzle to collector distance (10000x).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7734190/v1/d0ef713e4335c6eabcfd9c83.png"},{"id":93927661,"identity":"3198c256-4545-4491-b32a-a4e5a6e79985","added_by":"auto","created_at":"2025-10-20 10:56:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1407262,"visible":true,"origin":"","legend":"\u003cp\u003eThe SEM images of a) surface of EYO-loaded TG hydrogel; b) surface and c) cross-section of the hybrid structure (SF-Ge-PVA nanofibers/ EYO-loaded TG hydrogel).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7734190/v1/474e03a0b0c1a1b42171785c.png"},{"id":93926903,"identity":"3ed6d937-9d83-4136-a078-1136838e90a2","added_by":"auto","created_at":"2025-10-20 10:48:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4153993,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of A) silk fibroin (SF), PVA, Gelatin (Gث) and SF-Ge-PVA nanofibers and B) Egg yolk oil (EYO), Tragacanth (TG) and EYO-loaded TG.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7734190/v1/e0c6afe25ea832656791574b.png"},{"id":93926884,"identity":"2fb6a66f-8d40-41ce-b0af-56641ae8b113","added_by":"auto","created_at":"2025-10-20 10:48:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1471294,"visible":true,"origin":"","legend":"\u003cp\u003eContact angle images of A) TG hydrogel and B) The hybrid structure of SF-Ge-PVA nanofibers/EYO-TG hydrogel; C) Swelling percentage of the only hydrogel layer and nanofiber/hydrogel hybrid scaffold; D) Release profile of EYO from the hybrid scaffold.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7734190/v1/fa0812e161a62965e7f23d27.png"},{"id":93927936,"identity":"5a6971bc-d2d6-48ca-bea2-7f796226cb01","added_by":"auto","created_at":"2025-10-20 11:04:02","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3749691,"visible":true,"origin":"","legend":"\u003cp\u003eA) MTT assay and HFF-2 cell viability after 48 hours exposed both EYO loaded and unloaded hybrid scaffold under different concentrations; SEM images of cell adhesion and proliferation on the surface of B) the hydrogel and C) the nanofibers/hydrogel hybrid scaffold.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-7734190/v1/957cb6efbab837046d031ff6.png"},{"id":109068292,"identity":"2f237d3d-4130-4052-b7b5-517d5581bfef","added_by":"auto","created_at":"2026-05-12 10:05:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":25851324,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7734190/v1/b8651649-b1bd-4a37-b49c-6a5a2425f3a9.pdf"},{"id":93926878,"identity":"5d5b3ea0-92c6-4684-a1b2-fd3cb0457071","added_by":"auto","created_at":"2025-10-20 10:48:02","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2299336,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-7734190/v1/3e247fa70f569f965f4134d6.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"A Hybrid Bi-layered Scaffold from Silk Fibroin-Gelatin nanofibers and Egg yolk oil-loaded Tragacanth Hydrogel as a promising wound dressing","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe clinical priority for tissue regeneration of different kinds of skin defects like major traumatic wounds, burns, pressure ulcers, and chronic diabetic wounds has increased substantially in recent times. Although, cellular sheets and cultured scaffolds represent current medical approaches, but they have not successfully rebuilt the entire natural structure and functional aspects of skin tissue [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Over the past decades, tissue engineering approaches have focused on mimicking the extracellular matrix (ECM) environment to guide cellular repair [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In particular, biomaterial scaffolds that replicate the composition and three-dimensional fibrous network of the dermal ECM can direct cell adhesion, migration, and differentiation in wound sites [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Thus, there is growing interest in designing materials that reproduce the nano-scale structure and biochemical cues of native skin to improve healing outcomes.\u003c/p\u003e\u003cp\u003eElectrospun nanofibrous membranes possess high porosity and large specific surface area, providing an optimal environment for the complex and dynamic wound healing process and numerous sites for carrying wound healing agents. Such nanofibrous mats have an extremely large surface area and pore volume, creating a microenvironment that can support complex healing processes and accommodate high loadings of bioactive molecules [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Furthermore, the fine fibrous structure closely resembles the dermal ECM, which has been shown to enhance hemostasis, reduce inflammation, and accelerate tissue repair [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Polymer biomaterials demonstrate excellent biocompatibility and biodegradability, together with native ECM-like morphology and suitable physicochemical mechanical and biological controls, which help establish an optimal wound-healing microenvironment [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Burn healing and skin tissue repairing face significant challenges in medical science such as severe infection, surface dehydration and scar formation. Modern wound dressings are thus designed to fulfill key functions \u0026ndash; they should maintain a moist, oxygenated environment, absorb exudate, allow gas exchange, and provide a barrier against infection [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Combinations of fiber mats and hydrogels have gained attention because they can incorporate multiple therapeutic functions (e.g. antimicrobial agents, growth factors, antioxidants) and thus accelerate healing while minimizing scar formation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNatural biopolymers in particular are attractive for skin repair because they inherently resemble ECM components and support cell function [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Examples include protein-based materials (e.g. collagen [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], keratin [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], gelatin [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and silk fibroin [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]) and polysaccharides (e.g. chitosan [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], alginate [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], tragacanth gum [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]). Gelatin as a collagen-derivative possesses most of collagen properties such as its biocompatibility and gel-forming ability [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, as a low-cost, non-immunogenic material, gelatin promotes hemostasis and absorbs wound exudate in the inflammatory phase [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. It made of composition of amino acids such as proline, glycine and hydroxyproline which can mimic the ECM fibril structure similar to collagen [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. So, it can stimulate the fibroblast migration to enhance the formation of granulation tissue in proliferation phase of healing process [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In chronic wound models, gelatin-based matrices have been shown to enhance platelet aggregation and support vascular and epithelial cell regeneration through its hydrophilic and bioactive properties as well as non-antigenicity [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Silk-fibroin as a protein derived from natural silk has been considered in wound healing application due to its highly anti-inflammatory and pro-angiogenic properties followed by sufficient biocompatibility and biodegradability [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Numerous studies have shown that SF accelerates wound closure by stimulating angiogenesis and modulating inflammation; compared to many conventional dressings, SF-based materials significantly speed skin regeneration [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Moreover, bilayer skin-like scaffolds incorporating silk fibroin have demonstrated scar-inhibition effects in animal models. For example, a recent study reported by Zhou et al. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] showed that the SF\u0026ndash;hyaluronic acid bilayer scaffold promoted full-thickness skin regeneration while reducing wound contraction and abnormal collagen deposition, thereby suppressing hypertrophic scar formation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. A research by Yerra et al. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] developed a nanofibrous scaffold based on silk fibroin loaded with antibiotics for burn wound treatment by showing suitable cell adherence properties and a proper morphology for wound dressing. These data highlight SF\u0026rsquo;s potential both to rebuild dermal structure and to improve healing quality.\u003c/p\u003e\u003cp\u003ePolysaccharide-based hydrogels also play a crucial role in advanced wound dressings. One promising example is Tragacanth gum (TG), a natural hydrocolloid derived from \u003cem\u003eAstragalus\u003c/em\u003e plants. TG is a highly branched, anionic polysaccharide containing galacturonic acid, xylose, arabinose, galactose, and so on [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. It shows inherent properties in wound healing process due to its biocompatibility, biodegradability, ability to create a moist environment and promote cell growth [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Recent studies have demonstrated that TG-based hydrogels can naturally accelerate wound closure and improve tissue regeneration in vivo, while also lowering infection risk (likely due to TG\u0026rsquo;s ability to incorporate antimicrobial agents) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In addition, as a natural hydrogel, tragacanth has a great potential to be an excellent carrier matrix. It can encapsulate drugs, growth factors, or bioactive extracts for sustained release at the wound site [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBioactive lipids and natural extracts are often incorporated into dressings to further enhance healing. Egg yolk oil (EYO) as a traditional remedy for third degree burns has been considered due to its anti-inflammatory, anti-microbial and tissue regenerative properties [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. It contains saturated and unsaturated fatty acids, fat miscible vitamins, phospholipids, immunoglobulin, antioxidants and cholesterol [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Shadman-manesh et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] developed a nanofibrous scaffold based on PCL which was loaded with EYO for third degree burns in rat models. From in vivo results, they concluded that the rate of wound closure, re-epithelialization and collagen deposition after 21-day treatment with EYO-loaded-scaffold were improved significantly.\u003c/p\u003e\u003cp\u003eThis study has developed a new bi-layered scaffold based on gelatin-silk fibroin nanofibers combined with egg yolk oil loaded-tragacanth hydrogel layered as a promising structure for burn wound treatment. The inner layer is an electrospun nanofibrous mat composed of gelatin and silk fibroin, chosen to mimic the dermal ECM structure and to leverage the inherent wound-healing activity of these proteins. The fibrous mat provides mechanical strength, cellular attachment sites, and hemostatic capability. The outer layer is a TG hydrogel loaded with egg yolk oil, forming a moist, protective covering. This hydrogel maintains wound hydration and slowly releases EYO\u0026rsquo;s antibacterial and regenerative factors. In this design, the two layers work in concert: the top hydrogel blocks dehydration and infection, while the bottom fibrous layer promotes cell adhesion and tissue regeneration. The hybrid scaffolds have been characterized using different physicochemical studies such as FESEM, FTIR, contact angle, swelling and release study as well as in vitro studies including cytocompatibility, fibroblast adhesion and proliferation, and antibacterial activity to investigate their efficiency for skin repairing and wound healing applications.\u003c/p\u003e\u003cp\u003eIn summary, this study presents a fully biomimetic, multifunctional bilayer dressing for burn wounds, combining gelatin/SF nanofibers with an EYO-loaded TG hydrogel. By uniting the favorable properties of these natural materials, the scaffold is expected to provide a moist, antimicrobial, and ECM-like environment that supports rapid, scar-minimizing skin repair.\u003c/p\u003e"},{"header":"2. Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. Materials\u003c/h2\u003e\n \u003cp\u003eTragacanth (TG) and Silk (\u003cem\u003eBombyx mori\u003c/em\u003e) cocoons were purchased from Attarak Company (Iran). Egg yolk oil was purchased from Giah Taghdis Company (Iran). Gelatin (Ge) (type B, CAS No.: 9000-70-8) and glutaraldehyde (GA) were purchased from Sigma-Aldrich (Germany). Polyvinyl alcohol (PVA) (M\u003csub\u003ew\u003c/sub\u003e=72000 g.mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), calcium chloride dehydrate, lithium bromide (LiBr) and sodium carbonate (Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e) were supplied by Merck (USA).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. Extraction of silk fibroin\u003c/h2\u003e\n \u003cp\u003eExtraction of silk fibroin from silk cocoons was performed according to the guidelines reported by Cao et al. [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. Briefly, 5 g of crushed, dried \u003cem\u003eB. mori\u003c/em\u003e cocoons were boiled in 0.02 M Na₂CO₃ solution for 20 min to remove sericin. The fibers were rinsed three times with cold distilled water and dried at ambient temperature for 24 h. The degummed silk was dissolved in 9.3 M LiBr at 60\u0026deg;C for 4 h with stirring. The resulting solution was dialyzed (molecular weight cutoff: 12\u0026ndash;14 kDa) against deionized water for 3 days, with water changes every 6 h, to remove residual LiBr. The dialyzed solution was centrifuged at 9000 rpm for 20 min at 4\u0026deg;C to yield a concentrated SF solution, which was stored at 4\u0026deg;C until use. The process is shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. The extracted fibroin was analyzed in our previous study [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3. Preparation of SF-Ge-PVA nanofibers\u003c/h2\u003e\n \u003cp\u003eAqueous solutions of 10% (w/v) Ge and 10% (w/v) PVA were prepared at 40\u0026deg;C and blended at Ge:PVA ratios of 1:1 and 1:2 (v/v). SF solution was subsequently added to achieve SF:Ge:PVA ratios of 1:1:1 and 1:1:2 (v/v/v). The mixtures were stirred at 25\u0026deg;C for 2 h to obtain clear, homogeneous solutions, which were loaded into syringes for electrospinning. Electrospinning was performed using a conventional setup (FNM Duos Electroris, HV35P OV, Fanavaran Nanomeghyas Co., Iran) under voltages of 15 kV and 18 kV, nozzle-to-collector distances of 12 cm and 15 cm, and a flow rate of 1 mLh⁻\u0026sup1;.\u003c/p\u003e\n \u003cp\u003eTo improve structural stability in aqueous media, fibers were crosslinked via glutaraldehyde (GA) vapor treatment. Following a previous report [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e], nanofibrous mats were exposed to the vapor of 30 mL GA in a sealed desiccator for 18 h at room temperature. Crosslinked mats were washed three times with phosphate-buffered saline (PBS) and dried in a vacuum oven at 40\u0026deg;C for 2 h.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4. Preparation of SF-Ge-PVA nanofibers/EYO-loaded TG hydrogel hybrid\u003c/h2\u003e\n \u003cp\u003eA 4 wt% TG solution was prepared by dissolving TG in distilled water at 40\u0026deg;C with stirring for 3 h. EYO (15 wt% of the dried polymer weight) was incorporated and stirred for 20 min. Subsequently, CaCl₂ (1.5 wt%) was added as a crosslinker, and the solution was stirred for an additional 30 min. The mixture was cast into 80 mm Petri dishes (4 mm thickness) and dried at 50\u0026deg;C for 1 h in a vacuum oven. Finally, the crosslinked SF-Ge-PVA nanofibrous mat was placed on the surface of the hydrogel and left to dry overnight at room temperature to form the hybrid bilayer scaffold. Figure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e shows the preparation of both EYO loaded and unloaded hybrids.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5. Physico-chemical and morphological characterization\u003c/h2\u003e\n \u003cp\u003eATR-FTIR (Attenuated Total Reflection Fourier Transform Infrared) spectroscopy was performed using EQUINOX 55 spectrophotometer (Bruker, Germany) to study the chemical bonds of the extracted SF and the hybrid scaffolds. Briefly, the analyses was used to investigate the interactions between components in the scaffolds using KBr tablets in the range of 4000\u0026ndash;400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eMorphological studies were performed using field emission scanning electron microscopy (FESEM, XL30 PHILIPS SEM, Scanning electron microscope) to determine the optimum conditions of the electro spun nanofibers and hybrid hydrogels. Digimizer software (v. 6.3.0) was used to measure the average fiber diameters.\u003c/p\u003e\n \u003cp\u003eContact angle measurements were performed according to ASTM D7334-08 to investigate the hydrophilic properties of the produced scaffolds using a static optical contact angle measurement setup (CAG-10, Jikan, Iran) by the sessile drop method. A 1cm\u0026times;1cm piece of each scaffolds was cut and placed on the instrument\u0026rsquo;s sample holder and exposure to a single droplet of distilled water (volume\u0026sim;2\u0026micro;l). The shape of the drops on the scaffold surfaces was recorded using the camera attached to the drop shape analyzer after 3 s. The test was repeated three times and the average value was reported.\u003c/p\u003e\n \u003cp\u003eSwelling studies were carried on both TG hydrogel and the hybrid scaffold to measure the water uptake capacity of the scaffolds. Regards, the samples were cut into squares with an area of 1 cm\u003csup\u003e2\u003c/sup\u003e and dried at 60\u0026deg;C until constant weight (\u003cem\u003eW\u003c/em\u003e\u003csub\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sub\u003e) was obtained. The specimens were placed in 10 ml water (7.4 pH and 37\u0026deg;C) during 24 h and removed in two time points of 12 and 24 h and after drying their surface they were weighted (\u003cem\u003eW\u003c/em\u003e\u003csub\u003e\u003cem\u003es\u003c/em\u003e\u003c/sub\u003e). The swelling ratio (SR) was calculated using the following Eq.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, where, \u003cem\u003eW\u003c/em\u003e\u003csub\u003e\u003cem\u003ed\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eW\u003c/em\u003e\u003csub\u003e\u003cem\u003es\u003c/em\u003e\u003c/sub\u003e were the weights of dried and swollen samples, respectively.\u003c/p\u003e\n \u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\n \u003cdiv class=\"EquationNumber\"\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6. Release study\u003c/h2\u003e\n \u003cp\u003eEYO release from hydrogels was evaluated using UV\u0026ndash;Vis spectroscopy (UNICO, USA). Pre-weighed EYO-loaded hydrogels were immersed in 2 mL PBS (pH 7.4) at 37\u0026deg;C. At predetermined intervals (0.5, 1, 2, 4, 6, 12, 24, 48, and 72 h), 1 mL of medium was withdrawn and replaced with fresh PBS. Absorbance was recorded between 270\u0026ndash;480 nm. Each experiment was performed in triplicate.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7. Antibacterial activity\u003c/h2\u003e\n \u003cp\u003eThe antibacterial activities of both EYO-loaded and unloaded bi-layered scaffolds were evaluated against \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e, ATCC 25922) as a gram-negative bacteria and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (\u003cem\u003eS. aureus\u003c/em\u003e, ATCC 6538) as a gram-positive bacteria which were purchased from Pasteur Institute of Iran (Tehran, Iran). In this study, the logarithmic count reduction test was performed by the microbial count method according to ASTM E2180 to examine the antibacterial activities of the scaffolds. In order to perform the analysis, the bacterial strains were grown on Mueller Hinton Agar medium plates (Merck, Germany) at 37\u0026deg;C for 18 h. Using distilled salinized water (0.9% NaCl), suspensions from \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e at a final concentration of 2.2 \u0026times; 10⁸ CFU/mL and 3.9 \u0026times; 10⁸ CFU/mL were prepared respectively. Each hybrid scaffold was cut into a 10 mm in diameter discs and exposing to the prepared bacteria suspensions in petri dishes. The petri dishes were incubated for 24 h at 37˚C. Each experiment was performed in triplicate.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8. Cytotoxicity and cell attachment\u003c/h2\u003e\n \u003cp\u003eCytotoxicity was evaluated via MTT assay using human foreskin fibroblast cells (HFF-2; purchased from Pasteur Cell Bank (Pasteur Institute, Tehran, Iran) \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://en.pasteur.ac.ir/Department-of-Cell-Bank\u003c/span\u003e\u003c/span\u003e). Cells (1 \u0026times; 10⁴ per well) were seeded in 96-well plates in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 50 U\u0026middot;mL⁻\u0026sup1; penicillin, and 50 U\u0026middot;mL⁻\u0026sup1; streptomycin, and incubated at 37\u0026deg;C for 24 h. Extracts from scaffolds (90 \u0026micro;L) and 10 \u0026micro;L FBS were added and incubated for another 24 h. MTT reagent (0.5 mg\u0026middot;mL⁻\u0026sup1;, 100 \u0026micro;L) was then added for 4 h. The formazan crystals were dissolved in isopropanol, and absorbance was measured at 570 nm using a microplate reader (BioTek ELx808, USA). Cell viability (%) was calculated relative to control wells by using Eq.\u0026nbsp;2.\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e\n \u003cp\u003eFor cell attachment, scaffolds seeded with HFF-2 were fixed after 3 days with 3.5% GA for 2 h at 4\u0026deg;C, dehydrated in graded ethanol (50\u0026ndash;96%), air-dried, and imaged by SEM.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.9. Statistical analysis\u003c/h2\u003e\n \u003cp\u003eAll data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analyses were performed using SPSS v16.0. One-way ANOVA with Tukey\u0026rsquo;s post hoc test was applied, and differences were considered significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cp\u003e\u003cstrong\u003e3.1. Morphological Analysis of SF-Ge-PVA Nanofibers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDue to the negative electrostatic charge within silk fibroin chains in aqueous solutions, and the repulsion of like charges in an electric field, the possibility of electrospinning it purely is very poor [22]. Blending SF with a carrier polymer such as polyvinyl alcohol (PVA) is a common strategy to increase chain entanglement and improve spinnability. Fig. 3 presents SEM micrographs of SF:Ge:PVA nanofibers prepared at blend ratios of 1:1:1 and 1:1:2 (v/v/v) electrospun at an applied voltage of 15 kV and a nozzle-to-collector distance of 12 cm.\u0026nbsp;\u003cbr\u003e\u0026nbsp;Increasing the PVA fraction yielded smoother fibers with greater morphological uniformity [23]. Concomitantly, the mean fiber diameter decreased from 402 \u0026plusmn; 54 nm (Fig. 3a) to 294 \u0026plusmn; 37 nm (Fig. 3b). This trend is consistent with modest reductions in effective solution viscosity and enhanced jet stretching in the electric field (within a regime that avoids bead formation), in line with general electrospinning principles and prior reports on SF-blend systems [24]. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe blend ratio of 1:1:2 (SF:Ge:PVA) was selected for further optimization of electrospinning conditions. In order to evaluate the influence of voltage and nozzle-to-collector distance, fibers were electrospun at applied voltages of 15 kV and 18 kV and distances of 12 cm and 15 cm and the SEM results are shown in figure 4.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAt a constant distance of 12 cm, increasing the applied voltage from 15 kV to 18 kV significantly reduced the mean fiber diameter from 294 \u0026plusmn; 37 nm to 201 \u0026plusmn; 18 nm (Figs. 4a and 4b). A similar trend was observed at 15 cm, where fiber diameter decreased under higher voltage (Figs. 4c and 4d). This behavior can be explained by the dominant effect of increased electric field strength, which enhances jet acceleration and stretching, overriding the tendency for a thicker jet at higher voltages. Stronger fields promote more intense whipping instabilities, resulting in finer and more uniform fibers [25]. Under the condition of 18 kV and 12 cm, highly uniform fibers with smooth morphology were obtained, representing the optimal electrospinning condition.\u003c/p\u003e\n\u003cp\u003eComparison of Figs. 4a vs. 4c and 4b vs. 4d illustrates the influence of increasing the nozzle-to-collector distance at constant voltage. At 18 kV, increasing the distance from 12 cm to 15 cm led to an increase in fiber diameter from 201 \u0026plusmn; 18 nm to 298 \u0026plusmn; 30 nm. This is attributed to the reduction in effective field strength at greater distances, leading to less jet stretching during flight and consequently thicker fibers [25]. These findings confirm that an applied voltage of 18 kV and a collection distance of 12 cm represent the most favorable electrospinning conditions for producing uniform SF:Ge:PVA fibers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. Morphological study of TG hydrogel and the hybrid structure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 5 depicts the SEM micrographs of TG hydrogel and the hybrid scaffold comprising SF-Ge-PVA nanofibers on an EYO-TG hydrogel in both surface and cross-sectional views. The TG hydrogel exhibited a relatively smooth surface morphology, while the hybrid sample displayed increased surface roughness due to the nanofibrous layer.\u003c/p\u003e\n\u003cp\u003eAccording to Figs. 5b and 5c,\u0026nbsp;confirmed strong adhesion between the two layers of hybrid scaffold. It can be seen that the TG hydrogel penetrated into the pores of the SF-Ge-PVA nanofibrous mat prior to complete crosslinking, thereby forming a coherent bilayer structure. These results are consistent with the findings of Irantash et al., [21] who reported similar interpenetration of nanofibers into polysaccharide hydrogel matrices\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. FTIR results of SF-Ge-PVA Nanofibers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFig. 6A shows the FTIR spectra of SF, PVA, gelatin and the SF-Ge-PVA nanofibrous mat. For silk fibroin, characteristic peaks at 1642 cm⁻\u0026sup1; (C=O stretching, amide I), 1530 cm⁻\u0026sup1; (N\u0026ndash;H bending, amide II), and 1233 cm⁻\u0026sup1; (C\u0026ndash;N stretching/C=O bending, amide III) confirm a random coil conformation [26]. Additional peaks at 1069 cm⁻\u0026sup1; and 1441 cm⁻\u0026sup1; correspond to C\u0026ndash;O\u0026ndash;C stretching and C\u0026ndash;H bending vibrations, respectively. A broad band at 3000\u0026ndash;3600 cm⁻\u0026sup1; is assigned to N\u0026ndash;H and O\u0026ndash;H stretching [21]. The PVA spectrum exhibited a broad hydroxyl band centered at 3412 cm⁻\u0026sup1;, asymmetric and symmetric C\u0026ndash;H stretching at 2920 cm⁻\u0026sup1; and 2840 cm⁻\u0026sup1;, and a carbonyl band at 1730 cm⁻\u0026sup1;, attributable to residual acetate groups [21]. Gelatin displayed a strong N\u0026ndash;H stretching band at 3284 cm⁻\u0026sup1;, indicating hydrogen bonding, and amide I, II, and III bands at 1636 cm⁻\u0026sup1;, 1539 cm⁻\u0026sup1;, and 1237 cm⁻\u0026sup1;, respectively [10, 27].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe FTIR spectrum of the SF-Ge-PVA nanofibrous mat (Fig. 6A) showed characteristic peaks of all three components with slight shifts, indicating the formation of physical interactions, particularly hydrogen bonding, between functional groups. A broad absorption band centered at 3429 cm⁻\u0026sup1; corresponds to the overlapping O\u0026ndash;H and N\u0026ndash;H stretching vibrations of hydroxyl and amine groups. As a result of hydrogen bond formation, the ester C=O stretching of PVA, the amide I (C=O stretching) of gelatin and SF, and the amide II (N\u0026ndash;H bending) of both gelatin and SF shifted to 1720, 1647, and 1533 cm⁻\u0026sup1;, respectively. A peak observed at 1244 cm⁻\u0026sup1; can be assigned to C\u0026ndash;O stretching in esters or alcohols and may also correspond to amide III (C\u0026ndash;N stretching) vibrations. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTogether, these spectra confirm the coexistence of SF, PVA, and gelatin within the hybrid scaffold.\u003c/p\u003e\n\u003cp\u003eFig. 6B presents the spectra of TG, EYO, and the EYO-loaded TG hydrogel. TG displayed characteristic absorptions near 3312 cm⁻\u0026sup1; (O\u0026ndash;H stretching) and 1621 cm⁻\u0026sup1; (carboxylate stretching and H\u0026ndash;O\u0026ndash;H bending), which shifted to 3296 and 1632 cm⁻\u0026sup1;, respectively, in the EYO-loaded hydrogel. This shift confirms intermolecular interactions between TG hydroxyl/carboxylate groups and the N\u0026ndash;H/C=O groups of EYO, in agreement with earlier findings [19]. TG also exhibited peaks associated with C\u0026ndash;H stretching in methylene groups, asymmetric and symmetric stretching of the carboxylate anion, and a pyranose ring vibration at 632 cm⁻\u0026sup1;, which shifted to 627 cm⁻\u0026sup1; in the EYO-loaded hydrogel [21].\u003c/p\u003e\n\u003cp\u003eThe FTIR spectrum of EYO showed features typical of lipids and proteins: a broad O\u0026ndash;H/N\u0026ndash;H stretching band at 3294 cm⁻\u0026sup1;, strong symmetric and asymmetric C\u0026ndash;H stretching at 2850 and 2919 cm⁻\u0026sup1;, and an ester C=O stretching peak at 1742 cm⁻\u0026sup1;, slightly shifted to 1744 cm⁻\u0026sup1; in the hydrogel. These observations confirm the successful incorporation of EYO into the TG hydrogel, consistent with previous reports [19].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4. Surface hydrophilicity and swelling behavior\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSurface wettability as an important property that actively affects biological properties such as cell adhesion and compatibility was evaluated by contact angle test and the results can be seen in Figure 7A and B. Both hydrogel-only and hybrid scaffolds exhibited significant hydrophilicity, with contact angles below 60\u0026deg;. The hybrid scaffold showed lower contact angles than the hydrogel alone, likely due to the increased surface roughness imparted by the nanofibrous layer (SEM images, Fig. 4). Such hydrophilic surfaces are beneficial for wound dressings, especially in burn care where moisture balance is crucial. These results are consistent with the findings of Mohammadi et al. [28].\u003c/p\u003e\n\u003cp\u003eSwelling studies (Fig. 7C) revealed water uptake capacities of 1106% for TG hydrogel and 902% for the hybrid scaffold after 24 h. These high swelling values indicate that both systems can absorb substantial wound exudate, maintaining a moist healing environment and minimizing desiccation-related complications. The slightly lower swelling of the hybrid reflects the structural contribution of the nanofibrous layer, which restricts excessive hydrogel expansion while maintaining fluid absorption\u0026mdash;an advantage for handling and stability. Similar results have been reported for other polysaccharide-based hydrogels with nanofibrous reinforcements [29].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5. Drug Release Results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe release of EYO from the TG hydrogel was monitored by UV\u0026ndash;Vis spectroscopy over 72 h (Fig. 7D). Based on the reports, crosslinked polysaccharide based hydrogels can provide a proper substrate for encapsulating oily extracts [30].\u0026nbsp;The release curve exhibited a biphasic pattern. An initial slow diffusion phase released ~22% of the encapsulated oil within the first 18 h. This was followed by a more pronounced release, reaching 56% at 24 h, before gradually plateauing at ~88% by 72 h. This behavior reflects a combination of diffusion through the swollen hydrogel matrix and polymer relaxation/degradation, typical of polysaccharide hydrogels [29]. Such sustained release is advantageous in wound management, providing prolonged antibacterial and regenerative activity at the wound site without repeated dressing changes [31].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5. Antibacterial Results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAntibacterial performance was assessed against \u003cem\u003eE. coli\u003c/em\u003e (Gram-negative) and \u003cem\u003eS. aureus\u003c/em\u003e (Gram-positive) using colony reduction assays (Table 1). Even the unloaded hybrid scaffold showed notable antibacterial effects, particularly against \u003cem\u003eS. aureus\u003c/em\u003e, likely attributable to the inherent antibacterial activity of SF [16]. Upon EYO loading, antibacterial efficacy was substantially enhanced: 96.1% reduction in \u003cem\u003eE. coli\u003c/em\u003e colonies and 99.8% reduction in \u003cem\u003eS. aureus\u003c/em\u003e. These findings highlight the synergistic antibacterial action of EYO with the SF-based matrix, in line with previous studies demonstrating the antimicrobial potency of egg yolk oil in burn wound models [31].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Antibacterial activities of EYO-loaded and unloaded hybrid structures.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBacteria Type\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMaterial Type\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eT₀ (CFU/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eT\u003csub\u003e24\u003c/sub\u003eh (CFU/mL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eColony Reduction (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e2.2 \u0026times; 10⁸\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e1.15 \u0026times; 10⁸\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e47.7%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003eUnloaded hybrid scaffold\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e2.2 \u0026times; 10⁸\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e3.9 \u0026times; 10⁷\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e82.2%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003eEYO-loaded hybrid scaffold\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e2.2 \u0026times; 10⁸\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e8.5 \u0026times; 10⁶\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e96.1%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003eControl\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e3.9 \u0026times; 10⁸\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e3.1 \u0026times; 10⁸\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e20.5%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003eUnloaded hybrid scaffold\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e3.9 \u0026times; 10⁸\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e4.7 \u0026times; 10⁶\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e98.76%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 198px;\"\u003e\n \u003cp\u003eEYO-loaded hybrid scaffold\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e3.9 \u0026times; 10⁸\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e7.9 \u0026times; 10⁵\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e99.79%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e3.6. Cell viability and attachment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBiocompatibility was evaluated using MTT assays with HFF-2 fibroblasts (Fig. 8A). Both unloaded and EYO-loaded hybrid scaffolds exhibited excellent cell viability, with no cytotoxic effects compared to controls.\u0026nbsp;Although at concentrations of 1 to 5 \u0026mu;g/mL, cell viability in the unloaded sample was significantly higher than that in the oil-containing sample, with increasing concentration from 5 to 100 \u0026mu;g/mL, cell viability on the EYO-loaded scaffold showed a further increase.\u0026nbsp;This suggests that EYO incorporation does not impair, and may even promote, fibroblast growth at therapeutically relevant concentrations. These observations are consistent with previous reports of EYO-enhanced wound healing [19, 32].\u003c/p\u003e\n\u003cp\u003eSEM images (Fig. 8B,C) further confirmed favorable cell\u0026ndash;material interactions. Fibroblasts adhered well, flattened, and spread extensively on the nanofibrous surface of the hybrid scaffold, resembling behavior on natural ECM. Enhanced attachment and spreading, together with high viability, demonstrate that the hybrid construct provides a supportive environment for dermal regeneration.\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eA bilayer hybrid scaffold composed of SF–Ge–PVA nanofibers and an EYO-loaded TG hydrogel was successfully fabricated and characterized as a candidate wound dressing. Optimal electrospinning conditions (18 kV, 12 cm) produced bead-free, uniform nanofibers. FTIR confirmed the coexistence of all polymeric and bioactive components with hydrogen bonding interactions. Contact angle and swelling analyses demonstrated favorable hydrophilicity and high water uptake, ensuring a moist healing environment. Controlled EYO release provided sustained delivery, while antibacterial assays revealed nearly complete elimination of \u003cem\u003eS. aureus\u003c/em\u003e and strong inhibition of \u003cem\u003eE. coli\u003c/em\u003e. Cytocompatibility and SEM analyses confirmed excellent fibroblast viability and attachment. Taken together, these findings establish the SF-Ge-PVA/EYO-TG bilayer scaffold as a promising material for burn wound healing, combining structural support, moisture regulation, antibacterial protection, and cytocompatibility.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParisa Malekian-Nouri performed experiments and acquisition of data and contributed to write the draft and final approval. Adeleh Gholipour-Kanani contributed to supervision, conception and design of the study, preformed interpretation of data and writing the manuscript as well as revising the draft and final approval.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;There is no Funding to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003cbr\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHama, R., Reinhardt, J. W., Ulziibayar, A., Watanabe, T., Kelly, J., Shinoka, T. 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Chitosan gel formulations containing egg yolk oil and epidermal growth factor for dermal burn treatment. \u003cem\u003ePharmazie\u003c/em\u003e. \u003cstrong\u003e70(2)\u003c/strong\u003e, 67-73 (2015).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Silk fibroin nanofibers, Tragacanth hydrogels, Egg yolk oil, Hybrid bi-layered scaffolds, Burn Dressing","lastPublishedDoi":"10.21203/rs.3.rs-7734190/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7734190/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHybrid wound dressings such as nanofibers-hydrogel structures have been developed to prevent infection and accelerate wound healing. The main objective of this study was to fabricate a hybrid scaffold composed of silk fibroin (SF)\u0026ndash;gelatin (Ge) nanofibers and tragacanth(TG) hydrogel loaded with egg yolk oil(EYO) for burn wound applications.\u003c/p\u003e\u003cp\u003eThe TG hydrogel, serving as a flexible drug carrier, was loaded with egg yolk oil, which is well known for its burn-healing efficacy. Silk fibroin was employed for its regenerative properties and low scarring potential. FESEM results revealed that SF-Ge-PVA nanofibers, prepared at a volume ratio of 1:1:2 and electrospun under 18kV with a 12cm nozzle-to-collector distance, exhibited bead-free and uniform morphology with an average diameter of 201\u0026thinsp;\u0026plusmn;\u0026thinsp;18 nm. Contact angle and swelling studies demonstrated that both the hydrogel layer and the hybrid nanofiber\u0026ndash;hydrogel structure were hydrophilic, with swelling ratios of 1106% and 902%, respectively. MTT assay results confirmed the absence of cytotoxicity in both EYO-loaded and unloaded hybrid structures. Furthermore, the SF-Ge-PVA nanofibers combined with EYO-loaded hydrogel exhibited excellent antibacterial activity against Gram-positive bacteria, achieving 99.97% elimination of bacterial colonies. Overall, these findings suggest that the developed hybrid scaffold is a highly promising material for burn wound healing applications.\u003c/p\u003e","manuscriptTitle":"A Hybrid Bi-layered Scaffold from Silk Fibroin-Gelatin nanofibers and Egg yolk oil-loaded Tragacanth Hydrogel as a promising wound dressing","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-20 10:47:57","doi":"10.21203/rs.3.rs-7734190/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"190109fe-c7cb-4b32-9891-7b2b6188afe2","owner":[],"postedDate":"October 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":56539015,"name":"Biological sciences/Biotechnology"},{"id":56539016,"name":"Physical sciences/Chemistry"},{"id":56539017,"name":"Physical sciences/Materials science"},{"id":56539018,"name":"Physical sciences/Nanoscience and technology"}],"tags":[],"updatedAt":"2026-05-12T05:42:47+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-20 10:47:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7734190","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7734190","identity":"rs-7734190","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}