Topically Applied Biopolymer-Based Tri-Layered Hierarchically Structured Nanofibrous Scaffold with a Self-Pumping Effect for Accelerated Full-Thickness Wound Healing in a Rat Model

Wound healing has grown to be a significant problem at a global scale. The lack of multifunctionality in most wound dressing-based biopolymers prevents them from meeting all clinical requirements. Therefore, a multifunctional biopolymer-based tri-layered hierarchically nanofibrous scaffold in wound dressing can contribute to skin regeneration. In this study, a multifunctional antibacterial biopolymer-based tri-layered hierarchically nanofibrous scaffold comprising three layers was constructed. The bottom and the top layers contain hydrophilic silk fibroin (SF) and fish skin collagen (COL), respectively, for accelerated healing, interspersed with a middle layer of hydrophobic poly-3-hydroxybutyrate (PHB) containing amoxicillin (AMX) as an antibacterial drug. The advantageous physicochemical properties of the nanofibrous scaffold were estimated by SEM, FTIR, fluid uptake, contact angle, porosity, and mechanical properties. Moreover, the in vitro cytotoxicity and cell healing were assessed by MTT assay and the cell scratching method, respectively, and revealed excellent biocompatibility. The nanofibrous scaffold exhibited significant antimicrobial activity against multiple pathogenic bacteria. Furthermore, the in vivo wound healing and histological studies demonstrated complete wound healing in wounded rats on day 14, along with an increase in the expression level of the transforming growth factor-β1 (TGF-β1) and a decrease in the expression level of interleukin-6 (IL-6). The results revealed that the fabricated nanofibrous scaffold is a potent wound dressing scaffold, and significantly accelerates full-thickness wound healing in a rat model.

healing [35]. AMX is a semi-synthetic antibiotic with a broad-spectrum activity against several Gram-positive and Gram-negative micro-organisms [36]. Nonetheless, several reports revealed that systemic administration of antibiotics did not have a significant outcome and was accompanied by several side effects. In addition, the local application of antibiotic powder at the wound site is easily detached from the wound area, causing severe inflammation [36]. The sustained release of antibiotics promoted wound healing by enhancing the nanofiber's ability to inhibit bacterial growth.
Due to their varying hydrophilicity and hydrophobicity, unidirectional fluids discharging from electrospun nanofibrous membranes have been extensively used in a variety of biomedical applications [37][38][39]. Utilizing the hydrophilic-hydrophobic gradient structure to generate an additional pressure difference between the hydrophobic region (middle layer) and the hydrophilic region (top layers) could be exploited to achieve unidirectional fluid discharge and self-pumping effects capable of absorbing the excessive wound exudates [40][41][42].
Nevertheless, the majority of electrospun nanofibrous membranes have a bi-layered structure, which limits their applications in preventing reverse osmosis [43]. Therefore, it is anticipated that tri-layered electrospun nanofibrous membranes accelerate the wound healing process due to their improved fluid pumping and the ability of reverse osmosis prevention [44]. Many researchers have developed a tri-layered nanofibrous composite for a wide variety of biomedical applications such as, wound healing [45][46][47], bone tissue regeneration [48], cardiac tissue engineering [49], and tendon rupture repair [50].
To our knowledge, this paper describes, for the first time, the fabrication and design of a tri-layered hierarchically structured nanofibrous scaffold with potent properties based on an electrospinning strategy and sequential layered stacking. The thin layer of electrospun nanofibrous membrane rapidly degrades. The bottom layer consists of a permeable thin layer of electrospun hydrophilic SF, which serves as a source of nutrition for the myofibroblasts differentiation and proliferation in the skin extracellular matrix (ECM). The middle layer consists of hydrophobic polymer PHB loaded with antimicrobial drug AMX, respectively, to offer prolonged sustained drug release at the wound site. The hydrophilic polymer COL on the top layer is a component that simulates the ECM to enhance cell growth and absorb the excessive exudates discharged by the unidirectional fluid discharge effect.
The designed tri-layered hierarchically structured nanofibrous scaffold exhibited prolonged drug release, slow in vitro degradation, and a potent antimicrobial effect. The scaffold also exhibited good in vitro biocompatibility. Furthermore, in vivo wound closure, histological and Q-RT-PCR studies on rat models treated with the tri-layered hierarchically structured nanofibrous scaffold demonstrated a complete wound closure, accompanied by increasing the expression level of TGF-β1 and alleviating the expression level of interleukin-6 (IL-6). As a result, we believe that the designed tri-layered hierarchically structured nanofibrous scaffold is a promising candidate for accelerated wound healing applications.

Animal and Ethical Approval
Using 21 adult male Wistar rats, the in vivo wound healing rating for the prepared nanofibers was determined (8 weeks old; 180-200 g). The in vivo experimentation complied with the guidelines and protocols and was confirmed by the Research Ethical Committee of Institutional Animal Care and Use Committee at Alexandria University (ALEXU-IACUC) with approval number; AU14-210126-3-3.

Preparation of SF
The SF was prepared according to the previous strategy conducted by Chen,W. et al. [46]. Briefly, raw silk cocoons were degummed in triplicate for 30 min in boiling water containing 0.5% (w/w) Na 2 CO 3 , followed by washing in distilled water each time. The degummed silk cocoons were dissolved in a CaCl 2 /H 2 O/EtOH solution (molar ratio 1:8:2) at 70 • C under continuous stirring for 1 h. In cellulose dialysis tubes with a 14 Kda cut-off, the solution was dialyzed against distilled water for 3 days at room temperature. Every 5 h, freshly distilled water was changed. The preparation steps ended with filtering, centrifugation, and freeze-drying the solution to obtain a regenerated sf sponge.

Preparation of COL from Tilapia Fish Skin
The fresh tilapia skin was purchased from a local fish market and stored at −20 • C until usage. Fins, fat, and muscle fragments were scraped from the skins before being cut into small sections (0.5 cm 2 × 0.5 cm 2 ) and mixed well. Following Treesin Potaros et al.'s method, acid-solubilized collagen was extracted from the skin of Tilapia fish [51]. Briefly, the fragments were dissolved in 10 volumes of 0.1 M NaOH, and the suspension was agitated with a magnetic stirrer overnight. The skin fragments were re-suspended after decanting in 20 volumes of 0.1 M NaOH solution. The alkaline-insoluble components were filtered through a cloth and repeatedly rinsed with distilled water to achieve a neutral pH. The insoluble parts of the collagen were removed using 10 volumes of 0.5 M acetic acid over the course of three days. Furthermore, the resulting viscous solution was centrifuged at 10,000× g for 20 min at 4 • C. The residue was extracted again using 10 volumes of 0.5 M acetic acid for three days, and the extract was then centrifuged. The two extracts' supernatants were mixed and salted by adding NaCl at a final concentration of 0.9 M. The precipitate was obtained by centrifuging at 10,000× g for 20 min after standing overnight. Afterward, it was dissolved in 10 volumes of 0.5 M acetic acid. The solubilization and salting-out processes were carried out three times. The resulting solution was dialyzed against 0.1 M acetic acid in a membrane with a 14,000 kDa cut-off, followed by lyophilization to obtain an acid-solubilized collagen sponge.

Fabrication of a Tri-Layered Nanofibrous Scaffold
The electrospun nanofibrous scaffold was prepared using an electrospinning machine (Nano NC laboratory machine, South Korea Republic, ESR 100) with a grounded aluminum foil-covered drum collector. Briefly, the tri-layered hierarchically structured nanofibrous scaffold was prepared by sequentially layering stacking electrospinning with three different polymeric solutions of SF/PEO, AMX-loaded PHB, and COL/PEO in three different solvents. The bottom layer was made according to the method of Chen et al. [52]; SF/PEO was mixed at a mass ratio of 8:2 and then blended and dissolved in 5 mL distilled water with constant stirring for 24 h at room temperature to obtain the total polymeric concentration of 14% (w/v). SF/PEO polymeric solution was filled into a 2.5 mL plastic syringe with a bluntended needle 7G (ID = 3.81 mm) at a distance of 20 cm from the rotating drum collector at speed = 460 rpm, dispensing rate of 1 mL/h and under applied high voltage of 18-20 Kv. The middle layer was made one according to the method of El-shanshory et al. [21], with some modifications.
In brief, 0.35 g of PHB was dissolved in 5 mL TFE with constant stirring for 4 h at 50 • C. Subsequently, a PHB with a concentration of 7% (w/v) was mixed with two concentrations of AMX 5% (w/w) and 10% (w/w), depending on the total polymeric solution concentration. PHB and AMX-containing PHB polymeric solution was filled into a 5 mL plastic syringe with a blunt-ended needle 23G (ID = 0.34 mm) at a distance of 20 cm from the rotating drum collector at a speed = 460 rpm, dispensing rate of 1 mL/h and under applied high voltage of 18 Kv. Furthermore, for the top layer, COL/PEO at a weight ratio of 8:2 was blended and dissolved in a 5 mL mixed solvent of glacial acetic acid/DMSO (4.65:0.35) (v/v) under gentle stirring for 24 h at ambient temperature to obtain a total polymeric concentration of 12% (w/v). COL/PEO polymeric solution was loaded into a 5 mL plastic syringe with a blunt-ended needle 7G (ID = 3.81 mm) at a distance of 20 cm from the rotating drum collector at speed = 460 rpm, dispensing rate of 1 mL/h and under applied high voltage of 18-20 Kv. For the stabilization and crosslinking of the tri-layered hierarchically structured nanofibrous scaffold against dissolution in fluids, the bottom layer SF/PEO and the top layer COL/PEO were sequentially placed in a sealed desiccator containing 75% EtOH vapor for two days and another sealed desiccator containing 10% (w/w) GTA vapor for two days, respectively. The stabilized tri-layered hierarchically structured nanofibrous scaffold was dried in a vacuum oven at 40 • C for two days to remove any solvents or crosslinker residuals.

Physicochemical Characterizations
The morphology and tri-layered hierarchically structured nanofibrous scaffold were examined utilizing SEM (JEOL -JSM-6360LA, Tokyo, Japan). Before observation, nanofibrous scaffolds were sputtered with gold. Then, the average diameters of nanofibers were measured using Image analysis software (Image J, National Institute of Health, Bethesda, MD, USA) by randomly selecting 100 nanofibers from the SEM micrographs. FTIR (FT-IR, Shimadzu FTIR-8400 S, Kyoto, Japan) with a wavelength range of 4000-400 cm −1 was used to evaluate the composition and chemical structure of the samples.

Swelling, Porosity, and Surface Wettability
The swelling capacity of the samples was determined according to the method conducted by El-shanshory et al. [21]. The initial weight (Wd) of the synthesized nanofibrous sample was recorded, and the sample was placed in phosphate buffer at room temperature. After 24 h, samples were collected, and any excess surface water was gently wiped with filter paper. At this point, the sample's weight was recorded as (Ww). The following formula was used to calculate the swelling (%).
The porosity of the samples was measured according to the liquid displacement method [53]. A divided cylinder containing a given volume (V1) of absolute ethanol was submerged in a known weight (W) of the sample. When no bubbles were discovered, the resulting volume was then reported (V2). Finally, the absolute ethanol volume remaining after the sample was removed from the absolute ethanol was determined (V3). The porosity of the sample was determined according to the following equation: The surface wettability of the nanofibrous scaffold samples was evaluated using a contact angle meter, model VCA 2500 XE, with a CCD camera and software (AST Products, Billerica, MA, USA). After 0.03 mL of deionized water was dropped onto the surface of the nanofibrous scaffolds for 1 s, images were captured using the connected camera.

Mechanical Properties Evaluation
The tensile strength of nanofibrous scaffolds at room temperature was measured using a universal testing machine (Shimadzu UTM, Kyoto, Japan). Crossheads were moved at a constant rate of 5 mm/min at room temperature until samples rupture (n = 5), while tensile strength was measured automatically. The elongation at rupture and tensile strength were calculated.

In Vitro Antimicrobial Activity
Antimicrobial agents can kill or inhibit bacterial growth. Several techniques, including agar dilution, disc -diffusion, and well diffusion, have been successfully applied to evaluate and screen antimicrobial activity [54]. In this study, the antimicrobial activities of the fabricated nanofibers (SF/PEO/PHB/COL/PEO, SF/PEO/AMX/PHB/COL/PEO) were evaluated against Staphylococcus epidermidis ATCC 12228, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, and Enterococcus faecalis ATCC 29212, according to previously reported methods [55]. Briefly, the previously refreshed bacteria suspensions were diluted with sterile 1% LB broth medium up to 100 fold, then 100 µL of the diluted bacterial suspension was incubated with 10 mL of sterile 1% LB medium containing 0.030 g of tested sample (nanofibers) while shaking for 24 h at 37 • C. Evaluation of the absorbance of the culture medium at 600 nm with visible spectroscopy revealed the percentage of bacterial growth inhibition. The percentage of bacterial inhibition was calculated according to the following equation.

% inhibition
where A and B are the absorbances of bacterial culture in the absence and the presence of tested nanofiber, respectively.

Drug Release Assessment
To evaluate the in vitro drug release of nanofibrous scaffolds containing AMX, a calibration curve was generated by measuring the absorbance values of progressively diluted AMX concentrations at 273 nm. In brief, 3 mL of PBS buffer was added to two cassettes containing 10 mg of two concentrations of AMX-loaded nanofibrous scaffold. The cassettes were then placed in a shaker incubator set to 100 rpm at 37 • C. The buffer was removed, and the same amount of new PBS buffer was added at a predetermined time. The UV spectrophotometer-double beam (T80+, PG Instruments Ltd., England, UK) was utilized for absorbance detection. Calculations were performed based on the AMX concentration in the buffer, the AMX release percentage, and the cumulative release curve.

Cytotoxicity Assay of Nanofibrous Scaffolds
Following treatment, the effect of nanofibrous scaffolds on normal human cells of the HSF (Primary skin fibroblasts) and HFB-4 (melanocytes) cell lines was evaluated using the MTT cell viability assay. In 24-well sterile flat-bottom tissue culture plates, HSF and HFB-4 cells (1.0 × 10 3 each) were cultivated for 24 h in a CO 2 incubator. HSF and HFB-4 cells were maintained in full DMEM media supplemented with 10% FBS. In triplicates, the discs from each manufactured NF were cultured in the monolayer cells for two and four days at weights of 0.5, 1.0, 2.0, and 4.0 mg/mL. After three rounds of washing with a new medium, cells were treated with a 0.5 mg/mL MTT solution to remove dead cells and debris before being cultured for approximately 2-3 h in 5% CO 2 . The formed formazan crystals were dissolved in DMSO, and the optical density of each well was measured at 590 nm using an ELISA reader and a microplate. Without including the prepared NFs, the relative cell viability (%) in comparison to reference cells was calculated using the formula (X) test/(Y) reference 100%.

Cell Scratching Assay of Nanofibrous Scaffolds
The effect of cell healing on the manufactured nanofibrous scaffold was determined using the cell scratching method. Briefly, sterile 24-well cell culture plate (HFB-4, 1.0 × 10 5 ) cells were cultivated and then incubated in 5% CO 2 until the cell monolayers reached about 90% confluence. The cells were washed with fresh medium after being scratched by a sterile micro-tip on the monolayers. After adding different discs of the nanofibrous scaffolds to each well individually, they were incubated in 5% CO 2 for 24 and 48 h to allow for cell migration in the medium. Scratching healing and cell migration were then visualized and recorded using a phase contrast microscope. Each experiment was carried out three times, and the results were compared to untreated scratched cells.

In Vivo Wound Healing
The in vivo wound healing rating for the nanofibrous scaffolds was executed using 21 adult male Wistar rats (8 weeks old; 180-200 g). The in vivo experimentation followed the guidelines and protocols and was approved by the Research Ethical Committee of the Institutional Animal Care and Use Committee at Alexandria University (ALEXU-IACUC) with approval number; AU14-210126-3-3. The animals were divided into three groups, with seven animals in each one. Group one received sterile gauze, group two received SF/PEO/PHB/COL/PEO (blank), and group three received SF/PEO/AMX/PHB/COL/PEO. All groups were covered with plaster to secure the wound dressing at the wound place. The rats were housed in separate stainless-steel cages, supplied with a standard laboratory diet and mineral water ad libitum, and kept under planned environmental conditions (50-60% humidity and 12 h light/dark cycle at 25 ± 2 • C) for 7 days prior to the experiment to allow for adaptation. The surgical operation was performed as previously reported [2,4,56,57]. The rats were anesthetized with intramuscular injection of 10% Ketamine Hydrochloride "(Dopalen ® -Sespo Indústria e Comércio Ltda, Vetbrands Saúde Animal Division, Paulínia, Brazil, 0.1 mL/100 g body weight) and 2%xylazine hydrochloride (Calmium ® -Agener União, União Química, Embu-Guaçu, SP, Brazil, 0.1 mL/100 g body weight)", the back hair was shaved using an electric animal shaver, followed by sterilization of the skin using ethanol solution (70%) and chlorhexidine. Then, using sterile surgical scissors, a surgical scalpel, and forceps, a 1.5 cm diameter, a full-thickness circular excisional wound was created in the center of the hairless skin. The dressed nanofibrous scaffolds and sterile gauze were substituted for new ones every 3 days for 14 days. The wound areas were calculated with the help of a digital caliper. The macroscopic photos of the wounds were captured to evaluate the % contraction of the wound area using a digital camera on days 0, 3, 7,10, and 14 immediately after the surgical operation. The percentage of wound area reduction was estimated according to the following equation.
For histological examination, the skins from the wound sites were removed before scarification on the 7 and 14 days and then fixed in 10% formalin prior to slide preparation. Subsequently, the skin slides were stained with both hematoxylin and eosin (H&E) and Masson's trichrome (MTS) and then examined under an optical microscope for epithelialization, keratinization, and collagen deposition [17,53]. Finally, a section of the skin tissue was preserved at −80 • C for further molecular analysis.

Quantitative Real-Time Polymerase Chain Reaction (RT PCR) Gene Expression Assay for Interleukin-6 and TGF-β1
The total tissue RNA was isolated from the wounded skin samples collected on day 14 using the Easy-spinTM Total RNA extraction kit (cat. No. 17221, South Korea, iNtRON Biotechnology) following the manufacturer's instructions. The purity and the concentration of the extracted total RNA were evaluated utilizing a NanoDrop™ UV-vis spectrophotometer. Using the cDNA synthesis kit TOPscriptTM (cat. No. EZ005S, Daejeon, South Korea, Enzynomics, Inc.), the reverse-transcription step for converting RNA to complementary DNA (cDNA) was performed. The RT-PCR amplification reactions were performed using SYBR green qPCR Master Mix (Thermo Fisher Scientific, Inc., Waltham, MA, USA, cat. No. K0251) utilizing real-time PCR system, Applied Biosystems with 2.5 µL of cDNA and 1.5 µL)of each primer in a 25 µL reaction mixture final volume. The expression level of the house-keeping gene Beta-actin (β-actin) was applied as the internal control for the amplified samples. The sequences for the primers used for the amplifi- cation of (cDNA) were as follows: 5-TTTCTCTCCGCAAGAGACTTCC-3 (forward) and 5-TGTGGGTGGTATCCTCTGTGA-3 (reverse) for IL-6; 5-TGACATGAACCGACCCTTCC-3 (forward) and 5-TGTGGAGCTGAAGCAGTAGT-3 (reverse) for TGF-β1 and 5-AGATCAAG ATCATTGCTCCTCCT-3 (forward) and 5-ACGCAGCTCAGTAACAGTCC-3 (reverse) for β-actin. The alterations in the level of gene expression were estimated using a delta delta comparative Ct (2-∆∆Ct) analysis technique. For the examined genes, the amplification technique consisted of one cycle of initial denaturation at 95 • C for 10 min, followed by 40 cycles of 15 s at 95 • C, 30 s at 57 • C and 30 s at 72 • C. Melting curve analyses were performed for all amplifications to ensure a single product was generated from each reaction [58].

Statistical Analysis
The collected data were statistically analyzed using costate software. One-way ANOVA was performed, followed by the LSD test for multiple comparisons. Data were expressed using the mean and standard deviation M ± SD. p ≥ 0.05 was regarded as statistically significant across all analyses.

Preparation and Physicochemical Characterization
The morphological appearance and tri-layered hierarchical structures of the as-prepared nanofibrous scaffolds ( Figure 1) were evaluated under magnification, and the distribution of their average diameters is depicted in supplementary data (Figures S1-S3). Smooth surfaces, beadless, and no spindle on a string behavior were detected. Moreover, no AMX accumulations were detected on the surface of nanofibrous scaffolds. According to these results, the size distribution and average diameters are 250 ± 82 nm, 261 ± 49 nm, as well as 266 ± 55 nm for SF/PEO, PHB, and COL/PEO, respectively.
FT-IR was evaluated to detect the characteristic peaks and functional groups for the ingredients of the as-prepared nanofibrous scaffolds SF, PHB, and COL. The basic characteristic peaks of SF/PEO are explained by the appearance of amide I, C=O stretching bands and were assisted by the presence of amide II and III at 1535 and 1238 cm −1, respectively. Furthermore, the random coil of amide I appeared at 1654 cm −1 , corresponding to the vibration band. PHB exhibited infrared absorption peaks at 1262 and 1725 cm −1 , corresponding to -CH and C=O, respectively, present in the ester group in the molecular chain. Additionally, sharp peaks at 1034 and 1097 cm −1 represent C-O stretching. Furthermore, absorption peaks at 2926 and 2963 are attributed to C-H stretching vibrations of the methyl and methylene groups [59]. AMX relevant major peaks are present at 1490, 1509-1520, 1685-1692, 2050, 3000, 3175, 3366, and 3448-3458 cm −1 corresponding to N-H, C=C benzene ring stretching, C=O stretching, amide I, C-C and C-N stretching, C-H benzene ring stretching and amide N-H and phenol O-H stretching, respectively [60,61]. Collagen-derived tilapia fish skin showed characteristic peaks at 3292-3315 cm −1 corresponding to peptide N-H groups. There are COL amide I band at 1656 cm −1 , amide II at 1538-1548 cm −1 , and amide III at 1232-1238 cm −1 . Therefore, these results confirmed that the extracted COL is collagen type I which are in concordance with the previously reported results by Elbaily et al. [62]. The successful loading of AMX within the nanofibrous composite can be confirmed by the appearance of the peaks in AMX powder at 1509 cm −1 and 1685 cm −1 , which are assigned to amide I and Amide II bond of AMX, respectively [63]. Moreover, the peaks at 1618 cm −1 , 1774 cm −1 and 3448 cm −1 are present due to the absorption band of benzene ring, the vibration of carboxylic group and the stretching vibration of hydroxyl and amino group in the AMX structure [64,65]. Additionally, the appearance of the AMX peak at 1509 cm −1 in both SF/PEO/5%AMX/PHB/COL/PEO and SF/PEO/10%AMX/PHB/COL/PEO confirms the presence of AMX in both composites due to some weak van der Waals interactions between AMX and nanofibrous composite. However, the detection of other AMX signals was difficult perhaps due to some overlapping between vibration bands of AMX and the nanofibrous composites [66]. The results obtained indicated the successful incorporation of AMX into the nanofibrous scaffold. FT-IR graphs are demonstrated and plotted in Figure 2. FT-IR was evaluated to detect the characteristic peaks and functional groups for the ingredients of the as-prepared nanofibrous scaffolds SF, PHB, and COL. The basic characteristic peaks of SF/PEO are explained by the appearance of amide I, C=O stretching bands and were assisted by the presence of amide II and III at 1535 and 1238 cm −1, respectively. Furthermore, the random coil of amide I appeared at 1654 cm −1 , corresponding to the vibration band. PHB exhibited infrared absorption peaks at 1262 and 1725 cm −1 , corresponding to -CH and C=O, respectively, present in the ester group in the molecular chain. Additionally, sharp peaks at 1034 and 1097 cm −1 represent C-O stretching. Furthermore, absorption peaks at 2926 and 2963 are attributed to C-H stretching vibrations of the methyl and methylene groups [59]. AMX relevant major peaks are present at 1490, 1509-1520, 1685-1692, 2050, 3000, 3175, 3366, and 3448-3458 cm −1 corresponding to N-H, C=C benzene ring stretching, C=O stretching, amide I, C-C and C-N stretching, C-H benzene ring stretching and amide N-H and phenol O-H stretching, respectively [60,61]. Collagen-derived tilapia fish skin showed characteristic peaks at 3292-3315 cm −1 corresponding to peptide N-H groups. There are COL amide I band at 1656 cm −1 , amide II at 1538-1548 cm −1 , and amide III at 1232-1238 cm −1 . Therefore, these results confirmed that the extracted COL is collagen type I which are in concordance with the previously reported results by Elbaily et al. [62]. The successful loading of AMX within the nanofibrous composite can be confirmed by the appearance of the peaks in AMX powder at 1509 cm −1 and 1685 cm −1 , which are assigned to amide I and Amide II bond of AMX, respectively [63]. Moreover, the peaks at 1618 cm −1 , 1774 cm −1 and 3448 cm −1 are The nanofibrous scaffold's ability to manage wound exudates and drug delivery depends on its ability to absorb fluids. Fluids uptake is affected by the hydrophilic nature of the applied materials and their porosity. The swelling ability of the material affects its weight, which decreases due to erosion during prolonged exposure to fluids, whereas weight gain is due to fluid uptake during short exposure to fluids. The weight change obtained after 72 h of immersion in PBS pH 7.4 at room temperature with partial weight loss in the first 24 h was 25%, 5%, and 65% for SF/PEO, PHB, and COL/PEO, respectively. Furthermore, All nanofibrous scaffolds maintained their structural stability for at least 48 h. These results demonstrate the potency of the nanofibrous scaffold for various biomedical applications. Additionally, the porosity % of the nanofibrous scaffolds is a key factor affecting their biomedical applications. The porosity % for SF/PEO, PHB, SF/PEO/PHB/COL/PEO (blank), and COL/PEO are 75%, 80%, 85.71%, and 82.2%, respectively. These results demonstrate the suitability of the materials for use in biomedical applications and the capacity of the nanofibrous scaffold to facilitate the self-pumping effect and fluid movement from the interior to the exterior surface of the matrix.
The hydrophilic behavior of nanofibrous scaffold significantly enhances cell differentiation and adhesion. In general, the water contact angle of the SF/PEO is 79.2 • , while that for PHB is 102.8 • . Moreover, the water contact angles for the COL/PEO, SF/PEO/COL/PEO, 5% AMX-loaded nanofibrous scaffold, and 10% AMX-loaded nanofibrous scaffold were 47 • , 43 • , 62 • , and 92 • , respectively. The photographs of water contact angles of the different nanofibrous scaffolds are shown in Figure 3. The obtained results indicate the hydrophobic nature of PHB, the moderate hydrophilicity of SF, COL, SF/PEO/COL/PEO, and the change in behavior of the 10% AMX-incorporated nanofibrous scaffold towards hydrophobicity. SF/PEO/5%AMX/PHB/COL/PEO and SF/PEO/10%AMX/PHB/COL/PEO confi presence of AMX in both composites due to some weak van der Waals int between AMX and nanofibrous composite. However, the detection of other AM was difficult perhaps due to some overlapping between vibration bands of AMX nanofibrous composites [66]. The results obtained indicated the successful inco of AMX into the nanofibrous scaffold. FT-IR graphs are demonstrated and p Figure 2.
(a) The nanofibrous scaffold's ability to manage wound exudates and drug deli depends on its ability to absorb fluids. Fluids uptake is affected by the hydrophilic na of the applied materials and their porosity. The swelling ability of the material affec weight, which decreases due to erosion during prolonged exposure to fluids, whe weight gain is due to fluid uptake during short exposure to fluids. The weight cha obtained after 72 h of immersion in PBS pH 7.4 at room temperature with partial we loss in the first 24 h was 25%, 5%, and 65% for SF/PEO, PHB, and COL/PEO, respectiv Furthermore, All nanofibrous scaffolds maintained their structural stability for at lea h. These results demonstrate the potency of the nanofibrous scaffold for var biomedical applications. Additionally, the porosity % of the nanofibrous scaffolds is a factor affecting their biomedical applications. The porosity % for SF/PEO, P SF/PEO/PHB/COL/PEO (blank), and COL/PEO are 75%, 80%, 85.71%, and 82 respectively. These results demonstrate the suitability of the materials for us biomedical applications and the capacity of the nanofibrous scaffold to facilitate the pumping effect and fluid movement from the interior to the exterior surface of the ma The hydrophilic behavior of nanofibrous scaffold significantly enhances differentiation and adhesion. In general, the water contact angle of the SF/PEO is 7 while that for PHB is 102.8°. Moreover, the water contact angles for the COL/P SF/PEO/COL/PEO, 5% AMX-loaded nanofibrous scaffold, and 10% AMX-loa Materials used in biomedical applications should have suitable mechanical properties to serve well as a scaffold. The results summarized in Table 1 indicated that the SF/PEO has a tensile strength of 9.1911 N/mm 2 and the value of elongation at break was 2.70500%, whereas the tensile strength of COL/PEO is 27.777 N/mm 2 , and the value of elongation at break was 1.1100%, Moreover, adding PHB and COL/PEO to SF/PEO layers exhibited a tensile strength value for SF/PEO/PHB/COL/PEO scaffold to be 4.1666 N/mm 2 and the value of elongation at break decreased to be 0.6000%. The incorporation of AMX (5%) in SF/PEO/PHB/COL/PEO increased the values of tensile strength and elongation at break, respectively, from 4.1666 N/mm 2 to 35.1732 N/mm 2 and 0.6000% to 1.84500%. The addition of AMX(10%) in SF/PEO/PHB/COL/PEO increased the tensile strength and elongation at break values, respectively, from 4.1666 N/mm 2 to 86.137 N/mm 2 and 0.6000% to 2.50250% [67][68][69].  Materials used in biomedical applications should have suitable mechanic properties to serve well as a scaffold. The results summarized in Table 1

In Vitro Antimicrobial Activity
In several cases, wound infection may lead to severe problems and complications associated with delayed healing as well as mortality. Consequently, it is necessary for the newly developed wound dressing to estimate its ability to inhibit the growth of pathogenic bacteria. The antibacterial activity was assessed regarding the % growth inhibition of Staphylococcus epidermidis ATCC 12228, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, and Enterococcus faecalis ATCC 29212bacteria after one day of contact with the nanofiber. These bacteria are prevalent in wound discharges, especially in post-operative patients. SF/PEO/PHB/COL/PEO (blank) did not inhibit bacterial growth because it lacked antimicrobial ingredients. Conversely, SF/PEO/5%AMX/PHB/COL/PEO demonstrated highly significant antimicrobial activity against Staphylococcus epidermidis, Escherichia coli, Staphylococcus aureus, and Enterococcus faecalis, with the calculated reduction in the number of CFU of these bacterial cells reaching maximum of 83, 76.5, 83, and 89.5%, respectively. whereas at week 6, it was 43.9%. In contrast, the percentage of weight loss at week 1 and week 6 for the nanofibrous scaffold containing 10% AMX was 38.8% and 50%, respectively.

In Vitro Drug Release and In Vitro Weight Loss
Previous studies reported that AMX is more soluble in acidic media resulting in a decrease in the swelling of the polymeric matrix with increasing AMX concentration [70]. Moreover, electrospinning process allows hydrophobic components of AMX to face the surface of polymers due to the rapid solvent evaporation rate and this results in polymer degradation retardment in the presence of AMX. These results are in agreement with the results reported previously by Mollo et al. [71]. Based on the obtained results, we hypothesize that a nanofibrous scaffold containing 5% AMX can serve as an effective scaffold against bacterial invasion for wound healing applications.

In Vitro Cytotoxicity and Cell Scratching Assay
Cell viability and the effect of cell healing by the cell scratching method, as depicted in Figures 5 and 6, are binding assays for scaffolds used in wound healing and other biomedical applications. According to Figure 5, the cell viability of all tested nanofibrous scaffolds after two and four days of incubation ranged between 50 and 80%, indicating that these nanofibrous scaffolds have some cytotoxic effects on cells exposed to them for four consecutive days. Consequently, based on the obtained results, it can be concluded that blank nanofibrous scaffolds under study are cytocompatible, allowing more than 80% cell viability, while the nanofibrous scaffold containing 5% and 10% AMX were found to be almost 35-50% cell viability.
Moreover, from the results obtained from the cell scratching assay, the blank and Additionally, the degradation rate of the nanofibrous scaffolds incubated in PBS solution at 37 • C was conducted for up to 6 weeks, as shown in Figure 4b. For the nanofibrous scaffold containing 5% AMX, the weight loss percentage at week 1 was 22.4%, whereas at week 6, it was 43.9%. In contrast, the percentage of weight loss at week 1 and week 6 for the nanofibrous scaffold containing 10% AMX was 38.8% and 50%, respectively.
Previous studies reported that AMX is more soluble in acidic media resulting in a decrease in the swelling of the polymeric matrix with increasing AMX concentration [70]. Moreover, electrospinning process allows hydrophobic components of AMX to face the surface of polymers due to the rapid solvent evaporation rate and this results in polymer degradation retardment in the presence of AMX. These results are in agreement with the results reported previously by Mollo et al. [71]. Based on the obtained results, we hypothesize that a nanofibrous scaffold containing 5% AMX can serve as an effective scaffold against bacterial invasion for wound healing applications.

In Vitro Cytotoxicity and Cell Scratching Assay
Cell viability and the effect of cell healing by the cell scratching method, as depicted in Figures 5 and 6, are binding assays for scaffolds used in wound healing and other biomedical applications. According to Figure 5, the cell viability of all tested nanofibrous scaffolds after two and four days of incubation ranged between 50 and 80%, indicating that these nanofibrous scaffolds have some cytotoxic effects on cells exposed to them for four consecutive days. Consequently, based on the obtained results, it can be concluded that blank nanofibrous scaffolds under study are cytocompatible, allowing more than 80% cell viability, while the nanofibrous scaffold containing 5% and 10% AMX were found to be almost 35-50% cell viability.  Moreover, from the results obtained from the cell scratching assay, the blank and nanofibrous scaffold incorporated with 5%AMX at different cell concentrations performed well for wound healing. In contrast, the nanofibrous scaffold incorporated with 10%AMX showed relatively lower wound healing and some toxic effect on the cells. Based on these findings, the tri-layered hierarchically structured nanofibrous scaffold is recommended for wound healing applications. Furthermore, The viability of PIEC cultured on pure SF, SF/P(LLA-CL) and SF/HBC nanofibrous scaffolds were good and beneficial to cell growth in comparison with coverslips as reported by Zhang et al. [72,73]. Further, the previous studies conducted by Tian Zhou et al. have demonstrated that the fish collagen/BG nanofibers induced proliferation on HaCaTs, indicating that fish collagen nanofibers could effectively promote wound healing [74]. Moreover, the cell viability of HPDLCs cultured was en-hanced upon seeding on the Col/BG/CS membrane for periodontal tissue regeneration as reported by Zhou et al. [75]. Additionally, it has been reported that the biocompatibility results of the PHB/gelatin nanofibers against NIH-3T3 fibroblast cell lines were found to be non-toxic and aid in greater cell viability [76]. Additionally, it has been reported that the combination of PHB with SF supported the cell attachment and proliferation of L929 and HaCaT cell lines [77].

In Vivo Wound Healing
The effect of different treatment groups on in vivo wound healing efficiency is illustrated in Figure 7a and Table 1. Topical delivery of the antibiotics leads to bulk collection of higher drug doses at the target site, thus reducing side effects such as systemic toxicity associated with high drug doses and bacterial resistance. Therefore, the optimal wound dressing material should contain antimicrobial agents with a sustained release behavior in order to accelerate wound healing [78]. Additionally, in contrast to free antibiotics that cannot arrive and spread evenly over infected areas, antibiotic-loaded nano-carriers are characterized by their higher penetration efficiency and equal distribution around the infected spots [79]. In this regard, AMX, a broad-spectrum antimicrobial compound, was incorporated into the SF/PEO/PHB/COL/PEO nanofibrous scaffold to boost wound repair. In comparison to the sterile gauze group, the SF/PEO/PHB/COL/PEO (blank)-and 5% AMX incorporated SF/PEO/5%AMX/PHB/COL/PEO nanofibrous scaffold-treated groups displayed significant progress in wound healing associated with normal tissue, with no exudates at all healing stages.  In addition, the incorporation of AMX into the SF/PEO/PHB/COL/PEO nanofibrous scaffold accelerated the wound healing process. The wounds treated with the SF/PEO/5%AMX/PHB/COL/PEO nanofibrous scaffold were partially healed after 7 days. The % contraction of the wound area was studied as shown in Table 2 and Figure 7b. It was found that after 3 days from treatment, the percentage of wound closure was 9.09%, 24.18%, and 53.73% for the sterile gauze group, the SF/PEO/PHB/COL/PEO nanofibrous scaffold, and the SF/PEO/5%AMX/PHB/COL/PEO nanofibrous scaffold, respectively. Interestingly, at day 14 after treatment, both the SF/PEO/5%AMX/PHB/COL/PEO nanofibrous scaffold and the SF/PEO/PHB/COL/PEO nanofibrous scaffold showed 99.63% and 98.40% wound closure, respectively. Conversely, the sterile gauze group showed a wound closure of 86.54%. The previous results showed that the wound closure was slower in the sterile gauze group than in the nanofiber-treated groups. The histopathological examination of the skin sections from different treated groups was performed to estimate the degree of vascularization, inflammation, and skin regeneration. During the early stage of wound healing, the lower inflammation level can promote factors responsible for wound healing. On the other hand, severe inflammation in wound sites hindered tissue repair. Figure 8a,b showed the histopathological features of H&E and MTS stained skin sections of NF-treated wounds compared with untreated control and normal skin on days 7 and 14 post-wounding, respectively. As depicted in Figure 8a, the normal skin image presented the typical structural elements of healthy skin, such as intact epidermis, dermis (connective tissue layer), muscles, sebaceous glands (SG), blood vessels, and hair follicles. It is evident that the healing of the skin section treated with the SF/PEO/PHB/COL/PEO nanofibrous scaffold and the SF/PEO/5%AMX/PHB/COL/PEO nanofibrous scaffold was higher compared to the sterile gauze group. Furthermore, the nanofibers loaded with AMX showed considerable healing over the early stages of post-operation. On day 7, the skin section from groups treated with sterile gauze showed ulcers, inflammatory cells, no SGs, and intact hair follicles. On the contrary, the SF/PEO/PHB/COL/PEO and SF/PEO/5%AMX/PHB/COL/PEO nanofibrous scaffold-treated group begins forming a thin coat of neo-epithelium associated with mild inflammatory cells, mild new vessel, and fibroblasts. On day 14, the wound section from the sterile-gauze-treated group showed a small ulcer, scab formation with a smaller number of fibroblasts and vasculature, and ahigh number of inflammatory cells with minimal re-epithelialization and the absence of both regenerated SGs, hair follicles, and other adnexa in the renovated dermis. The wound site also showed loosely arranged connective tissue bundles in the dermis. Conversely, the wound sections of SF/PEO/PHB/COL/PEO and SF/PEO/5%AMX/PHB/COL/PEO nanofibrous scaffold-treated group showed relatively normal skin appearance loaded with connective tissues (CT) and enveloped by a new dermal layer. The tissues contained a fully developed epidermis (2-3 layers) and dermis (completely organized connective tissue layer). The wound section also displayed the most basic skin structures, such as hair follicles, SG, and blood vessels. The degree of collagen formation and deposition from the wound section were estimated among various groups by using Masson's trichrome staining (MTS) Figure 8b. Sterile-gauze-treated groups showed loose collagenous fiber deposition at the lesion site on both day 7 and day 14. In contrast, the highest collagen fiber deposition was observed in both SF/PEO/PHB/COL/PEO and SF/PEO/5%AMX/PHB/COL/PEO nanofibrous scaffold-treated groups characterized by mild collagen deposition on day 7, which became denser on day 14 and resembled the pattern of collagen deposition in the normal skin (basketweave), which confers both flexibility and pliability [22]. The photographic images and histopathological examination results suggested that the SF/PEO/5%AMX/PHB/COL/PEO nanofibrous scaffolds accelerate wound healing by promoting complete re-epithelialization, collagen deposition, and arrangement. The efficient in vivo wound healing performance was previously reported for collagen and zein nanofibrous membranes loaded with berberine [80]. Another study observed an accelerated in vivo wound healing, re-epithelization, and collagen deposition for collagen nanofibers loaded with AgNPs due to their intrinsic antibacterial activity [81]. The hybrid between collagen and silk fibroin might improve both the physical and biological characteristics of the scaffold for both tissue engineering and biomedical applications. This result may be due to the hybrid's resemblance to the ECM skin, which promotes cell adhesion and proliferation [82].

Gene Expression of IL-6 and TGF-β1
The wound repair process comprises successive steps that initiate immediately after an injury and develop into a complete skin reconstruction. The process involves the coordinated action of multiple cell types (resident and circulating cells homing to the wound site), the ECM, and soluble mediators named cytokines [83]. The wound repair process begins with coagulation and hemostasis, which halt bleeding and initiate the cellular response. In addition, the activated platelets within the clot release significant growth factors and cytokines that stimulate the resident cells to initiate angiogenesis, re-epithelialization, and connective tissue restoration. Any perturbation to these steps leads to chronic wounds [84]. IL-6 is a potent immunologic mediator that plays a fundamental role in inflammation, representing a substantial physiological phase in normal wound healing. The study of the expression of IL-6 in wound healing revealed that its expression was upregulated following injury in human, animal, and in vitro models. It was reported that overexpression of IL-6 following injury stimulates the production of various pro-inflammatory cytokines from existing cells, including stromal cells, keratinocytes, endothelial cells, and macrophages. In combination with the expression of anti-inflammatory mediators, macrophages are reconfigured from M1 pro-inflammatory to M2 tissue repairing [85].The delay in this conversion step results in delayed healing, an increased risk of infection, and scar formation [86]. The results revealed that both blank SF/PEO/PHB/COL/PEO and SF/PEO/5%AMX/PHB/COL/PEO nanofibrous scaffold-treated wounds significantly inhibited (p < 0.05) the expression level of IL-6 gene compared to sterile-gauze-treated wounds (Figure 9a). The faster a wound heals, the lower the expression of inflammatory factors, and vice versa. This significant decrease in IL-6 gene expression can be attributed to the potent anti-inflammatory and immune-enhancing properties of the SF/COL nanofibrous scaffold. A hybrid SF/COL nanofibrous scaffold has been reported to hasten wound closure and tissue restoration in vivo [82,87]. Moreover, the level of IL-6 gene expression was lower in wounds treated with SF/PEO/5%AMX/PHB/COL/PEO nanofibrous scaffolds compared to wounds treated with blank SF/PEO/PHB/COL/PEO nanofibrous scaffolds. Through incorporation into the SF/PEO/PHB/COL/PEO nanofibrous scaffold, AMX is slowly released at the wound site, preventing bacterial infections and accelerating the healing process, contributing to the superior anti-inflammatory activity of AMX-loaded nanofibers. Furthermore, the released AMX is readily engulfed at the inflammation site by immune cells such as macrophages and produces localized effects on the wound [78,88].
Growth factors are crucial for proper wound repair. TGF-β is a type of pluripotent cytokine that is produced primarily by macrophages and plays a significant role in cell proliferation and migration, immune regulation, apoptosis, and inflammatory response. It is also involved in all healing processes, such as the stimulation offibroblast generation, differentiation of fibroblasts into myofibroblasts, and synthesis of collagen I and II [89,90]. In addition, TGF-β1 can stimulate the healing process via activation of angiogenesis and augment the ECM production via both fibroblast and differentiated myofibroblast [62]. As shown in (Figure 9b), the expression level of the TGF-β1 gene in the group topically treated with SF/PEO/5%AMX/PHB/COL/PEO nanofibrous scaffold was significantly enhanced (p < 0.05) in comparison to SF/PEO/PHB/COL/PEO nanofibrous and sterile-gauze-treated group. This enhanced expression level in the case of treatment with AMX incorporated SF/PEO/5%AMX/PHB/COL/PEO nanofibrous scaffold might be due to induction in the formation of contractile bundles of normal fibroblasts [91].

Gene Expression of IL-6 and TGF-β1
The wound repair process comprises successive steps that initiate immediately after an injury and develop into a complete skin reconstruction. The process involves the coordinated action of multiple cell types (resident and circulating cells homing to the wound site), the ECM, and soluble mediators named cytokines [83]. The wound repair   A significant increase in both wound closure size and wound healing percentage was previously reported by Abo El-Ela, F.I, and his colleagues after topical application of AMX loaded into Layered Double Hydroxide (LDH) nanocomposite (AMOX/LDH) compared to the non-treated and control group [78]. They attributed this result to the excellent penetration capacity of the LDH nanocomposites carrying the antimicrobial agent that accelerate the healing process via preventing infections. Moreover, a higher expression level for the TGF-β1 gene was recorded for groups treated with blank nanofiber (collagen/silk fibroin nanofibers) compared to the sterile-gauze-treated group. Due to the activation of macrophages to produce chemotactic growth factors (GF), angiogenesis, and fibroblast proliferation via upregulation of TGF-β1, bFGF (primary fibroblast growth factor), and α-SMA (α-small muscle actin) genes, topical application of collagen isolated from tilapia skin resulted in optimal and standard cutaneous wound healing in the rat model [62]. It was previously reported that diabetic wounds in mice treated with silk fibroin/poly-(L-lactide-co-caprolactone) nanofiber scaffolds had a significantly higher TGF-β1 gene expression level than wounds in untreated mice.

Conclusions
Wound dressing nanofibrous scaffolds fabricated from natural components have garnered significant interest as promising wound dressings. In the current research, three layers of a biocompatible, multifunctional, antibacterial, biopolymer-based, hierarchically structured nanofibrous scaffold were fabricated. The bottom and top layers contain hydrophilic silk fibroin from natural SF and fish skin COL for accelerated wound healing, interspersed with a middle layer of hydrophobic PHB containing AMX as an antibacterial drug. The fabricated hierarchically structured nanofibrous scaffold elucidated sustained in vitro AMX release, good mechanical properties, high cytocompatibility, and enhanced antimicrobial effect. This hierarchically structured nanofibrous scaffold improves in vivo wound healing in rats, alleviating inflammation and increasing tissue epithelization. Therefore, it can be concluded that this nanofibrous scaffold can be utilized as an effective wound dressing.

Conflicts of Interest:
The authors declare no conflict of interest.