Preparation method and application of hydrogel patch with Janus asymmetric adhesion

Abstract

The invention discloses a preparation method and application of a hydrogel patch with Janus asymmetric adhesion, and the preparation method is characterized by comprising the following steps: uniformly mixing GelMA-T, OHA, photoinitiators LAP, AAc and AAc-NHS, injecting a solution into a glass plate mold under the protection of nitrogen, carrying out ultraviolet irradiation crosslinking to form gel, and drying to obtain an adhesion layer; the preparation method comprises the following steps: adding SBMA, GelMA, MBAA and ammonium persulfate APS into water, fully stirring and mixing to obtain a mixed solution, and immersing one side of the adhesion layer obtained in the step 1 into the mixed solution for thermal crosslinking to obtain the hydrogel patch with the Janus asymmetric adhesion characteristic. Meanwhile, the composition has multiple biological functions of resisting bacteria, resisting oxidation, resisting inflammation, promoting proliferation and the like, and the complication risk after lung leakage repair can be remarkably reduced.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, and in particular relates to a method for preparing a hydrogel patch with Janus asymmetric adhesion and its application. Background Technology

[0002] Persistent air leakage is a common and challenging complication following pulmonary surgery (such as pulmonary nodules, pneumothorax, and pleural fistula), especially in patients on continuous positive airway pressure (CPAP). Air leakage can lead to serious complications such as pneumothorax and respiratory failure, ultimately resulting in death, prolonged hospital stays, increased medical costs, and poor postoperative recovery. Therefore, preventing air leakage has always been a primary clinical goal after pulmonary surgery. Current clinical practice for managing air leakage relies mainly on traditional surgical sutures, staples, and surgical sealing materials. However, these methods have significant limitations: traditional surgical sutures and staples are difficult to provide an effective seal on fragile lung tissue and carry the risk of secondary damage to fragile lung tissue.

[0003] Pneumopulmonary leakage is a common and challenging complication following thoracic surgery, and its healing process is often severely hampered by surgical trauma, mechanical friction, and potential bacterial contamination. The dynamic and moist environment of the wound site not only easily triggers local inflammation and generates high levels of reactive oxygen species (ROS), hindering fibroblast proliferation and collagen deposition, but also provides conditions for bacterial colonization, further exacerbating tissue damage and delaying the healing process. Therefore, achieving effective closure and rapid repair of pneumopulmonary leakage wounds urgently requires a multifunctional material that combines excellent sealing performance with active anti-inflammatory, antioxidant, antibacterial, and tissue regeneration-promoting properties.

[0004] Hydrogels, due to their excellent three-dimensional network structure and high water content, have been widely used in the field of tissue engineering. Among them, adhesive hydrogels have developed rapidly due to their excellent biocompatibility and tunable physicochemical properties, and have been applied in medical fields such as tissue wound closure and repair. However, unfortunately, currently used adhesive hydrogels such as cyanoacrylate and fibrin glue have limited clinical efficacy due to their limited adhesion strength, especially poor stability in wet environments, and high cost. Therefore, developing a novel sealing material that can achieve strong adhesion to wet tissue, excellent biocompatibility, and asymmetric adhesion function has become an urgent need in the field of thoracic surgery. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing a hydrogel patch with Janus asymmetric adhesion that has good biocompatibility, mechanical properties and sealing performance, and has multiple biological functions such as antibacterial, antioxidant, anti-inflammatory and proliferative effects, and its application, which can significantly reduce the risk of complications after lung leak repair.

[0006] The technical solution adopted by this invention to solve the above-mentioned technical problems is as follows: a method for preparing a hydrogel patch with Janus asymmetric adhesion, comprising the following steps: Step 1: Preparation of the Adhesion Layer After mixing GelMA-T, oxidized hyaluronic acid (OHA), photoinitiator (LAP), acrylic acid (AAc), and N-hydroxysuccinimide acrylate (AAc-NHS) evenly, the solution is injected into a glass plate mold under nitrogen protection. After cross-linking by ultraviolet light to form a gel and drying, the adhesion layer is obtained. Step 2: Preparation of hydrogel patches [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide SBMA, gelatin methacrylate GelMA, N,N'-methylenebisacrylamide MBAA, and ammonium persulfate APS are added to water and thoroughly mixed to obtain a mixed solution. The adhesive layer obtained in step 1 is immersed on one side of the mixed solution and thermally crosslinked to obtain a hydrogel patch with Janus asymmetric adhesion properties.

[0007] Further, step 1, preparation of the adhesion layer Add 0.05-0.2g GelMA-T, 0.1-0.3g OHA, and 0.01-0.03g LAP to 5-20g of water, dissolve completely at 40℃ and stir thoroughly. Then add 1-5g AAc and 0.01-0.03g AAc-NHS and continue stirring until homogeneous. After purging the mixture with nitrogen to remove oxygen, inject the solution into a glass plate mold under nitrogen protection. Crosslink the solution by irradiation with 365nm ultraviolet light for 20-40 minutes to form a gel. Then immerse the gel in deionized water to remove biotoxic monomer molecules. After drying at 30-50℃ for 10-30 minutes, the adhesion layer is obtained. Step 2: Preparation of hydrogel patches Add 1-3 g SBMA, 0.1-0.3 g GelMA, 0.04-0.08 g MBAA and 0.05-0.2 g APS to 5-20 g of water and stir thoroughly to obtain a mixed solution. Place the adhesive layer obtained in step 1 into a mold and mix the solution on one side for 20-40 minutes. Then, perform thermal crosslinking at 50-70℃ for 50-70 minutes to obtain a hydrogel patch with Janus asymmetric adhesion properties.

[0008] Further, step 1, preparation of the adhesion layer 0.1g GelMA-T, 0.2g OHA and 0.02g LAP were added to 10g water, dissolved completely at 40℃ and stirred thoroughly. Then, 3g AAc and 0.02g AAc-NHS were added and stirred until homogeneous. Nitrogen gas was introduced into the mixture to remove oxygen. The solution was then injected into a glass plate mold under nitrogen protection. After crosslinking by 365 nm ultraviolet light for 30 minutes, a gel was formed. The gel was then immersed in deionized water to remove biotoxic monomer molecules. After drying at 40℃ for 20 minutes, the adhesion layer was obtained. Step 2: Preparation of hydrogel patches Add 2 g SBMA, 0.2 g GelMA, 0.06 g MBAA and 0.1 g APS to 10 g water and stir thoroughly to obtain a mixed solution. Place the adhesive layer obtained in step 1 into a mold, mix the solution on one side for 30 minutes, and then thermally crosslink at 60°C for 60 minutes to obtain a hydrogel patch with Janus asymmetric adhesion properties.

[0009] Further, the synthesis of the gelatin methacrylate (GelMA) is as follows: 8-12 g of gelatin is dissolved in 100 mL of phosphate buffer at 60°C until completely dissolved. Then, 1-3 mL of methacrylic anhydride is slowly and evenly added dropwise. The temperature is then adjusted to 50°C, and the reaction is continued for 3 hours. Finally, 400 mL of phosphate buffer is added to terminate the reaction. After the reaction is completed, the gelatin methacrylate (GelMA) is obtained by dialyzing in deionized water using a dialysis bag with a molecular weight cutoff of 8-14 KD and then freeze-drying.

[0010] Furthermore, the synthesis steps of GelMA-T are as follows: 3-5 g of GelMA is dissolved in 200 mL of deionized water and stirred continuously at 60°C until completely dissolved. Then, 1.63 g of EDC and 1.35 g of NHS are added to the solution, and the reaction is carried out for 20-40 minutes. Then, 2.54 g of Tris is added, and the reaction is continued at 25°C for 48 hours. After the reaction is completed, the product is dialyzed in deionized water for 4 days using a dialysis bag with a molecular weight cutoff of 8-14 KD. Finally, the product is obtained by freeze-drying.

[0011] Furthermore, the oxidized hyaluronic acid (OHA) is prepared by periodate oxidation, with the following steps: 0.2-0.4 mol / L sodium periodate solution is added dropwise to 8-12 mg / mL hyaluronic acid solution, and the reaction system is stored in the dark for 4-8 hours. Then, ethylene glycol is added to terminate the reaction. After stirring, the mixture is dialyzed in deionized water for 4 days using a dialysis bag with a molecular weight cutoff of 3500D, and then freeze-dried to obtain the OHA product.

[0012] The present invention also provides the application of the hydrogel patch prepared by the above method in the preparation of lung leak repair agent.

[0013] Compared with existing technologies, the advantages of this invention are as follows: This invention provides a natural polymer hydrogel patch with Janus asymmetric adhesion properties for lung leak repair and prevention of postoperative tissue adhesion. The patch consists of an adhesion layer (GTO) and an anti-adhesion layer (p-SBMA): the GTO layer achieves strong adhesion to wet tissues through the introduction of tris(hydroxymethyl)aminomethane (Tris) and the use of hydrogen bonds, electrostatic interactions, and Schiff base reactions with active groups on tissue surfaces; the p-SBMA layer effectively resists cell and protein adhesion by forming a hydration layer, thereby reducing the risk of postoperative tissue adhesion.

[0014] In terms of mechanical properties, S-GTO hydrogel exhibits excellent tensile and compressive properties. Its bilayer network structure (containing oxidized hyaluronic acid (OHA) and SBMA networks) significantly improves compressive modulus, toughness, cohesion and energy dissipation capacity, enabling it to adapt to the complex mechanical environment inside the thoracic cavity and maintain stable adhesion and sealing properties during dynamic breathing, while reducing mechanical stimulation to surrounding tissues.

[0015] In terms of biological function, GelMA and OHA, as key functional components, not only possess good biocompatibility but also synergistically scavenge excess ROS, alleviate oxidative stress, and promote the proliferation and migration of lung epithelial cells and fibroblasts. This hydrogel also exhibits significant immunomodulatory effects, guiding macrophages from pro-inflammatory M1 to reparative M2 polarization within the lung molecular microenvironment, thereby reducing inflammatory factors, increasing repair-related factors, and improving the healing process. Histological and immunofluorescence analyses further confirmed that S-GTO treatment can reduce inflammation, enhance collagen deposition, and promote the complete repair of tissue structure.

[0016] Furthermore, S-GTO hydrogel possesses antibacterial properties and effectively sealed air leaks, reduced inflammatory responses, and accelerated wound healing in a rat model of air leaks. In summary, the S-GTO hydrogel patch combines excellent mechanical properties, asymmetric adhesion, multiple biological activities, and good microenvironment regulation capabilities, demonstrating promising application prospects in the field of air leak repair.

[0017] In summary, this invention presents a novel natural polymer hydrogel patch (S-GTO) with Janus asymmetric adhesion properties for lung leak repair and prevention of postoperative tissue adhesion. The adhesive layer (GTO) of the S-GTO hydrogel patch significantly enhances its wet tissue adhesion by introducing tris(hydroxymethyl)aminomethane (Tris). Furthermore, it achieves strong adhesion to wet tissue through hydrogen bonding, electrostatic interactions, and Schiff base reactions with tissue surface active groups. The S-GTO hydrogel patch exhibits good biocompatibility, mechanical properties, and sealing performance, significantly reducing the risk of complications after lung leak repair. In addition, in vitro and in vivo experiments demonstrate that the S-GTO hydrogel possesses multiple biological functions, including antibacterial, antioxidant, anti-inflammatory, and proliferative effects, effectively regulating the wound microenvironment and accelerating the tissue healing process. In conclusion, the S-GTO hydrogel patch, as an innovative biomaterial, demonstrates outstanding performance and potential in the field of lung leak repair, showing broad clinical application prospects. Attached Figure Description

[0018] Figure 1 shows the X-ray photoelectron spectroscopy analysis of the N 1s and S 2p of the adhesive layer (GTO-2), the anti-adhesion layer (pSBMA), and the hydrogel patch (S-GTO-2); Figure 2 shows a transmission electron microscope image of the transition layer between the adhesion layer (GTO-2), the anti-adhesion layer (pSBMA), and the hydrogel patch (S-GTO-2). Figure 3 shows the adhesion performance of hydrogel patches before and after GelMA modification and with different concentrations of GelMA-T without the addition of OHA. In the figure, a is the stress-displacement curve of the overlap shear test between different concentrations of GelMA-T and dry pigskin, and b is the bar chart of the maximum stress of the overlap shear test between different concentrations of GelMA-T and dry pigskin. Figure 4 The adhesion properties of hydrogel patches before and after the addition of OHA and at different concentrations of OHA were compared. In the figure, a is the stress-displacement curve of the overlap shear test between different concentrations of OHA and dry pigskin, and b is the bar chart of the maximum stress of the overlap shear test between different concentrations of OHA and dry pigskin. Figure 5 Mechanical property analysis of hydrogel patches, where a is the tensile test result, b is the Young's modulus test result, c is the compression test result, and d is the compression modulus test result; Figure 6 The sealing performance of the hydrogel was analyzed, where a represents the liquid sealing performance of the hydrogel in the damaged pig stomach; b represents the liquid sealing performance of the hydrogel in the damaged pig heart; and c represents the liquid sealing performance of the hydrogel in the damaged pig lung. Figure 7The swelling properties of GTO hydrogels with different OHA concentrations are analyzed, where a is the swelling curve of different concentration components and b is the comparison of swelling rates of different concentration components. Figure 8 shows the water retention analysis of GTO hydrogels with different OHA concentrations, where a is the water retention curve of different concentration components and b is the comparison of water retention rates of different concentration components. Figure 9 Live / dead cell staining images of A549 and BEAS-2B cells after co-culturing with pure culture medium, GTO-0, GTO-2 and S-GTO-2 gel for 24 hours; Figure 10 shows the CCK-8 assay results of cell viability after treatment with S-GTO-2 extract at different concentration gradients, where a is the BEAS-2B cell line and b is the A549 cell line. Figure 11 shows the hemolysis test results of the positive control group (Triton-X100), GTO-0, GTO-2 and S-GTO-2 gel; Figure 12 The effect of hydrogel patches on cell migration was shown in Figure a, where a represents the migration ability of A549 and BEAS-2B cells after co-incubation with pure culture medium, GTO-0, GTO-2 and S-GTO-2 hydrogel extraction media for 24 hours; and b represents the migration rate of A549 and BEAS-2B cells in the control group, GTO-O, GTO-2 and S-GTO-2 groups. Figure 13 The analysis of the antibacterial properties of hydrogel patches is as follows: a) represents the antibacterial effect of different groups against Staphylococcus aureus and Escherichia coli, and b) represents the survival rate of Staphylococcus aureus and Escherichia coli in different groups. Figure 14 shows the ROS flow cytometry results for H2O2, GTO-0, GTO-2, S-GTO-2, and the untreated group; Figure 15a shows Western blot images of CD86, iNOS, CD206, and β-actin in the control group, GTO-0, GTO-2, and S-GTO-2 groups; bd shows the relative expression of CD86, CD206, and iNOS in the control group, GTO-0, GTO-2, and S-GTO-2 groups. Figure 16 shows the expression levels of inflammatory factors in the control group, GTO-0, GTO-2, and S-GTO-2 groups, where a represents IL-1, b represents IL-6, c represents IL-10, and d represents TNF-α. ; Figure 17 The results show the in vivo degradation of the hydrogel patch, where a is a schematic diagram of the hydrogel degradation model under the skin, b is the biodegradation curve of S-GTO-2 and GTO-2 hydrogel in vivo, and c is a gross image of S-GTO-2 and GTO-2 hydrogel at 1, 2, 3 and 4 weeks after implantation. Figure 18 shows the anti-adhesion results of hydrogel patch in the cecum, where a is HE and Masson staining in the untreated group, b is HE and Masson staining in the GTO-2 group, and c is HE and Masson staining in the S-GTO-2 group. Figure 19 shows the pleural adhesion of the hydrogel patch 7 days after implantation in the lung leak model, where a is the negative control group, b is the positive control group, and c is S-GTO-2. Figure 20 shows the gross lung healing and HE staining results (H&E, 20×) 7 days after the hydrogel patch was implanted in the live lung leak model. In the figure, a is the negative control group, b is the positive control group, c is S-GTO-0, and d is S-GTO-2. Figure 21 The study aimed to demonstrate the repair and anti-inflammatory effects of hydrogel patches on lung leak rats, where ad represents the immunohistochemical staining of lung sections for α-SMA, IL-1β, IL-6, TNF-α, and IL-10; and f–i represents the levels of IL-1β, IL-6, TNF-α, and IL-10 in the blood. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0020] Materials and Animals: Acrylic acid (AAc, purity > 99%), N-hydroxysuccinimide acrylate (AAc-NHS, purity > 98%), photoinitiator lithium phenyl-2,4,6-trimethylbenzoyl phosphate (LAP, purity > 98%), gelatin, methacrylic anhydride (purity 94%), [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (SBMA, purity > 97%), sodium periodate (NaIO4), tris(hydroxymethyl)aminomethane (Tris), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), hyaluronic acid (HA, molecular weight = 0.69 MDa) and Triton X-100 were all provided by Aladdin Biochemical Technology Co., Ltd. (China). Ammonium persulfate (APS, 99.99% purity) and N,N'-methylenebisacrylamide (MBAA, 99% purity) were supplied by Shanghai Maclean Biotechnology Co., Ltd. (China). All chemicals met analytical grade purity requirements and were ready for use without further purification.

[0021] Human alveolar epithelial cells (A549 cells) and human bronchial epithelial cells (BEAS-2B) were purchased from Wuhan Yunclone Technology Co., Ltd. (Wuhan, China). Sprague-Dawley (SD) rats (male, 200-250 g, 7-8 weeks old) and C57BL / 6 rats (male, 18-21 g, 7-8 weeks old) were purchased from the Laboratory Animal Center of Ningbo University (Ningbo, China). All animal research experiments were conducted in accordance with the National Research Council's Guidelines for the Care and Use of Laboratory Animals, and all animal experimental protocols were approved by the ethics committee of the Affiliated Taizhou Hospital of Wenzhou Medical University (Approval No.: tzyy2025219). The experimental procedures were strictly performed in accordance with scientific standards. In vivo studies followed the 3Rs animal welfare guidelines.

[0022] A549 cells and BEAS-2B cells were cultured in RPMI 1640 medium (Gibco, USA) containing 10% fetal bovine serum (FBS; HyClone, USA) and 1× penicillin-streptomycin, and DMEM medium (Dibco, USA) containing 10% FBS, respectively. All cells were cultured in a 37°C, 5% CO2 incubator.

[0023] I. Specific Embodiment: A method for preparing a hydrogel patch with Janus asymmetric adhesion, comprising the following steps: Step 1: Preparation of the adhesion layer (GTO) First, 0.1g GelMA-T, 0.2g OHA, and 0.02g LAP were added to 10g of water and completely dissolved at 40℃. After thorough stirring, 3g AAc and 0.02g AAc-NHS were added and stirring was continued until homogeneous. Nitrogen gas was introduced into the mixture to remove oxygen. Under nitrogen protection, the solution was injected into a glass plate mold and crosslinked by 365nm ultraviolet light for 30 minutes to form a gel. The gel was then immersed in deionized water to remove biotoxic monomer molecules. After drying at 40℃ for 20 minutes, the adhesion layer was obtained, denoted as GTO-2.

[0024] Step 2: Preparation of the anti-adhesion layer (pSBMA) 2 g SBMA, 0.2 g GelMA, 0.06 g MBAA and 0.1 g APS were added to 10 g water and stirred thoroughly to obtain a mixed solution. The solution was then thermally crosslinked at 60°C for 60 minutes to obtain an anti-adhesion layer, denoted as pSBMA. Step 3: Preparation of hydrogel patches The adhesive layer obtained in step 1 is placed in a mold, and one side is immersed in the mixed solution obtained in step 2 for 30 minutes. Then, it is thermally crosslinked at 60°C for 60 minutes to obtain a hydrogel patch with Janus asymmetric adhesion properties, denoted as S-GTO-2.

[0025] The synthesis of the above-mentioned gelatin methacrylate (GelMA) was as follows: 10 g of gelatin was dissolved in 100 mL of phosphate buffered saline (PBS) at 60°C until completely dissolved. Then, 2 mL of methacrylic anhydride (MA) was slowly and evenly added dropwise. The temperature was then adjusted to 50°C, and the reaction was continued for 3 hours. 400 mL of PBS was added to terminate the reaction. After the reaction, the gel was dialyzed in deionized water for 4 days using a dialysis bag with a molecular weight cutoff of 8-14 KD. The solution was changed every 6 hours for the first 24 hours of dialysis, and then the water was changed at least three times a day thereafter. After dialysis, GelMA was obtained by freeze-drying and stored in a sealed container at -20°C for later use.

[0026] The synthesis steps of GelMA-T are as follows: 4 g of GelMA was dissolved in 200 mL of deionized water and stirred continuously at 60°C until completely dissolved. Then, 1.63 g of EDC and 1.35 g of NHS were added to the solution, and the reaction was carried out for 30 minutes. Next, 2.54 g of Tris was added, and the reaction was continued at 25°C for 48 hours. After the reaction was completed, the mixture was dialyzed in deionized water for 4 days using a dialysis bag with a molecular weight cutoff of 8-14 KD. Finally, the GelMA-T product was obtained by freeze-drying and stored in a sealed container at -20°C for later use.

[0027] Oxidized hyaluronic acid (OHA) was prepared by periodate oxidation, and the steps were as follows: Hyaluronic acid was dissolved in deionized water to obtain a 10 mg / mL hyaluronic acid solution, and 0.3 mol / L sodium periodate solution was added dropwise. The reaction system was stored in the dark for 6 hours, and then 1 mL of ethylene glycol was added to terminate the reaction. After stirring for 1 hour, the solution was dialyzed in deionized water for 4 days using a dialysis bag with a molecular weight cutoff of 3500D. After freeze-drying for 48 hours, the OHA product was obtained and stored in a sealed container at -20℃.

[0028] II. Analysis of Experimental Results All data in this study are expressed as mean ± standard deviation (mean ± SD). Unpaired t-tests, one-way ANOVA, and Tukey's test were performed using GraphPad Prism 8.0 software (GraphPad Software, USA). Data graphs were generated using GraphPad Prism 8.0 and Origin 2021 software (Origin Labs, USA). All experiments were repeated at least three times per group. p < 0.05 was considered statistically significant, and ns indicated no significant difference.

[0029] 1. Structural characterization of hydrogel patches (1) X-ray photoelectron spectroscopy (XPS, K-Alpha type, Thermo Fisher Scientific) was used to analyze the changes in chemical composition of the adhesive layer (GTO-2), the anti-adhesion layer (pSBMA) and the hydrogel patch (S-GTO).

[0030] like Figure 1 XPS results showed that the surfaces of the adhesive and anti-adhesion layers had two peaks at 401.48 eV (NH) and 399.87 eV (CN), while the peak intensity on the hydrogel patch surface was significantly enhanced at 402.07 eV (NH). Furthermore, compared to the surfaces of the adhesive and anti-adhesion layers, the hydrogel patch surface exhibited a higher intensity due to the presence of -SO3. - Two new sulfur (S 2p) peaks appeared in the group. This indicates that SBMA successfully infiltrated the original network and formed an interpenetrating network after crosslinking, thus constructing the asymmetric two-sided structure of S-GTO.

[0031] (2) To evaluate the microstructure of pSBMA gel, S-GTO transition layer and GTO, the adhesion layer (GTO-2), anti-adhesion layer (pSBMA gel) and hydrogel patch (S-GTO-2) prepared in Example 1 were frozen in a -80°C freezer for 8 hours and then freeze-dried to obtain dried samples. The internal microstructure of S-GTO was observed using a scanning electron microscope (SEM, TM-3000, Hitachi).

[0032] The cross-sectional morphology of the hydrogel was observed using transmission electron microscopy (SEM). For example... Figure 2 As shown, pSBMA exhibits a loose and porous structure, while GTO exhibits a smooth and flat structure. The transformation of the S-GTO capping layer from a smooth and flat structure to a loose and porous structure also indicates the successful synthesis of the hydrogel.

[0033] 2. Tissue Adhesion Analysis of Hydrogel Patches Experimental Methods: To determine the tissue adhesion properties of the test samples, an overlap shear test was used for evaluation. Pigskin samples were cut to 25 mm × 75 mm. Two pieces of pigskin were bonded together with an overlap area of ​​25 mm × 30 mm. Using a soft material mechanical testing instrument (INSTRON) equipped with a 1000 N force sensor, the pigskin samples were fixed to both ends of the test fixture at a constant displacement rate of 20 mm / min to test the peel strength. Each experiment was repeated at least three times to ensure accuracy. Adhesion strength was calculated using the following formula: Adhesion Strength = FMax / S, where FMax represents the maximum tensile stress (N) during the overlap shear process, and S represents the hydrogel bonding area (m²). 2 ).

[0034] To determine the adhesion properties of the test sample on moist tissue, two pieces of pigskin were moistened with an appropriate amount of liquid and then bonded to the test sample. The sample was then fixed to both ends of the testing machine fixture at a constant displacement rate of 20 mm / min. The overlap shear strength was calculated according to the aforementioned formula.

[0035] (1) Comparison of the adhesion properties of hydrogel patches before and after GelMA modification and with different concentrations of GelMA-T. To investigate the relationship between the performance of the adhesive layer and the concentration of GelMA-T, different GelMA-T concentrations were prepared according to the method in step 1 of the specific embodiment without the addition of OHA. These were designated as G1T0, GT1, GT2, GT3, GT4, and GT5, respectively. The adhesion strength of each adhesive layer on the pigskin surface was then tested. The results are as follows: Figure 3 a and Figure 3 As shown in Figure b, the adhesion strength of GT-2 (i.e., GTO-2) at a concentration of 1% GelMA-T on the pigskin surface was significantly higher than that of the other groups, while the adhesion strength of 1 wt% unmodified GelMA (GTO-0) was significantly lower than that of GTO-2. This indicates that the hydrogel has the best adhesion strength at a concentration of 1% GelMA-T, and that Tris-grafted GelMA can significantly improve the adhesion strength.

[0036] (2) Comparison of the adhesion properties of hydrogel patches before and after the addition of OHA and at different concentrations of OHA To investigate the relationship between the performance of the adhesive layer and the concentration of OHA, different OHA concentrations were prepared according to the method in step 1 of the specific embodiment, calculated by dividing the mass of OHA by the mass of water. These were designated as GTO-0, GTO-1, GTO-2, GTO-3, and GTO-5, respectively. The adhesion strength of each adhesive layer on the pigskin surface was then tested. The results are as follows: Figure 4 a and Figure 4 As shown in Figure b, GTO-0 without OHA exhibits lower adhesion than GTO-2. Notably, GTO-2 displays the highest adhesion strength—adhesion performance is closely related to the migration ability of polymer molecular chains: compared to GTO-2, the higher concentrations of OHA in GTO-3 and GTO-5 form tighter molecular chain entanglements, restricting chain migration and ultimately weakening the interfacial interaction strength.

[0037] 3. Mechanical property analysis of hydrogel patches Suitable mechanical properties (tensile and compressive properties) are crucial for achieving strong and durable adhesion within the thoracic cavity. To investigate the effect of OHA concentration on the mechanical properties of GTO hydrogels and to evaluate the enhancing effect of the PSBMA hydrogel layer on the mechanical properties of S-GTO bilayer hydrogels, we conducted mechanical tests including tensile, compressive, and cyclic compression.

[0038] (1) Experimental methods Tensile testing: The hydrogel was prepared into dumbbell-shaped specimens with dimensions of 17 mm × 2 mm × 2 mm. The dumbbell-shaped specimens were tested using a soft material mechanical testing instrument (INSTRON) equipped with a 10N force sensor at a constant displacement rate of 20 mm / min.

[0039] Compression test: The hydrogel was made into a cylindrical sample with a diameter of 16 mm and a height of 6 mm. The cylindrical sample was compressed at a constant displacement rate of 20 mm / min using a soft material mechanical tester with a 1000 N mechanical sensor (INSTRON). The stress value when the strain reached 80% was taken as the compressive strength of the hydrogel sample. Cyclic compression test: To evaluate the durability of the hydrogel under repeated compression, the specimen was compressed to 80% of the strain at a rate of 20 mm / min, and then three repeated compression-unloading cycles were performed at the same rate.

[0040] (2) Analysis of experimental results: like Figure 5 As shown, the tensile test results indicate that ( Figure 5 a): With the increase of OHA content, the stretchability of the material first increases and then decreases, while Young's modulus first decreases and then increases. Figure 5 b) In compression tests, GTO-0 exhibited poor compression performance due to the lack of OHA. The compression performance gradually improved with increasing OHA content. Figure 5 c). S-GTO-2 exhibits greater toughness due to the introduction of the SBMA network, resulting in a significantly improved compressive modulus. Figure 5 d).

[0041] 4. Sealing performance analysis of hydrogel patches To evaluate the sealing performance of S-GTO, liquid tightness and air tightness tests were conducted. Liquid tightness and air tightness tests: Standard-sized wounds were made on the surface of water-injected pig hearts, stomachs, and lungs, and S-GTO was applied before observing the sealing effect.

[0042] The results are as follows Figure 6As shown in Figure ac, S-GTO-2 can achieve rapid sealing on the surface of water-filled pig stomachs and hearts, and maintain its integrity even after continuous water injection. Similarly, S-GTO-2 can tear leaking lung lobes, demonstrating rapid airtight sealing, and maintain its integrity even after continuous inflation.

[0043] 5. Swelling analysis of hydrogel patches To evaluate the swelling behavior of each component of GTO and S-GTO, hydrogel patches of specific sizes were immersed in phosphate-buffered saline (PBS, pH=7.4) and incubated at 37°C with shaking at 60 rpm. At specific time points, the gels were removed, and residual moisture was gently blotted off with absorbent paper. The mass (Wt) after moisture absorption was measured. The swelling ratio (SR) was calculated using the following formula: SR = (Wt - W0) / W0 × 100%, where W0 represents the initial weight of the freshly prepared hydrogel patch on day 0, and Wt represents the weight of the hydrogel patch after swelling on day t. Each experiment was repeated four times.

[0044] The results are as follows Figure 7 As shown in ab, all hydrogels initially exhibited rapid water absorption, with GTO-5 showing the highest absorption rate. The absorption rate then gradually decreased until swelling equilibrium was reached. S-GTO-2 showed a significant difference in swelling ratio compared to GTO-2. S-GTO-2 had a lower swelling ratio, which contributes to long-term tissue adhesion, primarily due to its more stable bilayer network structure.

[0045] 6. Analysis of the water retention performance of hydrogel patches A hydrogel sample of a specific size was completely dried in a freeze dryer. Then, deionized water was added to swell the sample until equilibrium was reached. After measuring the total water absorption, the sample was placed in an empty beaker, and the residual mass of the hydrogel was measured every 24 hours at 37°C. The water retention rate (Wr) was calculated using the following formula: Wr = (W0 - Wt) / (W0 - W) × 100%, where Wr is the water retention rate, W0 is the mass of the hydrogel at swelling equilibrium, Wt is the remaining mass of the hydrogel after different times, and W is the absolute dry mass of the hydrogel.

[0046] The results are as follows Figure 8 As shown in Figure ab, the water retention performance curves of the hydrogels are illustrated: In the first few days, the hydrogels experienced rapid water loss, with GTO-0 showing the highest water loss rate and GTO-5 showing the slowest. Compared to monolayer GTO-2 hydrogels, the water retention performance of bilayer S-GTO-2 hydrogels is significantly improved.

[0047] 7. In vitro cell compatibility analysis of hydrogel patches Cell compatibility is a key indicator for wound dressing materials, especially when applied to damaged tissues. Cytotoxicity and proliferation behavior are crucial in biocompatibility assessment. A549 and BEAS-2B cells were tested using a live / dead cell staining method and a CCK8 assay kit to analyze the cytotoxicity and cell proliferation behavior of hydrogel patches.

[0048] To evaluate the in vitro cell compatibility of GTO and S-GTO, different masses of hydrogels were weighed and immersed in 37°C culture medium for 24 hours to prepare extraction media. Cells were then treated with GTO extraction media, S-GTO extraction media, and the original culture medium, respectively.

[0049] (1) Live / dead cell staining: A549 cells and BEAS-2B cells were stained at 1×10⁻⁶ cells per well. 4 Cells were seeded in 96-well plates and cultured for 24 h with 100 μg / ml GTO-0, GTO-2, S-GTO-2 hydrogel extraction media or LPS. Cell viability was assessed using a live / dead cell staining kit (Beyotime Biotechnology, China): under a fluorescence microscope (Olympus, Japan), live cells stained with calcein-AM showed green fluorescence, while dead cells stained with propidium iodide (PI) showed red fluorescence. Results are as follows: Figure 9 Fluorescence images of A549 and BEAS-2B cells showed that the hydrogels in all groups were non-toxic.

[0050] (2) CCK-8 assay: A549 cells and BEAS-2B cells were respectively stored at 1×10⁻⁶ cells per well. 4 Each sample was inoculated into a 96-well plate and co-incubated with different concentrations of S-GTO-2 for 24 and 48 hours (10, 20, 50, 100, 200, and 500 μg·mL⁻¹, respectively). -1 Add 10 μL of CCK-8 solution (MCE, New Jersey, USA) to each well and incubate for 1 hour. Detect absorbance (OD) at 450 nm using a Varioskan LUX system (Thermo Fisher Scientific, Massachusetts, USA). Cell viability is calculated using the following formula: Cell viability (%) = [(OD) / (Cell viability)] 样品 - OD 培养基 ) / (OD 对照 - OD 培养基 )] × 100%, where: OD 样品 OD values ​​representing different concentration groups of GTO-0, GTO-2, and S-GTO-2; OD 培养基 The OD value represents the pure culture medium; OD 对照 This represents the OD value of the control group.

[0051] The results are as follows Figure 10 As shown in Figures a and b, cell compatibility tests on the free gel concentration gradient showed that when the free gel concentration increased to 200 μg·mL⁻¹, the cell compatibility was significantly improved. -1 At 48 h, the viability of both A549 and BEAS-2B cells decreased. The CCK8 assay further quantified the cell compatibility of the hydrogels: the results showed that GTO-0, GTO-2, and S-GTO-2 gels did not significantly inhibit the growth of A549 and BEAS-2B cells; on the contrary, they promoted cell proliferation to some extent. 8. Blood compatibility analysis of hydrogel patches Hemolysis test: 100 mg of GTO-0, GTO-2, and S-GTO-2 were placed in 1 mL of PBS (10×) and soaked at 37°C for 24 hours to prepare extracts. 1 mL of fresh rat blood was placed in an EDTA anticoagulant tube, centrifuged at 2000 rpm and 4°C for 10 minutes, the supernatant was discarded, and the blood was washed four times with 0.9% physiological saline to remove excess serum. A 2% erythrocyte suspension was then prepared with PBS. The erythrocyte suspension and hydrogel extract were mixed at a 9:1 ratio. PBS was used as a negative control, and 1% Triton X-100 was used as a positive control. The mixture was incubated at 37°C for 2 hours. After incubation, the mixture was centrifuged at 2000 rpm and 4°C for 10 minutes. 100 μL of the supernatant from each group was added to a 96-well plate, and the absorbance (OD value) at 541 nm was measured using a microplate reader. The hemolysis rate was calculated as follows: Hemolysis rate (%) = (OD value) / (GTO-0, GTO-2, and S-GTO-2) Sa - OD Ne ) / (OD Po -OD Ne ) × 100%, where OD Sa OD value of the experimental group Ne The OD value is for the negative control group. Po The OD value is for the positive control group.

[0052] Hemolysis rate is another important indicator of biocompatibility, and the results are as follows: Figure 11 As shown, the hemolysis rates of the gels in the GTO-0, GTO-2, and S-GTO-2 groups were all between 0.9% and 2.0%, which are far below the safety threshold of 5%.

[0053] 9. Analysis of the migration-promoting ability of hydrogel patches Cell scratch assay: 3 × 10 4 A549 cells and BEAS-2B cells / well were seeded in 6-well plates and cultured for 24 hours. First, a scratch was made at the bottom of each well using a pipette tip, and then the cells were gently washed with PBS to remove free cells. Then, FBS-free medium was added to each well, and 100 μg / mL of the medium was added. -1Cells were co-cultured with hydrogel extraction medium or LPS. Cells with serum-free medium were used as a control group. Images were taken at 0 h and 24 h using a fluorescence microscope (Olympus, Japan), and scratch width was measured using ImageJ software. The migration rate was calculated as follows: Migration rate (%) = [(S0 - S...] 24 ) / S0] × 100%, where: S0 represents the initial scratch area, S 24 The area of ​​the scratch represents the area after 24 hours of cultivation.

[0054] like Figure 12 As shown in Figure a, the A549 and BEAS-2B cell migration experiments demonstrated that GTO-0, GTO-2, and S-GTO-2 gels not only maintained good biocompatibility but also effectively promoted cell migration. Meanwhile, as... Figure 12 As shown in Figure b, the cell migration rate in the GTO-2 and S-GTO-2 groups was significantly higher than that in the control group. All results confirm that the S-GTO-2 gel possesses both excellent biocompatibility and migration-promoting ability, meeting the application standards for wound dressings.

[0055] 10. In vitro antibacterial activity analysis of hydrogel patches In cases of air leakage from lung wounds, bacterial infection is highly likely, making the antibacterial properties of the patch crucial. To assess the antibacterial properties of GTO and S-GTO-2, we tested them against Gram-positive Staphylococcus aureus and Gram-negative E. coli.

[0056] Experimental methods: This method targets Gram-positive Staphylococcus aureus (Staphylococcus aureus) S.aureus ) and Gram-negative Escherichia coli ( E. coli The antibacterial properties of the hydrogel were tested to evaluate its antibacterial effect. The simplified procedure is as follows: The logarithmic growth phase bacterial culture was diluted with PBS (pH 7.4) to a concentration of 5 × 10⁻⁶. 6 CFU / mL bacterial suspensions were evenly and smoothly spread across the entire surface of culture dishes. A cylindrical hydrogel (6 mm × 6 mm) was then placed on top of the dish, and the dishes were incubated at 37°C for 24 hours to observe the results. Experimental groups included a control group (pure bacterial suspension), pSBMA group, GTO-2 group, and S-GTO-2 group.

[0057] The results are as follows Figure 13 As shown in ab, except for PSBMA, no obvious bacterial colonies grew around the other hydrogels, demonstrating good antibacterial properties.

[0058] 11. Analysis of ROS scavenging ability of hydrogel patches Bacterial colonization in wound tissue not only triggers inflammatory responses but also leads to the overexpression of reactive oxygen species (ROS). It is well known that overexpressed ROS in infected wound tissue damages normal cells and exacerbates the inflammatory response, creating a vicious cycle. Therefore, antioxidant capacity is crucial for wound healing.

[0059] Reactive oxygen species (ROS) analysis: Bone marrow-derived macrophages (BMDMs) were stimulated with 0.5 mM H2O2 as follows: A549 cells and BEAS-2B cells were mixed at a ratio of 1 × 10⁶ cells per well. 4 Cells were seeded in 96-well plates. Cells stimulated with H2O2 were inoculated with 100 μg / mL water. -1 After co-incubation for 6 hours in hydrogel extraction media (GTO-0, GTO-2 groups, and S-GTO-2), 10 μM MDFH-DA stock solution was diluted 1:1000 with serum-free medium and incubated with cells at 37°C for 20 minutes. After washing three times, the cells were analyzed and observed using FlowJo flow cytometry and fluorescence microscopy.

[0060] The results are as follows Figure 14 As shown, the ROS expression rates of the positive control group, GTO-0 group, GTO-2 group, S-GTO-2 group and negative control group were 24.96%, 7.85%, 2.00%, 2.19% and 0.35%, respectively.

[0061] 12. In vitro anti-inflammatory and macrophage phenotype-regulating effects of hydrogel patches Macrophages are the main immune cells in lung wound tissue, secreting pro-inflammatory factors (TNF-α, IL-6, IL-1β) and anti-inflammatory factors (IL-10) according to their phenotype. While M1 macrophages in infected wounds can clear phagocytic bacteria, overactivated M1 macrophages can exacerbate the inflammatory response and delay collagen deposition, thus hindering wound healing. Therefore, regulating macrophage phenotype is crucial for promoting wound healing. We first tested their regulatory capacity by co-culturing lipopolysaccharide (LPS, 200 ng / ml)-activated M1 macrophage membrane dendritic cells (BMDMs) with gel extraction media. The relative expression levels of CD86, CD206, and inducible nitric oxide synthase (iNOS) were detected by Western blotting (WB).

[0062] (1) Experimental methods Macrophage polarization: C57BL / 6 cells were induced for 3-5 days by intraperitoneal injection of 1.5-2 mL of 4% mercaptoacetate broth. After mouse sacrifice, the peritoneal cavity was aseptically flushed with 8-10 mL of pre-cooled PBS, and the flushing fluid was collected and centrifuged at 1200 rpm for 8 minutes. The pellet was resuspended in complete culture medium. A concentration of 5 × 10⁻⁶ cells was then used. 5Peritoneal macrophages were seeded per well in 6-well plates and cultured at 37°C and 5% CO2 for 2–4 hours. Non-adherent cells were washed away to obtain adherent macrophages. M1 polarization was then induced by stimulation with LPS (200 ng / mL) and IFN-γ (20 ng / mL) for 24 hours. Subsequently, 100 μg / mL... -1 Hydrogel extraction media (GTO-0, GTO-2, and S-GTO-2 groups) were co-incubated with cells for 24 hours. Peritoneal macrophages without any other treatment were stimulated with LPS as a control group.

[0063] Western Blot (WB) Experiment: Cells were collected and total protein was extracted according to methods described in previous literature. Antibodies used included: rabbit anti-CD206 (1:1000; CST), rabbit anti-CD86 (1:1000; CST), rabbit anti-iNOS (1:1000; CST), rabbit anti-tubulin (1:1000; CST), and goat anti-rabbit secondary antibody (1:10000; Beyotime Biotechnology). β-actin was used as an internal control protein to standardize the protein expression levels of CD206, CD86, and iNOS.

[0064] In the enzyme-linked immunosorbent assay (ELISA), the supernatant from each well was collected immediately, and the protein levels of the inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-10 (IL-10) were detected using an ELISA kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions. To evaluate the synergistic inhibitory effect of the hydrogel on pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6), the above ELISA method was used, with control, GTO-0, GTO-2, and S-GTO-2 groups. All data were standardized with respect to the control group, and each group was tested three times.

[0065] (2) Experimental results The results are as follows Figure 15 As shown in a, the WB results showed that S-GTO-2 gel significantly downregulated CD86 and iNOS expression, while upregulating CD206 expression. Figure 15 a). Figure 15 Quantitative analysis showed that the relative expression levels of CD86 and iNOS decreased by 2-3 times in the S-GTO-2 group, while the expression level of CD206 increased by 5 times. The experimental results indicate that S-GTO-2 gel exhibits significant phenotypic regulatory function by regulating the transformation of macrophages from M1 to M2 phenotypes and inhibiting inflammatory responses.

[0066] The regulatory effect was further verified by detecting classic pro-inflammatory factors (TNF-α, IL-6, IL-1β) and anti-inflammatory factors (IL-10) using an ELISA kit. Figure 16 As shown in the diagram, the levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β in the S-GTO-2 group were significantly lower than those in the control group; conversely, the level of anti-inflammatory factor IL-10 was significantly higher than that in the control group. Based on the synergistic antioxidant effects of GelMA-T and OHA, we further explored their synergistic anti-inflammatory effects: ELISA results showed that both the GTO-2 and GTO-0 groups significantly inhibited the release of pro-inflammatory factors and promoted the release of anti-inflammatory factors, but the effect was more pronounced in the GTO-2 group than in the GTO-0 group, confirming the synergistic effect of GelMA-T and OHA in inhibiting pro-inflammatory factors.

[0067] 13. Biocompatibility and anti-adhesion ability of hydrogel patches in vivo (1) Experimental methods In vivo degradation assay: To assess the in vivo biocompatibility of GTO and S-GTO, hydrogels were implanted subcutaneously into the back tissue of SD rats. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital. After disinfection of the back skin, the hydrogels were implanted, and penicillin was administered postoperatively to prevent infection. Rats were sacrificed at 1, 2, 3, and 4 weeks post-implantation, and back tissue was collected for photographic recording and H&E staining to observe material degradation.

[0068] Anti-adhesion experiment: To evaluate the anti-adhesion effect of S-GTO in rats, a rat cecal adhesion model was used. SD rats were anesthetized by intraperitoneal injection of sodium pentobarbital. The skin was incised in the left lower abdomen, and the cecum was gently exposed. The surface of the cecum was repeatedly rubbed with sterile gauze until obvious bleeding points appeared. The experimental group had S-GTO and GTO covered on the surface of the damaged cecum, while the control group received no treatment. Penicillin was administered postoperatively to prevent infection. Two weeks later, the rats were sacrificed, and the adhesion was observed by laparotomy. Tissue was collected for H&E staining and Masson staining.

[0069] (2) Experimental results In addition to in vitro cell compatibility assessment, this study further investigated the in vivo biocompatibility and in vivo degradation performance by subcutaneously implanting S-GTO-2 and GTO-2-based materials into the backs of rats. Figure 17 a) At weeks 1, 2, 3, and 4 post-implantation, the gel was separated from the surrounding tissue and weighed to assess its in vivo degradation behavior. From Figure 17 As can be seen from b, a fibrous capsule structure had formed on the tissue surface during the first week, encapsulating S-GTO-2 and GTO-2. During the first week, the hydrogel increased in volume due to swelling. With the progress of metabolism, by the fourth week, both S-GTO-2 and GTO-2 showed good degradation, decreasing to 25% of their weight in the first week after implantation (4 weeks later). Figure 17 c).

[0070] To evaluate the anti-adhesion properties of S-GTO-2, a cecal injury model was established. Figure 18 Masson staining results in ac showed that fibrous tissue was present between the skin and cecum in the untreated group and the GTO-2 group, while no obvious fibrous tissue was observed in the S-GTO-2 group due to the effect of PSBMA. These results demonstrate the good anti-tissue adhesion properties of SBMA material.

[0071] 14. Performance of hydrogel patches in repairing and healing air leaks in living lungs To evaluate the repair effect of S-GTO hydrogel patches on lung leak wounds, a rat lung leak model was established in this study. All rats survived to day 7 post-surgery throughout the experimental period, and no adverse reactions or complications related to S-GTO were observed, indicating that the material has good biocompatibility.

[0072] (1) Experimental methods Lung Leak Repair Experiment: To evaluate the lung wound repair and healing capabilities of hydrogels, a lung leak model was used. SD rats were placed in a closed induction chamber connected to a small animal anesthesia machine (such as a Matrx product or an equivalent system). The initial induction concentration was 3%-3.5% isoflurane mixed with oxygen (flow rate: 2-3 L / min) until the rats lost consciousness. After induction, the rats were quickly transferred to the operating table, and anesthesia was maintained using a face mask. The isoflurane concentration was reduced to 1.7%-2.5% (adjusted according to the operation time), and the oxygen flow rate was adjusted to 0.3-0.5 L / min. The body temperature was maintained at (37 ± 0.5)℃ throughout the procedure using an animal thermometer. The rats were fixed in a supine position, and the anterior neck area was shaved and disinfected. A longitudinal incision was made along the midline of the neck, and the subcutaneous tissue and sternohyoid muscle were separated to expose the trachea. The trachea was incised between the two cartilaginous rings (approximately 1 / 3 of the circumference), and an indwelling intravenous catheter (18G) was quickly inserted. The catheter was connected to the ventilator and secured with tape to prevent dislodgement. The rats were mechanically ventilated at a ventilation rate of 40 breaths / min and a tidal volume of 4 mL. After respiratory stability, a left lateral thoracotomy was performed in the fifth intercostal space. A 23-gauge needle was used to puncture the left lung 2 mm from the lung surface to create an air leak wound; air leakage was confirmed by observing air bubbles forming at the wound site. In the experimental group, the damaged lung surface was covered with S-GTO-2 or S-GTO-0, while the positive control group received no treatment. In the negative control group, the thoracic cavity was closed directly after a left lateral thoracotomy in the fifth intercostal space without creating an air leak wound. On postoperative day 7, the rats were euthanized with a high dose of anesthetic (pentobarbital, 0.3 mg / g), and a left lateral thoracotomy was performed to examine the treated area. Left lung tissue, including the surgical site, was harvested and fixed in a 4% formaldehyde solution.

[0073] H&E staining and Masson staining: Major organ and tissue samples were fixed in 4% paraformaldehyde and then embedded in paraffin. Depending on experimental requirements, paraffin sections were dewaxed to water, or frozen sections were thawed and fixed before hematoxylin-eosin (H&E) staining or Masson trichrome staining, respectively. The H&E staining procedure included pretreatment, hematoxylin staining, differentiation and blueing, eosin counterstaining, and a series of ethanol dehydration steps, followed by xylene clearing and mounting with neutral resin. Masson trichrome staining involved overnight soaking in solution A, staining with a mixture of solutions B and C, immersion in solutions D, E, and F, differentiation with 1% acetic acid, dehydration with anhydrous ethanol, clearing with xylene, and mounting. All stained sections were examined under a microscope and images were acquired and analyzed (Nikon, Japan).

[0074] Immunohistochemical staining: The expression of α-SMA, TNF-α, IL-6, IL-10, and IL-1β in lung tissue obtained from the lung leak repair experiment was detected. Immunohistochemical analysis was performed on paraffin-embedded lung tissue sections. Sections were dewaxed using a gradient of xylene and ethanol, and antigen retrieval was performed with 0.1 M citrate buffer. After treatment with peroxidase blocking solution for 25 minutes, non-specific binding sites were blocked with donkey serum. The sections were then incubated overnight at 4°C with diluted primary antibodies (IL-1β, 1:800; TNF-α, 1:500; α-SMA, 1:500; IL-6, 1:200; IL-10, 1:500). Following incubation with horseradish enzyme-labeled goat anti-mouse polymer secondary antibody (Zhongshan Jinqiao, Beijing, China), a colorimetric reaction was performed using DAB chromogenic solution (Zhongshan Jinqiao, Beijing, China). After hematoxylin counterstaining, the slides were mounted with neutral resin and finally observed using an upright optical microscope (Nikon, Japan).

[0075] (2) Analysis of experimental results like Figure 19 Gross observation on day 7 post-ac surgery showed significant adhesions between the lung tissue and pleural cavity in the positive control group, while no significant adhesions were observed in any of the S-GTO treatment groups. Among them, the wound closure of the S-GTO-2 group was the most significant, superior to that of the positive control group.

[0076] like Figure 20 As shown in the ad, hematoxylin and eosin (H&E) staining results further revealed the tissue-level repair status: the negative control group showed intact lung tissue structure and clear cell boundaries; the positive control group exhibited typical pathological features of spontaneous healing of lung leaks, including nuclear condensation, deep staining, and cytoplasmic shrinkage, accompanied by significant inflammatory infiltration. In contrast, the S-GTO treatment groups only induced mild local inflammation at the application site, with the S-GTO-2 group showing the mildest inflammatory response. The preparation method of the hydrogel product in the S-GTO-0 group was the same as that in S-GTO-2, the difference being the absence of OHA.

[0077] Collagen deposition and myofibroblast activation are key steps in the wound healing process. Figure 21 Immunohistochemical staining results showed that, compared with the untreated control group (positive), the expression of α-smooth muscle actin (α-SMA) in the wound margin and granulation tissue region of the S-GTO treatment group was significantly upregulated. The positive staining cells were dense and of high intensity, suggesting that myofibroblasts were effectively activated and recruited, thereby enhancing tissue contraction and repair capabilities and accelerating wound closure. To elucidate the regulatory role of adhesive treatment on the inflammatory response during the healing of air leak wounds, we analyzed the expression changes of key inflammatory factors at both the local tissue and systemic circulation levels. Figure 21 Immunohistochemical staining of lung tissue showed that, compared with the untreated control group, the expression of pro-inflammatory factors TNF-α, IL-1β, and IL-6 in the wound area of ​​the treated group was significantly downregulated, with a significant decrease in both positive signal intensity and the number of positive cells. Conversely, the expression of the anti-inflammatory factor IL-10 was specifically upregulated, exhibiting a stronger staining signal and more positive cells. This indicates that the adhesive treatment effectively remodeled the local immune microenvironment of the wound, transforming it from a pro-inflammatory state to an anti-inflammatory and repair-promoting state.

[0078] Meanwhile, ELISA testing of serum samples on postoperative day 7 further validated this anti-inflammatory effect at the systemic level. Figure 21 As shown in Figure 1, the concentrations of TNF-α, IL-1β, and IL-6 in the circulatory system of rats in the adhesive patch group were significantly reduced, while the concentration of IL-10 was significantly increased. This result is highly consistent with the findings at the tissue level, jointly indicating that adhesive patch intervention not only inhibits excessive local inflammation but also induces a beneficial systemic anti-inflammatory response, creating a favorable immune environment for wound regeneration.

[0079] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. A method for preparing a hydrogel patch with Janus asymmetric adhesion, characterized in that... Includes the following steps: Step 1: Preparation of the Adhesion Layer After mixing GelMA-T, oxidized hyaluronic acid (OHA), photoinitiator (LAP), acrylic acid (AAc), and N-hydroxysuccinimide acrylate (AAc-NHS) evenly, the solution is injected into a glass plate mold under nitrogen protection. After cross-linking by ultraviolet light to form a gel and drying, the adhesion layer is obtained. Step 2: Preparation of hydrogel patches [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide SBMA, gelatin methacrylate GelMA, N,N'-methylenebisacrylamide MBAA, and ammonium persulfate APS are added to water and thoroughly mixed to obtain a mixed solution. The adhesive layer obtained in step 1 is immersed on one side of the mixed solution and thermally crosslinked to obtain a hydrogel patch with Janus asymmetric adhesion properties.

2. The method for preparing a hydrogel patch with Janus asymmetric adhesion according to claim 1, characterized in that: Step 1: Preparation of the Adhesion Layer Add 0.05-0.2g GelMA-T, 0.1-0.3g OHA, and 0.01-0.03g LAP to 5-20g of water, dissolve completely at 40℃ and stir thoroughly. Then add 1-5g AAc and 0.01-0.03g AAc-NHS and continue stirring until homogeneous. After purging the mixture with nitrogen to remove oxygen, inject the solution into a glass plate mold under nitrogen protection. Crosslink the solution by irradiation with 365nm ultraviolet light for 20-40 minutes to form a gel. Then immerse the gel in deionized water to remove biotoxic monomer molecules. After drying at 30-50℃ for 10-30 minutes, the adhesion layer is obtained. Step 2: Preparation of hydrogel patches Add 1-3 g SBMA, 0.1-0.3 g GelMA, 0.04-0.08 g MBAA and 0.05-0.2 g APS to 5-20 g water and stir thoroughly to obtain a mixed solution. Place the adhesive layer obtained in step 1 into a mold and mix the solution on one side for 20-40 minutes. Then, perform thermal crosslinking at 50-70℃ for 50-70 minutes to obtain a hydrogel patch with Janus asymmetric adhesion properties.

3. The method for preparing a hydrogel patch with Janus asymmetric adhesion according to claim 2, characterized in that: Step 1: Preparation of the Adhesion Layer 0.1g GelMA-T, 0.2g OHA and 0.02g LAP were added to 10g water, dissolved completely at 40℃ and stirred thoroughly. Then, 3g AAc and 0.02g AAc-NHS were added and stirred until homogeneous. Nitrogen gas was introduced into the mixture to remove oxygen. The solution was then injected into a glass plate mold under nitrogen protection. After crosslinking by 365 nm ultraviolet light for 30 minutes, a gel was formed. The gel was then immersed in deionized water to remove biotoxic monomer molecules. After drying at 40℃ for 20 minutes, the adhesion layer was obtained. Step 2: Preparation of hydrogel patches Add 2 g SBMA, 0.2 g GelMA, 0.06 g MBAA and 0.1 g APS to 10 g water and stir thoroughly to obtain a mixed solution. Place the adhesive layer obtained in step 1 into a mold, mix the solution on one side for 30 minutes, and then thermally crosslink at 60°C for 60 minutes to obtain a hydrogel patch with Janus asymmetric adhesion properties.

4. A method for preparing a hydrogel patch with Janus asymmetric adhesion according to any one of claims 1-3, characterized in that... The synthesis steps of GelMA are as follows: Dissolve 8-12 g of gelatin in 100 mL of phosphate buffer at 60°C until completely dissolved. Then, slowly and evenly add 1-3 mL of methacrylic anhydride. Adjust the temperature to 50°C and continue the reaction for 3 hours. Add 400 mL of phosphate buffer to terminate the reaction. After the reaction is completed, dialyze the gelatin in deionized water using a dialysis bag with a molecular weight cutoff of 8-14 KD. After dialysis, freeze-dry to obtain gelatin methacrylic acid.

5. The method for preparing a hydrogel patch with Janus asymmetric adhesion according to claim 4, characterized in that... The synthesis steps of GelMA-T are as follows: 3-5 g of GelMA is dissolved in 200 mL of deionized water and stirred continuously at 60°C until completely dissolved. Then, 1.63 g of EDC and 1.35 g of NHS are added to the solution and reacted for 20-40 minutes. Next, 2.54 g of Tris is added and the reaction is continued at 25°C for 48 hours. After the reaction is completed, the mixture is dialyzed in deionized water for 4 days using a dialysis bag with a molecular weight cutoff of 8-14 KD. Finally, the GelMA-T product is obtained by freeze-drying.

6. A method for preparing a hydrogel patch with Janus asymmetric adhesion according to any one of claims 1-3, characterized in that... The oxidized hyaluronic acid was prepared by periodate oxidation, and the steps are as follows: 0.2-0.4 mol / L sodium periodate solution was added dropwise to 8-12 mg / mL hyaluronic acid solution, and the reaction system was stored in the dark for 4-8 hours. Then, ethylene glycol was added to terminate the reaction. After stirring, the mixture was dialyzed in deionized water for 4 days using a dialysis bag with a molecular weight cutoff of 3500D. The product was then freeze-dried to obtain OHA.

7. The use of a hydrogel patch prepared by any one of claims 1-3 in the preparation of a lung leak repair agent.

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