Swim bladder-based bioactive patch for rotator cuff tendon repair and preparation method of swim bladder-based bioactive patch

Abstract

The invention provides a swim bladder-based bioactive patch for rotator cuff tendon repair and a preparation method thereof, and relates to the technical field of biological materials.The swim bladder-based bioactive patch is composed of a swim bladder substrate and a metal-phenol toughening network component, has good tensile strength and biological activity and can be used for repairing rotator cuff tendons. The compound can support cell adhesion and proliferation, stimulate differentiation and phenotype maintenance of various cells derived from tendon-bone tissue, and is expected to be used for repairing rotator cuff tendon injury and promoting tendon-bone healing. Besides, the invention provides a preparation method of the swim bladder-based rotator cuff patch, a special microstructure of directionally arranged nano-matrix fibers is reserved, and a metal-phenol network further plays roles in toughening the patch and enhancing the biological activity of the material. Particularly, the metal-network has an active oxygen free radical response function, excessive free radicals at the shoulder joint part can be removed, and metal ions are released, so that the patch has the effects of resisting bacteria, resisting inflammation, resisting oxidation and improving healing.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and particularly to a swim bladder-based bioactive patch for rotator cuff tendon repair and a preparation method thereof. Background Art

[0002] Rotator cuff tendon injury is a common sports medicine disease, often requiring surgical repair. Due to the poor blood supply of the rotator cuff tendon itself and degenerative changes, etc., existing surgical repairs face problems such as gaps between tendon and bone, long healing cycles, tendon degeneration, and poor tendon-bone healing. In addition, due to the certain tension of the tendon itself, the tendon after surgical repair also faces the problem of excessive tension, which can lead to difficult tendon healing or even re-rupture. And for some severe rotator cuff tendon injuries, such as massive rotator cuff tears, etc., it is difficult to suture the tendon through surgery, resulting in gaps between the tendon and the bone interface.

[0003] To address the above problems, using tendon patches or tendon grafts for adjuvant treatment of damaged tendons and tendon transplantation is a feasible treatment method. Tendon patches can reduce the tension between the tendon and the bone, provide an attachment point for cell growth, and thus improve the healing of the tendon and the tendon-bone interface. Artificial tendon patches are increasingly favored by clinicians due to factors such as wide sources, avoidable donor site injury, low immunogenicity, and no risk of infectious diseases. Although a series of preparation methods of artificial tendon patches have been proposed in recent years, it is still difficult for existing artificial tendon patches to balance high strength and high bioactivity, and this problem remains to be solved. Summary of the Invention

[0004] The purpose of the present invention is to solve the problem that existing artificial tendon patches are difficult to balance high strength and high bioactivity.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] A swim bladder-based bioactive patch for rotator cuff tendon repair, characterized in that: the bioactive patch is obtained by modifying a swim bladder substrate through a metal-polyphenol network.

[0007] Preferably, the swim bladder substrate is prepared from natural swim bladders.

[0008] Preferably, the swim bladder substrate is prepared from crucian carp swim bladders.

[0009] Preferably, the metal-polyphenol network is a combination of polyphenols and metal ions, wherein the polyphenols are at least one of tannic acid, epigallocatechin gallate, ellagic acid, catechin, anthocyanin, resveratrol.

[0010] Preferably, the metal ion is at least one of gallium ion, strontium ion, calcium ion, zinc ion, magnesium ion, copper ion, and manganese ion.

[0011] The present application also provides an application of a fish bladder-based bioactive patch for rotator cuff tendon repair in the preparation of a rotator cuff tendon repair patch, and the bioactive patch is the above-mentioned bioactive patch.

[0012] The present application also provides a preparation method of a fish bladder-based bioactive patch for rotator cuff tendon repair, which is used to prepare the above-mentioned fish bladder-based bioactive patch for rotator cuff tendon repair, and the preparation method includes the following steps:

[0013] S1: Fish bladder pretreatment:

[0014] Collect fresh fish bladders, wash and strip off soft tissues such as fascia, soak in 75% medical alcohol for 1 hour for disinfection, wash with deionized water, and then soak in a 0.25% concentration of EDTA-trypsin solution, incubate at 37 °C for 1 hour for decellularization treatment. Subsequently, after washing with deionized water, soak in an SDS solution with a concentration of 1 mg / mL and a DNA enzyme solution for 1 hour each for decellularization / nucleic acid treatment. This process is incubated at 37 °C, and then washed with deionized water; after the washing is completed, the fish bladders are placed at -80 °C and frozen for 1 hour, and then freeze-dried to form the fish bladder substrate of the patch.

[0015] S2: Metal-polyphenol precursor toughening solution:

[0016] Dissolve polyphenols and soluble salts of metal ions in a buffer solution, adjust the pH to 7.5 - 9.5, and after the polyphenols and metal salts are dissolved, centrifuge at 10000 rpm to remove the precipitate. The supernatant is the metal-polyphenol precursor toughening solution;

[0017] S3: Fish bladder toughening treatment:

[0018] Place the fish bladder substrate in S1 into the metal-polyphenol precursor toughening solution in S2 for 6 hours;

[0019] S4: Freeze-drying:

[0020] After the fish bladder substrate after toughening is washed and freeze-dried for the second time, a fish bladder-based rotator cuff repair patch is obtained.

[0021] Preferably, the total concentration of polyphenols contained in the metal-polyphenol precursor toughening solution in S2 is 0.1 M - 5 M; the total concentration of metal ions contained in the metal-polyphenol precursor toughening solution is 0.1 M - 0.5 M.

[0022] Preferably, the buffer solution is one of Tris-HCl buffer solution, citrate buffer solution, or MES buffer solution, and the pH is 7.5 - 9.5.

[0023] Preferably, 10 mL of toughening solution needs to be prepared for every 1 g of fish bladder substrate in S3.

[0024] Compared with the prior art, the present application has the following beneficial effects:

[0025] 1. The bioactive patch prepared in the present application retains the special microstructure of the nano-matrix fibers arranged in an oriented manner. Further, the method of toughening with a metal-phenol network is adopted. The phenolic hydroxyl groups in the polyphenol component form hydrogen bond linkages, Schiff base reactions, and Michael addition reactions with groups such as amino groups and hydroxyl groups in the fish bladder. At the same time, metal ions combine with phenolic hydroxyl groups under weakly alkaline conditions, and finally a uniform metal-polyphenol cross-linked network is formed in the fish bladder matrix. Thus, it is possible to successfully prepare fish bladder-based rotator cuff biological patches with different metal ion component contents.

[0026] 2. Through the setting of the metal-polyphenol network, it not only further enhances the strength of the fish bladder, but also improves the biological activity of the material. In particular, this network has a reactive oxygen species radical response function, which can scavenge excessive free radicals in the shoulder joint area and release metal ions, enabling the patch to play antibacterial, anti-inflammatory, antioxidant, and healing improvement roles.

[0027] 3. The bioactive patch prepared in the present application has excellent mechanical properties and biological activity. When performing rotator cuff tendon repair surgery, one end can be fixed to the humeral head and the other end can be sutured to the stump of the rotator cuff tendon. This fish bladder-based patch can stimulate the differentiation of various cells, can achieve the regulation of rotator cuff tendon regeneration, repair injuries, and improve the healing and biomechanics of the tendon-bone interface. Description of the Drawings

[0028] Figure 1It is the characterization data graph of the fish bladder-based rotator cuff patches modified by different gallium-tannic acid toughening networks in Embodiments 1-3 of the present invention. Among them, (A) is the scanning / transmission electron micrograph, fiber diameter statistics and EDS energy spectrum analysis of each group of fish bladder rotator cuff patches (each component is, FSB: fish bladder substrate after preliminary preparation of natural fish bladder; PP@FSB: fish bladder substrate treated with polyphenol solution without metal ions; 1GaPP@FSB, 2GaPP@FSB, 3GaPP@FSB are fish bladder-based rotator cuff patches modified by various gallium-tannic acid toughening networks in Embodiments 1-3), (B) is the infrared spectrum (ATR-FTIR) data of the fish bladder-based rotator cuff patches in Embodiments 1-3, (C) is the X-ray photoelectron spectroscopy analysis (XPS) data of the fish bladder-based rotator cuff patches in Embodiments 1-3. The above data illustrate that the gallium-tannic acid toughening network has been successfully modified on the fish bladder substrate. (D) is the degradation data of the fish bladder-based rotator cuff patches in Embodiments 1-3, that is, 200 mg of the patch sample is immersed in 10 ml of type I collagenase-PBS solution (the concentration of type I collagenase is 0.1 mg / ml), incubated in a water bath at 37 °C, and the samples are taken out and dried at each time point and weighed. The degradation rate can be analyzed according to the mass loss of the samples at each time point.

[0029] Figure 2 It is the Mapping element analysis of the fish bladder substrate (FBS), the polyphenol-treated fish bladder substrate (PP@FSB), and the fish bladder-based rotator cuff patches modified by various gallium-tannic acid toughening networks in Embodiments 1-3 (1GaPP@FSB, 2GaPP@FSB, 3GaPP@FSB). It can be seen that after treatment with the metal-polyphenol toughening solution, the metal element (gallium) is evenly deposited between the nanofibers.

[0030] Figure 3 It is the detection of the mechanical properties of the materials of the fish bladder substrate (FBS), the polyphenol-treated fish bladder substrate (PP@FSB), and the fish bladder-based rotator cuff patches modified by various gallium-tannic acid toughening networks in Embodiments 1-3 (1GaPP@FSB, 2GaPP@FSB, 3GaPP@FSB). (A) is the maximum tensile strength, elastic modulus and maximum elongation of each group of materials, and (B) is the digital photo of 1GaPP@FSB suspending a 500 g weight.

[0031] Figure 4The effects of various fish swim bladder-based rotator cuff patches on the cell adhesion, migration, and proliferation of various cells derived from the tendon-bone interface (bone marrow mesenchymal stem cells BMSCs, chondrocytes chondrocyte, and tendon stem cells TSPC). (A) Fluorescent images of live / dead staining of various cells seeded on the patch, showing that BMSCs, chondrocyte, and TSPC can adhere to the patch directionally; (B) CCK-8 data showing that the cells seeded on the patch can proliferate in the long term, indicating that the patch has good biocompatibility; (C) Schematic diagram of the experimental design for the effect of the patch on cell migration, that is, the solution after complete enzymatic digestion of the patch is co-cultured with cells to observe cell migration; (D) Quantitative data of the 24-hour cell scratch test; (E) Observation of cell migration under an optical microscope (*P<0.05; **P<0.01; ***P<0.001);

[0032] Figure 5 For (A) Intraoperative photos of the modeling and repair surgery for repairing rat rotator cuff injury using the fish swim bladder-based rotator cuff patch (GaPP@FSB), (B) Schematic diagram of the model for rotator cuff tendon injury modeling and repair;

[0033] Figure 6 Rotator cuff pathological tissue section staining at 4 weeks and 8 weeks after repair with the fish swim bladder-based rotator cuff patch, including HE staining, toluidine blue staining, and safranin-fast green staining, as well as immunofluorescence labeling images of the rotator cuff tissue (TNF-α, Arg-1, and ColII);

[0034] Figure 7 Comparison of the mechanical properties of the fish swim bladder-based rotator cuff patches prepared in Examples 4-7 of this application. Detailed Description of the Invention

[0035] The present invention will be further described in detail below in conjunction with specific embodiments.

[0036] A fish swim bladder-based bioactive patch for rotator cuff tendon repair, the bioactive patch is prepared based on natural fish swim bladder. Specifically, the bioactive patch is obtained by modifying the fish swim bladder substrate with a metal-polyphenol network. The biological activity of the metal-polyphenol network is manifested as having a reactive oxygen species radical response function, which can scavenge excessive free radicals in the shoulder joint area and release metal ions, playing antibacterial, anti-inflammatory, antioxidant, and healing improvement roles.

[0037] The bioactive patch provided in this application retains the special microstructure of the nano matrix fibers arranged directionally, and at the same time the metal-polyphenol network plays a role in toughening the patch and enhancing the biological activity of the material.

[0038] The fish swim bladder substrate is prepared from natural fish swim bladder. Preferably, the fish swim bladder substrate is prepared based on the fish swim bladder of crucian carp.

[0039] The metal-polyphenol network is a combination of polyphenols and metal ions. In one embodiment, the polyphenol is at least one of tannic acid, epigallocatechin gallate, ellagic acid, catechin, anthocyanin, resveratrol; the metal ion is at least one of gallium ion, strontium ion, calcium ion, zinc ion, magnesium ion, copper ion, manganese ion.

[0040] This application also provides the use of the above-mentioned bioactive patch in the preparation of a rotator cuff tendon repair patch. The mechanical strength of the bioactive patch can match that of the natural rotator cuff tendon (maximum destructive force, patch: 26.4 Mpa, natural rotator cuff tendon: 4 - 24 MPa); moreover, the maximum tensile force of the bioactive patch is 15.0 - 26.4 MPa, and the Young's modulus is 87 - 102 Mpa.

[0041] The application method of the bioactive patch is as follows: First, fix one end of the bioactive patch to the humeral head, and suture the other end to the stump of the rotator cuff tendon. The bioactive patch plays a role in bridging the gap between the tendon and bone and acting as a bridge.

[0042] In addition, this application also provides a preparation method of the above-mentioned bioactive patch, which includes the following steps:

[0043] S1: Pre-treatment of fish bladder

[0044] Collect fresh fish bladders. After washing and peeling off soft tissues such as fascia, soak them in 75% medical alcohol for 1 hour for disinfection. After washing with deionized water, soak them in a 0.25% concentration of EDTA-trypsin solution and incubate at 37°C for 1 hour for decellularization treatment. Subsequently, after washing with deionized water, soak them in a 1 mg / mL sodium dodecyl sulfate (SDS) solution and a DNA enzyme solution for 1 hour each for decellularization / nucleic acid treatment. This process is maintained at 37°C for incubation, and then washed with deionized water;

[0045] After the washing is completed, place the fish bladder at -80°C for 1 hour, and then perform freeze-drying to form the fish bladder substrate of the patch;

[0046] S2: Metal-polyphenol precursor toughening solution:

[0047] According to the composition of the polyphenol and metal ion components, prepare a precursor toughening solution containing polyphenols and metal ions at a certain concentration. The solution preparation method is to dissolve soluble salts of polyphenols and metal ions at a certain concentration in a buffer solution, adjust the pH to 7.5 - 9.5. After the polyphenol and metal salt are dissolved, centrifuge at 10000 rpm to remove the precipitate, and the supernatant is the metal-polyphenol precursor toughening solution.

[0048] In one embodiment, the polyphenol is at least one of tannic acid, epigallocatechin gallate, ellagic acid, catechin, anthocyanin, resveratrol; the metal ion is at least one of gallium ion, strontium ion, calcium ion, zinc ion, magnesium ion, copper ion, manganese ion.

[0049] The total concentration of the polyphenol contained in the metal-polyphenol precursor toughening solution is 0.1M - 5M; the total concentration of the metal ion contained in the metal-polyphenol precursor toughening solution is 0.1M - 0.5M.

[0050] The buffer solution is one of Tris-HCl buffer solution, citric acid buffer solution or MES buffer solution, and the pH is 7.5 - 9.5.

[0051] S3: Toughening treatment of fish swim bladder

[0052] Place the fish swim bladder substrate obtained in S1 into the metal-polyphenol precursor toughening solution in S2 for 6 hours. For every 1 g of fish swim bladder substrate, 10 mL of toughening solution needs to be prepared. After soaking in the metal-polyphenol precursor toughening solution, the metal-polyphenol toughening network can be modified on the fish swim bladder substrate, playing a role in toughening and improving biological activity.

[0053] S4: Freeze-drying

[0054] After the fish swim bladder substrate is toughened, washed, and secondarily freeze-dried, a fish swim bladder glenoid repair patch is obtained.

[0055] The above content is elaborated below in conjunction with specific embodiments:

[0056] Experimental materials and sources:

[0057]

[0058]

[0059] Example 1

[0060] Fresh crucian carp swim bladders were washed with running water. After stripping off soft tissues such as fascia, they were soaked in 75% medical alcohol for 1 hour for disinfection. After washing with deionized water, they were soaked in a 0.25% concentration of EDTA-trypsin solution and incubated at 37 °C for 1 hour for decellularization treatment. Subsequently, after washing with deionized water, they were successively soaked in a 1 mg / mL sodium dodecyl sulfate (SDS) solution and a DNA enzyme solution for 1 hour each for decellularization / nucleic acid treatment. This process was carried out with incubation at 37 °C. Subsequently, they were washed with deionized water. Then the swim bladders were placed in a -80 °C low-temperature refrigerator and frozen for 1 hour and then freeze-dried to form the swim bladder substrate for the patch. Subsequently, a gallium-tannic acid precursor toughening solution was prepared. Gallium nitrate and tannic acid were dissolved in Tris-HCl buffer. The concentration of gallium nitrate was 0.1 M, the concentration of tannic acid was 2 M, and the pH of the solution was adjusted to 8.5. The mixed solution was centrifuged at 10,000 rpm to obtain the gallium-tannic acid precursor toughening solution. Subsequently, 3 g of the processed swim bladder substrate was placed in a centrifuge tube, and 30 mL of the gallium-tannic acid precursor toughening solution was added to soak the swim bladder substrate for 6 hours. Subsequently, the patch was washed with deionized water, placed in a -80 °C low-temperature refrigerator and frozen for 1 hour again and then freeze-dried to obtain the swim bladder-based rotator cuff biological patch, named 1GaPP@FSB. Through scanning electron microscopy, transmission electron microscopy, EDS energy spectrum and Mapping analysis, it was found that the microstructure of the oriented arrangement of nanofibers in 1GaPP@FSB was well preserved, the gallium-tannic acid network was evenly deposited between the nanofibers, and the gallium element content was 0.18%. Through mechanical testing, it was found that the maximum breaking tensile force of 1GaPP@FSB was 26.0 ± 0.9 MPa, and the elastic modulus was 107.6 ± 9.7 MPa.

[0061] Example 2

[0062] Fresh crucian carp swim bladders were washed with running water. After stripping off soft tissues such as fascia, they were soaked in 75% medical alcohol for 1 hour for disinfection. After washing with deionized water, they were soaked in a 0.25% concentration of EDTA-trypsin solution and incubated at 37°C for 1 hour for decellularization treatment. Subsequently, after washing with deionized water, they were successively soaked in a 1mg / mL sodium dodecyl sulfate (SDS) solution and a DNA enzyme solution for 1 hour each for decellularization / nucleic acid treatment. This process was maintained at 37°C incubation. Subsequently, they were washed with deionized water. Then the swim bladders were placed in a -80°C low-temperature refrigerator and frozen for 1 hour and then freeze-dried to form the swim bladder substrate of the patch. Subsequently, a gallium-tannic acid precursor toughening solution was prepared. Gallium nitrate and tannic acid were dissolved in Tris-HCl buffer. The concentration of gallium nitrate was 0.2M, and the concentration of tannic acid was 2M. The pH of the solution was adjusted to 8.5. The mixed solution was centrifuged at 10000rpm to obtain the gallium-tannic acid precursor toughening solution. Subsequently, 3g of the processed swim bladder substrate was placed in a centrifuge tube, and 30mL of the gallium-tannic acid precursor toughening solution was added to soak the swim bladder substrate for 6 hours. Subsequently, the patch was washed with deionized water, placed in a -80°C low-temperature refrigerator and frozen for 1 hour again and then freeze-dried to obtain the swim bladder-based rotator cuff biological patch, named 2GaPP@FSB. Through scanning electron microscopy, transmission electron microscopy, EDS energy spectrum and Mapping analysis, it was found that the oriented arrangement microstructure of the nanofibrous matrix in 2GaPP@FSB was well preserved, the gallium-tannic acid network was evenly deposited between the nanofibers, and the gallium element content was 0.24%. Through mechanical testing, it was found that the maximum breaking tensile force of 2GaPP@FSB was 26.4±0.6MPa, and the elastic modulus was 102.0±11.0MPa.

[0063] Example 3

[0064] Fresh crucian carp swim bladders were washed with running water. After stripping off soft tissues such as fascia, they were soaked in 75% medical alcohol for 1 hour for disinfection. After washing with deionized water, they were soaked in a 0.25% concentration of EDTA-trypsin solution and incubated at 37°C for 1 hour for decellularization treatment. Subsequently, after washing with deionized water, they were successively soaked in a 1 mg / mL sodium dodecyl sulfate (SDS) solution and a DNA enzyme solution for 1 hour each for decellularization / nucleic acid treatment. This process was maintained at 37°C for incubation. Subsequently, they were washed with deionized water. Then, the swim bladders were placed in a -80°C low-temperature refrigerator and frozen for 1 hour, followed by freeze-drying to form the swim bladder substrate for the patch. Subsequently, a gallium-tannic acid precursor toughening solution was prepared. Gallium nitrate and tannic acid were dissolved in Tris-HCl buffer. The concentration of gallium nitrate was 0.5 M, the concentration of tannic acid was 2 M, and the pH of the solution was adjusted to 8.5. The mixed solution was centrifuged at 10,000 rpm to obtain the gallium-tannic acid precursor toughening solution. Subsequently, 3 g of the processed swim bladder substrate was placed in a centrifuge tube, and 30 mL of the gallium-tannic acid precursor toughening solution was added to soak the swim bladder substrate for 6 hours. Subsequently, the patch was washed with deionized water, placed in a -80°C low-temperature refrigerator and frozen for 1 hour again, followed by freeze-drying to obtain the swim bladder-based rotator cuff biological patch, named 3GaPP@FSB. Through scanning electron microscopy, transmission electron microscopy, EDS energy spectrum and Mapping analysis, it was found that the nano matrix fibers in 3GaPP@FSB were thickened to some extent, but their oriented arrangement microstructure was well preserved. The gallium-tannic acid network was evenly deposited between the nanofibers, and the gallium element content was 0.58%. Through mechanical testing, it was found that the maximum breaking tensile force of 3GaPP@FSB was 22.4 ± 0.7 MPa, and the elastic modulus was 102.2 ± 5.0 MPa.

[0065] The performance of the swim bladder-based rotator cuff biological patch was evaluated using the bioactive patch prepared through the above examples. Specifically, the performance to be evaluated included mechanical properties, cell compatibility activity, and the function of repairing tendon injuries in animals. The above performance evaluations characterized the microstructure and compositional changes of the swim bladder rotator cuff biological patch through means such as scanning electron microscopy, transmission electron microscopy, infrared spectroscopy, and XPS spectroscopy. The results are shown as follows:

[0066] From Figure 1 it can be seen that the swim bladder-based bioactive patches prepared in the present invention, namely Examples 1-3, still retained the microstructure of the nano matrix fibers arranged in an oriented manner. In addition Figure 1 B The infrared spectroscopy data showed characteristic peaks from phenolic hydroxyl groups, and the XPS spectrum ( Figure 1 C) showed Ga2p and Ga3d characteristic peaks from gallium elements. And Figure 1 A The EDS data in showed that with the increase in the concentration of gallium ions in the gallium-tannic acid toughening precursor solution, the deposition amount of gallium ions in the patch increased. In addition, Figure 1D shows the degradation data of the fish swim bladder-based rotator cuff patch. Figure 2 The Mapping data shows that gallium elements are evenly deposited on the nanofibers of the patch. The above data illustrate that gallium-tannic acid is successfully modified on the matrix, and the deposition amount of metal ions on the patch can be adjusted by controlling the concentration of the toughening precursor solution.

[0067] From Figure 3 it can be seen that the fish swim bladder-based rotator cuff biological patch exhibits good maximum tensile strength and elastic modulus, both of which can match the natural rotator cuff tendon (4 - 24 Mpa), showing good mechanical properties. Since the mechanical strength of the fish swim bladder matrix FSB is poor, it indicates that in a metal-polyphenol precursor toughening solution with a certain concentration, the mechanical properties of the fish swim bladder-based bioactive patch are greatly enhanced, thereby enhancing its applicability in rotator cuff tendon repair.

[0068] Verification Experiment 1: Cell Experiment

[0069] Rat BMSCs, primary rat TSPCs, and primary rat chondrocytes were used. All fish swim bladder patch samples were cut into discs with a diameter of 1.2 cm, immersed in 75% ethanol, sterilized by ultraviolet irradiation, and then attached to the bottom of the wells of a 24-well cell culture plate. Subsequently, BMSCs, TSPCs, and chondrocytes were seeded onto the scaffolds at a density of 2×10 4 cells per well. After 24 hours, the cells were fluorescently labeled with a live / dead staining kit, and cell adhesion was observed by fluorescence microscopy. In addition, a CCK-8 kit was used to analyze the viability of BMSCs and TSPCs 1, 3, and 7 days after they were seeded onto each sample. In addition, the effect of the material on the migration of these three types of cells was verified by a scratch experiment. First, a part of the 2GaPP@FSB scaffold in Example 2 was taken and digested with a pepsin solution (concentration 1 mg / ml, 1 ml of pepsin solution corresponding to 100 mg of scaffold sample). BMSCs, TSPCs, and chondrocytes were seeded into a 24-well cell culture plate at a density of 5×10 4 cells per well. After 24 hours, the cells were scratched with a 1000 μL sterile pipette tip, and the scratch images were taken with a microscope and the positions were marked. Subsequently, 100 μL of the digested solution was added to the medium of each well of the culture plate, and after incubating for another 24 hours, the scratch site images were taken with a microscope, and the cell fusion situation was counted.

[0070] From Figure 4It can be seen that the fish bladder-based rotator cuff biological patches in Examples 1-3 show good cell compatibility with various cells, such as bone marrow mesenchymal stem cells (BMSCs), chondrocytes, and tendon stem / progenitor cells (TSPCs). They can support cell adhesion, proliferation, and cell migration. After the cells were seeded on the patches for 1, 4, and 7 days, obvious cell proliferation and directional adhesion were presented, and cell migration was strongly promoted, indicating that the fish bladder-based biological active patches have good cell compatibility.

[0071] Verification experiment 2: Animal modeling of rotator cuff injury:

[0072] All animal experiments were conducted in accordance with the procedures approved by the Experimental Animal Ethics Committee of the Experimental Animal Center of Nantong University (S20240116-004). All experimental procedures complied with the ARRIVE guidelines and were carried out in accordance with the 1986 UK Animals (Scientific Procedures) Act and related guidelines. Briefly, for the steps of creating a rotator cuff injury model and surgical repair in rats, adult male Sprague-Dawley rats (260±22 g) were anesthetized with isoflurane gas and fixed in the supine position. After shaving the skin over the shoulder joint, the skin was disinfected. The acromial skin was incised with a scalpel to expose the supraspinatus tendon of the rotator cuff, and the supraspinatus tendon was separated with a Kirschner wire. After the supraspinatus tendon was transected, each group of scaffolds (3 mm×6 mm) was used for repair, that is, one end of the scaffold was sutured to the stump of the supraspinatus tendon, and the other end was fixed to the humeral head. The suture used during the operation was 4-0 Ethibond suture. A 1-mm-wide hole was drilled in the humeral head with a Kirschner wire, and the suture passed through this hole to firmly fix the scaffold to the humeral head. After the operation, the wound was sutured and disinfected, and the rats were allowed to move freely in the cage. In this verification experiment, the fish bladder-based rotator cuff biological patch prepared in Example 1 was used.

[0073] From Figure 5 It can be seen that the fish bladder-based rotator cuff biological patch (GaPP@FSB) is applicable to rotator cuff repair surgery. When the patch is cut into a size of 0.5×0.2 cm, it can be well transplanted through surgery to the injured site of the rotator cuff tendon in rats. One end of the patch can be fixed to the humeral head through surgery, and the other end is sutured to the stump of the rotator cuff tendon. The patch plays a role in bridging the gap between the tendon and bone.

[0074] Verification experiment 3: Staining of rotator cuff pathological tissue sections

[0075] Rats were sacrificed by isoflurane gas anesthesia at 4 weeks and 8 weeks after repair with the fish swim bladder-based rotator cuff patch. The supraspinatus tendon attached to the humeral head in each rat model was collected, and other soft tissues were excised from the humeral head. Subsequently, all samples were fixed in 10% neutral formalin solution for three days, and then immersed in EDTA decalcifying solution for one month for decalcification. After complete decalcification, the samples were dehydrated through a gradient alcohol solution (30%-100%). Subsequently, the samples were treated with xylene and further embedded in paraffin. Paraffin sections were made along the direction from tendon to bone, and the section thickness was 5 μm. After dewaxing the paraffin sections, safranin-fast green staining, H&E staining, and toluidine blue staining were performed. The stained sections were observed under an optical microscope and images were taken. In addition, the sections were treated with primary antibody solutions of CoL II, TNF-α, and Arg-1. Subsequently, after washing with PBS, the sections stained with the primary antibody were treated with corresponding fluorescently labeled secondary antibody solutions. After washing with PBS and mounting with an anti-fluorescence quencher, the sections were observed and photographed under a fluorescence microscope.

[0076] This application also verified the application effect of the fish swim bladder-based rotator cuff patch by performing a repair operation on rats using the fish swim bladder-based rotator cuff patch. From Figure 6 It can be seen that at 4 weeks and 8 weeks after the repair of the rat rotator cuff injury, the pathological section staining pictures (HE staining, toluidine blue staining, and safranin-fast green staining) showed that the fish swim bladder-based rotator cuff biological patch (GaPP@FSB) could fuse well with bone and tendon, and significantly promoted the healing of the tendon-bone interface, that is, significantly promoted the regeneration of the fibrocartilage layer. This shows that the fish swim bladder-based rotator cuff biological patch can enhance the healing after rotator cuff injury.

[0077] As can be seen above, the fish swim bladder-based bioactive patch has excellent mechanical properties and biological activities, indicating that the fish swim bladder-based bioactive patch can be applied to clinical rotator cuff tendon-related injuries.

[0078] Example 4

[0079] Fresh bighead carp swim bladders were washed with running water. After stripping off soft tissues such as fascia, they were soaked in 75% medical alcohol for 1 hour for disinfection. After washing with deionized water, they were soaked in a 0.25% concentration of EDTA-trypsin solution and incubated at 37°C for 1 hour for decellularization treatment. Subsequently, after washing with deionized water, they were successively soaked in a 1mg / mL sodium dodecyl sulfate (SDS) solution and a DNA enzyme solution for 1 hour each for decellularization / nucleic acid treatment. This process was maintained at 37°C incubation. Subsequently, they were washed with deionized water. Then the swim bladders were placed in a -80°C low-temperature refrigerator and frozen for 1 hour and then freeze-dried to form the swim bladder substrate for the patch. Subsequently, a strontium-EGCG (epigallocatechin gallate) precursor toughening solution was prepared. Strontium chloride and EGCG were dissolved in MES buffer. The concentration of strontium chloride was 0.2M, and the concentration of EGCG was 3M. The pH of the solution was adjusted to 8.0. The mixed solution was centrifuged at 10,000 rpm to obtain the strontium-EGCG precursor toughening solution. Subsequently, 3g of the processed swim bladder substrate was placed in a centrifuge tube, and 30mL of the gallium-tannic acid precursor toughening solution was added to soak the swim bladder substrate for 6 hours. Subsequently, the patch was washed with deionized water, placed in a -80°C low-temperature refrigerator and frozen for 1 hour again and then freeze-dried to obtain the strontium-EGCG modified swim bladder-based rotator cuff biological patch, named SrEG@FSB. As Figure 7 shown, through mechanical tests, it was found that the maximum breaking tensile force of SrEG@FSB was 21.3±3.5MPa, and the elastic modulus was 91.6±9.5MPa.

[0080] Example 5

[0081] Fresh carp swim bladders were washed with running water. After stripping soft tissues such as fascia, they were soaked in 75% medical alcohol for 1 hour for disinfection. After washing with deionized water, they were soaked in a 0.25% concentration of EDTA-trypsin solution and incubated at 37°C for 1 hour for decellularization treatment. Subsequently, after washing with deionized water, they were successively soaked in a 1 mg / mL sodium dodecyl sulfate (SDS) solution and a DNA enzyme solution for 1 hour each for decellularization / nucleic acid treatment. This process was maintained at 37°C incubation. Subsequently, they were washed with deionized water. Then the swim bladders were placed in a -80°C low-temperature refrigerator and frozen for 1 hour and then freeze-dried to form the swim bladder substrate for the patch. Subsequently, a magnesium-anthocyanin precursor toughening solution was prepared. Magnesium chloride and anthocyanin were dissolved in MES buffer. The concentration of magnesium chloride was 0.3 M, and the concentration of anthocyanin was 2 M. The pH of the solution was adjusted to 8.5. The mixed solution was centrifuged at 10,000 rpm to obtain the magnesium-anthocyanin precursor toughening solution. Subsequently, 3 g of the processed swim bladder substrate was placed in a centrifuge tube, and 30 mL of the magnesium-anthocyanin precursor toughening solution was added to soak the swim bladder substrate for 6 hours. Subsequently, the patch was washed with deionized water, placed in a -80°C low-temperature refrigerator and frozen for 1 hour again and then freeze-dried to obtain a magnesium-anthocyanin-modified fish swim bladder rotator cuff biological patch, named MgAN@FSB. As Figure 7 shown, through mechanical testing, it was found that the maximum breaking tensile force of MgAN@FSB was 25.8 ± 4.1 MPa, and the elastic modulus was 101.6 ± 12.5 MPa.

[0082] Example 6

[0083] Fresh bighead carp swim bladders were washed with running water. After stripping fascia and other soft tissues, they were soaked in 75% medical alcohol for 1 hour for disinfection. After washing with deionized water, they were soaked in a 0.25% concentration of EDTA-trypsin solution and incubated at 37°C for 1 hour for decellularization treatment. Subsequently, after washing with deionized water, they were successively soaked in a 1mg / mL sodium dodecyl sulfate (SDS) solution and a DNA enzyme solution for 1 hour each for decellularization / nucleic acid treatment. This process was carried out while incubating at 37°C. Subsequently, they were washed with deionized water. Then the swim bladders were placed in a -80°C low-temperature refrigerator and frozen for 1 hour and then freeze-dried to form the swim bladder substrate for the patch. Subsequently, a calcium / copper-ellagic acid precursor toughening solution was prepared. Calcium chloride, copper chloride, and ellagic acid were dissolved in Tris-HCl buffer. The concentration of calcium chloride was 0.2M, the concentration of copper chloride was 0.1M, and the concentration of ellagic acid was 2M. The pH of the solution was adjusted to 9. The mixed solution was centrifuged at 10,000 rpm to obtain the calcium / copper-ellagic acid precursor toughening solution. Subsequently, 3g of the processed swim bladder substrate was placed in a centrifuge tube, and 30mL of the magnesium-anthocyanin precursor toughening solution was added to soak the swim bladder substrate for 6 hours. Subsequently, the patch was washed with deionized water, placed in a -80°C low-temperature refrigerator again and frozen for 1 hour and then freeze-dried to obtain the calcium / copper-ellagic acid-modified swim bladder-based rotator cuff biological patch, named CaCuEA@FSB. As Figure 7 shown, through mechanical testing, it was found that the maximum failure tensile force of CaCuEA@FSB was 23.1±1.7MPa, and the elastic modulus was 98.3±5.2MPa.

[0084] Example 7

[0085] Fresh silver carp swim bladders were washed with running water. After stripping off soft tissues such as fascia, they were soaked in 75% medical alcohol for 1 hour for disinfection. After washing with deionized water, they were soaked in a 0.25% concentration of EDTA-trypsin solution and incubated at 37°C for 1 hour for decellularization treatment. Subsequently, after washing with deionized water, they were successively soaked in a 1 mg / mL sodium dodecyl sulfate (SDS) solution and a DNA enzyme solution for 1 hour each for decellularization / nucleic acid treatment. This process was maintained at 37°C incubation. Subsequently, they were washed with deionized water. Then the swim bladders were placed in a -80°C low-temperature refrigerator and frozen for 1 hour, followed by freeze-drying to form the swim bladder substrate for the patch. Subsequently, a calcium / copper-ellagic acid precursor toughening solution was prepared. Manganese chloride and resveratrol were dissolved in MES buffer. The concentration of manganese chloride was 0.15 M and the concentration of resveratrol was 4 M. The pH of the solution was adjusted to 8.5. The mixed solution was centrifuged at 10,000 rpm to obtain the manganese-resveratrol precursor toughening solution. Subsequently, 3 g of the processed swim bladder substrate was placed in a centrifuge tube, and 30 mL of the manganese-resveratrol precursor toughening solution was added to soak the swim bladder substrate for 6 hours. Subsequently, the patch was washed with deionized water, placed in a -80°C low-temperature refrigerator and frozen for 1 hour again, followed by freeze-drying to obtain the manganese-resveratrol-modified swim bladder-based rotator cuff biological patch, named MnRV@FSB. As Figure 7 shown, through mechanical tests, it was found that the maximum breaking tensile force of MnRV@FSB was 22.2 ± 1.9 MPa, and the elastic modulus was 90.2 ± 10.5 MPa.

[0086] In summary, this application provides a swim bladder-based rotator cuff patch for rotator cuff tendon repair and its preparation method. The swim bladder-based bioactive patch provided by the present invention is composed of a swim bladder substrate and a metal-polyphenol toughening network component, has good tensile strength and biological activity, can support cell adhesion and proliferation, stimulate the differentiation and phenotype maintenance of various cells derived from tendon-bone tissues, and is expected to be used for the repair of rotator cuff tendon injuries and promote tendon-bone healing. In addition, the present invention proposes a preparation method for the swim bladder-based rotator cuff patch, which retains the special microstructure of the nano-matrix fibers arranged in an oriented manner, and the metal-polyphenol network further plays a role in toughening the patch and enhancing the biological activity of the material. In particular, the metal-polyphenol network has a reactive oxygen species free radical response function, can scavenge excessive free radicals at the shoulder joint site, and release metal ions, enabling the patch to play antibacterial, anti-inflammatory, antioxidant and improve healing effects.

Claims

1. A swim bladder-based bioactive patch for rotator cuff tendon repair, characterized in that: The bioactive patch is obtained by modifying a fish bladder substrate through a metal-polyphenol network.

2. The bioactive patch based on fish bladder for rotator cuff tendon repair according to claim 1, wherein: The fish bladder substrate is prepared from natural fish bladders.

3. The bioactive patch based on fish bladder for rotator cuff tendon repair according to claim 2, wherein: The fish bladder substrate is prepared from crucian carp bladders.

4. The bioactive patch based on fish bladder for rotator cuff tendon repair according to claim 1, wherein: The metal-polyphenol network is a combination of polyphenols and metal ions. Among them, the polyphenols are at least one of tannic acid, epigallocatechin gallate, ellagic acid, catechin, anthocyanin, and resveratrol.

5. A swim bladder-based bioactive patch for rotator cuff tendon repair according to claim 4, characterized in that: The metal ions are at least one of gallium ions, strontium ions, calcium ions, zinc ions, magnesium ions, copper ions, and manganese ions.

6. Use of a swim bladder-based bioactive patch for rotator cuff tendon repair in the preparation of a rotator cuff tendon repair patch, characterized in that: The bioactive patch is the bioactive patch described in any one of claims 1-5.

7. A preparation method of a swim bladder-based bioactive patch for rotator cuff tendon repair, characterized in that: For preparing the fish bladder-based bioactive patch for rotator cuff tendon repair described in any one of claims 1-5, the preparation method comprises the following steps: S1: Fish bladder pretreatment: Collect fresh fish bladders. After washing and peeling off soft tissues such as fascia, soak them in 75% medical alcohol for 1 hour for disinfection. After washing with deionized water, soak them in a 0.25% concentration of EDTA-trypsin solution and incubate at 37 °C for 1 hour for decellularization treatment. Subsequently, after washing with deionized water, soak them in an SDS solution with a concentration of 1 mg / mL and a DNA enzyme solution for 1 hour each for decellularization / nucleic acid treatment. This process is kept at 37 °C for incubation, and then washed with deionized water. After the washing is completed, the fish bladders are placed at -80 °C for freezing for 1 hour, and then freeze-dried to form the fish bladder substrate of the patch. S2: Metal-polyphenol precursor toughening solution: Dissolve polyphenols and soluble salts of metal ions in a buffer solution, adjust the pH to 7.5-9.

5. After the polyphenols and metal salts are dissolved, centrifuge at 10000 rpm to remove the precipitate, and the supernatant is the metal-polyphenol precursor toughening solution. S3: Fish bladder toughening treatment: Place the fish bladder substrate in S1 into the metal-polyphenol precursor toughening solution in S2 for 6 hours. S4: Freeze-drying: The fish bladder substrate after toughening is washed and then freeze-dried for a second time to obtain the fish bladder-based rotator cuff repair patch.

8. The preparation method of a swim bladder-based bioactive patch for rotator cuff tendon repair according to claim 7, characterized in that: In the metal-polyphenol precursor toughening solution in S2, the total concentration of the polyphenols contained is 0.1M-5M; the total concentration of the metal ions contained in the metal-polyphenol precursor toughening solution is 0.1M-0.5M.

9. The preparation method of a swim bladder-based bioactive patch for rotator cuff tendon repair according to claim 8, characterized in that: The buffer solution is one of Tris-HCl buffer solution, citrate buffer solution, or MES buffer solution, and the pH is 7.5-9.

5.

10. The preparation method of a swim bladder-based bioactive patch for rotator cuff tendon repair according to claim 9, characterized in that: In S3, 10 mL of toughening solution needs to be prepared for every 1 g of fish bladder substrate.

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