Synthesis and formation mechanism of functionalized HA hydrogels

As shown in Fig. 1A, CA was grafted onto HA by EDC/NHS chemistry to produce the conjugate HA-CA. The copolymer structure was validated by 1H NMR spectra and FTIR spectrum, as shown in Fig. 1B and C. The peak observed at δ 1.8–2.0 corresponds to the proton in methylene of HA. The conjugation of CA to HA was confirmed by the presence of aromatic-proton peaks at δ 6.70–6.85 ppm and methylene-proton peaks at δ 3.1 and 2.8 ppm. According to the calculation, the substitution degree of CA in HA was 12.9%. In comparison with the FTIR spectrum of HA, the absorption peak at 1732 cm−1 may assigned to the stretching vibration peak of − COOH. In addition, the asymmetric and symmetric stretching vibration intensities of − COONa at 1619 cm−1 and 1411 cm−1 were significantly reduced. Even more important, HA-CA showed a stronger absorption peak at 1565 cm−1, which was formed by N–H bending vibration of amide II band. These findings confirm successful conjugation between HA and CA via amide bonds. The schematic of HP-Ag structure is shown in Fig. 1D. CA loses two electrons and two protons to become quinone, which reacts highly with the mercaptan group through the Michael addition reaction. CA groups can form physical or chemical bonds with different surfaces, and quinone groups promote the cohesion of hydrogels [28].

Fig. 1

Structure and schematic of hydrogel formation. A Synthesis of HA-CA conjugates. B 1H NMR spectra and C FTIR spectrum of HA-CA conjugates. D Schematic of the HP-Ag structure.

Physicochemical properties of the hydrogel

The micromorphology and average pore size of freeze-dried hydrogels with different Ag NPs contents are shown in Fig. 2A and B. All hydrogels exhibit a uniform network structure and porous interpenetration. The crosslinking density can be reflected by hydrogel pore size to some extent. With the increase of Ag NPs content, the pore size of hydrogel gradually decreases. The average pore sizes of HP, HP-AgL, HP-AgM and HP-AgH were 242 ± 54.4 μm, 234 ± 48.3 μm, 211 ± 46.2 μm and 122 ± 25.3 μm, respectively. The porous structure allows the hydrogel to absorb blood and exudate from the wound tissue, providing enough space for cell proliferation and thus speeding up hemostasis [29]. The addition of Ag NPs enhances the gel network structure and occupies the ordered pores in the hydrogel, leading to a reduction in pore size. Two possible causes of gel network enhancement are: First, CA on the HA-CA polymer deprotonates to form quinone groups, which help the deposition of HA-CA on the Ag surface through hydrophobic action; Secondly, sulfophilic Ag can react with available sulfhydryl groups in 4-arm PEG-SH to form Ag–S bond, which attach the PEG-SH to Ag NPs surface [30,31,32].

Fig. 2

Physicochemical properties of HP-Ag/bFGF hydrogel. A Scanning electron microscope images of the freeze-dried HP-Ag hydrogel. Scale bar: 500 μm. B The pore size of the hydrogels prepared by different concentrations of Ag NPs. C Morphology of of Ag NPs. D TEM micrograph of Ag NPs in HP-Ag hydrogel. E DPPH radical scavenging rate of HP hydrogel. F Cumulative release curve of bFGF in HP-Ag/bFGF hydrogel. G Cumulative release curve of Ag+ in HP-Ag hydrogel. (H) Bioactivity of released bFGF. I G′ of HP hydrogels with different matrix contents measured in frequency sweep. J G′ of HP-Ag hydrogel measured in frequency sweep. K Compressive stress–strain curves of HP-Ag hydrogel. Data are presented as mean ± S.D, n = 3

Ag NPs have a large specific surface area, and their antibacterial activity is affected by their particle size. From Fig. 2C and Additional file 1: Fig. S1, it can be observed that Ag NPs are in regular cube shape with a particle size of 100 nm and a potential of − 23.87.

As shown in Fig. 2E, the DPPH-clearance of the HP hydrogel was 59.53%. The clearance was lower than expected, considering that there were fewer CA groups available on HA, as some of them may have been oxidized to quinone or attached to other groups.

The release behavior of bFGF in HP-Ag/bFGF was studied, and it can be clearly observed from Fig. 2F that bFGF was released rapidly in HP hydrogel, while the incorporation of Ag NPs slowed down the release rate. For HP hydrogel, a burst release of bFGF (51.3%) was observed on day 1, while 46.2% and 39.9% bFGF release were observed in HP-AgM and HP-AgH hydrogels, respectively. The release in HP hydrogel reached 73.9% on day 4, and the release rate in HP-AgM and HP-AgH hydrogels were 64.4% and 53.4%, respectively. These pieces of evidence suggest that long-term stable release can be achieved by adding Ag NPs to the hydrogel to enhance the network structure, thus simplifying drug administration.

The antibacterial activity of HP-Ag hydrogel depends on the release of Ag from the hydrogel into the infected site. Ag NPs can not only cross-link networks in hydrogel, but also act as reservoirs for Ag. As shown in Fig. 2G, a steady and sustained release of Ag was observed throughout the test time. The quantitative method of Ag release used does not allow the differentiation of its form (ion or nanoparticle) [33]. The cumulative Ag releases of the HP-AgL, HP-AgM and HP-AgH after 14 days were 0.143 mg L−1, 0.203 mg L−1 and 0.171 mg L−1, respectively. The relatively high release on the first day ensured a strong antibacterial effect during the initial phase of tissue healing, and a certain amount of Ag remained at the wound site although the release of Ag was reduced later [34]. The current release was slightly lower than expected [35,36,37], possibly because some Ag NPs participated in the network as a crosslinking agent, and the surface of Ag NPs was attached to HA-CA or 4-arm PEG-SH, thus delaying Ag release. This low concentration ensures effective sterilization above subinhibitory concentrations to avoid the formation of bacterial biofilms, while being safe for cells and tissues [32, 33, 36].

In general, Ag NPs tend to accumulate and can lead to large increases in local Ag concentrations, which may affect cell activity. The result shows that Ag NPs were distributed uniformly in HP-Ag (Fig. 2D). It is speculated that the hydroxyl and sulfhydryl groups deposited on the surface of Ag NPs cause the stable and uniform distribution of Ag NPs in the network structure.

The bioactivity of released bFGF was evaluated by the stimulating effect on NIH/3T3 cell viability. From Fig. 2H, compared with maintenance medium group and HP-Ag group, the cell viability of the bFGF release group was significantly increased, indicating that bFGF in hydrogel maintained bioactive.

The dynamic rheological analysis was conducted to investigate the mechanical performance of hydrogels. Figure 2I and J shows the storage modulus (G′) of hydrogels with different contents of HA-CA, 4-arm PEG-SH and Ag NPs measured in frequency sweep. All hydrogels showed elastic solid-like hydrogel after gelation. The G' of hydrogel increased with the increasing concentration of HA-CA and 4-arm PEG-SH. Due to the introduction of HA-CA and 4-arm PEG-SH, the network cross-linking of hydrogels was enhanced through multiple interactions, and the stiffness of hydrogels was improved. Similarly, the G' of HP-Ag hydrogel gradually increased with the increase of Ag NPs content. The G' increased with the increase of strain frequency within the angular frequency of 0.1–100 rad s−1. The increase in modulus with strain frequency is the strain-stiffening behavior of hydrogels[38]. This property enables hydrogel to produce a better bonding effect when high strain and strong stress are generated at the bonding site[39].

Compressive stress–strain tests were performed on the hydrogels to assess its mechanical strength (Fig. 2K). Consistent with the rheological results, the stresses of HP-Ag/bFGF hydrogels were higher than that of HP hydrogel. Specifically, the compressive strength of HP hydrogel was as low as 3.3 kPa, while the compressive strength of HP-AgL, HP-AgM and HP-AgH increased by 4.7 kPa, 4.9 kPa and 5.3 kPa, respectively. With the increase content of Ag NPs, the compressive rupture strain of hydrogel also increased slightly. The compressive rupture strain of HP hydrogel was 119.7%, and that of HP-AgL, HP-AgM and HP-AgH were 133.6%, 129.3% and 130.9%, respectively. From Fig.S2A, the HP hydrogel has an additional weight loss temperature range compared to the weight loss of HA, with complete thermal decomposition occurring at the 300–420 °C stage. From Fig.S2B, the total residual weight of HP-AgH was always higher than that of the HP-AgL composite system at each weight loss stage. These results indicate that Ag NPs are embedded in the hydrogel matrix and the hydrogels with higher Ag NPs content can form more stable structures. In summary, these results indicate that HP-Ag/bFGF hydrogel holds several merits including porous microstructure, bFGF release regulation, Ag release and adjustable stiffness capability.

in vitro biocompatibility and antibacterial activity

The versatility of hydrogels inspired us to explore the biological properties required for their application in wound dressings. Good biocompatibility is an essential prerequisite for hydrogel dressings because they are in direct contact with tissues and blood in practical applications. The cytotoxicity of HP-Ag hydrogel on NIH/3T3 was evaluated using a CCK-8 assay in vitro, with PBS as the control group. As shown in Fig. 3A, after 12 h and 24 h culture, the relative cell growth rate of each group of hydrogels exceeded 86%, far higher than the minimum non-toxic standard of 70%. Similar results were obtained in proliferation experiments (Fig. 3B). There was no significant difference in cell proliferation rate between the HP-Ag hydrogel and control group, indicating that hydrogels had good cytocompatibility. After 3 days and 5 days of incubation, the cell activity of HP-AgM decreased slightly compared with PBS group, which may be due to the inactivation of some cells exposed to residual Ag NPs. Most conventional cross-linkers used in catechol polymers, such as Fe3+ and NaIO4, are cytotoxic, limiting their use as biomaterials. HP-Ag prepared using 4-arm PEG-SH and Ag NPs as cross-linkers had no cytotoxicity and is expected to be used in clinical wound dressings.

Fig. 3

Biocompatibility and antibacterial properties of hydrogels. Cell viability of NIH/3T3 co-cultured with hydrogel extracts for A 12 h and 24 h. and B 1d, 3d and 5d. C Hemolytic activity assay of HP and HP-AgM hydrogel. Inset is the corresponding photograph. D Photographs of survival bacteria colonies (S. aureus, white dots in plates) growing on agar plates after direct contact incubation with HP and HP-Ag hydrogels. PBS was used as control. E OD values at 600 nm of S. aureus solution co-cultured with the hydrogels for 12 h and 24 h. All data are presented as mean ± SD, n = 4, **p < 0.01, ***p < 0.001, ****p < 0.0001

Hemocompatibility is an important factor to evaluate the compatibility between blood and biomaterials. The standard hemolysis activity test was used to evaluate the hemolysis ratio between homogeneous hydrogel and red blood cells (RBCs). As shown in Fig. 3C, hemolysis rates of HP and HP-AgM hydrogels were 1.83% and 1.13%, respectively. The hemolysis rates were similar to that of the PBS negative control, but much lower than that of the positive control and well below the allowable limit of 5%. Direct visual observation showed that the supernatant of the hydrogel group was yellowish and almost indistinguishable from the PBS group. In the positive group, the supernatant was bright red caused by RBCs lysis, indicating that the hemolysis of HP and HP-AgM hydrogels was negligible. The low hemolysis rate is due to the hydrophilicity of HA and 4-arm PEG-SH, which reduces RBCs destruction.

The antibacterial activity of HP-Ag hydrogel against gram-positive S. aureus, the representative cause of skin infections, was evaluated by colony counting. As shown in Fig. 3D, a large number of bacterial colonies appeared in the agar plate of PBS group, while no viable colonies appeared in HP-Ag groups, indicating strong bactericidal effects. Similarly, OD values of the S. aureus solutions co-cultured with HP-Ag hydrogel were significantly lower than that in PBS group, and the suspensions were clear after 12 and 24 h (Fig. 3E). These results suggest that HP-Ag hydrogel was effective in killing S. aureus and had negligible damage to cells. It should be emphasized that the highly effective antibacterial properties of HP-Ag hydrogel also greatly reduce the side effects of wound treatment that may be caused by high doses[40, 41]. As for possible antibacterial mechanisms, it is speculated that first the available phenol hydroxyl groups in HP-Ag hydrogel trap the bacteria, and then the positively charged Ag+ from Ag NPs interact electrostatic with the phosphoric groups of the phospholipids in the cell membrane, killing the bacteria by interfering with DNA replication and denaturing microbial proteins [42,43,44]. Overall, HP-Ag hydrogel showed superior cytocompatibility, hemocompatibility and strong antibacterial activity, which has great potential for clinical application.

Accelerate wound healing process of acute wound

The wound healing processes of acute full-thickness skin defects were monitored photographically (Fig. 4A), and the corresponding calculation of the existing wound area were presented in Fig. 4B. The wound size of each group showed a decreasing trend over time. After 14 days of treatment, the wound area of HP-Ag/bFGF treatment was the smallest, and the wound became smooth and some new epidermal and dermal tissues appearing. In contrast, the skin surface of the wound in other groups was red with obvious scabs, suggesting the formation of early scar tissues. The redness of these scars is usually due to incomplete angiogenesis [19]. Wound closure varied significantly during the first 7 days after modeling due to self-contraction of the skin. The wound closure area of the control group (6.6%) was significantly lower than that of the HP-Ag/bFGF group (37.7%) on day 3 and was only 48.7% on day 7. The wound closure rate of HP-Ag /bFGF group reached 68.2%, higher than that of HP/bFGF group (54.7%). Compared with other groups, the HP-Ag/bFGF composite hydrogel group was more effective in promoting wound healing. On the one hand, the hydrogel formed in situ can fit the wound well and adhere closely to the wound site to avoid microbial infection. On the other hand, the water-retaining ability of HA preserves the moist environment required for wound healing, and HP-Ag/bFGF can sustainably provide bFGF for tissue regeneration.

Fig. 4

A Photographs of the skin wounds of various groups on day 0, 3, 7, 11and 14 (Scale: 10 mm). B Wound closure analysis from the existing wound area on day 3, 7, 11 and 14. C H&E staining of the wound sections on day 3, 7 and 14 (Scale: 100 μm). D Masson staining of the wound sections on day 7 and 14 (Scale: 50 μm). E Semi-quantification of skin granulation tissue thickness on day 7. F Collagen volume fraction of the wound sections on day 7. All data are presented as mean ± SD, *p < 0.05, **p < 0.01, ****p < 0.00001, n = 3

In order to observe the process of wound healing more directly and accurately, the histological changes of skin were evaluated. H&E staining images (Fig. 4C) show that there was no or only a thin layer of granulation tissue under the skin of the control, HP and HP/bFGF groups, and a large number of inflammatory cells infiltrated. The semi-quantitative analysis (Fig. 4E) shows that the granulation tissue thickness in HP-Ag/bFGF group was 776 μm, which was obviously higher than that in other groups. On day 14, the new epidermis was obviously thickened and uneven in control group, and the inflammatory cells were obviously gathered under the epidermis, both of which could lead to the formation of scar at the wound site [45, 46]. However, the HP-Ag/bFGF group showed uniform epidermal tissue thickness, orderly granulation tissue, no evident inflammation occurred, and the regenerated dermis tissue with appendages like hair follicles and sebaceous glands was detected, all of which were important indicators of skin scarless healing.

As the support of cell growth, collagen can promote the proliferation and differentiation of tissue cells and create a better microenvironment. However, excessive production and disorderly deposition of collagen in the dermis can lead to scar production [5, 47]. Masson staining was used to evaluate collagen deposition at wound sites (Fig. 4D). The collagen deposition amount of HP-Ag/bFGF group was higher than that of other groups on day 7, and the collagen fibers were loosely and well-organized distributed in dermal tissue on day 14. This is consistent with the characteristics of scarless skin repair, where collagen accumulates in large quantities in the early stages and then partially fades and rearranges into the tissue [48]. Semi-quantitative analysis (Fig. 4F) shows that the collagen deposition was only 15.2% in control group on day 7, while it was 81.7% in HP-Ag/bFGF group, significantly higher than that in other groups. These results indicate that HP-Ag/bFGF composite hydrogels can accelerate wound regeneration and promote wound scarless healing.

TNF-α, a typical proinflammatory cytokine, was selected to evaluate the anti-inflammatory effect of hydrogels in vivo. The expression of TNF-α at the wound site on day 3 was shown in Additional file 1: Fig.S3 and Fig.S4. The expression of pro-inflammatory factors in control group was significantly higher than that in HP/bFGF and HP-Ag/bFGF group. In addition, TNF-α expression was lowest in HP-Ag/bFGF group, which was related to the continuous release of Ag to provide an anti-inflammatory micoenvironment. As shown in Additional file 1: Fig.S5, HP /bFGF and HP-Ag/bFGF group showed more mature blood vessels in the wound bed than the other groups. Long-term release of bFGF and Ag promotes the formation of mature blood vessels and the regression of immature blood vessels, thus providing adequate oxygen, nutrients, and growth factors for tissue regeneration.

Transforming growth factor TGF-β can induce fibroblasts to differentiate into myofibroblasts, which is closely related to scar formation in tissues. From Additional file 1: Fig.S6 and S7, the expression of TGF-β in HP-Ag/bFGF group was significantly lower than that in other groups. These results indicate that HP-Ag/bFGF composite hydrogel can facilitate scarless wound healing by down-regulating the expression of TGF-β.

Evaluation of bacteria-infected wound healing in vivo

The HP-Ag/bFGF hydrogel has the ability to continuously release Ag, and in vitro experiments have shown that it has a strong antibacterial effect. To further expand its clinical application, a bacterial infected wound model was used. Figure 5A shows that S. aureus at the wound site of the control group and the bFGF group grew wantonly. From day 4 to day 7, the wound was covered by a large number of bacterial colonies, which seriously hindered its closure process. There was no significant change in the rate of wound closure until the bacterial layer and blood scab were shed. However, the HP-Ag/bFGF group experienced a more regular closure process and was less affected by bacterial infection. On day 14, the wound was smooth and closed and even vanished for treatment group of HP-Ag/bFGF, whereas the wound boundaries were still observed for other groups. In the process of skin repair, the wound closure rate of HP-Ag/bFGF group was significantly higher than that of other groups (Fig. 5B).

Fig. 5

A Photographs of the skin wounds of various groups on day 0, 4, 7, 11and 14 (Scale: 10 mm). B Wound closure analysis from the existing wound area on day 4, 7, 11 and 14. C Photographs of survival bacteria colonies (S. aureus, yellow dots in plates) growing on agar plates from wound sites on day 7 after different treatments. D Counting of survival bacteria colonies growing on agar plates from wound sites on day 7. E H&E and Masson staining of the wound sections on day 7 and 14. (Scale: 100 μm). F Collagen volume fraction of the wound sections on day 7 (red arrow: hair follicles). All data are presented as mean ± SD, **p < 0.01, ***p < 0.001, n = 3

On day 7, skin tissues were homogenized and diluted at an appropriate ratio before being smeared on the agar plate. Photographs and colony counts of infected skin in different groups are shown in Fig. 5C and D. The number of colonies in the HP-Ag/bFGF group was significantly less than that in the other two groups, which was consistent with the results of in vitro antibacterial experiments. These results confirm that HP-Ag/bFGF hydrogel was capable of destroying bacterial as an antibacterial platform, which is consistent with the results of previous studies [49].

As shown in Fig. 5E, similar to the acute wound, the epidermis in control group was thickened, with mastoid processes, and fibroblasts were abundant and irregularized. Meanwhile, the collagen fibers were numerous, dense and thick, with disordered arrangement, and the boundary between dermal reticular layer and epidermal layer was blurred. In contrast, the epidermal layer in HP-Ag/bFGF group was flat and thin, and there are fewer fibroblasts, and most of them are arranged in parallel. A key feature of optimal wound healing is remodeling the ECM by depositating collagen in a well-organized network to restore the normal structure of tissue. The loose and orderly arrangement of collagen and the presence of regenerated skin appendages such as hair follicles and sebaceous glands further confirm that HP-Ag/bFGF leads to scarless and effective regeneration. Collagen content in the HP-Ag/bFGF group was significantly higher than that in the other two groups on day 7, as shown in Fig. 5F.

Regeneration and remodeling of the blood supply system is critical for tissue regeneration. New blood vessels can provide necessary nutrients and oxygen supply for tissue reconstruction and carry away metabolic waste in time [50]. CD31 and VEGF were detected to evaluate wound angiogenesis. As shown by the CD 31 staining results in Fig. 6A, the immature capillaries in the control group were dense and disordered. Figure 6B and C show that HP-Ag/bFGF comprised significantly more mature vessels than the other two groups, while the temporarily constructed immature vessels degenerated over time. This control of vessel density by partially blocking capillary growth may lead to a reduced but fully functional vascular system, which has been shown to improve long-term healing outcomes and avoid scarring [51, 52].

Fig. 6

A Immunohistochemistry staining images of CD31(red arrow: mature blood vessels) and VEGF in the wound tissues on day 14. Semi-quantitative analysis of the expression level of CD31-positive ( +) mature vessels B, VEGF C in the wound tissues on day 14. D Immunohistochemistry staining images of TGF-β and α-SMA in the wound tissues on day 14. Semi-quantitative analysis of the expression level of TGF-β E and α-SMA F in the wound tissues on day 14 (Scale: 100 μm). All data are presented as mean ± SD. *p < 0.05, **p < 0.01, n = 3

TGF-β directly induces α-SMA expression in recruited fibroblasts, promoting myoblast differentiation into myoblasts, which in turn promotes uncontrolled ECM production, leading to scarring [53]. Therefore, TGF-β activity was associated with increased scar formation and fibrosis induction at the later stage of wound healing. Figure 6D and E show that the expression of TGF-β in HP-Ag/bFGF group was significantly less than that of the other two groups. These results suggest that HP-Ag/bFGF has greater potential to promote scarless wound healing. The low expression of α-SMA in Fig. 6F also confirmed this conclusion. In addition, as shown in Additional file 1: Fig.S8 and Fig.S9, the representative organs (heart, liver, spleen, lung, and kidney) were examined by H&E staining, and no obvious pathological abnormalities or damage were found in the organ sections, verifying the biosafety of HP-Ag/bFGF hydrogel for wound healing.

Modulated macrophages polarization and inflammation response

Interleukin (IL) is a group of cytokines that play a crucial role in the inflammatory process. IL-6, a pro-inflammatory cytokine, is released early in inflammation and has been shown to be an effective stimulant of fibroblast proliferation [54]. IL-10, an anti-inflammatory and antifibrotic cytokine, has been reported to reduce the expression of IL-6, IL-8, and other inflammatory genes, and significantly improve collagen patterns [55, 56]. The content of IL-6 in tissues of HP-Ag/bFGF group was significantly lower than that in other groups, as shown in Fig. 7A and C. Meanwhile, the content of IL-10 in tissues of HP-Ag/bFGF group was significantly higher than that in other groups (Fig. 7B and D). These results suggest that compared with the other groups, the tissue inflammation was the weakest in HP-Ag/bFGF group on day 4 and the anti-inflammatory effect was the strongest on day 7.

Fig. 7

Regulation of inflammation and immune microenvironment. A Immunohistochemistry staining images of IL-6 in the wound tissues on day 4. B Immunohistochemistry staining images of IL-10 in the wound tissues on day 7. Semi-quantitative analysis of the expression level of IL-6 C and IL-10 D. Immunofluorescence staining of M1 and M2 type macrophage in the spleen of infected mice on day 4 E and day 7 F. Multicolor flow cytometry G and quantitative analysis H to detect the M1-type and M2-type macrophages in the spleen of infected mice on day 4. Scale bar: 20 μm (n = 3, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Innate immunity is a crucial component of the body’s defense against pathogen invasion and plays a vital role in mediating tissue repair. The transformation of macrophages from M1 phenotype to M2 phenotype is believed to be necessary for wound healing [57]. On day 4 of acute wound infection, different macrophages subtypes were labeled by immunofluorescence and multicolor flow cytometry. M1-type macrophages (F4/80 + /CD11c +) are pro-inflammatory immune cells that secrete inflammatory cytokines during the acute phase of infection, while M2-type macrophages (F4/80 + /CD206 +) have anti-inflammatory and repair effects [58]. From Fig. 7E, the number of M1-type macrophages of HP-Ag/bFGF group was higher than that of the other two groups. Similarly, flow cytometry confirmed the results. As can be seen from Fig. 7G and H, M1-type macrophages in the HP-Ag/bFGF group were significantly higher than those in the other two groups, while M2-type macrophages were significantly lower than those in control group. The results indicate that the immune response in the control group and the bFGF group was relatively slow at the initial stage of wound infection, while the HP-Ag/bFGF group successfully stimulated the immune response through the release of Ag, accelerating the process of inflammation after infection and injury. Figure 7F shows the staining of M1-type and M2-type macrophages labeled by immunofluorescence 7 days after the infection. At this time, with the progress of tissue repair, the number of M2-type macrophages in HP-Ag/bFGF group was higher than that in other groups, while the number of M1-type macrophages was the lowest. The results indicate that the inflammatory period of the infected skin tissue in the HP-Ag/bFGF group had transitioned to the subsequent tissue repair period, while the control group and the bFGF group were still experiencing the inflammatory period. This observation is consistent with previous immunohistochemical results of IL-6 and IL-10.