Introduction

Wound healing is a complex and dynamic process that unfolds through four consecutive, overlapping, and tightly regulated phases: hemostasis, inflammation, proliferation, and remodeling1,2,3. Each phase involves distinct tissue and cellular responses. The hemostatic phase begins immediately after injury, where platelets and coagulation factors accumulate at the wound site. These factors release and activate a cascade of growth factors, attracting leukocytes, epithelial cells, and vascular endothelial cells to the wound to initiate clot formation and stop bleeding. This phase typically lasts 1–2 days. The inflammatory phase follows, during which the injury activates both the coagulation system and the immune system, triggering the release of vasoactive mediators and chemokines. This recruits inflammatory cells, such as leukocytes, to the wound site. These cells not only clean the wound but also release growth factors that amplify the local inflammatory response. However, excessive inflammation can lead to complications such as hypertrophic scars or keloids. Once hypertrophic scars or keloids form, they will persist for a long time and will not diminish over time without specific treatment or surgical intervention4,5. This phase usually occurs 2–3 days post-injury. The proliferative phase, spanning days 3–14, is characterized by granulation tissue formation. Fibroblasts and endothelial cells migrate to the wound area, producing a vascular-rich extracellular matrix. Keratinocytes proliferate and migrate from the wound edges to the center, forming a new epidermal layer6. Finally, the remodeling phase begins around day 14, during which the extracellular matrix undergoes reorganization. Collagen fibers and proteoglycans are restructured, leading to scar maturation and increased tensile strength7. Given this intricate process, many wound repair strategies aim to shorten the proliferation or remodeling phases. Currently, various hydrogels, such as those containing human epidermal growth factor, keratin, or chitosan, have been developed for wound repair8,9,10,11. However, these hydrogels often face limitations due to complex preparation processes, the need for organic chemical reagents, or high material costs, hindering their widespread application12,13,14. Consequently, there is a significant demand for cost-effective and efficient wound repair hydrogels. Developing such hydrogels using abundant and inexpensive raw materials remains a critical challenge.

Ovalbumin (OVA), a major storage protein in egg whites, constitutes 54-69% of the total protein content in egg whites15. It is a globular glycoprotein with a molecular weight of 44.5 kDa, containing four free sulfhydryl groups and 385 amino acid residues16. OVA’s secondary structure is predominantly composed of α-helices and β-sheets, stabilized by one disulfide bond and four sulfhydryl groups. When heated, these groups are exposed, enhancing the protein’s colloidal stability through intermolecular interactions. OVA has been shown to possess several beneficial properties17,18. First, it can enhance both humoral and cellular immunity, as immune functions are largely protein-dependent. Second, OVA improves skin quality by enhancing hydration, regulating sebum production, and promoting tighter tissue binding. Third, it is effective in managing hypoproteinemia and maintaining plasma colloid osmotic pressure, making it useful as a blood volume expander in conditions such as hemorrhagic shock, severe burns, and protein deficiency. Due to these properties, OVA has found applications in biomedicine, food, and cosmetics. Dendrobium officinale polysaccharides (DOPs) are bioactive compounds derived from the Chinese herb dendrobium officinale. They exhibit a wide range of pharmacological effects, including immunomodulation, antioxidant activity, anti-fatigue effects, and hepatoprotection19,20. Notably, DOPs have strong antioxidant properties, protecting cell membranes and internal structures from free radical damage, thereby slowing the aging process. While previous studies have focused on DOPs’ immunomodulatory and antitumor effects, their potential in wound healing remains underexplored. This study investigates DOPs’ role in skin wound repair. Erythromycin (EM), a macrolide antibiotic with the chemical formula C37H67NO13, is widely used to treat infections caused by Gram-positive bacteria, including streptococcal and staphylococcal infections21,22. Although OVA exhibits some antimicrobial properties, EM was incorporated into the composite hydrogel to enhance its antimicrobial efficacy23,24.

Given the abundance, low cost, and accessibility of OVA, DOPs, and EM, these materials are ideal for constructing a cost-effective composite hydrogel25,26,27. This study addresses the unmet need for affordable and effective wound repair hydrogels by developing a simple yet efficient OVA/EM/DOPs composite hydrogel. In vitro and in vivo studies have demonstrated that this hydrogel accelerates wound healing while reducing inflammation, offering a promising alternative for future wound repair applications. The successful development of this hydrogel not only meets the demand for low-cost wound repair solutions but also provides a novel approach for designing other functional hydrogels.

Materials and methods

Materials

Ovalbumin, dendrobium polysaccharide, erythromycin, and Cell Counting KIT-8 (CCK-8) were purchased from Solarbio Science & Technology Co. Ltd., Beijing, China. Fetal bovine serum, RPMI Medium 1640 and Trypsin-EDTA solution were purchased from Gibco, Thermo Fisher Scientific Inc., USA. HEK293T and L929 cells were purchased from National Collection of Authenticated Cell Cultures, China. SD rats were purchased from SPF Biotechnology Co. Ltd., Beijing, China. CD31, VEGF, IL-10, TNF-α antibody and Alexa Fluor 555, Alexa Fluor 488 secondary antibody were purchased from Beyotime Biotechnology Co. Ltd., Shanghai, China. Live and Dead Cell Double Staining Kit, Hematoxylin and Eosin (H&E) Staining Kit and Modified Masson’s Trichrome Staining Kit were purchased from Solarbio Science & Technology Co. Ltd., Beijing, China. Human Epidermal Growth Factor Gel (HEGF hydrogel) was purchased from Jingdong Pharmacy, China.

Preparation of composite hydrogel

4 g ovalbumin dissolved in 10 ml of ddH2O, and the concentration of ovalbumin solution was 0.4 g/ml. Then, an appropriate amount of dendrobium polysaccharide was dissolved into the ovalbumin solution, and the concentration of dendrobium polysaccharide was 50 mg/ml. Next, appropriate amount of erythromycin was added to the composite solution, the working concentration of erythromycin was 100 µg/ml. Finally, the complex solution containing ovalbumin, dendrobium polysaccharide and erythromycin was mixed thoroughly. Incubation at specific temperatures promotes the formation of hydrogels. In this experiment, after analyzing and comparing different temperatures and durations, it was ultimately determined that incubating at 53°C for 40 min successfully produced the hydrogel28. All raw materials were sterile, the hydrogel was constructed within a super-clean workbench, and after the final product was formed, it was irradiated under UV sterilization for 45 min to ensure the sterility of the hydrogel.

The preparation steps for OVA hydrogel and OVA/EM hydrogel are consistent with those for above OVA/EM/DOPs composite hydrogel, except that they do not contain erythromycin and dendrobium polysaccharides, or dendrobium polysaccharides, respectively.

Characterization

OVA hydrogel, OVA/EM hydrogel and OVA/EM/DOPs hydrogel (dendrobium polysaccharide composite hydrogel) were sequentially dried in a vacuum freeze dryer (Capable, China). The dried hydrogel samples of each type were sprayed with gold, and then the micro-morphology was visualized by Scanning Electron Microscope (SEM, Hitachi, Japan) and pictures were collected. In this study, the specific pore sizes of the various types of hydrogels were not determined.

Each type of dried hydrogel sample was ground separately to be powder, and each type of hydrogel powder was analyzed by Fourier Infrared Spectroscopy (FTIR, Thermo Fisher Scientific, USA).

The test of contact angle

Dried powder of OVA hydrogel, OVA/EM hydrogel and OVA/EM/DOPs were sequentially placed on slides, a drop of water was then applied to each of the 3 types of slides. Next, a contact angle meter (DataPhysics Instruments, German) was utilized to measure the angle presented by the water droplet, and figures of water droplets were also collected by the contact angle meter.

The assay of hydrogel regeneration

Samples of OVA hydrogel, OVA/EM hydrogel and OVA/EM/DOPs hydrogel were sequentially ground to be powder. Then, an appropriate amount of the powder was taken and added to an equal volume of water to observe if the hydrogel could be generated again.

Antibacterial assay

Solid LB medium was prepared by adding 10 g tryptone, 5 g yeast extract, 10 g NaCl and 15 g agar per liter of water. LB solid medium was configured in plastic petri dishes. The surface of the medium was coated with E. coli or S. aureus. Then, OVA, OVA/EM, and OVA/EM/DOPs hydrogels were dropped in petri dishes, respectively. 12 h later, the growth of E. coli or S. aureus around each type of hydrogel was observed and analyzed. The specific steps for measuring the inhibition zone are as follows. Using the Image J software, randomly measure the radius of the inhibition zone five times, take the average value, and then proceed with further statistical analysis.

Analysis of water retention

The OVA, OVA/EM and OVA/EM/DOPs hydrogels were placed at 4–37°C, respectively, and these hydrogels were allowed natural evaporation of water. All kinds of hydrogels were weighed at 0, 6, 12, 18, 24 and 30 h respectively. Then, the collected data was analyzed.

Analysis of hydrogel adhesion and injectability

OVA, OVA/EM and OVA/EM/DOPs hydrogels were weighed equally for further analysis. Each hydrogel sample was then dabbed up by hand and stretched to observe its adhesion, and photographs were taken. To further demonstrate the adhesiveness of the prepared hydrogel, the repair effects on damaged heart and kidney were observed by collecting images of the damaged heart and kidney before and after repair.

The injectability of hydrogels is crucial for their broader applications. In this study, the injectability of the prepared hydrogels was evaluated by drawing them into a 5-milliliter syringe and then expelling them.

Hemolytic assay

The experimental group of saline, triton X-100, OVA hydrogel, OVA/EM hydrogel, HEGF hydrogel and OVA/EM/DOPs hydrogel were set up for the hemolytic experiment. 6 ml of blood from SD rats was taken, and the blood was divided into 6 portions of 1 ml each. Six portions of blood were centrifuged at 12,000 rpm at high speed to obtain erythrocyte precipitates. 700 µl of saline, triton X-100, OVA hydrogel solution, OVA/EM hydrogel solution, HEGF hydrogel solution and OVA/EM/DOPs hydrogel solution were added to the erythrocyte precipitates, respectively. The concentrations of triton X-100, OVA, OVA/EM, HEGF and OVA/EM/DOPs hydrogel solutions were all 40 mg/ml. 30 min later, hemolysis of erythrocytes in each experimental group was observed and the absorbance of the solution in each experimental group was determined by UV spectrophotometer (SHIMADZU, Japan). Finally, the absorbance measured in each experimental group was statistically analyzed to determine the level of hemolysis.

Cell scratch assay

L929 cells were cultured in a cell culture dish and the almost full-grown L929 cells were gently scratched with a yellow tip, and the width of the scratches is in the range of 250–400 μm. Next, the OVA, OVA/EM and OVA/EM/DOPs hydrogel lyophilized samples were diluted to 10, 50, 100, 200, 300 and 400 µg/ml with complete medium. Complete medium containing different concentrations of hydrogels were added to the constructed scratch-model of L929 cells. Scratch closure was observed at 0, 12, 24 and 36 h after the start of incubation and photographs were taken. Finally, the closure of scratches in each experimental group was analyzed and quantified.

The specific method for measuring cell migration rate is as follows. Regularly capturing images of the wound healing process using a microscope, then using Image J to randomly measure the wound diameter five times on the collected images, and further calculating the average wound diameter. Finally, comparing and analyzing the average diameter obtained from regular measurements with the original average diameter to calculate the relative migration.

Cytotoxicity analysis

First, the three types of hydrogels were freeze-dried into dry samples. Then, each dried hydrogel sample was ground into powder, dissolving the powder in complete culture medium to form medium solutions containing lyophilized hydrogel samples at different concentrations, and then co-culture them with normal cells to achieve the goal of exploring the cytotoxicity of the hydrogel.

Cytotoxicity was analyzed by CCK8 assay29,30. 5 × 105 L929 cells and 5 × 105 HEK293T cells were used as normal cells for cytotoxicity experiments. The specific experimental steps are as follows. First, 100 µl cell suspension was prepared in a 96-well plate and the cell suspension was pre-incubated in an incubator for 24 h at 37℃ with 5% CO2. Second, 10 µl of different concentrations of the samples to be tested were added to each experimental well, then the 96-well plate was mixed gently and incubation was continued in the incubator for 6 h. Three parallel experiments were done for each concentration. Third, 10 µl of CCK8 solution was added to each well, air bubbles that could affect the OD value should be avoided. Finally, the incubation was continued in an incubator for 4 h and then the absorbance at 450 nm was measured by an enzyme marker.

Live and dead cell staining

First, L929 cells and HEK293T cells were respectively inoculated in a 24-well plate and each well had 5 × 105 cells. Then cells were co-cultured with OVA/EM/DOPs hydrogel samples of the appropriate concentration. After 6 h of incubation, the medium was aspirated from each experimental well and each well was washed 3 times with sterilized PBS buffer. Then, an appropriate amount of live/dead cell staining work-solution was added to each well and incubated at 37°C for 30 min away from light. After the 30 min incubation, L929 or HEK293T cells was observed by fluorescence microscopy or laser confocal microscopy to analyze apoptosis. The whole process of observation needs to be operated away from light.

Animal modeling

All SD rats were purchased from SPF (Beijing) Biotechnology Co., Ltd. A circular wound whose diameter is 16 mm was created on the back of SD rats, and the wound was deep to fascia. When establishing the SD rat trauma model, isoflurane was applied to anesthetize the rats to reduce their pain. Six rat models were established for each experimental group, and a total of 30 SD rats were used. The five experimental groups were control, OVA hydrogel, OVA/EM hydrogel, HEGF hydrogel and OVA/EM/DOPs hydrogel. When wounds of SD rats in the group of OVA/EM/DOPs hydrogel healed, rats in each experimental group were euthanized. SD rats were anesthetized by inhalation of isoflurane (RWD, RWD Life Science Co., Ltd, China) in oxygen (5% for induction; 2–3% for maintenance). Then, rats were euthanized immediately by cervical dislocation. At last, samples of trauma from each experimental group were taken for further analysis.

All animal procedures used in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of Laboratory Animal Center of Nantong University (Protocol No. S20250420-005). We also confirmed that all procedures were carried out in accordance with the ARRIVE guidelines (https://arriveguidelines.org) and other relevant guidelines and regulations, such as the standard operating procedures for Laboratory Animal Center of Nantong University.

Wound healing therapy

Wound healing therapy were divided into five groups, namely control, OVA hydrogel, OVA/EM hydrogel, HEGF hydrogel and OVA/EM/DOPs hydrogel. The remaining groups, except for the blank control group, applied hydrogels or drug every day. The wound was covered with the appropriate amount of hydrogel or positive drug (HEGF hydrogel) to a thickness of about 2 mm in each group. Wound healing was observed at day 0, 5, 8, 11 and 14, respectively, and measured and statistically analyzed. There was a total of 6 rats in each experimental group, one of which was euthanized on day 7 in every group. Wound samples at day 7 were used for further Hematoxylin and Eosin (H&E) staining, Masson’s Trichrome staining, or immunofluorescence staining. At day 14, the wound healing therapy was terminated because wounds of animals in the group of OVA/EM/DOPs hydrogel had completely healed.

H&E staining and masson’s trichrome staining

Skin samples from each experimental group, along with the heart, liver, spleen, lungs, and kidneys, were further subjected to H&E staining analysis. This was done to assess the hydrogel’s efficacy in promoting wound healing through pathological sections and to determine whether it exhibited any toxicity to the major organs. Specific procedure for H&E staining is as follows. Frozen slices were fixed for 20 s, and they were washed gently with PBS buffer for 2 s. Frozen slices were then stained for 60 s at 60℃ using hematoxylin staining solution. After hematoxylin staining was completed, frozen slices were washed under running water for 10 s. Next, frozen sections were treated with 1% ethanol-hydrochloride for 2 s, and then frozen slices were gently washed with PBS buffer for 1 s. Frozen sections were treated for 10 s in order to re-blue by the blue-promoting solution, and frozen slices were then gently washed under running water for 15 s. Frozen slices were then stained with 0.5% eosin staining solution for 30 s, and then they were washed slightly for 2 s using distilled water. Next, frozen sections were sequentially treated with 80% ethanol, 95% ethanol and anhydrous ethanol for 2 s. Then, they were sequentially treated with (carbonic acid)-xylene, xylene (I) and xylene (II) for 3 s. Finally, frozen sections were sealed with neutral gum and then ready for further observation. When all staining is complete, nuclei are blue and collagen fibers, myofibers, cytoplasm, and erythrocytes are shades of red.

Specific procedure for masson staining is as follows. Sequentially, paraffin sections were put into xylene (I) for 20 min, xylene (II) for 20 min, anhydrous ethanol (I) for 10 min, anhydrous ethanol (II) for 10 min, 95% alcohol for 5 min, 90% alcohol for 5 min, 80% alcohol for 5 min and 70% alcohol for 5 min, then they were washed by distilled water. Next, slices were stained by Weigert’s iron hematoxylin for 5 min and washed using water, then they were treated by 1% (hydrochloric acid)-alcohol for 10 s and rinsed by running water for a few minutes to return to blue. Then, the slices were stained with Ponceau S acid Fuchsin Stain for 10 min and rinsed quickly by distilled water. Slices were treated with aqueous phosphomolybdic acid for 3 min, and then the slices were re-stained with aniline blue solution for 5 min. Then they were treated with 1% acetic acid for 1 min. The slices were sequentially placed in 95% alcohol (I) for 5 min, 95% alcohol (II) for 5 min, anhydrous ethanol (I) for 5 min, anhydrous ethanol (II) for 5 min, xylene (I) for 5 min, xylene (II) for 5 min for dehydration and transparency, and then they were taken out of xylene to be dried, and the slices were sealed with neutral gum. Finally, slices were observed under a microscope and pictures were collected. Collagen fibers, mucus, and cartilage are blue. Myofibrils, fibrils, and erythrocytes are red. Nuclei are blue-black.

Immunofluorescence

Slices or cells were fixed on slides, and the samples were then cell permeabilized. Next, the samples are uncoated to enhance antigen surface expression and make it easy to bind to the antibody. Samples were then blocked using 3% BSA to prevent non-specific proteins from binding to the antibody for 30 min. Next, the appropriate concentration of primary antibody was added to samples and incubated at 4°C overnight. Then, samples were washed with sterilized PBS buffer to remove unbound primary antibodies. Next, the appropriate concentration of specific fluorescently labeled secondary antibody was added to samples to bind to the primary antibody. Samples were then washed with sterilized PBS buffer to remove unbound secondary antibodies. Finally, samples were observed under a fluorescence microscope or laser confocal microscope and pictures are collected.

Statistical analysis

All data are presented as means ± standard deviation. Specific number of samples was indicated in the caption of each figure. Statistical analyses were carried out using unpaired t-test or one-way ANOVA with Tukey’s multiple comparison test in GraphPad Prism 8 (GraphPad Software Inc., USA). P < 0.05 was considered statistically significant, and P > 0.05 was considered statistically nonsignificant.

Results and discussion

Preparation and characterization of dendrobium polysaccharide composite hydrogel

In previously published studies, dendrobium polysaccharides have been reported to exhibit potential benefits, such as promoting cell proliferation and migration, enhancing immune function, and possessing anti-tumor properties31,32,33. However, these findings have not yet been fully validated through animal experiments. Therefore, in this study, dendrobium polysaccharides (DOPs) and a dendrobium polysaccharide composite hydrogel were utilized to investigate their effects on cell proliferation, migration, and skin wound healing. The OVA, OVA/EM, and OVA/EM/DOPs solutions were all capable of forming hydrogels when incubated at 53°C for 40 min (Fig. 1A). The pH values of the prepared hydrogels were all approximately 7.0. The pH value was close to that of skin and would not cause damage to the skin. The OVA hydrogel and OVA/EM hydrogel appeared milky white, opaque, and possessed a certain degree of viscosity and injectability. The OVA/EM/DOPs composite hydrogel exhibited a yellowish-brown color, was opaque, and had stronger viscosity and injectability. As shown in Fig. 1B, scanning electron microscopy (SEM) images revealed that all three hydrogels (OVA, OVA/EM, and OVA/EM/DOPs) exhibited a porous structure, indicating their potential for good air-permeability. This porous microstructure is conducive to the hydrogel’s further loading of various small or large molecular drugs34. Compared to the OVA hydrogel, the pores in the OVA/EM/DOPs and OVA/EM hydrogels are significantly larger. Generally, larger pores help to enhance the adsorption rate and capacity, especially for the adsorption of macromolecular substances. On the other hand, smaller pores contribute to improved selective adsorption, particularly for substances with smaller molecular sizes. This implies that the OVA/EM/DOPs and OVA/EM hydrogels have a better ability to adsorb macromolecular drugs, while also maintaining superior water retention and breathability. Cytotoxicity assays further demonstrated that both L929 and HEK293T cells maintained over 80% viability when exposed to the hydrogels at a concentration of 400 µg/ml (Fig. 1C and D). These results are consistent with the cytotoxicity profile of a hydrogel previously developed by Wen et al., suggesting that the OVA, OVA/EM, and OVA/EM/DOPs hydrogels possess excellent biocompatibility35.

To evaluate the hydrophilicity of the OVA, OVA/EM, and OVA/EM/DOPs hydrogels, contact angle measurements were conducted. The results revealed contact angles of 46.097°, 48.413°, and 44.646° for the OVA, OVA/EM, and OVA/EM/DOPs hydrogels, respectively (Fig. 1E and F). Since a contact angle below 90° is generally indicative of hydrophilicity36,37, all three hydrogels can be classified as hydrophilic. Notably, the OVA/EM/DOPs hydrogel exhibited slightly greater hydrophilicity compared to the OVA and OVA/EM hydrogels. According to Fourier-transform infrared (FTIR) spectroscopy, C-O and C=O typically appear between 1000 and 1475 cm−1, C=C usually appears between 1450 and 1650 cm− 1, and N-H generally appears between 3300 and 3500 cm−1. FTIR analysis of the hydrogels revealed that, compared to OVA and OVA/EM, the OVA/EM/DOPs hydrogel exhibited additional peaks corresponding to C–O, C=O, C=C, and N–H bonds (Fig. 1G). This suggests that OVA, EM, and DOPs have successfully formed a stable composite hydrogel.

Fig. 1
figure 1

Construction and characterization of OVA, OVA/EM and OVA/EM/DOPs hydrogel. (A) Preparation of OVA, OVA/EM and OVA/EM/DOPs hydrogel. (B) SEM images of OVA, OVA/EM and OVA/EM/DOPs hydrogel, and the scale bar is 100 μm. (C) Cytotoxicity analysis of OVA, OVA/EM and OVA/EM/DOPs hydrogel on L929 cells, n = 3. (D) Cytotoxicity analysis of OVA, OVA/EM and OVA/EM/DOPs hydrogel on HEK293T cells, n = 3. (E) Analysis of hydrophilicity of OVA, OVA/EM and OVA/EM/DOPs hydrogel by measuring contact angle. (F) Statistics results of contact angle. “N.S.” means no significant differences, n = 3. (G) FTIR spectra of OVA, OVA/EM and OVA/EM/DOPs hydrogel.

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The hydrogel exhibits excellent regeneration capability, which significantly enhances its storage and usability38,39. To evaluate the regeneration capacity of OVA, OVA/EM, and OVA/EM/DOPs hydrogels, regeneration experiments were conducted on these three types of hydrogels. As illustrated in Fig. 2A, upon the addition of an equal volume of ddH2O, the powdered forms of all three hydrogels rapidly reverted to their respective hydrogel states. All regenerated hydrogels show no significant differences in color, texture, transparency, and other aspects compared to their original forms. These results indicate that all three hydrogels possess remarkable reproducibility, a crucial advantage that facilitates their subsequent storage and application. Following this, antimicrobial experiments were performed on the three hydrogels. OVA, OVA/EM, and OVA/EM/DOPs hydrogels all demonstrated varying degrees of antibacterial activity, with particularly notable effects observed in the OVA/EM and OVA/EM/DOPs hydrogel groups (Fig. 2B). The results revealed that the OVA hydrogel inherently possesses antimicrobial properties, aligning with the findings reported by Zeng et al.40. Furthermore, when erythromycin was incorporated to form the dendrobium polysaccharide composite hydrogel, a significant enhancement in antimicrobial efficacy was observed. This improvement is clearly demonstrated in Fig. 3A. Statistical analysis of the antimicrobial experiment results revealed that the P-values for both the OVA/EM and OVA/EM/DOPs hydrogel groups, when compared to the OVA hydrogel group, were less than 0.05, indicating significant differences. However, no significant differences were observed between the OVA/EM and OVA/EM/DOPs hydrogel groups.

Fig. 2
figure 2

Regeneration capacity and antibacterial property. (A) Experiments on regeneration of OVA, OVA/EM and OVA/EM/DOPs hydrogel, and regeneration method is shown in the figure. (B) Antibacterial assays of OVA, OVA/EM and OVA/EM/DOPs hydrogel against E. coli and S. aureus.

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Evaluation of dendrobium polysaccharide composite hydrogel in vitro

To thoroughly assess the water-retention capacity of the three hydrogels, a comprehensive analysis was conducted. As illustrated in Fig. 3B and C, all three hydrogels maintained over 80% of their water content within the first 6 h, both at 4℃ and 37℃. After 30 h at 4℃, the water retention for all hydrogels remained above 60%. However, when exposed to 37°C for the same duration, the water retention slightly decreased to just under 60%. These findings confirm that the OVA, OVA/EM, and OVA/EM/DOPs hydrogels exhibit robust water-retention capabilities over a two-day period, ensuring their effectiveness within this timeframe41,42. This suggests that a daily application of the composite hydrogel could suffice to maintain its efficacy. Additionally, live/dead cell staining was employed to further evaluate the biocompatibility of the OVA/EM/DOPs composite hydrogel (Fig. 3D and E). In these images, red indicates dead cells while green signifies live cells43,44. The results revealed that even at a concentration of 400 µg/ml, the OVA/EM/DOPs composite hydrogel induced minimal apoptosis in L929 and HEK293T cells, underscoring its low cytotoxicity and excellent biocompatibility. These cytotoxicity findings are consistent with those reported by Cheng et al. for similar hydrogels30.

Fig. 3
figure 3

Evaluation of antimicrobial ability, water retention and biocompatibility. (A) Statistics results of antibacterial radius of OVA, OVA/EM and OVA/EM/DOPs hydrogel, n = 5. (B) Water retention rate of OVA, OVA/EM and OVA/EM/DOPs hydrogel at 4℃, and “*” means P < 0.05, n = 3. (C) Water retention rate of OVA, OVA/EM and OVA/EM/DOPs hydrogel at 37℃, and “*” means P < 0.05, n = 3. (D) Results of live and dead cell staining of L929 cells after co-culture with different concentrations of OVA/EM/DOPs hydrogel, and the scale bar is 100 μm. (E) Results of live and dead cell staining of HEK293T cells after co-culture with different concentrations of OVA/EM/DOPs hydrogel, and the scale bar is 100 μm.

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Given the critical importance of viscosity, injectability, and hemocompatibility for the biomedical application of composite hydrogels, comprehensive experiments were conducted to evaluate these properties45,46. The study initially focused on the tissue repair capability of the composite hydrogel through adhesion. As illustrated in Fig. 4A, the OVA/EM/DOPs composite hydrogel demonstrated remarkable efficacy in repairing damaged kidney and heart tissues, achieving results comparable to the original state. This indicates that the hydrogel possesses sufficient adhesive strength to effectively bond broken tissues. The adhesive performance of the OVA/EM/DOPs composite hydrogel aligns with the established criteria for polymer hydrogels as outlined by Guo et al.47. Subsequently, the injectability of the OVA/EM/DOPs composite hydrogel was examined. Figure 4B demonstrates that the hydrogel could be smoothly extruded through a syringe to form the letters “NTU”, confirming its excellent injectability. This characteristic makes it suitable for various injection-based applications, including in-situ drug delivery for tumor therapy, tissue repair, and orthopedic treatments48,49,50,51. Finally, hemocompatibility testing was performed to assess the safety profile of the hydrogel. As depicted in Fig. 4C, erythrocyte integrity was well maintained in the OVA hydrogel, OVA/EM hydrogel, HEGF hydrogel, and OVA/EM/DOPs hydrogel groups, with clear supernatants similar to the saline control group after centrifugation. This observation, coupled with absorbance measurements (Fig. 4D), confirms that the OVA/EM/DOPs composite hydrogel does not induce significant hemolysis or destructive effects on red blood cells52,53. The absence of hemolytic activity ensures the safety of this composite hydrogel for biomedical applications, in contrast to hemolytic hydrogels which could potentially cause severe adverse effects in biological systems.

Fig. 4
figure 4

Evaluation of Adhesion, injectability and hemolytic property. (A) Adhesion of OVA/EM/DOPs hydrogel, as exemplified by the repair of kidney and heart injuries. (B) “NTU” was written by OVA/EM/DOPs hydrogel, which indicated that OVA/EM/DOPs hydrogel has good injectability. (C) Results of hemolytic assay of saline, triton X-100, OVA hydrogel, OVA/EM hydrogel, HEGF hydrogel and OVA/EM/DOPs hydrogel. (D) Statistical results of absorbance of each experimental group in hemolytic assay, and “***” means P < 0.001, n = 5.

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The wound-healing potential of the OVA/EM/DOPs composite hydrogel was evaluated through cell scratch assays54,55. In line with standard practices in the field, L929 cells were selected as the model system for this investigation56,57,58. As illustrated in Fig. 5A and B, comparative analysis revealed that 200 µg/ml concentrations of OVA hydrogel, OVA/EM hydrogel, and OVA/EM/DOPs composite hydrogel significantly enhanced cell migration rates, with the OVA/EM/DOPs composite hydrogel demonstrating particularly pronounced effects. To further characterize the concentration-dependent effects, additional experiments were conducted using hydrogel concentrations of 50, 100, 300, 400, and 500 µg/ml (Fig. S1-5). The results consistently demonstrated that all tested formulations (OVA hydrogel, OVA/EM hydrogel, and OVA/EM/DOPs hydrogel) effectively promoted cell migration and accelerated scratch wound closure across various concentrations, with the OVA/EM/DOPs composite hydrogel showing superior performance. These findings provide robust evidence supporting the wound-healing potential of these hydrogel systems, particularly the OVA/EM/DOPs composite formulation. The observed effects are consistent with previous studies by Chen et al. and Guan et al., who reported enhanced cell migration using mechano-active nanocomposite hydrogels and reactive oxygen species-scavenging hydrogels, respectively54,59. Notably, the OVA/EM/DOPs composite hydrogel developed in this study demonstrated comparable or superior cell migration-promoting capabilities relative to these previously reported systems, suggesting its potential as an effective wound-healing material.

Fig. 5
figure 5

Cell scratching experiments cocultured with 200 µg/ml OVA hydrogel, OVA/EM hydrogel or OVA/EM/DOPs hydrogel. (A) Images were collected at 0, 12, 24 and 36 h in cell scratching assay, and the scale bar is 100 μm. (B) Relative migration ratio of L929 cells in the group of control, OVA hydrogel, OVA/EM hydrogel and OVA/EM/DOPs hydrogel, and “*” means P < 0.05, n = 5.

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Wound repair experiments in SD rats

In this study, five experimental groups were established, control, OVA hydrogel, OVA/EM hydrogel, HEGF hydrogel, and OVA/EM/DOPs hydrogel. Wound healing experiments were conducted over a period of 14 days (Fig. 6A). At the end of this period, all experimental rats were euthanized, and relevant samples were collected. Wound images from each group were captured on days 0, 5, 8, 11, and 14 (Fig. 6B). The images revealed that wounds in the OVA/EM/DOPs hydrogel group were completely healed by day 14, demonstrating the most effective wound healing among all groups. Additionally, the wound healing effects in the OVA hydrogel and OVA/EM hydrogel groups were slightly superior to those in the HEGF hydrogel group and significantly better than the control group. These results indicate that the composite hydrogel significantly accelerates wound healing. Figure 6C illustrates the simulated wound areas for each group at different time points, clearly showing that the OVA/EM/DOPs hydrogel group exhibited the fastest healing rate. Further statistical analysis of wound area and healing rate at days 5, 8, 11, and 14 (Fig. 6D and E) confirmed that treatment of the OVA/EM/DOPs hydrogel resulted in the most rapid reduction in wound area, underscoring its efficacy in promoting wound healing. Generally, wounds with a diameter of 10 mm or larger can heal rapidly within 14 days, suggesting that the applied hydrogel significantly promotes wound healing55,60,61. If a 10-mm wound takes more than 21 days to heal, the hydrogel used is considered ineffective. In the study by Ding et al., a photopolymerizable and immunomodulatory hydrogel was shown to completely heal a 10-mm rat wound within 14 days, demonstrating a strong wound healing capability similar to the OVA/EM/DOPs hydrogel developed in this study62. In 2023, researchers from Nanjing University reported a living microecological hydrogel that promotes wound healing63. This hydrogel contains Chlorella, which produces oxygen through photosynthesis to alleviate wound hypoxia and support Bacillus subtilis, which inhibits pathogenic bacteria growth and prevents infection through rapid reproduction and antimicrobial secretion. The preparation of this living microecological hydrogel is simple, cost-effective, and controllable, much like the OVA/EM/DOPs hydrogel. By optimizing the ratio of Chlorella to Bacillus subtilis, the hydrogel achieves optimal oxygen release and antimicrobial effects. In 2024, Zhong et al. developed a multifunctional spray hydrogel that promotes rapid wound healing within 13 days and prevents infection64. In summary, the OVA/EM/DOPs hydrogel demonstrated excellent wound repair capabilities, completely healing rat wounds within 14 days, comparable to other reported hydrogels. In future clinical applications, wet hydrogel formulations directly as products can be a key consideration. Not only are wet hydrogels convenient for patient use, but they are also easy to produce and transport65. Under the premise of large-scale raw material procurement, the price of OVA is approximately 10 $/100 g, with a usage of 0.4 g/ml in the wet hydrogel. The price of EM is about 10 $/25 g, and its dosage in the wet hydrogel is 100 µg/ml. The price of DOPs is roughly 10 $/20 g, with a usage of 50 mg/ml in the wet hydrogel. Since the various raw materials required for the preparation of this composite hydrogel are widely available and inexpensive, we anticipate that the cost of mass-producing this composite hydrogel in the future may not exceed 5 $/20 ml.

Fig. 6
figure 6

Wound repair experiments. (A) The schematic diagram of wound healing experiments. (B) Representative images of wound at day 0, 5, 8, 11 and 14. (C) Traces of wound closure during 14 days for five groups. (D) Statistics results of wound area for the group of control, OVA hydrogel, OVA/EM hydrogel, HEGF hydrogel and OVA/EM/DOPs hydrogel. “N.S.” means no significant differences, “*” means P < 0.05, “**” represents P < 0.01 and “***” indicates P < 0.001, n = 5. (E) Statistics results of wound healing rate for five experimental groups, and “*” means P < 0.05. Day 0, 5: n = 6. Day 8, 11 and 14: n = 5.

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Assessment of wound repair

To thoroughly evaluate the wound repair efficacy of the OVA/EM/DOPs hydrogel, histological analyses including H&E staining, Masson’s Trichrome staining, and immunofluorescence staining were conducted. H&E staining, a fundamental histological technique, utilizes hematoxylin to stain nuclear components a deep blue due to its affinity for basic cellular elements, and eosin to color cytoplasmic and extracellular matrix components pink, highlighting acidic structures. This differential staining allows for clear visualization and differentiation of tissue architecture under microscopic examination66,67,68. In our study, wound tissue specimens were sectioned and subjected to H&E staining (Fig. 7A). Remarkably, by day 7 post-treatment with OVA/EM/DOPs hydrogel, H&E staining sections revealed substantial wound improvement. By day 14, the stained sections of rat wound skin exhibited complete wound closure with well-organized tissue regeneration. Comparative analysis with control groups demonstrated that OVA/EM/DOPs hydrogel treatment yielded more physiologically normalized skin architecture, strongly suggesting its potent wound-healing properties. Furthermore, H&E staining was employed to assess potential systemic toxicity by examining major organs (Fig. S6). Histopathological evaluation of heart, liver, spleen, lung, and kidney tissues across all experimental groups showed no significant pathological alterations, indicating excellent biocompatibility of the hydrogel. This safety profile aligns with findings from other medical-grade hydrogels. For instance, Wang et al. developed a thermosensitive hydrogel encapsulating triptolide, known for its hepatotoxicity and nephrotoxicity, yet demonstrated no significant organ toxicity69. Similarly, recent advancements by Korean researchers in 2023 introduced injectable hydrogels incorporating camptothecin-loaded mesoporous silica nanoparticles for sustained cancer therapy, which also exhibited excellent organ biocompatibility70. These consistent findings across various hydrogel systems reinforce the safety profile of our OVA/EM/DOPs hydrogel for biomedical applications. Complementary Masson’s Trichrome staining (Fig. 7B) provided additional evidence of the hydrogel’s therapeutic efficacy, showing significantly enhanced collagen deposition in the OVA/EM/DOPs hydrogel-treated group compared to control group. This increased collagen synthesis further substantiates the superior wound-healing capacity of the developed hydrogel system. In conclusion, comprehensive histological evaluation through multiple staining techniques has demonstrated that the OVA/EM/DOPs hydrogel not only significantly accelerates wound healing but also maintains excellent biocompatibility with major organs, making it a promising candidate for advanced wound care applications.

Fig. 7
figure 7

H&E and Masson’s Trichrome staining. (A) H&E staining images of wound in the group of saline, HEGF hydrogel, OVA hydrogel, OVA/EM hydrogel and OVA/EM/DOPs hydrogel at day 7 and 14. Scale bar is shown in the figure. (B) Masson’s Trichrome staining images of wound for five experimental groups at day 7 and 14, and scale bar is shown in the figure.

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To further evaluate the wound-healing efficacy of the OVA/EM/DOPs composite hydrogel, immunofluorescence staining was conducted to assess the expression of platelet endothelial cell adhesion molecule-1 (CD31), vascular endothelial growth factor (VEGF), interleukin-10 (IL-10), and tumor necrosis factor-α (TNF-α) (Fig. 8A and B). CD31, a marker predominantly found in vascular endothelial cells, platelets, macrophages, Kupffer cells, granulocytes, lymphocytes, megakaryocytes, osteoclasts, and neutrophils71, was utilized to evaluate vascular regeneration in the wound area. VEGF, a specialized cytokine that exclusively regulates endothelial cell activity72, plays a dual role in inducing mesenchymal production and promoting vascular endothelial cell growth. Additionally, VEGF enhances vascular permeability, facilitating the deposition of plasma proteins in the extracellular matrix, thereby providing a provisional matrix for fibroblast and vascular endothelial cell proliferation73. Consequently, VEGF is widely used as a clinical indicator for monitoring angiogenesis. IL-10, a multifunctional cytokine derived from multiple cell types, regulates cell growth and differentiation and modulates inflammatory and immune responses. It is recognized as a key anti-inflammatory and immunosuppressive factor74. In this study, IL-10 levels were measured to assess the inflammatory status of wounds and reflect the progression of wound healing. TNF-α, primarily secreted by activated monocyte-macrophages, exhibits diverse biological functions, including tumor cell cytotoxicity, neutrophil phagocytosis enhancement, anti-infection activity, fever induction, acute-phase protein synthesis in hepatocytes, and promotion of myeloid leukemia cell differentiation into macrophages. As a pivotal inflammatory mediator, TNF-α is implicated in the pathogenesis of certain autoimmune diseases75. Thus, TNF-α serves as a critical biomarker for evaluating immune function, with significant implications for disease diagnosis, disease progression monitoring, treatment efficacy assessment, and cytokine therapy evaluation.

As illustrated in Fig. 8A, immunofluorescence staining revealed significantly higher expression of CD31 and VEGF in wounds treated with the OVA/EM/DOPs composite hydrogel compared to the saline control group. This finding suggests that the OVA/EM/DOPs composite hydrogel markedly enhances neovascularization and accelerates wound healing. These results align with previous studies. For instance, Lan et al. developed a self-pumping organo-hydrogel that significantly upregulated CD31 expression and promoted neovascularization compared to conventional hydrogels, homogeneous organo-hydrogels, or Tegaderm™76. Similarly, Chen et al. designed a composite hydrogel composed of ε-polylysine, calcium peroxide, and borosilicate glass to facilitate diabetic wound healing77. This hydrogel provided a sustained oxygen supply to the wound site, restoring normal cellular functions, enhancing CD31 expression, and promoting vascular regeneration. Quantitative analysis of CD31 and VEGF expression further confirmed that the OVA/EM/DOPs composite hydrogel significantly upregulated these markers, underscoring its exceptional wound-healing properties.

To investigate the inflammatory response in each experimental group, immunofluorescence staining of IL-10 and TNF-α was performed (Fig. 8A). Notably, minimal expression of IL-10 and TNF-α was observed in wounds treated with OVA hydrogel, OVA/EM hydrogel, or OVA/EM/DOPs hydrogel, indicating that these hydrogels promote wound healing while mitigating inflammation. Statistical analysis corroborated these findings, revealing negligible levels of IL-10 and TNF-α in wounds treated with OVA-based hydrogels (Fig. 8B). Consistent results were obtained from immunofluorescence staining of wound samples collected on day 7 (Fig. S7), which demonstrated elevated expression of CD31 and VEGF in wounds treated with OVA hydrogel, OVA/EM hydrogel, or OVA/EM/DOPs hydrogel. Additionally, minimal IL-10 and TNF-α expression was observed in wounds treated with the OVA/EM/DOPs composite hydrogel on day 7. These findings further validate that the OVA/EM/DOPs composite hydrogel significantly enhances wound healing while reducing inflammation. In the research, wet hydrogel (0.4 g/ml OVA, 100 µg/ml EM and 50 mg/ml DOPs) demonstrates excellent wound-healing promotion effects. In the future, clinical products will also be presented in the form of wet hydrogel rather than powder. Compared to powder form, the wet hydrogel is ready-to-use and more convenient.

Fig. 8
figure 8

Immunofluorescence staining at day 14. (A) CD31, VEGF, IL-10 and TNF-α immunofluorescence staining images of healed skin in the group of saline, HEGF hydrogel, OVA hydrogel, OVA/EM hydrogel and OVA/EM/DOPs hydrogel. Scale bar is 100 μm. (B) Statistical analysis of relative expression of CD31, VEGF, IL-10 and TNF-α for five experimental groups. “N.S.” means no significant differences and “*” represents P < 0.05, n = 3.

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Conclusion

In summary, this work developed a rapid and straightforward method for preparing a composite hydrogel based on OVA. The resulting OVA/EM/DOPs hydrogel demonstrated remarkable wound-healing properties at both cellular and animal levels, alongside effective antibacterial, anti-inflammatory, and pro-angiogenic activities. Specifically, in vitro analyses revealed that the OVA/EM/DOPs hydrogel exhibited excellent mechanical strength, antimicrobial efficacy, water retention, and regenerative capabilities. At the cellular level, the hydrogel showed high biocompatibility and significantly accelerated the closure of cellular scratch wounds. In animal experiments, the OVA/EM/DOPs hydrogel promoted the expression of CD31 and VEGF while reducing levels of IL-10 and TNF-α, thereby facilitating high-quality and rapid wound healing. The successful development of this composite hydrogel not only offers a cost-effective and efficient solution for wound repair but also broadens the application scope of dendrobium polysaccharide. This innovative approach holds great promise for providing a simple, low-cost, and high-quality strategy for accelerated wound healing.