Preparation method of decellularized extracellular matrix patch with antibacterial function, its products and applications

Abstract

This application discloses a method for preparing an antibacterial acellular extracellular matrix patch, its product, and its application, belonging to the field of medical materials technology. The preparation method includes: providing an acellular extracellular matrix patch matrix, and immersing the matrix in a sodium hypochlorite solution for chlorination treatment to obtain an antibacterial acellular extracellular matrix patch. This method enables N-haloamine functionalization at the molecular level of the acellular extracellular matrix patch matrix, not only endowing the prepared patch with excellent antibacterial properties and reducing the likelihood of bacterial resistance, but also allowing it to intelligently switch to an antibacterial effect after partial chlorination consumption, achieving long-lasting antibacterial action. Simultaneously, N-haloamine functionalization effectively maintains the good cell compatibility of the acellular extracellular matrix, making the prepared patch material promising for efficient repair of abdominal wall defects, with excellent application prospects.

Description

Technical Field

[0001] This application belongs to the field of medical materials technology, and in particular relates to a method for preparing a decellularized extracellular matrix patch with antibacterial function, its product and application. Background Technology

[0002] Acellular extracellular matrix (ACM) patches possess excellent biocompatibility, good mechanical properties, and the ability to induce cell growth and proliferation. They can significantly improve repair outcomes for abdominal wall defects and show promising application prospects in this field. However, when using ACM patches to repair infected abdominal wall defects clinically, infection is prone to occur before neovascularization, affecting patch durability and potentially leading to complications such as local abscesses and postoperative recurrence. Therefore, antibacterial modification of ACM patches is necessary to improve their antibacterial / bacterial properties.

[0003] Currently, the main method for antibacterial modification of decellularized extracellular matrix materials in related research is to combine the decellularized extracellular matrix materials with antibacterial agents (antibiotics, quaternary ammonium salts, silver nanoparticles). For example, Xu Cong et al. prepared antibacterial decellularized extracellular matrix materials by combining them with silver nanoparticles.

[0004] However, existing methods for modifying acellular extracellular matrix materials with composite antibacterial agents have the following problems in clinical applications: First, antibiotic-modified acellular extracellular matrix materials are prone to drug resistance, which cannot meet the repair needs of infected abdominal wall defects; second, metal nanoparticle-modified acellular extracellular matrix patches are prone to dissolution, causing serious cytotoxicity and endangering human safety. Summary of the Invention

[0005] This application discloses a method for preparing an antibacterial decellularized extracellular matrix patch, its product, and its application, which solves the technical problems of existing methods for modifying decellularized extracellular matrix materials with composite antibacterial agents, which damage biocompatibility and easily lead to drug resistance and cytotoxicity.

[0006] To achieve the above objectives, the first aspect of this application provides a method for preparing a decellularized extracellular matrix patch with antibacterial function. The preparation method of this application includes the following steps:

[0007] Provide a matrix for decellularized extracellular matrix patches;

[0008] The decellularized extracellular matrix patch substrate was chlorinated in a sodium hypochlorite solution;

[0009] In addition, an antibacterial decellularized matrix patch is harvested from the chlorination product of the previous step.

[0010] In some embodiments, the matrix source of the decellularized extracellular matrix patch includes the small intestine, heart valves, and bladder basement membrane of mammals.

[0011] In some implementations, the acellular extracellular matrix patch matrix is ​​derived from porcine small intestine.

[0012] In some embodiments, when the decellularized extracellular matrix patch matrix is ​​chlorinated in a sodium hypochlorite solution, the concentration of the sodium hypochlorite solution is 1-10 wt%.

[0013] In some embodiments, when the decellularized extracellular matrix patch substrate is chlorinated in a sodium hypochlorite solution, the volume ratio of the sodium hypochlorite solution to the decellularized extracellular matrix patch substrate is 20-100:1.

[0014] In some embodiments, when the decellularized extracellular matrix patch matrix is ​​chlorinated in sodium hypochlorite solution, the chlorination treatment time is 4-48 hours.

[0015] In some embodiments, the process further includes, after harvesting the decellularized extracellular matrix patch with antibacterial function from the chlorination product of the previous step:

[0016] The decellularized extracellular matrix patch with antibacterial function was sequentially drained, pre-frozen, vacuum freeze-dried, and laminated.

[0017] In some implementations, the lamination process includes vacuum lamination or hot pressing.

[0018] In some implementations, the parameters of the hot pressing process include: 1-12 hot pressing layers, 5-50 MPa hot pressing pressure, 45-70°C hot pressing temperature, and 1-20 min hot pressing time.

[0019] The second aspect of this application provides a decellularized extracellular matrix patch with antibacterial function prepared by the preparation method described in the first aspect of this application.

[0020] The third aspect of this application provides the application of the decellularized extracellular matrix patch with antibacterial function in the repair of abdominal wall defects.

[0021] Compared with the prior art, the advantages or beneficial effects of the embodiments of this application include at least the following:

[0022] The preparation method provided in this application involves immersing the decellularized extracellular matrix patch substrate in a sodium hypochlorite solution for chlorination treatment, thereby grafting an N-halogenated amine antibacterial coating onto the decellularized extracellular matrix patch substrate at the molecular level in one step. Firstly, the N-Cl bonds can be used to directly adsorb onto the cell membrane of infected bacteria and effectively inhibit their metabolic processes, achieving contact antibacterial / antimicrobial effects. Furthermore, the oxidatively active chloride ions released from the interaction of N-Cl bonds with water molecules can selectively react with certain active functional groups in the infected bacteria, thus endowing the prepared decellularized extracellular matrix patch with a dual-mechanism synergistic antibacterial / antimicrobial effect. This improves antibacterial / antimicrobial performance, avoids drug resistance issues, and effectively maintains good biocompatibility. Secondly, the N-halogenated amine antibacterial coating and the decellularized extracellular matrix patch... The matrix is ​​stably and firmly bonded together by covalent bonds, which can effectively prevent the dissolution of the N-haloamine antibacterial coating, ensuring the antibacterial / antimicrobial efficacy is durable and does not produce obvious cytotoxicity problems. Thirdly, this application proposes for the first time to use the decellularized extracellular matrix as the matrix for N-haloamine antibacterial modification treatment. The uniform and high-density amino groups distributed on its collagen backbone can be used to directly achieve the grafting modification of the N-haloamine coating. The preparation is simple and can effectively improve the grafting uniformity while effectively avoiding the introduction of components with poor biocompatibility, thereby achieving long-lasting antibacterial effect and ensuring good biocompatibility. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 Scanning electron microscope images of the surface morphology of SIS and SIS-NCl-1 samples provided for embodiments of this application;

[0025] Figure 2 X-ray photoelectron spectrum of the SIS-NCl-1 sample provided in the embodiments of this application;

[0026] Figure 3 Figures showing the test results of fracture strength and elongation at break of the SIS sample and the SIS-NCl-1 sample provided in the embodiments of this application;

[0027] Figure 4 Scanning electron microscope images of the surface and cross-sectional morphology of the Hp8-SIS-NCl-5 sample provided in the embodiments of this application;

[0028] Figure 5Physical images of the SIS-NCl-1 sample and the Hp8-SIS-NCl-1 sample provided in the embodiments of this application;

[0029] Figure 6 Cell compatibility diagram of Hp8-SIS-NCl-5 mouse embryonic fibroblasts (L929) provided in the embodiments of this application;

[0030] Figure 7 The image shows the antibacterial test results of Hp8-SIS-NCl-5 against Staphylococcus aureus, Escherichia coli, and methicillin-resistant Staphylococcus aureus provided in the embodiments of this application.

[0031] Figure 8 The image shows the antibacterial test results of Hp8-SIS-NCl-5 against Staphylococcus aureus, Escherichia coli, and methicillin-resistant Staphylococcus aureus provided in the embodiments of this application. Detailed Implementation

[0032] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0033] In the following description of this embodiment, the term "and / or" is used to describe the association relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, B existing alone, and A and B existing simultaneously. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0034] In the following description of this embodiment, the term "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0035] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms "a" and "the" as used in the embodiments of this application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0036] Those skilled in the art should understand that, in the following description of the embodiments of this application, the sequence of numbers does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0037] Those skilled in the art will understand that the numerical ranges in the embodiments of this application should be understood to specifically disclose each intermediate value between the upper and lower limits of the range. Each smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0038] Unless otherwise stated, the technical / scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. While this application describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this application. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0039] In a first aspect, embodiments of this application provide a method for preparing a decellularized extracellular matrix patch with antibacterial function, the method comprising the following steps:

[0040] Provide a matrix for decellularized extracellular matrix patches;

[0041] The decellularized extracellular matrix patch substrate was chlorinated in a sodium hypochlorite solution;

[0042] In addition, an antibacterial decellularized matrix patch is harvested from the chlorination product of the previous step.

[0043] The preparation method provided in this application involves immersing the decellularized extracellular matrix patch substrate in a sodium hypochlorite solution for chlorination treatment, thereby grafting an N-haloamine antibacterial coating onto the decellularized extracellular matrix patch substrate at the molecular level in one step. Firstly, the N-Cl bond can be used to directly adsorb onto the cell membrane of infected bacteria and effectively inhibit the metabolic process of infected bacteria, achieving a contact antibacterial / antimicrobial effect. Furthermore, the oxidatively active chloride ions released from the interaction of N-Cl bonds with water molecules can selectively react with certain active functional groups in infected bacteria, thus endowing the prepared decellularized extracellular matrix patch with antibacterial function with a dual-mechanism synergistic antibacterial / antimicrobial effect. This improves antibacterial / antimicrobial performance and avoids drug resistance problems while effectively maintaining good biocompatibility. Secondly, the N-haloamine antibacterial coating and the decellularized extracellular matrix patch... The matrix is ​​stably and firmly bonded together by covalent bonds, which can effectively prevent the dissolution of the N-haloamine antibacterial coating, ensuring the antibacterial / antimicrobial efficacy is durable and does not produce obvious cytotoxicity problems. Thirdly, this application proposes for the first time to use the decellularized extracellular matrix as the matrix for N-haloamine antibacterial modification treatment. The uniform and high-density amino groups distributed on its collagen backbone can be used to directly achieve the grafting modification of the N-haloamine coating. The preparation is simple and can effectively improve the grafting uniformity while effectively avoiding the introduction of components with poor biocompatibility, thereby achieving long-lasting antibacterial effect and ensuring good biocompatibility.

[0044] It should be noted that the specific source of the decellularized extracellular matrix patch substrate is not particularly limited in the embodiments of this application, and it can be obtained through commercial purchase or common methods known in the art. Specifically, the embodiments of this application provide a method for preparing the decellularized extracellular matrix patch substrate, which preferably includes:

[0045] Step A: After rinsing the fresh pig small intestine with water and removing the adhering substances on the inner and outer walls, select small intestine segments with uniform lumen diameter, no wall damage, and no lymph nodes, and cut the selected small intestine segments into tubular precursors with a length of 5-15cm.

[0046] Step B: After removing the mucosa, serosa and muscular layer of the tubular precursor obtained in Step A along the longitudinal axis of the small intestine, cut it longitudinally into sheet-like samples and rinse it with water until there is no tissue residue on the surface.

[0047] Step C: Immerse the washed flake sample obtained in Step B in a mixed aqueous solution of 100 mmol / L EDTA (29.2 g / L) and 200 mmol / L NaOH (8 g / L) at a solid-liquid volume ratio of 1:100 for 12-16 h.

[0048] Step D: After rinsing the sheet-like sample obtained in step C with deionized water, immerse it in a mixed aqueous solution (pH = 1-2) of 1 mol / L HCl (36.5 g / L) and 1 mol / L NaCl (58.5 g / L) at a solid-liquid volume ratio of 1:100 for 6-10 hours.

[0049] Step E: After rinsing the sheet-like sample obtained in step D with deionized water, immerse it in a 10 mmol / L PBS solution (pH = 7-7.4) containing 1 mol / L NaCl at a solid-liquid volume ratio of 1:100 for 12-16 hours.

[0050] Step F: After rinsing the sheet-like sample obtained in step E with deionized water, immerse it in 10 mmol / L PBS solution (pH = 7-7.4) at a solid-liquid volume ratio of 1:100 for 1.5-4 hours.

[0051] Step G: The sheet-like sample treated in step F is immersed in a mixed solution of 0.1% peracetic acid and 20% ethanol at a solid-liquid volume ratio of 1:100 for sterilization for 6-10 hours. After rinsing with deionized water and draining, the decellularized extracellular matrix patch substrate is obtained and stored in sterile distilled water.

[0052] In this embodiment, the acellular extracellular matrix patch matrix is ​​derived from, but is not limited to, the small intestine, heart valves, and bladder basement membrane of mammals. These tissues possess suitable porosity, good three-dimensional structure, necessary plasticity, and biodegradability, making them suitable for preparing acellular extracellular matrix patches with good plasticity and effectively improving the repair effect of abdominal wall defects.

[0053] In this embodiment, the decellularized extracellular matrix patch substrate is preferably derived from porcine small intestine. Studies have shown that decellularized extracellular matrix patch substrates prepared using porcine small intestine possess good biocompatibility, anti-adhesion properties, bioactivity, mechanical properties, and low cost, and are effective for repairing wall defects.

[0054] In this embodiment, when the decellularized extracellular matrix patch matrix is ​​chlorinated in a sodium hypochlorite solution, the concentration of the sodium hypochlorite solution is preferably 1-10 wt%, for example, 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, or any concentration within the range described above. By selecting the above-mentioned concentration of sodium hypochlorite solution, this embodiment ensures that the decellularized extracellular matrix patch matrix (collagen) reaches a suitable degree of chlorination, thereby enabling the prepared decellularized extracellular matrix patch with antibacterial function to possess excellent antibacterial properties while improving its safety and durability in vivo.

[0055] In this embodiment, when the decellularized extracellular matrix patch substrate is chlorinated in sodium hypochlorite solution, the volume ratio of sodium hypochlorite solution to the decellularized extracellular matrix patch substrate is preferably 20-100:1, for example, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or any one within the aforementioned volume ratio range. By selecting the above volume ratio, this embodiment ensures that an N-haloamine antibacterial coating can be uniformly formed on the surface of the decellularized extracellular matrix patch substrate, thereby improving the stability of the antibacterial properties.

[0056] In this embodiment, when the decellularized extracellular matrix patch matrix is ​​chlorinated in sodium hypochlorite solution, the chlorination time is preferably 4-48 hours, for example, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, or any time range thereof. This embodiment controls the chlorination time to ensure the decellularized extracellular matrix patch matrix (collagen) reaches a suitable degree of chlorination, thereby improving the in vivo safety and durability of the prepared antibacterial decellularized extracellular matrix patch while maintaining excellent antibacterial properties.

[0057] In this embodiment of the application, preferably after harvesting the decellularized extracellular matrix patch with antibacterial function from the chlorination product in the previous step, the following method is further included:

[0058] The decellularized extracellular matrix patch with antibacterial function was sequentially drained, pre-frozen, vacuum freeze-dried, and laminated.

[0059] In this application, the specific processes of pre-freezing and vacuum freeze-drying are not particularly limited. Those skilled in the art can operate according to conventional methods to prepare decellularized extracellular matrix patches with antibacterial function that meet the requirements of void space and three-dimensional network space.

[0060] In this embodiment, the lamination process is either vacuum lamination or hot pressing. Specifically, by performing vacuum lamination or hot pressing on a single-layer decellularized extracellular matrix patch with antibacterial function, this embodiment not only effectively optimizes the surface smoothness of the prepared patch product but also improves the interlayer bonding strength, resulting in a better patch product for abdominal wall defect repair.

[0061] In this embodiment, the preferred parameters for the hot-pressing process include: 1-12 hot-pressing layers, 5-50 MPa hot-pressing pressure, 45-70°C hot-pressing temperature, and 1-20 min hot-pressing time. By selecting these hot-pressing conditions, this embodiment not only effectively improves the surface smoothness of the prepared patch product but also transforms it from a single-layer, slightly yellowish, opaque layer to a semi-transparent surface, thus significantly improving the quality of the patch product.

[0062] Secondly, embodiments of this application provide a decellularized extracellular matrix patch with antibacterial function prepared by the preparation method described in the first aspect. Specifically, the preparation method described above enables the surface of the decellularized extracellular matrix patch substrate to be firmly and stably bonded by covalent bonds to form an N-haloamine antibacterial coating with synergistic antibacterial / antimicrobial effects and high biocompatibility. Therefore, the decellularized extracellular matrix patch with antibacterial function prepared in this application embodiment possesses excellent antibacterial / antimicrobial properties and good biocompatibility, while effectively avoiding drug resistance and cytotoxicity issues.

[0063] Thirdly, the embodiments of this application provide the above-described antibacterial acellular extracellular matrix patch for use in preventing infection of abdominal wall defects and treating infectious abdominal wall defects. Specifically, because the antibacterial acellular extracellular matrix patch of this application possesses excellent antibacterial / antimicrobial properties, good biocompatibility, and can effectively avoid drug resistance and cytotoxicity, its use in preparing repair materials for preventing infection of abdominal wall defects and treating infectious abdominal wall defects can effectively solve the problems of infection during abdominal wall defect treatment and postoperative recurrence, showing promising clinical application prospects.

[0064] The technical solution of this application will be further described below with reference to specific embodiments.

[0065] Example 1

[0066] This embodiment provides a method for preparing a single-layer decellularized extracellular matrix patch (SIS-NCl-1) with antibacterial function, comprising the following steps:

[0067] S101: After rinsing the commercially available fresh pig small intestine with water and removing the adhering substances on the inner and outer walls, select small intestine segments with uniform lumen diameter, no wall damage, and no lymph nodes, and cut the selected small intestine segments into tubular precursors with a length of 10cm.

[0068] S102: After removing the mucosa, serosa and muscular layer of the tubular precursor obtained in step S101 along the longitudinal axis of the small intestine, cut it longitudinally into sheet-like samples and rinse it with water until there is no tissue residue on the surface.

[0069] S103: The washed flake sample obtained in step S102 was immersed in a mixed aqueous solution of 100 mmol / L EDTA (29.2 g / L) and 200 mmol / L NaOH (8 g / L) at a solid-liquid volume ratio of 1:100 for 16 h.

[0070] S104: After rinsing the sheet-like sample obtained in step S103 with deionized water, immerse it in a mixed aqueous solution of 1 mol / L HCl (36.5 g / L) and 1 mol / L NaCl (58.5 g / L) at a solid-liquid volume ratio of 1:100 for 8 hours.

[0071] S105: After rinsing the sheet-like sample obtained in step S104 with deionized water, immerse it in a PBS (10 mmol / L) solution of 1 mol / L NaCl at a solid-liquid volume ratio of 1:100 for 16 h.

[0072] S106: After rinsing the sheet-like sample obtained in step S105 with deionized water, immerse it in 10 mmol / L PBS solution at a solid-liquid volume ratio of 1:100 for 2 hours.

[0073] S107: The sheet-like sample processed in step S106 is immersed in a mixed solution of 0.1% peracetic acid and 20% ethanol at a solid-liquid volume ratio of 1:100 for sterilization for 8 hours. After rinsing with deionized water and draining, the decellularized extracellular matrix patch substrate (SIS) is obtained and stored in sterile distilled water.

[0074] S108: The decellularized extracellular matrix patch substrate (SIS) was immersed in a 1wt% sodium hypochlorite aqueous solution at a solid-liquid volume ratio of 1:100, and then placed in a refrigerator at 4°C for chlorination treatment for 12 hours. After that, it was taken out and drained, spread evenly in a petri dish, and placed in a refrigerator at -20°C for pre-freezing for 3 hours. After that, it was taken out and vacuum freeze-dried to obtain a single-layer decellularized extracellular matrix patch (SIS-NCl-1) with antibacterial function.

[0075] Example 2

[0076] This embodiment provides a method for preparing an 8-layer thermo-pressed decellularized extracellular matrix patch (Hp8-SIS-NCl-1) with antibacterial function, comprising the following steps:

[0077] S201: After rinsing the commercially available fresh pig small intestine with water and removing the adhering substances on the inner and outer walls, select small intestine segments with uniform lumen diameter, no wall damage, and no lymph nodes, and cut the selected small intestine segments into tubular precursors with a length of 10cm.

[0078] S202: After removing the mucosa, serosa and muscular layer of the tubular precursor obtained in step S201 along the longitudinal axis of the small intestine, cut it longitudinally into sheet-like samples and rinse it with water until there is no tissue residue on the surface.

[0079] S203: The washed flake sample obtained in step S202 is immersed in a mixed aqueous solution of 100 mmol / L EDTA (29.2 g / L) and 200 mmol / L NaOH (8 g / L) at a solid-liquid volume ratio of 1:100 for 16 h.

[0080] S204: After rinsing the sheet-like sample obtained in step S203 with deionized water, immerse it in a mixed aqueous solution of 1 mol / L HCl (36.5 g / L) and 1 mol / L NaCl (58.5 g / L) at a solid-liquid volume ratio of 1:100 for 8 hours.

[0081] S205: After rinsing the sheet-like sample obtained in step S204 with deionized water, immerse it in a PBS (10 mmol / L) solution of 1 mol / L NaCl at a solid-liquid volume ratio of 1:100 for 16 h.

[0082] S206: After rinsing the sheet-like sample obtained in step S205 with deionized water, immerse it in 10 mmol / L PBS solution at a solid-liquid volume ratio of 1:100 for 2 hours.

[0083] S207: The sheet-like sample processed in step S206 is immersed in a mixed solution of 0.1% peracetic acid and 20% ethanol at a solid-liquid volume ratio of 1:100 for sterilization for 8 hours. After rinsing with deionized water and draining, the decellularized extracellular matrix patch substrate (SIS) is obtained and stored in sterile distilled water.

[0084] S208: The decellularized extracellular matrix patch substrate (SIS) was immersed in a 1wt% sodium hypochlorite aqueous solution at a solid-liquid volume ratio of 1:100, and then placed in a refrigerator at 4°C for chlorination treatment for 12 hours. After that, it was taken out and drained, spread evenly in a petri dish, and placed in a refrigerator at -20°C for pre-freezing for 3 hours. After that, it was taken out and vacuum freeze-dried to obtain a single-layer decellularized extracellular matrix patch (SIS-NCl-1) with antibacterial function.

[0085] S209: After stacking 8 layers of antibacterial single-layer decellularized extracellular matrix patch (SIS-NCl-1), place it in a hot press with a hot pressing pressure of 30MPa and a hot pressing temperature of 60℃ for 10min to obtain an 8-layer hot-pressed decellularized extracellular matrix patch (Hp8-SIS-NCl-1) with antibacterial function.

[0086] Example 3

[0087] This embodiment provides a method for preparing an 8-layer thermo-pressed decellularized extracellular matrix patch (Hp8-SIS-NCl-5) with antibacterial function, comprising the following steps:

[0088] S301: After rinsing the commercially available fresh pig small intestine with water and removing the adhering substances on the inner and outer walls, select small intestine segments with uniform lumen diameter, no wall damage, and no lymph nodes, and cut the selected small intestine segments into tubular precursors with a length of 10cm.

[0089] S302: After removing the mucosa, serosa and muscular layer of the tubular precursor obtained in step S301 along the longitudinal axis of the small intestine, cut it longitudinally into sheet-like samples and rinse it with water until there is no tissue residue on the surface.

[0090] S303: The cleaned sheet sample obtained in step S302 is immersed in a mixed aqueous solution of 100 mmol / L EDTA (29.2 g / L) and 200 mmol / L NaOH (8 g / L) at a solid-liquid volume ratio of 1:100 for 16 h.

[0091] S304: After rinsing the sheet-like sample obtained in step S303 with deionized water, immerse it in a mixed aqueous solution of 1 mol / L HCl (36.5 g / L) and 1 mol / L NaCl (58.5 g / L) at a solid-liquid volume ratio of 1:100 for 8 hours.

[0092] S305: After rinsing the sheet-like sample obtained in step S304 with deionized water, immerse it in a PBS (10 mmol / L) solution of 1 mol / L NaCl at a solid-liquid volume ratio of 1:100 for 16 h.

[0093] S306: After rinsing the sheet-like sample obtained in step S305 with deionized water, immerse it in 10 mmol / L PBS solution at a solid-liquid volume ratio of 1:100 for 2 hours.

[0094] S307: The sheet sample processed in step S306 is immersed in a mixed solution of 0.1% peracetic acid and 20% ethanol at a solid-liquid volume ratio of 1:100 for sterilization for 8 hours. After rinsing with deionized water and draining, the decellularized extracellular matrix patch substrate (SIS) is obtained and stored in sterile distilled water.

[0095] S308: The decellularized extracellular matrix patch substrate (SIS) was immersed in a 5wt% sodium hypochlorite aqueous solution at a solid-liquid volume ratio of 1:100, and then placed in a refrigerator at 4°C for chlorination treatment for 12 hours. After that, it was taken out and drained, spread evenly in a petri dish and placed in a refrigerator at -20°C for pre-freezing for 3 hours. After that, it was taken out and vacuum freeze-dried to obtain a single-layer decellularized extracellular matrix patch (SIS-NCl-5) with antibacterial function.

[0096] S309: After stacking 8 layers of antibacterial single-layer decellularized extracellular matrix patch (SIS-NCl-5), place it in a hot press with a hot pressing pressure of 40MPa and a hot pressing temperature of 60℃ for 5 minutes to obtain an 8-layer hot-pressed decellularized extracellular matrix patch (Hp8-SIS-NCl-5) with antibacterial function.

[0097] Example 4

[0098] This embodiment provides a 12-layer heat-pressed decellularized extracellular matrix patch (Hp) with antibacterial function. 12 The preparation method of (-SIS-NCl-10) includes the following steps:

[0099] S401: After rinsing the commercially available fresh pig small intestine with water and removing the adhering substances on the inner and outer walls, select small intestine segments with uniform lumen diameter, no wall damage, and no lymph nodes, and cut the selected small intestine segments into tubular precursors with a length of 10cm.

[0100] S402: After removing the mucosa, serosa and muscular layer of the tubular precursor obtained in step S401 along the longitudinal axis of the small intestine, cut it longitudinally into sheet-like samples and rinse it with water until there is no tissue residue on the surface.

[0101] S403: The washed flake sample obtained in step S402 is immersed in a mixed aqueous solution of 100 mmol / L EDTA (29.2 g / L) and 200 mmol / L NaOH (8 g / L) at a solid-liquid volume ratio of 1:100 for 16 h.

[0102] S404: After rinsing the sheet-like sample obtained in step S403 with deionized water, immerse it in a mixed aqueous solution of 1 mol / L HCl (36.5 g / L) and 1 mol / L NaCl (58.5 g / L) at a solid-liquid volume ratio of 1:100 for 8 hours.

[0103] S405: After rinsing the sheet-like sample obtained in step S404 with deionized water, immerse it in a PBS (10 mmol / L) solution of 1 mol / L NaCl at a solid-liquid volume ratio of 1:100 for 16 h.

[0104] S406: After rinsing the sheet-like sample obtained in step S405 with deionized water, immerse it in 10 mmol / L PBS solution at a solid-liquid volume ratio of 1:100 for 2 hours.

[0105] S407: The sheet-like sample processed in step S406 is immersed in a mixed solution of 0.1% peracetic acid and 20% ethanol at a solid-liquid volume ratio of 1:100 for sterilization for 8 hours. After rinsing with deionized water and draining, the decellularized extracellular matrix patch substrate (SIS) is obtained and stored in sterile distilled water.

[0106] S408: The decellularized extracellular matrix patch substrate (SIS) was immersed in a 10wt% sodium hypochlorite aqueous solution at a solid-liquid volume ratio of 1:100, and then placed in a refrigerator at 4℃ for chlorination treatment for 12 hours. After that, it was taken out and drained, spread evenly in a petri dish and placed in a refrigerator at -20℃ for pre-freezing for 3 hours. After that, it was taken out and vacuum freeze-dried to obtain a single-layer decellularized extracellular matrix patch (SIS-NCl-10) with antibacterial function.

[0107] S409: A single-layer decellularized extracellular matrix patch (SIS-NCl-10) with antibacterial function is stacked in 12 layers and then placed in a hot press at a pressure of 50 MPa and a temperature of 50°C for 10 minutes to obtain a 12-layer hot-pressed decellularized extracellular matrix patch (Hp) with antibacterial function. 12 -SIS-NCl-10).

[0108] To further illustrate the performance of the antibacterial decellularized extracellular matrix patch prepared in the embodiments of this application, the following characterization tests will be performed using a single-layer antibacterial decellularized extracellular matrix patch (SIS-NCl-1), an 8-layer antibacterial thermo-pressed decellularized extracellular matrix patch (Hp8-SIS-NCl-1), and an 8-layer antibacterial thermo-pressed decellularized extracellular matrix patch (Hp8-SIS-NCl-5) as test samples.

[0109] 1. Surface morphology characterization of SIS and SIS-NCl-1 samples

[0110] The surface morphology of the SIS and SIS-NCl-1 samples was characterized using a scanning electron microscope (S-4800, 5.0 kV), and the results were as follows: Figure 1 As shown. Among them, Figure 1 Scanning electron microscope images of the surface morphology of SIS and SIS-NCl-1 samples.

[0111] according to Figure 1 It can be seen that after N-halogen modification, the SIS in the SIS-NCl-1 sample can still maintain its natural porous structure, indicating that N-halogen modification does not damage the three-dimensional network structure of SIS, and it still has a natural loose porous structure.

[0112] 2. X-ray photoelectron spectroscopy analysis

[0113] The SIS-NCl-1 sample was characterized using an X-ray photoelectron spectroscopy (ESCALAB 250), and the results were as follows: Figure 2 As shown. Among them, Figure 2 The image shows the X-ray photoelectron spectrum of the SIS-NCl-1 sample.

[0114] according to Figure 2 It can be seen that a significant Cl2p absorption peak appeared in the XPS full spectrum of the N-haloamine modified SIS-NCl-1 sample. Since there is no Cl element in SIS, this indicates that the N-haloamine modified decellularized extracellular matrix patch was successfully prepared by the chlorination of sodium hypochlorite.

[0115] 3. Fracture performance test

[0116] Tensile tests were conducted using a universal testing machine (WD-5A) to characterize the tensile strength and elongation at break of the SIS and SIS-NCl-1 samples. The specific testing method involved cutting the SIS and SIS-NCl-1 samples into 2cm × 2cm rectangular strips and then testing them at 30 mm·min⁻¹. -1 Tensile properties were tested at a tensile speed, and the results were as follows: Figure 3 As shown in the figure. Among them, a is the fracture strength test result of SIS sample and SIS-NCl-1 sample; b is the fracture elongation test result of SIS sample and SIS-NCl-1 sample.

[0117] according to Figure 3 It can be seen that the fracture strength of the SIS sample is 0.54 MPa and the elongation at break is 115%; the fracture strength of the SIS-NCl-1 sample modified with N-haloamine is 0.50 MPa and the elongation at break is 133%, indicating that N-haloamine modification does not significantly affect its mechanical properties.

[0118] 4. Surface and cross-sectional morphology characterization of Hp8-SIS-NCl samples

[0119] The surface and cross-sectional morphology of the Hp8-SIS-NCl-1 sample were characterized using a scanning electron microscope (S-4800, 5.0 kV), and the results are as follows: Figure 4 As shown. Among them, Figure 4 Scanning electron microscope images of the surface and cross-sectional morphology of the Hp8-SIS-NCl-1 sample.

[0120] according to Figure 4 It can be seen that the surface of the Hp8-SIS-NCl-1 sample after 8-layer hot pressing is smooth; no obvious cross-section was observed in the cross-sectional scanning electron microscope image, indicating that there is good bonding strength between the layers of SIS-NCl-1.

[0121] 5. Macroscopic morphological characterization

[0122] The macroscopic morphology of SIS-NCl-1 and Hp8-SIS-NCl-1 samples was characterized using a digital camera, and the results are shown in Figure 5. Figure 5 Images of SIS-NCl-1 and Hp8-SIS-NCl-1 samples.

[0123] according to Figure 5 It can be seen that the SIS-NCl-1 sample is slightly yellow and opaque but has a smooth surface, while the Hp8-SIS-NCl-1 sample has a semi-transparent and smooth surface. This indicates that hot pressing can change the SIS-NCl-1 sample from slightly yellow and opaque to semi-transparent.

[0124] 6. Cell compatibility test

[0125] The effect of Hp8-SIS-NCl-5 on cell proliferation was detected using 2-(2-methoxy-4-nitrobenzene)-3-(4-nitrobenzene)-5-(2,4-disulfonylbenzene)-2H-tetrazole monosodium salt (WST-8). The assay method was as follows:

[0126] Mouse embryonic fibroblasts (L929) were used at 5 × 10⁻⁶ 3 After seeding cells at a density of / wells into 96-well cell culture plates, complete culture medium containing Hp8-SIS-NCl-5 (DMEM medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibody) and blank complete culture were cultured for 7 days at 37°C, 5% CO2, and 90% relative humidity. After 24 hours, the culture medium was replaced with complete culture medium containing 10% WST-8 reagent, and incubated at 37°C in the dark for 1 hour. 100 μL was then aspirated from each well, and the absorbance at 450 nm was measured using a microplate reader. The results were as follows: Figure 6 As shown. Among them, Figure 6 This is a cell compatibility diagram of mouse embryonic fibroblasts (L929) of Hp8-SIS-NCl-5.

[0127] according to Figure 6 It can be seen that the absorbance at 450 nm of the complete culture medium containing Hp8-SIS-NCl-5 was not statistically different from that of the blank complete culture medium group, which proves that the N-haloamine modified Hp8-SIS-NCl-5 has no adverse effect on the proliferation of L929 cells and has good cell compatibility.

[0128] 7. Antibacterial performance test

[0129] The antibacterial activity of Hp8-SIS-NCl-5 was tested using a bacterial co-culture plate counting method. The test method was as follows:

[0130] Using Staphylococcus aureus, Escherichia coli, and methicillin-resistant Staphylococcus aureus (MRSA) as model bacteria, the bacterial concentration was adjusted to 1×10⁻⁶. 7 / mL, after co-culturing Hp8-SIS-NCl-5 with the bacterial culture for 12h, collect the bacterial culture and dilute it 10⁻⁶. 6 The number of coated plates was multiplied by 1, and the result was 1. Figure 7 As shown. Among them, Figure 7 The image shows the antibacterial test results of Hp8-SIS-NCl-5 against Staphylococcus aureus, Escherichia coli, and methicillin-resistant Staphylococcus aureus.

[0131] according to Figure 7 It can be seen that the N-haloamine-modified Hp8-SIS-NCl-5 has an antibacterial rate of 99.65% against Staphylococcus aureus, an antibacterial rate of 92.45% against Escherichia coli, and an antibacterial rate of 96.34% against methicillin-resistant Staphylococcus aureus, proving that the N-haloamine-modified Hp8-SIS-NCl-5 has good antibacterial properties against Staphylococcus aureus, Escherichia coli, and methicillin-resistant Staphylococcus aureus.

[0132] 8. Long-lasting antibacterial performance test

[0133] The long-lasting antibacterial activity of Hp8-SIS-NCl-5 was tested using a bacterial co-culture plate counting method. The test method was as follows:

[0134] Hp8-SIS-NCl-5 patches were immersed in PBS and placed in a shaker at 37°C and 200 rpm. The PBS solution was replaced every 24 hours to consume the antibacterial components released by the Hp8-SIS-NCl-5 patches. After 31 days of immersion, Staphylococcus aureus, Escherichia coli, and methicillin-resistant Staphylococcus aureus (MRSA) were used as model bacteria, and the bacterial concentration was adjusted to 1×10⁻⁶. 7 / mL, after co-culturing Hp8-SIS-NCl-5 with the bacterial culture for 12h, collect the bacterial culture and dilute it 10⁻⁶. 6 The number of coated plates was multiplied by 1, and the result was 1. Figure 8 As shown. Among them, Figure 8 The image shows the antibacterial test results of Hp8-SIS-NCl-5 against Staphylococcus aureus, Escherichia coli, and methicillin-resistant Staphylococcus aureus.

[0135] according to Figure 8It can be seen that Hp8-SIS-NCl-5, after long-term PBS immersion, has an antibacterial rate of 96.23% against Staphylococcus aureus, an antibacterial rate of 91.54% against Escherichia coli, and an antibacterial rate of 90.95% against methicillin-resistant Staphylococcus aureus. This proves that Hp8-SIS-NCl-5, after long-term PBS immersion, can still maintain good antibacterial properties against Staphylococcus aureus, Escherichia coli, and methicillin-resistant Staphylococcus aureus. Its antibacterial cycle can cover the high-incidence infection period (14 days) of abdominal wall defect repair, achieving long-term tolerance and treatment of abdominal wall defect infection.

[0136] The various embodiments in this specification are described in a progressive manner. The same or similar parts between the various embodiments can be referred to each other. Each embodiment focuses on describing the differences from other embodiments.

[0137] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit this application. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of this application.

Claims

1. A method for preparing a decellularized extracellular matrix patch with antibacterial function, characterized in that, Includes the following steps: Provide a matrix for decellularized extracellular matrix patches; The decellularized extracellular matrix patch substrate was chlorinated in a sodium hypochlorite solution; In addition, an antibacterial decellularized matrix patch is harvested from the chlorination product of the previous step; The matrix sources of the decellularized extracellular matrix patch include the small intestine, heart valves, and bladder basement membrane of mammals; When the decellularized extracellular matrix patch substrate is chlorinated in a sodium hypochlorite solution, the concentration of the sodium hypochlorite solution is 1-10 wt%, the volume ratio of the sodium hypochlorite solution to the decellularized extracellular matrix patch substrate is 20-100:1, and the chlorination treatment time is 4-48 h.

2. The preparation method according to claim 1, characterized in that, The matrix of the decellularized extracellular matrix patch is derived from pig small intestine.

3. The preparation method according to claim 1 or 2, characterized in that, Following the harvesting of the antibacterial decellularized extracellular matrix patch from the chlorination product in the previous step, the procedure further includes: The decellularized extracellular matrix patch with antibacterial function was sequentially drained, pre-frozen, vacuum freeze-dried, and laminated.

4. The preparation method according to claim 3, characterized in that, The lamination process includes vacuum lamination or hot pressing.

5. The preparation method according to claim 4, characterized in that, The parameters for hot pressing include: 1-12 hot pressing layers, 5-50 MPa hot pressing pressure, 45-70℃ hot pressing temperature, and 1-20 min hot pressing time.

6. A decellularized extracellular matrix patch with antibacterial function, characterized in that, Prepared according to any one of the preparation methods described in claims 1-5.

7. The application of the acellular extracellular matrix patch with antibacterial function according to claim 6 in the preparation of abdominal wall defect repair materials.

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