WO2024114050A1

WO2024114050A1 - Biological tissue material, preparation method therefor, and use thereof

Info

Publication number: WO2024114050A1
Application number: PCT/CN2023/119105
Authority: WO
Inventors: 裴歌; 于世河; 何海红; 刘世红; 陈国明
Original assignee: 上海微创心通医疗科技有限公司
Priority date: 2022-11-30
Filing date: 2023-09-15
Publication date: 2024-06-06
Also published as: CN118105544A

Abstract

A biological tissue material and a preparation method therefor. The method comprises the following steps: soaking a biological tissue in a protective agent solution and then decellularizing the biological tissue; and subjecting the decellularized biological tissue to fixing, cross-linking, drying, and degreasing. The method can effectively remove calcified substances from biological tissue materials, ensuring the long-term stability of the biological tissue materials for in vivo use.

Description

Biological tissue material and its preparation method and application

Related Applications

This application claims priority to Chinese patent application number 2022115206552, filed on November 30, 2022, entitled “Biological Tissue Materials, Preparation Methods and Applications Thereof”, the entire text of which is hereby incorporated by reference.

Technical Field

The present application relates to the technical field of biomedical materials, and in particular to a biological tissue material and a preparation method and application thereof.

Background technique

Animal-derived biomaterials (such as animal pericardium, heart valves, etc.) are widely used in clinical practice to make artificial bio-valves, bio-patches, etc. Since animal-derived biomaterials have the characteristics of wide sources and wide application range, their market share has gradually increased, playing an important role in the medical device industry. However, this type of material also faces many challenges. Natural animal-derived biological tissues are soft and rich in a large number of cells, biological factors, etc., which makes it difficult to meet the requirements of biocompatibility and long-term application with implant materials. Therefore, it is necessary to process natural animal-derived biological tissues to improve their biocompatibility and long-term effectiveness. However, the current methods for processing animal-derived biomaterials are not only easy to damage biological tissues, but also have poor anti-calcification capabilities, resulting in a high calcium content in biological tissues, making it difficult to solve the problem of poor long-term effectiveness.

Summary of the invention

Based on this, it is necessary to provide a biological tissue material and a preparation method and application thereof.

In a first aspect, a method for preparing a biological tissue material is provided, comprising the following steps:

The biological tissue is immersed in a protective agent solution and then decellularized; and

The biological tissue after decellularization is fixed, cross-linked, dried and defatted.

In some embodiments, the biological tissue includes one or more of pericardium, valve, blood vessel, jugular vein, skin, small intestinal submucosa, meninges, bladder lining, ligament, tendon and swim bladder.

In some embodiments, the protective agent in the protective agent solution includes one or more of neomycin sulfate, tannic acid, quercetin, curcumin and polyethylene glycol;

The concentration of the protectant in the protectant solution is about 0.1 mM to about 1 mM.

In some embodiments, the decellularization method includes physical method, chemical method and biological method. One or more of the methods;

The physical method includes one or more of ultrasound, mechanical stirring, perfusion, pressure treatment, supercritical fluid, repeated freezing and thawing, and osmotic pressure;

The chemical method is to use chemical reagents for decellularization, and the chemical reagents include one or more of chaotropic agents, detergents, acids, bases, chelating agents, protease inhibitors, tributyl phosphate and antibiotics;

The biological method is to use enzyme to carry out decellularization treatment.

In some embodiments, the chemical reagent satisfies at least one of the following characteristics:

(1) The chaotropic agent comprises one or both of urea and thiourea;

(2) The detergent includes one or more of an ionic detergent, a nonionic detergent and a zwitterionic detergent;

(3) The acid includes one or more of acetic acid, peracetic acid, hydrochloric acid and sulfuric acid;

(4) The base includes one or more of ammonium hydroxide, sodium hydroxide and ammonia water;

(5) The chelating agent includes one or both of ethylenediaminetetraacetic acid and ethylene glycol bis(2-aminoethyl ether)tetraacetic acid.

In some embodiments, the detergent satisfies at least one of the following characteristics:

(1) The ionic detergent includes one or more of sodium dodecyl sulfate, sodium deoxycholate, sodium cholate, sarcosine, sodium dodecyl polyoxyethylene ether sulfate and glucose lipid quaternary ammonium salt;

(2) the nonionic detergent comprises one or more of Triton, Tween, glycosyl lithocholic acid ester, glycoenzyme, digitonin, glucosides, alkyl glycosides and cocoyl monoethanolamide;

(3) The zwitterionic detergent includes one or more of 3-(3-(cholamidopropyl)dimethylamino)propanesulfonic acid inner salt, sulfobetaines, carboxylic acid betaines, oleyl sulfate ester type imidazoline, dodecylaminopropionic acid, amino acids and cocamidopropylamine oxide.

In some embodiments, the chemical reagent is a solvent containing a detergent, and the concentration of the detergent in the solvent is about 0.01 wt % to about 1 wt %.

In some embodiments, the fixation cross-linking is performed in a fixation solution containing a cross-linking agent, and the cross-linking agent includes one or both of a chemical cross-linking agent and a natural cross-linking agent;

The chemical cross-linking agent includes one or more of glutaraldehyde, formaldehyde, carbodiimide and polyepoxide;

The natural cross-linking agent includes one or two of genipin and proanthocyanidin.

In some embodiments, the concentration of the crosslinking agent in the fixing solution is about 0.05 wt % to about 10 wt %.

In some embodiments, the degreasing treatment method is solvent immersion; the solvent includes one or more of dichloromethane, n-hexane, cyclohexane and petroleum ether.

In some of the embodiments, after the fixation and cross-linking, a step of using a capping agent solution to perform end-capping is also included.

In some embodiments, the capping agent in the capping agent solution includes one or more of amino acids, aminoamides, alkylamides, saturated alkylamines, unsaturated alkylamines, aminoalcohols, polyetheramines, aminoalkyl acids, aminopolysaccharides, aminoalkyldiacids, amino surfactants and fatty diamines.

In some embodiments, the concentration of the capping agent in the capping agent solution is about 1 mM to about 50 mM.

In a second aspect, a biological tissue material is provided which is prepared by the preparation method described in the first aspect.

In a third aspect, there is provided a use of the biological tissue material as described in the second aspect as an artificial valve or a biological patch, or in the preparation of an artificial valve or a biological patch.

The details of one or more embodiments of the present application are set forth in the description below. Other features, objects, and advantages of the present application will become apparent from the description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to better describe and illustrate the embodiments and/or examples of the inventions disclosed herein, reference may be made to one or more drawings. The additional details or examples used to describe the drawings should not be considered to limit the scope of the disclosed inventions, the embodiments and/or examples currently described, and any of the best modes of these inventions currently understood.

FIG1 is a schematic diagram of a method for preparing a biological tissue material of the present application;

FIG2 is a graph showing the calcium content of bovine pericardial tissue treated in Example 1 and Comparative Example 1 after implantation into rats for 60 days;

FIG3 is the HE staining results of bovine pericardial tissues of Example 1 and Comparative Example 4 of the present application.

Detailed ways

The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

Except as shown in the operating examples or otherwise indicated, all numbers used in the specification and claims to indicate the amount of ingredients, physicochemical properties, etc. are understood to be adjusted by the term "about" in all cases. For example, therefore, unless otherwise indicated, the numerical parameters listed in the above specification and the appended claims are approximate values, and those skilled in the art will be able to use the teachings disclosed herein to seek to obtain the desired properties and appropriately change these approximate values. The use of numerical ranges expressed as endpoints includes all numbers within the range and any range within the range, for example, 1 to 5 includes 1, 1.1, 1.3, 1.5, 2, 2.75, 3, 3.80, 4 and 5, etc.

In this document, the terms "first aspect", "second aspect", "third aspect", "fourth aspect", etc., are used interchangeably. "Second", "third", "fourth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

The term “plurality” used herein refers to a number that is equal to or greater than 2.

In this article, 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.

The temperature parameters herein, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument. For example, fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

Traditional methods for processing biological tissue materials will damage the cytoplasmic matrix during decellularization and reduce the mechanical properties of biological tissue materials. In addition, they cannot effectively remove calcified substances in biological tissue materials, making it difficult to meet the requirements of long-term stable use in vivo. To this end, the present application provides a method for preparing biological tissue materials to improve the above problems.

In a first aspect, a method for preparing a biological tissue material is provided, comprising step S100, step S200, step S300 and step S400.

The preparation method of the biological tissue material provided in the present application adopts a multi-step processing process to remove or reduce the sites of possible calcification of biological tissues from many aspects, and improve the physical properties and durability of biological tissues. In particular, the decellularization treatment is combined with drying and defatting, which is simple to operate, and on the basis of avoiding damage to biological tissues, the removal of lipids in biological tissues is maximized, the anti-calcification effect of biological tissues is improved, its physical and mechanical properties are guaranteed, and the service life is extended. Moreover, the degreasing treatment can reduce the lipids inside the biological tissues on the one hand, and on the other hand, it can also dissolve the chemical reagents remaining on the surface of the biological tissues, thereby improving the convenience and safety of storage and transportation of biological tissue materials. Drying before defatting can also reduce the requirements of biological tissue materials for storage and transportation conditions, and further improve the anti-calcification performance of biological tissues.

In addition, before decellularization, treating biological tissue with a protective agent can protect the cytoplasmic matrix of the biological tissue, avoiding the problem of structural damage during subsequent decellularization and fixation and cross-linking treatments, and ensuring that the original structure of the biological tissue material is not destroyed.

Step S100: cleaning the biological tissue. This step can remove redundant tissues in the biological tissue, such as fat, thick blood vessels, etc.

It is understandable that in some implementations, step S100 may be omitted.

In some embodiments, the biological tissue is of non-human origin, for example, the biological tissue may be xenogeneic biological tissue.

Optionally, the biological tissue may include pericardium (porcine pericardium, bovine pericardium, etc.), valves (aortic valve, mitral valve, The invention relates to a method for preparing the fish maw of a fish, wherein the fish maw is selected from the group consisting of: tricuspid valve, blood vessel, jugular vein, skin, small intestinal submucosa, cerebrospinal meninges, bladder lining, ligament, tendon and fish bladder. Exemplarily, the fish maw can be taken from carp, grass carp, crucian carp, silver carp, bighead carp, bream, yellow croaker, sturgeon, Spanish mackerel or kingfish.

Step S200: soaking the biological tissue treated in step S100 in a protective agent solution and performing a decellularization treatment.

In some embodiments, the protective agent in the protective agent solution refers to an agent that can protect the cytoplasmic matrix in biological tissues. For example, the protective agent can include but is not limited to one or more of neomycin sulfate, tannic acid, quercetin, curcumin and polyethylene glycol.

In some embodiments, the concentration of the protectant in the protectant solution is about 0.1 mM to about 1 mM, for example, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM.

In the present application, the soaking time and temperature are not limited, as long as the protective agent can be immersed in the biological tissue. Exemplarily, the soaking time can be about 1 hour to about 36 hours, for example, about 2 hours, about 5 hours, about 8 hours, about 10 hours, about 15 hours, about 20 hours, about 25 hours, about 30 hours; the temperature can be room temperature, for example, about 18°C to about 30°C, and can also be about 20°C, about 25°C.

It is understandable that during the soaking process, an oscillation method can be used to allow the protective agent to fully immerse the biological tissue.

In the present application, the method used for decellularization can be selected from the commonly used methods in the field of decellularization to reduce or remove cell components, antigen activity, immunogenicity, etc. in biological tissues. In some embodiments, the method used for decellularization includes, but is not limited to, one or more of physical methods, chemical methods, and biological methods. Among them, physical methods may include one or more of ultrasound, mechanical stirring, perfusion, pressure treatment, supercritical fluid, repeated freezing and thawing (freeze/thaw cycle) and osmotic pressure (hypotonic/hypertonic alternation); chemical methods are to use chemical reagents for decellularization, and chemical reagents may include one or more of chaotropic agents, detergents, acids, bases, chelating agents, protease inhibitors, tributyl phosphate and antibiotics; biological methods are to use enzymes for decellularization.

Usually, several methods can be combined, such as physical method and chemical method, or physical method, chemical method and biological method. Any combination can be made without violating the concept of this application, and all belong to the protection scope of this application.

In some embodiments, the physical methods used for decellularization, such as ultrasound, mechanical stirring, repeated freezing and thawing, and osmotic pressure, and chemical methods need to be performed in a solution, wherein the solution used may include one or more of phosphate buffer, Tris-HCL buffer, Hepes buffer, ethanol, water, and physiological saline.

In some embodiments, the chaotropic agent includes one or more of urea and thiourea.

In some embodiments, the detergent comprises one or more of an ionic detergent, a nonionic detergent and a zwitterionic detergent; wherein the ionic detergent may include sodium dodecyl sulfate, sodium deoxycholate, sodium cholate, The detergent may include one or more of amino acids, sodium lauryl polyoxyethylene ether sulfate and glucose lipid quaternary ammonium salt; the non-ionic detergent may include Triton X-100, Tween, glycosyl lithocholic acid ester amphiphilic molecules (such as GLC-1, GLC-2 and GLC-3), glycoenzyme (GDN), digitonin, glucosides, alkyl glycosides and cocoyl monoethanolamide; the zwitterionic detergent may include one or more of 3-(3-(cholamidopropyl) dimethylamino) propane sulfonic acid inner salt (CHAPS), sulfobetaines, carboxylic acid betaines, oleyl sulfate ester type imidazoline, dodecylaminopropionic acid, amino acids and cocoamidopropylamine oxide.

In some embodiments, the acid includes one or more of acetic acid, peracetic acid, hydrochloric acid, and sulfuric acid.

In the present application, the base is mainly an inorganic base. In some embodiments, the base includes one or more of ammonium hydroxide, sodium hydroxide and ammonia water.

In some embodiments, the chelating agents include ethylenediaminetetraacetic acid (EDTA) and ethylene glycol bis(2-aminoethyl ether)tetraacetic acid (EGTA).

In some embodiments, the enzyme includes one or more of trypsin, pepsin, nuclease (such as RNase A, DNase), dispase, lipase and disaccharidase.

In some embodiments, a solvent containing a detergent is used for decellularization. In the present application, the temperature and time of the decellularization treatment are not limited. For example, the temperature of the decellularization treatment can be about 4°C to about 37°C, and can also be about 5°C, about 10°C, about 15°C, about 20°C, about 25°C, and about 30°C; the time can be about 1h to about 48h, and can also be about 2h, about 5h, about 10h, about 12h, about 15h, about 20h, about 25h, about 30h, about 35h, about 40h, and about 45h.

In some embodiments, the concentration of the detergent in the solvent containing the detergent is about 0.01 wt% to about 1 wt%, for example, about 0.02 wt%, about 0.05 wt%, about 0.08 wt%, about 0.1 wt%, about 0.2 wt%, about 0.5 wt%, about 0.8 wt%.

It is understood that shaking can be performed during the decellularization process. For example, a shaker can be used to shake the decellularization process more fully.

Step S300: washing the biological tissue after the decellularization treatment in step S200.

It is understood that the residual reagent in the biological tissue can be removed by washing. In some embodiments, this step can be omitted.

Step S400: Fixing, cross-linking, drying, and degreasing the biological tissue processed in step S300.

In the present application, the method of fixation and crosslinking is not limited, and the commonly used fixation and crosslinking methods in the art can be selected, for example, the fixation and crosslinking method can be ultraviolet crosslinking or crosslinking using a solution containing a crosslinking agent. In some embodiments, the fixation and crosslinking is performed in a fixation solution containing a crosslinking agent.

In some embodiments, the crosslinking agent includes one or both of a chemical crosslinking agent and a natural crosslinking agent; The chemical cross-linking agent may include one or more of glutaraldehyde, formaldehyde, carbodiimide and polyepoxides; the natural cross-linking agent may include one or two of genipin and proanthocyanidins.

In some embodiments, the concentration of the cross-linking agent in the fixing solution is about 0.05 wt % to about 10 wt %, for example, about 0.08 wt %, about 0.1 wt %, about 0.5 wt %, about 1 wt %, about 1.5 wt %, about 2 wt %, about 2.5 wt %, about 3 wt %, about 3.5 wt %, about 4 wt %, about 4.5 wt %, about 5 wt %, about 5.5 wt %, about 6 wt %, about 6.5 wt %, about 7 wt %, about 7.5 wt %, about 8 wt %, about 8.5 wt %, about 9 wt %, about 9.5 wt %.

In some embodiments, after the cross-linking is fixed, a step of using a capping agent solution to perform end-capping is further included.

The anti-calcification performance and stability of biological tissue materials can be improved by end-capping treatment. In particular, when an aldehyde cross-linking agent is used, the aldehyde group can be capped and reduced by the end-capping agent, thereby improving the anti-calcification performance of the biological tissue material. Among them, end-capping refers to the reaction of a reagent containing a primary amino group with a free aldehyde group on the biological tissue material to generate a Schiff base, which can play a role in blocking the aldehyde group; reduction refers to the use of a reducing agent to reduce the Schiff base, which plays a role in stabilizing chemical bonds. Among them, the choice of reducing agent is not limited, and a reducing agent commonly used in the art can be used. For example, the reducing agent can include one or more of sodium borohydride, sodium cyanoborohydride, potassium borohydride and ammonium formate.

In some embodiments, the capping agent in the capping agent solution includes one or more of amino acids, aminoamides, alkylamides, saturated alkylamines, unsaturated alkylamines, aminoalcohols, polyetheramines, aminoalkyl acids, aminopolysaccharides, aminoalkyldiacids, amino surfactants, alkyldiamines and fatty diamines.

In some embodiments, the concentration of the capping agent in the capping agent solution is about 1 mM to about 50 mM, for example, about 2 mM, about 5 mM, about 8 mM, about 10 mM, about 12 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM.

In the present application, the drying method is not limited, and any drying method commonly used in the art can be selected, for example, freeze drying can be used. The freeze drying pressure is less than about 100 Pa, the temperature can be about -60°C to about 25°C, the drying time can be about 2h to about 48h, and the number of freeze drying times can be 1 to 10 times.

In some embodiments, the degreasing treatment method is solvent immersion. In the present application, the choice of solvent is not limited, and a low-toxic, non-polar and volatile solvent can be selected. Exemplary, the solvent may include one or more of dichloromethane, n-hexane, cyclohexane and petroleum ether. Among them, the immersion time can be about 12h to about 48h, the temperature is about 15°C to about 37°C, and the number of immersions can be 1 to 20 times.

It is understood that after degreasing, the biological tissue may be sealed or sterilized, or the steps of sealing and sterilizing may be performed in sequence. The sterilization process may be a commonly used process in the art, for example, the sterilization process may be ethylene oxide (EO) sterilization.

Referring to FIG1 , according to one embodiment, a method for preparing a biological tissue material comprises the following steps:

1) Soaking the biological tissue in a protective agent solution and then performing a decellularization treatment;

2) placing the biological tissue after decellularization in a fixative solution for fixation and cross-linking;

3) drying the fixed and cross-linked biological tissue; and

4) Soaking the dried biological tissue in a solvent for degreasing.

In a fourth aspect, a method for treating heart valve disease or repairing valve defects is provided, comprising implanting an artificial valve into a subject, wherein the material of the artificial valve comprises the biological tissue material described in the second aspect.

In order to make the purpose, technical solutions and advantages of the present application more concise and clear, the present application is described with the following specific embodiments, but the present application is by no means limited to these embodiments. The embodiments described below are only preferred embodiments of the present application and can be used to describe the present application, and cannot be understood as limiting the scope of the present application. It should be pointed out that any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

In order to better illustrate the present application, the present application is further described below in conjunction with the embodiments. The following are specific embodiments.

Example 1

1) Place the cleaned bovine pericardial tissue in a 1mM solution of neomycin sulfate dissolved in PBS buffer and shake for 2 hours at room temperature. Then soak the bovine pericardial tissue in a hypotonic PBS buffer (concentration of 0.1M, pH 7.4), add 0.5wt% TritonX-100, 20μg/mL RNase A and 0.2mg/mL DNase, mix and shake for 24 hours, take out, and wash the bovine pericardial tissue 8 times with sterile saline;

2) preparing a 0.625wt% glutaraldehyde aqueous solution with a pH of 7.4 as a fixative solution, and placing the bovine pericardial tissue cleaned in step 1) in the fixative solution for 24 hours. Subsequently, the bovine pericardial tissue was washed in a 0.1M PBS buffer with a pH of 7.4 for three times, each washing for 5 minutes;

3) preparing 1 L of PBS buffer (concentration of 0.1 M, pH 7.4) containing 10 mM lysine, and immersing the bovine pericardial tissue cleaned in step 2) in the PBS buffer and placing it on a constant temperature shaker at 25° C. and 90 rpm for 48 hours, and then washing the bovine pericardial tissue with a sterile saline solution for 30 minutes;

4) The bovine pericardial tissue washed in step 3) is freeze-dried at -20°C and 100Pa for 4 hours, and then heated to 20°C for analysis to obtain dried bovine pericardial tissue. The dried bovine pericardial tissue is soaked in cyclohexane at room temperature, and each time is allowed to stand for 12 hours, and repeated 6 times in total, and then the bovine pericardial tissue is taken out and placed in a fume hood for 48 hours to allow the cyclohexane to volatilize, thereby obtaining the treated bovine pericardial tissue. The bovine pericardial tissue can then be sealed and sterilized with EO.

Example 2

1) Place the cleaned bovine pericardial tissue in a 1 mM quercetin solution dissolved in Tris buffer at room temperature. The bovine pericardial tissue was then immersed in a hypotonic PBS buffer (concentration of 0.1 M, pH 7.4), and then 0.5 wt% sodium deoxycholate and 2 wt% sodium dodecyl sulfate (SDS) were added and mixed and shaken for 12 hours, then taken out, and treated with 0.5 wt% trypsin for 2 hours, and the bovine pericardial tissue was washed 8 times with sterile saline;

2) Soak the bovine pericardial tissue obtained in step 1) in a mixed solution of EDC and N-hydroxysuccinimide (NHS) (concentration of 15mM:15mM) for 24 hours. Prepare a 0.625wt% glutaraldehyde solution with a pH of 7.4 as a fixative solution, and place the bovine pericardial tissue soaked in step 1) in the fixative solution for 24 hours. Then wash the bovine pericardial tissue in a PBS buffer with a concentration of 0.1M and a pH of 7.4 for 3 times, each wash for 5 minutes;

3) The bovine pericardial tissue washed in step 2) is freeze-dried at -20°C and 100Pa for 4 hours, and then heated to 20°C for analysis to obtain dried bovine pericardial tissue. The dried bovine pericardial tissue is soaked in dichloromethane at room temperature, and each time is allowed to stand for 12 hours, and repeated 6 times in total, and then the bovine pericardial tissue is taken out and placed in a fume hood for 48 hours to allow the dichloromethane to volatilize, thereby obtaining the processed bovine pericardial tissue. The bovine pericardial tissue can then be sealed and sterilized with EO.

Example 3

1) Place the cleaned bovine pericardial tissue in a 1 mM curcumin solution dissolved in PBS buffer and shake for 1 hour at room temperature. Then soak the bovine pericardial tissue in a hypotonic PBS buffer (concentration of 0.1 M, pH 8), add 1wt% alkyl glycoside, 20μg/mL RNase A and 0.2mg/mL DNase, mix and shake for 48 hours, take out, and wash the bovine pericardial tissue 8 times with sterile saline;

2) preparing a 0.625wt% glutaraldehyde solution with a pH of 7.4 as a fixing solution, and placing the bovine pericardial tissue washed in step 1) in the fixing solution for 4 hours, and then placing it in a 5wt% proanthocyanidin solution (PBS buffer) for 72 hours. Subsequently, the bovine pericardial tissue was washed three times in a 0.1M PBS buffer with a pH of 7.4, each washing for 5 minutes;

4) The bovine pericardial tissue washed in step 3) is freeze-dried at -40°C and 60Pa for 2 hours, and then heated to 10°C for analysis to obtain dried bovine pericardial tissue. The dried bovine pericardial tissue is soaked in n-hexane at room temperature, and each time is allowed to stand for 12 hours, and repeated 6 times in total, and then the bovine pericardial tissue is taken out and placed in a fume hood for 48 hours to allow the n-hexane to volatilize, thereby obtaining the treated bovine pericardial tissue. The bovine pericardial tissue can then be sealed and sterilized with EO.

Example 4

The processing method of this embodiment is basically the same as that of embodiment 1, except that the biological tissue material is a porcine aortic valve, and the specific steps are as follows:

1) Place the cleaned porcine aortic valve tissue in a 5 mM solution of neomycin sulfate dissolved in PBS buffer and shake for 2 hours at room temperature. Then soak the tissue in a hypotonic PBS buffer (concentration 0.1 M, pH 7.4), add 1wt% TritonX-100, 20 μg/mL RNase A and 0.2 mg/mL DNase, mix and shake for 24 hours, take out, and wash 8 times with sterile saline;

2) preparing a 0.625wt% glutaraldehyde solution with a pH of 7.4 as a fixative solution, and placing the porcine aortic valve tissue cleaned in step 1) in the fixative solution for 24 hours. The tissue was then washed three times in a 0.1M PBS buffer with a pH of 7.4, each washing for 5 minutes;

3) preparing 1 L of PBS buffer (concentration of 0.1 M, pH 7.4) containing 10 mM lysine, and immersing the tissue washed in step 2) in the above PBS buffer and placing it on a constant temperature shaker at 37° C. and 90 rpm for 24 h, and then washing the tissue with sterile saline solution for 30 min;

4) The tissue washed in step 3) was freeze-dried at -20°C and 100Pa for 4 hours, and then heated to 20°C for analysis to obtain dried porcine aortic tissue. The dried tissue was soaked in cyclohexane at room temperature, and each time was allowed to stand for 12 hours, and repeated 3 times in total. Then the tissue was taken out and placed in a fume hood for 48 hours to allow the cyclohexane to volatilize, and the treated porcine aortic tissue was obtained. The porcine aortic tissue can then be sealed and sterilized with EO.

Example 5

The processing method of this embodiment is basically the same as that of embodiment 1, except that the biological tissue material is jugular vein, and the specific steps are as follows:

1) Place the cleaned jugular vein tissue in a 3mM solution of neomycin sulfate dissolved in PBS buffer and shake for 12 hours at room temperature. Then soak the tissue in a hypotonic PBS buffer (concentration 0.1M, pH 7.4), add 3wt% TritonX-100, 20μg/mL RNase A and 0.2mg/mL DNase, mix and shake for 24 hours, remove, and wash 8 times with sterile saline;

2) Prepare a 0.625wt% glutaraldehyde solution with a pH of 7.4 as a fixative solution, and place the jugular vein tissue cleaned in step 1) in the fixative solution for 24 hours. Then wash the tissue in a 0.1M PBS buffer with a pH of 7.4 for 3 times, each time for 5 minutes;

3) preparing 1 L of PBS buffer (concentration of 0.1 M, pH 7.4) containing 10 mM lysine, and immersing the tissue washed in step 2) in the PBS buffer and placing it on a constant temperature shaker at 25° C. and 90 rpm for 24 h, and then washing the tissue with sterile saline solution for 30 min;

4) The tissue washed in step 3) is freeze-dried at -20°C and 100 Pa for 4 hours, and then heated to 20°C for analysis to obtain dried jugular vein tissue. The dried tissue is immersed in cyclohexane at room temperature, and each time is allowed to stand for 12 hours, and repeated 3 times in total, and then the tissue is taken out and placed in a fume hood for 48 hours to allow the cyclohexane to evaporate, thereby obtaining the treated jugular vein tissue. The jugular vein tissue can then be sealed and sterilized with EO.

Example 6

The treatment method of this embodiment is basically the same as that of embodiment 1, except that the decellularization treatment technology is different. The specific steps are as follows:

1) The cleaned bovine pericardial tissue was placed in a 1 mM solution of neomycin sulfate dissolved in PBS buffer and shaken for 2 hours at room temperature. The bovine pericardial tissue was then frozen at -20°C for 4 hours, taken out and thawed at room temperature for 4 hours, cycled 3 times, and then 1 wt% CHAPS was added, mixed and shaken for 24 hours, taken out, and ultrasonicated for 2 hours using a trypsin solution (PBS buffer, pH = 7.4, 1 mM), and then washed with saline, ultrasonicated in an EDTA solution (Tris buffer, pH = 7.4, 1 mM) for 2 hours, and the bovine pericardial tissue was washed 8 times with sterile saline;

2) preparing a 0.625wt% glutaraldehyde solution with a pH of 7.4 as a fixative solution, and placing the bovine pericardial tissue cleaned in step 1) in the fixative solution for 24 hours. Subsequently, the bovine pericardial tissue was washed in a 0.1M PBS buffer with a pH of 7.4 for three times, each washing for 5 minutes;

Example 7

1) Place the cleaned bovine pericardial tissue in a 1mM neomycin sulfate solution at room temperature and shake for 2 hours. Then soak the bovine pericardial tissue in a hypotonic Tris solution, add 1wt% tributyl phosphate and 0.5wt% sodium cholate, shake for 24 hours, and then soak it in a hypertonic PBS solution after cleaning, add 1wt% glucoside, and shake for 24 hours. After the reaction is completed, wash the bovine pericardial tissue 8 times with sterile saline;

3) The bovine pericardial tissue washed in step 2) is freeze-dried at -20°C and 100Pa for 4 hours, and then heated to 20°C for analysis to obtain dried bovine pericardial tissue. The dried bovine pericardial tissue is soaked in cyclohexane at room temperature, and each time is allowed to stand for 12 hours, and repeated 6 times in total, and then the bovine pericardial tissue is taken out and placed in a fume hood for 48 hours to allow the cyclohexane to volatilize, thereby obtaining the treated bovine pericardial tissue. The bovine pericardial tissue can then be sealed and sterilized with EO.

Comparative Example 1

In this comparative example, the bovine pericardial tissue was not subjected to decellularization and degreasing. The specific steps are as follows:

1) Prepare a 0.625wt% glutaraldehyde solution with a pH of 7.4 as a fixative solution, place the cleaned bovine pericardial tissue in the fixative solution at room temperature, and fix for 24 hours. Then, soak the bovine pericardial tissue in a 0.1M PBS buffer with a pH of 7.4 and wash it three times, each time for 5 minutes;

2) Prepare 1 L of PBS buffer (concentration of 0.1 M, pH 7.4) containing 10 mM lysine, and immerse the bovine pericardial tissue cleaned in step 1) in the above PBS buffer and place it on a constant temperature shaker at 25° C. and 90 rpm for 48 hours, then use sterile saline solution to wash the bovine pericardial tissue for 30 minutes, and then store it in glutaraldehyde solution.

Comparative Example 2

The treatment method of this comparative example is basically the same as that of Example 2, except that no protective agent is added before the decellularization treatment.

1) The cleaned bovine pericardial tissue was immersed in a hypotonic PBS buffer (concentration of 0.1 M, pH 7.4) at room temperature, and then 0.5wt% sodium deoxycholate and 2wt% sodium dodecyl sulfate (SDS) were added and mixed and shaken for 12 hours, then taken out, and then treated with 0.5wt% trypsin for 2 hours, and the bovine pericardial tissue was washed 8 times with sterile saline;

2) Soak the bovine pericardial tissue in a mixed solution of EDC and N-hydroxysuccinimide (NHS) (concentration of 15mM:15mM) for 24 hours. Prepare a 0.625wt% glutaraldehyde solution with a pH of 7.4 as a fixative solution, and place the bovine pericardial tissue soaked in step 1) in the fixative solution for 24 hours. Then wash the bovine pericardial tissue in a PBS buffer with a concentration of 0.1M and a pH of 7.4 for 3 times, each wash for 5 minutes;

Comparative Example 3

The treatment method of this comparative example is basically the same as that of Example 1, except that no degreasing treatment is performed.

1) Place the cleaned bovine pericardial tissue in a 1 mM neomycin sulfate solution (formed by dissolving neomycin sulfate in PBS buffer) and shake for 2 hours at room temperature. Then soak the bovine pericardial tissue in a hypotonic PBS buffer (concentrated 0.1 M, pH 7.4), then add 0.5 wt% TritonX-100, 20 μg/mL RNase A and 0.2 mg/mL DNase, mix and shake for 24 hours, take out, and wash the bovine pericardial tissue 8 times with sterile saline;

4) freeze-drying the bovine pericardial tissue washed in step 3) at -20°C and 100 Pa for 4 hours, and then heating to 20°C for desorption to obtain dried bovine pericardial tissue. The bovine pericardial tissue can then be sealed and sterilized by EO.

Comparative Example 4

The treatment method of this comparative example is basically the same as that of Example 1, except that cross-linking is performed first and then decellularization is performed. The specific steps are as follows:

1) Place the cleaned bovine pericardium in a 1mM neomycin sulfate solution (formed by dissolving neomycin sulfate in PBS buffer) and shake for 2 hours. Then prepare a 0.625wt% glutaraldehyde aqueous solution with a pH of 7.4 as a fixative solution, and place the cleaned bovine pericardium tissue in the fixative solution at room temperature for 24 hours. Then wash the bovine pericardium tissue in a 0.1M PBS buffer with a pH of 7.4 for 3 times, each wash for 5 minutes;

2) Then, the bovine pericardial tissue fixed and cross-linked in step 1) was immersed in a hypotonic PBS buffer (concentration of 0.1 M, pH 7.4), and then 0.5wt% TritonX-100, 20μg/mL RNase A and 0.2mg/mL DNase were added and mixed and shaken for 24 hours, then taken out and washed with sterile saline for 8 times;

3) placing the bovine pericardial tissue cleaned in step 2) into 1 L of PBS buffer (concentration of 0.1 M, pH 7.4) containing 10 mM lysine, and immersing the cleaned bovine pericardial tissue in the above PBS buffer and placing it on a constant temperature shaker at 25° C. and 90 rpm for 48 hours, and then washing the bovine pericardial tissue with a sterile saline solution for 30 minutes;

The mechanical properties of the bovine pericardial tissues treated in Examples 1 to 7 and Comparative Examples 1 to 2 were evaluated in the following specific steps:

The treated bovine pericardial tissue was rehydrated for 5 min, cut into 5 mm × 50 mm rectangles, and tested for its mechanical properties on a universal material testing machine. The test gauge length was set to 25 mm, the tensile rate was 20 mm/min, and the test was performed. The tensile test was carried out until the specimen broke and the test results were recorded as shown in Table 1.

Table 1 Mechanical properties of bovine pericardium tissue

It can be seen from the test results in the above table that, compared with Comparative Example 1, the treatment method used in the present application will not affect the mechanical properties of biological tissues; compared with Comparative Example 2, after using the protective agent, decellularization will not cause damage to the mechanical properties of the tissue.

The calcification performance of the bovine pericardial tissue treated in Example 1 and Comparative Examples 1 and 3 was evaluated, and the specific steps were as follows:

The samples of Example 1 and Comparative Examples 1 and 3 were cut into discs with a diameter of 10 mm, and each group of samples was washed twice with anhydrous physiological saline for use.

Nine 3-week-old Wistar rats in good health and with basically the same body shape were selected as subcutaneous implantation animal models (named as rat No. 1, rat No. 2, rat No. 3, ..., rat No. 8 and rat No. 9, respectively). After anesthesia, two samples of different components were implanted in the surgical area of their backs. Six parallel samples were implanted in each group, and the samples were ensured to be flat. The schematic diagram of subcutaneous implantation in rats is as follows:

Table 2 Schematic diagram of subcutaneous implantation in rats

The implantation time is 60 days, and the bovine pericardial tissue is taken out after 60 days and dried at a constant temperature of 60°C for 48h. Subsequently, the calcium content in the bovine pericardial tissue is determined by inductively coupled plasma spectroscopy, and the test results are shown in Figure 2. As shown in Figure 2, the calcium content in the bovine pericardial tissue in Example 1 is only 1.03μg/mg, the calcium content in the bovine pericardial tissue in Comparative Example 1 is as high as 19.94μg/mg, and the calcium content in the bovine pericardial tissue in Comparative Example 3 is 7.86μg/mg. It can be seen from Example 1 and Comparative Example 3 that when the other conditions are basically the same, Comparative Example 3 is only not defatted, and the calcium content in the bovine pericardial tissue obtained by the treatment is higher than that in Example 1, indicating that the treatment method provided in Comparative Example 3 cannot effectively reduce the lipid content in the biological tissue material, resulting in a high calcium content and poor anti-calcification performance. It can be seen that the treatment method provided in the present application can significantly remove or reduce the lipid content in the biological tissue material, reduce the calcium content in the biological tissue material, and significantly improve its anti-calcification performance.

HE staining was performed on the bovine pericardial tissue obtained in Example 1 and Comparative Example 4, and the processing steps were as follows: The bovine pericardial tissues obtained in Example 1 and Comparative Example 4 were selected, embedded in paraffin blocks, and a soft tissue slicer was used to select the tissue cross-section direction, and a 3 μm thick slice was cut, and hematoxylin and eosin dye was used for staining. The staining results are shown in Figure 3; Figure 3a is a HE staining result of the bovine pericardial tissue obtained in Example 1, and Figure 3b is a HE staining result of the bovine pericardial tissue obtained in Comparative Example 4. As shown in Figure 3, the bovine pericardial tissue obtained by the treatment process of Example 1 can achieve decellularization, while the bovine pericardial tissue in Comparative Example 4 has no decellularization effect.

The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

The above-described embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be construed as limiting the scope of the patent application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent application shall be subject to the attached claims.

Claims

A method for preparing a biological tissue material, comprising the following steps:

The biological tissue is immersed in a protective agent solution and then decellularized; and

The biological tissue after decellularization is fixed, cross-linked, dried and defatted.
The preparation method according to claim 1, wherein the biological tissue comprises one or more of pericardium, valve, blood vessel, jugular vein, skin, small intestinal submucosa, meninges, bladder lining, ligament, tendon and swim bladder.
The preparation method according to claim 1 or 2, wherein the protective agent in the protective agent solution includes one or more of neomycin sulfate, tannic acid, quercetin, curcumin and polyethylene glycol;

The concentration of the protectant in the protectant solution is about 0.1 mM to about 1 mM.
The preparation method according to any one of claims 1 to 3, wherein the method used for the decellularization treatment comprises one or more of a physical method, a chemical method and a biological method;

The physical method includes one or more of ultrasound, mechanical stirring, perfusion, pressure treatment, supercritical fluid, repeated freezing and thawing, and osmotic pressure;

The chemical method is to use chemical reagents for decellularization, and the chemical reagents include one or more of chaotropic agents, detergents, acids, bases, chelating agents, protease inhibitors, tributyl phosphate and antibiotics;

The biological method is to use enzyme to carry out decellularization treatment.
The preparation method according to claim 4, wherein the chemical reagent satisfies at least one of the following characteristics:

(1) The chaotropic agent comprises one or both of urea and thiourea;

(2) The detergent includes one or more of an ionic detergent, a nonionic detergent and a zwitterionic detergent;

(3) The acid includes one or more of acetic acid, peracetic acid, hydrochloric acid and sulfuric acid;

(4) The base includes one or more of ammonium hydroxide, sodium hydroxide and ammonia water;

(5) The chelating agent includes one or both of ethylenediaminetetraacetic acid and ethylene glycol bis(2-aminoethyl ether)tetraacetic acid.
The preparation method according to claim 5, wherein the detergent satisfies at least one of the following characteristics:

(1) The ionic detergent includes one or more of sodium dodecyl sulfate, sodium deoxycholate, sodium cholate, sarcosine, sodium dodecyl polyoxyethylene ether sulfate and glucose lipid quaternary ammonium salt;

(2) the nonionic detergent comprises one or more of Triton, Tween, glycosyl lithocholic acid ester, glycoenzyme, digitonin, glucosides, alkyl glycosides and cocoyl monoethanolamide;

(3) The zwitterionic detergents include 3-(3-(cholamidopropyl)dimethylamino)propanesulfonic acid inner salt, sulfobetaines, carboxylic acid betaines, oleyl sulfate ester type imidazoline, dodecylaminopropionic acid, amino acids and cocamide One or more of propylamine oxides.
The preparation method according to any one of claims 4 to 6, wherein the chemical reagent is a solvent containing a detergent, and the concentration of the detergent in the solvent is about 0.01 wt % to about 1 wt %.
The preparation method according to any one of claims 1 to 7, wherein the fixation cross-linking is carried out in a fixation solution containing a cross-linking agent, and the cross-linking agent includes one or both of a chemical cross-linking agent and a natural cross-linking agent;

The chemical cross-linking agent includes one or more of glutaraldehyde, formaldehyde, carbodiimide and polyepoxide;

The natural cross-linking agent includes one or two of genipin and proanthocyanidin.
The preparation method according to any one of claim 8, wherein the concentration of the crosslinking agent in the fixing solution is about 0.05 wt % to about 10 wt %.
The preparation method according to any one of claims 1 to 9, wherein the degreasing treatment method is solvent immersion; the solvent comprises one or more of dichloromethane, n-hexane, cyclohexane and petroleum ether.
The preparation method according to any one of claims 1 to 10, wherein after the fixation and cross-linking, it further comprises a step of end-capping with an end-capping agent solution.
The preparation method according to claim 11, wherein the capping agent in the capping agent solution comprises one or more of amino acids, aminoamides, alkylamides, saturated alkylamines, unsaturated alkylamines, aminoalcohols, polyetheramines, aminoalkyl acids, aminopolysaccharides, aminoalkyldiacids, amino surfactants and fatty diamines.
The preparation method according to claim 11 or 12, wherein the concentration of the capping agent in the capping agent solution is about 1 mM to about 50 mM.
A biological tissue material obtained by the preparation method according to any one of claims 1 to 13.
The use of the biological tissue material as claimed in claim 14 as an artificial valve or a biological patch or in the preparation of an artificial valve or a biological patch.