Microenvironment response type hydrogel for accurately and sequentially repairing acute infectious wound

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to microenvironment response type hydrogel for accurately and sequentially repairing acute infectious wounds, which is prepared from the following components in proportion: 4% w / v of sodium alginate, 2% w / v of carboxymethyl chitosan, 0.5% w / v of tannic acid, 10mg / mL of zinc-doped bioglass and 10% w / v of sodium citrate as cross-linking agents. The structural characteristics of the hydrogel are dynamically adjusted by responding to local pH value changes, it is ensured that antibacterial tannic acid and tissue repair ions are released in good time, bacterial loads are effectively relieved, inflammatory response is adjusted, and oxidative stress is weakened. The two-phase mechanism can promote the optimal wound microenvironment, accelerate tissue regeneration and enhance the overall healing effect. Danging in-vitro and in-vivo evidences highlight the clinical potential of the microenvironment response type hydrogel as a next-generation wound dressing, and the microenvironment response type hydrogel can be used for realizing targeted and sequential repair of complex infectious wounds.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to a microenvironment-responsive hydrogel for precisely and sequentially repairing acute infectious wounds. Background Art

[0002] The skin, the largest organ in the human body, plays a vital role in defending against external physical, chemical, and biological threats, as well as maintaining fluid and electrolyte balance. However, as the outermost layer, the skin is particularly vulnerable to injury. Wounds are generally categorized as acute or chronic based on the length of their healing process. Acute, infected wounds are common in clinical practice, severely impacting patients' health and quality of life and posing significant challenges to their treatment.

[0003] Traditional wound dressings typically act as a passive barrier, covering the injury site and adhering to the surface of damaged tissue. However, these dressings have limited ability to control wound exudate, resulting in local moisture and temperature instabilities. Over time, their ability to absorb exudate and act as a protective barrier decreases, failing to continuously isolate external pathogens, thereby increasing the risk of infection. In addition, traditional dressings lack inherent antimicrobial properties, cannot actively inhibit microbial growth, and cannot provide the moist environment necessary for cell migration and new tissue formation. In addition, they lack the bioactive ingredients necessary to promote tissue repair, thereby hindering the healing process of infected wounds. Summary of the Invention

[0004] The purpose of the present invention is to provide a microenvironment-responsive hydrogel with a simple structure and reasonable design for accurately and sequentially repairing acute infectious wounds in order to solve the above problems.

[0005] The present invention achieves the above-mentioned purpose through the following technical solutions: A microenvironment-responsive hydrogel for precise sequential repair of acute infected wounds, comprising the following components in proportion: sodium alginate 4% w / v, carboxymethyl chitosan 2% w / v, tannic acid 0.5% w / v, zinc-doped bioglass 10 mg / mL, and sodium citrate 10% w / v as a cross-linker; the hydrogel forms an interpenetrating network structure through hydrogen bonding and hydrophobic interactions, can shrink and rapidly release tannic acid in an acidic environment, and expand and continuously release Zn in an alkaline environment. 2+ and Ca 2+ .

[0006] As a further optimized solution of the present invention, the molecular weight of the sodium alginate is 200,000, the molecular weight of the carboxymethyl chitosan is 100,000, and the degree of acetylation is ≥75%.

[0007] As a further optimization solution of the present invention, the zinc-doped bioglass is uniformly dispersed in the carboxymethyl chitosan solution by ultrasonic treatment at a frequency of 20-25 kHz and a power of 150 W for 60 minutes.

[0008] As a further optimization scheme of the present invention, the hydrogel releases ≥50% of tannic acid within 48 hours in an acidic environment of pH 5.5, and releases ≥30 mg / L of Ca in an alkaline environment of pH 7.4 within 48 hours. 2+ and ≥20 mg / L Zn 2+ .

[0009] A method for preparing a microenvironment-responsive hydrogel for precise sequential repair of acute infected wounds comprises the following steps: (1) Dissolve 4% w / v sodium alginate in distilled water and stir until completely dissolved; (2) Dissolve 2% w / v carboxymethyl chitosan in 1-2% v / v acetic acid to form a transparent solution; (3) Add 10 mg / mL zinc-doped bioglass to carboxymethyl chitosan solution and disperse evenly by ultrasonic treatment; (4) Mix the sodium alginate solution and carboxymethyl chitosan solution in a volume ratio of 2:1 and stir for two minutes; (5) Add 0.5% w / v tannic acid and stir evenly, then add 10% w / v sodium citrate for cross-linking to form a three-dimensional network hydrogel at room temperature.

[0010] The beneficial effects of the present invention are: 1. The hydrogel dynamically adjusts its structural properties in response to local pH changes, ensuring the timely release of antimicrobial tannins and tissue-repairing ions, effectively reducing bacterial load, modulating inflammatory responses, and attenuating oxidative stress. This biphasic mechanism promotes an optimal wound microenvironment, accelerates tissue regeneration, and enhances overall healing. Encouraging in vitro and in vivo evidence highlights the clinical potential of this microenvironment-responsive hydrogel as a next-generation wound dressing for targeted, sequential repair of complex infected wounds.

[0011] 2. Tannic acid (TA) is quickly released in an acidic environment, effectively inhibiting bacterial growth and biofilm formation.

[0012] 3. Zinc ion (Zn 2+ ) and calcium ions (Ca 2+ ) is continuously released in an alkaline environment, promoting angiogenesis, cell proliferation and tissue remodeling.

[0013] 4. The hydrogel has good cell compatibility and blood compatibility and will not cause hemolysis or cytotoxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Schematic diagram of the preparation process and pH-responsive release of the hydrogel of the present invention; Figure 2 is a characterization diagram of the SA / CMSC-based hydrogel with tannic acid and zinc-doped bioglass added in the present invention; Figure 3 is a graph showing the in vitro biocompatibility of the SA / CMSC / TA / BAG hydrogel of the present invention; Figure 4 This is a graph showing the in vitro antibacterial evaluation of the SA / CMSC / TA / BAG hydrogel of the present invention; Figure 5 This is a graph evaluating the in vitro immunomodulatory properties of the SA / CMSC / TA / BAG hydrogel of the present invention; Figure 6 This is a diagram of the wound healing state in the in vivo experiment of the present invention; Figure 7 It is the histological and immunofluorescence analysis diagram of the tissue section of the present invention. DETAILED DESCRIPTION

[0015] The present application is described in further detail below in conjunction with the accompanying drawings. It is necessary to point out that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technicians in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0016] Example: Figure 1 As shown, a microenvironment-responsive hydrogel for precise sequential repair of acute infected wounds is constructed. It is composed of an interpenetrating network of sodium alginate (SA) and carboxymethyl chitosan (CMSC), with tannic acid and zinc-doped bioglass bound together by hydrogen bonding and hydrophobic interactions. Under typical acidic conditions of infection, the carboxyl groups of SA are protonated, while the enhanced hydrogen bonding between the -COOH and -OH groups in CMSC causes the network to contract, promoting the rapid release of tannic acid, which has strong antimicrobial properties. During the healing phase, as the pH shifts to a weak alkaline state, the hydrogel swells, absorbing exudate and promoting the release of growth factors that aid tissue repair. Furthermore, the sustained release of zinc and calcium ions promotes angiogenesis and provides anti-inflammatory effects, thereby accelerating wound healing.

[0017] Reagents including carboxymethyl chitosan (CMSC, molecular weight approximately 100,000, degree of acetylation ≥75%), sodium alginate (SA, molecular weight approximately 200,000), tannic acid (TA, molecular weight approximately 1,701.2), and Bioglass® 45S5 (zinc-modified) were purchased from Sigma-Aldrich (Darmstadt, Germany). Sodium citrate (analytical grade) was obtained from the same supplier and used for solution preparation. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, trypsin-EDTA (0.25%), and phosphate-buffered saline (PBS) were purchased from Gibco (Grand Island, NY, USA). Antibodies against vascular endothelial growth factor (VEGF), CD86, CD206, inducible nitric oxide synthase (iNOS), arginase-1, and F4 / 80 were provided by Proteintech (Wuhan) and Santa Cruz Biotechnology (USA). Cell counting kit-8 (CCK-8) was purchased from Biosharp (Hefei), and 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was purchased from Solarbio (Beijing). All other reagents were purchased from China National Pharmaceutical Group Corporation (Shanghai). Deionized water was used for all solutions and washes. Hydrogel construction: Sodium alginate (SA) / carboxymethyl chitosan (CMSC) hydrogel was prepared using tannic acid (TA) and bioglass (BAG) as functional agents ( Figure 1 A). Sodium alginate (4% w / v) was dissolved in distilled water and stirred until completely dissolved. Chitosan (2% w / v) was dissolved in dilute acetic acid (1-2% v / v) to form a transparent uniform solution. Bioglass (10 mg / mL) was added to the chitosan (CMSC) solution and ultrasonically treated at a frequency of 20–25 kHz and a power of 150 W for 60 minutes to ensure that BAG was fully dispersed in the solution. Sodium alginate and treated chitosan solution were mixed in a ratio of 2:1 and stirred continuously at room temperature for 2 minutes. Tannic acid (0.5% w / v) was then added to the mixed solution and stirred evenly. Finally, sodium citrate (10% w / v) was added as a crosslinker. Finally, under the weakly acidic environment provided by sodium citrate, sodium alginate and chitosan formed a stable three-dimensional cross-linked hydrogel network at room temperature through enhanced hydrogen bonding and electrostatic interactions.

[0018] To evaluate the effect of tannic acid (TA) in hydrogel and zinc (Zn) in bioglass (BAG) 2+ ) and calcium (Ca 2+) ions, the prepared samples were incubated in buffer solutions of pH 5.5 and pH 7.4. These pH conditions were chosen to replicate different physiological environments: pH 5.5 simulates the acidic microenvironment typically associated with infected or inflamed tissue, where bacterial activity and metabolic byproducts lower the pH value, while pH 7.4 corresponds to the neutral physiological environment of healthy tissue in the late stage of wound healing or in a non-infected state. The hydrogel samples were cut into pieces of 10 mm × 10 mm × 2 mm and placed in 10 mL of buffer solution in a centrifuge tube. These samples were incubated at 37°C in a thermostatic shaker. 1 mL of solution was withdrawn at specific time intervals (0, 3, 5, 7, 9, 11 and 14 days) and replaced with fresh buffer. The collected solutions were then analyzed using inductively coupled plasma mass spectrometry (ICP-MS) or atomic absorption spectroscopy (AAS) to quantify Zn 2+ and Ca 2+ The cumulative ion release at each time point was calculated, and release curves were generated to compare the ion release characteristics of the hydrogels under acidic and neutral pH conditions.

[0019] Wound healing assay: The effect of hydrogel extract on the migration of human umbilical vein endothelial cells (HUVEC) was studied using a scratch test. HUVEC were plated at 5×10 4 Cells were seeded at a density of 1000 μL in a 6-well plate and cultured to form a confluent monolayer. A 1000 μL pipette tip was then used to vertically scratch the cell layer. After discarding the culture medium, the plate was rinsed with phosphate-buffered saline (PBS) to remove cell debris. The experimental group used complete culture medium containing different hydrogel extracts, while the control group used culture medium without any hydrogel extracts. Images of the scratch area were taken at 0 and 24 hours using an inverted microscope. The scratch closure area was measured, and the cell migration rate was calculated using ImageJ software.

[0020] Cytoskeleton staining: The cytoskeleton and cell nucleus were fluorescently stained to evaluate the effect of hydrogel extract on cell viability and morphology. L929 cells were cultured at 4×10 5 Cells were seeded at a specific density in 12-well culture plates. After an 8-hour incubation period to allow for cell adhesion, the culture medium was replaced with the hydrogel extract corresponding to the experimental group. The cells were then incubated for an additional 24 hours under standard culture conditions. Following incubation, the cells were stained according to the manufacturer's instructions to simultaneously visualize the cytoskeleton and nuclei. This method allows for qualitative and quantitative analysis of the effects of the hydrogel extract on cell morphology and viability.

[0021] Bacterial live / dead staining assay: To evaluate cytotoxicity and cell viability, HUVEC treated with hydrogel extracts were double stained with Calcein-AM / PI. 4Cells were seeded at a specific density in 24-well plates and allowed to adhere. After attachment, the cells were treated with hydrogel extracts from different experimental groups for 24 hours. Subsequently, a staining solution containing propidium iodide (PI) and calcein-AM was applied, and the cells were incubated in the dark at room temperature for 30 minutes. Using a fluorescence microscope, fluorescent images of live cells (stained green with calcein-AM) and dead cells (stained red with PI) were acquired. ImageJ software was used to quantify the number of live and dead cells, and cell viability and cytotoxicity were calculated. This analysis provided a comprehensive assessment of the biocompatibility of the hydrogel extracts.

[0022] In vitro antibacterial performance: To evaluate the inhibitory effect of the hydrogel on bacterial biofilm formation, Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were cultured in LB broth at 37°C and shaken at 220 rpm until they reached the logarithmic growth phase. 7 CFU / mL) were inoculated onto glass coverslips placed in 24-well plates and incubated at 37°C for 24 hours to allow mature biofilms to form. After 48 hours, the bacterial culture medium was replaced with hydrogel extracts from different experimental groups: PBS (control), SA / CMSC, SA / CMSC / TA, SA / CMSC / BAG, and SA / CMSC / TA / BAG. The samples were then incubated at 37°C for another 12 hours. To observe biofilm formation and assess bacterial viability, we used confocal laser scanning microscopy (CLSM, Zeiss LSM880) to perform three-dimensional (3D) imaging of the biofilms, which were first stained with DMAO (green, indicating live bacteria) and EthD-III (red, indicating dead bacteria). Biofilm thickness and bacterial viability were analyzed, and the live / dead bacteria ratio was quantified using imaging software; To further evaluate the bacterial viability after hydrogel treatment, Escherichia coli and Staphylococcus aureus were cultured in LB broth at 37°C and 220 rpm to the logarithmic growth phase. The culture medium in each well was then replaced with the hydrogel extract of each experimental group. Subsequently, the samples were incubated at 37°C for another 12 hours. After co-culture, the bacteria were collected and stained using a bacterial live / dead staining kit according to the manufacturer's instructions. Live bacteria were stained with DMAO, and dead bacteria were labeled with EthD-II dye. Bacterial viability was then observed using a confocal laser scanning microscope (CLSM, Olympus FV1000), and the antibacterial activity of the hydrogel was quantified based on the ratio of live to dead bacteria. This analysis can be used to comparatively evaluate the antibacterial effect of the hydrogel; To evaluate the antibacterial efficacy of the hydrogel extracts, suspensions of Escherichia coli and Staphylococcus aureus treated with the hydrogel extracts were diluted tenfold. 100 µL of each dilution was plated onto LB agar plates. The plates were incubated at 37°C for 12 hours, and then colony-forming units (CFU) were counted. The CFU counts for each group were used to evaluate the antibacterial properties of the hydrogels and to compare the antibacterial effects of different formulations. Evaluation of anti-inflammatory effects: Immunofluorescence staining was used to assess the expression of inflammatory markers (CD86 and Arginase-1) in cells treated with the hydrogel extract. Cells were seeded on sterile glass coverslips in 24-well plates and incubated until confluence reached 70%-80%. After 24 hours of incubation with the hydrogel extract, cells were fixed with 4% paraformaldehyde at room temperature, permeabilized with 0.1% Triton X-100, and blocked with 5% bovine serum albumin (BSA) for 1 hour. Specific primary antibodies against CD86, CD206, and iNOS were added (1:200 dilution) and incubated overnight at 4°C. After washing with PBS, cells were incubated with Alexa Fluor 488-conjugated secondary antibodies (1:500 dilution) for 1 hour at room temperature in the dark. Cell nuclei were counterstained with DAPI for 5 minutes. Immunofluorescence images were acquired using a confocal laser scanning microscope (CLSM, Zeiss LSM880), and fluorescence intensity was quantified using ImageJ software. This allowed assessment of the expression levels of these inflammatory markers and provided insights into the potential of the hydrogel to modulate immune responses; Western blotting was used to quantify the expression of inflammatory proteins (CD86, CD206, Arg-1, and iNOS) in cell samples treated with hydrogel extracts. After 24 hours of incubation, total proteins were extracted with RIPA buffer and subjected to SDS-PAGE electrophoresis. Proteins were then transferred to a PVDF membrane and incubated with primary antibodies specific for CD86, CD206, Arg-1, and iNOS (1:1000 dilution) overnight at 4°C. The membrane was then treated with an HRP-conjugated secondary antibody (1:5000 dilution) for one hour at room temperature. Protein bands were visualized using a chemiluminescence detection system and detected using enhanced chemiluminescence (ECL) reagent. Band intensities were quantified using ImageJ software. Simultaneous immunofluorescence staining was performed to further validate the anti-inflammatory effects of the hydrogel and confirm its potential to modulate inflammatory responses.

[0023] Evaluation of antioxidant capacity: Flow cytometry was used to evaluate the effect of hydrogel extracts on intracellular reactive oxygen species (ROS) levels. Cells were exposed to hydrogel extracts for 24 hours and then incubated with the ROS-sensitive fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA) at 37°C in the dark for 30 minutes. After incubation, the cells were washed with PBS to remove excess probes, and then the cells were collected for analysis. The fluorescence intensity of oxidized DCFH-DA is proportional to the ROS level and can be quantitatively analyzed using flow cytometry. The data were analyzed to compare the ROS generation between the treated and untreated groups to quantitatively evaluate the antioxidant properties of the hydrogels.

[0024] In vitro angiogenesis assay: Tube formation assay was performed using human umbilical vein endothelial cells (HUVEC) to evaluate the potential angiogenesis-promoting effects of the hydrogel. Matrigel (Corning, USA) was thawed overnight at 4°C, and 50 μL Matrigel was dispensed into each well of a 96-well plate and then incubated at 37°C for 30 minutes to solidify. HUVEC were cultured at 2×10 4 After seeding onto solidified Matrigel layers at a density of 100 cells / mL, cells were cultured under various conditions, including control groups and experimental groups treated with hydrogel extracts. The cells were incubated at 37°C in an atmosphere of 5% CO2 for 6 hours. During incubation, the cells were stained with Calcein-AM, and the formation of tubular structures was observed using an inverted fluorescence microscope (Zeiss AxioObserver A1, Zeiss, Germany). Key parameters of angiogenesis, including total branch length, number of branch points, and mesh count, were analyzed using the "Angiogenesis Analyzer" plugin in ImageJ software (National Institutes of Health). Each experiment was repeated three times, and the results are presented as the mean ± standard deviation (SD). Statistical analysis was performed to determine data significance.

[0025] Hemolysis Assay: To evaluate the hemolytic potential of the hydrogel, a red blood cell (RBC) hemolysis assay was performed. Whole blood was collected from healthy Sprague-Dawley rats (purchased from the Experimental Animal Center of Shantou University Medical College) and placed in EDTA-coated tubes. The blood was centrifuged at 1500 rpm for 10 minutes at 4°C to separate RBCs. The blood was then washed three times with PBS (pH 7.4) and diluted to a 2% (v / v) RBC suspension. Both the experimental and hydrogel extract-treated groups were accompanied by a positive control (0.1% Triton X-100 for complete hemolysis) and a negative control (PBS). The hydrogel extract was prepared by incubating the hydrogel in PBS at 37°C for 24 hours. 200 µL of the reaction solution was added to a 96-well plate, incubated at 37°C for 1 hour, and centrifuged at 1500 rpm for 5 minutes at 4°C. The absorbance of the supernatant was measured at 540 nm using a microplate reader (Thermo Fisher Scientific, USA). The hemolysis rate was calculated as follows: hemolysis rate (%) = [(OD_sample−OD_PBS) / (OD_Triton−OD_PBS)] × 100%; where ODsample is the absorbance of the sample group, ODPBS is the absorbance of the negative control (hemolysis baseline), and ODTriton is the absorbance of the positive control (hemolysis 100%).

[0026] All experiments were performed in triplicate, and the results are expressed as mean ± standard deviation (SD). A hemolysis rate of less than 5% was considered to be inactive; In vivo full-thickness skin defect rat model: To evaluate the therapeutic effects of the hydrogel on wound healing, a full-thickness skin defect model was established using male Sprague-Dawley (SD) rats (8 weeks old). Rats were housed under standard conditions and acclimated for one week before the experiment. Preoperatively, rats were put to sleep by intraperitoneal injection of sodium pentobarbital (50 mg / kg). After shaving and cleaning the dorsal area with iodine solution, a 10 mm diameter full-thickness skin defect was created using a biopsy punch. The animals were randomly divided into five groups of three each: a blank control group (PBS-treated) and four experimental groups treated with SA / CMSC, SA / CMSC / TA, SA / CMSC / BAG, and SA / CMSC / TA / BAG hydrogels, respectively. Equal amounts of each hydrogel were evenly applied to the wound site and covered with a breathable dressing. Wound photographs were taken on days 0, 3, 5, 7, 9, 11, and 14 postoperatively to monitor changes in wound area. Wound closure rate was calculated using ImageJ software (National Institutes of Health) according to the following formula: Wound closure rate (%) = [(initial wound area − current wound area) / initial wound area] × 100%.

[0027] Initial wound area (Initial Wound Area) refers to the wound area measured immediately after injury (day 0), and current wound area (Current Wound Area) refers to the wound area at subsequent time points. Rats were sacrificed on day 14, and wound tissue was excised and preserved in 4% paraformaldehyde, embedded in paraffin, and sectioned. Histological analysis and collagen deposition were performed using Masson's trichrome and hematoxylin and eosin (H&E) staining. Tissue sections were observed under an inverted microscope.

[0028] Statistical Analysis: All experimental data are presented as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 9.0 (GraphPad Software, USA). Pairwise comparisons were performed using an unpaired two-tailed Student's t-test, and comparisons between multiple groups were performed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. A p-value of less than 0.05 (P < 3C < 0.05) was considered statistically significant. Graphs were generated to visualize the data, and the statistical tests used are indicated in the figure legends.

[0029] like Figure 2 Shown: Preparation and characterization of hydrogel: To cooperate with the physiological processes of infection control and wound healing, a pH-responsive hydrogel was developed by utilizing the electrostatic interaction between the amino groups of carboxymethyl chitosan (CMSC) and the carboxyl groups of sodium alginate (SA). FTIR spectrum of SA / CMSC / TA / BAG hydrogel ( Figure 2 A) displays several characteristic absorption peaks. Stretching vibrations of amino (-NH2) and hydroxyl (-OH) groups were identified as the cause of the large peak at approximately 3250 cm⁻¹, reflecting the interaction between chitosan (CS) and tannic acid (TA). Furthermore, prominent peaks around 1600 cm⁻¹ correspond to the C=O stretching vibrations of carboxyl (-COOH) groups and the amide I band, indicating cross-linking between the carboxyl groups of SA and the amino groups of CS, forming the hydrogel network. Peaks in the 1000–1100 cm⁻¹ range correspond to C-O-C stretching vibrations, further confirming the cross-linked polymer structure of SA and CS. Furthermore, a peak around 500 cm⁻¹ is attributed to the bending vibrations of Si-O-Si bonds, indicating that bioglass (BAG) has been successfully incorporated into the hydrogel matrix. These spectral features confirm the chemical functionality and structural stability of the hydrogel.

[0030] The chemical composition of the hydrogel was further investigated using XPS, and its chemical structure was analyzed ( Figure 2B). The C1s signal observed at 284.8 eV indicates the presence of C–C / C–H bonds, which is a common feature of all organic components. The peaks at 286.5 eV and 288.2 eV are attributed to C–O and O=C–N bonds, respectively, confirming the presence of tannic acid (TA) and chitosan (CS). In addition, the peaks of Zn2p and Ca2p were observed at 1020–1045 eV and 346–352 eV, respectively, which are the Zn2p and Ca2p peaks in bioglass (BAG). 2+ ) and calcium ions (Ca 2+ ), bioglass is known for its antibacterial and angiogenic properties.

[0031] Further analysis of the XPSN1s spectrum ( Figure 2 E) Peaks at approximately 399.5 eV and 400.5 eV were found, corresponding to free amino groups (-NH2 / -NH3⁺) and amide bonds (-CONH2), respectively. These peaks indicate that the carboxyl groups of SA and the amino groups of CMSC cross-link to form a stable hydrogel network. Notably, less than 50% of the amino groups in chitosan were protonated, indicating the presence of a mixture of protonated and deprotonated chitosan during the binding process. Chemical shifts observed in the N1s spectrum support the presence of hydrogen bonding and electrostatic interactions between TA and CS, as well as possible weak interactions with the bioglass.

[0032] C1s spectrum ( Figure 2 D) Peaks appear at 284.8 eV (CC / CH) and 286.5 eV (CN / CO), indicating the existence of potential interactions between CMSC and TA, such as hydrogen bonding or electrostatic forces. O1s spectrum ( Figure 2 F) Peaks appear at 531.0 eV and 532.5 eV, which are associated with carbonyl (C=O) and hydroxyl (-OH) or siloxane (Si-O) bonds, indicating that BAG has been successfully incorporated into the composite.

[0033] XRD analysis of SA / CMSC / TA / BAG group ( Figure 2 C) shows a broad diffraction peak around 20°–30°, consistent with the amorphous nature of the hydrogel. In contrast, the SA / CMSC / BAG group exhibits a weaker diffraction peak in the 2θ range of 30°–35°, confirming the integration of the BAG microcrystalline structure into the hydrogel matrix. The amorphous nature of the hydrogel enables it to maintain high flexibility and processability.

[0034] SEM images of the composite hydrogel ( Figure 2G) reveals significant microstructural changes, further demonstrating the successful integration of the individual components into the SA / CMSC / TA / BAG system. The SA / CMSC hydrogel exhibits a porous structure, which facilitates biocompatibility and promotes nutrient exchange. The addition of tannic acid (TA) results in a denser surface morphology, indicating stronger intermolecular interactions. The presence of bioactive glass (BAG) introduces a particulate feature to the surface, enhancing the hydrogel's rigidity and providing active sites for bioactivities such as ion release and cellular interactions.

[0035] Both SA / CMSC / BAG and SA / CMSC / TA / BAG exhibited a granular surface morphology, highlighting the influence of BAG on the hydrogel structure. These granular inclusions likely facilitate the formation of micropores, thereby increasing the surface area and enhancing the release of bioactive ions. The synergistic effect of TA and BAG enhanced the mechanical strength and bioactivity of the hydrogel, making it conducive to cell adhesion, proliferation, and nutrient exchange.

[0036] Energy dispersive X-ray spectroscopy (EDS) Figure 2 H) Further analysis reveals the uniform distribution of organic (C, N, O) and inorganic (Ca, Si, Zn) elements within the SA / CMSC / TA / BAG hydrogel. This distribution indicates that TA and BAG have successfully integrated into a stable network structure, with the SA / CMSC / TA / BAG hydrogel exhibiting the most complex elemental distribution. The uniform dispersion of Ca, Si, and Zn throughout the hydrogel matrix highlights the synergistic effect of TA and BAG, significantly enhancing the mechanical properties and multifunctional bioactivity of the material.

[0037] To optimize the hydrogel's response to continuous physiological processes, we evaluated its drug release behavior under different pH conditions. At pH 5.5 (simulating the acidic environment of infected wounds) and pH 7.4 (simulating the slightly alkaline environment of healing tissue), the hydrogel exhibited a distinct phased release profile (Figures L and M).

[0038] In an acidic environment (pH 5.5), the hydrogel rapidly releases tannic acid (TA), with a cumulative release of approximately 50% within the first two days. By day 8, the release rate slowed to nearly 85%, ultimately reaching nearly 100% by day 14. The rapid release of TA is attributed to its structural characteristics, as its phenolic hydroxyl (-OH) groups exhibit increased solubility under acidic conditions due to reduced ionization. Furthermore, protonation of the carboxyl (-COOH) groups on the sodium alginate (SA) chains enhances hydrogen bonding with chitosan (CMSC) through -COOH and -OH interactions, leading to contraction of the hydrogel network and accelerated TA release. This behavior is consistent with the early, acute inflammatory stage of infected wounds, when effective pathogen inhibition is crucial.

[0039] In contrast, at pH 5.5, the zinc ions (Zn 2+ ) and calcium ions (Ca 2+ ) release is minimal, ensuring that the hydrogel's primary function during this phase remains antimicrobial, rather than repair. This design strategy effectively prioritizes infection control while retaining bioactive ions for release during the tissue repair phase at pH 7.4. These phase-specific release profiles highlight the hydrogel's potential to provide a personalized therapeutic response throughout the wound healing process.

[0040] At pH 5.5 and pH 7.5, the calcium ions (Ca 2+ ) and zinc ions (Zn 2+ ) showed significant differences in the release patterns of calcium ions (Figures L and M). In an acidic environment of pH 5.5, calcium ion release was relatively slow, reaching concentrations of approximately 10 mg / L in the first two days. Over the next 14 days, concentrations gradually increased to approximately 25 mg / L. In contrast, at pH 7.5, calcium ion release accelerated significantly, reaching nearly 30 mg / L within 48 hours and maintaining high release levels for several days thereafter. This suggests that calcium ion release is significantly affected by pH, with faster release under alkaline conditions supporting endothelial cell activation and angiogenesis during wound healing.

[0041] Similarly, zinc ion release showed a similar pH-dependent trend. In an acidic environment, zinc ion release was relatively low, reaching approximately 15 mg / L within 14 days, but under alkaline conditions, zinc ion release was significantly enhanced, reaching approximately 20 mg / L within two days and continuing to increase thereafter. This accelerated release is consistent with the key role of zinc ions in promoting fibroblast proliferation and inhibiting microbial growth, thereby creating a favorable microenvironment for tissue repair.

[0042] Wounds typically undergo gradual changes in pH during the healing process. Due to inflammation and tissue damage, acute wounds are initially acidic (pH 5.5-6.5). As healing progresses, tissue regeneration and repair are typically accompanied by an increase in pH, gradually moving toward a neutral or slightly alkaline environment (pH 7.0-7.5). This natural shift in pH supports cell proliferation and tissue remodeling.

[0043] In the early stages, tannic acid (TA) exerts its antimicrobial effect by binding to bacterial cell wall proteins, disrupting their structure and inhibiting microbial growth. Despite its acidic nature, TA's antimicrobial activity does not significantly alter the overall pH of the wound. By reducing the bacterial load and its associated acidic byproducts, TA indirectly contributes to an increase in wound pH. This pH-responsive release mechanism ensures that TA exerts a potent antimicrobial effect during the early, acute inflammatory phase, preventing excessive pathogen proliferation without creating an environment that is overly favorable for bacterial survival.

[0044] To optimize the bioactivity of bioglass, its composition can be adjusted by reducing the SiO2 content and increasing the ZnO and CaO content. This modification weakens the silicate network, causing it to dissolve and release ions more quickly in an alkaline environment. 2+ and Ca 2+ The release of ions promotes angiogenesis, enhances cell migration, and supports tissue repair, thereby accelerating wound healing and providing sustained repair support.

[0045] Based on the above, although the release of TA may lead to local acidification, the pH-responsive hydrogel is designed to maintain its antimicrobial efficacy while preventing conditions that are overly favorable for bacterial growth. This balanced approach effectively meets the needs of controlling infection and promoting wound healing.

[0046] like Figure 3 To determine the optimal concentrations of tannic acid (TA) and bioglass (BAG) in sodium alginate-chitosan hydrogels, the CCK-8 assay was used to assess cell viability and proliferation. The results showed that BAG was most effective at 10 mg / mL, while the most effective TA concentration was 0.5% (w / v). Figure 2 I). At a 0.5% TA concentration, cell proliferation and viability were significantly enhanced, indicating that the antioxidant and antibacterial properties of TA are optimally expressed at this level. It also promoted cell adhesion and proliferation within the hydrogel matrix. Conversely, TA concentrations below or above 0.5% resulted in reduced cell viability. Low concentrations of TA were insufficient to produce the desired biological effects, while higher concentrations exhibited cytotoxicity, disrupting the cellular microenvironment and hindering normal cell growth. Similarly, BAG exhibited the highest bioactivity at a concentration of 10 mg / mL, where the release of bioactive ions was optimal for promoting cell proliferation and differentiation. Concentrations below this level resulted in insufficient ion release, thereby reducing cell viability; whereas excessively high BAG concentrations overstimulated the cellular environment and compromised the mechanical integrity of the hydrogel. Therefore, the combination of 0.5% TA and 10 mg / mL BAG optimized cell viability and proliferation without compromising the structure or mechanical properties of the hydrogel.

[0047] The biocompatibility of the hydrosol was further evaluated using an in vitro scratch test, and the SA / CMSC / TA / BAG formulation showed significant scratch closure within 24 hours ( Figure 3A) highlights the hydrogel's ability to enhance HUVEC cell migration and proliferation. As a three-dimensional scaffold, the hydrogel provides the necessary physical support for cell adhesion and migration, mimicking the structural characteristics of the extracellular matrix (ECM) and creating an environment conducive to cell growth. The addition of TA to the hydrogel further supports this microenvironment through its potent antioxidant and anti-inflammatory properties, thereby reducing levels of reactive oxygen species (ROS) and inflammatory cytokines. This regulation promotes an environment conducive to cell proliferation. TA can also modulate signaling pathways such as PI3K / Akt, further enhancing cell growth and migration.

[0048] BAG also releases calcium ions (Ca 2+ ) and zinc ions (Zn 2+ ) plays a crucial role in promoting cell proliferation. Calcium ions are crucial for cell signaling, influencing cell cycle progression and division; zinc ions, through their antioxidant and anti-inflammatory effects and involvement in protein synthesis, play a crucial role in cell growth. Furthermore, ions released by BAGs support angiogenesis, enhancing the delivery of nutrients and oxygen to regenerating tissues, thereby accelerating tissue repair.

[0049] In the cytoskeleton staining experiment ( Figure 3 B) Compared with the control group, cells in the SA / CMSC / TA / BAG group showed more organized and intact F-actin structure (FITC staining, green). The cytoskeleton structure of this group was well-organized and tightly arranged, indicating that the hydrogel significantly enhanced the integrity of the cytoskeleton, thereby improving the mechanical properties and migration ability of the cells. Cytoskeleton stabilization and improved cell-matrix adhesion are essential for effective cell migration. TA contributed by reducing inflammation and reducing ROS levels, which helped maintain the integrity of the cytoskeleton. In addition, calcium ions (Ca) released by BAG 2+ ) plays a key role in intracellular signaling, promoting cytoskeletal remodeling and enhancing cell migration.

[0050] In the live / dead cell assay, minimal cytotoxicity was observed in all experimental groups, with almost all cells alive (green fluorescence from Calcein-AM staining) and very few dead cells detected (red fluorescence from PI staining) ( Figure 3 B). These results confirm the excellent biocompatibility of the hydrogel with HUVEC cells. Specifically, the SA / CMSC / TA / BAG group exhibited high cell viability, while also promoting cell migration and cytoskeletal remodeling. Overall, the results demonstrate that the SA / CMSC / TA / BAG hydrogel exhibits excellent biocompatibility, as demonstrated by enhanced cell viability and minimal cytotoxicity in all assays, making it highly suitable for biomedical applications, particularly in wound healing and tissue regeneration.

[0051] like Figure 4 As shown: In order to evaluate whether SA / CMSC / TA / BAG hydrogel meets the antibacterial requirements and effectively destroys the formation of biofilm, we further studied the mechanism of hydrogel killing bacteria.

[0052] The antibacterial properties of the hydrogels were evaluated by various methods, including confocal laser scanning microscopy ( Figure 4 A and Figure 4 B) Live / dead bacterial staining ( Figure 4 C and Figure 4 D) and colony forming unit (CFU) assay ( Figure 4 E and Figure 4 F). Across all experiments, the control group consistently demonstrated robust, intact biofilms with high bacterial viability, as evidenced by strong green fluorescence under microscopy, minimal red fluorescence in live / dead bacterial staining, and numerous bacterial colonies in CFU assays. The SA / CMSC hydrogel exhibited limited antibacterial activity, with only slight reductions in biofilm density, green fluorescence, and CFU counts, indicating that the hydrogel matrix alone lacked sufficient antibacterial efficacy.

[0053] When tannic acid (TA) was added to the hydrogel (SA / CMSC / TA), the antibacterial activity was significantly enhanced in all tested methods. This included an increase in red fluorescence in live / dead staining, a decrease in green fluorescence under microscopy, and a significant decrease in CFU counts. The enhanced antibacterial effect can be attributed to the multifaceted mechanism of TA, which can disrupt bacterial membranes and inhibit bacterial survival. The polyphenolic structure of TA enables it to form hydrogen bonds with bacterial membrane proteins and phospholipids, causing the membrane to become unstable and intracellular substances such as proteins and ions to leak out, ultimately leading to bacterial cell death. In addition, TA chelates essential metal ions such as Fe 2+ and Zn 2+ , depriving bacteria of key nutrients required for metabolism and growth. TA also induces oxidative stress by producing reactive oxygen species (ROS), which damages bacterial DNA, proteins, and lipids. In addition, TA interferes with bacterial adhesion and hinders the establishment and persistence of biofilms. Notably, the antibacterial activity of TA is enhanced under the acidic conditions of biofilms (pH 4.0-6.5), where the protons released by the phenolic groups of TA enhance its interaction with bacterial membranes, thereby enhancing its bactericidal effect. This pH-dependent behavior explains why TA exhibits stronger antibacterial activity than BAG under the same experimental conditions.

[0054] SA / CMSC / BAG hydrogel also showed significant antibacterial effect, with an increase in bacterial cell death rate and a decrease in colony forming unit (CFU) count compared with the control group. 2+) exerts its antimicrobial effects, with zinc ions interfering with bacterial enzymatic processes, disrupting membrane integrity, and inhibiting biofilm formation. However, in the acidic environment of biofilms, BAG's antimicrobial performance was slightly lower than TA, indicating that pH plays an important role in regulating the relative effectiveness of these materials. TA's ability to release protons under acidic conditions provides it with a distinct advantage, enabling it to outperform BAG in this setting.

[0055] Among all formulations, the SA / CMSC / TA / BAG hydrogel exhibited the strongest antimicrobial performance, with minimal bacterial viability detected in all tests. This exceptional efficacy was primarily attributed to the potent antimicrobial mechanism of TA, particularly at low pH. These results highlight the critical role of the biofilm microenvironment in influencing the antimicrobial efficacy of hydrogels, with TA emerging as the most effective agent in hydrogel systems against biofilm-associated infections.

[0056] Inflammation is a crucial component of the wound healing process, recruiting immune cells such as neutrophils and macrophages to help clear pathogens, cellular debris, and damaged tissue. However, if inflammation persists or becomes excessive, it can impede healing, leading to delayed cell proliferation, prolonged tissue damage, and hindering subsequent healing phases, including proliferation and remodeling. Furthermore, excessive production of reactive oxygen species (ROS) and proinflammatory cytokines such as IL-6 and TNF-α is associated with chronic inflammation, which exacerbates tissue damage and prolongs the inflammatory phase. The addition of anti-inflammatory agents, particularly in advanced hydrogel formulations, can help modulate the inflammatory response by reducing the overproduction of inflammatory mediators, promoting macrophage polarization toward a pro-repair M2 phenotype, and scavenging reactive oxygen species (ROS). This controlled regulation of the immune response promotes effective tissue repair, helps prevent chronic inflammation, and reduces the risk of complications such as scarring and non-healing wounds.

[0057] like Figure 5 As shown in our anti-inflammatory experiments, immunofluorescence analysis showed that SA / CMSC / TA / BAG hydrogels exhibited significant anti-inflammatory effects. Specifically, the expression of pro-inflammatory M1 macrophage-related marker CD86 ( Figure 5 A), indicating effective inhibition of M1 macrophage activation. In contrast, the same hydrogel formulation showed significantly higher expression levels of CD206 and arginase-1 (Arg-1), both markers of M2 macrophages, indicating a strong promotion of M2 polarization ( Figure 5 B). Western blot analysis ( Figure 5I) These results were further validated, demonstrating significant downregulation of iNOS and CD86, and increased expression of CD206 and Arg-1, highlighting the ability of the hydrogel to inhibit M1 macrophage activation and enhance M2 macrophage polarization. These results are consistent with the immunofluorescence results.

[0058] The anti-inflammatory properties of the SA / CMSC / TA / BAG hydrogel are primarily attributed to the action of tannic acid (TA). TA acts as a potent antioxidant by scavenging ROS and blocking proinflammatory signaling pathways, including the NF-κB pathway, which triggers the release of proinflammatory cytokines such as TNF-α and IL-6. This enables TA to reduce oxidative stress and inflammation while also preventing the activation of M1 macrophages. Furthermore, TA promotes a microenvironment conducive to tissue repair by disrupting the oxidative stress cascade and indirectly promoting M2 macrophage polarization. While calcium and zinc ions released from the SA / CMSC / BAG hydrogel also contribute to tissue repair and macrophage regulation (zinc inhibits proinflammatory cytokine production, while calcium aids macrophage function), the primary anti-inflammatory effect is driven by TA.

[0059] In summary, the SA / CMSC / TA / BAG hydrogel effectively regulates inflammation through a dual mechanism: inhibiting M1 macrophage activation and promoting M2 macrophage polarization. This dual action makes the hydrogel a promising candidate for accelerating wound healing and enhancing tissue regeneration because it can provide a balanced anti-inflammatory response.

[0060] Excessive reactive oxygen species (ROS) in wounds can cause oxidative stress, which can damage cells, delay healing, inhibit angiogenesis, and interfere with collagen synthesis. To address this, neutralizing ROS is crucial to mitigate oxidative damage, reduce inflammation, promote angiogenesis, and promote effective tissue repair. Adding antioxidants such as tannic acid (TA) to wound dressings can help restore the balance of ROS, thereby creating an environment that accelerates wound healing.

[0061] Flow cytometry results demonstrated the differential effects of different hydrogel formulations on ROS levels (Figure S1). The blank control group displayed minimal signal intensity, establishing a baseline, while the positive control group confirmed proper functioning of the experimental system and provided a reference for maximal ROS scavenging. The untreated control group exhibited moderate signal intensity, reflecting unchanged cellular responses. SA / CMSC hydrogels resulted in a modest decrease in signal intensity, suggesting limited antioxidant efficacy. In contrast, SA / CMSC / TA hydrogels significantly reduced the signal, highlighting the potent antioxidant capacity of TA. TA acts as a powerful ROS scavenger due to its polyphenolic structure, which directly neutralizes ROS and interferes with oxidative stress pathways. Furthermore, TA inhibits prooxidant signaling, such as the NF-κB pathway, which contributes to reduced oxidative damage and inflammation.

[0062] Similarly, SA / CMSC / BAG hydrogel also reduced ROS levels, although the effect was less pronounced than SA / CMSC / TA. The reduced antioxidant efficacy of SA / CMSC / BAG hydrogel may be due to the limited release of bioactive ions from bioglass (BAG) under the acidic conditions typical of the early stages of wound healing. The small amount of calcium (Ca) released by BAG 2+ ) and zinc (Zn 2+ ) ions play a role in stabilizing mitochondrial function and enhancing antioxidant enzyme activity, which helps regulate oxidative stress. Notably, the SA / CMSC / TA / BAG hydrogel exhibited the most significant reduction in ROS signal intensity, highlighting its excellent antioxidant properties.

[0063] Angiogenesis is a critical process in wound healing as it ensures an adequate supply of oxygen and nutrients, supporting fibroblast activity, collagen synthesis, and epithelialization. Angiogenesis also promotes the recruitment of immune cells and the delivery of growth factors, such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF), which promote the proliferation of endothelial cells and fibroblasts, stimulate granulation tissue formation, and contribute to wound remodeling. Furthermore, angiogenesis helps wounds transition from the inflammatory to the proliferative phase by resolving inflammation and supporting the polarization of macrophages toward an M2 phenotype. Impaired angiogenesis can lead to chronic wounds, hypoxia, and delayed healing, highlighting its critical role in effective tissue repair.

[0064] Angiogenesis assay results showed that the SA / CMSC / TA / BAG hydrogel significantly promoted the formation of capillary-like structures within 6 hours, demonstrating its robust angiogenic potential (Figures S2A and S2B). In contrast, the SA / CMSC / BAG hydrogel also exhibited some angiogenic activity, but its effect was slightly weaker than that of the SA / CMSC / TA / BAG group. The SA / CMSC / TA hydrogel exhibited a moderate angiogenic effect, while the SA / CMSC and control groups showed minimal to no capillary formation. This enhanced angiogenic effect can be attributed to the synergistic effects of TA and BAG at different stages of wound healing.

[0065] In the early stages of inflammation, TA plays a crucial role by utilizing its antioxidant and anti-inflammatory properties. TA scavenges excess ROS and inhibits pro-inflammatory pathways such as NF-κB, thereby reducing oxidative stress and protecting endothelial cells from damage. These actions create a favorable microenvironment for angiogenesis. In the subsequent proliferation phase, BAGs promote angiogenesis by releasing bioactive ions such as silicon (Si 4+ ) and calcium (Ca 2+)) further supports angiogenesis. Silicon ions enhance the formation of the extracellular matrix (ECM), providing a stable and functional scaffold for the growth of capillary networks. Calcium ions stimulate endothelial cell proliferation and migration by activating angiogenesis-related signaling pathways (such as PI3K / Akt), thereby accelerating the formation of capillary-like structures.

[0066] Crucially, the pH-responsive release properties of BAG ensure sustained and stage-specific delivery of bioactive ions. In the acidic environment of the early stages of wound healing, BAG releases small amounts of silicon and calcium ions, which gradually increase as the pH shifts toward neutral or alkaline. This dynamic ion release supports angiogenesis and tissue regeneration at multiple stages of the healing process. Therefore, the excellent angiogenic capacity of SA / CMSC / TA / BAG hydrogel stems from the antioxidant and anti-inflammatory effects of TA during the inflammatory phase and the direct angiogenic effect of BAG during the proliferative phase.

[0067] like Figure 6 Shown: In vitro hemolysis assay. Hemolysis, the rupture of red blood cells (RBCs) and the subsequent release of hemoglobin, is a key parameter for assessing the biocompatibility of wound-healing materials. Excessive hemolysis damages RBCs, hinders oxygen transport, disrupts the wound microenvironment, and ultimately impedes the healing process. Therefore, wound-healing hydrogels must exhibit minimal hemolytic activity to avoid exacerbating tissue damage or delaying healing. Hemolysis assay results showed that the SA / CMSC / TA / BAG hydrogel had an extremely low hemolysis rate of less than 5%, demonstrating excellent hemocompatibility. In comparison, the SA / CMSC / BAG hydrogel had a slightly higher hemolysis rate of approximately 7%, while the SA / CMSC / TA hydrogel also had a low hemolysis rate (less than 6%). The SA / CMSC hydrogel, as a matrix, exhibited negligible hemolysis, similar to that of the negative control. The minimal hemolysis observed in the SA / CMSC / TA / BAG hydrogel can be attributed to the antioxidant and membrane-stabilizing properties of tannic acid (TA), which helps protect RBCs from damage caused by oxidative stress. The phenolic hydroxyl groups in TA can neutralize reactive oxygen species (ROS), thereby reducing oxidative damage and preventing lipid peroxidation on erythrocyte membranes.

[0068] In addition, bioglass (BAG) minimizes damage to the red blood cell membrane due to its controlled ion release properties, thus helping the hydrogel reduce hemolytic activity. BAG can continuously release bioactive ions such as calcium ions (Ca 2+ ) and silicon ions (Si 4+ ), ensuring a slow release of ions and minimizing potential damage to red blood cells. However, under acidic conditions, the hemolytic potential of BAG may be slightly increased, which may explain the higher hemolysis rate observed in SA / CMSC / BAG hydrogels compared with SA / CMSC / TA / BAG hydrogels.

[0069] The rapid release of calcium and silicon ions from BAGs could alter the osmotic balance surrounding red blood cells, leading to a degree of hemolysis. However, the controlled-release system in the hydrogel mitigates these effects by regulating ion release, ensuring that ion concentrations remain within a safe range for maintaining red blood cell integrity. This controlled-release system not only reduces the risk of red blood cell damage but also effectively unleashes the therapeutic benefits of BAGs without compromising blood compatibility. Negligible hemolysis was observed when the SA / CMSC matrix was used alone, further demonstrating the non-hemolytic nature of the hydrogel network. When combined with TA and BAGs, the hydrogel exhibits both protective and therapeutic benefits, ensuring it maintains blood compatibility while promoting wound healing.

[0070] In summary, the SA / CMSC / TA / BAG hydrogel achieves an optimal balance between therapeutic function and blood compatibility. Its extremely low hemolytic activity indicates that it will not negatively impact the wound microenvironment, thus showing promise as a biocompatible material for wound healing applications.

[0071] To evaluate the effectiveness of the hydrogel in promoting wound healing, we used a rat acute skin infection wound model. The results showed that the SA / CMSC / TA / BAG composite hydrogel significantly accelerated wound healing. Compared with the control group, the SA / CMSC / TA / BAG hydrogel exhibited higher wound contraction rates at all time points, and the wound was nearly completely healed by day 14 ( Figure 6 D, E, F). This accelerated healing process suggests that the hydrogel can improve the speed and quality of wound repair. The heat map further confirms these findings ( Figure 6 F), the SA / CMSC / TA / BAG group showed the most significant reduction in wound area, indicating effective tissue regeneration and repair. The excellent healing performance of the SA / CMSC / TA / BAG hydrogel can be attributed to the synergistic effect of its components. Tannic acid (TA) has anti-inflammatory and antioxidant properties, creating a favorable microenvironment for tissue regeneration. In addition, calcium (Ca) released by bioglass (BAG) 2+ ) and zinc (Zn 2+ ) and other bioactive ions can enhance angiogenesis and promote cell proliferation, further accelerating the healing process. Histological analysis using H&E staining ( Figure 7 A and Figure 7 C) shows that by day 14, the skin tissue of the SA / CMSC / TA / BAG group closely resembled normal tissue, with an intact epidermal layer and well-organized dermal fibers, indicating near-complete wound healing. In contrast, the other groups, especially the control group, exhibited severe tissue loss and disorganized fiber arrangement.

[0072] Masson trichrome staining further showed that the SA / CMSC / TA / BAG group had the highest collagen fiber density, indicating effective collagen deposition and enhanced tissue repair. However, the SA / CMSC group and the control group had lower collagen production and irregular and incomplete collagen structure, indicating poor healing. Immunofluorescence staining results ( Figure 7 B and Figure 7 D) Confirming the above results, the SA / CMSC / TA / BAG group showed a significant decrease in the expression of iNOS, a pro-inflammatory M1 macrophage marker, indicating effective suppression of local inflammation. Simultaneously, the expression of CD206, an anti-inflammatory M2 macrophage marker, was significantly increased, demonstrating that the hydrogel promotes M2 macrophage polarization, thereby promoting wound healing and tissue reconstruction.

[0073] In summary, the SA / CMSC / TA / BAG composite hydrogel effectively accelerated the healing of acutely infected wounds through multiple mechanisms, including anti-inflammatory and antioxidant effects, angiogenesis promotion, and enhanced collagen deposition. These results support the potential of this hydrogel as a biocompatible material for promoting wound healing in clinical applications.

[0074] like Figure 7 Shown: (A) Hematoxylin-eosin (HE) staining of tissue sections from different treatment groups (control group, SA / CMSC, SA / CMSC / TA, SA / CMSC / BAG, SA / CMSC / TA / BAG). (B) Masson trichrome staining of tissue sections from different treatment groups.

[0075] It is important to note that this microenvironment-responsive hydrogel, designed for precise and sequential repair of acute infected wounds, dynamically adjusts its structural properties in response to local pH changes, ensuring the timely release of antimicrobial tannins and tissue-repairing ions, effectively reducing bacterial load, modulating inflammatory responses, and attenuating oxidative stress. This biphasic mechanism promotes an optimal wound microenvironment, accelerates tissue regeneration, and enhances overall healing. Encouraging in vitro and in vivo evidence highlights the clinical potential of this microenvironment-responsive hydrogel as a next-generation wound dressing for targeted, sequential repair of complex infected wounds.

[0076] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. A microenvironment-responsive hydrogel for precise and sequential repair of acute infected wounds, characterized by: The hydrogel is composed of the following components in proportion: sodium alginate 4% w / v, carboxymethyl chitosan 2% w / v, tannic acid 0.5% w / v, zinc-doped bioglass 10 mg / mL, and sodium citrate 10% w / v as a cross-linking agent; the hydrogel forms an interpenetrating network structure through hydrogen bonding and hydrophobic interactions, which can shrink and quickly release tannic acid in an acidic environment, and expand and continuously release Zn in an alkaline environment. 2+ and Ca 2+ .

2. The microenvironment-responsive hydrogel for precise sequential repair of acute infectious wounds according to claim 1, characterized in that: The molecular weight of the sodium alginate is 200,000, the molecular weight of the carboxymethyl chitosan is 100,000, and the acetylation degree is ≥75%.

3. The microenvironment-responsive hydrogel for precise sequential repair of acute infectious wounds according to claim 1, characterized in that: The zinc-doped bioglass is uniformly dispersed in the carboxymethyl chitosan solution by ultrasonic treatment at a frequency of 20-25 kHz and a power of 150 W for 60 minutes.

4. The microenvironment-responsive hydrogel for precise sequential repair of acute infectious wounds according to claim 1, characterized in that: The hydrogel releases ≥50% of tannic acid in an acidic environment of pH 5.5 within 48 hours, and releases ≥30 mg / L of Ca in an alkaline environment of pH 7.4 within 48 hours. 2+ and ≥20 mg / L Zn 2+ .

5. The method for preparing a microenvironment-responsive hydrogel for precise sequential repair of acute infectious wounds according to claim 1, characterized in that: The following steps are involved: (1) Dissolve 4% w / v sodium alginate in distilled water and stir until completely dissolved; (2) Dissolve 2% w / v carboxymethyl chitosan in 1-2% v / v acetic acid to form a transparent solution; (3) Add 10 mg / mL zinc-doped bioglass to carboxymethyl chitosan solution and disperse evenly by ultrasonic treatment; (4) Mix the sodium alginate solution and carboxymethyl chitosan solution in a volume ratio of 2:1 and stir for two minutes; (5) Add 0.5% w / v tannic acid and stir evenly, then add 10% w / v sodium citrate for cross-linking to form a three-dimensional network hydrogel at room temperature.

Images