Polyvinyl alcohol hydrogel patch with double-disc structure as well as preparation method and application of polyvinyl alcohol hydrogel patch

Abstract

The invention provides a polyvinyl alcohol hydrogel patch with a double-disc structure as well as a preparation method and application of the polyvinyl alcohol hydrogel patch. The preparation method comprises the following steps: S1, preparing a PVA solution; s2, the PVA solution in the step S1 is poured into a mold of a double-disc structure, and t-PVA is obtained through a freezing pouring method; s3, the t-PVA in the step S2 is subjected to salting-out treatment, and a reinforced hydrogel patch a is obtained; s4, performing solvent exchange assisted wet annealing on the reinforced hydrogel patch a in the step S3 to obtain a reinforced hydrogel patch b; and S5, carrying out swelling equilibrium treatment on the reinforced hydrogel patch b in the step S4 to obtain e-PVA. According to the preparation method provided by the invention, the crystallinity and molecular entanglement degree of the hydrogel patch are remarkably improved, so that the hydrogel patch has a compact porous structure, tough mechanical firmness, excellent elasticity and excellent water locking property. The polyvinyl alcohol hydrogel patch obtained by the preparation method provided by the invention has good biocompatibility and a full-layer repair effect.

Description

Technical Field

[0001] The present invention relates to the field of biomaterials, and particularly relates to a polyvinyl alcohol hydrogel patch with a double-disk structure, a preparation method thereof, and an application thereof. Background Art

[0002] Tracheal fistula (TF) refers to a pathological state in which there is communication between the trachea and adjacent cavities or organs such as the pleural cavity, esophagus, and stomach, forming a fistula or fistulous opening. Currently, the clinical treatment methods for TF mainly include surgical treatment and stent occlusion. Surgical treatment usually involves resection of the tissue around the fistulous opening, followed by suture or reconstruction. For those with larger tissue defects around the fistulous opening, in addition to directly suturing the fistulous opening, autologous tissues such as pericardium, muscle tissue, and pedicled skin flaps are often required for repair. In addition to conventional surgery, thoracoscopic and mediastinoscopic surgeries also have successful cases. Surgeons often need to design personalized surgical methods according to the nature and size of the fistulous opening. When necessary, partial tracheal resection and anastomosis, lobar anastomosis, etc. are also required. The process is not only cumbersome but also has certain limitations, such as severe local infection after surgery, edema and fibrosis of the tissue around the fistulous opening, and non-durable repair effects. Stent occlusion of the fistulous opening can quickly restore the integrity of the digestive or respiratory tract, quickly relieve symptoms, and this method has less trauma, simple operation, and is relatively safe in the short term, and has become an important treatment method for TF.

[0003] Gastric perforation (GP) refers to a serious clinical symptom in which the integrity of the gastric wall is damaged, resulting in the leakage of gastric contents (such as gastric acid, food, etc.) into the abdominal cavity, thereby causing acute peritonitis. Gastric perforation is an acute abdomen with a relatively high mortality rate and complication rate. When gastric perforation occurs, gastric contents, bile, and gastric acid are easily leaked into the abdominal cavity, causing abdominal cavity pollution, peritonitis, abdominal abscess, etc. In severe cases, toxic shock may occur, leading to multiple organ failure. Surgery is the main means of treating gastric perforation. Different surgical methods are selected according to the location, size, and cause of the perforation. Common surgical methods include simple suture, partial gastrectomy, gastroenterostomy, etc. For some small perforations, endoscopic treatment can be attempted, such as endoscopic clipping, covered stent implantation, etc., to reduce trauma and postoperative complications. Gastric perforation or local gastric defects can now be repaired by a variety of synthetic materials, including polyglycolic acid, polylactic acid, poly(lactic-co-glycolic acid), polyester polyurethane, and polyhydroxyalkanoates. However, these synthetic materials have many problems in terms of mechanical strength, acid resistance, plasticity, and tissue adhesion, and most of them are non-degradable materials, making it difficult to achieve the characteristics of tissue repair, remodeling, and growth promotion.

[0004] Tracheoesophageal fistula (TEF) refers to the pathological connection caused by the abnormal communication between the posterior wall of the trachea or bronchus and the anterior wall of the adjacent esophagus. TEF can occur in the cervical trachea, the junction of the neck and chest, or the mediastinal trachea. TEF usually causes dysphagia, recurrent aspiration pneumonia, and severe weight loss in patients, having a huge impact on the health and life of patients. The current treatment plan for TEF is still mainly surgery. With the development of clinical technology, there are currently more options for surgical repair of TEF, including tracheoesophageal fistula resection, separate repair of the tracheoesophageal fistula opening, resection of the diseased ends of the trachea / esophagus and anastomosis, etc. In addition to traditional surgical repair methods, the continuous innovation of biomaterials has also brought great help to the treatment of TEF. Stent implantation, including tracheal stent implantation, esophageal stent implantation, synchronous double-stent implantation of the esophagus and trachea, etc., is an important treatment method for tracheal obstruction and fistulas caused by malignant or benign lung and esophageal diseases. In recent years, some scientists have extracted fibrinogen, thrombinogen, thrombin, calcium chloride and other substances from animal blood and made them into biological protein glue to repair TEF. Although it has the advantages of less local irritation, rapid solidification, high glue bonding strength, and high antibacterial property, when using biological protein glue for TEF with a larger fistula opening, the risk of the glue flowing into the trachea greatly increases, and the viscosity of the glue is difficult to control, and the consequences brought by these are unimaginable.

[0005] Currently, there are few materials available on the market for repairing defects in organs such as the stomach, trachea, and esophagus. Most of them are metal stent materials with poor biocompatibility and non-degradability. Clinical needs have prompted researchers to strive to develop biomaterials with lasting stability, leak prevention, toughness, bioabsorbability, and compatibility with the dynamic environment of the respiratory tract.

[0006] The current biomaterials have the following problems: (1) Insufficient biocompatibility: Biomaterials should have good biocompatibility, not cause obvious foreign body reactions and rejection reactions, and be able to combine well with surrounding tissues and promote healing.

[0007] (2) Insufficient mechanical properties: Biomaterials should have sufficient mechanical strength and stability to withstand the deformation during tracheal breathing and movement, be not easily broken or deformed, and ensure the stability and durability of the repair layer.

[0008] (3) Acid and enzyme intolerance: The biomaterials of the trachea should have certain properties to tolerate the multi-mucus environment of the respiratory tract. In some tracheoesophageal fistulas, the gastric acid in the digestive tract also puts certain requirements on the acid tolerance of the biomaterials.

[0009] (4)Biodegradation resistance: Biomaterials should have controllable degradation properties, be able to gradually degrade within a certain period of time and fuse with natural tissues, thereby promoting the regeneration and repair of the defect site.

[0010] (5)Difficulty in achieving tissue repair: How to achieve the structural and functional repair of the defect site has always been a major challenge in repair.

[0011] (6)Difficulty in the combination of the repair area with the surrounding natural tissues: Biomaterials should be able to combine well with the surrounding natural tissues to form a continuous and seamless repair layer, reduce the risk of repair layer shedding, and improve the stability and durability of the repair effect.

[0012] Therefore, there is an urgent need for a new type of repair material with excellent biocompatibility, adjustable degradation properties and strong mechanical properties.

[0013] Polyvinyl alcohol (PVA) hydrogel, as a new type of biomaterial, can be enhanced in terms of mechanical properties and biological properties through some improvement techniques and become an ideal repair material for gastric, tracheal and esophageal defect sites. However, PVA hydrogel still has some disadvantages and deficiencies. First, the hydrogel prepared by using PVA alone may perform poorly in terms of elasticity and hardness. The mechanical strength of PVA hydrogel is not high and its anti-creep performance is poor, which may limit its use in some application fields that require higher mechanical properties. Second, PVA itself does not have properties such as acid and enzyme resistance, which means that the application of PVA hydrogel in the respiratory tract and digestive tract may have certain limitations. Summary of the Invention

[0014] Aiming at the defects of the prior art, the purpose of the present invention is to provide a polyvinyl alcohol hydrogel patch with a double-disk structure, its preparation method and application.

[0015] The technical solution of the present invention is as follows: The first aspect of the present invention provides a preparation method of a polyvinyl alcohol hydrogel patch with a double-disk structure, and the specific steps are as follows: S1. Prepare a PVA solution; S2. Pour the PVA solution in step S1 into a mold with a double-disk structure, and obtain t-PVA by the freeze-casting method; S3. Subject the t-PVA in step S2 to salting-out treatment to obtain a strengthened hydrogel patch a; S4. Subject the strengthened hydrogel patch a in step S3 to solvent exchange-assisted wet annealing to obtain a strengthened hydrogel patch b; S5. Subject the strengthened hydrogel patch b in step S4 to swelling equilibrium treatment to obtain e-PVA.

[0016] Further, in step S1, the steps of preparing the PVA solution are specifically as follows: The PVA powder is dispersed in deionized water, stirred evenly, and ultrasonically treated for 1 h to obtain the PVA solution.

[0017] Further preferably, the mass percentage of PVA in the PVA solution is 10 wt%.

[0018] Further, step S2 specifically includes the following steps: S21. Place the mold filled with the PVA solution in a freezer at -80 °C or below, and then thaw it at 15 °C - 25 °C for 3 h; S22. Repeat step S21 three times, and demold to obtain t-PVA.

[0019] Further, step S3 specifically includes the following steps: Immerse t-PVA in a sodium citrate solution with a concentration of 1 mol / L - 2 mol / L for 12 h - 24 h to complete the first hydrogel strengthening, and obtain the strengthened hydrogel patch a.

[0020] Further, step S4 specifically includes the following steps: Immerse the strengthened hydrogel patch a in glycerol at room temperature for at least 48 h to obtain the PVA / glycerol organogel. Place the obtained PVA / glycerol organogel in a dryer at 100 °C - 150 °C for wet annealing for 30 minutes to complete the second hydrogel strengthening, and obtain the strengthened hydrogel patch b.

[0021] Further, step S5 specifically includes the following steps: Immerse the strengthened hydrogel patch b in deionized water for more than 48 h until swelling equilibrium is reached to obtain e-PVA.

[0022] The second aspect of the present invention provides a polyvinyl alcohol hydrogel patch with a double-disk structure, and the hydrogel patch is obtained by the above preparation method.

[0023] The third aspect of the present invention provides the application of the polyvinyl alcohol hydrogel patch with a double-disk structure, and the polyvinyl alcohol hydrogel patch with a double-disk structure has at least the following applications: 1) The application of the polyvinyl alcohol hydrogel patch with a double-disk structure in the preparation of gastric perforation repair products.

[0024] 2) The application of the polyvinyl alcohol hydrogel patch with a double-disk structure in the preparation of tracheal fistula repair products.

[0025] 3) The application of the polyvinyl alcohol hydrogel patch with a double-disk structure in the preparation of tracheoesophageal fistula repair products.

[0026] Compared with the prior art, the present invention has the following beneficial effects: 1. The polyvinyl alcohol hydrogel patch with a double-disk structure provided by the present invention is obtained by combining two techniques: freeze-casting assisted salting-out and solvent-exchange assisted wet annealing. Characterization and mechanical property testing of the polyvinyl alcohol hydrogel patch with a double-disk structure show that the combination of these two techniques significantly improves the crystallinity and molecular entanglement of the hydrogel patch, endowing it with a dense porous structure, strong mechanical robustness, excellent elasticity, and outstanding water-locking property.

[0027] 2. The polyvinyl alcohol hydrogel patch with a double-disk structure provided by the present invention can effectively perform in-situ repair of tracheal fistula defect sites, providing mechanical support structurally to maintain airway patency and avoid airway stenosis, and promoting effective healing and re-epithelialization of tracheal defect tissues functionally.

[0028] 3. The polyvinyl alcohol hydrogel patch with a double-disk structure provided by the present invention can effectively perform in-situ full-thickness repair of gastric / esophageal wall defects and promote tissue healing. It enables the use of the same biomaterial for defects in the walls of two different organs, namely the stomach and the esophagus, and the polyvinyl alcohol hydrogel patch with a double-disk structure has a good full-thickness repair effect on both the gastric and tracheal walls. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Other features, objectives, and advantages of the present invention will become more apparent by reading the detailed description of the non-limiting embodiments with reference to the following drawings: Figure 1 Schematic diagram of the preparation method of e-PVA provided in Example 1 of the present invention (e-PVA refers to the polyvinyl alcohol hydrogel patch with a double-disk structure; t-PVA refers to the traditional polyvinyl alcohol hydrogel); Figure 2 Schematic diagram of the characterization results of e-PVA provided in Experimental Example 1 of the present invention (e-PVA refers to the polyvinyl alcohol hydrogel patch with a double-disk structure; t-PVA refers to the traditional polyvinyl alcohol hydrogel; Figure 2 B: Display diagrams of t-PVA and e-PVA at different angles; Figure 2 C: SEM images of t-PVA and e-PVA; Figure 2 D: Water content of t-PVA and e-PVA (n = 3). * p < 0.05; Figure 2 E: FTIR spectral analysis of t-PVA and e-PVA; Figure 2 F: X-ray diffraction of t-PVA and e-PVA; Figure 2 G: Degradation curves of t-PVA and e-PVA in lipase (n = 3); Figure 2 H: Degradation curves of t-PVA and e-PVA in gastric acid (n = 3)); Figure 3Schematic diagram of the mechanical property results of t-PVA and e-PVA provided for the experimental examples of the present invention (e-PVA refers to a polyvinyl alcohol hydrogel patch with a double-disk structure; t-PVA refers to a traditional polyvinyl alcohol hydrogel; Figure 3 A: Wet tensile test of t-PVA and e-PVA. Scale bar: 1 cm; Figure 3 B: Tensile stress-strain curves of t-PVA and e-PVA (n = 3); Figure 3 C: Elastic modulus in the tensile test, * p < 0.05; Figure 3 D: Maximum stress in the tensile test, * p < 0.05; Figure 3 E: Maximum strain in the tensile test, * p < 0.05; Figure 3 F: Compressive stress-strain curves of t-PVA and e-PVA (n = 3); Figure 3 G: Maximum stress in the compression test, * p < 0.05; Figure 3 H: Compression modulus in the compression test, * p < 0.05); Figure 4 Schematic diagram of the bioabsorbability and biocompatibility results of t-PVA and e-PVA provided for the experimental examples of the present invention (e-PVA refers to a polyvinyl alcohol hydrogel patch with a double-disk structure; t-PVA refers to a traditional polyvinyl alcohol hydrogel; Figure 4 A: Schematic diagram of the degradation experiment under the skin of nude mice; Figure 4 B: Subcutaneous degradation results of t-PVA and e-PVA on the 1st, 7th, 14th, and 21st days; Figure 4 C: HE staining images of t-PVA and e-PVA after 21 days of subcutaneous degradation. Scale bar: 1 mm, 200 μm; Figure 4 D: Schematic diagram of CCK-8 proliferation experiment and live / dead cell staining; Figure 4 E: CCK-8 detection results after 24 / 48 h of culture; Figure 4 F: Live / dead staining of fibroblasts cultured with the extracts of t-PVA and e-PVA for 24 / 48 h: Figure 4 G: Even when the rabbit TF model is stretched and deformed, e-PVA still maintains good sealing stability on the rabbit TF model; Figure 4 H: e-PVA has a good plugging effect on rabbit TF, preventing liquid leakage; Figure 4 I: Finite element analysis of the mechanical environment of e-PVA during the repair of tracheal fistula); Figure 5 Schematic diagram of the effect of t-PVA and e-PVA in repairing TF provided for the experimental examples of the present invention (suture refers to surgical suture, and absorbable suture is used for surgical suture; e-PVA refers to a polyvinyl alcohol hydrogel patch with a double-disk structure; t-PVA refers to a traditional polyvinyl alcohol hydrogel; Figure 5A: Surgical images and bronchoscope images of repairing TF using suture, t-PVA, and e-PVA; Figure 5 B: Survival curves of rabbit TF models in the t-PVA repair group (n = 10), e-PVA repair group (n = 10), and suture repair group (n = 10) 4 weeks after the experiment; Figure 5 C: CT imaging and three-dimensional reconstruction 4 weeks after surgery. The red arrow indicates the repair area. The meanings of the letters in the CT imaging are as follows: L: left, R: right, H: head, F: foot, A: front, P: back; Figure 5 D、 Figure 5 E: Evaluation of the tracheal morphology after repair in the suture repair group, t-PVA repair group, and e-PVA repair group ( Figure 5 D: Diameter stenosis rate; Figure 5 E: Cross-sectional area ratio; * p < 0.05); Figure 5 F: Observation images of the damaged parts of rabbit TF models 4 weeks after repair in the suture repair group, t-PVA repair group, and e-PVA repair group); Figure 6 Schematic diagram of the tissue regeneration effect of t-PVA and e-PVA in the repair of rabbit TF models provided by the experimental examples of the present invention (suture refers to surgical suture, using absorbable suture for surgery; e-PVA refers to a polyvinyl alcohol hydrogel patch with a double-disk structure; t-PVA refers to a traditional polyvinyl alcohol hydrogel; Figure 6 A: Results of HE staining and Masson staining, fluorescence staining results of β-Tubulin, Muco5AC PCK, FoxJ1, vwf, and α-sma. Scale bar: 1 mm, 500 μm; Figure 6 B、 Figure 6 C: Evaluation of the infiltration of regenerated tissue in the repair area ( Figure 6 B: β-Tubulin ratio; Figure 6 C: Muco5AC ratio, * p < 0.05); Figure 6 D: Re-epithelialization evaluation, * p < 0.05; Figure 6 E: Angiogenesis evaluation: ratio of vwf to α-sma, * p < 0.05); Figure 7 Schematic diagram of the effect of e-PVA in repairing rat gastric perforation for 4 weeks provided by the experimental examples of the present invention (surgery refers to surgical suture, using absorbable suture; e-PVA refers to a polyvinyl alcohol hydrogel patch with a double-disk structure; Repair refers to e-PVA repair; Natural refers to normal tissue without injury; Figure 7 A: Schematic diagram of e-PVA in repairing rat gastric perforation; Figure 7 B: Physical display of e-PVA:Figure 7 C: Schematic diagram before repairing the gastric perforation of rats; Figure 7 D: Schematic diagram of repairing the gastric perforation of rats with e-PVA; Figure 7 E: Macroscopic observation of the repair effect of rats after 4 weeks of repair; Figure 7 f: Histological staining results of the gastric wall after 4 weeks of repair; Figure 7 G- Figure 7 L: The repair effect after 4 weeks of e-PVA repair was compared with the normal tissue without injury to evaluate the repair effect); Figure 8 Schematic diagram of the 4-week effect of e-PVA in repairing rabbit esophageal fistula provided by the experimental example of the present invention (Repair refers to e-PVA repair; Natural refers to the normal tissue without injury; e-PVA refers to the polyvinyl alcohol hydrogel patch with a double-disk structure; Figure 8 A: Schematic diagram of repairing rabbit esophageal fistula with e-PVA; Figure 8 B: Physical display of e-PVA; Figure 8 C: Schematic diagram before repairing the esophageal fistula of rabbits; Figure 8 D: Schematic diagram of repairing rabbit esophageal fistula with e-PVA; Figure 8 E: Anatomical diagram and barium meal radiograph of the rabbit esophagus after 4 weeks of surgical suture repair; Figure 8 F: Anatomical diagram and barium meal radiograph of the rabbit esophagus after 4 weeks of e-PVA repair; Figure 8 G: Finite element analysis result diagram of e-PVA; Figure 8 H: Histological staining results of the rabbit esophageal wall after 4 weeks of e-PVA repair; Figure 8 I- Figure 8 N: The repair effect after 4 weeks of e-PVA repair was compared with the normal tissue without injury to evaluate the repair effect); Figure 9 Schematic diagram of the 4-week effect of e-PVA in repairing rabbit tracheoesophageal fistula provided by the experimental example of the present invention (Repair refers to e-PVA repair; Natural refers to the normal tissue without injury; e-PVA refers to the polyvinyl alcohol hydrogel patch with a double-disk structure; Figure 9 A: Physical observation diagram of repairing rabbit tracheoesophageal fistula with e-PVA. The two disk-shaped structures of the double-disk e-PVA are respectively located on the inner walls of the esophagus and the trachea, playing a sealing role; Figure 9 B: Endoscopic observation of the inner wall of the trachea after e-PVA repair in vivo; Figure 9 C: Endoscopic observation of the inner wall of the esophagus after e-PVA repair in vivo; Figure 9 D: Endoscopic observation of the inner wall of the trachea after 4 weeks of e-PVA repair in vivo; Figure 9 E: Endoscopic observation of the inner wall of the esophagus after 4 weeks of e-PVA repair in vivo; Figure 9 F: Barium meal radiograph after 4 weeks of e-PVA repair; Figure 9G: Three-dimensional CT imaging of the upper esophagus 4 weeks after e-PVA repair; Figure 9 H: Three-dimensional CT imaging of the esophago-tracheal junction 4 weeks after e-PVA repair; Figure 9 I: Narrowing rate of the diameter of the e-PVA repair area in CT imaging; Figure 9 J: Percentage of the cross-sectional area of the e-PVA repair area in CT imaging; Figure 9 K: Histological staining results of the tracheo-esophageal wall connection area in rabbits 4 weeks after repair; Figure 9 L- Figure 9 Q: The repair effect 4 weeks after e-PVA repair was compared with normal tissues without injury to evaluate the repair effect); Figure 10 Schematic diagram of the exploration results of the clinical application of e-PVA provided by the experimental example of the present invention (e-PVA refers to a polyvinyl alcohol hydrogel patch with a double-disk structure; Figure 10 A: CT image of a patient with tracheal fistula; Figure 10 B: Three-dimensional reconstruction model of the patient's tracheal fistula; Figure 10 C: Development of a personalized e-PVA suitable for the shape of the fistula according to the reconstruction model; Figure 10 D: Preparation process of personalized e-PVA; Figure 10 E: Schematic diagram of in-situ non-invasive repair assisted by fibrobronchoscope; Figure 10 F: Physical map of personalized customized e-PVA suitable for human size; Figure 10 G: Schematic diagram of simulating e-PVA positioning repair on goat esophagus; Figure 10 H: Schematic diagram of simulating e-PVA repair of tracheo-esophageal fistula on goat trachea and esophagus. The upper figure refers to the external view, and the lower figure refers to the internal view of the trachea and esophagus). Detailed implementation mode

[0030] The present invention will be described in detail below with reference to specific embodiments.

[0031] The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several changes and improvements can be made, and these all belong to the protection scope of the present invention.

[0032] Embodiment This embodiment provides a polyvinyl alcohol hydrogel patch with a double-disk structure, and the preparation method includes the following steps: S1. Prepare a PVA solution: Disperse PVA powder in deionized water, stir evenly at 95 °C, and ultrasonically treat for 1 h to fully dissolve it to obtain a PVA solution with a mass percentage of 10 wt%.

[0033] S2. Pour the PVA solution in step S1 into a mold with a double-disk structure, and obtain a traditional polyvinyl alcohol hydrogel (t-PVA) by the freeze-casting method. The specific steps are as follows: The PVA solution is evenly injected into an acrylic mold with a double-disk structure, and the acrylic mold is assembled from acrylic plates; place the acrylic mold containing the PVA solution in a freezer at -80 °C for 8 h, and then thaw it at 25 °C - 15 °C for 3 h; after repeating the freeze-thaw cycle 3 times, take it out of the mold to obtain a traditional polyvinyl alcohol hydrogel (t-PVA).

[0034] S3. Subject the t-PVA in step S2 to salting-out treatment to obtain a reinforced double-disk hydrogel patch a. The specific steps are as follows: Immerse the t-PVA in a sodium citrate solution with a concentration of 1.5 mol / L for 12 h - 24 h to complete the first hydrogel strengthening and obtain a reinforced double-disk hydrogel patch a. The concentration of the sodium citrate solution can be controlled at 1 mol / L - 2 mol / L, and the specific concentration is selected according to the actual situation. This invention does not make specific requirements.

[0035] S4. Subject the reinforced double-disk hydrogel patch a in step S3 to solvent exchange-assisted wet annealing to obtain a reinforced double-disk hydrogel patch b. The specific steps are as follows: Immerse the reinforced double-disk hydrogel patch a in glycerol at room temperature for 48 h. During this period, the water molecules in the network of the reinforced double-disk hydrogel patch a are gradually replaced by glycerol molecules. After 48 h, the obtained PVA / glycerol organic gel is placed in a vacuum dryer at 120 °C for wet annealing for 30 minutes to complete the second gel strengthening and obtain a reinforced double-disk hydrogel patch b. The temperature of the vacuum dryer can be controlled at 100 °C - 150 °C, and the specific temperature can be selected according to the actual situation. This invention does not make specific requirements.

[0036] S5. Subject the reinforced double-disk hydrogel patch b in step S4 to equilibrium swelling to obtain a polyvinyl alcohol hydrogel patch with a double-disk structure (e-PVA). The specific steps are as follows: Immerse the reinforced hydrogel patch b in deionized water for 48 h until equilibrium swelling is achieved to obtain a tough and bioabsorbable e-PVA in an aqueous environment.

[0037] Experimental Example 1.1 Characterize and test the mechanical properties of the e-PVA obtained by the preparation method provided in the examples, and conduct a series of performance evaluations on it together with t-PVA to verify the enhancement effect of e-PVA.

[0038] 1.2 Verify the biological characteristics of the e-PVA obtained by the preparation method provided in the examples through the following experiments.

[0039] 1) Live / dead cell staining The e-PVA and t-PVA were separately placed in centrifuge tubes containing conventional cell culture medium, and continuously oscillated in a constant temperature shaker at 37 °C and 120 rpm for 72 h. The extracts of the t-PVA and e-PVA patches were centrifuged at 2000 rpm for 10 min, and the supernatant was collected for the determination of the cytotoxicity of t-PVA and e-PVA. Fibroblasts cultured in the extracts of t-PVA and e-PVA for 24 h and 48 h were collected. The cells were soaked and rinsed 3 times with PBS to eliminate the residual esterase activity in the culture medium, and the survival status of the cells on the extract was analyzed. Under light-proof conditions, the cells were immersed in 100 μL of dead / live cell staining working solution (dead / live cell staining kit, Invitrogen, USA), and incubated in a constant temperature incubator at 37 °C for 30 min. Subsequently, a confocal microscope (Nikon, Japan) was used to observe live cells with yellow-green fluorescence and dead cells with red fluorescence under the excitation wavelength condition of 490 ± 10 nm. In addition, dead cells could be observed separately using an excitation wavelength of 545 nm.

[0040] 2) CCK-8 cell proliferation assay The e-PVA and t-PVA were separately placed in centrifuge tubes containing conventional cell culture medium and continuously shaken in a constant temperature shaker at 37 °C and 120 rpm for 72 h to prepare the extracts of t-PVA and e-PVA; the extracts of t-PVA and e-PVA were centrifuged at 2000 rpm for 10 min, and the supernatant was collected for the determination of the cytotoxicity of t-PVA and e-PVA. The second-generation fibroblasts were collected and accurately counted, and inoculated into a 96-well plate at a cell density of 2000 cells per well. The wells around the edge of the 96-well plate were selected, and 100 μL of sterile PBS was added to each well to maintain the humidity in the culture environment and avoid affecting the experimental results due to water evaporation. After the cells adhered, the conventional cell culture medium was discarded and replaced with the supernatant of the corresponding extract of e-PVA and t-PVA. In addition, a group was added with conventional culture medium as a normal control. The cells were cultured in vitro for 11 days, and during the in vitro culture period, the corresponding extract supernatant or conventional culture medium was replaced in full every other day. Starting from the first day, 10 μL of CCK-8 reagent (Shanghai Beyotime Biotechnology Co., Ltd.) was added to 5 replicate wells in each group every other day, and incubated in a constant temperature incubator at 37 °C for 120 min. Subsequently, the absorbance value (OD) was measured at a wavelength of 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader (Thermo Fisher Scientific, USA). Among them, a blank control group (only adding the corresponding extracts of t-PVA and e-PVA or conventional culture medium without fibroblasts) was set in each group, and each experiment was repeated three times. After the data were summarized, a cell growth curve was plotted to evaluate the growth and proliferation of fibroblasts in the extracts of t-PVA and e-PVA, and further indirectly evaluate the cytotoxicity of t-PVA and e-PVA.

[0041] 3) Repair experiment of PVA hydrogel patch for tracheal fistula A rabbit tracheal fistula model was made, and the tracheal fistula site was repaired by three methods: surgical suture repair, t-PVA repair, and e-PVA repair. When t-PVA and e-PVA were used to repair the tracheal fistula, the following operation method was used: both t-PVA and e-PVA were in a double-disk shape, one disk was embedded into the inner wall of the trachea through the tracheal defect site, and the other disk remained on the outer wall of the trachea; after 4 weeks, the internal morphology of the repaired trachea was photographed using a tracheoscope.

[0042] 4) HE staining After the specimens were fixed, they were embedded in paraffin and sectioned; baked in an oven at 70 °C for 2 hours, and then dewaxed and dehydrated conventionally (immersed in three xylenes for 10 minutes each for dewaxing; immersed in five gradient alcohols from high to low for hydration, 10 seconds each). Next, the sections were immersed in hematoxylin staining solution for 9 minutes, differentiated in hydrochloric acid alcohol for 2 seconds, and the color was observed in a timely manner; blued in running water for 40 minutes, the longer the time, the darker the hematoxylin color, and then immersed in alcohol for 2 seconds; stained with eosin for 2 minutes, and then immersed in four gradient alcohols from low to high, immersed in three xylenes, and finally sealed with resin.

[0043] 5) Masson staining After conventional dehydration and dewaxing, the staining agent was prepared first: prepare copper ferrocyanide stock solution, alizarin yellow iron sulfate stock solution, phosphotungstic acid and 1% formaldehyde. Immerse the sections in the copper ferrocyanide stock solution for 10 minutes, then take them out and rinse with distilled water for 5 minutes. Then immerse the sections in 1% formaldehyde for 5 minutes, then take them out and rinse with distilled water for 5 minutes.

[0044] Next, immerse the sections in the alizarin yellow iron sulfate stock solution for 5 - 10 minutes (depending on the thickness of the specimen), take them out and rinse with distilled water for 5 minutes. Then immerse the sections in phosphotungstic acid for 2 - 5 minutes, take them out and rinse with distilled water for 5 minutes. Decolorize with absolute alcohol and iodobenzene for 1 - 2 minutes. After washing off the excess dye, immerse in xylene for 3 - 5 minutes, and then mount the slides with lignin glue for microscopic examination.

[0045] 6) Immunofluorescence detection method The tissue specimens were first fixed in 4% paraformaldehyde solution at 4 °C for 24 hours. After sufficient fixation, dehydration and paraffin embedding were carried out, and 5 µm sections were cut along the tracheal axis (longitudinal section direction of the tracheal wall) for standby. After the specimens were fixed, they were embedded in paraffin and sectioned; baked in an oven at 70 °C for 2 hours, and then conventionally dewaxed and dehydrated (immersed in three xylenes for 10 minutes each for dewaxing; then immersed in five gradient alcohols from high to low for hydration, 10 seconds each). Antigen retrieval by microwave: Place the dewaxed and hydrated sections in an antigen retrieval box and add enough 1x antigen retrieval solution. Place the antigen retrieval box in a microwave oven and boil it at high fire. Maintain low - grade fire for 10 - 15 minutes (note to supplement the antigen retrieval solution to prevent dry slices caused by excessive evaporation). Take out the antigen retrieval box and let it cool to room temperature naturally.

[0046] Inactivate endogenous peroxidase: Wash the sections with PBS 3 times, 5 minutes each time. After slightly shaking off the water on the sections, circle the sample area on the slide with a histochemical pen and add 3% H 2 0 2 Cover the tissue and incubate at room temperature in the dark for 25 minutes to block endogenous peroxidase and reduce non - specific background staining.

[0047] Blocking: The sections were washed 3 times with PBS for 5 min each time. After slightly shaking off the moisture on the sections, 3% BSA or serum was added dropwise onto the tissues and blocked for 30 min. The blocking reagent was specifically determined according to the species of the primary antibody and the secondary antibody.

[0048] Primary antibody incubation: Remove the blocking solution on the sections. Select the corresponding primary antibodies for the target proteins (CK19, CD31, LYVE-1, PGP9.5, β-Tubulin, Muco5AC, SMA, Myosin-6, CK-5, CK-14). Add an appropriate amount of the primary antibody solution to cover the sample area. The sections were incubated overnight at 4°C in a wet box with water added. Wash 3 times with PBS for 5 min each time.

[0049] Secondary antibody incubation: Remove the remaining washing solution on the sections. Directly add the fluorescence-labeled secondary antibody solution to cover the sample area. Incubate at room temperature for 50 min. Wash 3 times with PBS for 5 min each time; Fluorescent staining to amplify the signal: Remove the remaining washing solution on the sections. Add an appropriate amount of 1x dye working solution dropwise onto the tissues. The monochromatic fluorescent dye was diluted 100 times with the signal amplification solution to ensure complete coverage of the tissues. Incubate at room temperature in the dark for 10 min. Wash 3 times with PBS for 5 min each time.

[0050] Microwave treatment: Perform microwave heating treatment according to step 2 to remove the already bound primary and secondary antibodies. Naturally cool to room temperature at room temperature. Wash 3 times with PBS for 5 min each time.

[0051] Nucleus staining and mounting: Remove the remaining washing solution on the sections. Add DAPI staining solution and incubate in the dark at room temperature for 10 min. Wash 3 times with PBS for 5 min each time. After slightly shaking off the moisture on the sections, add an enhanced anti-quenching mounting agent dropwise onto the tissues and cover with a coverslip, avoiding air bubbles.

[0052] Imaging and reading slides: Collect images of the stained tissue sections in a condenser imaging device, observe, and interpret. 7) e-PVA experiment on gastric wall defect in rat GP model To test the actual effect of e-PVA in the repair process of gastric / esophageal wall defects, the feasibility of repairing gastric wall defects was preliminarily verified on a rat gastric perforation (GP) model, and the repair effects of e-PVA and conventional surgical suture were evaluated 4 weeks after e-PVA repair.

[0053] Preparation of rat gastric perforation (GP) model The rats were anesthetized and their skin was prepared. An incision was made in the middle of the upper abdomen to fully expose the stomach. A defect with a diameter of 3 mm was created in front of the rat's stomach using a trephine to simulate a gastric perforation in clinical practice. In the experimental group, e-PVA was used for immediate repair in situ; in the control group, conventional surgical suture repair was performed. Four weeks after the repair was completed, barium meal imaging was performed on the rats with gastric fistula repaired by conventional surgical suture (surgical suturing rat, SSR) and the rats with gastric fistula repaired by e-PVA (EPR) to evaluate the repair of the perforation site and the position and status of e-PVA through imaging.

[0054] When using e-PVA to repair the gastric perforation in rats, the following operating method was used: e-PVA was in a double-disk shape. One disk was inserted into the inner wall of the stomach through the gastric defect, and the other disk remained on the outer wall of the stomach. Four weeks later, subsequent experiments were carried out.

[0055] 8) Experiment on the repair of tracheoesophageal wall defect in rabbit TEF model with e-PVA Preparation of rabbit TEF model The skin and fascial tissues of the rabbit's neck were incised to expose the trachea and the adjacent esophagus. A circular defect was created behind the trachea using a 3-mm skin trephine, and the esophageal wall was incised in front of the esophagus using a 3-mm skin trephine to obtain a TEF defect model. e-PVA was used to repair it. The hydrogel patch was in a double-disk shape. One disk entered the inner wall of the trachea through the tracheal defect, and the other disk was inserted into the inner wall of the esophagus through the esophageal defect. Four weeks after the repair was completed, a full evaluation was carried out.

[0056] 9) Experiment on the repair of goat tracheal model with personalized e-PVA First, a tracheal fistula with a special shape was constructed on the goat trachea, and the personalized e-PVA was used to block it. On the goat model, the process of minimally invasive interventional surgery to repair TEF using e-PVA was simulated. Subsequently, the in situ repair of TEF with e-PV was further simulated using the goat's trachea and esophagus.

[0057] Results and analysis Figure 1 A shows the preparation process of e-PVA. The prepared PVA solution was placed in a customized mold and repeatedly frozen-thawed to obtain traditional polyvinyl alcohol hydrogel (t-PVA), and then it was immersed in citrate for salting out to achieve the first step of strengthening. Subsequently, it was immersed in glycerol, and the second step of strengthening was completed through solvent exchange-assisted wet annealing. After immersion in deionized water, equilibrium swelling was carried out to obtain a tough and bioabsorbable polyvinyl alcohol hydrogel patch with a double-disk structure (e-PVA).

[0058] As Figure 2As shown in Figure B, macroscopically, t-PVA is semi-transparent, while e-PVA becomes milky white and opaque after a series of improvement treatments.

[0059] As Figure 2 shown in Figure C, the SEM results show that the surfaces and cross-sections of t-PVA and e-PVA are still porous structures. Through the further optimization of the pore size distribution and porosity of the hydrogel patch by salting out and solvent exchange, it can be seen that the enhanced e-PVA has smaller pore sizes and a denser pore structure.

[0060] Figure 2 Figure D illustrates the change in water content before and after the enhancement of the PVA hydrogel. The water content of the PVA hydrogel before enhancement is approximately 90% (t-PVA), and after enhancement it is approximately 70% (e-PVA). Therefore, t-PVA is more structurally similar to a hydrogel because it has a higher water content, while e-PVA exhibits a lower water content and is somewhat similar to a porous scaffold. In other words, e-PVA reduces the water retention capacity, reduces the resistance of the internal pores, and increases the number of internal pores. This modification makes the penetration path of tissues and cells smoother, ultimately improving the biopermeability and absorption capacity of e-PVA.

[0061] Fourier transform infrared spectroscopy (FTIR) was used to characterize the chemical composition, bonding, and changes in functional groups of t-PVA and e-PVA. From the FTIR results, it can be seen that t-PVA and e-PVA have the same peaks, while the absorption peak intensity of e-PVA exceeds that of t-PVA, indicating that this enhancement method does not change the types of chemical bonds inside the PVA hydrogel, but the concentration of the corresponding chemical bonds or functional groups in e-PVA has increased ( Figure 2 Figure E).

[0062] The XRD results show that t-PVA and e-PVA have the same diffraction angles, but e-PVA shows higher peaks, which also indicates that the lattice structure inside the hydrogel has not changed substantially, but the internal lattice structure has become more compact and tough ( Figure 2 Figure F). These results indicate that the chemical structure inside e-PVA has not changed substantially, but its internal structure has become more compact and stable after enhancement, and the mechanical strength has also increased significantly.

[0063] By plotting the mass change curves of e-PVA and t-PVA in lipase and gastric acid ( Figure 2G, 2H), exploring the stability of PVA hydrogel. t-PVA will gradually decompose under the action of lipase and gastric acid, while the enhanced and improved e-PVA can better resist the erosion of pH and biological enzymes and slow down the decomposition rate. This means that e-PVA can maintain structural stability in the trachea for a longer time, thereby improving the durability and stability of the repair effect, better protecting the defect area, promoting tissue healing and repair, and reducing the risk of postoperative complications.

[0064] Mechanical tests such as Figure 3 As shown, e-PVA can significantly improve the tensile strength ( Figure 3 A) to counteract the pressure difference between the inside and outside of the trachea. Compared with t-PVA, e-PVA shows significant advantages in tensile properties, elastic modulus, maximum elastic stress and maximum elastic strain, and compressive stress-strain. The tensile curve results show that ( Figure 3 B), e-PVA showed higher tensile strength and ductility during the stretching process. Compared with t-PVA, the breaking strength of e-PVA was significantly improved, and the stretching process before breaking was more stable, showing better tensile properties. The elastic modulus test results showed ( Figure 3 C), the elastic modulus of e-PVA is significantly higher than that of t-PVA. This indicates that e-PVA has better deformation resistance and resilience, and can maintain a more stable shape and structure under external force. Further analysis of the maximum elastic stress ( Figure 3 D) and the maximum elastic strain ( Figure 3 E) The test results further verified the superiority of e-PVA mechanics. e-PVA can maintain higher elastic stress and greater elastic strain when subjected to external force, showing better stress-strain response characteristics. The compression curve results show ( Figure 3 F), compared with t-PVA, e-PVA shows higher compressive strength during compression. Compression modulus test results ( Figure 3 H) and maximum compressive stress ( Figure 3 G) The test results show that the compression modulus and maximum stress of e-PVA are significantly higher than those of t-PVA, which indicates that e-PVA has better compression resistance and can maintain a more stable shape and structure under external forces. Therefore, e-PVA is superior to t-PVA in terms of tensile and compression properties, elastic modulus, etc. These excellent properties make e-PVA have a wider application prospect.

[0065] Bioabsorption performance and biocompatibility results such as Figure 4As shown, currently for tracheal fistulas, most of the tubular stents used clinically are non-degradable, which can only play a role in temporary occlusion and support, unable to further promote the regenerative repair of tissues, and may also cause chronic inflammatory reactions and lifelong rejection problems. Therefore, in order to verify the bioabsorbability of t-PVA and e-PVA, a subcutaneous degradation experiment in nude mice was conducted ( Figure 4 A). t-PVA and e-PVA were respectively implanted subcutaneously in nude mice and removed after 1 / 7 / 14 / 21 days ( Figure 4 B). It can be seen that both t-PVA and e-PVA can maintain their original shapes in the first week, proving that t-PVA and e-PVA can tolerate the internal physiological environment of living tissues in the initial stage in vivo, maintain their original morphological structures, and thus effectively achieve stable occlusion of tracheal fistulas. Two weeks after subcutaneous implantation, both t-PVA and e-PVA began to be absorbed by tissues and gradually incorporated into the subcutaneous fibrous connective tissue, proving that t-PVA and e-PVA have good bioabsorbability. Further histological verification also showed that t-PVA and e-PVA were absorbed by subcutaneous tissues 3 weeks after implantation in nude mice, and the subcutaneous tissues infiltrated into the double-disk hydrogel patches, providing favorable conditions for t-PVA and e-PVA to promote the regenerative repair of tracheal fistula defect tissues ( Figure 4 C).

[0066] Analysis of live / dead cell staining and cck-8 results. Since chemical reagents such as sodium citrate and glycerol will be used during the enhancement process, these chemical reagents may remain in t-PVA and e-PVA, affecting the regenerative repair of tissues. Therefore, in order to verify the biocompatibility of t-PVA and e-PVA, live / dead cell staining and cck-8 cell proliferation experiments were conducted. Figure 4 D shows the experimental procedure for verifying the biocompatibility of t-PVA and e-PVA by using the extracts of t-PVA and e-PVA. Figure 4 E shows the detection results of fibroblasts after 24 hours and 48 hours in the cck-8 cell proliferation experiment. Compared with the control group, there were no significant differences in the t-PVA and e-PVA experimental groups. Figure 4 F shows the live / dead cell staining results of fibroblasts cultured in culture dishes for 24 hours and 48 hours respectively. From the cell staining, it can be seen that the extracts of t-PVA and e-PVA do not have a significant impact on the growth of fibroblasts.

[0067] In order to verify the effectiveness of e-PVA in repairing TF defects, first, the sealing ability of e-PVA for perforated TF defects and its resistance to deformation during respiratory movement were evaluated. As Figure 4As shown in Fig. G, e-PVA maintained the stability of its sealing performance even under stretching and deformation in the rabbit TF model. As Figure 4 shown in Fig. H, e-PVA achieved stable occlusion in the experimental group of the in vitro rabbit TF model, effectively preventing liquid leakage.

[0068] To simulate the in vivo repair environment of e-PVA, theoretical calculations and finite element analyses were performed to study the mechanical behavior of e-PVA in the context of repairing TF. As Figure 4 shown in Fig. I, during exhalation, the pressure inside the trachea is positive and the pressure outside is negative. This pressure difference generates a force from the inside to the outside, causing the part of e-PVA inside the trachea to be mainly affected. The stress calculation on the surface of this part shows that the force is mainly concentrated on the disk edge, thus providing effective support. On the contrary, during inhalation, a negative pressure is generated inside the trachea and a positive pressure exists outside, resulting in a force from the outside to the inside on the part of e-PVA outside the trachea. Analyzing the stress distribution on the surface of this part reveals that the force is also concentrated on the disk edge, and e-PVA cannot be inhaled into the respiratory tract by offsetting the pressure difference. Through theoretical calculations and finite element analyses, it is beneficial to enhance the understanding of the behavior of e-PVA in the trachea under different conditions, considering factors such as the flexibility and stress distribution of e-PVA. These insights are crucial for optimizing the design of e-PVA to ensure proper function after implantation, thus maintaining the stability and integrity of the structure while improving the repair effect.

[0069] The experimental results of using t-PVA and e-PVA to repair tracheal fistulas are as Figure 5 shown Figure 5 From left to right in Fig. A are the surgical images and macroscopic observation images of bronchoscopy of the rabbit TF model, surgical suture repair, t-PVA repair, and e-PVA repair. It can be seen that surgical suture repair will cause changes in the tracheal morphology, lumen stenosis, and continuous bleeding; although t-PVA maintained the lumen morphology, due to insufficient mechanical strength, it was difficult to fully adhere to the tube wall, and bleeding was likely to occur at the defect; e-PVA not only maintained the lumen morphology but also tightly sealed the tracheal fistula gap and inhibited bleeding.

[0070] Taking surgical suture repair as a control, the survival curves of the three groups of rabbit TF models were statistically analyzed ( Figure 5 Fig. B). Four weeks after surgical suture repair, the survival rate of the rabbit TF model was 60% (n = 10), and the survival rate of the t-PVA repair was only 20% (n = 10) four weeks after surgery. And serious postoperative complications such as wound infection, dyspnea, and eating disorders began to appear around 1 week after surgery, indicating that t-PVA could not effectively improve the survival rate of rabbits. The e-PVA repair effectively improved the survival rate of rabbits, with a survival rate as high as 70% (n = 10) four weeks after surgery, and the vital signs were normal and the mental state was good.

[0071] After autopsy, it was found that the rabbits that died in the early and middle stages of t-PVA repair died from the recurrence of the fistula due to the shedding of t-PVA. The rabbits that died 1 week after the operation died from severe suppurative secondary infection, with a large amount of yellow purulent matter accumulating at the tracheal defect. This is because the trachea has been in a state of being connected to the external environment and lacks the protection of the airway mucosal epithelium. The mechanical and elastic strength of t-PVA is insufficient to form a tight seal and is easily attacked by pathogenic bacteria in the environmental air, resulting in secondary chronic infection and inflammatory reaction. This reaction will gradually spread to other parts of the body and the blood, causing systemic infection and even septic shock, ultimately damaging multiple organs, leading to organ failure and death. In contrast, e-PVA can effectively seal the tracheal fistula with its excellent elasticity and mechanical strength, and at the same time adhere tightly to the tracheal wall to maintain the patency of the airway.

[0072] Four weeks after the repair, CT imaging was performed on the surviving rabbits to evaluate the tracheal repair effect structurally ( Figure 5 C). The three-dimensional CT imaging of the rabbits showed obvious tracheal deformation and stenosis in the left front of the trachea repaired by surgical suture. This was due to the tension of the tracheal cartilage ring, which caused an increase in the wall tension during direct suture, resulting in distortion and stenosis. Over time, the stenosis of the tracheal wall caused by the tension will gradually worsen, and may even lead to scar fibrosis. If the tension is too high, the wound may crack again and secondary infection may occur. In contrast, the tracheas repaired with t-PVA and e-PVA basically remained patent, without obvious deformation and stenosis. In addition, through the three-dimensional CT imaging reconstruction of the tracheas repaired by the three repair methods, it was found that although the three-dimensional CT reconstruction of the trachea repaired by surgical suture showed that the defect was successfully repaired, a trend of inward stenosis of the tracheal wall could be clearly observed. The defect in the anterior midline of the trachea repaired with t-PVA was still visible because at this time t-PVA had not been completely degraded, and the surrounding tissue cells had not fully grown in, resulting in a certain density difference between the repair area and the surrounding tissue. Four weeks after e-PVA repair, the tracheal wall was more patent, without obvious stenosis and gaps, and the tracheal fistula gap disappeared after three-dimensional CT reconstruction. This was because after 4 weeks, the full degradation of e-PVA and the continuous infiltration of the surrounding tissues made relatively mature tissues form inside e-PVA. ImageJ was used to statistically quantify the imaging to evaluate the effects of t-PVA, e-PVA, and surgical suture in repairing tracheal fistulas: the narrowing rate of the diameter of the tracheal fistula repair area on CT imaging ( Figure 5 D) and the percentage of the cross-sectional area occupying the cross-sectional area of the normal trachea ( Figure 5 E) showed that e-PVA repair was significantly better than t-PVA repair and surgical suture in structural repair; The tracheas of the three groups of rabbits were dissected ( Figure 5F), it can be seen that although the repaired area of the surgical suture has started to heal, due to the tension of the trachea itself, obvious stenosis has occurred in the lower tracheal lumen, and the repaired area shows slightly white fibrotic tissue after dissection. After 4 weeks of t-PVA repair, the repaired area is smooth and smoothly connected to the surrounding normal tissues, but it is more transparent in color and lacks the color of normal tissues. After 4 weeks of e-PVA repair, the tissue color and properties of the repaired area are very close to those of the surrounding normal tissues, and there is basically no difference.

[0073] The results of histological section staining (HE and Masson) and immunofluorescence detection are as Figure 6 shown. To further evaluate the internal repair effect, axial histological section staining and immunofluorescence detection were performed on three groups of tracheal repair samples after 4 weeks of experiment ( Figure 6 A) to observe the internal tissue repair situation in the tracheal fistula area. HE and Masson staining showed that there was basically no new tissue in the surgical suture, and deformation and stenosis occurred due to tracheal tension. Initial degradation and absorption occurred in the t-PVA repair, and a small amount of tissue cells began to grow into the t-PVA, mainly composed of collagen and fibers, but the tissue structure was loose and disordered. The e-PVA repair degraded and absorbed more completely, and there was more abundant tissue formation inside, proving that e-PVA has better tissue compatibility, and its internal pore structure is conducive to the migration and growth of surrounding tissue cells. The results of immunofluorescence detection showed that the degree and quantity of fibroblast and mucous cell infiltration shown by β-Tubulin and Muco5AC staining were significantly higher than those of t-PVA repair and surgical suture. A large number of fibroblasts and mucous cells crawled into the e-PVA along the internal pore structure, fully infiltrating the inside of the e-PVA and promoting tissue regeneration at the defect site. This may be because the water content of t-PVA is too high, closer to a hydrogel, which hinders the infiltration and growth of surrounding tissues to a certain extent. The enhanced e-PVA has a reduced water content, is closer to a porous scaffold, and is more conducive to cell and tissue regeneration. Regarding the problem of re-epithelialization, immunofluorescence staining was performed on the airway epithelial marker pan-cytokeratin PCK protein. The results showed that at 4 weeks, due to tracheal tension, the fistula opening in the surgical suture group could not be completely opposed, which affected the re-epithelialization of the trachea to a certain extent. Only the edge area of the t-PVA repair showed epithelial cells migrating from the surrounding area, and the re-epithelialization effect in the center of the repair area was insufficient, which was related to the insufficient cell adhesion caused by the high water content of t-PVA. The e-PVA repair showed excellent re-epithelialization effect, and the regenerated epithelium crawling from around the airway was continuously distributed, indicating a high degree of re-epithelialization of airway basal cells crawling from the surrounding normal mucosa in the repair area. Immunofluorescence staining of vascularization-specific indicators vWF and α-SMA also found that the degree of neovascularization of the e-PVA repair was significantly higher than that of the t-PVA repair group and the surgical suture group.

[0074] Accordingly, ImageJ was used for statistical quantification of imaging and immunofluorescence staining to evaluate the effects of t-PVA repair, e-PVA repair, and surgical suture in repairing tracheal fistulas: the fluorescence proportion of β-Tubulin ( Figure 6 B) showed that the infiltration degree of fibroblasts in e-PVA repair was significantly higher than that in the other two groups; the fluorescence proportion of Muco5AC ( Figure 6 C) showed that the infiltration degree of goblet cells in e-PVA repair was significantly higher than that in the other two groups; the positive coverage rate of PCK ( Figure 6 D) showed that the degree of epithelialization in e-PVA repair was higher than that in the surgical suture group; the fluorescence proportion of vWF&α-SMA ( Figure 6 E) showed that the degree of neovascularization in e-PVA repair was significantly higher than that in the surgical suture.

[0075] It can be seen that in-situ repair of tracheal fistulas with e-PVA has shown excellent repair effects. Compared with t-PVA repair and surgical suture repair, using e-PVA for repair has significant effects in terms of tracheal stenosis degree, infection prevention, infiltration degree of regenerated tissue, degree of epithelialization, and tissue vascularization.

[0076] The effect of e-PVA in repairing rat gastric perforation at 4 weeks is as Figure 7 shown Figure 7 A: Schematic diagram of the method for e-PVA to repair rat gastric perforation. e-PVA is in a double-disk shape. One disk is embedded into the inner wall of the stomach through the gastric defect site, and the other disk remains on the outer wall of the stomach; Figure 7 B: Physical display of e-PVA. e-PVA is in a milky white double-disk shape; Figure 7 C: A round hole was made in the normal gastric wall of the rat using a 3.0 mm trephine to simulate clinical gastric perforation; Figure 7 D: e-PVA repairs rat gastric perforation. The double-disk structure of e-PVA, located on both sides of the gastric wall respectively, can fit well with the perforated part of the gastric wall and play an effective sealing role; According to the barium meal radiography results ( Figure 7E) The results showed that there was no leakage at the repair site in both the surgery (referring to surgical suture using absorbable suture) and e-PVA repair groups, indicating that both e-PVA repair and direct surgical suture can effectively seal the perforation. Using e-PVA can avoid the traction on the gastric wall during direct suture and maintain the original volume and size of the stomach. Further observation of the images clearly showed that the position of e-PVA in the e-PVA repair did not change and it was not completely degraded temporarily, verifying the good biostability and biocompatibility of e-PVA. Anatomical observation of the rat GP model after 4 weeks of repair showed that, macroscopically, compared with surgical suture, e-PVA repair significantly avoided liver adhesion and connective tissue hyperplasia caused by direct surgical suture. When observing the surface of the gastric wall repair site, it was obvious that there was still significant extensive bleeding and swelling on the surface of the gastric wall repaired by surgical suture after four weeks, while there was no abnormality in the surface of the gastric wall repaired by e-PVA and the surrounding normal tissues. Observation of the inner gastric wall also showed that there was a certain degree of swelling and redness at the site of direct suture in surgical suture repair, while the inner gastric wall repaired by e-PVA was normal.

[0077] Analysis of histological staining (HE staining and immunofluorescence staining) results: For the composition of the gastric wall, histological staining of the gastric wall at the e-PVA repair site was performed using various specific dyes and compared with the staining results of normal tissues to evaluate the effect of e-PVA on the full-thickness repair of the gastric wall ( Figure 7 F). CK-19 was selected to stain the tissues to evaluate the regeneration and integrity of epithelial cells in the gastric mucosa layer: The observation results showed that the e-PVA repair samples after 4 weeks of repair showed that the mucosa layer of the gastric wall had recovered normal epithelial cell coverage, which helped to ensure the protective function of the mucosa layer. CD31 staining was used to evaluate angiogenesis and vascular density, LYVE-1 staining was used to evaluate the distribution and density of lymphatic vessels, PGP9.5 staining was used to evaluate nerve distribution and regeneration, and Muco5AC staining was used to evaluate the number of goblet cells and mucus secretion. Through histological staining analysis of various components in the submucosa layer of the gastric wall, e-PVA showed good repair effects, enabling the submucosa layer of the gastric wall to restore its structure and function, maintain its supportive and nutritive functions, and at the same time maintain the flexibility of the gastric wall during expansion and contraction. Moreover, the analysis results of Masson and SMA staining showed that e-PVA repair has the potential for the reconstruction and repair of the muscular layer of the gastric wall, providing important support for the restoration of normal gastric function.

[0078] In summary, through histological and cytological staining analysis using these dyes and markers, the restoration of the organizational structure and function of the gastric wall at various levels after e-PVA repair was comprehensively evaluated, including the regeneration and repair of epithelial cells, blood vessels, lymphatic vessels, nerves, mucus secretion, as well as smooth muscle and connective tissues. The observation results of fluorescence staining microscopy (Figure 7 F) and the fluorescence quantitative statistical results ( Figure 7 G-7L) showed that the repair effects of the gastric wall mucosa layer and submucosa layer 4 weeks after e-PVA repair were close to the level of normal tissues (gastric wall) without injury. Although fewer smooth muscle cells were generated in the muscular layer repair, an obvious repair trend was shown. The above analysis indicated that e-PVA could effectively block the perforation site, first repair the mucosa layer and submucosa layer of the gastric wall, restore its protective function and provide nerve and vascular support, and then gradually promote the recovery of the muscular layer, thereby reconstructing the peristaltic functions of the stomach for stirring and pushing food.

[0079] The effect of e-PVA in repairing rabbit esophageal fistula for 4 weeks was as Figure 8 shown Figure 8 A: Schematic diagram of the method for repairing rabbit esophageal fistula with e-PVA. e-PVA was in a double-disk shape. One disk was embedded into the inner wall of the rabbit esophagus through the defect site of the rabbit esophagus, and the other disk remained on the outer wall of the rabbit esophagus; Figure 8 B: Physical display of e-PVA. e-PVA was in a milky white double-disk shape; Figure 8 C: A round hole was drilled on the side wall of the normal esophagus using a 3.0 mm trephine to simulate a clinical esophageal fistula opening; Figure 8 D: Repairing rabbit esophageal fistula with e-PVA. The double-disk structure of e-PVA was located on both sides of the esophageal wall respectively, and could fit well with the esophageal wall, playing an effective blocking role; as shown by the barium meal contrast X-ray, it was observed that the surgically sutured ( Figure 8 E) and e-PVA-repaired ( Figure 8 F) esophagi were both shown to be unobstructed, and the contrast agent could pass through smoothly. However, the surgically sutured esophagus showed obvious stenosis, which might be caused by fibrosis and scar formation at the fistula site, and might cause dilation of the lower segment of the esophagus during swallowing. In contrast, the esophagus of the rabbit repaired with e-PVA did not show obvious stenosis or deformity, indicating that the enhanced hydrogel patch had good elasticity and anti-stretching ability.

[0080] In the anatomical observation, the surgically sutured esophagus ( Figure 8 E) showed obvious redness, swelling and bleeding, while the esophagus repaired with e-PVA ( Figure 8 F) showed a smooth tube wall, and the surrounding tissues were almost the same as normal tissues. Further, finite element analysis ( Figure 8 G) was used to simulate the mechanical environment of the esophagus during swallowing after repair with the e-PVA patch. The stress analysis of the inner surface of the esophagus showed that during swallowing and the passage of food through the esophagus, e-PVA and the esophageal wall could evenly disperse the pressure, and e-PVA effectively supported the structure of the esophageal wall, ensuring normal swallowing function and stability during the passage of food.

[0081] The esophageal wall is composed of the mucosa layer, submucosa layer, muscular layer, and adventitia layer. These layers work together to enable the esophagus to effectively transport food and protect the internal tissues. To further compare the internal repair effect of e-PVA in repairing EF, histological staining was performed on samples at 4 weeks after repair ( Figure 8 H). HE and CK-14 staining showed that a large number of squamous epithelial cells had formed in the mucosa layer of the repaired rabbit esophagus, indicating that the epithelial structure and barrier function in this area had returned to normal.

[0082] Markers such as CD31, SMA, LYVE-1, PGP9.5, and Muco5AC were used to evaluate the repair of the submucosa layer. Through microscopic observation and ImageJ fluorescence quantitative statistics ( Figure 8 I-8N), it was found that the histological staining results of the submucosa layer of the esophageal wall after e-PVA repair were very close to those of the uninjured normal tissue (esophageal wall). Fluorescence quantitative analysis showed that the expression levels of PGP9.5 and Muco5AC in the repaired area were slightly higher than those in the normal esophageal wall, indicating that e-PVA could more effectively promote esophageal wall nerve regeneration, regulate esophageal motility, and increase mucus secretion to enhance the immune defense function. From the fluorescence results of Masson staining, it could be seen that a rich surrounding connective tissue had formed at the defect of the repaired esophageal wall, mainly composed of fibers and collagen. And the Myosin-6 staining results showed that a large number of smooth muscle cells had regenerated in the esophageal wall after e-PVA repair, and the fluorescence intensity of Myosin-6 was higher than that of the normal tissue, further indicating that e-PVA had a good repair and reconstruction effect in the muscular layer In summary, e-PVA had successfully achieved the repair of all layers of the esophageal wall at 4 weeks after repair. Fluorescence quantitative statistics showed that after repair, it was even superior to normal tissue in terms of submucosal nerve regeneration, mucus secretion, and muscular layer reconstruction, and did not cause obvious immune reactions and infections. These results fully demonstrated that the prepared e-PVA effectively achieved the full-thickness repair of the tracheal wall.

[0083] The effect of e-PVA in repairing rabbit tracheoesophageal fistula at 4 weeks was as Figure 9 shown, Figure 9 A: Macroscopic observation diagram of e-PVA repairing rabbit tracheoesophageal fistula. e-PVA was used to repair it, and the hydrogel patch was in a double-disk shape. One disk entered the inner wall of the trachea through the tracheal defect, and the other disk was embedded into the inner wall of the esophagus through the esophageal defect. When using e-PVA to repair rabbit TEF, after repair, in vivo endoscopic observation showed that e-PVA had blocked the fistula between the trachea and the esophagus, and the hydrogel patch was clearly visible on the inner wall of the trachea ( Figure 9 B) and the inner wall of the esophagus ( Figure 9 C). After 4 weeks of repair experiments, all rabbits receiving repair survived. Repeated in vivo tracheoscopy imaging showed that originally on the inner wall of the trachea (Figure 9 D) and the inner wall of the esophagus ( Figure 9 E) the e-PVA has been completely degraded, and a mature tissue has formed at the tracheoesophageal fistula, the lumen inner wall is smooth and continuous, and the mucosa is completely covered.

[0084] Subsequently, in vivo X-ray and CT imaging were performed to evaluate the repair effect. Lateral X-ray showed that the repaired trachea and esophagus of the rabbit were unobstructed, without obvious defects or stenosis segments ( Figure 9 F), and the barium meal imaging results further confirmed this. CT three-dimensional imaging observation showed that the repaired esophagus ( Figure 9 G) and the tracheoesophageal junction ( Figure 9 H) did not have obvious stenosis or distortion, the e-PVA trace had basically disappeared, replaced by a new tube wall, which was consistent with the previous bronchoscope imaging results.

[0085] CT imaging analysis of the repaired area showed that the narrowing rate ( Figure 9 I) and the percentage of the cross-sectional area of the e-PVA repaired tracheoesophageal fistula site accounting for the normal cross-sectional area ( Figure 9 J) were almost no different from normal tracheoesophageal tissues. This indicates that e-PVA not only successfully blocked the tracheoesophageal fistula, but also promoted the regeneration of the inner wall at the defect, enabling the trachea and esophagus to restore integrity without causing any tracheoesophageal stenosis or malformation.

[0086] Further observation of the staining results of each layer of markers ( Figure 9 K) and ImageJ fluorescence quantitative statistics ( Figure 9 L-9Q), in the regenerated tissue at the tracheoesophageal junction after e-PVA repair, the mucosal epithelial layer (CK-5, CK-14), submucosa (CD31&SMA, LVYE-1, PGP9.5, Muco5AC) and muscular layer (Masson, Myosin-6) all had fluorescence staining, and compared with the uninjured normal tissue (tracheoesophageal tissue), the mucosal epithelial layer, submucosa and muscular layer were all effectively repaired, and the intensity was almost no different from that of normal tracheoesophageal tissue, indicating that e-PVA has achieved full-layer repair of the defect after 4 weeks of TEF repair. These results show that e-PVA has good effects and broad clinical application prospects in TEF repair.

[0087] The exploration of the application of e-PVA in clinical practice is as Figure 10 shown. In clinical practice, the size, shape and location of the fistula of each TEF patient may be different. Therefore, personalized customized e-PVA can better meet the specific needs of patients and ensure a perfect match between e-PVA and the fistula. The personalized customized e-PVA was used in the goat model ( Figure 10The process of minimally invasive interventional surgery for TEF repair using e-PVA was simulated on G-10H).

[0088] In the thoracic surgery database of Shanghai Ruijin Hospital, a typical TEF patient was screened. The patient developed tracheoesophageal fistula 3 weeks after esophageal cancer surgery. CT images showed that the fistula was located in the left main bronchus, opening at the membranous part, connected to the esophagus, with an irregular shape and a maximum diameter of about 2.5 cm ( Figure 10 A). Through clinical CT images, three-dimensional reconstruction of the patient's TEF fistula and its surrounding tissues was performed ( Figure 10 B): Green represents the trachea, yellow represents the esophagus, and the red part in the middle indicates the tracheoesophageal defect caused by TEF. According to the special shape and characteristics of the fistula, a personalized e-PVA model was designed and prepared ( Figure 10 C). To deal with various clinically diverse fistulas of different sizes and shapes, the mold reaction method ( Figure 10 D) was used to customize e-PVA patches with special shapes. First, a mold of PVA hydrogel was prepared using 3D printing technology and silicone mold turning. Then, the prepared PVA solution was injected into the mold to obtain t-PVA. Subsequently, through the combination of cryogenic casting-assisted salting out and solvent exchange-assisted wet annealing technology for enhancement, personalized e-PVA was finally prepared. This method can not only easily prepare e-PVA of different sizes and shapes, but also the whole process is safe, efficient and convenient, with broad prospects for clinical transformation.

[0089] Schematic diagram of fiberoptic bronchoscope-assisted in-situ non-invasive repair ( Figure 10 E): With the assistance of a bronchoscope, the customized e-PVA can be precisely implanted into the fistula position to achieve in-situ minimally invasive repair. e-PVA has excellent elasticity and can be compressed in a large volume for loading into the working channel of the bronchoscope. After reaching the predetermined position, the patch is pushed out of the channel using a guide wire and quickly expands to its original shape to effectively block the fistula. Once the e-PVA is implanted, the doctor can observe its fitting condition through the bronchoscope and make fine adjustments if necessary to ensure the best fitting effect.

[0090] To verify the feasibility of e-PVA patches in personalized customized repair of TEF, first, a tracheal fistula with a special shape was constructed on a goat trachea, and the customized e-PVA ( Figure 10 F) was used to block it, and the patch completely fit the fistula ( Figure 10 G). Subsequently, the in-situ repair of TEF with the hydrogel patch was further simulated using the trachea and esophagus of goats. The unique double-disk structure of e-PVA enables the upper and lower disks to fully cover the fistula, achieving in-situ minimally invasive repair of TEF in goats ( Figure 10 H).

[0091] In the future, with the assistance of bronchoscope technology, doctors can fully utilize e-PVA to achieve minimally invasive and efficient repair of clinical diseases caused by gastric / esophageal wall defects such as GP, EF, and TEF. Therefore, the e-PVA disk hydrogel patch has important clinical transformation value.

[0092] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. Without conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily.

Claims

1. A method for preparing a polyvinyl alcohol hydrogel patch with a double-disc structure, characterized in that: The following steps are involved: S1, prepare PVA solution; S2, pouring the PVA solution in step S1 into a mold with a double-disc structure to obtain t-PVA by freeze casting; S3, subjecting the t-PVA in step S2 to salting-out treatment to obtain a reinforced hydrogel patch a; S4, subjecting the reinforced hydrogel patch a in step S3 to a solvent exchange-assisted wet annealing method to obtain a reinforced hydrogel patch b; S5. Subject the reinforced hydrogel patch b in step S4 to swelling equilibrium treatment to obtain e-PVA.

2. The method for preparing the polyvinyl alcohol hydrogel patch with a double-disc structure according to claim 1, characterized in that: In step S1, the steps of preparing the PVA solution are as follows: PVA powder is dispersed in deionized water, stirred evenly, and ultrasonically treated for 1 hour to obtain a PVA solution.

3. The method for preparing the polyvinyl alcohol hydrogel patch with a double-disc structure according to claim 1, characterized in that: The mass percentage of PVA in the PVA solution is 10wt%.

4. The method for preparing the polyvinyl alcohol hydrogel patch with a double-disc structure according to claim 1, characterized in that: Step S2 specifically includes the following steps: S21, freezing the mold filled with PVA solution at -80°C or below for 8 h, and then thawing it at 15-25°C for 3 h; S22. Repeat step S21 three times, demold, and obtain t-PVA.

5. The method for preparing a polyvinyl alcohol hydrogel patch with a double-disc structure according to claim 1, characterized in that: Step S3 specifically includes the following steps: immersing t-PVA in a sodium citric acid solution for 12 h to 24 h to complete the first hydrogel strengthening and obtain a strengthened hydrogel patch a.

6. The method for preparing the polyvinyl alcohol hydrogel patch with a double-disc structure according to claim 5, characterized in that: The concentration of the sodium citrate solution is 1 mol / L-2 mol / L.

7. The method for preparing a polyvinyl alcohol hydrogel patch with a double-disc structure according to claim 1, characterized in that: Step S4 specifically includes the following steps: immersing the reinforced hydrogel patch a in glycerol at room temperature for at least 48 hours to obtain a PVA / glycerol organogel, placing the obtained PVA / glycerol organogel in a dryer at 100°C-150°C for wet annealing for 30 minutes to complete the second hydrogel reinforcement to obtain a reinforced hydrogel patch b.

8. The method for preparing a polyvinyl alcohol hydrogel patch with a double-disc structure according to claim 1, characterized in that: Step S5 specifically includes the following steps: immersing the reinforced hydrogel patch b in deionized water for more than 48 hours until swelling equilibrium is reached to obtain e-PVA.

9. A polyvinyl alcohol hydrogel patch with a double-disc structure, characterized in that: Prepared according to the preparation method according to any one of claims 1 to 8.

10. Use of the polyvinyl alcohol hydrogel patch with a double-disc structure according to claim 9 in the preparation of gastric perforation repair products, tracheal fistula repair products, and tracheoesophageal fistula repair products.

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