Acellular matrix repair material as well as preparation method and application thereof

Abstract

The invention relates to an acellular matrix repair material as well as a preparation method and application thereof. The preparation method of the acellular matrix repair material comprises the following steps: providing an acellular matrix of a peritoneal tissue of a mammal, and cross-linking the acellular matrix with a cross-linking agent to prepare a cross-linked acellular matrix; preparing an antibacterial drug into nano drug-loaded particles, and connecting the nano drug-loaded particles with the cross-linked acellular matrix to prepare a drug-loaded acellular matrix; and carrying out freeze-drying treatment on the drug-loaded acellular matrix to prepare the acellular matrix repair material. The invention provides the preparation method of the acellular matrix repair material, the obtained repair material can be used for repairing urethral defects, the excellent degradation resistance of the repair material can prolong the degradation rate of the material in vivo, effectively prevent contracture and reduce the recurrence rate of urethrostenosis, and the good antibacterial ability can enhance the anti-infection ability, so that the repair material can be used for repairing urethral defects. And the risk of urethral infection of postoperative patients is reduced.

Description

Technical Field

[0001] This application relates to the technical field of biomimetic materials, and particularly to a decellularized matrix repair material, its preparation method, and application. Background Art

[0002] Urethral defect is a common disease of soft tissue defect in clinic. Generally, it is caused by the injury or loss of urethral epithelium or cavernous tissue, resulting in symptoms such as urethral stricture, hypospadias, and urethral fistula. Its incidence rate is about 2 / ‰, which is common in men. There is 1 case in every 250 - 300 male infants on average.

[0003] Traditional methods for treating urethral defects generally use autologous foreskin tissue or oral mucosa tissue as soft tissue repair materials, which are rolled and implanted to replace the defective urethral soft tissue. However, autologous tissues are limited and cannot meet the needs of one - stage or multi - stage transplantation. It will also cause secondary damage to the soft tissue of the donor area, and there are obvious postoperative complications and a high recurrence rate.

[0004] Therefore, some studies have used materials such as silicone, polyethylene, and gelatin sponge for urethral repair. However, the above - mentioned materials have many deficiencies in mechanical properties, tissue structure, and biocompatibility.

[0005] The microstructure and biomechanical properties of decellularized matrix materials are similar to those of natural urethra, which well solve the shortcomings of traditional urethral repair materials. However, natural decellularized matrix materials degrade relatively fast. After being implanted into the urethra, it may cause contracture at the defect site, resulting in complications such as recurrence of urethral stricture and urethral fistula. In addition, due to the complex healing environment of urethral soft tissue, postoperative infection is more likely to occur, making the implanted site difficult to heal or even necrotic, increasing the pain of patients. Therefore, in addition to excellent biocompatibility and appropriate mechanical properties, good anti - degradation and antibacterial properties are also crucial for an ideal urethral repair material. However, the currently developed urethral repair materials have not yet overcome the above - mentioned technical difficulties. For example, the domestic patent application CN113750297A provides a structure - and - function - biomimetic urethral stent and its preparation method, which uses polymer materials cellulose and decellularized matrix to prepare a porous stent. The preparation method is complex, and the material lacks antibacterial ability and is difficult to cope with the complex environment of the urethral wound surface. The domestic patent application CN116870252A provides a double - layer microchannel structure design of a decellularized matrix urethral material. The preparation method is also quite complex, difficult to mass - produce, and the urethral material degrades relatively fast and lacks antibacterial properties. The foreign patent application US11596512B2 provides a decellularized tubular graft, but its degradation performance is relatively fast, and the formed tubular material is not conducive to clinical operation. Summary of the Invention

[0006] Based on this, the present application provides a decellularized matrix repair material suitable for urethral defect repair, having good anti-degradation and antibacterial properties, and a preparation method thereof.

[0007] The present application provides a preparation method of a decellularized matrix repair material, comprising the following steps:

[0008] Providing a decellularized matrix of mammalian peritoneal tissue, crosslinking the decellularized matrix with a crosslinking agent to prepare a crosslinked decellularized matrix;

[0009] Preparing the antibacterial drug into nano-drug-loaded particles, and connecting them with the crosslinked decellularized matrix to prepare a drug-loaded decellularized matrix; and

[0010] Performing freeze-drying treatment on the drug-loaded decellularized matrix to prepare a decellularized matrix repair material.

[0011] In some embodiments, the step of crosslinking the decellularized matrix with a crosslinking agent comprises:

[0012] Placing the decellularized matrix in a solution containing the crosslinking agent for a first immersion.

[0013] In some embodiments, the conditions of the first immersion satisfy one or more of the following conditions:

[0014] (1) The crosslinking agent includes one or more of glutaraldehyde, formaldehyde, butanediol diglycidyl ether, and genipin;

[0015] (2) In the solution containing the crosslinking agent, the mass percentage concentration of the crosslinking agent is 0.1% - 5%;

[0016] (3) The time of the first immersion is 4h - 48h.

[0017] In some embodiments, the step of preparing the drug-loaded decellularized matrix comprises:

[0018] Using amino-polyethylene glycol-poly(lactic acid) to encapsulate the antibacterial drug to prepare nano-drug-loaded particles, and placing the crosslinked decellularized matrix in a solution containing the nano-drug-loaded particles for a second immersion.

[0019] In some embodiments, the conditions of the second immersion satisfy one or more of the following conditions:

[0020] (1) The antibacterial drug includes one or more of levofloxacin, ciprofloxacin, moxifloxacin, azithromycin, clarithromycin, doxycycline, minocycline, and clindamycin;

[0021] (2) In the solution containing the antibacterial agent, the mass percentage concentration of the antibacterial agent is 0.01% to 0.5%;

[0022] (3) The solution for the second immersion includes a promoter, and 0.001 mmol to 0.1 mmol of the promoter is added per milligram of the crosslinked acellular matrix;

[0023] Optionally, the promoter includes one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide;

[0024] (4) The time for the second immersion is 2 h to 24 h.

[0025] In some embodiments, the freeze-drying conditions satisfy one or more of the following conditions:

[0026] (1) The freeze-drying temperature is -30°C to -50°C;

[0027] (2) The freeze-drying time is 24 h to 48 h.

[0028] In some embodiments, the mammal is selected from one or more of pigs, cows, and sheep.

[0029] In some embodiments, the preparation of the acellular matrix includes the following steps:

[0030] Place the pretreated peritoneal tissue of the mammal in a strong alkali solution for the third immersion to prepare the peritoneal tissue after the third immersion;

[0031] Optionally, the third immersion satisfies one or more of the following conditions:

[0032] (1) The alkali in the strong alkali solution includes one or more of sodium hydroxide and potassium hydroxide;

[0033] (2) The mass percentage concentration of the alkali in the strong alkali solution is 0.3% to 10%;

[0034] (3) The pH value of the strong alkali solution is 11 to 14;

[0035] (4) The time for the third immersion is 4 h to 24 h;

[0036] Place the peritoneal tissue after the third immersion in an acidic solution for the fourth immersion to prepare the peritoneal tissue after the fourth immersion;

[0037] Optionally, the fourth immersion satisfies one or more of the following conditions:

[0038] (1) The acid in the acidic solution includes one or more of hydrochloric acid, lactic acid, citric acid, and acetic acid;

[0039] (2) The mass percentage concentration of the acid in the acidic solution is 1% - 8%;

[0040] (3) The pH value of the acidic solution is 0.5 - 1.5;

[0041] (4) The time of the fourth immersion is 4h - 24h;

[0042] Place the peritoneal tissue after the fourth immersion in a neutral salt solution for a fifth immersion to prepare the peritoneal tissue after the fifth immersion;

[0043] Optionally, the fifth immersion satisfies one or more of the following conditions:

[0044] (1) The neutral salt in the neutral salt solution includes one or more of sodium chloride, potassium chloride, and sodium sulfate;

[0045] (2) The concentration of the neutral salt in the neutral salt solution is 5% - 10%;

[0046] (4) The time of the fifth immersion is 4h - 24h;

[0047] Place the peritoneal tissue after the fifth immersion in a weak base salt solution for a sixth immersion to prepare the peritoneal tissue after the sixth immersion; and

[0048] Optionally, the sixth immersion satisfies one or more of the following conditions:

[0049] (1) The weak base salt in the weak base salt solution includes one or more of sodium bicarbonate, sodium acetate, and disodium hydrogen phosphate;

[0050] (2) The mass percentage concentration of the weak base salt in the weak base salt solution is 0.5% - 1.5%;

[0051] (3) The time of the sixth immersion is 15h - 30h;

[0052] Wash the peritoneal tissue after the sixth immersion, remove the surface fat, and place it in an enzyme solution for a seventh immersion to prepare the acellular matrix;

[0053] Optionally, the seventh immersion satisfies at least one of the following conditions:

[0054] (1) The enzyme in the enzyme solution includes one or more of lipase, trypsin, pepsin, and neutral protease;

[0055] (2) The mass percentage concentration of the enzyme in the enzyme solution is 0.2% - 2.0%;

[0056] (3) The time of the seventh immersion is 5h - 36h.

[0057] The present application also provides a decellularized matrix repair material, which is prepared by the preparation method described in any one of the above embodiments.

[0058] The present application also provides the application of the decellularized matrix repair material described in any one of the above embodiments in the preparation of urethral repair products.

[0059] The present application provides a preparation method of a decellularized matrix repair material. First, the decellularized matrix of mammalian peritoneal tissue is cross-linked with a cross-linking agent to form a cross-linked network structure of the decellularized matrix, improving the anti-degradation ability of the decellularized matrix. Then, an antibacterial drug is loaded to endow the decellularized matrix with good antibacterial properties. The repair material obtained after freeze-drying and solidification molding can be used for urethral defect repair. The excellent anti-degradation performance of the repair material can prolong the degradation rate of the material in vivo, effectively prevent contracture, and reduce the recurrence rate of urethral stricture. The good antibacterial ability can enhance the anti-infection ability and reduce the risk of urethral infection in patients after surgery. Description of the Drawings

[0060] Figure 1 is the process route diagram for the preparation of the decellularized matrix repair material in some embodiments;

[0061] Figure 2 is the SEM scan picture of the decellularized matrix repair material prepared in Example 1; among them Figure 2 A in is the SEM scan picture of the rough surface, Figure 2 B in is the SEM scan picture of the smooth surface, Figure 2 C in is the SEM scan picture of the cross-section;

[0062] Figure 3 is the cytotoxicity test result of the decellularized matrix repair material prepared in Example 1;

[0063] Figure 4 is the anti-degradation performance test result of the decellularized matrix repair material prepared in Example 1. Detailed Embodiments

[0064] For the convenience of understanding the present application, the present application will be described more comprehensively below in conjunction with embodiments. However, the present application can be implemented in many different forms and is not limited to the embodiments described in the present application. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosed content of the present application more thorough and comprehensive.

[0065] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used in this application includes any and all combinations of one or more related listed items.

[0066] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0067] The terms "first", "second", "third", "fourth", etc. (if any) used in this application are used to distinguish similar objects and are only used for descriptive purposes, and are not necessarily used to describe a specific order or sequence, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of this application, the meaning of "multiple" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0068] In the present application, the terms “preferred”, “better”, “more preferred” and “suitable” are only used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute limitations on the scope of protection of the present application.

[0069] In the present application, the terms “further”, “furthermore”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0070] In this application, "optionally", "optional" and "optional" refer to optional, that is, to any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0071] In this application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval and includes the two numerical endpoints (i.e., the minimum value and the maximum value) of the numerical range, as well as each numerical value between the two numerical endpoints. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all sub-ranges included therein.

[0072] like Figure 1As shown in the figure, the present application provides a method for preparing a decellularized matrix repair material, including the following steps S300 to S500.

[0073] Step S300: Provide a decellularized matrix of mammalian peritoneal tissue, crosslink the decellularized matrix with a crosslinking agent, and prepare a crosslinked decellularized matrix.

[0074] Step S400: Prepare the antibacterial drug into nano-drug-loaded particles, connect them with the above crosslinked decellularized matrix, and prepare a drug-loaded decellularized matrix.

[0075] Step S500: Perform freeze-drying treatment on the drug-loaded decellularized matrix to prepare a decellularized matrix repair material.

[0076] The present application provides a method for preparing a decellularized matrix repair material. First, crosslink the decellularized matrix of mammalian peritoneal tissue with a crosslinking agent to form a crosslinked network structure of the decellularized matrix, improving the anti-degradation ability of the decellularized matrix. Then, load the antibacterial drug to endow the decellularized matrix with good antibacterial properties. The repair material obtained after freeze-drying and solidification molding can be used for urethral defect repair. The excellent anti-degradation performance of the repair material can prolong the degradation rate of the material in the body, effectively prevent contracture, and reduce the recurrence rate of urethral stricture. And the good antibacterial ability can enhance the anti-infection ability and reduce the risk of urethral infection in patients after surgery.

[0077] Furthermore, the peritoneal tissue is used in the present application. The peritoneal tissue has a bilayer structure, and one layer is a rough layer and the other layer is a smooth layer. The rough layer has a three-dimensional porous structure, which can provide a scaffold template for cell repair and regeneration, guide the rapid growth of cells around the urethral defect site, and complete in-situ repair. The smooth layer faces away from the urethra, isolating the invasion of external urethral soft tissues and preventing tissue adhesion at the defect site.

[0078] Furthermore, the peritoneal tissue of mammals is used in the present application. After the mammalian peritoneal tissue is treated by decellularization technology to remove the antigenic factors, the formed decellularized matrix has excellent biocompatibility, wide sources, can be mass-produced, can replace the transplantation of donor mucosa and skin flaps in traditional surgeries, and reduce the pain of patients.

[0079] Specifically:

[0080] Step S300: Provide a decellularized matrix of mammalian peritoneal tissue, crosslink the decellularized matrix with a crosslinking agent, and prepare a crosslinked decellularized matrix. After the crosslinking reaction between the decellularized matrix and the crosslinking agent, a crosslinked network can be formed, greatly improving the anti-degradation ability of the decellularized matrix, being beneficial to reducing the occurrence of contracture at the defect site after implantation into the urethra, and reducing complications.

[0081] In some of these embodiments, the step of crosslinking the acellular matrix with a crosslinking agent includes step S310.

[0082] Step S310: Place the acellular matrix in a solution containing a crosslinking agent for a first immersion.

[0083] Furthermore, the crosslinking agent includes one or more of glutaraldehyde, formaldehyde, butanediol diglycidyl ether, and genipin. The above crosslinking agent can carry out a rapid and efficient crosslinking reaction with the acellular matrix.

[0084] Furthermore, in the solution containing the crosslinking agent, the mass percentage concentration of the crosslinking agent is 0.1% - 5%. Within the above concentration range, the crosslinking agent can fully carry out a crosslinking reaction with the acellular matrix, and it also avoids excessive cytotoxicity caused by too high a concentration of the crosslinking agent.

[0085] Furthermore, the time of the first immersion is 4h - 48h. Within the above immersion time, the crosslinking effect is better. If the immersion time is too short, the crosslinking reaction is not sufficient. If the immersion time is too long, excessive crosslinking is likely to cause excessive cytotoxicity.

[0086] Furthermore, after step S310, there is also step S320: cleaning the crosslinked acellular matrix.

[0087] Even further, the cleaning method can be, for example, cleaning with injection water, and the number of cleaning times can be, for example, but not limited to, 6 - 8 times.

[0088] Step S400: Prepare the antibacterial drug into nano - drug - loaded particles, connect them with the above - crosslinked acellular matrix, and prepare the acellular matrix loaded with the drug. After loading the antibacterial drug on the acellular matrix, the antibacterial ability of the acellular matrix can be greatly enhanced, which is beneficial to reducing postoperative infections after implantation into the urethra.

[0089] In some of these embodiments, the step of preparing the acellular matrix loaded with the drug includes step S410.

[0090] Step S410: Use amino - polyethylene glycol - polylactic acid to encapsulate the antibacterial drug to prepare nano - drug - loaded particles, and place the above - crosslinked acellular matrix in a solution containing the above nano - drug - loaded particles for a second immersion.

[0091] Furthermore, the antibacterial drug includes one or more of levofloxacin, ciprofloxacin, moxifloxacin, azithromycin, clarithromycin, doxycycline, minocycline, and clindamycin. The above antibacterial drugs have good antibacterial effects and can effectively prevent postoperative infections. It can be understood that in other embodiments, other antibacterial drugs with good antibacterial effects can also be used.

[0092] It is understandable that an antibacterial drug is encapsulated with amino-polyethylene glycol-polylactic acid to prepare nano-drug-loaded particles. This step can be prepared by a common nano-precipitation method.

[0093] Further, in the solution containing the antibacterial drug, the mass percentage concentration of the antibacterial drug is 0.01% to 0.5%. If the concentration of the antibacterial drug is too low, the amount of the drug loaded on the acellular matrix is small and cannot achieve a good antibacterial effect. If the concentration of the antibacterial drug is too high, it will increase the cytotoxicity of the final product and is not conducive to biosafety. Therefore, it is optimal within the above concentration range.

[0094] Further, the solution for the second soaking includes a promoter, and 0.001 mmol to 0.1 mmol of the promoter is added per milligram of the crosslinked acellular matrix.

[0095] In some embodiments, the above promoter includes one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide.

[0096] It is understandable that a chemical reaction occurs between the amino group of the antibacterial drug nanoparticles and the carboxyl group of the acellular matrix to form an amide bond, thereby connecting the antibacterial drug to the acellular matrix, improving the stability of the antibacterial drug, increasing the loading capacity of the acellular matrix for the antibacterial drug, and obtaining a drug-loaded acellular matrix with relatively high stability.

[0097] Further, the time for the second soaking is 2 h to 24 h. If the soaking time is too short, the antibacterial drug nanoparticles cannot be successfully connected to the acellular matrix, and the improvement of the antibacterial effect of the product is limited. If the soaking time is too long, the content of the antibacterial drug loaded on the acellular matrix is too high, which is likely to cause drug overflow, resulting in excessive cytotoxicity of the product and poor biocompatibility.

[0098] Further, after step S410, there is also step S420: cleaning the drug-loaded acellular matrix.

[0099] Furthermore, the cleaning method can be, for example, but not limited to, cleaning with a disodium hydrogen phosphate-sodium dihydrogen phosphate solution, and the number of cleaning times can be, for example, but not limited to, 1 to 4 times.

[0100] Step S500: Freeze-drying the drug-loaded acellular matrix to prepare an acellular matrix repair material. After freeze-drying, the acellular matrix solidifies and forms a sheet, which can be used as a repair material to be implanted into a patient's body for repairing urethral defects.

[0101] In some embodiments, the freeze-drying temperature is -30°C to -50°C.

[0102] In some of these embodiments, the lyophilization time is 24 h to 48 h.

[0103] In some of these embodiments, the mammal is selected from one or more of pigs, cows, and sheep. The peritoneal tissue of the present application is derived from common mammals, is convenient to obtain materials, is easy to produce on a large scale, and the acellular matrix obtained after removing antigen factors by decellularization has excellent biocompatibility.

[0104] In some of these embodiments, in the preparation method of the present application, step S200 is further included before step S300.

[0105] Step S200: Prepare the acellular matrix of mammalian peritoneal tissue. Specifically, step S200 includes the following steps S210 to step S240.

[0106] Step S210: Place the pretreated peritoneal tissue of the mammal in a strong base solution for a third immersion to prepare the peritoneal tissue after the third immersion.

[0107] Furthermore, the base in the strong base solution includes one or more of sodium hydroxide and potassium hydroxide.

[0108] Furthermore, the mass percentage concentration of the base in the strong base solution is 0.3% to 10%.

[0109] Furthermore, the pH value of the strong base solution is 11 to 14.

[0110] Furthermore, the time of the third immersion is 4 h to 24 h.

[0111] Step S220: Place the peritoneal tissue after the third immersion in an acidic solution for a fourth immersion to prepare the peritoneal tissue after the fourth immersion.

[0112] Furthermore, the acid in the acidic solution includes one or more of hydrochloric acid, lactic acid, citric acid, and acetic acid.

[0113] Furthermore, the mass percentage concentration of the acid in the acidic solution is 1% to 8%.

[0114] Furthermore, the pH value of the acidic solution is 0.5 to 1.5.

[0115] Furthermore, the time of the fourth immersion is 4 h to 24 h.

[0116] Step S230: Place the peritoneal tissue after the fourth immersion in a neutral salt solution for a fifth immersion to prepare the peritoneal tissue after the fifth immersion.

[0117] Furthermore, the neutral salt in the neutral salt solution includes one or more of sodium chloride, potassium chloride, and sodium sulfate.

[0118] Further, the concentration of the neutral salt in the neutral salt solution is 5% - 10%.

[0119] Further, the time of the fifth immersion is 4h - 24h.

[0120] Step S240: Place the peritoneal tissue after the fifth immersion into a weak base salt solution for a sixth immersion to prepare the acellular matrix.

[0121] Further, the weak base salt in the weak base salt solution includes one or more of sodium bicarbonate, sodium acetate, and disodium hydrogen phosphate.

[0122] Further, the mass percentage concentration of the weak base salt in the weak base salt solution is 0.5% - 1.5%.

[0123] Further, the time of the sixth immersion is 15h - 30h.

[0124] It can be understood that through Step S230 and Step S240, the strong acid in Step S220 can be neutralized to achieve the effect of cleaning the acellular matrix.

[0125] In some embodiments, in the preparation method of the present application, Step S100 is further included before Step S200.

[0126] Step S100: Pretreat the peritoneal tissue of a mammal. Specifically, Step S100 includes Step S110 - Step S120.

[0127] Step S110: Clean the peritoneal tissue of a mammal and remove the surface fat.

[0128] Step S120: Place the peritoneal tissue after removing the surface fat into an enzyme solution for a seventh immersion to remove the residual fat.

[0129] Further, the enzyme in the enzyme solution includes one or more of lipase, trypsin, pepsin, and neutral protease;

[0130] Further, the mass percentage concentration of the enzyme in the enzyme solution is 0.2% - 2.0%;

[0131] Further, the time of the seventh immersion is 5h - 36h.

[0132] In some embodiments, in the preparation method of the present application, Step S600 is further included after Step S500.

[0133] Step S600: Package and sterilize the freeze-dried acellular matrix.

[0134] Further, the sterilization method can be, for example, irradiation sterilization. Further still, the irradiation dose can be, for example, but not limited to, 20 KGy to 25 KGy.

[0135] The present application also provides a decellularized matrix repair material prepared by the preparation method in any of the above embodiments.

[0136] The present application also provides the application of the decellularized matrix repair material in any of the above embodiments in the preparation of urethral repair products.

[0137] The following further elaborates on the present application through specific embodiments. The following embodiments are relatively specific. It can be understood that in other embodiments, it is not limited to this. In the following specific embodiments, the instruments, reagents, and materials involved, unless otherwise specified, are all conventional instruments, reagents, and materials existing in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, are all conventional experimental methods and detection methods existing in the prior art.

[0138] Example 1

[0139] A preparation method of a decellularized matrix repair material includes the following steps:

[0140] (1) Pretreatment: Take fresh bovine peritoneal tissue, rinse it twice with purified water, and then use mechanical scraping to remove the excess fat on the peritoneal surface.

[0141] Place the rinsed bovine peritoneal tissue in a lipase solution with a mass percentage of 0.5% for soaking for 24 hours to remove the residual fat, and then wash it 4 times with purified water to prepare the pretreated peritoneal tissue.

[0142] (2) Decellularization treatment: Place the pretreated peritoneal tissue obtained in step (1) in a sodium hydroxide solution with a pH value of 11.2 and a mass percentage concentration of 0.5% for soaking for 8 hours to prepare the peritoneal tissue after strong alkali soaking.

[0143] Place the peritoneal tissue after alkali soaking in a hydrochloric acid solution with a pH value of 1 and a mass percentage concentration of 1% for acid soaking for 24 hours to prepare the peritoneal tissue after acid soaking.

[0144] Place the peritoneal tissue after acid soaking in a sodium chloride solution with a mass percentage concentration of 1% for soaking for 24 hours to prepare the peritoneal tissue after neutral salt soaking.

[0145] Then place the peritoneal tissue after neutral salt soaking in a sodium bicarbonate solution with a mass percentage concentration of 1% for soaking for 24 hours to prepare the decellularized treatment matrix.

[0146] (3) Crosslinking: Immerse the acellular matrix prepared in step (2) in a glutaraldehyde solution with a mass percentage concentration of 2% to crosslink the acellular matrix with the glutaraldehyde crosslinking agent. The immersion time is 10 h. After the immersion, wash the acellular matrix 4 times with injection water to prepare the crosslinked acellular matrix.

[0147] (4) Drug loading: Prepare levofloxacin into nanoparticles by the nanoprecipitation method using amino-polyethylene glycol-polylactic acid. Immerse the crosslinked acellular matrix prepared in step (3) in a levofloxacin solution with a mass percentage concentration of 0.2%. Add 0.05 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 0.05 mmol of N-hydroxysuccinimide per milligram of the crosslinked acellular matrix. The immersion time is 24 h. After the immersion, wash the acellular matrix 2 times with a disodium hydrogen phosphate-sodium dihydrogen phosphate solution to prepare the drug-loaded acellular matrix.

[0148] (5) Freeze-drying: First freeze the drug-loaded acellular matrix prepared in step (4), and then freeze-dry it. The specific conditions are a freeze-drying temperature of -45°C and a freeze-drying time of 36 h.

[0149] Prepare an acellular matrix repair material.

[0150] (6) Sterilization: Package the peritoneum after freeze-drying in step (5) and sterilize it by irradiation with an irradiation dose of 20 KGy to 25 KGy.

[0151] Example 2

[0152] A method for preparing an acellular matrix repair material, comprising the following steps:

[0153] (1) Pretreatment: Take fresh sheep peritoneal tissue, rinse it 2 times with purified water, and then use a mechanical method to scrape off the excess fat on the peritoneal surface.

[0154] Immerse the rinsed sheep peritoneal tissue in a lipase solution with a mass percentage of 0.5% for 24 h to remove residual fat, and then wash it 4 times with purified water to prepare the pretreated peritoneal tissue.

[0155] (2) Decellularization treatment: Immerse the pretreated peritoneal tissue obtained in step (1) in a sodium hydroxide solution with a pH value of 11.5 and a mass percentage concentration of 0.5% for 6 h to prepare the peritoneal tissue after strong base immersion.

[0156] Immerse the peritoneal tissue after strong base immersion in a hydrochloric acid solution with a pH value of 1 and a mass percentage concentration of 1% for 20 h to prepare the peritoneal tissue after acid immersion.

[0157] The peritoneal tissue after acid immersion was immersed in a sodium chloride solution with a mass percentage concentration of 1% for 24 h to prepare peritoneal tissue after neutral salt immersion.

[0158] Then, the peritoneal tissue after neutral salt immersion was immersed in a sodium bicarbonate solution with a mass percentage concentration of 1% for 24 h to prepare a decellularized matrix.

[0159] (3) Crosslinking: The decellularized matrix prepared in step (2) was immersed in a 1,4-butanediol diglycidyl ether solution with a mass percentage concentration of 5% to crosslink the decellularized matrix with the 1,4-butanediol diglycidyl ether crosslinking agent. The immersion time was 16 h. After the immersion, the decellularized matrix was washed 6 times with injection water to prepare a crosslinked decellularized matrix.

[0160] (4) Drug loading: Ciprofloxacin was prepared into nanoparticles by the nanoprecipitation method using amino-polyethylene glycol-poly(lactic acid). The crosslinked decellularized matrix prepared in step (3) was immersed in a ciprofloxacin solution with a mass percentage concentration of 0.01%. For every milligram of the crosslinked decellularized matrix, 0.001 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 0.001 mmol of N-hydroxysuccinimide were added. The immersion time was 12 h. After the immersion, the decellularized matrix was washed 2 times with a disodium hydrogen phosphate-sodium dihydrogen phosphate solution to prepare a drug-loaded decellularized matrix.

[0161] (5) Freeze-drying: The drug-loaded decellularized matrix prepared in step (4) was first frozen and then freeze-dried. The specific conditions were a freeze-drying temperature of -30 °C and a freeze-drying time of 48 h.

[0162] Prepare a decellularized matrix repair material.

[0163] (6) Sterilization: The peritoneum after freeze-drying in step (5) was packaged and sterilized by irradiation with an irradiation dose of 20 KGy to 25 KGy.

[0164] Comparative Example 1

[0165] It was substantially the same as Example 1, except that the decellularized matrix was not crosslinked and drug-loaded. Specifically:

[0166] A method for preparing a decellularized matrix repair material, comprising the following steps:

[0167] (1) Pretreatment: Take fresh bovine peritoneal tissue, rinse it 2 times with purified water, and then use the mechanical method to scrape off the excess fat on the peritoneal surface.

[0168] The rinsed bovine peritoneal tissue was immersed in a lipase solution with a mass percentage of 0.5% for 24 hours to remove residual fat, and then washed 4 times with purified water to prepare the pretreated peritoneal tissue.

[0169] (2) Decellularization treatment: The pretreated peritoneal tissue obtained in step (1) was immersed in a sodium hydroxide solution with a pH value of 11.5 and a mass percentage concentration of 0.5% for 8 hours to prepare the peritoneal tissue after strong alkali immersion.

[0170] The peritoneal tissue after alkali immersion was acid-soaked in a hydrochloric acid solution with a pH value of 1 and a mass percentage concentration of 1% for 24 hours to prepare the peritoneal tissue after acid soaking.

[0171] The peritoneal tissue after acid soaking was immersed in a sodium chloride solution with a mass percentage concentration of 1% for 24 hours to prepare the peritoneal tissue after neutral salt soaking.

[0172] Then the peritoneal tissue after neutral salt soaking was immersed in a sodium bicarbonate solution with a mass percentage concentration of 1% for 24 hours to prepare the decellularized matrix.

[0173] (3) Freeze-drying: The decellularized matrix prepared in step (2) was first frozen and then freeze-dried. The specific conditions were a freeze-drying temperature of -45°C and a freeze-drying time of 36 h. A decellularized matrix repair material was prepared.

[0174] (4) Sterilization: The peritoneal membrane after freeze-drying in step (3) was packaged and sterilized by irradiation, with an irradiation dose of 20 KGy - 25 KGy.

[0175] Performance tests were carried out on the decellularized matrix repair materials prepared in Examples 1 - 2 and Comparative Example 1:

[0176] (1) SEM scanning: The decellularized matrix repair material prepared in Example 1 was subjected to SEM scanning, and the scanning results are as Figure 2 shown. It can be seen that one side of the decellularized matrix repair material is rough and the other side is smooth. Figure 2 A in Figure 2 is the rough surface, and the rough surface has a loose structure, which is beneficial for urethral cells to grow in. Figure 2 B in

[0177] (2)Cytotoxicity detection: The test method is based on GB / T 16886.5-2017 Biological evaluation of medical devices - Part 5: Tests for in vitro cytotoxicity. The test results are shown in Figure 3 and Table 1;

[0178] Table 1

[0179]

[0180] As can be seen from Figure 3 and Table 1, the cell survival rates of the samples in Example 1, Example 2, and Comparative Example 1 are all higher than 80%, indicating that the acellular matrix repair material has no cytotoxicity and good biocompatibility.

[0181] (3)Degradation performance detection: Test method: Treat the acellular matrix repair material with a collagenase solution of 200 U / mL for 4, 8, 16, 24, 48, and 96 h, respectively measure the mass before and after treatment, and calculate the degradation residue rate of the acellular matrix repair material. The test results are shown in Figure 4 and Table 2;

[0182] Table 2

[0183]

[0184] As can be seen from Figure 4 and Table 2, compared with the uncrosslinked acellular matrix repair material in Comparative Example 1, the acellular matrix repair materials in Example 1 and Example 2 have a slower degradation rate and good anti-degradation performance, which can provide a continuous template scaffold for cell repair and regeneration.

[0185] (4)Antibacterial effect detection: The test method is based on GB / T 20944.2-2007 Textiles - Evaluation of antibacterial properties - Part 2: Absorption method. The test results are shown in Table 3 below.

[0186] As can be seen from Table 3, compared with the uncrosslinked acellular matrix repair material in Comparative Example 1, the acellular matrix repair materials in Example 1, Example 2, and Comparative Example 1 have a more obvious antibacterial effect against Escherichia coli and Staphylococcus aureus.

[0187] Table 3

[0188]

[0189] (5)Mechanical property detection: The test method is based on GB / T 529-2008 Determination of tear strength of vulcanized rubber or thermoplastic rubber (trouser, right-angle and crescent specimens). The test results are shown in Table 4 below.

[0190] Table 4

[0191]

[0192] As can be seen from Table 4, compared with the non-crosslinked acellular matrix repair material of Comparative Example 1, the acellular matrix repair materials of Example 1, Example 2 and Comparative Example 1 have more excellent mechanical properties and meet the clinical suture and repair requirements.

[0193] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0194] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of this application. Therefore, the protection scope of the patent of this application should be subject to the appended claims.

Claims

1. A preparation method of a decellularized matrix repair material, characterized in that It includes the following steps: Provide a decellularized matrix of mammalian peritoneal tissue, crosslink the decellularized matrix with a crosslinking agent to prepare a crosslinked decellularized matrix; Prepare the antibacterial drug into nano-drug-loaded particles, connect them with the crosslinked decellularized matrix to prepare a drug-loaded decellularized matrix; and perform lyophilization treatment on the drug-loaded decellularized matrix to prepare a decellularized matrix repair material.

2. The preparation method of the acellular matrix repair material according to claim 1, wherein The step of crosslinking the decellularized matrix with a crosslinking agent includes: Place the decellularized matrix in a solution containing the crosslinking agent for a first immersion.

3. The preparation method of the acellular matrix repair material according to claim 2, characterized in that, The conditions of the first immersion satisfy one or more of the following conditions: (1) The crosslinking agent includes one or more of glutaraldehyde, formaldehyde, butanediol diglycidyl ether, and genipin; (2) In the solution containing the crosslinking agent, the mass percentage concentration of the crosslinking agent is 0.1% - 5%; (3) The time of the first immersion is 4h - 48h.

4. The preparation method of the acellular matrix repair material according to claim 1, wherein, The step of preparing the drug-loaded decellularized matrix includes: Use amino-polyethylene glycol-poly(lactic acid) to encapsulate the antibacterial drug to prepare nano-drug-loaded particles, and place the crosslinked decellularized matrix in a solution containing the nano-drug-loaded particles for a second immersion.

5. The preparation method of the acellular matrix repair material according to claim 4, characterized in that, The conditions of the second immersion satisfy one or more of the following conditions: (1) The antibacterial drug includes one or more of levofloxacin, ciprofloxacin, moxifloxacin, azithromycin, clarithromycin, doxycycline, minocycline, and clindamycin; (2) In the solution containing the antibacterial drug, the mass percentage concentration of the antibacterial drug is 0.01% - 0.5%; (3) The solution for the second immersion includes a promoter, and 0.001 mmol - 0.1 mmol of the promoter is added per milligram of the crosslinked decellularized matrix; Optionally, the promoter includes one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide; (4) The time of the second immersion is 2h - 24h.

6. The preparation method of the acellular matrix repair material according to claim 1, wherein, The conditions of lyophilization satisfy one or more of the following conditions: (1) The freeze-drying temperature is -30°C to -50°C; (2) The freeze-drying time is 24 h to 48 h.

7. The preparation method of the acellular matrix repair material according to any one of claims 1 to 6, characterized in that, The mammalian is selected from one or more of pigs, cows, and sheep.

8. The preparation method of the acellular matrix repair material according to any one of claims 1 to 6, characterized in that, The preparation of the decellularized matrix includes the following steps: Place the pretreated mammalian peritoneal tissue in a strong base solution for a third immersion to prepare the peritoneal tissue after the third immersion; Optionally, the third immersion satisfies one or more of the following conditions: (1) The base in the strong base solution includes one or more of sodium hydroxide and potassium hydroxide; (2) The mass percentage concentration of the base in the strong base solution is 0.3% - 10%; (3) The pH value of the strong base solution is 11 - 14; (4) The time of the third immersion is 4h - 24h; Place the peritoneal tissue after the third immersion in an acidic solution for a fourth immersion to prepare the peritoneal tissue after the fourth immersion; Optionally, the fourth immersion satisfies one or more of the following conditions: (1) The acid in the acidic solution includes one or more of hydrochloric acid, lactic acid, citric acid, and acetic acid; (2) The mass percentage concentration of the acid in the acidic solution is 1% - 8%; (3) The pH value of the acidic solution is 0.5 - 1.5; (4) The time of the fourth immersion is 4h - 24h; Place the peritoneal tissue after the fourth immersion in a neutral salt solution for a fifth immersion to prepare the peritoneal tissue after the fifth immersion; Optionally, the fifth immersion satisfies one or more of the following conditions: (1) The neutral salt in the neutral salt solution includes one or more of sodium chloride, potassium chloride, and sodium sulfate; (2) The concentration of the neutral salt in the neutral salt solution is 5% - 10%; (4) The time of the fifth immersion is 4h - 24h; Place the peritoneal tissue after the fifth immersion in a weak base salt solution for a sixth immersion to prepare the peritoneal tissue after the sixth immersion; and Optionally, the sixth immersion satisfies one or more of the following conditions: (1) The weak base salt in the weak base salt solution includes one or more of sodium bicarbonate, sodium acetate, and disodium hydrogen phosphate; (2) The mass percentage concentration of the weak base salt in the weak base salt solution is 0.5% - 1.5%; (3) The time of the sixth immersion is 15h - 30h; Wash the peritoneal tissue after the sixth immersion to remove the surface fat, and place it in an enzyme solution for a seventh immersion to prepare the acellular matrix; Optionally, the seventh immersion satisfies at least one of the following conditions: (1) The enzyme in the enzyme solution includes one or more of lipase, trypsin, pepsin, and neutral protease; (2) The mass percentage concentration of the enzyme in the enzyme solution is 0.2% - 2.0%; (3) The time of the seventh immersion is 5h - 36h.

9. An acellular matrix repair material, characterized in that, Prepared by the preparation method according to any one of claims 1 - 8.

10. Use of the acellular matrix repair material according to claim 9 in the preparation of urethral repair products.

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