Introduction

Diabetes mellitus (DM) is a prevalent metabolic disease that affects over 400 million individuals worldwide. Among its numerous complications, diabetic ulcer (DU) stands out as a prominent issue. Chronic DU wound with serious infection may lead to necrosis of lower limb and increase the risk of amputation in diabetic patients by 50% compared to the general population, significantly impacting the life quality of patients1. The treatment of DU poses a considerable challenge, thus making it a global concern2.

The normal wound healing process comprises four overlapped and distinct stages: hemostasis, inflammation, proliferation, and remodeling3. However, due to the complex wound environment in DU, these physiological processes are disrupted, leading to a stagnation of wound recovery in the inflammatory stage and preventing transition to the proliferative stage. As a result, DU become difficult to heal and disordered microenvironment has been the important issue. Extensive research suggests that mitigating excessive inflammation is crucial for accelerating diabetic wound healing. Macrophages play a pivotal role in the healing of chronic wounds, especially in DU. Due to the extended inflammatory phase in DU, macrophages persistently release pro-inflammatory cytokines, such as tumor necrosis factor α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and interferon γ (IFN-γ), which contribute to the prolonged inflammatory phase and delayed wound healing4,5,6. Furthermore, the high glucose level in diabetic patients can easily lead to an increase in advanced glycosylation end products (AGEs), which further causes cell damage or tissue injury. These AGEs bind to the receptor of advanced glycation end products (RAGE) on the cell membrane, thereby activating downstream signaling pathways to elevate the body’s oxidative stress levels and induce chronic inflammation. This results in increased production of Reactive Oxygen Species (ROS) and the release of pro-inflammatory factors, ultimately delaying the healing process of wounds7,8. Furthermore, the persistent high glucose environment in wounds makes them highly susceptible to bacterial infections. When diabetic ulcer wounds are infected by bacteria in the early stages, the levels of indicators such as nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3 inflammasome), interleukin-18, and interleukin-1β increase, which can promote the release of inflammatory factors and elevate oxidative stress levels, leading to endothelial cell damage and thereby exacerbating inflammation9. Although antibiotics are commonly employed to address the issue of infection, the multi-target microenvironment regulation remained difficult to treat, and their prolonged usage often results in antibiotic resistance10. Consequently, the treatment of DU remains exceedingly challenging in clinical settings.

Hydrogels have shown great potential as a favorable form of dressing for promoting wound healing in DU11. The three-dimensional network structure of hydrogel dressings closely mimics the natural extracellular matrix, both physically and functionally, providing a suitable environment for wound healing. The development of the hydrogel materials has been greatly advanced in the latest decades, which enable good physical and chemical properties when applying the hydrogel dressings to the wound, however, the biological and pharmaceutical function of the hydrogel remained undesirable due the lack of efficient and reliable drugs that incorporated to the hydrogel, resulting as unsatisfactory clinical treatment. In seeking a good pharmaceutical ingredient, natural polysaccharide has attracted extensive attention of many researches. For instance, Wang et al. prepared a composite hydrogel loading natural polysaccharides derived from Periplaneta americana herbal residue for the treatment of DU12. Similarly, Hao et al. developed a hydrogel composed of chitosan, sodium alginate, and velvet blood peptides to promote DU healing by regulating angiogenesis, inflammatory response, and skin microbiota13.

Chinese herbal medicine offers the advantages of low toxicity and side effects, multi-target action, and excellent therapeutic efficacy. Bletilla striata, a traditional Chinese herbal medicine, has a long history in clinical treatments for its astringent, hemostatic, detumescence and promoting granulation. Bletilla striata polysaccharide (BSP), an essential active component of Bletilla striata, exhibits remarkable abilities in promoting coagulation, regulating inflammation, and enhancing cell proliferation. Extensive application has been recorded in the clinical treatment of acute and chronic skin wounds14. The application of BSP in preparation of hydrogel has attracted attention of many researches. Jin et al. prepared BSP/konjac glucomannan blend hydrogel, which can effectively promote wound healing15. Chen et al. prepared BSP-waterborne polyurethane hydrogel for wound healing. However, problems remained in the existing studies regarding BSP hydrogels16. Firstly, the preparation method was cumbersome and the hydrogel lacked toughness and self-healing properties during application. Secondly, BSP alone falls short as an effective drug for treating DU, especially due to its limited antibacterial effect.

Borax (sodium tetraborate, BOX), being an inorganic compound, serves as an excellent chemical crosslinking agent for hydrogels due to its low toxicity, affordability, and good water solubility17. Ma et al. successfully developed a tough, adhesive, self-healing, and antibacterial plant-based hydrogel through the dynamic cross-linking of pyrotriol-borax via borate18 and hydrogen bonds between borax and oxidized dextrose (OD)19. This dual crosslinking offers exceptional mechanical strength, self-healing properties, injectability, and pH responsiveness to hydrogels. The existence of abundant hydroxyl groups in BSP20 also facilitate the reaction of forming borate bonds, thus realizing the preparation of hydrogels.

Berberine (BER), the primary active compound found in Coptis chinensis, possesses a wide spectrum of antibacterial, anti-inflammatory, antioxidant, and hypoglycemic effects21. It exhibits excellent wound healing properties, particularly in infectious and chronic wounds, which is the strong support to pro-cell proliferation and anti-inflammatory properties of BSP and help to achieve multi-target biological function and complementary efficacy in promoting the healing of DU. However, the optimum dosage of the BER is a considerable issue to achieve its best performance. Wang et al. found that BER is the most important active ingredient in berberine and the main toxic ingredient22. Li et al. found that excessive use of BER may have toxic side effects on the spleen23. Therefore, exploration the optimum dosage of BER in preparation the hydrogel turns out another goal of the development of the hydrogel.

In this study, a series of BSP/BER composite hydrogels were prepared. The graphical abstract of this paper is shown in Fig. 1. The physicochemical properties of the composite hydrogel were evaluated using scanning electron microscopy (SEM), infrared spectroscopy (FTIR), and rheology tests. Furthermore, in vitro anti-inflammatory, antioxidant, cell migration activity, and antibacterial properties were investigated. Animal experiments were conducted to verify its ability to promote wound healing in DU. The results showed that the BSP/BER composite hydrogels exhibited excellent antimicrobial, anti-inflammatory and antioxidant properties, which provided a suitable microenvironment for wound healing and significantly promoted the healing of DU wounds. These hydrogels have unique properties and adaptability, and have a promising future in the field of DU care.

Fig. 1
figure 1

Schematic diagram of the preparation of the hydrogel and its promotion effect to the healing of diabetic wound.

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Results and discussion

Preparation and characterization of the BSP/BER hydrogels

Based on the material ratios presented in Table 1, four distinct types of hydrogels were prepared. The internal structural characteristics of a wound dressing play a pivotal role in its effectiveness in promoting wound healing24. Scanning electron microscopy (SEM) results, as depicted in Fig. 2(a), revealed that the microstructures of these four composite hydrogels closely resemble the extracellular matrix, featuring a porous network structure, and alterations of the drug dosage did not significantly alter the hydrogel structure.

Fig. 2
figure 2

Representative SEM images of the hydrogel (a); FTIR spectra of BSP, BER, Box and BSP/BER hydrogel (b).

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Table 1 The sample codes and compositions of the BSP/ BER hydrogels.
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The FTIR measurements were shown in Fig. 2(b), the peak of 3338 cm− 1 corresponded to the hydroxyl group of BSP, and the peak of 2920 cm− 1 corresponded to the carbon stretching methyl or methylene group of BSP, absorbing the stretching vibration and C-O-C glycosidic bond at the 1000–1200 cm− 1 C-O-H end group, and the stretching vibration of 810 cm− 1 indicates the characteristic absorption of mannose. The absorption peak of 1509 cm− 1, 1634 cm− 1, 1263 cm− 1, and 1028 cm− 1 were from the bending vibration of the benzene ring skeleton of BER, the imine group, the C-N stretching vibration, and the C-O-C characteristic vibration, respectively. The absorption peak of borax at 1000–1300 cm− 1 was due to the in-plane bending vibration of OH, and the absorption peak at 1300–1500 cm− 1 was from the asymmetric stretching vibration of BO3. The spectrum of BSP/BER composite hydrogels exhibited characteristic peaks of BSP, BER, and borax. Moreover, the infrared spectra of the four types of composite hydrogels were basically consistent, indicating that the BER did not significantly alter the structure of BSP/BER composite hydrogels.

To investigate the rheological properties of the hydrogels, amplitude scanning was performed to measure the energy storage modulus (G’) and loss modulus (G”) of BSP/BER hydrogels in the strain range of 0.1 to 1000%. As shown in the Fig. 3(a), at low strains, the G’ value was higher than G” and the hydrogel maintained a solid-like shape. At high strains, a crossover occurred between two curves and G” exceed G’, indicating a transition to the liquid-like behavior. This transition from solid-like behavior to liquid-like behavior at high frequencies demonstrated the hydrogels’ ability to adapt to mechanical stimuli. Additionally, frequency scanning was performed on BSP/BER hydrogels. The energy storage modulus (G’) and loss modulus (G”) of the hydrogels were measured over a range of angular velocities from 0 to 20 rad/s. As shown in the Fig. 3(b), all hydrogels exhibited a higher G’ value than G”, indicating their solid-like nature and stability.

An exceptional wound dressing should have the ability to absorb wound exudates effectively, and the swelling rate serves as a significant indicator in evaluating its performance25. As depicted in the Fig. 3(c), the freeze-dried hydrogel BSP/BER5 demonstrated a maximum swelling rate with an absorption capacity of approximately 1000% of its dry weight. The other freeze-dried hydrogels exhibited an absorption capacity exceeding 1000%. Therefore, the BSP/BER hydrogel dressing possesses a considerable absorption capacity, enabling it to absorb tissue exudates and maintain a moist environment that aids in reducing infection and promoting the healing of infected wounds.

Hydrogel dressings are widely recognized for their ability to retain water on the wound surface, providing a moist environment that is conducive to wound angiogenesis and cell proliferation26. This, in turn, promotes optimal wound healing. To assess the water retention capabilities of these dressings, the water loss rate of different hydrogels was tested. The results, presented in Fig. 3(d), indicate that the water content of the four hydrogels gradually decreased over time, with complete water loss observed at 12 h. Specifically, the hydrogels exhibited an hourly water loss rate of approximately 8.3%, suggesting that they possess commendable water retention properties. The porosity of the hydrogel may have an effect on the swelling properties and water content of the hydrogel. Therefore, we determined the porosity of four kinds of composite hydrogels. As shown in the Figure S1, the porosity of all four kinds of composite hydrogels was above 90%, and there was no significant difference among them. The above results demonstrate that the three-dimensional structure of hydrogels is beneficial for maintaining moisture balance and providing a suitable environment for wound healing27. This structural advantage of hydrogels enables them to act as effective platforms wound dressings.

Fig. 3
figure 3

Rheological properties of BSP/BER hydrogel in strain scanning (a), angular frequency scanning (b); The swelling ratio of BSP/BER hydrogel (c); The water loss rate of BSP/BER hydrogel within 12 h at 37 °C (d). Data presented as mean ± SD, n = 3. Statistically significant: * p < 0.05, ** p < 0.01. a: There is no statistical difference compared with each group.

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Cytocompatibility

Excellent cellular compatibility is the fundamental requirement for biomedical dressings. It necessitates wound dressings to be safe, non-toxic, and non-damaging to the wound while maximizing their efficacy28. Therefore, the in vitro cytocompatibility of BSP/BER hydrogels was assessed using the CCK8 method. As shown in the Fig. 4(a), the extracts from the four hydrogels did not significantly impede the proliferation of L929 cells, with cell viability exceeding 80%. This indicates that BSP/BER hydrogels exhibit no apparent cytotoxicity.

Fig. 4
figure 4

Cytocompatibility of the hydrogel: cell viability of L929 cells (a) treated with BSP/BER hydrogel extracts after 24 h; live–dead cell staining of L929 cells (b) treated with BSP/BER hydrogel extracts after 48 h. Green: live, red: dead, scar bar: 100 μm.

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Furthermore, the cytotoxicity of BSP/BER hydrogels was further examined through live/dead experiments. Figure 4(b) showed the results under an inverted fluorescence microscope that, compared to the control group, the number of living cells (green) cultured with hydrogel extract did not change significantly, nor did their cell morphology (spindle or oval shape). Moreover, there was no significant difference in the number of dead cells (red) between the hydrogel extract treatment group and the control group. Given that wound dressings inevitably contact with red blood cells upon application29, the cytotoxicity of BSP/BER hydrogels on red blood cells was also evaluated through a red blood cell compatibility test. The experimental results in the Fig. 5(a) and (b) showed noticeable hemolysis in the positive group, while the supernatant in the negative group and the four hydrogel groups remained clear and transparent, implying no apparent hemolysis. The hemolysis rates of the four hydrogels on red blood cells were less than 5%, indicating excellent red blood cell compatibility30. Based on the CCK8 results, live/dead cell staining, and erythrocyte compatibility test, it can be concluded that BSP/BER hydrogels exhibit satisfactory cytocompatibility.

Fig. 5
figure 5

Hemolytic photographs (a) and ratio (b); Quantitative results of the migratory effect on L929 cells (c); L929 cell (d) migration. The black frame: schematic diagram of the middle free area of cell. Data presented as mean ± SD, n = 3. Statistically significant: ** p < 0.01, * p < 0.05.

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Cell migration

DU is notorious for their recalcitrance to treatment, and one of the critical factors influencing the healing process is the cell migration capability31. Therefore, the ability to enhance cell migration serves as a pivotal metric for evaluating hydrogel dressings. These dressings have demonstrated effectiveness in promoting cell proliferation, which consequently facilitates wound healing. The experimental results, as depicted in the Fig. 5(c) and (d), indicated that the cell scratch area decreased over time for all five experimental groups. Notably, the cell activity in the four hydrogel extract treatment groups was significantly elevated compared to the blank control group (P < 0.05). Among these groups, the BSP/BER1 hydrogel group exhibited the most pronounced difference, with a cell mobility rate of 48.17 ± 1.68%. These findings underscore that the BSP/BER composite hydrogel is effective in promoting cell migration towards the wound site, thereby accelerating the wound healing process. This enhanced cell migration capability is pivotal in promoting rapid and efficient wound closure, highlighting the potential utility of these hydrogels in managing chronic wounds, particularly DU.

Anti-oxidation

Studies have shown that the hyperglycemic environment at a wound site can lead to an increase in the level of oxidative stress at the wound, resulting in the production of a large amount of reactive oxygen species (ROS)32,33. These ROS can cause sustained inflammation at the wound site, which can significantly hinder the healing process. Therefore, the ability of a hydrogel dressing to exhibit good antioxidant properties is a crucial evaluation criterion. As shown in the Fig. 6(a) and (b), the ROS levels in the model group were significantly higher than those in the normal group (P < 0.05), indicating that the hyperglycemic oxidative stress model was successfully established. All four hydrogels were effective in reducing ROS levels, with BSP/BER1 hydrogels exhibiting the most significant effect (P < 0.05). After skin injury, the local oxidation/antioxidant system at the wound site was disrupted, leading to DNA fragmentation, lipid peroxidation, enzymatic inactivation, and delayed wound healing. Antioxidant-containing dressings can counteract oxidative stress and improve wound healing by restoring the redox balance34. In conclusion, BSP/BER composite hydrogels can effectively reduce ROS levels, alleviate the hyperglycemic oxidative stress state of DU wounds.

Fig. 6
figure 6

Flow cytometry of intracellular reactive oxygen species (ROS) in Raw cells (a); mean FITC in Raw cells (b). Data presented as mean ± SD, n = 3. Statistically significant: * p < 0.05, ** p < 0.01.

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Anti-inflammation

Inflammation plays a crucial role in the wound healing process and is a crucial link in the process of chronic wound healing. During the inflammatory phase of wound healing, macrophages are recruited to the wound area to resist the irritation of pathogens. Normally, this phase is transient, and inflammation subsides within a specific period, facilitating the transition to the proliferative phase. However, prolonged and excessive inflammation can impede the proper progression of wound healing35,36. Moreover, excessive bacteria and endotoxins existed in the diabetic wound environment often lead to excessive secretion of pro-inflammatory cytokines (such as MCP-1 and TNF-α), making it difficult for inflammation to subside. The prolonged inflammatory state hinders the transition from the wound healing phase to the proliferative phase, resulting in difficult wound healing. The experimental results in the Fig. 7(a, b, c) showed that after LPS stimulation, the levels of cellular inflammatory factors were significantly increased compared to the control group, indicating that the cellular inflammatory model was successfully established. The extracts of four hydrogels significantly reduced the levels of LPS-induced inflammatory factors IL-6, MCP-1, and TNF-α in RAW 264.7 cell supernatants, indicating that the composite hydrogels were able to inhibit LPS-induced cellular inflammation. Among them, BSP/BER10 hydrogel group had the best anti-inflammatory effect, reducing the level of MCP-1 from 858.52 ± 55.01 pg/mL to 124.68 ± 71.80 pg/mL, the level of IL-6 from 496.6075 ± 162.74 pg/mL to 295.6075 ± 9.63 pg/mL, and the level of TNF - α from 10428.03 ± 2127.28 pg/mL to 1696.42 ± 69.47 pg/mL. Meanwhile, the level of nitric oxide (NO) in the cell supernatant was measured. The experimental results in the Fig. 7(d) showed a notable elevation in the NO level in the MOD group, suggesting that inflammation significantly increased NO levels. Importantly, the extracts from the four hydrogels exhibited substantial reductions in the NO level, indicating their potential to exert a considerable anti-inflammatory effect. These findings highlight the potential utility of these hydrogel extracts in mitigating inflammatory responses. The anti-inflammatory activity may be attributed to the pharmacological effects of BSP and BER in the hydrogel. For instance, Lin et al. discovered that BSP effectively mitigates inflammation and oxidative stress in LPS-induced knee osteoarthritis models by inhibiting COX-2 and activating p-Smad2/3 and p-Erk1/237. Wu et al. found that BSP can significantly inhibit the inflammation caused by LPS/Nigericin38. Additionally, Lin et al. found that BER inhibits LPS-induced inflammation by regulating ER stress-mediated ERK1/2 activation in macrophages and hepatocytes39. The low inflammatory state can reduce the risk of bacterial infection and the level of oxidative stress on the wound surface, which can promote DU healing in multiple ways. Firstly, a lower inflammatory response can reduce the recruitment of inflammatory cells and the release of inflammatory cytokines, which can interfere with wound healing. Secondly, a low inflammatory state can also protect the wound from further damage and promote the formation of new tissue. Finally, the reduction of oxidative stress can enhance the activity of antioxidant enzymes and promote cell proliferation and migration, thereby promoting wound healing.

Fig. 7
figure 7

Effects of different hydrogel extracts on tumor necrosis factor (TNF)-α (a), monocyte chemotactic protein (MCP)-1 (b), interleukin IL-6 (c) factors, and NO (d) in lipopolysaccharide (LPS)-induced RAW 264.7 cell supernatant. Data presented as mean ± SD, n = 3. Statistically significant: * p < 0.05, ** p < 0.01.

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Antibacterial activity

Repeated bacterial infections are a major contributor to the chronicity and difficulty in healing DU40,41. These infections lead to sustained inflammation, which in turn fosters further bacterial infections, creating a vicious cycle that prolongs the healing process. Consequently, assessing the antibacterial properties of hydrogel dressings is paramount. In this study, the antibacterial efficacy of the hydrogels was evaluated through co-culture experiments with S. aureus and E. coli. The findings, as illustrated in the Fig. 8, revealed that while the BSP hydrogel group did not exhibit a reduction in the number of colonies compared to the normal saline group, BSP/BER1, BSP/BER5, and BSP/BER10 demonstrated notable antibacterial activity against S. aureus. Specifically, the colony count was significantly reduced, with the bacterial survival rate in the BSP/BER5 and BSP/BER10 groups dropping below 5%. Similarly, for E. coli, both BSP/BER5 and BSP/BER10 exhibited pronounced antibacterial effects, leading to a substantial decrease in the number of colonies. The antibacterial efficacy of the composite hydrogel was observed to be dependent on the BER content, with higher BER concentrations resulting in enhanced antibacterial activity, aligning with the established antibacterial properties of BER42. The proposed antibacterial mechanism involves altering the permeability of the bacterial cell membrane, inhibiting bacterial biofilm synthesis, disrupting bacterial cell structure and function, and consequently impeding bacterial growth and reproduction, thereby impacting normal bacterial physiological metabolism. In conclusion, both BSP/BER5 and BSP/BER10 hydrogels have been shown to possess potent antibacterial properties. The highly effective antibacterial activity of the composite hydrogel can effectively protect the wound from bacterial invasion and proliferation, promoting wound healing. Meanwhile, the composite hydrogel can alleviate the inflammatory response caused by microbial infection, reducing the risk of chronic inflammation and promoting a healthy healing process. In summary, the composite hydrogel was found to be effective in protecting DU from infection, thereby promoting rapid wound healing and alleviating associated healing difficulties.

Fig. 8
figure 8

Antibacterial properties of BSP/BER hydrogel. Photographs (a) and antibacterial activity of S. aureus (b) and E. coli (c) treated with BSP/BER hydrogel. Data presented as mean ± SD, n = 3. Statistically significant: * p < 0.05, ** p < 0.01.

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Wound healing effect

To evaluate the effect of hydrogel on wound healing of DU, an animal model of DU was established43. The wound healing results were shown in Fig. 9. With the passage of time, the wound area of mice from all groups was gradually reduced. The model group showed a consistently slower rate of wound healing at all stages of the actual healing process compared to the other groups. At day 4, there was no significant difference in wound healing rate between the composite hydrogel group and the model group (P > 0.05). For day 7 and day 10, the wound healing rates of the BSP, BSP/BER1 and BSP/BER5 hydrogel groups were significantly different from that of the model group (P < 0.05). There were significant differences between BSP/BER1 and BSP/BER5 hydrogel groups and model group at day 14 (P < 0.05). The healing rate of BSP/BER5 hydrogel group was the highest, reaching 94.9%, which was much higher than the model group.

Fig. 9
figure 9

Effect of BSP/BER hydrogel on the healing of diabetic wounds in mice. (a) Schematic diagram of the establishment of DW mice and treatment with different BSP/BER hydrogels. (b) Representative digital photographs of days 0, 4, 7, 10 and 14 under different treatment methods. (c) Schematic diagram of DW healing(day 14). (d) Wound closure rate analyzed via Image J. Data presented as mean ± SD, n = 5. Statistically significant: * p < 0.05, ** p < 0.01.

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Pathological examination of wound tissue was performed after the day 14. Figure 10 showed the results of H&E staining and Masson tri-color staining of skin tissue. The model group retained a small area of scab, while the hydrogel-treated group formed a complete, dense, and thin epidermal layer. The model group had obvious inflammatory cell infiltration and thicker epidermis, while the hydrogel treatment group showed significantly reduced inflammatory cell infiltration, thinner epidermis and rich skin structure. Among them, the skin structure of the BSP/BER5 hydrogel group was closest to the normal skin structure, with clear tissue structure, good dermis, and abundant collagen, blood vessels and hair follicle structures. Collagen fibers fill the defects in a net-like form, arranged loosely and orderly in parallel to the direction of the skin surface. It can be seen that among the BSP/BER hydrogel composite hydrogels, BSP/BER5 hydrogels had the best wound healing performance in this experiment. However, further research is still needed to understand how BSP/BER composite hydrogels can be applied in clinical settings.

Fig. 10
figure 10

Histology analysis of the wounds treated with BSP/BER hydrogel at the 14th day. The images of hematoxylin and eosin (H&E) and Masson trichrome staining under 4× (scar bar: 500 μm).

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Generally, BSP/BER hydrogels demonstrate favorable physicochemical properties and biocompatibility. These hydrogels efficiently leverage their anti-inflammatory, antioxidant, bactericidal, and moisturizing effects, creating an optimal healing environment that facilitates the rapid healing of DU. Despite their beneficial properties, the underlying mechanisms of hydrogel dressings requires further exploration to better understand their full potential and limitations.

Conclusion

In summary, a series of hydrogels prepared from BSP, BER and borax are beneficial for promoting wound healing. The experimental results show that the prepared hydrogel has a porous network structure and good moisture retention. The hydrogel has good cytocompatibility, anti-inflammatory and antioxidant properties, and has good antibacterial activity against E. coli and S. aureus. According to the results of animal experiments, BSP/BER5 hydrogels have the highest wound healing rate among all prepared hydrogels, and may have the greatest potential to promote DU healing, which has been further confirmed by histological studies. The results of this study proved the application prospect of BSP/BER composite hydrogel in the treatment of DU wounds, and also provided a basis for the development and application of BSP and BER in the treatment of DU. However, the underlying mechanisms of how BSP/BER composite hydrogels exert their anti-inflammatory, antioxidant, and antibacterial effects are not sufficient, which requires further studies in the future. In addition, we believe that BSP/BER composite hydrogel has potential application value in cosmetic and dermatological applications. Due to their excellent biocompatibility and moisturizing properties, BSP/BER composite hydrogels can be utilized in cosmetics and dermatological products. They can be formulated into creams, masks, or patches to improve skin hydration, reduce wrinkles, or address specific skin issues such as acne or pigmentation. However, further research is needed to fully explore their potential in these areas.

Materials and methods

Materials and reagents

BSP (Product number: S27914), BER (Product number: S30593), and Borax (Product number: S30082) were purchased from Yuanye biotechnology Company (Shanghai, China). Analytical grade KBr was also supplied by Yuanye biotechnology (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, penicillin, and streptomycin were purchased from Gibco (Waltham, MA, USA). Bead Array (CBA) Mouse Cytokine Kit (Product number: 552364) was purchased from BD Biosciences (NJ, USA). The Cell CountingKit-8 (Product number: BS350C) was procured from Biosharp (Hangzhou, China). ROS kit (Product number: S0033M) and NO kit (Product number: S0021S) were purchased from Beyotime biotechnology Company (Shanghai, China). Flow cytometry kit were purchased from Becton Dickinson company (Waltham, MA, USA). Staphylococcus aureus (S. aureus, ATCC25923) and Escherichia coli (E. coli, ATCC 8739) were obtained from the American type culture collection. Lipopolysaccharide (Product number: l2880) was purchased from Sigma–Aldrich Co. (St., Louis, MO, USA). All other reagents used were of analytical grade and deionized water was obtained from the Milli-Q water purification system (Milford, MA, USA).

Preparation and characterizations of BSP/BER hydrogels

Preparation of BSP/BER hydrogel

Four 10 mL BSP solutions at 20 mg/mL were prepared by dissolving BSP powder in deionized water. Subsequently, 0 mg, 1 mg, 5 mg, and 10 mg of BER powder were sequentially added to the BSP solutions to prepare BSP, BSP/BER1, BSP/BER5, and BSP/BER10 hydrogel precursor solutions, respectively. After complete dissolution, 50 mg of borax was added to each of the four precursor hydrogel solutions, which were then stirred at room temperature until uniform and transparent composite hydrogels were formed. The hydrogels were stored at 4 °C before use.

Scanning electron microscopy (SEM)

The microstructures of the lyophilized hydrogel products (BSP, BSP/BER1, BSP/BER5 and BSP/BER10) were observed by a Hitachi SU-8010 scanning electron microscope (Tokyo, Japan) under an accelerating voltage of 20 kV.

Fourier transform infrared spectroscopy (FTIR)

BSP, BER, Borax, and lyophilized hydrogel were milled into powder, mixed with KBr at a ratio of 1:100, and pelletized by a hydraulic press. FTIR analysis of pellets was performed on a Thermo Fisher Nicolet iS50 Scientifc Nicolet 6700 spectrometer (Waltham, MA, USA) with a resolution of 2 cm− 1 and a wavenumber range of 4000 –400 cm− 1.

Rheology characteristics of the hydrogel

The rheology characteristics of the hydrogel were measured by Anton Paar MCR 302 Rotational Rheometer (Austria). At a temperature of 25 ± 1 °C, the storage modulus G′ and loss modulus G″ of the hydrogel were measured over a strain range of 0.1–1000% and an angular frequency range of 0–20 rad/s using a parallel plate with a diameter of 50 mm.

Water loss rate

The initial water content of 1 g hydrogel was recorded as M1 and placed in a centrifuge tube. The test tube containing the hydrogel was then placed in an incubator at 37 °C. The weight of the test tube M2 was recorded every hour, and the water loss rate of the hydrogel was calculated according to the following equation.

$${\text{Water\, loss\, rate}}\, (\%) = \frac{{M}_{1}-{M}_{2}}{{M}_{1}}\times 100\%$$

Swelling behavior

The freeze-dried hydrogel (weight M0) sample was immersed in PBS(pH = 7.4, 0.38 mol/L) at room temperature. Periodically, the swollen hydrogel was removed, and the surface moisture was blotted using filter paper. Subsequently, the hydrogel was weighed and denoted as Ms. The swelling rate of the hydrogel was calculated by the following equation,

$${\text{Swelling\, ratio}}\, (\%) = \frac{{M}_{S}-{M}_{0}}{{M}_{0}}\times 100\%$$

In vitro studies

Cell culture

Mouse fibroblasts (L929, Chinese Academy of Sciences Cell Bank, Shanghai, China) and mouse RAW 264.7 macrophages (RAW 264.7, Chinese Academy of Sciences Cell Bank, Shanghai, China) were cultured in modified culture medium containing 90% DMEM, 10% (V/V) fetal bovine serum and 1% (V/V) penicillin–streptomycin at 37 °C with 5% CO2. The hydrogel extracts were sterilized with a 0.22 μm filter.

Cytocompatibility of BSP/BER hydrogel

L929 cells were seeded on the modified culture medium at the cell density of 1 × 104 per well in 96-well cell culture plates. After cell attachment, the experimental groups were added with different hydrogel extracts. Medium was used as blank group and medium with cells was used as control group. After 24 h of incubation, CCK8 was added and incubation was conducted for another 1 h. Finally, the absorbance at 450 nm was measured by a microplate spectrophotometer. The absorbance of the test groups, blank group, and control group were recorded as ODA, ODB, ODC, and the cell viability was calculated as follows.

$${\text{Cell\, viability}}\, (\%) = \frac{{OD}_{A}-{OD}_{B}}{{OD}_{C}-{OD}_{B}}\times 100\%$$

For the dead/live assay, L929 cells were cultured in the medium containing correspondingly different hydrogel extracts for 24 h. The cells were then stained using the Calcein-AM/PI Double Stain Kit, and the survival images of L929 cells were observed under an inverted fluorescence microscope.

For the hemolysis rate experiment, the mixture of 1 mL hydrogel sample and 20 µL 2% red blood cell suspension was prepared and incubated at 37 °C for 1 h. After incubation, the mixture was centrifuged at 2000 rpm for 5 min and the absorbance of supernatants was measured at 545 nm. The absorbance of the positive group, negative group and experimental groups were recorded as ODp, ODn and ODs, respectively. The cell viability was calculated as follows:

$${\text{Hemolysis\, ratio}} = \frac{{OD}_{s}-{OD}_{n}}{{OD}_{p}-{OD}_{n}}\times 100\%$$

Cell migration activity of BSP/BER hydrogel

L929 cells were inoculated on 12-well cell culture plates at a cell density of 6 × 104 per well. After the cells were attached to the wall, a sterile straight ruler and a 200 µL pipette tip were used to make scratches on the bottom surface of the 12-hole plate, and 3 vertical lines were drawn in parallel in each hole. In the control group, 1% of serum DMEM culture solution was added, while in the drug experimental group, hydrogels extract was added to culture cells. At 0 h, 24 h and 48 h, the plate was observed under an inverted microscope at the same magnification and brightness. ImageJ was used to analyze the cell mobility of each experimental group.

Antioxidation performance of BSP/BER hydrogel

RAW 264.7 cells were seeded on the modified culture medium at a cell density of 3 × 105 per well in 6-well cell culture plates. After cell attachment, the experimental groups were added with different hydrogel extracts for 2 h. The LPS was added to a final concentration of 1 µg/mL. A control group and a model group were set-up by medium and medium with LPS, respectively. After 24 h, DCFH-DA (2,7-Dichlorofluorescein diacetate) was diluted with serum-free medium at a ratio of 1:1000 to achieve a final concentration of 10 µmol/L. Approximately 500 µL of the prepared solution was added to each well to cover the cells and incubated at 37 °C for 20 min. Following incubation, the cells were washed three times with serum-free medium and subsequently treated with trypsin for 2 min for digestion. DMEM culture solution was added to stop the digestion process, and the corresponding cell samples were collected. Flow cytometry was used to detect the fluorescence of each group of cells and to determine their reactive oxygen species (ROS) levels.

Antiinflammation performance of BSP/BER hydrogel

RAW 264.7 cells were seeded on the modified culture medium at a cell density of 3 × 105 per well in 6-well cell culture plates. After cell attachment, the experimental group was added with four different hydrogel extracts and incubated for 2 h. The LPS was added to a final concentration of 1 µg/mL. A control group and a model group were set-up by medium and medium with LPS, respectively. After 24 h, the culture medium was collected. The procedures included mixing the culture medium, cytokine capture microspheres, and PE (phycoerythrin) labeled cytokine detection antibodies together, and then incubating the mixtures in the dark at room temperature for 3 h. Concentrations of TNF-α, MCP-1, and IL-6 were measured using FAC scanning flow cytometry. The anti-inflammatory effect of hydrogel was evaluated by detecting the levels of inflammatory factors and NO in the cell supernatant.

Antibacterial performance of BSP/BER hydrogel

Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were inoculated in LB medium and cultured in a shaker table at 37 °C and 150 rpm for 12 h. Two strains were harvested by centrifugation and diluted to 1 × 105-106 CFU/mL with sterile physiological saline. 200 mg BSP, BSP/BER1, BSP/BER5, and BSP/BER10 hydrogels were mixed with 1 mL bacterial suspension in respective groups, and no hydrogel was added in the control group. The mixed liquid was cultured for 12 h under the same conditions. The cultured suspensions were inoculated on the medium and cultured for 12 h to form colony units. 100 µL bacterial suspension was mixed with 1mL Luria-Bertani (LB) liquid medium and cultured in a shaking table at 37 °C and 200 rpm for 12 h, and the absorbance of the bacterial solution at 625 nm was measured to investigate the survival rate of the bacteria.

In vivo wound healing

Wound healing evaluation

35 ICR mice (male, 6–8 weeks, 20–30 g) were purchased from the Animal Experiments Center of Zhejiang Chinese Medical University and randomly divided into 7 groups, which included control (negative control), recombinant epidermal growth factor gel (GF), MOD (model), BSP, BSP/BER1, BSP/BER5, and BSP/BER10. After adaptive feeding for a week, the mice were fasted for 24 h and diabetic model was established by intraperitoneally injection of Streptozotocin (STZ) as 120 mg/kg. Mice in the Control group were injected intraperitoneally with the same amount of citric acid buffer. After continuous intraperitoneal injection for 2 days, the blood glucose levels of mice were measured, and the modeling was considered successful when the blood glucose of the mice was ≥ 16.7 mmol/L44. Throughout the modeling period, the mice were administered a high-fat and high-sugar diet. After anesthetizing the mice with atropine and Zoletil 50, the back hair was removed and sterilized with 75% (V/V) ethanol. Circular wounds with a diameter of 1.5 cm were made on the back of mice. The control group and MOD group were treated with corresponding physiological saline, the GF group was treated with GF, and the other groups were treated with corresponding hydrogel. The wound size was meaesured at 0, 4, 7, 10, and 14 days after the operation, and the wound healing rate was calculated according to the follow equation:

$${\text{Wound\, Closure}}\, (\%) = \frac{{A}_{0}-{A}_{T}}{{A}_{0}}\times 100\%$$

where A0 and AT represent the initial wound area and the wound area at a time interval, respectively.

Histopathological analysis

Mice were sacrificed on day 14, and the skin of the whole wound including the adjacent normal skin was excised and fixed in 4% buffered paraformaldehyde. The implanted samples were divided into sections of 4 μm thickness and stained with Masson’s trichrome and hematoxylin and eosin (H&E). Sections were examined using a light microscope (Nikon, Japan) and captured on camera (×100). The National Institutes of Health’s guide (NIH Publications No. 8023, revised 1978) for the care and use of laboratory animals was followed in all experimental methods. All reasonable efforts were made to minimize the animals’ suffering.

Statistical analysis

All results were statistically analyzed using SPASS v.20.0 software (IBM SPSS Statistics, USA). The significance level for the statistical test analysis was set at p < 0.05. Values were expressed as mean ± standard deviation.