Fabrication of a core–shell nanofibrous wound dressing with an antioxidant effect on skin injury

Abstract

Oxidative stress is one of the obstacles preventing wound regeneration, especially for chronic wounds. Herein, designing a wound dressing with an anti-oxidant function holds great appeal for enhancing wound regeneration. In this study, a biocompatible and degradable nanofiber with a core–shell structure was fabricated via coaxial electrospinning, in which polycaprolactone (PCL) was applied as the core structure, while the shell was composed of a mixture of silk fibroin (SF) and tocopherol acetate (TA). The electrospun PST nanofibers were proven to have a network structure with significantly enhanced mechanical properties. The PSTs exhibited a diameter distribution with an average of 321 ± 134 nm, and the water contact angle of their surface is 124 ± 2°. The PSTs also exhibited good tissue compatibility, which can promote the adhesion and proliferation of L929 cells. Besides, the dissolution of silk fibroin encourages the release of TA, which could play a synergistic effect and regulate the oxidative stress effect in the damaged area, for it promotes the adhesion and proliferation of skin fibroblasts (L929), reduces the cytotoxicity of hydrogen peroxide to cells, and lowers the level of reactive oxygen species. The animal experiment indicated that the PSTs would promote the reconstruction of skin. These nanofibers are expected to repair skin ulcers related to diabetes.

---

1. Introduction

The skin is the largest organ of the human body,¹ which plays a vital role in isolating the external environment and maintaining the homeostasis of the internal environment.² Thus, skin damage and wound healing are everyday clinical activities in our daily lives.³ Various commonly used commercial wound dressings, such as gauze, wound bandages, and hydrocolloids,⁴ are applied for wound healing; most of these dressings could cover the damaged areas and prevent them from external invasion.⁵ However, several clinical issues persist with these, warranting attention: (1) most commercial wound dressings would adhere to the wound site, causing tissue adhesion and discomfort when changing the dressing;^6,7 (2) according to previous research, the imbalance between oxidation and antioxidation during spontaneous physiological healing creates a destructive microenvironment when skin is damaged,^8,9 further causing cell damage and aging.¹⁰ Therefore, designing a wound dressing that can guide the adhesion and proliferation of wound-repairing cells and regulate oxidative/antioxidative stress is an immediate requirement in clinical application.

We applied electrospinning technology to design wound dressing materials with bioactive and biocompatible properties, as it is a practical and mature technology for producing nanofibers with porous structures.^11,12 The electrospun nanofiber dressings have a three-dimensional grid structure, allowing water and oxygen exchange.^13,14 Moreover, the electrospun nanofiber dressing with a bioactive substance could simulate the extracellular matrix and promote cell adhesion and migration on the fiber surface.^15,16

The critical factors that hinder wound healing are the focus of an increasing number of wound healing studies today.¹⁷ These factors include skin damage, diabetic neuropathy, ischemia, infection, insufficient glycemic control, poor nutritional status, and severe morbidity.¹⁸ It is believed that oxidative stress plays a vital role in diabetic and chronic wound healing.¹⁹ The body produces too many reactive oxygen species due to an imbalance between free radicals and antioxidants,^20,21 which damages cells and tissues and slows the healing of wounds.^22–24 Antioxidant systems may help improve healing by lowering ROS levels and reducing oxidative stress-related damage.²⁵ Thus, designing a multifunctional nanofiber with regulatory antioxidant properties is a promising strategy to accelerate wound healing, especially for treating diabetic and chronic wounds.

Tocopheryl acetate (TA) is a synthetic form of vitamin E, which has the practical ability of anti-oxidation, anti-aging, and elimination of free radicals in the body during human metabolism,²⁶ and is often widely used as medicine,²⁷ nutrition, and cosmetic additives.²⁸ A crucial problem for applying TA-based biomaterials is to properly use TA as an effective anti-oxidative agent to deliver toward the wound site and regulate oxidative stress precisely. No reports about this issue or an effective TA wound-curing strategy have been reported.

In this study, coaxial electrospinning technology²⁹ is applied to create a TA-loaded nanofiber (PCL-SF-TA, termed PST) with a core–shell porous structure;³⁰ TA is mixed with silk fibroin (SF) to form a shell structure (SF-TA), PCL (polycaprolactone) is applied as a core structure. These nanofibers have a unique structure; PCL contributes to the fibrous skeleton, and SF-TA constitutes the coating layer, in which TA is blended with SF. This coating technology could protect TA from oxidation in advance and ensure TA's slow release into the wound sites. Moreover, the SF contents in the coating layer could enhance the biocompatibility³¹ of those nanofibers, as SF is a widely used biopolymer in the application of wound healing.³² This unique structure would promote wound healing and synergize between SF and TA for regulating wound regeneration and oxidative/reductive stress from the wound site (Fig. 1).

Fig. 1 An electrospun nanofiber membrane used as a skin wound repair dressing. (a) Schematic illustration of the PST (PCL/SF/TA) electrospun nanofiber membrane preparation. (b) Antioxidant regulation of PST electrospun nanofiber dressing in skin wound healing.

2. Experimental section

2.1 Materials

Commercial silk was purchased from Shenzhou Union Silk Co. Ltd (Zhejiang, China). PCL (M_w = 80 000 Da), sodium carbonate, and calcium chloride were purchased from Aladdin Co., Ltd (Shanghai, China); DMSO was purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). The L929 mouse lung fibroblast cell line was obtained from the Shanghai Institutes for Biological Science (SIBS), Chinese Academy of Sciences (Shanghai, China). DMEM medium (DMEM basic), fetal bovine serum (FBS), trypsin, penicillin–streptomycin, DAPI, Alexa Fluor TM488 phalloidin, H2DCFDA (DCFH-DA), and PBS buffer purchased from Gibco Thermo Fisher Scientific (Shanghai, China). Tocopheryl acetate (TA), ethanol and hexafluoroisopropanol (HFIP) were purchased from Sigma-Aldrich Chemical Reagent Co., Ltd (China). The live/dead viability kit, Cell Counting Kit 8 (WST-8/CCK8), was supplied by Genecreate Biotech Co., Ltd (Hubei, China).

2.2 Fabrication of PS and PST

Silk fibroin (SF) is isolated and purified based on previous reports.³³ Briefly, 50 g of silkworm cocoon was boiled in 0.5% (w/v) Na₂CO₃ aqueous solution for at least 30 min. The silk fibroin was then thoroughly rinsed with water and dried, and the process was repeated at least three times. Then, the degummed SF was put into a ternary solution (CaCl₂ : C₂H₅OH : H₂O = 1 : 2 : 8 in mole ratio) and dissolved at 80 °C. After centrifuging and filtering the dissolved SF, the MWCO14000 dialysis bag was utilized for the dialysis process. Finally, the product was freeze-dried after one week of dialysis, and then the regenerative SF (RSF) was obtained. 0.25 g of TA and 1 g of RSF were dissolved in 10 mL of hexafluoroisopropanol (HFIP) as a coaxial shell spinning solution, and 1 g of PCL was dissolved in 10 mL of HFIP as the coaxial core spinning solution. Then, all the above solutions were added to the injection pump. The coaxial electrospinning parameters were set as follows: the voltage was 9 kV, the receiving distance was 9 cm, the spinning solution flow rate of the shell was 0.8 mL h⁻¹, and the flow rate of the core was 1.1 mL h⁻¹. The nanofibers were protected from light and stored at low temperatures following electrospinning.

The process for creating PCL-SF coaxial electrospinning nanofibers is similar to that of PST, except that the shell-spinning solution in PCL-SF coaxial electrospinning nanofibers only contained SF.

2.3 Physical and chemical characterizations of PST

The morphological characteristics of PST were observed via field emission scanning electron microscopy (SIGMA500, Carl Zeiss AG, Germany), using Image-J software, randomly selecting fiber filaments from SEM images and calculating nanofibers’ diameter distribution. The attenuated total reflection ATR-FTIR spectrometer (Nicolet^TM iS50, Thermo Fisher Scientific, America) was used to investigate the chemical structure of PST. They used an XRD diffractometer (D8 Advance, Bruker, Germany) for X-ray diffraction measurement at the scanning rate of 5° min⁻¹. To measure the hydrophilic property of PST, a water droplet (volume: 2 μL) was deposited on each sample surface, and the contact angle was captured using a video contact angle system. (FTA100, First Ten Angstroms, USA). The tensile strength and axial tensile modulus of PST (cut into thin strips with 1 cm × 5 cm) were measured using a microcomputer-controlled electronic universal material testing machine (CTM6050, Xie Qiang Instrument Manufacturing, Shanghai) with a load cell of 100 N and the elongation rate was set as 5 mm min⁻¹.

TA, SF, and PCL microspheres were respectively dispersed with water as a dispersant. The mass volume ratio for SF was 5%, for TA, it was 1%, and for PCL, it was 5%, and the surface charge is measured by zeta potential (NanoPlus, Micromeritics, Shanghai) after ultrasonic crushing.

2.4 Cytocompatibility evaluation of PST

The preparation method of the extracts of the PSTs refers to previous reports;³⁴ simply put, it means extracting PST in culture medium at a ratio of 2 mg mL⁻¹ for three days. The extraction solution of PS is prepared using PS fibers instead of PSTs, using the same proportion and process. The scratch assay is often used to test cell migration. Following incubation of the L929 cells at a density of 2 × 10⁴ cells per well in 96-well plates, sterile 200 L plastic pipette tips were used to scrape a linear scratching in the wells, and PBS was used to remove the floating cells. Then, the PST extract was added to the PST group, and the blank group was cultured using a regular medium, with each cell incubated for 24 hours. After 48 hours, photos of the scratch closure process were taken using an inverted fluorescent microscope (OLYMPUS IX73), and three representative images of the scratch area were analyzed.

For the cytocompatibility assay, L929 cells are planted on a 96-well plate with a cell culture density of 5000 cells per well. Add extracts of PSTs to the PST group for cell culture. Add the PS extract to the PS group for cell culture, the PCL extract to the PCL group for cell culture, and ordinary culture medium without the extract to the blank group of cultured cells as a control. All experiments were incubated at 37 °C and 5% carbon dioxide for 24 h, 48 h, and 72 h, respectively, and cell viability was assessed using a CCK-8 kit. The microplate reader (SpectraMax M2) was used to measure the absorbance at the wavelength of 450 nm. Use six parallel wells for each material extraction solution as a control.

For the live/dead staining assay, the L929 cells at a density of 5 × 10³ cells per well were separately cultured with extracts of PSTs, extracts of PS, and regular culture media in 96-well Tissue Culture Plates for 72 h. All cell-laden TCPs were rinsed by PBS buffer and then immersed in 50 μL of PBS containing 0.7 μM Calcein-AM stock as well as 1.5 μM PI for 20 min. After that, the stained TCPs were washed with PBS several times before observation using an inverted fluorescence microscope (E_x/E_m = 488/515 nm for AM; E_x/E_m = 493/636 nm for PI).

All the samples were cut into pieces for the cell adhesion test and then transferred into a 14 mm diameter cell culture disc. After that, the L929 cells were seeded into each disc, with 5 × 10⁵ cells per sample, and co-cultured with the sample for 72 h. Then, the cells were fixed with 4% paraformaldehyde for 10 min, rinsed with PBS three times, and then treatment is performed using 0.1% TritonX-100 for 4 min. After rinsing with PBS several times, cytoskeleton staining was performed with Alexa Fluor 488-labeled ghost pen cyclic peptide in the dark for 45 min; then the nucleus was stained in the dark with 1 μg mL⁻¹ DAPI for 10 min. Make three parallel sets for each material. After staining, an FV3000 confocal microscope was applied to observe cells on the surface of the nanofiber membrane (E_x/E_m = 368/461 nm for DAPI; E_x/E_m = 488/506 nm for FITC; Leica TCS SP8STED).

2.5 Oxidative-resistance evaluation

Slide glasses were placed into the 24-well plate and then seeded L929 cells on the slide glass at a density of 2 × 10⁵ cells per well and added the extract of PST culture medium and 3% w/v hydrogen peroxide solution into the cultured cells, keeping the H₂O₂ concentration in the culture medium at 0.5 mM using the hydrogen peroxide kit. The positive control of the experiment was set as a culture medium without H₂O₂ addition, and 0.5 mM H₂O₂ culture medium was selected as a negative control. After 24 hours of culture, the cells were stained with 10 μM H2DCFDA reagent, incubated away from visible light for another 30 min, then photographed by the FV3000 confocal microscope (E_x/E_m = 488/525 nm). Repeat the same experiment 6 times. After that, the Image-J software is applied to statistically analyze the AOD (Average Optical Density) value.

For in vitro antioxidative evaluation of TA, briefly, the L929 cells were cultured in 96 well plates with 1.7 × 10⁴ cells per well; the blank control group was applied with cell medium without the addition of any extracts; the negative control group added hydrogen peroxide³⁵ to the culture medium to achieve a hydrogen peroxide concentration of 0.5 mM, in the TA experimental group, H₂O₂ was added and its concentration was unified as 0.5 mM. On this basis, different concentrations of TA were added separately to achieve a final concentration of TA of 1 μM, 10 μM, 100 μM, 1 mM, and 10 mM. In the PST group, 40 mg of PST was added to a 10 mL medium for extraction for three days, and H₂O₂ was added to this culture medium to achieve a concentration of 0.5 mM. After one day of co-culture, the proliferation activity of cells was detected by the CCK-8 kit.

To validate the antioxidant effect, flow cytometry was performed. The L929 cells were placed into 6 well plates at a density of 5 × 10⁶ per well and 2 mL of culture medium per well. The blank group was culture cells with ordinary medium, while the negative control group added H₂O₂ to the culture medium to reach a concentration of 0.5 mM and then incubated the cells. In the PST group, use a culture medium that has been extracted with PST (extract PST in culture medium at a ratio of 2 mg mL⁻¹ for three days) and add H₂O₂ to achieve a concentration of 0.5 mM, after one-day co-culture, 10 μM H2DCFDA reagent was applied to stain the cells with high ROS expression, and 1.5 μM PI was applied to stain the dead cells. The staining process was performed in the incubator in the dark for 30 min and then using flow cytometry to analyze the level of reactive oxygen species and the number of dead cells. The test was repeated three times.

2.6 Animal experiment

The surgery operation was approved by the Animal Care & Welfare Committee at the Hubei Provincial Center for Disease Control and Prevention (No. 202210184). 6 Sprague Dawley (SD) male rats (about 200 g) were purchased from the Hubei Provincial Center for Disease Control and Prevention (HBCDC), China. Anesthesia of rats by the intraperitoneal injection of chloral hydrate according to published reports.³⁶ Four full-thickness excisional skin wounds (diameter: 20 mm) were created on the dorsum of each rat.³⁷ The wound beds were covered with PST, and silicon films were sutured to prevent movement, serving as the PST group. The PS group covered the wound with nanofiber membrane PS and re-covered it with silicone. The commercial product (CP) group applied a benzalkonium chloride bandage to cover the wound bed and re-covered it with silicone films, and the blank group does not take any treatment measures on the wound bed, only suturing silicone film on top to prevent touch.

A digital camera was used to capture the images of wound beds; the wound areas at different time intervals were analyzed using Image-J software. The following equation calculated the wound contraction rate: A₀ and A_t refer to the initial wound area and the wound area at predeterminate time intervals, respectively.

2.7 Histological analysis

Following a 14-day post-surgery period, all animals were euthanized. The regenerated skins were fixed in a 4% polyformaldehyde (PFA) solution for at least 48 hours. After selecting the sample, the samples underwent dehydration and were embedded in paraffin. Subsequently, they were sectioned into 4 μm slices, deparaffinized, and hydrated. HE staining was then performed to evaluate the wound-healing effect.

2.8 Statistical analysis

The mean ± standard deviation was used to present all data, with n ≥ 3. Statistical analysis was performed using SPSS 19.0 software. Statistical differences between the groups were calculated using the one-way ANOVA method with post hoc Tukey's test. The significance levels were indicated as *p < 0.05, **p < 0.01, and ***p < 0.001, representing statistical, significant, and very significant statistical differences, respectively.

3. Results and discussion

3.1 Physical and chemical characterization

The chemical composition of the electrospun membrane was characterized using an attenuated total reflection ATR-FTIR spectrometer (Nicolet^TM iS50, Thermo Fisher Scientific, America). The infrared spectrum of all the nanofiber membranes is presented in Fig. 2a. For the PCL nanofiber, the characteristic peaks of PCL were observed at 2944 cm⁻¹ (CH₂: asymmetric stretching vibration), 2864 cm⁻¹ (CH₂: symmetrical stretching vibration), 1730 cm⁻¹ (C=O: stretching vibration), 1239 cm⁻¹ (C–O–C: asymmetric stretching vibration), and 1181 cm⁻¹ (C–O–C: symmetrical stretching vibration), respectively. All the absorption peaks were in accord with the previous reports.³⁸ For silk fibroin, its characteristic peaks were presented at 1653 cm⁻¹ (amide I band), 1530 cm⁻¹ (amide II band), 1219 cm⁻¹ (amide III band), and 680 cm⁻¹ (amide V band). These peaks were in accord with the previous reports. For the spectrum of TA, the peaks located at 2920 cm⁻¹ and 2863 cm⁻¹ indicated the asymmetric and symmetrical stretching vibrations of the methylene group (CH₂), the peak located at 1756 cm⁻¹ indicated the stretching vibrations of the carbonyl group (C=O), the peaks located at 1458 cm⁻¹ and 1365 cm⁻¹ indicated the bending vibrations of the methyl group (CH₃), the peak located at 916 cm⁻¹ indicated the C–O vibrations that were related to the alkoxyl group (C–O–R), all of which were in accord with the previous reports.^39,40 For the electrospun nanofiber of PST, all the characteristic peaks for PCL, SF and TA were detected and exhibited a slight chemical shift. That was caused by the intermolecular interactions as well as hydrogen bonding between functional groups (such as NH₂ from SF) and HFIP during the electrospinning preparation. The results of FT-IR clearly indicated the chemical compositions of PST nanofibers. Besides, minor chemical shifts in SF in PST indicated that the electrospinning process did not alter the spatial conformation (random coil) of SF molecules.

Fig. 2 Physicochemical characterization of materials. (a) FT-IR spectra of PST, PS, and PCL; (b) XRD spectrum of PST, PS and PCL. (c) Water contact images of all membranes; (d) SEM images of PST, PS, and PCL. (f) Element composition of the fibrous membrane; (e) TEM images of PST, PS, and PCL; PST is a shell-wrapped inner core structure. (g) Diameter distribution of nanofibers. (h) Zeta potentials of various components. (i) The mechanical properties of PST were significantly enhanced with the addition of PCL of the core layer. (j) Picture of the electrospun nanofiber membrane. Bar graphs represent mean + SD. N.S. means there is no significant difference between these groups. *p < 0.05; **p < 0.01, ***p < 0.001, and ****p < 0.0001.

The XRD measurement was applied to evaluate the crystallographic structure of PST (Fig. 2b). The diffraction peaks (2θ) of PCL fibers appear at 21.5° and 23.8°, which is entirely consistent with the standard diffraction peaks of PCL. The SF and TA did not exhibit obvious diffraction peaks as SF was in a random coil structure, which was proved by the FT-IR spectra, and TA was liquid at room temperature and was not present as a crystal. The characteristic crystalline peaks of PCL were observed at the PST diffraction peaks, which indicated that the main crystalline structure of PST was originated from the PCL segment.

The water contact angle test reflects the contact behavior between pure water and the spinning membrane surface, which is one of the important surface properties affecting nanofiber membranes’ physical, chemical, and biochemical properties. The results show that the water contact angle of the coaxial electrospun material made by wrapping the SF shell outside PCL is smaller than that of pure PCL (Fig. 2c). SF itself is soluble in water. When used as the fiber shell, the electrospun membrane's surface changes from hydrophobic to hydrophilic form. However, TA is insoluble in water, and the surface of PST is loaded with TA, which causes the PST to change from hydrophilic to hydrophobic form; the water contact angles of the spinning membranes (n = 3) were as follows: PCL: 113.3 ± 3.5°, PS: 67.2 ± 8.5°, PST: 124.0 ± 2.0°. The variation in the water contact angle impacts the platelet and red blood cell adhesion to the dressing, as well as the leakage of blood and tissue fluid. Additionally, it influences wound repair by affecting cell proliferation and adhesion on the material's surface.

Scanning electron microscopy (SEM) images show the fiber structure of the nanofiber membrane with PCL exhibiting a diameter of 93 ± 31 nm, PS measuring 240 ± 70 nm, and PST displaying a diameter of 321 ± 134 nm (Fig. 2d and g). The increase in diameter is due to the PCL fiber wrapping the SF housing. Electrospun fibers interlace with each other to form films; the fiber diameter distribution can be changed by changing the conditions; for example, increasing the voltage will increase the fiber diameter. EDS elemental analysis was carried out to prove that the composition of electrospun film elements met the expectations, As shown in Fig. 2f. PCL membrane analysis results include elements C and O, and the ratio of C to O is 71.22 : 26.80, which is close to the element ratio of PCL itself. The PS and PST nanofibers have added SF, the main component of which is an amino acid, while the analysis results exhibit added N element, which is consistent with the composition of the material. When adding TA to PST, the main component of TA is C, so the content of C in PST is slightly higher than that in PS. To verify the successful synthesis of electrospun fibers with a core–shell structure, the films were photographed using a transmission electron microscope. According to the observation, it is proved that the PST nanofiber filament includes two parts: the core and the shell, and the shell core structure of the outer shell wrapping the inner core increases the nanofiber filament's diameter (Fig. 2e).

The zeta-potential of SF was −4.00 ± 0.18 mV (n = 5) because it was a natural polymer consisting of many hydroxyl and carboxyl groups from the peptide sequence, which attributed a negative charge. The zeta-potential of TA was −32.57 ± 2.56 mV (n = 5). The PCL charge is near neutral (Fig. 2h).

Thus, the mutual repulsion of the same charge between TA and SF is conducive to dispersing into tissue fluid after dissolution. To evaluate the mechanical properties of electrospun films, tensile tests were conducted on the electrospun films using a microcomputer-controlled electronic universal material testing machine (CTM6050, Xie Qiang Instrument Manufacturing, Shanghai). The stress–strain diagram (Fig. 2i) obtained showed that pure PCL had excellent tensile strength and maximum load elongation but was relatively easy to deform, while the mechanical properties of SF electrospun films were much more brittle, making them prone to fracture; the PCL core in PST materials can significantly improve the tensile strength and maximum load elongation of nanofiber membranes. The SF shell containing drugs also gives PST a certain hardness. Therefore, compared to using SF alone, PST exhibits significantly enhanced mechanical properties, making the dressing less prone to damage. Compared to using PCL alone, PST exhibits a higher anti-deformation ability, which somewhat reduces the interference of external forces on the wound. Adding TA to PST does not effectively alter the mechanical properties of electrospun fiber membranes.

3.2 In vitro effects of the PST

For clinical wound dressing applications, favorable biocompatibility is essential for wound dressings.⁴¹ To evaluate the biocompatibility of the nanofiber membrane, the in vitro biocompatibility of the nanofiber membrane was assessed by live/dead staining and the Cell Counting Kit (CCK-8). First, the CCK-8 (Cell Counting Kit) assay results indicated that the PST did not cause obvious cytotoxicity toward L929 cells (Fig. 3b). Both TA and SF are widely used in biomaterials, which employ excellent biocompatibility to promote cell proliferation. Overall, the CCK-8 assays of L929 cells proved that the PST was noncytotoxic and could have no side effect on cell proliferation.

Fig. 3 Effects of materials in vitro, utilizing L929 cells. (a)–(f) The growth status of cells on materials. (a) The cells were stained with DAPI and Alexa Fluor 488 Phalloidin and photographed using a confocal microscope (n = 3). (f) Counting the number of cells on the nanofiber membrane per unit area (n = 3); (b) cytocompatibility test of materials, cells proliferation curves obtained from the CCK-8 assay (n = 6). (c) The live/dead assay of cells (n = 3); (d)–(g) photos and statistics of the cell migration test in the material extract (n = 3); (e)–(i) flow cytometry was used to detect the anti-ROS ability of PST. (j) H2DCFDA was used to stain the cells cultured (n = 3); (h) semi-quantitative analysis of fluorescence intensity in (J), (n = 3). (k) CCK-8 Test to assess the cell activity and verify the antioxidant function of different concentrations of TA and PST (n = 4). The bar graphs represent mean + SD. N.S. means there is no significant difference between these groups. *p < 0.05; **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Planting cells on the nanofiber membrane, taking pictures for observation after three days of culture (Fig. 3a), and counting the cells adhered to each unit area (Fig. 3f), the cell density shows that PST is more conducive to cell adhesion growth and proliferation than the PCL nanofiber membrane (***p < 0.001), and there is no statistical difference between PST and PS.

The live/dead staining assay was performed after three days of culture. As shown in Fig. 3c, most fibroblasts exhibited good biocompability (green fluorescence) in the blank, PS, PST nanofiber membrane, with only a few dead cells present (red fluorescence), indicating that the PST could support cell adhesion and growth.

The scratch assays (Fig. 3d) are complementary methods for evaluating cell spreading and migration. The quantitative results of scratch assays were presented in terms of cell migration distance (Fig. 3g). At the 24th hour, the cell migration distance was 79 ± 6 μm (blank) and 99 ± 4 μm (PST) (**p < 0.01, n = 3); at the 48th hour, the distance was 146 ± 13 μm (blank) and 233 ± 13 μm (PST), (***p < 0.001, n = 3). Scratch assays revealed that the PST could significantly promote L929 cell migration.

The anti-ROS effect of the PST extract was quantitatively analyzed by flow cytometry (Fig. 3e). Using a culture medium containing H₂O₂ for 24 hours of cell culture, the cell mortality rate was 44% (Fig. 3i). Using a culture medium without H₂O₂ for the same duration of cell culture, the cell mortality rate was 23%. The addition of H₂O₂ led to an increase in the cell mortality rate, which is consistent with expectations. However, after adding the PST extract, the cell death rate was significantly reduced (18%), indicating that PST can effectively mitigate the lethal effect of ROS on cells. Upon ROS staining (H2DCFDA kit) and taking photos at the 48th hour of cell culture (Fig. 3j), it was found that the number of cells in the H₂O₂ group was small. Analysis of the H₂O₂ group shows that the ROS AOD (Average Optical Density) is 119 ± 6% (Fig. 3h) of the blank group, while the AOD of the PST group added with H₂O₂ and PST extract is only 77 ± 7% of the blank group, which suggests that the ROS expression level of the PST group is significantly reduced compared with that of the H₂O₂ group (**p < 0.01, n = 3). This proves that PST can protect cells from ROS damage.

Cells were cultured with different concentrations of TA and a medium containing 0.5 mM H₂O₂. Cell viability was detected using CCK-8 (Fig. 3k). Statistical calculation shows that the anti-ROS effect increases with the increase of TA concentration. When the concentration of TA was 1 mM, the cell viability reached 105.0 ± 8.2%, while the cell viability of the control group only treated with H₂O₂ was 54.9 ± 5.5%. There was a significant statistical difference between the two groups (****p < 0.0001, n = 4); however, when the concentration of TA was increased to 10 mM, the cell viability decreased significantly (83.4 ± 2.8%); therefore, when the concentration of TA is higher than 1 mM, it does not have a positive effect on cell proliferation with the increase of concentration. The antioxidant impact of TA has been demonstrated in this experiment, and under these experimental conditions, the best anti-ROS effect occurs at a TA concentration of 1 mM. In addition, the cells were co-cultured using the PST extract and 0.5 mM H₂O₂ medium in this experiment. The final cell viability was 110.7 ± 7.2%, which was significantly different from the H₂O₂ group (****p < 0.0001, n = 4); it can be deduced that PST can fully release TA into the culture medium and had an anti-ROS effect because this reveals that the PST extract can also effectively inhibit H₂O₂ damage to cells. The calculated inhibition effect is close to that of 1 mM TA.

3.3. In vivo animal evaluation

We created a rat model of whole skin layer defects to evaluate the healing effect of PST. The digital photos of representative images of wounds at different time points are shown in Fig. 4c.

Fig. 4 Effects of the electrospun nanofiber membrane in promoting wound healing in rats. (a) Picture of the PST used in animal experiment. (b) Process description of animal experiment. (c) Representative photographs of the wounds in different treatment groups.

By tracing the contour of the wound and stacking them together in Fig. 5a, the wound area repair statistics are shown in Fig. 5b. The healing area of the PST group was 64.6 ± 3.0% on the 7th day, which was significantly higher than that of the blank group, 40.3 ± 2.6% (***p < 0.001, n = 3). The healing area of the commercial product group (CP) was 24.4 ± 1.5% on the seventh day, thus the healing rate was significantly slower than that of the blank group (***p < 0.001, n = 3). The reason for this is that the adhesion of tissues and bandages hinders wound recovery. The overall recovery rate of wound area in the PS group was 42.5 ± 3.7%, and there was no significant difference between the PS group and the blank group on the 7th day (n = 3). It is proved that the electrospun nanofiber dressing used in this experiment solved the problem of inhibiting the growth caused by the adhesion of the dressing to the wound tissue.; On the 14th day, the healing area of the PST group was 87.8 ± 8.3%, which was also higher than that of the blank group, 71.5 ± 3.6%; thus, these findings exhibit statistical differences (*p < 0.05, n = 3). The healing area of the CP group was 74.4 ± 4.1%, which had no statistical difference with the blank group (n = 3); the PS group was 74.4 ± 9.6%, and there was no statistical difference with the blank group (n = 3). All these results indicate that the PS shell–core nanofiber dressing will not hinder wound healing, and the addition of TA can help wound repair.

Fig. 5 The therapeutic effect of the nanofiber membrane. (a) Wound recovery area map. (b) Statistical analysis of the wound healing rate (n = 3). Bar graphs represent mean + SD. N.S. means there is no significant difference between these groups. *p < 0.05; **p < 0.01, and ***p < 0.001.

3.4. Histological evaluation

The damaged skin will rebuild its epidermis before moving on to the subsequent healing phase.⁴² However, the reconstruction of blood vessels, hair follicles, and sweat glands is also essential as those accessory organs play a vital role⁴³ in the normal physiological behavior of skin tissue. The histological analysis of the wound bed at different time points was selected and presented on the 7th and 14th days (Fig. 6); the slices on the 7th day were stained with hematoxylin and eosin (H&E) and photographed. The image showed that there was a small amount of exudate around the tissues, and bleeding and tissue necrosis almost stopped; the CP group showed a lot of inflammatory infiltration and blood sinuses, considering the adverse effect of adhesion of commercial dressing and tissue on the wound surface. The H&E photos taken on the 14th day showed that the outer wound of each group was almost healed.

Fig. 6 H&E staining of wound tissues at various time points. The black box image represents the skin in the wound regeneration area (scale bar, 1 mm); the green box image represents a magnified display of the local tissue below the regenerated area epidermis (scale bar, 200 μm).

When observing the inside of the epidermis, the PS group exhibited more complete subcutaneous tissue reconstruction than the blank group and CP group, while the PST group exhibited more complete subcutaneous tissue reconstruction than all other groups, and sweat gland hair follicles were close to normal skin. Inflammatory cells around the wound were observed. Inflammatory reactions still existed in the blank control group and the CP group. At the same time, inflammatory cells and exudates were rarely observed in the PST group. Vascular reconstruction is a critical factor for wound healing.⁴⁴ On the 7th day, in the blank control group, PS group, and PST group, the expression of blood sinuses was dominant, and only mature blood vessels were observed in the PST group. On the 14th day, all experimental groups began to form blood vessels. In the blank control group, CP group, and PS group, there were still blood sinuses and unformed blood vessels, while in the PST group, newly formed mature blood vessels with compact structures and a large number of hair follicles were observed; the observation of a newly constructed and mature vascular structure shows that the PST has a prominent role in inducing angiogenesis.

4. Conclusions

This research utilized coaxial electrospinning to fabricate a shell–core nanofiber with anti-tissue oxidation properties. As the solution for spinning the core layer, PCL is utilized; the shell layer comprises TA and SF; and the core layer comprises PCL shell–core structure nanofibers. The research findings indicate that the nanofibers possessing this particular structure exhibit a commendable grid configuration, facilitating the exchange of oxygen and water. Moreover, these nanofibers retain the exceptional mechanical properties that are characteristic of conventional PCL nanofibers, thereby effectively safeguarding the wound surface against environmental influences. Notably, this wound dressing ingeniously combines TA and SF to form a fiber shell structure; this structure enhances the biocompatibility of the material, facilitates the growth of cells in the fiber, and maximizes the biological activity of SF. Surface migration and proliferation are facilitated; concurrently, TA that is susceptible to oxidation is encapsulated in a protective film, ensuring that the fiber's oxidative stress effect on the damaged area is regulated and TA is released for an extended period of time. Experiments on animals have demonstrated that this nanofiber effectively combines the synergistic effects of SF and TA. Furthermore, the PST nanofiber promotes hair follicle regeneration, remodels vascular system production, and expedites wound healing, all of which contribute to the induction of complete skin restoration. The PST as a whole is anticipated to constitute a novel dressing for diabetic wound therapy.

Author contributions

Kexin Feng: conceptualization, experiment, methodology, validation, analysis, investigation, data curation, and writing – original draft. Jinlan Tang: experiment, analysis, investigation. Ruiyang Qiu: investigation. Bin Wang: review, supervision and funding acquisition. Jianglin Wang: supervision and funding acquisition. Weikang Hu: supervision, project administration, writing – review & editing, and funding acquisition.

Conflicts of interest

There are no conflicts of interest to declare.

Acknowledgements

This work was supported by the Hubei Provincial Natural Science Foundation of China (2023AFB880) and the Guangdong High-level Hospital Construction Fund.

References

1. H. He, N. A. Fasoula, A. Karlas, M. Omar, J. Aguirre, J. Lutz, M. Kallmayer, M. Fuchtenbusch, H. H. Eckstein, A. Ziegler and V. Ntziachristos, Light: Sci. Appl., 2023, 12, 231.
2. N. Zarei and S. A. Hassanzadeh-Tabrizi, Int. J. Biol. Macromol., 2023, 253, 127249.
3. M. Farahani and A. Shafiee, Adv. Healthcare Mater., 2021, 10, e2100477.
4. V. Brumberg, T. Astrelina, T. Malivanova and A. Samoilov, Biomedicines, 2021, 9, 1235.
5. S. Tavakoli and A. S. Klar, Biomolecules, 2020, 10, 1169.
6. P. Choudhary, B. Ramalingam and S. K. Das, Int. J. Biol. Macromol., 2023, 246, 125347.
7. H. M. Nguyen, T. T. Ngoc Le, A. T. Nguyen, H. N. Thien Le and T. T. Pham, RSC Adv., 2023, 13, 5509–5528.
8. K. Xiang, J. Chen, J. Guo, G. Li, Y. Kang, C. Wang, T. Jiang, M. Zhang, G. Jiang, M. Yuan, X. Xiang, Y. Xu, S. Ren, H. Xiong, X. Xu, W. Li, X. Yang and Z. Chen, Mater. Today Bio, 2023, 23, 100863.
9. J. Zhang, Y. Zheng, J. Lee, J. Hua, S. Li, A. Panchamukhi, J. Yue, X. Gou, Z. Xia, L. Zhu and X. Wu, Nat. Commun., 2021, 12, 1670.
10. C. S. Cai, G. J. He and F. W. Xu, Int. J. Nanomed., 2023, 18, 6411–6423.
11. X. Cheng, X. Chang, X. Zhang, J. Dai, H. Fong, J. Yu, Y. T. Liu and B. Ding, Adv. Mater., 2023, e2307690,  DOI:10.1002/adma.202307690.
12. S. Wang, R. Ding, G. Liang, W. Zhang, F. Yang, Y. Tian, J. Yu, S. Zhang and B. Ding, Adv. Mater., 2023, e2313444,  DOI:10.1002/adma.202313444.
13. L. Li, Z. Zheng, J. Li, Y. Mu, Y. Wang, Z. Huang, Y. Xiao, H. Huang, S. Wang, G. Chen and L. Zeng, Small, 2023, 19, e2301261.
14. Y. Li, J. Wang, D. Qian, L. Chen, X. Mo, L. Wang, Y. Wang and W. Cui, J. Nanobiotechnol., 2021, 19, 131.
15. Z. Fu, D. Li, K. Lin, B. Zhao and X. Wang, Int. J. Biol. Macromol., 2023, 226, 1079–1087.
16. S. S. Rao, M. T. Nelson, R. Xue, J. K. DeJesus, M. S. Viapiano, J. J. Lannutti, A. Sarkar and J. O. Winter, Biomaterials, 2013, 34, 5181–5190.
17. X. Wang, R. Li and H. Zhao, Biomed. Pharmacother., 2024, 170, 116035.
18. T. Li, M. Sun and S. Wu, Nanomater., 2022, 12, 784.
19. Y. Wu, Y. Lyu, L. Li, K. Zhou, J. Cai, X. Wang, H. Wang, F. Yan and Z. Weng, Biomacromolecules, 2024, 25, 43–54.
20. D. Turcov, A. Zbranca-Toporas and D. Suteu, Int. J. Mol. Sci., 2023, 24, 17517.
21. G. A. Engwa, F. N. EnNwekegwa and B. N. Nkeh-Chungag, Altern. Ther. Health. Med., 2022, 28, 114–128.
22. T. Fukai and M. Ushio-Fukai, Cells, 2020, 9, 1849.
23. Y. Zhang, R. Wang, H. Fan, M. Wang, H. Liu, Y. Wang, X. Cui, E. Wang, B. Zhang, H. Gao, X. Liu, H. Li and Y. Cheng, ACS Appl. Mater. Interfaces, 2023, 15, 34451–34461.
24. G. Wang, W. Wang, Z. Chen, T. Hu, L. Tu, X. Wang, W. Hu, S. Li and Z. Wang, Chem. Eng. J., 2024, 148938,  DOI:10.1016/j.cej.2024.148938.
25. L. Deng, C. Du, P. Song, T. Chen, S. Rui, D. G. Armstrong and W. Deng, Oxid. Med. Cell. Longevity, 2021, 2021, 8852759.
26. M. Aparecida Stahl, F. Luisa Ludtke, R. Grimaldi, M. Lucia Gigante and A. Paula Badan Ribeiro, Food Chem., 2024, 439, 138149.
27. L. Saldanha and N. Vale, Pharmaceutics, 2023, 15, 2313.
28. F. Della Sala, A. Borzacchiello, C. Dianzani, E. Muntoni, M. Argenziano, M. T. Capucchio, M. C. Valsania, A. Bozza, S. Garelli, M. Di Muro, F. Scorziello and L. Battaglia, Pharmaceutics, 2023, 15, 1962.
29. N. Wang, B. Wang, W. Wang, H. Yang, Y. Wan, Y. Zhang, L. Guan, Y. Yao, X. Teng, C. Meng, H. Hu and M. Wu, J. Alloys Compd., 2023, 935, 167920.
30. A. Hiwrale, S. Bharati, P. Pingale and A. Rajput, Heliyon, 2023, 9, e18917.
31. X. Zhang, D. Zhu, Y. Cheng, X. H. Zhang, X. L. Guo, N. Lin and B. Q. Zuo, J. Macromol. Sci., Part B: Phys., 2021, 60, 603–615.
32. S. Meng, H. Hu, Y. Qiao, F. Wang, B. N. Zhang, D. Sun, L. Zhou, L. Zhao, L. Xie, H. Zhang and Q. Zhou, ACS Nano, 2023, 17, 24055–24069.
33. X. Zhang, H. Bao, C. Donley, J. Liang, S. Yang and S. Xu, BMC Chem., 2019, 13, 62.
34. C. H. Yao, C. Y. Lee, C. H. Huang, Y. S. Chen and K. Y. Chen, Mater. Sci. Eng., C, 2017, 79, 533–540.
35. G. W. Oh, S. C. Kim, K. J. Cho, S. C. Ko, J. M. Lee, M. J. Yim, K. W. Kim, H. S. Kim, J. Y. Kim, D. S. Lee, S. Y. Heo, Y. M. Kim and W. K. Jung, Int. J. Biol. Macromol., 2024, 255, 128047.
36. W. Hu, Z. Wang, Y. Zha, X. Gu, W. You, Y. Xiao, X. Wang, S. Zhang and J. Wang, Adv. Healthcare Mater., 2020, 9, e2000035.
37. E. A. Lucich, J. L. Rendon and I. L. Valerio, Regener. Med., 2018, 13, 443–456.
38. J. Han, C. J. Branford-White and L.-M. Zhu, Carbohydr. Polym., 2010, 79, 214–218.
39. X. M. Wu, C. J. Branford-White, D. G. Yu, N. P. Chatterton and L. M. Zhu, Colloids Surf., B, 2011, 82, 247–252.
40. S. Zahid, H. Khalid, F. Ikram, H. Iqbal, M. Samie, L. Shahzadi, A. T. Shah, M. Yar, A. A. Chaudhry, S. J. Awan, A. F. Khan and I. U. Rehman, Mater. Sci. Eng., C, 2019, 101, 438–447.
41. J. Su, J. Li, J. Liang, K. Zhang and J. Li, Life, 2021, 11, 1016.
42. E. M. Tottoli, R. Dorati, I. Genta, E. Chiesa, S. Pisani and B. Conti, Pharmaceutics, 2020, 12, 735.
43. H. L. Dingwall, R. R. Tomizawa, A. Aharoni, P. Hu, Q. Qiu, B. Kokalari, S. M. Martinez, J. C. Donahue, D. Aldea, M. Mendoza and I. A. Glass, Dev. Cell, 2024, 59, 20–32.
44. W. Wen, L. Yang, X. Wang, H. Zhang, F. Wu, K. Xu, S. Chen and Z. Liao, Int. Wound J., 2023, 20, 3606–3618.

---