WO2024114050A1 - Matériau de tissu biologique, son procédé de préparation et son utilisation - Google Patents

Matériau de tissu biologique, son procédé de préparation et son utilisation Download PDF

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WO2024114050A1
WO2024114050A1 PCT/CN2023/119105 CN2023119105W WO2024114050A1 WO 2024114050 A1 WO2024114050 A1 WO 2024114050A1 CN 2023119105 W CN2023119105 W CN 2023119105W WO 2024114050 A1 WO2024114050 A1 WO 2024114050A1
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preparation
tissue
solution
biological tissue
bovine pericardial
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PCT/CN2023/119105
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English (en)
Chinese (zh)
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裴歌
于世河
何海红
刘世红
陈国明
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上海微创心通医疗科技有限公司
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Publication of WO2024114050A1 publication Critical patent/WO2024114050A1/fr

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  • 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.
  • 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.
  • 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;
  • the biological tissue after decellularization is fixed, cross-linked, dried and defatted.
  • 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.
  • 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 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.
  • the chemical reagent satisfies at least one of the following characteristics:
  • the chaotropic agent comprises one or both of urea and thiourea
  • the detergent includes one or more of an ionic detergent, a nonionic detergent and a zwitterionic detergent;
  • the acid includes one or more of acetic acid, peracetic acid, hydrochloric acid and sulfuric acid;
  • the base includes one or more of ammonium hydroxide, sodium hydroxide and ammonia water;
  • the chelating agent includes one or both of ethylenediaminetetraacetic acid and ethylene glycol bis(2-aminoethyl ether)tetraacetic acid.
  • the detergent satisfies at least one of the following characteristics:
  • 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;
  • the nonionic detergent comprises one or more of Triton, Tween, glycosyl lithocholic acid ester, glycoenzyme, digitonin, glucosides, alkyl glycosides and cocoyl monoethanolamide;
  • 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.
  • 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 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.
  • the concentration of the crosslinking agent in the fixing solution is about 0.05 wt % to about 10 wt %.
  • the degreasing treatment method is solvent immersion; the solvent includes one or more of dichloromethane, n-hexane, cyclohexane and petroleum ether.
  • a step of using a capping agent solution to perform end-capping is also included.
  • 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.
  • the concentration of the capping agent in the capping agent solution is about 1 mM to about 50 mM.
  • a biological tissue material is provided which is prepared by the preparation method described in the first aspect.
  • 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.
  • plurality refers to a number that is equal to or greater than 2.
  • 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.
  • the present application provides a method for preparing biological tissue materials to improve the above problems.
  • a method for preparing a biological tissue material 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.
  • 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.
  • 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.
  • 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.
  • step S100 may be omitted.
  • the biological tissue is of non-human origin, for example, the biological tissue may be xenogeneic biological tissue.
  • the biological tissue may include pericardium (porcine pericardium, bovine pericardium, etc.), valves (aortic valve, mitral valve,
  • pericardium pericardium
  • bovine 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.
  • 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.
  • the protective agent in the protective agent solution refers to an agent that can protect the cytoplasmic matrix in biological tissues.
  • the protective agent can include but is not limited to 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, 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.
  • the soaking time and temperature are not limited, as long as the protective agent can be immersed in the biological tissue.
  • 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.
  • an oscillation method can be used to allow the protective agent to fully immerse the biological tissue.
  • 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.
  • the method used for decellularization includes, but is not limited to, one or more of physical methods, chemical methods, and biological methods.
  • 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.
  • 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.
  • the chaotropic agent includes one or more of urea and thiourea.
  • 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
  • the acid includes one or more of acetic acid, peracetic acid, hydrochloric acid, and sulfuric acid.
  • the base is mainly an inorganic base.
  • the base includes one or more of ammonium hydroxide, sodium hydroxide and ammonia water.
  • the chelating agents include ethylenediaminetetraacetic acid (EDTA) and ethylene glycol bis(2-aminoethyl ether)tetraacetic acid (EGTA).
  • EDTA ethylenediaminetetraacetic acid
  • EGTA ethylene glycol bis(2-aminoethyl ether)tetraacetic acid
  • the enzyme includes one or more of trypsin, pepsin, nuclease (such as RNase A, DNase), dispase, lipase and disaccharidase.
  • a solvent containing a detergent is used for decellularization.
  • the temperature and time of the decellularization treatment are not limited.
  • 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.
  • 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%.
  • shaking can be performed during the decellularization process.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 %.
  • 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.
  • the aldehyde group can be capped and reduced by the end-capping agent, thereby improving the anti-calcification performance of the biological tissue material.
  • 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.
  • the choice of reducing agent is not limited, and a reducing agent commonly used in the art can be used.
  • the reducing agent can include one or more of sodium borohydride, sodium cyanoborohydride, potassium borohydride and ammonium formate.
  • 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.
  • 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.
  • 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
  • the number of freeze drying times can be 1 to 10 times.
  • the degreasing treatment method is solvent immersion.
  • the choice of solvent is not limited, and a low-toxic, non-polar and volatile solvent can be selected.
  • the solvent may include one or more of dichloromethane, n-hexane, cyclohexane and petroleum ether.
  • the immersion time can be about 12h to about 48h
  • the temperature is about 15°C to about 37°C
  • the number of immersions can be 1 to 20 times.
  • 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.
  • EO ethylene oxide
  • a method for preparing a biological tissue material comprises the following steps:
  • a biological tissue material is provided which is prepared by the preparation method described in the first aspect.
  • a method for treating heart valve disease or repairing valve defects 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.
  • step 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;
  • step 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;
  • 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.
  • step 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.
  • NHS N-hydroxysuccinimide
  • 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.
  • step 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;
  • step 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;
  • 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.
  • 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:
  • step 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;
  • step 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;
  • step 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.
  • 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:
  • step 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;
  • step 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;
  • step 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.
  • 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:
  • step 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;
  • step 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;
  • 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.
  • 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:
  • step 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;
  • step 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;
  • 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.
  • the bovine pericardial tissue was not subjected to decellularization and degreasing.
  • the specific steps are as follows:
  • step 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.
  • PBS buffer concentration of 0.1 M, pH 7.4
  • sterile saline solution to wash the bovine pericardial tissue for 30 minutes, and then store it in glutaraldehyde solution.
  • 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.
  • 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.
  • the treatment method of this comparative example is basically the same as that of Example 1, except that no degreasing treatment is performed.
  • step 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;
  • step 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;
  • step 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.
  • 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:
  • step 2) 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, 20 ⁇ g/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;
  • a hypotonic PBS buffer concentration of 0.1 M, pH 7.4
  • step 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;
  • PBS buffer concentration of 0.1 M, pH 7.4
  • 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.
  • 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.
  • 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.
  • 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 1.03 ⁇ g/mg, the calcium content in the bovine pericardial tissue in Comparative Example 1 is as high as 19.94 ⁇ g/mg, and the calcium content in the bovine pericardial tissue in Comparative Example 3 is 7.86 ⁇ g/mg.
  • Example 1 and Comparative Example 3 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 ⁇ m 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
  • Figure 3b is a HE staining result of the bovine pericardial tissue obtained in Comparative Example 4.
  • 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.

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Abstract

La présente invention concerne un matériau de tissu biologique et son procédé de préparation. Le procédé comprend les étapes suivantes consistant à : tremper un tissu biologique dans une solution d'agent protecteur puis décellulariser le tissu biologique ; et soumettre le tissu biologique décellularisé à une fixation, une réticulation, un séchage et un dégraissage. Le procédé peut éliminer efficacement des substances calcifiées issues de matériaux de tissu biologique, garantissant la stabilité à long terme des matériaux de tissu biologique pour une utilisation in vivo.
PCT/CN2023/119105 2022-11-30 2023-09-15 Matériau de tissu biologique, son procédé de préparation et son utilisation WO2024114050A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001021228A1 (fr) * 1999-09-22 2001-03-29 Baxter International Inc. Valvule cardiaque et procede de preparation d'un tissu biologique
CN101224313A (zh) * 2008-02-04 2008-07-23 中国科学院上海硅酸盐研究所 采用槲皮素交联制备人工生物心脏瓣膜材料的方法
KR20100079908A (ko) * 2008-12-31 2010-07-08 서울대학교산학협력단 이종이식 보철편의 제조방법
CN104888274A (zh) * 2015-05-19 2015-09-09 暨南大学 一种具有天然水平糖胺聚糖的脱细胞基质及其制备与应用
CN109125810A (zh) * 2017-06-19 2019-01-04 上海微创心通医疗科技有限公司 一种生物瓣膜的制备方法
CN113874052A (zh) * 2019-05-22 2021-12-31 生物相容性创新责任有限公司 用于在生物基质中防止钙化沉积物形成和使异种抗原失活的方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001021228A1 (fr) * 1999-09-22 2001-03-29 Baxter International Inc. Valvule cardiaque et procede de preparation d'un tissu biologique
CN101224313A (zh) * 2008-02-04 2008-07-23 中国科学院上海硅酸盐研究所 采用槲皮素交联制备人工生物心脏瓣膜材料的方法
KR20100079908A (ko) * 2008-12-31 2010-07-08 서울대학교산학협력단 이종이식 보철편의 제조방법
CN104888274A (zh) * 2015-05-19 2015-09-09 暨南大学 一种具有天然水平糖胺聚糖的脱细胞基质及其制备与应用
CN109125810A (zh) * 2017-06-19 2019-01-04 上海微创心通医疗科技有限公司 一种生物瓣膜的制备方法
CN113874052A (zh) * 2019-05-22 2021-12-31 生物相容性创新责任有限公司 用于在生物基质中防止钙化沉积物形成和使异种抗原失活的方法

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