DEX was thoroughly dissolved in PBS, and then the prepared bFGF/BSA solution was added to the DEX solution to achieve a final bFGF concentration of 1 g/L and a final DEX concentration of 15% w/v (Zhang et al., 2020). The prepared DEX/bFGF/BSA solution was labeled the aqueous solution. Additionally, PLGA was dissolved in a mixture of trichloromethane and N,N-dimethylformamide (DMF) to prepare a 15% w/v PLGA solution to which 1.0% w/v wool keratin powder was added. Afterward, 0%, 0.2%, 0.5%, and 1.0% w/v nanosilver-loaded calcium alginate microsphere powders were thoroughly mixed to generate PLGA/WK solutions with different concentrations of nanosilver, which were labeled the oil phase solutions. Afterward, the aqueous solution was added to each oil phase solution dropwise at a water-to-oil volume ratio of 1:13 with stirring at 4 °C for 30 min to obtain four types of PLGA/WK electrospun solutions containing bFGF (0.07 g/L) and different concentrations of nanosilver.

Four types of PLGA/WK electrospun membranes containing bFGF and different amounts of nanosilver were obtained from four electrospinning solutions via emulsion electrospinning using a previously reported method (Zhang et al., 2022). Finally, the four types of membranes contained 0%, 0.2%, 0.5% and 1.0% nanosilver and were named the 0%, 0.2%, 0.5%, and 1.0% groups, respectively.

The surface morphologies of the four types of electrospun membranes were observed by scanning electron microscopy (SEM; JSM-6510, JEOL, Japan), and their fiber diameters were measured using Nano Measurer software. Additionally, the four types of electrospun fibers were collected on copper mesh and examined by transmission electron microscopy (TEM; JEM-3010, JEOL, Japan) to determine whether they had stable core–shell structures.

The crystal structures of the electrospun membranes were analyzed by X-ray diffraction (D8 ADVANCE, Bruker, Germany) at an accelerating voltage of 40 kV and a current of 40 mA and scanned at 5°/min from 5 to 90°.

The FTIR spectra of the electrospun membranes were acquired with a Fourier transform infrared spectrometer with an attenuated total reflection apparatus (Tensor 27, Bruker, Germany) in the range of 4,000 cm^-1 to 400 cm^-1.

The four types of electrospun membranes (n = 4) were weighed and added to a digestion tube containing 10 mL of a mixture of nitric acid and perchloric acid. After the samples had completely dissolved, the digestion tube was placed into a preheated instrument for acid digestion. When the bottom of the tube contained a crystalline salt, the tube was removed, the crystallized salt was dissolved, and the solution was diluted and subjected to further testing.

The content of nanosilver encapsulated in the membrane samples was determined with an inductively coupled plasma emission spectrometer (710-ES, Varian, United States). The encapsulation rate of nanosilver was subsequently calculated using the following formula: Encapsulation rate % = Amount   of   nanosilver   in   the   membrane Amount   of   nanosilver   added × 100 % .

The four types of electrospun membranes (n = 4) were weighed and placed in a centrifuge tube with 10 mL of PBS and then oscillated at a rate of 100 r/min at 37 °C. After 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 4 days, 7 days, 10 days and 14 days (14 days in total), the liquid in the centrifuge tube was removed as the test solution, and a new 10 mL of fresh PBS was added. After the test solution was subjected to microwave digestion, the silver ion content was determined using an inductively coupled plasma optical emission spectrometer.

The four types of electrospun membranes (n = 4) were cut, sterilized, and placed on nutrient agar plates coated with an S. aureus or E. coli solution. Afterward, the plates were incubated at 37 °C for 14 days. Photographs were taken after 1, 4, 7, 10 and 14 days of incubation, at which time the diameter of the inhibition zone was measured.

Disks of the four types of membranes (diameter: 1.5 cm; n = 4) were cocultured with L-929 cells at a density of 2.5 × 10⁴ cells/well in 24-well plates. CCK-8 assays were subsequently performed after 1, 3, 5 and 7 days of incubation. The cell survival rate was calculated using the following formula, and the cytotoxicity of the membranes was evaluated according to the ISO 10993–5 standard (Loloei et al., 2024): Cell survival rate % = OD   value   in   the   experimental   group OD   value   in   the   control   group × 100 %

The four types of sterilized membranes (n = 4) were placed at the bottom of 24-well plates. L-929 cells were subsequently inoculated on the membranes at a density of 1 × 10⁵ cells/mL and cultured in a CO₂ incubator. After 3 days, the adhesion of the cells to the electrospun membranes was observed by SEM.

Chitosan sponges were prepared by vacuum freeze-drying according to our previously reported method (Ma et al., 2024). The chitosan sponges were then soaked in 95% ethanol for 2 h and then in a 10% sodium hydroxide solution for another 2 h. After being cleaned with ultrapure water, the washed chitosan sponges were placed in a 24-well plate, prefrozen, and immediately transferred to a vacuum freeze dryer (LGJ-12 A, Sihuanqihang, China) for 12 h. Afterward, the sponges were soaked in 500 μL of solutions containing 0, 500, 1,000 or 1,500 ng of bFGF at 4 °C for 2 h, after which the sponges were freeze-dried for 12 h in a vacuum freeze dryer. Finally, four types of chitosan sponges containing 0, 500, 1,000 and 1,500 ng of bFGF were obtained and named the 0 ng, 500 ng, 1,000 ng and 1,500 ng groups, respectively.

The four types of dried sponge samples were attached to copper stubs using a conductive adhesive. After the samples were sputtered with gold for 55 s, their surface and cross-sectional morphologies were observed by SEM. The pore size and porosity of the samples were measured with an automatic mercury porosimeter (Auto Pore IV 9500, Micromeritics, United States).

Samples of the chitosan sponges containing bFGF (n = 4) were placed in centrifuge tubes filled with 4 mL of PBS and shaken at 37 °C and 100 r/min. At predetermined time points (1 h, 4 h, 8 h, 24 h, 3 days, 7 days and 14 days), 1 mL of solution was removed for storage at −20 °C. Additionally, 1 mL of fresh PBS solution was added to each tube after sampling. The amount of bFGF in each sample was measured with an ELISA kit, and these data were used to construct a release curve. The cumulative release rate of bFGF was calculated via the following formula: Cumulative release rate % = bFGF   content   in   sample   solution total   bFGF   content × 100 %

The cytotoxicity evaluation was performed according to the procedure described in Section 2.3.9.

The cell adhesion evaluation was performed according to the procedure described in Section 2.3.10.

The PLGA/WK solution containing bFGF and the optimal content of nanosilver prepared in Section 2.3 was used as the electrospinning solution to construct the dressing, and the chitosan sponge containing the optimal content of bFGF prepared in Section 2.4 was fixed on the roller of the electrospinning apparatus to collect the nanofibers. A bilayer dressing coloaded with nanosilver and bFGF was subsequently prepared by emulsion electrospinning. The upper layer was composed of a PLGA/WK electrospun membrane containing 0.2% nanosilver and bFGF, and the lower layer was composed of a chitosan sponge containing 500 ng of bFGF.

The cross-sectional morphology of the bilayer dressing was observed via SEM.

The water absorption rate, water retention rate and WVTR of the three types of materials (the bilayer dressing, the PLGA/WK electrospun membrane containing 0.2% nanosilver and bFGF, and the chitosan sponge containing 500 ng of bFGF; n = 4) were determined using previously reported methods (Ma et al., 2024). In brief, the PLGA/WK electrospun membrane containing 0.2% nanosilver and bFGF and the chitosan sponge containing 500 ng of bFGF were considered the upper layer and the lower layer, respectively.

The contact angles of the upper and lower layers of the bilayer dressing were measured with a contact angle measuring instrument (JY-82B, DataPhysics, Germany).

Circular samples of the sterile gauze, upper layer, lower layer, and bilayer dressing with diameters of 15 mm were prepared, whereas the control group did not include any sample material (n = 3). Additionally, S. aureus and E. coli suspensions were diluted to a concentration of 1 × 10⁹ colony forming units (CFUs)/mL. Each of the above materials was sterilized and placed on an agar plate onto which 20 µL of bacterial suspension was added and evenly coated. After an incubation at 37 °C for 24 h, the agar blocks underlying each sample were removed and transferred to a centrifuge tube containing 2 mL of PBS, and the surfaces were washed repeatedly with PBS to detach the bacteria. The samples in the centrifuge tubes were diluted, and 100 µL of each diluted solution was routinely coated on new agar plates and cultured at 37 °C for 24 h. The number of bacterial colonies that formed on each plate was recorded, from which the amount of bacteria contained in the original PBS solution was calculated.

Several evenly spaced, parallel horizontal lines were drawn on the back of the 6-well plate, with at least 5 lines per well. L-929 cells were then inoculated into 6-well plates at a density of 5 × 10⁵ cells per well. When the cell confluence reached 100%, a sterilized pipette tip (200 μL) was used to make a scratch in the cell layer perpendicular to the horizontal lines, and then the cells were washed 3 times with PBS to remove the detached cells and cell debris. Next, samples of the upper layer, lower layer, bilayer dressing (n = 3) and 2 mL of serum-free medium were added to each well, and the plates were cultured in an incubator and photographed at 0, 6, 12 and 24 h. The images of each group were analyzed with ImageJ software, and the cell migration rate was calculated using the following formula: Cell migration rate % = scratch   area   at   0   h − scratch   area   at   a   particular   time   point scratch   area   at   0   h × 100 %

The upper layer, lower layer, and bilayer dressing samples (n = 3) were placed into a 12-well plate, and 1 mL of complete medium was added to each well. Afterward, the plate was incubated at 37 °C for 24 h to prepare the leaching solutions. Matrix glue was spread onto a 96-well plate, and HUVECs were seeded at a density of 3 × 10⁴ cells/well. Then, 100 μL of each type of leaching solution was added to the appropriate wells, and the samples were cultured at 37 °C. After 12 h, blood vessel formation was observed under an inverted optical microscope, and the cells were photographed. The total number of junctions in each group was determined with ImageJ software.

The upper layer, lower layer, and bilayer dressing samples (n = 3) were placed on a Petri dish, and 50 μL of SD rat whole blood was dropped onto the surface of each sample and incubated at 37 °C for 5 min. Then, 20 mL of deionized water was slowly poured from the edge of the Petri dish so that the red blood cells not embedded in blood clots would undergo hemolysis in the water. After 20 min, the absorbance value of each sample was measured at 540 nm with a microplate reader, and the coagulation index (BCI) was calculated using the following formula: BCI = OD   value   experimental   group OD   value   control   group × 100

The following six groups were included in this test (Table 1): group a, blank control; group b, bilayer dressing without drugs; group c, bilayer dressing loaded with nanosilver and bFGF in the upper layer; group d, bilayer dressing loaded with bFGF in both the upper layer and lower layer; group e, bilayer dressing loaded with bFGF in the upper layer; and group f, bilayer dressing loaded with nanosilver and bFGF (n = 3). A full-thickness skin defect with a diameter of 1 cm was prepared on the backs of the SD rats, disinfected bilayer dressings were applied to the wounds, and medical gauze was applied on top.

The skin wounds on the rats were observed after 3, 7, 14 and 21 days, and photographs were captured with a digital SLR camera. ImageJ software was used to analyze the wound healing rate, which was calculated using the following formula: Wound healing rate % = original   wound   area − wound   area   at   each   time   point original   wound   area × 100 %

At 3, 7, 14 and 21 days after wounding, the selected rats were anesthetized, and a 2–3 mm area of the wound edge on the rats’ back was removed. The samples were subjected to hematoxylin–eosin (HE) staining to observe the degree of inflammation in the new tissues and the formation of new blood vessels, epithelium and skin appendages. The expression of proliferating cell nuclear antigen (PCNA) in each group was evaluated by immunohistochemical staining, and the ratio of the positively stained area was calculated using the following formula: Positively stained area ratio = positively   stained   area tissue   area × 100 %

At 7 days after wounding, proteins were extracted from tissue samples from each group using a whole protein extraction kit and quantified with a protein quantification kit (Servicebio, China). The proteins from each sample were subsequently used to determine PCNA and CD34 expression via Western blot. The ratio of the intensity of the target band to the intensity of the internal reference band was used to determine the relative expression levels of the corresponding proteins.

Photographs and SEM images of the four types of membranes are shown in Figures 1A,B. The four types of membranes were composed of fibers with smooth surfaces and no obvious beaded structures. The diameter distributions of the fibers in each group, which were all on the nanoscale, are shown in Figure 1C. The average fiber diameters of the 0%, 0.2%, 0.5%, and 1.0% groups were 0.36 ± 0.05 μm, 0.32 ± 0.04 μm, 0.32 ± 0.04 μm, and 0.31 ± 0.05 μm, respectively. TEM images of each type of fiber are shown in Figure 1D. All of the fibers had a stable core–shell structure.

The X-ray diffraction patterns of the four types of membranes, the wool keratin powder and the nanosilver-loaded calcium alginate microsphere powder are shown in Figure 2A. The characteristic diffraction peaks of the nanosilver-loaded calcium alginate microsphere powder were observed at 2θ = 38.15°, 44.32°, 64.47° and 77.53°, which are the (111), (200), (220) and (311) crystal plane diffraction peaks of face-centered cubic silver crystals, respectively (JCPDS, 04-0783) (Chen et al., 2022). The wool keratin powder, which was amorphous, displayed no obvious diffraction peaks. Among the four types of membranes, small diffraction peaks were observed at 2θ = 38.15° in the 0.2%, 0.5%, and 1.0% samples, and these peaks became more intense with increasing nanosilver-loaded calcium alginate microsphere content, whereas no diffraction peak was observed at 2θ = 38.15° in the 0% group. A small diffraction peak was also observed at 2θ = 44.32° in the 1.0% group. These results suggested that nanosilver was successfully incorporated into the electrospun membranes.

The FTIR spectra of the four types of membranes, the wool keratin powder and the nanosilver-loaded calcium alginate microsphere powder are shown in Figure 2B. Strong characteristic peaks for the wool keratin powder were observed at 3,306 cm^-1 (N–H stretching vibration), and other characteristic peaks were observed at 1,658 cm^-1 (C=O and C–N amide I stretching vibrations), 1,533 cm^-1 (amide II N–H bending and C–N stretching vibrations) and 1,240 cm^-1 (amide III C–N vibration and C=O bending vibrations) (Maurizii et al., 2024). A wide absorption peak was observed at approximately 3,374 cm^-1 in the spectrum of the nanosilver-loaded calcium alginate microsphere powder, which was attributed to the stretching vibration caused by the intramolecular and intermolecular hydrogen bonds of the hydroxyl group (-OH). This peak also appeared in the spectra of the 0.2%, 0.5% and 1.0% groups, indicating the formation of hydrogen bonds between the hydroxyl groups. The four types of membranes also displayed peaks at 1758 cm^-1, 1,186 cm^-1, 1,089 cm^-1, 2,997 cm^-1 and 1,455 cm^-1, which are the characteristic C=O, C-O-C, CH₃ and CH₂ peaks of PLGA, respectively (Wang et al., 2019). Because only a small amount of wool keratin powder was added to the four types of membranes, the characteristic peak of wool keratin powder was not observed in the spectra of the membranes.

The amount of nanosilver encapsulated and the nanosilver encapsulation rate in the four types of membranes are shown in Figures 2C,D. With increasing nanosilver content, the amount of nanosilver wrapped in the membrane gradually increased; the amount of nanosilver in the 1.0% group was the highest (92.55 ± 10.13 μg), whereas the amount of nanosilver in the 0.2% group was the lowest (75.41 ± 6.91 μg). With increasing nanosilver content, the encapsulation rate of nanosilver in the membrane decreased gradually, with the 0.2% group having the highest encapsulation rate.

As shown in Figure 2E, the release of nanosilver from all three groups persisted for more than 14 days. Within the first 24 h, the release curves of all groups exhibited a rapid increase, presumably due to the nanosilver localized at or near the surface of the composite membranes. These nanosilver particles rapidly dissolved and diffused into the release medium, resulting in an initial burst release. In addition, water uptake caused the membranes to swell, increasing their volume and porosity, which provided more release channels for the nanosilver particles and thereby accelerated their release rate. Between days 2 and 14, the release curves gradually plateaued, indicating a decline in release rates, possibly due to the diminishing effects of diffusion and swelling. By day 14, the cumulative release rates for the 0.2%, 0.5%, and 1% groups were 78.92% ± 0.49%, 80.98% ± 0.29%, and 51.96% ± 0.20%, respectively. Notably, the 1% group exhibited a significantly slower release rate, which may be attributed to its higher nanosilver concentration, leading to reduced dispersibility and the formation of larger aggregates. These aggregates may form a saturated layer on the membrane surface, hindering further diffusion of nanosilver from the interior of the membrane to the surface and thus slowing the release rate.

The general observations of the four types of membranes cocultured with S. aureus and E. coli for 1, 4, 7, 10 and 14 days are shown in Figure 3A, and the diameters of the inhibition zones are shown in Figures 3B,C. No antibacterial zone was observed around the sample without nanosilver (0% group), whereas antibacterial zones appeared in the 0.2%, 0.5% and 1.0% groups, indicating that these membranes, but not the 0% membrane, inhibited the growth of S. aureus and E. coli. As the nanosilver content in the membrane increased, the diameter of the antibacterial zone also increased. The diameters of the inhibition zones for each of the four types of membranes against S. aureus and E. coli after 1 day are shown in Figure 3D. Among them, the 0.2% group presented stronger antibacterial activity against E. coli than S. aureus, whereas the opposite trend was observed in the 0.5% and 1.0% groups.

The OD values of the four types of membranes cocultured with L-929 cells are shown in Figure 4A. The OD value in each group increased with increasing culture duration, indicating that all four types of membranes promoted the adhesion and proliferation of L-929 cells. After 7 days, the 0% group and 0.2% group presented the greatest number of cells, followed by the 0.5% group and then the 1.0% group. The viability of the cells on the four types of membranes is shown in Figure 4B. Both the 0% and 0.2% groups were assigned Grade I cytotoxicity (mildly cytotoxic) after 1 day and Grade 0 (noncytotoxic) after 3, 5 and 7 days. Additionally, the 0.5% group was assigned Grade I after 1 and 3 days and Grade 0 after 5 and 7 days, whereas the cytotoxicity rating of the 1.0% group was determined to be Grade II (slightly cytotoxic) after 1 and 3 days and Grade I after 5 and 7 days.

SEM images of L-929 cells cultured on the four types of membranes for 3 days are shown in Figure 4C. Most cells were elongated spindle-shaped or polygonal, and cell numbers were highest in the 0% and 0.2% groups, intermediate in the 0.5% group, and lowest in the 1.0% group, which is consistent with the CCK-8 results.

In summary, the four types of PLGA/WK electrospun membranes containing bFGF and different amounts of nanosilver were successfully prepared by emulsion electrospinning. The 0.2% group had the best drug encapsulation rate and release rate, sustained antibacterial activity and best cytocompatibility. Therefore, this membrane was selected to prepare a multifunctional bilayer dressing loaded with nanosilver and bFGF.

Photographs of the four types of chitosan sponges are shown in Figure 5A. All of the sponges appeared milky white and spongy with dense structures and uniform textures and were approximately 1.3 cm in diameter and 0.3 cm in height. Cross-sectional SEM images of the four types of sponges are shown in Figure 5B. All the samples exhibited a porous network structure with pores that were uniform in size and interconnected. The pore sizes of the 0 ng, 500 ng, 1,000 ng and 1,500 ng groups were 47.46 μm, 49.57 μm, 46.34 μm and 43.02 μm, respectively, and their porosities were all greater than 90%, with values of 97.90%, 94.86%, 97.42% and 96.30%, respectively.

The cumulative release rates of bFGF in the 500 ng, 1,000 ng and 1,500 ng groups are shown in Figure 5C (because the 0 ng group did not contain bFGF, this group was not included in this experiment). bFGF release can be divided into three main stages. In the first stage (8 h), bFGF displayed burst release, with cumulative release rates of 30.56% ± 0.28%, 15.65% ± 0.13% and 11.72% ± 0.11% in the 500 ng, 1,000 ng and 1,500 ng groups, respectively. The second stage occurred between 8 h and 24 h, when bFGF release was slow and sustained. During the third stage, after 24 h, bFGF release was stable, and the cumulative release rates in the 500 ng, 1,000 ng and 1,500 ng groups were 31.91% ± 0.27%, 16.78% ± 0.09% and 13.39% ± 0.13%, respectively. Thus, the cumulative release rate of bFGF in the 500 ng group was the highest, followed by that in the 1,000 ng group, while the 1,500 ng group presented the lowest cumulative release rate.

The OD values of the four types of chitosan sponges cocultured with L-929 cells are shown in Figure 5D. The OD values in each group increased with increasing culture duration, indicating that each of the sponges promoted L-929 cell adhesion and proliferation. After 1 day of coculture, the OD values increased, and the group with the highest bFGF content also had the highest OD value. The number of cells in the 1,500 ng group was the greatest, whereas the number of cells in the 0 ng group was the lowest. After 3 days, the numbers of cells in the 500 ng, 1,000 ng and 1,500 ng groups were greater than the number of cells in the 0 ng group, and after 5 and 7 days of coculture, the 500 ng group had the most cells. These results indicated that a moderate amount of bFGF promoted cell adhesion and proliferation, but excess bFGF inhibited cell adhesion and proliferation. The viability of the cells cultured on the four types of chitosan sponges is shown in Figure 5E. The cytotoxicity rating of the 0 ng group was Grade I after 1 day and Grade 0 after 3, 5 and 7 days; moreover, the cytotoxicity ratings of the 500 ng, 1,000 ng and 1,500 ng groups were Grade 0 after 1, 3, 5 and 7 days, indicating that these materials were noncytotoxic.

SEM images of L-929 cells cultured on the four types of chitosan sponges for 2 days are shown in Figure 5F. The morphologies of the cells on the sponges were similar among the groups. Additionally, both cell number and density in the 500 ng, 1,000 ng, and 1,500 ng groups exceeded those in the 0 ng group, with the 500 ng group showing the highest and densest population.

In summary, four types of chitosan sponges containing bFGF were successfully prepared by vacuum freeze-drying. Among them, the 500 ng sponge displayed the best cumulative drug release rate and cytocompatibility. Therefore, this sponge was selected to prepare a multifunctional bilayer dressing coloaded with nanosilver and bFGF.

Photographs of the bilayer dressing, which was approximately 1.3 cm in diameter and 0.3 cm in height, are shown in Figure 6A. The upper layer was composed of a PLGA/WK electrospun membrane containing 0.2% nanosilver and bFGF, and the lower layer was composed of a chitosan sponge containing 500 ng of bFGF. The cross-sectional SEM image of the bilayer dressing is shown in Figure 6B. The upper and lower layers were close together and not easily separated.

The water absorption rate, water retention rate and WVTR of each sample are shown in Figure 6C. The water absorption rates of the bilayer dressing and lower layer were high, whereas the water absorption rate of the upper layer was low. This difference is because the bilayer dressing and lower layer have loose, spongy structures with a strong water absorption capability. Additionally, the water retention rate of the bilayer dressing was the highest, and the water retention rate of the upper layer was the lowest, whereas the WVTR of the lower layer was the highest and that of the bilayer dressing was the lowest.

Images from the bilayer dressing water contact angle evaluation are shown in Figure 6D. The water contact angle of the upper layer was 121.34° ± 2.58°, indicating its hydrophobicity, which can simulate the epidermis of the skin and prevent bacterial invasion. The lower layer was hydrophilic with a water contact angle of 0°, and this layer can simulate the dermis of the skin and better absorb exudate.

General observations from the S. aureus and E. coli penetration tests are shown in Figure 7A. Moreover, the numbers of S. aureus and E. coli colonies that penetrated each sample are shown in Figure 7B. Fewer S. aureus and E. coli colonies penetrated the upper layer, lower layer, and bilayer dressing than the blank control and sterile gauze. Except for the blank control group, the number of S. aureus colonies that penetrated the bilayer dressing was the lowest, with an average of 80 CFUs, followed by the upper layer and then the lower layer. The sterile gauze allowed most of the S. aureus colonies to penetrate, with an average of 7.07 × 10⁹ CFUs. Similarly, for E. coli, when the blank control group was excluded, the number of colonies that penetrated the bilayer dressing was the lowest, and the number of colonies that penetrated the sterile gauze was the greatest. The results indicated that the bilayer dressing had an excellent ability to prevent bacterial penetration.

Figure 8A shows microscopy images of the cell scratch wound healing, and Figure 8B shows the cell migration rates of each group. Compared with that in the blank control group, fibroblast migration was accelerated in the other three groups. After 6 and 12 h, the cells in the upper layer, lower layer and bilayer dressing groups tended to migrate to the scratch area. Notably, the cells in the bilayer dressing group migrated the fastest, followed by those in the lower layer group, and the cells in the blank control group migrated the slowest. After 24 h, the scratches in the lower layer and bilayer dressing groups had essentially healed.

Microscopy observations of angiogenesis and the number of vascular junctions in each group are shown in Figures 8C,D. Tubular structures formed in the three experimental groups but not in the blank control group. Among them, the bilayer dressing group had the greatest number of vascular junctions, followed by the lower layer group and then the upper layer group.

General observations from the coagulation test are shown in Figure 9A, and the blood clotting index of each group is shown in Figure 9B. The color of the lower layer and the bilayer dressing groups did not change significantly after the addition of deionized water, whereas the liquids in the other two groups were obviously pink, indicating that the former two groups had better coagulation performance. In addition, the blood clotting indices of the former two groups were significantly lower than those of the latter two groups. The lower the blood clotting index is, the better the coagulation performance. These results showed that the lower layer and bilayer dressing had good coagulation properties.

The gross observations during wound healing and the wound healing rates in each group at different time points are shown in Figures 10A,B. The wound healing rate of each group increased with time. After 3 days, the wound healing rates were the highest in groups d and f at 32.64% ± 2.83% and 35.28% ± 2.06%, respectively. After 7 days, group f displayed the highest wound healing rate. By day 14, the wounds in all the groups had essentially healed, and a significant difference in the wound healing rate was not observed among the groups. After 21 days, the wounds in all the groups had completely healed. The results showed that the bilayer dressing coloaded with nanosilver and bFGF (group f) synergistically accelerated wound healing.

Images of HE-stained sections from each group are shown in Figure 11A. Inflammatory cell infiltration was observed in all the groups after 3 days. At 7 days, fibroblasts and many new capillaries were observed in groups d and f, along with significantly fewer inflammatory cells, whereas fewer new capillaries were observed in the other groups. At 14 days, new hair follicles were observed in groups d and f, but fewer hair follicles were observed in the other groups. After 21 days, the structure of the epidermis in each group was continuous and complete and similar to that of normal skin, and skin appendages, such as hair follicles, were also observed in all the groups, indicating that wound repair was essentially complete.

Images of immunohistochemical staining for PCNA and the percentages of the area positively stained for PCNA in each group are displayed in Figures 11B, 10C. PCNA is one of the main markers of cell proliferation and plays a key role in the initiation of cell proliferation; thus, PCNA expression reflects the conditions of cell proliferation (Hao et al., 2020). Compared with that in the other groups, PCNA expression in each group increased with time over 7 days and was significantly higher at 3 and 7 days in group f. These findings indicate that the application of both nanosilver and bFGF promoted the maintenance of cell proliferation and effectively promoted fibroblast proliferation and migration in the early stage of wound healing. On days 14 and 21, the expression of PCNA in each group decreased, indicating that in the late stage of wound healing, the tissue had gradually been remodeled because of collagen deposition and extracellular matrix formation, and the demand for fibroblasts decreased.

The results of the Western blot analysis of PCNA and CD34 levels at 7 days are shown in Figure 12. PCNA is an indicator for assessing the proliferative status of cells, and its expression level objectively reflects cellular proliferative activity. CD34, a well-established marker of vascular endothelial cells, labels endothelial cells and immature small vessels, thereby reflecting the extent of angiogenesis (Zhang et al., 2022). Western blot analysis revealed that the bFGF-loaded wound dressings (groups d and f) significantly upregulated the expression of both PCNA and CD34. These findings are consistent with those of several previous studies: high PCNA expression reflects increased cellular proliferative activity, which can be attributed to the direct stimulation of growth factor signaling pathways, such as collagen synthesis and epidermal regeneration, by bFGF, thereby attenuating oxidative stress and increasing the levels of matrix metalloproteinases (MMPs) in the wound microenvironment to ultimately accelerate re-epithelialization (Yin et al., 2024). In addition, bFGF promotes the infiltration of CD34-positive cells, thereby enhancing angiogenesis and granulation tissue formation and accelerating wound closure (Oshima et al., 2024). Collectively, the concerted upregulation of PCNA and CD34 expression underscores the therapeutic potential of the bFGF-loaded dressings, which effectively shorten the wound-healing timeline by increasing angiogenesis and cellular proliferation.