In vivo and in vitro studies of a propolis-enriched silk fibroin-gelatin composite nanofiber wound dressing

Abstract

In this study, electrospun nanofibers (NFs) used in trauma dressings were prepared using silk fibroin (SF) and gelatin (GT) as materials and highly volatile formic acid as the solvent, with three different concentrations of propolis extracts (EP), which were loaded through a simple process. The resulting samples were characterized by surface morphology, scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), contact angle meter, water absorption, degradation rate, and mechanical property tests. The incorporation of propolis improved its antibacterial properties against Escherichia coli, and Staphylococcus aureus, compared to those of the silk gelatin nanofiber material (SF/GT) alone. In vitro biocompatibility assays showed that SF/GT-1%EP had good cytocompatibility and hemocompatibility. In addition, it can also significantly promote the migration of L929 cells. SF/GT-1%EP was applied to a mouse model of full thickness skin defects, and it was found to significantly promote wound healing. These results indicate that the SF/GT-EP nanofiber material has good biocompatibility, migrating-promoting capability, antibacterial properties, and healing-promoting ability, providing a new idea for the treatment of full thickness skin defects.

1. Introduction

The skin is a vital organ that protects the body, and various acute and chronic diseases, as well as external factors, can lead to large defects in or impaired function of this tissue, which can be life-threatening in severe cases. Wound healing is a dynamic, sequential process that involves four overlapping phases, specifically coagulation, inflammation, repair, and maturation [1,2]. To accelerate wound healing and to prevent serious complications, different biomaterials have been developed for use in wound dressings, including hydrogels [3], films [4], sponges [5], and nanofibers [6], among others. As natural fiber materials gradually fail to meet the needs of the population, regenerative fibrous materials synthesized from biopolymers have been widely used in many scenarios owing to their modifiability and multi-functionality. The electrospinning technique is one of the simplest methods to prepare nanofibers. Using electrospinning, ultra-long fibers with uniform diameters can be created, and the composition and properties can be adjusted. Electrospinning is a technique that uses a polymer solution or results in melting under the action of a strong electric field and forms Taylor cones for spinning processing. Depending on the selected material, conditional optimization (e.g., the flow rate, spinning distance and applied voltage), and medium parameters (e.g., concentration), fibers with optimal qualities can be produced. Compared to other methods, nanofibrous scaffolds prepared by electrospinning technology are closer to the structure of the extracellular matrix owing to their good mechanical properties, significant permeability, and relatively high porosity [7]. At the same time, electrostatic spun nanofibers are more suitable for drug adsorption and release owing to their high surface area-to-volume ratio. Therefore, electrospinning technology is widely used in the field of scaffold materials for tissue engineering [[8], [9], [10], [11]].

There are many natural compounds that have been developed and applied based on tissue engineering. Propolis has already been used in the treatment of many diseases because of its good biological activity. Propolis is a gelatinous solid substance formed from resin or other secretions collected by worker bees from tree trunks or plant shoots. The bee prepares propolis by mixing these materials with a substance secreted by its own epiglottal gland. At high temperatures, propolis is soft and strongly adhesive; however, at room temperature, it is a hard and brittle gelatinous solid that is brown, gray-brown, or black [12,13]. Compared with many other natural substances that exist in nature, propolis has several biological activities such as antioxidant, antibacterial, anti-inflammatory, anti-ulcer, anticancer, and immunomodulatory properties. In general, these physiological activities are associated with the complex ingredients in raw propolis [[14], [15], [16], [17]]. Propolis products are considered to be a potential source of natural antioxidants. On the whole, the compounds possessing flavonoids and phenolic acids character are mainly responsible for propolis product'antioxidant capacity [18,19]. Besides, numerous substances with antibacterial properties may be found in propolis, including phenolic aldehyde, flavonoids, phenolic acids and their ester derivatives, ketone bodies, and amino acids [20]. Several studies have been conducted on the application of propolis to wounds, and they have demonstrated its undeniable effect on accelerating the proliferation and remodeling phases of wound healing [21,22]. Propolis, or bee glue, has also been used in a variety of biological scaffolds to improve their functions because of its good bioactivity [[23], [24], [25]].

Silk fibroin (SF) is a fibrous biomacromolecule of natural origin, and it is a popular biomaterial for guided tissue engineering and regenerative medicine applications because of its low immunogenicity, good biocompatibility, modifiable biodegradability, and mechanical properties [26]. Interactions within the cell-extracellular matrix (ECM) are closely related to cell proliferation, differentiation, migration, and functional regulation. By modulating the properties of the SF material to mimic the major functions of the ECM, SF can play an active role in cellular regulation. It can also play a vital role in regenerative medicine by regulating cell adhesion, growth, and differentiation. It has been shown that SF can provide good structural support for the attachment, proliferation, and migration of keratinocytes and fibroblasts [[27], [28], [29]]. Studies have been conducted to produce different forms of trauma dressings (hydrogels, sponges, microgels, etc.) from SF, which have been applied to wounds with good treatment results [[30], [31], [32]]. Under general environmental conditions, even at the nanoscale, it is possible to flexibly process SF into various forms that can be easily combined with multiple active ingredients, providing a new idea for the preparation of structurally and functionally optimized SF nanofibrous materials.

Gelatin (GT) is a natural polymer produced by breaking down the natural triple helix structure of collagen into a single chain of molecules. GT is inexpensive, biodegradable, and biocompatible and can be used as an ingredient in important ECM-like components [33]. It has already been widely used in a variety of wound dressings and tissue engineering scaffold materials, including granular gels, sponges, scaffolds, and hydrogels [[34], [35], [36]]. GT hydrogels have slightly inferior mechanical properties and superior water solubility, and many researchers have been able to modify GT significantly by using other materials to adjust its mechanical and biochemical properties [37]. The use of plain GT electrospun nanofibers with their characteristics of strong water solubility and weak mechanical strength, is limited in some fields, and therefore, they are mainly used as a component of hybrid spinning with other materials.

In this study, a SF/GT nanofiber with good physical properties was prepared, and different concentrations of propolis were loaded into the nanofiber to improve its antibacterial and healing ability. Analysis was done on how the propolis concentration affected the properties of SF/GT nanofibers, mainly in terms of the physical properties, antibacterial activity, and biocompatibility of the nanofibers, and the pro-healing effect of the new wound dressing was also evaluated in vivo. The results showed that the addition of propolis changed the physicochemical characteristics of SF/GT nanofibers, significantly enhanced their antimicrobial properties, and conferred good pro-healing ability. The SF/GT-propolis extract (EP) nanofibers thus show good potential for skin tissue engineering and treatment of wounds.

2. Materials and methods

2.1. Materials

EP was purchased from Wuxi Hengsheng Health Food Co., Ltd. The cocoons were purchased from Suzhou Simeite Biological Co., Ltd. GT was purchased from Sigma-Aldrich (USA). Formic acid was purchased from Sinopharm Chemical Reagent Co., Ltd. L929 fibroblast cells were purchased from Procell Life Science&Technology Co., Ltd., and fresh goat blood was purchased from Hongquan Biotechnology Co., Ltd. A dialysis membrane (3500 MWCO, Solarbio, China) and live/dead assay kit were purchased from Biyuntian Biotechnology Co., Ltd. An electrospinning apparatus (PINTECH, China), syringe pump (Baoding DiChuang Electronic Technology Co., Ltd., China), contact-angle measurement instrument (POWEREACH, China), scanning electron microscopy (SEM) apparatus (TM-3030; Hitachi, Yokohama, Japan), microplate reader (Epoch, BioTek, USA), attenuated total reflectance–Fourier transform infrared (ATR-FTIR) spectrometer (Thermo Fisher Scientific. USA), mechanical testing machine (SFMIT, China), laser scanning confocal microscopy (Leica TCS SP5, Germany) apparatus, and digital slice scanner (Pannoramic MIDI, China) were used for the sample preparation and characterization.

2.2. Silk preparation

SF was isolated from the silk cocoons according to a previously reported method [38,39]. Briefly, sericin was eliminated by boiling 30 g of silk cocoons in 14 L of 0.02 M sodium carbonate for 30 min. The degummed SF was carefully rinsed in ultrapure water, dried overnight, and then dissolved in 9.3 M lithium bromide solution (37 mL) for 5 h at 60 °C. To eliminate the salt, the dissolved silk solution was dialyzed for 72 h against 30 L of ultrapure water. During dialysis, the water was replaced seven times. Subsequently, the impurities were isolated by two rounds of centrifugation (10000 g, 4 °C, 20 min). The sample was lyophilized for 72 h and then stored at −20 °C. Fig. 1A shows a schematic of the silk preparation.

Fig. 1.

Flow-process diagram. (A) Schematic of the silk production. (B) Schematic of the fabrication of SF/GT-EP nanofibrous membranes.

2.3. Fabrication of nanofiber specimen

SF (0.7 g) was dissolved in 5 mL of formic acid for 5 h at room temperature (25 °C) under magnetic stirring. To obtain a SF/GT blended solution, 0.3 g GT was added to the SF solution under magnetic stirring for 4 h at 45 °C. Subsequently, EP of different amounts, namely 0.025 g (SF/GT-0.5%EP), 0.05 g (SF/GT-1%EP), and 0.15 g (SF/GT-3% EP), was mixed into the SF/GT blended solution and agitated overnight. For electrospinning, a 1 mL syringe (with a blunt 17 G needle) was filled with the prepared SF/GT-EP mixed solution. Electrospinning was carried out at room temperature (25 °C), with a working distance of 20 cm, flow rate of 200 L/h, and applied voltage of 18 kV. Finally, four types of nanofibers (SF/GT, SF/GT-0.5%EP, SF/GT-1%EP, and SF/GT-3%EP) were obtained and characterized. Fig. 1B shows a schematic of the nanofiber production procedure.

2.4. FTIR

The functional groups of the SF, SF/GT, and SF/GT-EP samples were examined using an ATR-FTIR spectrophotometer in the range of 500–4000 cm⁻¹ at room temperature (25 °C).

2.5. SEM

SEM was used to analyze the surface morphology and diameter. Dried nanofibrous mats were sputter-coated with gold before the examination. SEM was used at an accelerating voltage of 10 kV, and images were acquired at magnifications of 5000 × and 10000 × . The diameter distributions of the nanofibers were measured using ImageJ image-processing software (n = 3).

2.6. Mechanical properties

The mechanical properties of the samples were measured using an Instron universal testing machine equipped with a 100 N load cell. Before the measurement, each sample was cut to a length of 3 cm and a width of 1 cm. The test speed was set to 5 mm/min. The test was conducted in triplicate (n = 3).

2.7. Water uptake capacity (WUC)

Completely dried circular specimens (diameter of 6 mm) were immersed in phosphate-buffered saline (PBS; pH = 7.4) at 37 °C in an oven for 24 h. Subsequently, the samples were weighed after moisture removal. Finally, the WUC was calculated using:

$$\mathrm{W}\mathrm{U}\mathrm{C}(\%)=\frac{Ww—Wd}{Wd}\times 100\%$$ (1)

where Wd and Ww are the weight of the dried and wet specimens, respectively (n = 3).

2.8. Water contact angle (WCA) measurement

To evaluate the effects of propolis on the hydrophilic or hydrophobic properties of the SF/GT nanofibrous mat, their wettability was evaluated using a contact angle analyzer [40]. The images were captured 5 s after a droplet (5 μL) was in contact with the nanofibrous mat, using a camera that was level with the surface. The measurement was conducted in triplicate (n = 3).

2.9. In vitro degradation studies (DD)

The biodegradation of the nanofibrous specimen was evaluated in PBS (pH 7.4), which was left to rest in an oven at 37 °C to simulate human skin [41]. First, the dry weights of the samples were recorded. At regular intervals (1, 3, 5, 10, 20, and 30 days), the samples were removed, dried in an oven at 60 °C, and weighed. Finally, the weight loss of the samples was calculated as

$$\mathrm{D}\mathrm{D}(\%)=\frac{Wi-Wt}{Wi}\times 100\%$$ (2)

where Wi is the initial dry weight of the sample and Wt is the final dry weight of the sample. The measurement was conducted in triplicate (n = 3).

2.10. Hemolysis assay

A hemolysis assay was performed with goat blood. Briefly, nanofiber samples of SF/GT and SF/GT-1%EP were dried for 48 h and mechanically ground to form a powder, following which 10 mg of this sample powder was dissolved in 1 mL of normal saline. After incubation at 37 °C for 12 h, fresh goat blood (20 μL) was added to each sample and incubated at 37 °C for another 2 h with 120 rpm shaking. Subsequently, the admixture suspension was centrifuged at 1000 rpm for 10 min. Finally, the absorbance value of the supernatant was measured at 545 nm using a microplate reader. Diluted blood (normal saline containing 10% goat blood, 200 μL) added into ultrapure (800 μL) water was used as the positive control group (100% hemolysis), whereas diluted blood (normal saline containing 10% goat blood, 200 μL) added into normal saline (800 μL) was used as the negative control group (0% hemolysis). The hemolysis rate (HR) of the samples was calculated using the following equation:

$$\mathrm{H}\mathrm{e}\mathrm{m}\mathrm{o}\mathrm{l}\mathrm{y}\mathrm{s}\mathrm{i}\mathrm{s}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}(\%)=\frac{\mathrm{t}\mathrm{e}\mathrm{s}\mathrm{t}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}-\mathrm{n}\mathrm{e}\mathrm{g}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}{\mathrm{p}\mathrm{o}\mathrm{s}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}-\mathrm{n}\mathrm{e}\mathrm{g}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}\times 100\%$$ (3)

2.11. In vitro cell studies

The viability of L929 fibroblasts cultured with different samples was monitored after 1, 3, and 5 days using a cell counting kit-8 (CCK8) colorimetric assay. First, all samples were sterilized by exposure to UV radiation for 24 h (12 h for each side), after which 5 × 10³ cells were seeded at 96 wells with different samples. The assay was performed according to the instructions. Briefly, after incubation in a humidified 5% CO₂ incubator (37 °C) at different durations (1, 3 and 5 days), the CCK-8 solution (10 μL per well of the 96-well plate) was added to each well of the L929 fibroblasts on a polystyrene tissue culture plate (TCP), SF/GT, and SF/GT-1% EP, respectively. The plates were incubated at 37 °C for 2 h. Finally, a fresh 96-well plate was then filled with an equal amount of supernatant from each well. A microplate reader was used to determine the absorbance of each well at 450 nm. Three wells were used for each sample.

A live/dead experiment was used to evaluate the cell viability. First, the liquors were leached according to the methods in the National Standards of the People's Republic of China (ISO 10993–5: 2009, IDT) with some modifications. Briefly, the samples (1 g) were immersed in a complete medium (1 mL) and incubated at 37 °C for 24 h. Subsequently, the leached liquors (100 μL) of SF/GT and SF/GT–1%EP were added to the 96-well plates (5 × 10³ cells/well). After incubation at 37 °C in a humidified 5% CO₂ incubator for 24 h, propidium iodide (1 μL) and calcein-AM (1 μL) were freshly mixed with 1 mL of normal saline to prepare a live/dead test solution. The 100 μL mixed solution was added to the experimental orifice plate and incubated in a 5% CO₂ incubator at 37 °C for 30 min. Fluorescent images were captured by laser scanning confocal microscopy, the live and dead cells were quantified by Image J software, and the cell viability was calculated as the proportion of live cells to all other cells.

2.12. Cell adhesion and morphology analysis

The nanofibrous sample was placed in a 96-well plate (covered with 5 × 10³ cells/well) and incubated for 3, 7 and 10 days (5% CO₂, 37 °C) in order to examine the adherence and morphology of the L929 cells on the wound dressing [42]. Subsequently, the samples were cleaned with PBS to remove non-adherent cells before being fixed in a 4% v/v paraformaldehyde aqueous solution for an hour to observe the morphology. The samples were thoroughly cleaned with PBS before being dehydrated in ethanol at various concentrations (30%, 70%, 90%, 95%, and 100%) and lyophilized for 24 h. SEM was used to analyze the cellular morphology, and the dried nanofibrous samples were sputter-coated with gold before the examination.

2.13. Transwell migration assay

The influence of SF/GT and SF/GT-1%EP on the chemotaxis of L929 cells was evaluated using a transwell assay [43]. For this, 3 × 10⁴ cells/well were seeded into the upper well of the transwell 24-well plates with 8 μm pores (Corning) after being cultured in a serum-free medium for 12 h. The serum-free medium (with or without SF/GT and SF/GT-1%EP) was added to the lower well of a 24-well plate. After culturing (5% CO₂, 37 °C) for 12 h, the cells that had adhered to the upper surface of the 8 μm pores were removed using a cotton swab. The chemotaxis of the L929 cells was assessed via 0.5% crystal violet staining for 10 min on the lower surface of the pores. Images of the stained cells were acquired using an optical microscope, and the stained cells were quantified by Image J software.

2.14. Anti-bacterial evaluation

The antibacterial activities of the prepared samples against gram-positive Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) bacteria were evaluated using the inhibition zone method [44]. S. aureus and E. coli were cultured overnight to obtain bacterial suspensions. All strains were grown in a nutrient broth medium adjusted to 1 × 10⁷ colony-forming units (CFU)/mL. The fabricated samples were cut to a diameter of 6 mm. Sterilization was performed with ultraviolet light (254 nm) for 1 h, for which the samples were placed on solid agar containing 100 μL of the diluted bacterial suspension in 90 mm-diameter plates. After 24 h of incubation, photos of the plate were acquired, and the diameters of inhibition zones were quantified by Image J software.

2.15. Antibacterial activity

Nanofiber samples of SF/GT and SF/GT-1%EP were dried for 48 h and completely smash, and then 100 mg sample powder dissolved in 1 mL normal saline To obtain the sample suspension. And then, bacterial suspensions (100 μL, 1 × 10⁶ CFU/mL) were added to 96- well culture plates with different samples (10 μL). A microplate reader was used to determine the absorbance of each well at 600 nm after incubating for 12 h at 37 °C.

$$=\frac{\mathrm{A}0-\mathrm{A}\mathrm{s}}{\mathrm{A}0}\times 100\%$$ (4)

where A0 is the OD value of the blank group, As is the experimental group OD value. The measurement was conducted in triplicate (n = 3).

2.16. In vivo experiment in mouse

Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China) supplied ten specific-pathogen-free-grade male BALB/c mice (6–8 weeks old). All in vivo experiments were approved by the Jiangnan University Institutional Animal Ethical Committee and carried out under the Guidelines for Care and Use of Laboratory Animals of Jiangnan University. The ethical review number is JN. No20220930b0100101 [376].

The mice were anaesthetized with urethane (concentration of 10%, dose of 0.1 mL/10 g) prior to the operation. An electric razor was used to remove the hair from the dorsal area. The residual hair was removed using a depilatory cream (Veet, China) and disinfected with iodophor. Full-thickness circular skin wounds with a diameter of 7 mm were inflicted using surgical scissors and tweezers. Two wounds in five mice were classified as the negative control group treated with saline and SF/GT nanofiber treatment group, respectively. Two wounds in the other five mice were classified as the negative control group treated with saline and SF/GT-1%EP nanofiber treatment group. After treatment, all wounds were covered with TegadermTM films to prevent wound contraction and then with an adhesive bandage. The size of the wounds was measured using a digital caliper, and images were captured at 0, 7, 12, and 17 days post the operation.

2.17. Histopathological staining

The mice were sacrificed for histological analysis after healing for 17 days. The tissue samples were embedded in paraffin after being fixed in 4% paraformaldehyde for 24 h, according to the standard protocol, before sectioning [45]. The paraffin sections were cut into 5 μm thin layers and mounted on slides for histopathological investigation. First, the morphological structure of the epidermis was observed by hematoxylin and eosin (H&E) staining. Next, Masson's trichrome staining was performed to observe collagen fiber deposition in the granulation tissue. Images of the stained sections were obtained using a digital slice scanner.

2.18. Statistical analysis

One-way analysis of variance (n ≥ 3) was used for the statistical analysis. To establish a statistically significant difference between the groups, Tukey's post-hoc test was performed using GraphPad Prism Software (V.6). Differences were considered significant at P < 0.05, P < 0.01, and P < 0.001, and are indicated by *，**, and ***, respectively.

3. Result and discussion

3.1. Structural properties

Fig. 2A shows the darker color of the nanofibers with increasing propolis content (propolis has a brown-black color). Fig. 2B shows the typical ATR-FTIR spectrum of the SF nanofiber samples from 4000 to 400 cm⁻¹. The spectrum of SF exhibits several characteristic absorption peaks that are strongly affected by its physical and chemical structures. In the band range of amide I (1600–1700 cm−1), the absorption peak of SF appear at approximately 1642 cm−1, which are mainly attributed to the C O stretching vibration. The absorption peak at 1530 cm−1 is related to amide II (N–H deformation in the SF amide groups). The absorption peak at 1239 cm−1 is related to amide III. The frequencies of the SF amide groups are consistent with previous research [46]. The FTIR spectra of nanofibrous dressings with different ingredients (SF/GT, SF/GT-0.5%EP, SF/GT-1%EP, and SF/GT–3%EP) are shown in Fig. 2C. All samples exhibit similar spectra. No other peaks are observed. This demonstrates that all samples have the same functional groups. However, all functional groups are related to SF, which could be due to the presence of the solvent and a low propolis concentration.

Fig. 2.

Characterization of electrospun nanofibers. Photos of the electrospun SF/GT (A i), SF/GT-0.5% EP (A ⅱ), SF/GT-1%EP (A ⅲ), and SF/GT-3%EP (A ⅳ) nanofibers. FTIR spectra of the SF film (B) and electrospun nanofibrous SF/GT membranes after treatment in different EP contents (C).

Fig. 3A–H SEM images show the morphology and diameter distribution of SF/GT (A, B), SF/GT-0.5%EP (C, D), SF/GT-1%EP (E, F), SF/GT-3%EP (G, H) nanofibers, indicating that all samples had a uniform, continuous, bead-free porous morphology, and this porous structure facilitate cell migration and material exchange between cells and the microenvironment. Fig. 3I–L show the diameter distribution of SF/GT, SF/GT-0.5%EP, SF/GT-1%EP, and SF/GT-3%EP samples, respectively. All samples had diameters between 0 and 1000 nm, and four of the samples, specifically SF/GT, SF/GT-0.5%EP, SF/GT-1%EP, and SF/GT-3%EP, had diameters distributed from 100 to 200 nm, 200–300 nm, 200–400 nm, and 500–600 nm, respectively. The results showed that the addition of propolis changed the diameter of SF/GT nanofibers under constant spinning voltage (18 KV) and flow rate (200 μL/h) conditions. As the propolis concentration increased, the diameter of the samples became larger. However, it can be seen that the fiber structure remained uniform without any beads as the propolis concentration increased.

Fig. 3.

Characterization of electrospun nanofibers. SEM images of the SF/GT (A, B), SF/GT-0.5%EP (C, D), SF/GT-1%EP (E, F), and SF/GT-3%EP (G, H) samples. I (SF/GT), J (SF/GT-0.5%EP), K (SF/GT-1%EP), and L (SF/GT-3%EP) show the corresponding size distributions.

The mechanical strength of the wound dressing and tissue scaffolds is a determinative parameter of their handling efficacy. Therefore, therefore, in this study we have tested the tensile strength of the sample. The tensile strengths of pure SF/GT and nanofibrous dressings with different EP concentrations (SF/GT-0.5%EP, SF/GT-1%EP, and SF/GT-3%EP) are shown in Fig. 4A. All samples exhibit elasticity. The SF/GT nanofibers exhibit the highest tensile strength (7.74 KPa) and minimum elongation at break (19.07%), whereas the SF/GT-3%EP nanofibers exhibit the highest elongation at break (35.74%) and lowest tensile strength (3.62 KPa). Owing to the presence of propolis, the tensile strength decreases from 7.74 KPa to 3.62 KPa, while the elongation at break increases from 19.07% to 35.74% because of the plasticizing property of propolis. The tensile strengths of the SF/GT-0.5%EP and SF/GT-1%EP samples are 5.89 and 4.72 KPa, respectively. In addition, the elongations at break of the SF/GT-0.5%EP and SF/GT-1%EP samples are 23.78% and 29.64%, respectively. Therefore, EP plays a plasticizing role; it increases the elongation at break of the nanofibrous mat and decreases the tensile strength, consistent with the results of previous studies [47,48]. Although the addition of EP to the nanofibrous dressing decreases the tensile strength, a sufficiently high tensile strength can still be applied to the wound dressings.

Fig. 4.

Characterization of electrospun nanofibers. (A) Stress-strain curves of the electrospun SF/GT nanofibrous mats with different contents of EP. (B) Degradation of different samples in PBS at 37 °C. (C) WUC of the samples after 24 h of incubation (left to right: SF/GT, SF/GT-0.5%EP, SF/GT-1%EP, and SF/GT-3%EP). (D) WCA of the electrospun SF/GT nanofibrous mats with different contents of EP. Data are expressed as mean ± SD (n = 3).

3.2. Degradation evaluation

The degradability of biomaterials is important for the renewal of new tissues and the release of loaded drugs. The degradation rate of nanofibers indirectly affects cell functions and tissue remodeling by influencing cell proliferation and ECM production [49,50]. In addition, the migration of cells in the wound bed and the communication between the cells and the microenvironment are also correlated with their biodegradation rate [51]. Fig. 4B shows the degradation behavior of the samples. The rate of sample weight loss gradually increased with the incubation time until day 30. All samples degraded rapidly in the first 3 days, and the degradation rate of SF/GT nanofibers reached its highest value of 43.72% on the third day, which might be related to the rapid degradation of amino groups in GT. The degradation rates of SF/GT, SF/GT-0.5%EP, SF/GT-1%EP, and SF/GT-3%EP, were 55.71%, 51.13%, 52.67%, and 46.33%, respectively, at day 30. Our results indicated that the incorporation of propolis reduced the degradation rate of the samples. These results are consistent with the results of the water absorption capacity. The propolis-incorporated samples had a lower absorption capacity and were degraded more slowly. Furthermore, it can be concluded that the SF/GT-EP nanofibers have promising degradation properties.

3.3. Water uptake capacity

The ability of a material to absorb water is one of the decisive parameters in determining its suitability as a trauma dressing. The ideal trauma dressing should have good water retention capacity, which plays an important role in maintaining a moist local environment, preventing wound dryness, and preventing wound infection by absorbing fluid and exudate from the wound bed [[52], [53], [54]]. Furthermore, since the main function of the exudate is to aid in the diffusion of multiple cytokines and growth factors, enhance intercellular communication, and promote the migration of keratinocytes and the proliferation of fibroblasts, good water retention capacity helps with maintenance of the exudate, thus accelerating wound healing.

The WUC of the samples was assessed 24 h later (Fig. 4C). The WUC values of the SF/GT, SF/GT-0.5%EP, SF/GT-1%EP, and SF/GT-3%EP samples were 352.67 ± 4.2, 346.26 ± 9.8, 322.74 ± 5.4, and 278.82 ± 11.7%, respectively. Our results indicate that as the concentration of propolis increased, the WUC of the samples gradually decreased. The WUC of SF/GT, SF/GT-0.5%EP, and SF/GT-3%EP samples exhibited significant differences. The WUC values were in good agreement with the WCA results. The addition of propolis decreased the hydrophilicity of the nanofibers and changed their WUC. This is consistent with the results of previous studies [24]. This can be explained by the hydrophobicity of propolis, which is caused by the presence of fatty acids and terpenes in the substance, among other additional hydrophobic groups [25]. The SF/GT-EP nanofibers provided good WUCs, and hence, are promising for use as wound dressings to capture exudates and keep the wound moist.

3.4. WCA measurement

The hydrophobic (WCA >90°) and hydrophilic (WCA <90°) properties of the dressing play a critical role in the interactions between the cellular microenvironment and cells [55]. The hydrophilicity of biomaterials is one of the most critical parameters affecting cell proliferation and adhesion. Previous studies have reported that highly hydrophilic biomaterials support cell attachment and proliferation [56,57], thereby promoting wound healing. A hydrophilic surface is essential for wound dressing and tissue engineering scaffolds.

Measurement of the WCA is a common procedure for measuring the wettability of a sample surface. Fig. 4D shows the WCA of different sample mats measured for a duration of 5s. The WCA of SF/GT is 38.27 ± 3.2°, demonstrating the high hydrophilicity of the samples, which can be attributed to the water sensitivity of GT. The SF/GT-0.5% EP, SF/GT-1%EP, and SF/GT-3%EP blends have higher WCAs (45.7 ± 4.7°, 51.18 ± 6.2°, and 75.24 ± 5.9°, respectively) than the SF/GT blend. Hence, the WCA of the SF/GT nanofiber gradually increases with increasing concentration of propolis. This is consistent with the results of previous studies [52]. The complex composition of propolis may explain the decreased hydrophilicity of the composite SF/GT-EP nanofibers. WCAs of less than 90° indicate the desired hydrophilicity of the synthesized nanofibers. Therefore, the synthesized SF/GT-EP nanofibers will be beneficial for the absorption of wound exudates as well as the maintenance of moisture, thereby providing a new type of wound dressing.

3.5. Biocompatibility of nanofiber dressings

3.5.1. Hemolysis analysis

During actual use, the nanofiber dressing will be in direct contact with the blood at the wound site, as well as with various cells, such as skin keratinocytes and fibroblasts. When a hemolysis reaction occurs, the red blood cell membrane is destroyed, resulting in the efflux of hemoglobin from the red blood cells and increase in free hemoglobin. Hemolysis causes various biologically harmful outcomes in organisms. Favorable hemocompatibility is an imperative feature of wound dressings in tissue engineering [58,59]. In various studies, the hemolysis test has been used as a reliable and simple method to evaluate the hemocompatibility of nanofiber samples. According to the requirements of the American Society for Testing and Materials (ASTM F 756–00), samples with hemolysis (%) higher than 5% are hemolytic, those with hemolysis ranging from 2 to 5% are slightly hemolytic, and those with hemolysis less than 2% are non-hemolytic.

We selected SF/GT-1%EP for the subsequent experiment based on the physical parameters of each sample that we investigated. The HR results are shown in Fig. 5A. Compared with the pure SF/GT sample, the addition of EP slightly increases the HR to less than 5% for SF/GT and SF/GT-1%EP (the HRs are 0.63% and 1.67%, respectively). This indicates that the SF/GT-EP nanofiber can be used as a novel wound dressing with favorable blood compatibility.

Fig. 5.

Biocompatibility evaluation of the nanofibers. (A) Hemolysis performance of different samples in vitro. (B) Cell viability of the control group, SF/GT, and SF/GT–1%EP in the live/dead assay kit after 3 days. (C) Cell viability histogram. The measurements were performed in triplicate. (D) L929 fibroblast cell viability assay result of the TCP (control) and fabricated nanofiber mats (SF/GT and SF/GT-1%EP) after 1, 3, and 5 days of incubation.

3.5.2. Cellular biocompatibility

Fibroblasts are one of the most common cells in connective tissue, which can determine the rate and quality of wound healing by affecting the release of active factors, collagen deposition, and inflammatory response [60,61]. L929 fibroblasts are widely accepted as cell lines for cytotoxicity testing (ISO 10993–5:2009). We evaluated the cell viability of the control, SF/GT, and SF/GT-1%EP groups on the third day using the live/dead assay kit. The results are shown in Fig. 5B. All tested cells grow well, and the survival rates are above 95% (Fig. 5C), suggesting that two types of samples support the survival and proliferation of L929.

To further investigate the biocompatibility of nanofiber dressings with different components as biomaterials for tissue regeneration, three experimental groups, including the control TCP, SF/GT, and SF/GT-1%EP, were used. The cell viability was investigated on days 1, 3, and 5 using CCK8 assays. Our results are shown in Fig. 5D. On day 1, no significant difference is observed between the cells cultured with SF/GT, SF/GT-1%EP, and the controls. With the extension of the culture time, the cells proliferate significantly on the 3rd and 5th days. However, there is still no significant difference between the SF/GT, SF/GT-1%EP, and control groups. The good cytocompatibility of the nanofibrous materials is attributed to the mildness of the composition and porosity of the nanomaterials, which enable nutrient transport, metabolite exchange, and cell migration.

The cell morphology on the nanofiber is shown in the SEM images. Fig. 6A–F illustrates the adequate proliferation of the L929 cells and their attachment to the surface of the nanofiber 3, 7 and 10 days after seeding. Therefore, the SF/GT-1%EP sample exhibits high biocompatibility with the L929 fibroblasts. Accordingly, the above results are consistent with the results of previous studies [62]. We can further predict the good performance of the SF/GT-1%EP wound dressing in accelerating the healing of defective skin wounds in animal models. Therefore, SF/GT–1%EP is a potential candidate for wound dressings and skin tissue engineering.

Fig. 6.

SEM images of the cultured L929 fibroblast cells on the electrospun nanofibers from: (A) SF/GT (3D), (B) SF/GT (7D), (C) SF/GT (10D), (D) SF/GT-1%EP (3D), (E) SF/GT-1%EP (7D), and (F) SF/GT-1%EP (10D). (Scale bars = 50 μm).

3.6. SF/GT-1%EP promotes the migration of L929 fibroblasts

Fibroblasts contribute significantly to the healing process as one of the primary repair cells in wound healing. Under the conditions of the wound microenvironment, cytokines and other factors cause fibroblasts to multiply and migrate, resulting in the secretion of collagen and additional cytokines, which participate in the formation of an extracellular matrix and granulation tissue, occurrence of wound contraction, and scar formation. The migration of L929 fibroblasts toward SF/GT and SF/GT-1%EP was examined in a transwell experimental assay (Fig. 7A). According to our results (Fig. 7B and C), the number of migrating cells is considerably larger in the presence of the SF/GT-conditioned medium (67 ± 9/visual fields) and SF/GT-1%EP-conditioned medium (107 ± 14/visual fields) compared to the control (12 ± 3 cells/visual fields, p < 0.001, respectively). Hence, our results are significant when compared to earlier research [43]. In conclusion, the data show that SF/GT-1%EP enhances the in vitro migration of L929 fibroblasts. Thus, SF/GT-1% EP nanofibers have potential therapeutic effects in wound healing.

Fig. 7.

Influence of SF/GT and SF/GT-1%EP on the migration of L929 cells. (A) Schematic of the experimental setup. (B) Migration of L929 cells through 8 μm pore-size transwell inserts toward the lower surface of the transwell pores. (C) Cells migrating through the porous membrane were quantified by Image J software (n = 3 samples/condition).

3.7. Antibacterial activity

Bacterial infection often leads to delayed wound healing. As special materials that can kill bacteria or inhibit the reproduction of microorganisms, antibacterial materials have been widely used in wound treatment. In this study, two different methods were carried out to determine the antibacterial capacities of the nanofibrous materials toward the S. aureus and E. coli. First, Fig. 8A and B shows the bacteriostatic activity of the SF/GT and SF/GT-EP nanofiber patches against S. aureus and E. coli, respectively. No inhibition zone is formed on the SF/GT solid plates, proving that SF and GT do not have any antibacterial effect against E. coli and S. aureus. However, the addition of propolis endows significant antibacterial activity and gradually increases the diameter of the bacterial inhibition zone with increasing amounts of propolis in the SF/GT nanofiber patches. Therefore, the SF/GT-1%EP and SF/GT-3%EP samples have the highest antibacterial activity, i.e., the largest clear area around the sample. Second, we investigated the antibacterial ratios of the nanofibrous materials using the OD method, and the results were showed in Fig. 8C and D. The SF/GT-3%EP samples exhibited excellent antibacterial ratios for both S. aureus (91.2%) and E. coli (83.4%), which was mainly attributed to the release of the propolis. The antibacterial activity of propolis is not the single effect of a certain substance but is a result of the synergistic effect of multiple components, such as various aromatic structures and other compounds, including flavonoids, aromatic acids, and esters in the resin. Previous research has indicated that different compounds within propolis may target various bacterial cell activities. Propolis can cause partial bacterial lysis and inhibit nucleic acid and protein synthesis [63]. In addition, the synergistic activity between propolis and antibiotics has been confirmed by numerous investigations. Antibiotic therapy can be enhanced with propolis, which increases the antibacterial effect and decreases the recovery period.

Fig. 8.

Inhibition zones of different samples (SF/GT, SF/GT-0.5%EP, SF/GT-1%EP, and SF/GT-3%EP) against (A) S. aureus and (B) E. coli after 24 h of incubation. Antibacterial ratio of different samples against S. aureus (C) and E. coli (D) via the OD method (mean ± SD, n = 5).

3.8. In vivo wound healing

The wound-healing effects of the nanofiber dressings were further investigated in vivo. First, a full-thickness wound was created on the back of the mouse. As full-thickness wounds are the most difficult to heal among all types of wounds, they are frequently used to evaluate the ability of novel biomaterials to accelerate wound healing. Second, the nanofiber material was fabricated into a circular mat with a diameter of 7 mm and thickness of 0.5 mm and was attached to the wound surface. Finally, according to the previous experimental results, three groups, including the control (treated with saline), SF/GT, and SF/GT-1%EP, were used in the experiment to investigate the ability of the SF/GT-EP dressing to accelerate wound healing.

Visual observation of the wound areas in the mice showed that there was no infection or necrosis during the experiment (Fig. 9A and B). Mice have a certain self-healing ability because the wound area of the control group continuously shrinks after the operation. On the 7th day after treatment, the wound area covered by SF/GT-1%EP exhibits the most significant reduction. On the 12th and 17th days, the SF/GT group has a significantly improved treatment effect than the control group. In addition, the SF/GT-1%EP group offers the highest wound closure speed and most efficient healing effect, due to which the wound is almost completely closed at 17 days. Our result is consistent with previous findings [45], and our results show that SF/GT-1%EP has a good ability to promote healing.

Fig. 9.

In vivo wound healing results. (A) Macroscopic photographs of wounds at 0, 7, 12, and 17 days after a full-thickness skin wound was created. (B) Wound diameter (0, 7, 12, and 17 days) of different groups (control, SF/GT, and SF/GT-1% EP). The data are presented as mean ± SD (n = 4). (C, D) Histology analyses of the wound area sections at different groups 17 days after the operation (control, SF/GT, and SF/GT-1% EP).

SF and GT are widely recognized as biomaterials that can accelerate wound healing. This study showed that the addition of EP further enhanced the healing ability of SF and GT, which may be attributed to the complex composition of propolis. Therefore, SF/GT-1%EP nanofibers are promising wound dressings that can effectively accelerate wound healing and improve the healing quality.

3.9. Histological analysis

Hematoxylin-eosin (H&E) staining was used to evaluate the healing quality of the wounds on the 17th postoperative day. As shown in Fig. 9C, the differences in wound closure length and regenerated granulation tissue thickness were statistically significant between the groups. Wounds treated with saline were covered with a thin layer of new epidermis and had the longest wounds, and there was minimal regeneration of the skin structure. The SF/GT and SF/GT-1%EP groups had thicker layers of granulation tissue and shorter wounds, and the newly formed skin in the SF/GT-1%EP group was more complete than that in the other two groups, a result consistent with the general results we had observed. At the same time, the propolis-loaded SF/GT nanofiber group had a higher degree of re-epithelialization compared to that in the other two groups, showing more regular epithelial and connective tissue. We also observed the regeneration of skin appendages, including neovascularization, hair follicles, and sebaceous glands, on the wound bed. The skin thickness and histological features were similar to those of normal skin. These results suggest that propolis-loaded SF/GT nanofibers promote skin epithelialization and skin appendage regeneration, thus exhibiting good pro-healing properties.

In the process of wound healing, collagen synthesized by fibroblasts is the main component of the ECM, and the formation of new ECM can provide a suitable microenvironment for cell proliferation, migration, and differentiation, as well as intercellular communication. Collagen deposition plays an important role in tissue regeneration and functional repair [64], as shown in Fig. 9D, wherein the Masson trichrome staining showed that collagen fibers in the control group were loosely arranged on day 17 after saline injection and their collagen deposition was mainly distributed at the base of the dermis and the area of wound defects. A denser distribution of collagen structures was observed in the new dermis of the SF/GT treatment group. However, the wounds in the SF/GT-1%EP group were a darker blue color compared to that in the other two groups, showing thicker and more uniform collagen deposition and with collagen deposition in all parts of the dermis and epidermis, similar to that in normal skin. Despite the presence of collagen in the wound bed of all three groups, the distribution and morphology of collagen observed suggested that the SF/GT-1%EP group had the best wound healing quality. This could be related to the synergistic effect among the components of the dressing, SF, gelatin, and propolis, as well as the moist environment provided by the dressing, which promoted fibroblast migration, proliferation, and deposition of collagen during epithelialization.

4. Conclusion

In summary, we prepared propolis-loaded silk gelatin nanofiber membranes via electrospinning, investigated the effect of propolis incorporation on the properties of SF/GT nanofibers, and applied them to a mouse model of full thickness skin defects. The results showed that the addition of propolis significantly changed the mechanical properties, hydrophobicity, and degradability of SF/GT nanofibers. The addition of propolis obviously conferred some antibacterial properties to SF/GT. In vitro experiments showed that SF/GT-1%EP has good cytocompatibility and blood compatibility and can support cell adhesion and growth for a long time. In vivo wound healing experiments showed that SF/GT-1%EP significantly accelerated wound healing, and histological experiments further confirmed its pro-healing properties. Therefore, SF/GT-1%EP shows good promise for skin regeneration therapy.

Author contribution statement

Pan Du, Xue Chen, Guozhong Lv: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the datas; Contributed reagents, materials, analysis tools or data. Yang Chen, Jin Li, Yichi Lu: Performed the experiments; Analyzed and interpreted the data; Wrote the paper. Xiaoxiao Li, Kai Hu, Junfeng Chen: Conceived and designed the experiment; Contributed reagents, materials, analysis tools or data.

Funding statement

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability statement

Data included in article/supplementary material/referenced in article.

Declaration of interest's statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.