Hydrogel adhesive for plugging and repairing through orificium fistulae as well as preparation method and application of hydrogel adhesive

Abstract

The invention belongs to the field of medical biological materials, and particularly relates to a hydrogel adhesive for plugging and repairing a penetrating fistula, a preparation method and application. The hydrogel adhesive has a double-layer structure and comprises an enzyme-loaded gel sheet serving as a supporting layer and a repair gel layer which is coated on the surface of the enzyme-loaded gel sheet and is formed by gel precursor liquid through interfacial enzymatic polymerization in situ. The hydrogel adhesive is subjected to enzymatic reaction through a glucose oxidase-peroxidase cascade catalytic system formed on the surface of the supporting gel layer to generate free radicals at the wound part to initiate precursor solution in-situ polymerization gel forming, and physical plugging and sealing are achieved. Compared with the prior art, the problems that in the prior art, a hydrogel adhesive cannot achieve good fistula plugging and repair promoting effects due to the fact that the hydrogel adhesive is difficult to be in high-strength adhesion and mechanical matching with dynamic wet tissues are solved; the hydrogel adhesive disclosed by the invention can be rapidly formed in situ at a penetrating fistula position, is simple and mild in preparation process, good in biocompatibility and strong in tissue specific adhesion, integrates physical plugging and biochemical repair, can be used for rapid plugging of the penetrating fistula, wound healing and long-acting mechanical support, and has wide application prospects and popularization values.

Description

Technical Field

[0001] This invention belongs to the field of medical biomaterials, specifically relating to a hydrogel adhesive for sealing and repairing penetrating fistulas, its preparation method, and its application. Background Technology

[0002] Rapid and efficient treatment of open penetrating wounds is of great significance to patients' healthy lives and the construction of a healthy, harmonious, and beautiful society. Effective treatment of penetrating wounds caused by combat injuries is particularly crucial for ensuring soldiers maintain high combat effectiveness. Penetrating wounds are characterized by exposure to moist, bacterial environments, complex physiological structures, and continuous inflammatory stimulation, resulting in high infection rates, slow healing speeds, and often accompanied by traumatic bleeding, serious complications, and even death. Currently, wound repair often uses iodoform, silicone, and imported collagen products for packing. However, these sealing materials typically suffer from drawbacks such as poor mechanical strength, low tissue adhesion, easy failure and detachment, susceptibility to inflammatory reactions, weak biodegradability, and lack of biochemical repair activity, leading to unsatisfactory treatment outcomes. Skin flap transplantation surgery, due to its significant trauma, generally has low acceptance rates. Therefore, there is an urgent need to develop novel biomaterials to address this problem.

[0003] Hydrogels with three-dimensional hydrophilic networks resembling extracellular matrix have enormous application potential in repairing tissue and organ damage in the human body. Based on different gel repair methods, they can generally be divided into two categories: one is soft, porous, and malleable injectable repair hydrogels, which can promote the penetration, adhesion, migration, and proliferation of host cells and are receiving continuous attention as implant materials. However, their relatively weak mechanical properties cannot match the mechanical strength of the surrounding dynamic tissues, resulting in insufficient support for the regeneration of healthy tissues and limiting their use as load-bearing scaffolds. The other category is sealing hydrogels with good mechanical properties, capable of resisting large mechanical deformations (>500%) and achieving stable wound sealing through synergistic covalent adhesion. However, their high degree of cross-linking makes it difficult to fill irregular penetrating wounds (>2cm), restricts the penetration and infiltration of host cells, and hinders effective promotion of tissue regeneration. Therefore, in penetrating wound environments, single-structure gel dressings cannot meet repair needs. Developing layered gel adhesives with mechanical properties that match large deformations and possess both sealing and biochemical repair functions remains a key focus and challenge.

[0004] Currently, there are no reports of biomaterials that can self-adhere, integrate physical sealing and biochemical repair, and achieve regeneration and repair of refractory penetrating fistulas with a "rigid-flexible" approach. Summary of the Invention

[0005] The purpose of this invention is to address at least one of the aforementioned problems by providing a hydrogel adhesive, its preparation method, and its application for sealing and repairing penetrating fistulas. This addresses the lack of self-organizing, in-situ adhesives and related preparation methods for enhancing the sealing and repair of dynamically penetrating fistulas in the prior art. The in-situ formed bilayer hydrogel adhesive possesses excellent biocompatibility, good fistula-sealing effect, and strong motion tolerance. Through enzymatic catalytic reactions of different gel layers, it can achieve asymmetric functions of antibacterial properties in the outer layer and repair-promoting properties in the inner layer, respectively. It can be used for rapid sealing, long-term chemical support, and wound healing of penetrating fistulas such as oral-oropharyngeal fistulas.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] The first aspect of this invention discloses a hydrogel adhesive for sealing and repairing penetrating fistulas. The hydrogel adhesive has a double-layer structure, including an enzyme-carrying gel sheet as a support layer and a repair gel layer formed in situ by interfacial enzymatic free radical polymerization of a gel precursor solution coated on the surface of the enzyme-carrying gel sheet.

[0008] The supporting layer has mechanical strength (modulus 10-100 kPa) that matches moving tissue, and is used to provide mechanical support and anti-infection for dynamic and moist tissue. The repair gel layer has weak mechanical properties (modulus 0.1-5 kPa) that are adaptable to cell proliferation and has antioxidant and anti-inflammatory functions, and is used to support cell proliferation and promote tissue regeneration and repair.

[0009] Preferably, the hydrogel adhesive is formed in situ by enzymatic free radical polymerization on the surface of the supporting gel layer (initiated by a glucose oxidase-peroxidase cascade catalytic system). When this hydrogel adhesive is used at a penetrating fistula, it is formed by free radical polymerization initiated by the glucose oxidase (GOx) loaded on the surface of the supporting layer and the peroxidase in the gel precursor solution contacting the blood glucose (Glu) at the fistula site.

[0010] Preferably, the peroxidase is selected from any one of horseradish peroxidase, catalase, a complex with peroxidase activity (such as ferrous glycine, which has been tested to have good peroxidase activity), and a nanozyme with peroxidase activity.

[0011] A second aspect of this invention discloses a method for preparing the hydrogel adhesive for sealing and repairing penetrating fistulas as described above, comprising the following steps:

[0012] S1: Add the gelling component and photoinitiator to deionized water, and polymerize into a gel by photoinitiation. After vacuum drying, a support layer gel sheet is obtained.

[0013] S2: Glucose oxidase is coated onto the surface of the support layer gel sheet obtained in S1 to obtain an enzyme-loaded gel sheet.

[0014] S3: Mix the functional monomer polymerization system, cross-linking agent, peroxidase and repair-promoting components to obtain the repair layer gel precursor solution;

[0015] S4: The repair layer gel precursor solution obtained in step S3 is coated onto the surface of the enzyme-carrying gel sheet obtained in step S2, and then free radical polymerization is initiated based on the glucose oxidase-peroxidase cascade catalytic system to form a gel, thereby obtaining the hydrogel adhesive.

[0016] Preferably, in step S1, the gelling component comprises a polysaccharide, a double-bond modified polysaccharide, and an acrylic monomer in a mass ratio of (2-20):(1-5):(10-40); the photoinitiator is α-ketoglutaric acid; and the mass ratio of the gelling component to the photoinitiator is (20-40):1.

[0017] Preferably, in step S3, the mass ratio of the functional monomer polymerization system to the peroxidase is (100-600):1.

[0018] Preferably, in step S3, the functional monomer polymerization system comprises component A and component B, wherein component A includes one or more of acrylamide (AAM), N,N-dimethylacrylamide (DMAA), N-hydroxyethylacrylamide (HEAA), N-isopropylacrylamide (NIPAM), acrylic acid (AA), hydroxyethyl methacrylate (HEMA), hydroxypropyl acrylate (PHPMA), and polyethylene glycol methacrylate (PEGMA), and component B is N-succinimide acrylate (AAc-NHS);

[0019] The cross-linking agent is a polysaccharide modified with double bonds;

[0020] The repair-promoting components include one or more of platelet-derived growth factor (PDGF), transforming growth factor (TGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), hepatocyte growth factor (HGF), and fibroblast growth factor (FGF).

[0021] Preferably, in step S3, the mass ratio of component A to component B and crosslinking agent is 2:(0.1-2):8.

[0022] Preferably, the polysaccharide is selected from any one or more combinations of gelatin, dextran, chitosan, chondroitin sulfate, and hyaluronic acid.

[0023] Preferably, the repair layer gel precursor solution further includes glucose, and the mass ratio of glucose oxidase, peroxidase and glucose is (0.1-5):1:(0-10). The addition of glucose can accelerate the gelation process, and the amount of glucose can be added appropriately according to different application scenarios.

[0024] The third aspect of this invention discloses the application of the hydrogel adhesive described above in the preparation of materials for sealing and repairing penetrating fistulas, including oropharyngeal fistulas, esophageal fistulas, intestinal fistulas, anal fistulas, etc. In practical applications, the precursor solution of the repair layer gel of this invention is drop-coated onto the surface of the support layer gel sheet, and then the inside of the penetrating fistula is pressed for 1-10 minutes to achieve rapid gelation and sealing of the defect site. This provides mechanical support for dynamic tissues while effectively preventing bacterial infection in moist environments. The soft repair layer, through the release of growth factors and the biocatalytic antioxidant effects of glucose oxidase / peroxidase, can also promote cell proliferation and tissue regeneration and repair, achieving the therapeutic purpose.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The hydrogel adhesive of this invention can be rapidly formed in situ at the site of a penetrating fistula. The preparation process is simple and mild, with good biocompatibility and strong tissue-specific adhesion. It integrates physical sealing and biochemical repair and can be used for rapid sealing of penetrating fistulas, wound healing and long-term chemical support. It has broad application prospects and promotional value.

[0027] 1. This invention uses a tissue surface enzymatic polymerization initiation system, which has advantages such as mild conditions, biofriendliness, and a wide range of applicable reaction systems. It can be used in combination with various support materials. It can also rapidly gel in situ at physiological blood glucose concentrations, achieving seamless filling and sealing of difficult-to-treat wounds such as irregular penetrating fistulas, and accelerating wound healing.

[0028] 2. The hydrogel adhesive of this invention can regulate the microstructure and mechanical strength of each layer of the gel network by adjusting the concentration and combination of polymeric monomers and the concentration of the enzyme / ferrous glycine system. The outer support layer has high mechanical strength and can effectively isolate infection from the moist environment inside the wound, such as contamination from food residue, saliva, and bacteria in the oral cavity, and provide mechanical support in the event of movement. The scaffold structure of the inner repair layer, which resembles the extracellular matrix, can support cell activity and ensure the biomechanical activity of cells. At the same time, through the cascade catalytic reaction of glucose oxidase and peroxidase (such as peroxidase-like ferrous glycine, which constitutes the GOx / Fe[Gly]2 / Glu system) and the loading of growth factors, it can reshape the wound repair microenvironment, reduce intracellular oxidative stress, promote cell growth, and further accelerate tissue regeneration and repair. It integrates physical sealing and biochemical repair, providing a new method for treating penetrating fistulas (such as oropharyngeal fistulas, esophageal fistulas, intestinal fistulas, anal fistulas, etc.), which has important significance and promotional value.

[0029] 3. The preparation process of this invention is simple, convenient to operate, and the materials used are inexpensive and readily available. Through in-situ polymerization and covalent interaction with tissue, it achieves strong adhesion and sealing of wound tissue, which can supplement or replace the function of sutures, avoid secondary damage to tissues, and improve the patient's treatment tolerance. Attached Figure Description

[0030] Figure 1 A schematic diagram of a hydrogel adhesive used for sealing and repairing penetrating fistulas;

[0031] Figure 2 Mechanical properties of the support layer hydrogel material prepared in Example 1: a) tensile curves of support layer hydrogels prepared with different contents of acrylic acid; b) tensile modulus and elongation at break of support layer hydrogels prepared with different contents of acrylic acid.

[0032] Figure 3 The storage modulus (G') and loss modulus (G”) curves of the repair layer hydrogel prepared in Example 2 under oscillation time scanning mode at 37°C;

[0033] Figure 4 Images showing the sealing effect of the hydrogel adhesive prepared in Example 3 in a New Zealand rabbit oral fistula model;

[0034] Figure 5 These are representative photographs of wounds from different treatment groups in the New Zealand rabbit oral fistula model during days 0-12 of Example 4;

[0035] Figure 6 These are representative photographs of wounds from different treatment groups in the beagle oral fistula model during days 0-22 of Example 5;

[0036] Figure 7Images of H&E staining on the skin and mucous membrane sides of the wound in beagle dogs 22 days after treatment in Example 5;

[0037] Figure 8 This is a comparison of the mechanical strength of normal tissue and repaired wound tissue in a beagle dog in Example 5. Detailed Implementation

[0038] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0039] Unless otherwise specified in the following examples, all reagents can be commercially available products that are routinely available to those skilled in the art.

[0040] A hydrogel adhesive for sealing and repairing penetrating fistulas, with the structure as follows: Figure 1 As shown, the hydrogel adhesive has a bilayer structure. First, a support layer gel with good mechanical properties is prepared by photo-initiated polymerization of polysaccharides modified with double bonds and olefin comonomers. Then, glucose oxidase is applied to the surface of the support layer gel and finally pressed at the fistula. The precursor solution is polymerized by interfacial enzymatic free radical polymerization to obtain the hydrogel adhesive.

[0041] Example 1

[0042] 1) Polysaccharide grafting with double bonds: Dissolve 14g of chondroitin sulfate (CS) in 100mL of deionized water, stir at room temperature for 0.5h to form a homogeneous solution, then add 1M HCl dropwise to adjust the pH to 3.5. Add 4.5mL of GMA to the solution and react at 50℃ for 2-3h. After the reaction is complete, add the solution to anhydrous ethanol while stirring, producing a white flocculent substance. Wash three times with anhydrous ethanol, then dialyze in deionized water (MWCO 8000-12000) for 3 days. Freeze-dry the dialysate, and grind it after complete drying to obtain a white powder CSMA (double-bond modified CS).

[0043] 2) Preparation of the support layer gel material: 0.6 g CSMA, 0.9 g chondroitin sulfate, and acrylic acid (AA) with contents of 10% (w / w), 15% (w / w), 20% (w / w), 25% (w / w), and 30% (w / w) were added to deionized water, mixed thoroughly with 0.15 g photoinitiator (α-ketoglutarate), and the pH was adjusted to 5.5. The mixture was then placed under ultraviolet light (365 nm, 25 mW / cm²). 2 After being exposed to light for 20 minutes, the gel polymerizes to form a series of support layer gel materials.

[0044] Mechanical properties of support layer gel materials with different acrylic acid contents were tested using a tensile testing machine. The tensile test speed was 20 mm / min, and the sample was rectangular, 1 cm wide and 1 mm thick. The results are as follows: Figure 2As shown in a and 2b, the elongation at break and tensile modulus of the support layer hydrogel increase with the increase of acrylic acid content. When the acrylic acid content is 30%, the tensile modulus of the support layer gel material can reach 49 kPa and the elongation at break is 1970%. The hydrogel used for penetrating fistulas needs to adapt to tissue movement and deformation while also having excellent mechanical strength. Therefore, different acrylic acid contents can be selected to prepare support layer gel materials according to different tissues.

[0045] Example 2

[0046] 1) Grafting double bonds onto polysaccharides: The steps are the same as step 1 in Example 1.

[0047] 2) Preparation of enzyme-supporting gel material: 0.6 g CSMA, 0.9 g chondroitin sulfate and 30% (w / w) acrylic acid were added to deionized water and mixed thoroughly with 0.15 g photoinitiator (α-ketoglutarate). The pH was adjusted to 5.5, and then placed under ultraviolet light (365 nm, 25 mW / cm²). 2 After 20 minutes of light exposure, the mixture was polymerized into a gel, yielding a support layer gel material. Subsequently, 100 μL of 100 U / mL glucose oxidase was uniformly coated onto the surface of the freeze-dried support layer gel material, allowing the glucose oxidase to penetrate into the support layer gel network, thus obtaining an enzyme-loaded support layer gel material.

[0048] 3) Preparation of repair layer gel precursor solution: Add 8% (w / w) CSMA and 1% (w / w) N-succinimide acrylate (AAc-NHS) to deionized water, dissolve in a 50°C water bath and cool fully, then add 2% (w / w) N,N-dimethylacrylamide (DMAA) and Fe[Gly]2 (0.5 mg / mL), and vortex to form a uniform repair layer gel precursor solution.

[0049] To determine the gelation time of the hydrogel adhesive, the storage modulus (G') and loss modulus (G") of the hydrogel were measured using a Thermo Haake rheometer at a frequency of 1 Hz and a shear stress of 1 Pa. First, the enzyme-supported gel was laid flat on a parallel plate (20 mm in diameter, 0.3 mm gap) at 37°C. Then, glucose (1 mg / mL) was added to the repair layer gel precursor solution, mixed thoroughly, and an appropriate amount was dropped onto the surface of the flat enzyme-supported gel. Dynamic time scanning was performed, followed by dynamic frequency scanning at 10⁻⁰.⁻¹ Hz. The experimental results are as follows: Figure 3 As shown, the G′ and G″ curves intersect within a time of <60s, indicating that the system can gel in a short time and can be used for rapid adhesion and sealing of wounds.

[0050] Example 3

[0051] To evaluate the sealing performance of the hydrogel adhesive of this invention on penetrating fistulas, we selected the oropharyngeal fistula, characterized by its complex anatomical structure, variable physiological environment, repeated inflammatory stimulation, and continuous tissue movement, and which is extremely difficult to repair, as a model for animal experimental verification. First, a New Zealand rabbit oropharyngeal fistula model (POF) was established, forming a 2×1.5cm fistula in the rabbit's anterior cheek region. 2 For an elliptical, penetrating wound that runs through the skin and oral mucosa, an appropriate amount of the repair layer gel precursor solution prepared in step 3) of Example 2 is dropped onto the surface of the enzyme-carrying support layer gel material prepared in step 2), and then gently pressed onto the wound. After pressing for 1 minute, in-situ gelation is achieved, and efficient adhesion is achieved through the covalent coupling of NHS ester groups in the polymer network with amino groups on the tissue surface.

[0052] Experimental results are as follows Figure 4 As shown, after 1 minute of pressure, the hydrogel completely adhered to the defect, forming a tight seal at the tissue defect site. The adhered hydrogel maintained this adhesive seal for more than 3 days in a New Zealand rabbit POF model on a normal diet, without any sign of weakening adhesion. Even after 3 days of adhesion, oral injection of saline into the POF model did not result in any water seepage or leakage, demonstrating the excellent fluid-sealing and adhesion properties of this hydrogel adhesive.

[0053] Example 4

[0054] The repair-promoting effect of the hydrogel adhesive prepared in Example 2 was further evaluated using a New Zealand rabbit POF model, with a blank group, an iodoform group, and a gel group. For the gel group, an appropriate amount of the repair layer gel precursor solution obtained in step 3) of Example 2 was dropped onto the surface of the enzyme-carrying support layer gel material prepared in step 2), and then gently pressed onto the wound. After pressing for 1 minute, the hydrogel completely adhered to the damaged inner mucosa surface of the wound, requiring no further action. Figure 5 As shown.

[0055] In contrast, the clinically common iodoform treatment group requires suture fixation, which takes longer (>5 minutes) and carries the risk of secondary tissue damage due to the puncture of sutures and needles. Samples are collected and monitored every 4 days post-operatively. Wound healing progress is as follows: Figure 5 As shown, overall, the wound healing speed of the gel group and the iodoform group was significantly faster at different stages (days 4, 8, and 12), reaching wound closure rates of 78.4% and 60.2% respectively on day 8, significantly higher than the blank control group (31.8%). Notably, the wound treated with the hydrogel adhesive showed the best healing performance on day 12 (100%), with complete epithelialization of both the skin and mucous membrane at the wound site. In contrast, the other two groups still had unhealed penetrating defects, demonstrating the excellent repair effect of the hydrogel adhesive prepared in this invention on penetrating fistulas.

[0056] Example 5

[0057] A beagle model with POF defect (3×2.5cm) was further established. 2 As a proof of concept, the wound sealing and repair effects of the hydrogel adhesive prepared in Example 2 were evaluated. A blank group, an iodoform group, and a gel group were also set up. For the gel group, an appropriate amount of the repair layer gel precursor solution prepared in step 3) of Example 2 was dropped onto the surface of the enzyme-carrying support layer gel material prepared in step 2), and then gently pressed onto the wound for 1 minute. The fistula sealing and repair were then observed and recorded.

[0058] Experimental results are as follows Figure 6 As shown, the wound area reduction was most significant in the gel group from day 4 to day 12, with a wound healing rate of 95% on day 12, compared to only 75% and 80% in the blank and iodoform groups, respectively. By day 22, the fistula had completely healed, while the other two groups still had penetrating wounds, with epithelial defects ultimately leading to fistula formation. Tissue specimens were collected from the wounds of the three groups of beagle dogs, stored in 10% buffered formalin for at least 24 hours, and then fixed in paraffin. Sections (4 μm) were prepared and stained with hematoxylin and eosin (H&E). The experimental results are shown below. Figure 7 As shown in the figure (white dashed line: intact epithelial layer; red dashed line: defective epithelial layer; red asterisk: blood vessels; blue triangle: fibroblasts; yellow asterisk: striated muscle cells), the gel group showed obvious continuous epidermal tissue on both the mucosal and skin sides. The degree of epithelialization and the integrity of the mucosal epithelium were higher than those in the iodoform and control groups, indicating that these areas healed well. In contrast, the healing status of the control and iodoform groups was not ideal, with extensive epithelial necrosis and severe inflammation. This demonstrates that the hydrogel adhesive prepared in this invention can not only seal penetrating oral fistulas but also promote wound healing and tissue regeneration.

[0059] Figure 8 This image compares the preoperative and postoperative tissue strength at the fistula site in a beagle dog. As shown in the figure, compared with normal cheek tissue, the wound tissue strength was basically restored after repair. These results indicate that the described hydrogel adhesive possesses advantages such as strong adhesion to dynamically moist tissue, good fistula-sealing performance, mechanical compatibility with tissue, responsive wound repair, and ease of operation. It exhibits excellent sealing and repair effects on penetrating fistulas and has significant clinical application value.

[0060] Example 6

[0061] 1) Grafting double bonds onto polysaccharides: The steps are the same as step 1 in Example 1.

[0062] 2) Preparation of enzyme-carrying support layer gel material: The steps are the same as step 2 in Example 1.

[0063] 3) Preparation of repair layer gel precursor solution: Add 8% (w / w) CSMA and 1% (w / w) N-succinimide acrylate (AAc-NHS) to deionized water, dissolve in a 50°C water bath and cool thoroughly, then add 2% (w / w) N,N-dimethylacrylamide (DMAA), glucose (1 mg / mL), horseradish peroxidase (100 U / mL) and acetylacetone (0.05 mM), and vortex to form a uniform repair layer gel precursor solution.

[0064] Take 100 μL / cm 2 The repair layer gel precursor liquid is drop-coated onto the surface of the enzyme-supported gel material. After glucose oxidase and horseradish peroxidase come into contact, a cascade catalysis occurs, initiating free radical polymerization to obtain the target hydrogel adhesive.

[0065] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A hydrogel adhesive for sealing and repairing penetrating fistulas, characterized in that, The hydrogel adhesive has a bilayer structure, including an enzyme-carrying gel sheet as a support layer and a repair gel layer formed in situ by interfacial enzymatic free radical polymerization of a gel precursor solution coated on the surface of the enzyme-carrying gel sheet.

2. The hydrogel adhesive for sealing and repairing penetrating fistulas according to claim 1, characterized in that, The hydrogel adhesive is formed by in-situ gelation through free radical polymerization initiated by a glucose oxidase-peroxidase cascade catalytic system formed on the surface of the supporting gel layer. The peroxidase is selected from any one of horseradish peroxidase, catalase, complexes with peroxidase activity, and nanozymes with peroxidase activity.

3. A method for preparing a hydrogel adhesive for sealing and repairing penetrating fistulas as described in claim 1 or 2, characterized in that, Includes the following steps: S1: Add the gelling component and photoinitiator to deionized water, and polymerize into a gel by photoinitiation. After vacuum drying, a support layer gel sheet is obtained. S2: Glucose oxidase is coated onto the surface of the support layer gel sheet obtained in S1 to obtain an enzyme-loaded gel sheet. S3: Mix the functional monomer polymerization system, cross-linking agent, peroxidase and repair-promoting components to obtain the repair layer gel precursor solution; S4: The repair layer gel precursor solution obtained in step S3 is coated onto the surface of the enzyme-carrying gel sheet obtained in step S2, and then free radical polymerization is initiated based on the glucose oxidase-peroxidase cascade catalytic system to form a gel, thereby obtaining the hydrogel adhesive.

4. The method for preparing a hydrogel adhesive for sealing and repairing penetrating fistulas according to claim 3, characterized in that, In step S1, the gelling component includes polysaccharides, double-bond modified polysaccharides, and acrylic acid monomers in a mass ratio of (2-20):(1-5):(10-40); the photoinitiator is α-ketoglutaric acid; and the mass ratio of the gelling component to the photoinitiator is (20-40):

1.

5. The method for preparing a hydrogel adhesive for sealing and repairing penetrating fistulas according to claim 3, characterized in that, In step S3, the mass ratio of the functional monomer polymerization system to the peroxidase is (100-600):

1.

6. The method for preparing a hydrogel adhesive for sealing and repairing penetrating fistulas according to claim 3, characterized in that, In step S3, the functional monomer polymerization system includes component A and component B. Component A includes one or more of acrylamide, N,N-dimethylacrylamide, N-hydroxyethylacrylamide, N-isopropylacrylamide, acrylic acid, hydroxyethyl methacrylate, hydroxypropyl acrylate, and polyethylene glycol methacrylate. Component B is N-succinimide acrylate. The crosslinking agent is a double-bond modified polysaccharide. The repair-promoting component includes one or more of platelet-derived growth factor, transforming growth factor, vascular endothelial growth factor, epidermal growth factor, insulin-like growth factor, hepatocyte growth factor, and fibroblast growth factor.

7. The method for preparing a hydrogel adhesive for sealing and repairing penetrating fistulas according to claim 6, characterized in that, In step S3, the mass ratio of component A to component B and crosslinking agent is 2:(0.1-2):

8.

8. A method for preparing a hydrogel adhesive for sealing and repairing penetrating fistulas according to claim 4 or 6, characterized in that, The polysaccharide is selected from one or more of gelatin, dextran, chitosan, chondroitin sulfate, and hyaluronic acid.

9. A method for preparing a hydrogel adhesive for sealing and repairing penetrating fistulas according to claim 3, characterized in that, The repair layer gel precursor solution also includes glucose, and the mass ratio of glucose oxidase, peroxidase and glucose is (0.1-5):1:(0-10).

10. The application of the hydrogel adhesive as described in claim 1 or 2 in the preparation of a material for sealing and repairing penetrating fistulas, characterized in that, The penetrating fistulas include oral-oropharyngeal fistulas, esophageal fistulas, intestinal fistulas, and anal fistulas.

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