Introduction

The skin serves as a robust external protective barrier and initiates the first line of immune defense against various external stimuli, including mechanical, chemical, and pathogenic challenges1,2,3. Wounds, such as those resulting from surgery, trauma, superficial burns, or ulcers, can significantly impact patients’ physical and mental health, representing a major global health issue4,5,6. Despite advancements in wound dressing technologies designed to promote healing, significant challenges remain, especially in managing open wounds. These wounds are often complicated by bacterial infections, which can lead to tissue inflammation, necrosis, and, in severe cases, life-threatening conditions7,8,9. An excessive inflammatory response in wounds necessitates careful management. Furthermore, promoting effective revascularization is crucial for accelerating the healing and functional recovery of chronic infected wounds, as vascular tissues support normal cell metabolism by facilitating nutrient and oxygen transport10. Consequently, the development of multifunctional wound dressings that combine mechanical strength, high water retention, non-toxicity, good tissue compatibility, and antimicrobial properties offers significant potential for improving the healing of chronic infected wounds11,12.

Bacterial cellulose (BC) is a biopolymeric material composed of β-D-pyranose glucopyranosyl units linked by (1→4)-glycosidic bonds. Synthesized by bacteria, BC is a natural polysaccharide hydrogel with a unique structure that provides high water absorption, high water retention, and excellent permeability to liquids and gases, along with significant wet strength due to the “nano-effect”13,14. These characteristics make BC highly advantageous for medical dressing applications. However, BC lacks inherent antimicrobial properties and does not promote neovascularization, which limits its use in various biomedical applications. Therefore, it is essential to functionalize BC with antibacterial agents and neovascularization-promoting compounds to fully exploit its potential in wound dressings and prevent infections during the healing process.

Recent innovations in wound care have included the incorporation of growth factors into wound dressings to enhance healing15. Growth factors such as platelet-derived growth factor, epidermal growth factor, and transforming growth factor beta have demonstrated efficacy in improving wound healing16. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is produced by various cells involved in tissue repair, including keratinocytes, fibroblasts, endothelial cells, and macrophages. GM-CSF promotes keratinocyte proliferation and migration, facilitating re-epithelialization during wound healing17,18. It also enhances endothelial cell proliferation via NF-κB activation, thereby supporting neovascularization at the wound site. Neovascularization provides essential components for repair, while GM-CSF further promotes fibroblast differentiation, granulation tissue formation, and wound contraction19,20. Recent studies suggest that topical application of GM-CSF can significantly advance the wound healing process19.

The primary objective of this study was to develop an effective BC-based wound dressing. To achieve this, a BC/Ag composite was prepared to enhance the antimicrobial properties of the BC-based material. The BC/Ag composite was further enriched with GM-CSF to accelerate wound healing, improve re-epithelialization, and enhance granulation tissue formation compared to the unmodified BC/Ag composite. Systematic biological evaluations and animal model experiments were conducted to validate the effectiveness of the BC-based antimicrobial functional biomedical dressings. The results aim to provide a theoretical foundation for the development and application of these advanced wound dressings.

Materials and methods

Materials

BC was supplied from QiHong Technology Co Ltd (Guilin, China). Sodium hydroxide (NaOH), silver nitrate (AgNO3) was purchased from Sinopharm Chemical Reagent Co Ltd (Shanghai, China). GM-CSF was kindly provided by Xiamen Amoytop Biotech Co., Ltd. CCK-8 and pancreatic enzyme (0.25%) was purchased from InCellGene LLC. CD31 was provided by Abcam company, DMEM was purchased from Thermo Fisher Scientific, and fetal bovine serum was purchased from Sigma, Inc. All experimental procedures were conducted using deionized water, as the deionized water was obtained from the Millipore water purification system.

Preparation of BC/Ag/GM-CSF composite antimicrobial film

Synthesis of BC/Ag/GM-CSF composite film BC in gel form was boiled in a solution of NaOH at pH 13 for 4 h at 110 °C. After washing with deionized water to neutralize it, the BC was homogenized to obtain a BC homogenate. Then BC film was prepared by vacuum filtration and dried. With BC as a template, it was immersed in a 0.1 mol/L AgNO3 solution and placed in an autoclave with reaction conditions of 0.1 MPa and 121 °C for a hydrothermal reaction until the beige BC membrane turned brown, proving the successful synthesis of Ag nanoparticles. The prepared BC/Ag composite film was treated with high-pressure sterilization and then immersed in a 10 µg/mL GM-CSF aqueous solution. After 10 min of treatment, a BC/Ag/GM-CSF composite film wound dressing was formed.

Characterization

The Fourier transform infrared spectra (FTIR) of the thin films were acquired under ambient conditions using a Spotlight 400 spectrophotometer. The attenuated total reflection (ATR) mode was employed, with an average of 32 scans at a resolution of 4 cm−1 over a wavelength range of 400–4000 cm−1. A DX-2700BH X-ray diffractometer was used, with Cu Kα radiation (λ = 1.54 Å) under a 2θ scanning angle of 10–90°, to conduct X-ray diffraction (XRD) testing. The morphology and structure of the prepared composite films were characterized using a field emission scanning electron microscope (Quanta-450, FEI). For the water vapor transmittance test, we firstly cover the mouth of a test tube with a diameter of 2 cm with BC, BC/Ag, BC/GM-CSF, and BC/Ag/GM-CSF composite films, and secure them tightly with a rubber band. Place the sealed test tubes in the 37 °C constant temperature water bath and let them stand for 24 h. Record the amount of water reduction in the sealed test tubes and calculate the water vapor transmission rate (WVTR) of the film material using the following formula.

$$\:WVTR=\frac{\left({W}_{1}-{W}_{2}\right)\times\:24}{\text{A}}(g/{m}^{2} \,day)$$

W1—Initial mass of water; W2—Quality of water after heating at 37 °C for 24 h; A—film covering area of the test tube.

Antibacterial activity assay

S. aureus (ATCC 25923) is a typical Gram-positive bacterium, E. coli (ATCC 8739) and P. aeruginosa (ATCC 27853) representing typical Gram-negative bacteria, they were utilized to evaluate the antibacterial properties of various dressings using the disc diffusion method. The antimicrobial effectiveness was assessed by measuring the zone of inhibition (halo width) in a disk diffusion test. A 100-microliter suspension of either E. coli or S. aureus was uniformly spread onto Luria-Bertani (LB) agar plates. Circular BC, BC/Ag, BC/GM-CSF and BC/Ag/GM-CSF dressings (15 mm in diameter) were then placed on the LB agar plates and incubated at 37 °C. Bacterial growth was documented after three days. The halo width was determined with ImageJ software.

In vitro biocompatibility

Hemolysis assay

In the hemolysis experiment, collect 1 ml rabbit blood, add sodium heparin, and dilute with physiological saline. Centrifuge at 2,500 rpm for 10 min, discard supernatant, and isolate red blood cells. Wash the cells with physiological saline five times, then dilute with 10 ml saline. Combine 0.1 ml diluted red blood cells separately with 0.4 ml of negative control (physiological saline), positive control (deionized water), and extraction solutions from BC membrane, BC/Ag, BC/GM-CSF, and BC/Ag/GM-CSF. Mix and incubate at room temperature for 3 h. Centrifuge suspensions at 14,000 rpm for 3 min, transfer 100 µl supernatant to a 96-well plate, and complete within 4 h to minimize errors. Measure absorbance at 570 nm and 655 nm to assess hemolysis potential of extracted samples.

Cytotoxicity test

We initially selected serum-free DMEM culture medium with immersed material. Then, a membrane of 600 cm2 area was placed in a clean conical flask and 100 ml of serum-free culture medium was added. After soaking for 48 h at 37 °C, we obtained an extract at 6 cm2/ml. We then removed the membrane and performed low-speed centrifugation at 800 r/min for 5 min. The liquid after centrifugation was filtered through a 0.22 μm microporous filter and transferred into sterile test tubes. After filtration, the tubes were stored in a 4° refrigerator for later use. NIH3T3 cells in good state were inoculated at 6 × 103 cells/well into a 96-well plate and cultured for 24 h. DMEM was then added as a negative control group, while the other four groups were respectively introduced with extracts of BC membrane, BC/Ag, BC/GM-CSF, or BC/Ag/GM-CSF and cultured for another 24 h. Subsequently, 10 µL of CCK-8 was added in each well, and the culture was incubated for 2 h at 37 °C. Absorbance at 450 nm was measured in each well. Each group was replicated three times.

Scratch assay

The migratory behavior of NIH3T3 cells in the presence of the specified films was assessed using an in vitro scratch assay. NIH3T3 cells were seeded into 6-well plates at a density of 1 × 105 cells/well. After 24 h of incubation, the cells reached approximately 80% confluence, and a scratch was introduced into the cell monolayer using a 200 µl pipette tip. The films were prepared as for the CCK8 assay and placed into the appropriate wells. At time 0 h, the width of the scratch was recorded as 100%, and the initial scratch coverage by cells was considered 0%. The progressive cell migration into the scratched area was monitored using an Olympus microscope, with images captured at 0, 12, 24, and 48 h of incubation. Image analysis was performed using ImageJ software, with the percentage of scratch coverage calculated based on the ratio of white to black pixels. This was quantified over time by measuring the black pixel count in the images. Each experimental group was replicated three times.

In vivo wound healing

All animal-related experiments were conducted under the approval of the Ethical Committee of Shaanxi Provincial People’s Hospital. The wound healing ability of the prepared dressing film was tested on male BABL/C mice weighing 20–25 g. The mice were randomly divided into four groups (n = 5 per group): BC group, BC/Ag group, BC/GM-CSF group, and BC/Ag/GM-CSF group. After anesthetizing the mice with barbital sodium, their backs were shaved, and a full-thickness wound (1.0 cm) was made on each mouse. Subsequently, the wound was fully covered with the dressing and secured with Tegaderm (3 M). The dressings were used continuously for 2 days, followed by replacement with fresh dressings for another 2 days. The mice were then put in separate cages to enhance wound healing. Concurrently, photographs of the wounds were taken and their sizes measured at days 0, 3, 6, 9, 12, and 15, using a digital camera. Skin tissue samples were fixed in formalin solution (4%) and embedded in paraffin for histological observation. Histological micrographs after Hematoxylin and Eosin (H&E) and Masson’s Trichrome staining were recorded by the light microscope. To evaluate histology of wound tissues and collagen fibers deposition, at day 15, mice were euthanized, and wound tissues were collected and processed in 4% Polyformaldehyde (vol/vol) for 24 h. Each specimen was then dehydrated through an ethanol gradient and embedded in paraffin. Tissue sections of 5 millimeters thick were collected and stained with Hematoxylin and Eosin, Masson’s Trichrome, and CD31 immunofluorescent. Moreover, to assess neovascularization, tissues were immunofluorescent stained with CD31.

To further analyze the function of BC/Ag/GM-CSF in the infectious wound, we build the infectious wound on male BABL/C mice weighing 20–25 g. The dorsal surface of the rats was depilated, 100 µl S. aureus suspension (1 × 107 CFU/mL) was injected subcutaneously for infectious model fabrication. On the third day, abscess formation was observed. After anesthetizing the mice with barbital sodium, their backs were shaved, and a full-thickness wound (diameter 1.0 cm) was made on each mouse. The mice were randomly divided into four groups (n = 5 per group): BC, BC/Ag, BC/GM-CSF, and BC/Ag/GM-CSF group. Subsequently, the wound was fully covered with the dressing and secured with Tegaderm (3 M). The dressings were used continuously for 2 days, followed by replacement with fresh dressings for another 2 days. The mice were then put in separate cages to enhance wound healing. Concurrently, photographs of the wounds were taken and their sizes measured at day 0, 3, 6, 9, 12, and 15, using a digital camera.

Statistical analysis

After conducting statistical analysis using Student’s t-test and one-way analysis of variance (ANOVA), all experimental data were presented in the form of mean ± standard deviation. Statistical significance was considered when the P value was less than 0.05.

Results and discussion

Fabrication and characterization of BC/Ag/GM-CSF composite film

Combining the excellent biocompatibility and high water-holding properties of BC, it is a good choice to use them as a substrate for in situ growth of functional antibacterial particles. As shown in the process flow of Fig. 1, we prepared the BC/Ag/GM-CSF composite film. The stable load of silver nanoparticles and their excellent antibacterial properties endowed the prepared composite membrane with good biomedical performance. Combined with GM-CSF to enhance the proliferation and migration of keratinocytes, the wound healing process was greatly shortened. This not only ensured the rapid healing of the wound in the early stage but also significantly reduced the incidence of inflammation caused by bacterial invasion during the wound healing process. Based on the three-dimensional nano-network of BC, this composite antibacterial membrane has excellent breathability and oxygen permeability. Combined with the in-situ growth of silver nanoparticles and the impregnation load of GM-CSF, the BC/Ag/GM-CSF will effectively promote the healing of skin wounds.

Fig. 1
figure 1

Schematic representation of the fabrication of the BC/Ag/GM-CSF composite film.

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FT-IR analysis can be used to determine the surface material composition of composite membranes. The stretching vibration absorption peak of hydroxyl groups is located at approximately 3400 cm−1, while the stretching vibration absorption peaks of carboxylate anions and the C–O–C glycosidic bond are located at approximately 1613 cm−1 and 1090 cm−1, respectively, as shown in Fig. 2a. Silver ions are reduced by BC, which also acts as a stabilizer to prevent aggregation of the resulting Ag nanoparticles21. BC serves as a carrier in the hydrothermal reduction reaction, ensuring the uniform dispersion and fixation of Ag. Compared to the infrared curve of pure BC, there is no obvious characteristic peak at 1613 cm−1, indicating that the -OH on C6 of BC converts to C = O during the reduction process22. The strong characteristic peak of C=O at 1613 cm−1 in the BC/Ag curve confirms its presence.

To further confirm the presence of in-situ synthesized silver nanoparticles in the composite membrane, we conducted XRD testing on four types of films, as shown in Fig. 2b. Clear absorption peaks of the sample silver nanoparticles at 38.2°, 44.4°, 64.5°, and 77.5° indicate the corresponding crystal surfaces of cubic silver (111), (200), (220), and (311). The silver nanoparticles exhibit a face-centered cubic structure23,24. Through in-situ growth, utilizing the BC membrane as a template, Ag is successfully grafted onto the BC membrane, as evidenced by the XRD curve of BC/Ag and BC/Ag/GM-CSF film, which displays the absorption peaks of BC and three absorption peaks of Ag (111) and (200). The loading of silver particles on BC can also be observed from the color change of the BC film, turning from white to dark brown after in-situ growth of silver (Fig. 2c).

Fig. 2
figure 2

a FTIR curves of BC, BC/Ag, BC/GM-CSF and BC/Ag/GM-CSF film. b XRD curves of BC, BC/Ag, BC/GM-CSF and BC/Ag/GM-CSF film. c Digital image of film characteristics of BC, BC/Ag, BC/GM-CSF and BC/Ag/GM-CSF film.

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Scanning electron microscopy (SEM) images reveal the surface morphology of the composite film. Figure 3 depicts the smooth surface of the BC film. After the in-situ synthesis of silver nanoparticles, a large number of silver nanoparticles are attached to the surface of the BC/Ag film, demonstrating the effectiveness of the high-temperature, high-pressure hydrothermal process for creating silver nanoparticles on the BC film. From the SEM image of the BC/GM-CSF film, it can be observed that GM-CSF is distributed in a fibrous manner on the surface of BC. Similarly, GM-CSF can also adhere well to the surface of the BC/Ag film, forming a composite BC film with fibrous GM-CSF and spherical Ag nanoparticles jointly loaded.

EDS testing was conducted to analyze the surface elements of the films and confirm the distribution of silver nanoparticles and GM-CSF on the film surface. Figure 3 shows that the BC film only contains carbon and oxygen, while silver elements are uniformly distributed on the surface of the BC/Ag sample, confirming the successful synthesis of silver nanoparticles25. The EDS images of the BC/GM-CSF and BC/Ag/GM-CSF films also demonstrate the uniform distribution of nitrogen elements on the film surface which is attributed to the presence of GM-CSF. This indicates that both the in-situ synthesized silver nanoparticles and the GM-CSF loaded via immersion have successfully achieved even loading on the BC film, thereby playing a crucial role in the antibacterial and anti-inflammatory properties of the composite film.

Fig. 3
figure 3

SEM images and EDS spectra of BC film, BC/Ag film, BC/GM-CSF film, and BC/Ag/GM-CSF film. The elemental distribution of carbon, oxygen, silver, and nitrogen on the material surface, as detected by EDS spectrum testing, is represented by C, O, Ag, and N, respectively.

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Antibacterial activity

To further validate the antibacterial properties of the composite film, we evaluated the antibacterial activity of BC/GM-CSF, BC/Ag, and BC/Ag/GM-CSF composite films using the disc diffusion method. The antibacterial activity of these three composite films against S. aureus, E. coli, and P. aeruginosa was confirmed by the inhibition zones on the agar plates. As shown in Fig. 4a, both GM-CSF as a functional component and Ag nanoparticles exhibited antibacterial characteristics. GM-CSF activated the chemotaxis and activation abilities of neutrophils and macrophages, enhancing their phagocytic and bactericidal activities against necrotic tissue, deactivated fragments, and pathogenic foreign bodies26. In terms of the antibacterial mechanism of silver nanoparticles, positively charged silver ions adsorb to negatively charged microbial cells when they come into contact. Simultaneously, silver ions penetrate the cell membrane, disrupting cell DNA and inhibiting protein synthesis, which impairs the cell’s ability to metabolize and reproduce, ultimately leading to cell death and achieving antibacterial effects27,28. Based on the afore mentioned antibacterial mechanisms, the maximum inhibition zone diameters of BC/Ag/GM-CSF composite film against S. aureus, E. coli, and P. aeruginosa were 2.14 mm, 2.2 mm, and 1.98 mm, respectively, allowing the calculation of the antibacterial rates of BC/Ag/GM-CSF composite film against the afore mentioned three bacteria to be 99.09%, 99.79%, and 98.5% (as shown in Fig. 4b)29. The water vapor transmittance of several films is shown in Fig. 4c. Due to BC’s special 3D network structure, it is easy to penetrate water vapor, as the WVTR can reach 1088 g/m2/day. After adding Ag particles and GM-CSF, Ag particles or GM-CSF occupy the gap between the original BC networks, reducing water vapor’s transmittance.

Fig. 4
figure 4

a Agar plate diffusion method was used to test the bactericidal performance of BC/Ag film, BC/GM-CSF film, and BC/Ag/GM-CSF film against E. coli, S. aureus and P. aeruginosa in the natural environment. b Bacterial inhibition rate of BC/Ag film, BC/GM-CSF film, and BC/Ag/GM-CSF film against S. aureus, E. coli and P. aeruginosa in the natural environment. c Water vapor transmittance rate of BC, BC/Ag, BC/GM-CSF, and BC/Ag/GM-CSF composite films.

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In vitro biocompatibility

The hemolysis rate of a material reflects the degree of erythrocyte destruction after the material encounters blood. The larger the hemolysis rate, the greater the degree of erythrocyte disruption30,31,32. This can be applied to clinical biomaterials, with the hemolysis rate required to be less than 5%. Figure 5a shows samples from hemolysis experiments on different materials. The color intensity of the supernatant represents the degree of erythrocyte disruption, with the hemolysis rates of all dressing extraction groups less than 5%. There was no hemolysis phenomenon, and there was no significant difference from the negative control (physiological saline group), indicating good blood compatibility (Fig. 5b).

A good cell compatibility is a fundamental requirement for medical wound dressings. The BC membrane possesses excellent biocompatibility. The biocompatibility of Ag ions and GM-CSF infiltrated variants was validated via the CCK-8 method. NIH3T3 cells were cultured in 96-well plates, and the extract solutions from BC, BC/Ag, BC/GM-CSF, or BC/Ag/GM-CSF were added and incubated for 24 h. As shown in Fig. 5c and d, upon the addition of the extraction solutions, cell viability of each group slightly declined, but were all above 90%. There was no significant difference in cell viability between the BC/Ag/GM-CSF and the BC group. These findings suggest that in-situ deposition of silver particles on BC can greatly decrease the intrinsic cytotoxicity of silver particles, so that the produced antimicrobial film material can exert its excellent antimicrobial properties while maintaining good cell compatibility.

Scratch assay is very important to evaluated material have any wound healing potential. Thus, we performed scratch test to assay ability of these film for wound healing. Result showed as the area that cells closed during the 12 h, 24 h and 48 h of the experiment (Fig. 5e, f). The BC sample showed no significantly effect on cell migration. Moreover, there was no significant difference in cell migration abilities between the BC/Ag/GM-CSF and the BC group (p > 0.05).

Fig. 5
figure 5

a Hemolysis experiment image of the BC film, BC/Ag film, BC/GM-CSF film, and BC/Ag/GM-CSF film leachate; b histogram of the hemolysis ratio of the test samples; c microscopic photographs of cells exposed to BC film, BC/Ag film, BC/GM-CSF film and BC/Ag/GM-CSF film leachate for 24 h. d Cell viability of the test samples; e migration rate of BC film, BC/Ag film, BC/GM-CSF film, and BC/Ag/GM-CSF film leachate; f scratch experiment image of BC film, BC/Ag film, BC/GM-CSF film, and BC/Ag/GM-CSF film leachate.

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BC/Ag/GM-CSF enhanced the healing effect on normal wound model

The demonstrated intrinsic biocompatibility and antibacterial activity of the materials may facilitate effective wound healing in vivo. To evaluate the in vivo healing efficacy of BC/Ag/GM-CSF, we employed the commonly used traumatic skin injury model in BALB/c mice. The wound site was monitored on days 0, 3, 6, 9, 12, and 15 to record the healing process for detailed measurement in subsequent stages. On the day of the procedure, a full-thickness wound with a diameter of 10 mm was created. Macroscopic images (Fig. 6) illustrate a gradual decrease in wound area over time in all groups. The wound photographs (Fig. 6a) and closure histogram (Fig. 6b) indicate that by the 9th day, the BC/Ag/GM-CSF group exhibited a higher closure rate and narrower granulation tissue compared to the other groups. Previous studies highlight that the early reduction rate of wound area is critical for complete closure in later stages33,34. By the 15th day, the average remaining wound area in the BC/Ag/GM-CSF group had reduced to 6.5 ± 1.25% of the initial area, significantly smaller than that of the BC group (35 ± 5%), BC/Ag group (30 ± 2.5%), and BC/GM-CSF group (20 ± 3.5%), demonstrating a faster healing process. The wounds in both the BC/Ag/GM-CSF and BC/GM-CSF groups showed significant healing, with the remaining wound areas in the BC group being nearly invisible. These results suggest that GM-CSF-containing BC treatments significantly accelerate wound closure, with the BC/Ag/GM-CSF film showing the most pronounced effect due to the synergistic interaction between GM-CSF and Ag.

Fig. 6
figure 6

The effect of indicated film in the healing on acute wound model. a Wound conditions of the respective groups on day 0, 3, 6, 9, 12, and 15. The wounds without infection and treated with BC were noted in the control group. The bar was 10 mm; b wound areas closure of the respective groups on days 0, 3, 6, 9, 12, and 15. *P < 0.05 compared BC/Ag/GM-CSF film group with other groups.

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To evaluate wound healing and tissue regeneration at the histological level, H&E staining was employed, and the results are shown in Fig. 7A. The BC/Ag/GM-CSF group showed the thickest and most intact epidermis, with the smallest gap between the wound surfaces, while there were still unhealed cavities under the epidermis in the control group. In the process of wound healing, collagen synthesized by fibroblasts is the main component of the extracellular matrix, so the deposition of collagen at the wound site is of great significance for wound repair. Masson’s trichrome staining was used to evaluate the expression of collagen in the wound of different group and the results are shown in Fig. 7B. Collagen deposition in the BC and BC/Ag groups was lower than that in the other two groups. In addition, BC/Ag/GM-CSF group showed the largest amount of collagen deposition. The results further indicated that GM-CSF could promote the deposition of collagen around the wound and inhibit the growth of bacteria to prevent wound infection, leading to better wound healing35. Additionally, neovascularization at the injury site was observed via CD31 fluorescent staining (Fig. 7C). Compared to other groups, more neo vessels were observed in the BC/Ag/GM-CSF group. These newly formed vessels can provide nutrients and oxygen to the metabolically active wound bed, promoting granulation tissue formation, thus indicating that BC/Ag/GM-CSF can promote wound healing.

Fig. 7
figure 7

Staining results of wounds treated with BC, BC/Ag, BC/GM-CSF, and BC/Ag/GM-CSF films. A H&E staining of the respective groups on the 15th day post-operation; B Masson’s trichrome staining of the respective groups on the 15th day post-operation; C CD31 immunofluorescence staining of the respective groups on the 15th day post-operation. The blue color represents the cell nucleus, and the green color represents positive CD31 staining, indicating newly formed blood vessels.

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BC/Ag/GM-CSF enhanced the healing effect on infectious wound model

For infectious wounds, the inflammation phase can be significantly prolonged or even fail to progress to the proliferation phase36. The wound healing efficacy of BC-based membranes was evaluated in vivo using a mouse model with S. aureus-induced skin infections. All treatment groups demonstrated improved healing over time (Fig. 8a). Compared to normal wounds, infectious wounds exhibited a markedly slower healing rate. At 3 days, inflammatory exudate was present in all groups. By 6 days, scabs without exudate began to form in the BC/Ag and BC/Ag/GM-CSF groups. Notably, the BC/Ag/GM-CSF group had almost no visible blood scabs, indicating that BC/Ag/GM-CSF not only mitigates wound infection but also reduces hemorrhage during the early stages of healing. By 9 days, wounds treated with the BC/Ag/GM-CSF membrane had completely closed, with smooth new epidermal tissue formation and the initial growth of hair. In contrast, wounds in the BC, BC/Ag, and BC/GM-CSF groups were not fully closed and still had sporadic blood scabs. At 15 days, the wounds treated with BC/Ag/GM-CSF were fully healed, showing integrated skin structure. These results suggest that BC/Ag/GM-CSF demonstrates both strong antibacterial properties and enhanced healing effects (Fig. 8b).

Fig. 8
figure 8

The effect of indicated film in the healing on infectious wound. a Wound conditions of the respective groups on days 0, 3, 6, 9, 12, and 15; the infectious wound treated with BC were noted in the control group. The bar was 10 mm; b Wound areas closure of the respective groups on days 0, 3, 6, 9, 12, and 15. *P < 0.05 compared BC/Ag/GM-CSF film group with other groups.

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Conclusion

In this work, we utilized BC as the base material, silver nanoparticles as the antibacterial functional material, and GM-CSF as the cell growth promoting functional material. We prepared a medical material with antibacterial functionality that concurrently aids cell growth through vacuum filtration, in-situ synthesis, and impregnation coating techniques. The physicochemical properties and microscopic morphology of the materials showed that silver nanoparticles uniformly grew on the BC surface. The composite functional membrane has a good level of blood compatibility and cell compatibility, and due to the presence of silver nanoparticles and GM-CSF, the functional membrane can accelerate normal wound healing in early stage, while it can accelerate infectious wound healing in all stages. Through the impregnation process, GM-CSF also achieved a good load on the BC surface. The composite functional membrane has a certain level of blood compatibility and cell compatibility, and due to the presence of silver nanoparticles and GM-CSF, the functional membrane can accelerate wound healing while achieving antibacterial properties. Histological studies showed that in the wound tissue treated with the BC/Ag/GM-CSF composite membrane, more neovascularization was observed, supplying nutrients and oxygen to the metabolically active wound bed, promoting granulation tissue formation, ultimately proving beneficial in increasing wound healing speed. We believe the prepared BC/Ag/GM-CSF composite membrane could potentially serve as dressing material for clinical use in the field of skin wound healing.