CN118105544A - Biological tissue material and preparation method and application thereof - Google Patents
Biological tissue material and preparation method and application thereof Download PDFInfo
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- CN118105544A CN118105544A CN202211520655.2A CN202211520655A CN118105544A CN 118105544 A CN118105544 A CN 118105544A CN 202211520655 A CN202211520655 A CN 202211520655A CN 118105544 A CN118105544 A CN 118105544A
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- tissue
- biological tissue
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- acid
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- 238000002360 preparation method Methods 0.000 title claims abstract description 12
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- 238000005238 degreasing Methods 0.000 claims abstract description 20
- 239000003223 protective agent Substances 0.000 claims abstract description 19
- 238000004132 cross linking Methods 0.000 claims abstract description 18
- 238000001035 drying Methods 0.000 claims abstract description 12
- 210000001519 tissue Anatomy 0.000 claims description 150
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- 238000000034 method Methods 0.000 claims description 51
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- -1 glucose ester quaternary ammonium salt Chemical class 0.000 claims description 23
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 21
- 239000003795 chemical substances by application Substances 0.000 claims description 18
- 239000003431 cross linking reagent Substances 0.000 claims description 17
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 claims description 15
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 15
- 239000002904 solvent Substances 0.000 claims description 15
- PGBHMTALBVVCIT-VCIWKGPPSA-N framycetin Chemical compound N[C@@H]1[C@@H](O)[C@H](O)[C@H](CN)O[C@@H]1O[C@H]1[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](N)C[C@@H](N)[C@@H]2O)O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CN)O2)N)O[C@@H]1CO PGBHMTALBVVCIT-VCIWKGPPSA-N 0.000 claims description 12
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Classifications
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3625—Vascular tissue, e.g. heart valves
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- A—HUMAN NECESSITIES
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L2430/20—Materials or treatment for tissue regeneration for reconstruction of the heart, e.g. heart valves
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
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- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
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- General Chemical & Material Sciences (AREA)
- Cardiology (AREA)
- Heart & Thoracic Surgery (AREA)
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- Materials For Medical Uses (AREA)
Abstract
The application relates to the technical field of biomedical materials, in particular to a biological tissue material and a preparation method and application thereof. The preparation method of the biological tissue material comprises the following steps: soaking biological tissue in a protective agent solution, and then performing cell removal treatment; and performing fixed cross-linking, drying and degreasing on the biological tissue subjected to the decellularization treatment. The biological tissue material prepared by the preparation method has low lipid content, excellent calcification resistance and long-term stable use in organisms.
Description
Technical Field
The application relates to the technical field of biomedical materials, in particular to a biological tissue material and a preparation method and application thereof.
Background
Biological materials of animal origin (e.g., animal pericardium, heart valves, etc.) are widely used clinically to make prosthetic biological valves, biological patches, etc. Because the animal-derived biological material has the characteristics of wide sources and wide application range, the market occupation rate of the animal-derived biological material is gradually improved, and the animal-derived biological material plays an important role in the medical instrument industry. However, the materials also face a lot of challenges, natural animal-derived biological tissues are soft and rich in a large amount of cells, biological factors and the like, and the requirements of biocompatibility, long-acting application and the like between the materials and the implanted materials are difficult to meet. Therefore, it is necessary to treat natural animal-derived biological tissues to improve biocompatibility and long-lasting properties thereof. However, the current method for treating animal-derived biological materials is not only vulnerable to damage to biological tissues, but also poor in calcification prevention capability, which results in high calcium content in biological tissues, and is difficult to solve the problem of poor long-term effectiveness.
Disclosure of Invention
Based on the above, it is necessary to provide a biological tissue material capable of reducing calcium content and improving anti-calcification performance, and a preparation method and application thereof.
In one aspect of the present application, there is provided a method for preparing a biological tissue material, comprising the steps of:
soaking biological tissue in a protective agent solution, and then performing cell removal treatment; and
And (3) performing fixed cross-linking, drying and degreasing on the biological tissue subjected to the decellularization treatment.
In one embodiment, the biological tissue comprises one or more of pericardium, valve, blood vessel, jugular vein, skin, small intestine submucosa, meninges, bladder intima, ligament, tendon, and swim bladder.
In one embodiment, the protective agent comprises one or more of neomycin sulfate, tannic acid, quercetin, curcumin, and polyethylene glycol;
The concentration of the protective agent is 0.1 mM-1 mM.
In one embodiment, the decellularized treatment is performed by a method comprising one or more of a physical method, a chemical method, and a biological method;
the physical method comprises one or more of ultrasonic treatment, mechanical stirring, perfusion, pressure treatment, supercritical fluid, repeated freezing and thawing and osmotic pressure;
The chemical method is that a chemical reagent is adopted for cell removal treatment, wherein the chemical reagent comprises one or more of chaotropic agent, detergent, acid, alkali, chelating agent, protease inhibitor, tributyl phosphate and antibiotics;
The biological method is to adopt enzyme to carry out cell removal treatment.
In one embodiment, the chemical agent meets at least one of the following characteristics:
(1) The chaotropic agent comprises one or two of urea and thiourea;
(2) The detergent comprises one or more of an ionic detergent, a nonionic detergent and a zwitterionic detergent;
(3) The acid comprises one or more of acetic acid, peracetic acid, hydrochloric acid and sulfuric acid;
(4) The alkali comprises one or more of ammonium hydroxide, sodium hydroxide and ammonia water;
(5) The chelating agent comprises one or two of ethylenediamine tetraacetic acid and ethylene glycol bis (2-aminoethyl ether) tetraacetic acid.
In one embodiment, the detergent satisfies at least one of the following characteristics:
(1) The ionic detergent comprises one or more of sodium dodecyl sulfate, sodium deoxycholate, sodium cholate, sarcosine, sodium dodecyl polyoxyethylene ether sulfate and glucose ester quaternary ammonium salt;
(2) The nonionic detergent comprises one or more of triton, tween, glycocholate, glycosyl ferment, digitonin, glucosides, alkyl glycosides and cocoyl monoethanolamide;
(3) The zwitterionic detergent comprises one or more of 3- (3- (cholamidopropyl) dimethylamino) propane sulfonic acid inner salt, sulfobetaines, carboxylic acid betaines, oleic sulfuric acid ester salt type imidazolines, dodecylaminopropionic acid, amino acids and cocamidopropylamine oxides.
In one embodiment, the chemical agent is a detergent, and the chemical agent is a solvent containing the detergent, wherein the concentration of the detergent in the solvent is 0.01wt% to 1wt%.
In one embodiment, the immobilization crosslinking is performed in an immobilization solution containing a crosslinking agent comprising a chemical crosslinking agent comprising one or more of glutaraldehyde, formaldehyde, carbodiimide, and polyepoxide, and/or a natural crosslinking agent comprising one or both of genipin and procyanidins.
In one embodiment, the concentration of the cross-linking agent in the fixative solution is 0.05wt% to 10wt%.
In one embodiment, the degreasing treatment method is solvent soaking; the solvent comprises one or more of dichloromethane, n-hexane, cyclohexane and petroleum ether.
In one embodiment, after the fixing cross-linking, the step of capping with a capping agent solution is further included.
In one embodiment, the capping agent in the capping agent solution comprises one or more of an amino acid, an amino amide, an alkyl amide, a saturated alkyl amine, an unsaturated alkyl amine, an amino alcohol, a polyether amine, an amino alkyl acid, an amino polysaccharide, an amino alkyl diacid, an amino surfactant, an alkyl diamine, and a fatty diamine.
In one aspect of the present application, there is also provided a biological tissue material prepared by the preparation method as described above.
In another aspect of the application there is further provided the use of a biological tissue material as described above as a prosthetic valve or biological patch or in the preparation of a prosthetic valve or biological patch.
The above method for preparing biological tissue material employs a multi-step treatment process to remove or reduce sites of possible calcification of biological tissue from various aspects and to improve physical properties and durability of biological tissue. Particularly, the method combines decellularization treatment and drying degreasing, is simple to operate, and can maximally remove lipid in biological tissues on the basis of avoiding damage to the biological tissues, improve the calcification resistance of the biological tissues, ensure the physical and mechanical properties of the biological tissues and prolong the service life of the biological tissues. And on the one hand, the degreasing treatment can reduce the lipid in the biological tissue, on the other hand, the degreasing treatment can dissolve the residual chemical reagent on the surface of the biological tissue, and the convenience and the safety of the storage and the transportation of the biological tissue materials are improved. Drying before degreasing can also reduce the requirements of biological tissue materials on preservation and transportation conditions, and further improve the calcification resistance of biological tissues.
In addition, before the decellularization treatment, the biological tissue is treated by adopting the protective agent, so that the protective effect on the cytoplasm matrix of the biological tissue can be achieved, the problem that the structure is damaged when the subsequent decellularization treatment and the fixed crosslinking treatment are carried out is avoided, and the original structure of the biological tissue material can be prevented from being damaged.
Drawings
FIG. 1 is a graph showing the calcium content of the bovine pericardium tissue treated in example 1 and comparative example 1 after 60 days of implantation in rats;
FIG. 2 shows the HE staining results of bovine pericardium tissue according to example 1 and comparative example 4.
Detailed Description
Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.
Accordingly, it is intended that the present invention cover such modifications and variations as fall within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention will be disclosed in or be apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention.
Terminology
Unless otherwise indicated or contradicted, terms or phrases used herein have the following meanings:
In this context, "preferred" is merely to describe embodiments or examples that are more effective, and it should be understood that they are not intended to limit the scope of the invention.
Herein, the terms "first," "second," "third," "fourth," and the like are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor as implying an importance or quantity of a technical feature being indicated. Moreover, the terms "first," "second," "third," "fourth," and the like are used for non-exhaustive list description purposes only, and are not to be construed as limiting the number of closed forms.
Reference herein to "plurality" is to be equal in number to 2 or greater than 2.
In this context, the technical features described in open form include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.
The temperature parameter herein is not particularly limited, and may be a constant temperature process or may vary within a certain temperature range. It should be appreciated that the constant temperature process described allows the temperature to fluctuate within the accuracy of the instrument control. For example, it is allowed to fluctuate within a range such as + -5 ℃, + -4 ℃, + -3 ℃, + -2 ℃, + -1 ℃.
When the traditional method for treating the biological tissue material is used for cell removal treatment, the cytoplasmatic matrix is damaged, so that the mechanical property of the biological tissue material is reduced, calcified substances in the biological tissue material cannot be effectively removed, and long-term stable use in vivo is difficult to meet. To this end, the present application provides a method for preparing a biological tissue material, which employs a multi-step treatment process to remove or reduce sites of possible calcification of biological tissue from various aspects and to improve physical properties and durability of biological tissue. Particularly, the method combines decellularization treatment and drying degreasing, is simple to operate, and can maximally remove lipid in biological tissues on the basis of avoiding damage to the biological tissues, improve the calcification resistance of the biological tissues, ensure the physical and mechanical properties of the biological tissues and prolong the service life of the biological tissues. And on the one hand, the degreasing treatment can reduce the lipid in the biological tissue, on the other hand, the degreasing treatment can dissolve the residual chemical reagent on the surface of the biological tissue, and the convenience and the safety of the storage and the transportation of the biological tissue materials are improved. Drying before degreasing can also reduce the requirements of biological tissue materials on preservation and transportation conditions, and further improve the calcification resistance of biological tissues.
In addition, before the decellularization treatment, the biological tissue is treated by adopting the protective agent, so that the protective effect on the cytoplasm matrix of the biological tissue can be achieved, the problem that the structure is damaged when the subsequent decellularization treatment and the fixed crosslinking treatment are carried out is avoided, and the original structure of the biological tissue material can be prevented from being damaged.
The first object of the present application is to provide a method for preparing a biological tissue material, which includes steps S100, S200, S300 and S400:
Step S100: the biological tissue is washed. It will be appreciated that in some embodiments, step S100 may be omitted. By this step, unnecessary tissues such as fat, coarse blood vessels, etc. in the biological tissues can be removed.
In some embodiments, the biological tissue refers to a non-human origin, e.g., the biological tissue may be a xenogeneic biological tissue.
Alternatively, the biological tissue may include one or more of pericardium (porcine pericardium, bovine pericardium, etc.), valve (aortic valve, mitral valve, tricuspid valve), blood vessel, jugular vein, skin, small intestinal submucosa, meninges, intravesical membrane, ligament, tendon, and swim bladder. By way of example, the swim bladder may be taken from carp, grass carp, crucian carp, silver carp, bighead carp, bream, yellow croaker, sturgeon, spanish, or stone-head fish.
Step S200: and (3) immersing the biological tissue treated in the step (S100) in a protective agent solution, and then performing cell removal treatment.
In some embodiments, the protectant in the protectant solution refers to an agent that may protect the cytoplasmic matrix in biological tissue, and illustratively, the protectant may include, but is not limited to, one or more of neomycin sulfate, tannic acid, quercetin, curcumin, and polyethylene glycol.
Further, the concentration of the protecting agent in the protecting agent solution is 0.1 mM-1 mM.
In some embodiments, the time and temperature of the soaking are not limited, so long as the protective agent can be soaked on the biological tissue, for example, the soaking time can be 1h to 36h, and the temperature can be room temperature, namely 18 ℃ to 30 ℃. It will be appreciated that the protective agent may also be applied in an oscillating manner during the soaking process to allow adequate impregnation of the biological tissue.
In some embodiments, the methods employed for decellularization treatment can be selected from methods commonly used in the art, including but not limited to one or more of physical methods, chemical methods, and biological methods, to reduce or remove cellular components, antigenic activity, immunogenicity, and the like from biological tissues. Wherein the physical means may include one or more of ultrasound, mechanical agitation, perfusion, pressure treatment, supercritical fluid, repeated freeze thawing (freeze/thaw cycles), and osmotic pressure (hypotonic/hypertonic alternation); the chemical method is to adopt chemical reagent to carry out decellularization treatment, wherein the chemical reagent can comprise one or more of chaotropic agent, detergent, acid, alkali, chelating agent, protease inhibitor, tributyl phosphate and antibiotics; the biological method is to adopt enzyme to carry out cell removal treatment. Several methods may be generally selected and combined, such as a combination of physical methods and chemical methods, or a combination of physical methods, chemical methods and biological methods. Any combination can be made without departing from the spirit of the application, and the application is within the scope of protection.
In some embodiments, physical methods employed for decellularization treatment, such as sonication, mechanical agitation, repeated freeze thawing and osmotic pressure, and chemical methods, are desirably performed in solution, which may include one or more of phosphate buffer, tris-HCl buffer, hepes buffer, ethanol, water, and physiological saline.
In some embodiments, the chaotropic agent can include one or more of urea and thiourea; the detergent mainly comprises one or more of an ionic detergent, a nonionic detergent and a zwitterionic detergent; wherein the ionic detergent can comprise one or more of sodium dodecyl sulfate, sodium deoxycholate, sodium cholate, sarcosine, sodium dodecyl polyoxyethylene ether sulfate and glucose ester quaternary ammonium salt; the nonionic detergent may include one or more of triton (TritonX-100), tween, glycocholate amphipathic molecules (such as GLC-1, GLC-2 and GLC-3), sugar ferment (GDN), digitonin, glucosides, alkyl glycosides and cocoyl monoethanolamide; zwitterionic detergents may include one or more of 3- (3- (cholamidopropyl) dimethylamino) propane sulfonic acid inner salt (CHAPS), sulfobetaines, carboxybetaines, oleate sulfate salt imidazolines, dodecylaminopropionic acid, amino acids, and cocamidopropylamine oxide; the acid may include one or more of acetic acid, peroxyacetic acid, hydrochloric acid, and sulfuric acid; the base may include one or more of ammonium hydroxide, sodium hydroxide, and aqueous ammonia; chelating agents may include ethylenediamine tetraacetic acid (EDTA) and ethylene glycol bis (2-aminoethylether) tetraacetic acid (EGTA).
In some embodiments, the enzyme may include one or more of trypsin, pepsin, nucleases (e.g., RNase a, DNase), dispase, lipase, and biase.
In some embodiments, the decellularization treatment can be performed with a solvent containing a detergent, wherein the temperature of the decellularization treatment can be 4-37 ℃ and the time can be 1-48 hours, and the concentration of the detergent in the solvent containing the detergent can be 0.01-1 wt%. Further, shaking may be performed during the decellularization process, for example, shaking by a shaker may be used to make the decellularization process more complete.
Step S300: the biological tissue after the decellularization treatment in step S200 is washed. Residual reagents in biological tissue can be removed by washing. It will be appreciated that in some embodiments, this step may be omitted.
Step S400: and (3) performing fixation cross-linking, drying and degreasing on the biological tissue treated in the step (S300).
In the present application, the manner of the immobilization crosslinking is not limited, and immobilization crosslinking means commonly used in the art, such as ultraviolet crosslinking or crosslinking with a solution containing a crosslinking agent, may be selected. In some embodiments, the immobilization crosslinking is specifically performed in an immobilization solution containing a crosslinking agent, wherein the crosslinking agent may include a chemical crosslinking agent and/or a natural crosslinking agent; wherein the chemical crosslinking agent may include one or more of glutaraldehyde, formaldehyde, carbodiimide, and polyepoxide; the natural cross-linking agent may include one or both of genipin and procyanidins.
In some embodiments, the concentration of the cross-linking agent in the fixation solution may be 0.05wt% to 10wt%.
In some embodiments, after the fixing crosslinking, a step of capping with a capping agent solution may be further included; the calcification resistance and stability of the biological tissue material can be improved by the end capping treatment. In particular, when an aldehyde cross-linking agent is used, the aldehyde group can be blocked and reduced by a blocking agent, so that the calcification resistance of the biological tissue material is improved. Wherein, the end capping means that the reagent containing primary amino reacts with free aldehyde groups on biological tissue materials to generate Schiff base, thereby playing a role in closing the aldehyde groups; reduction means that the Schiff base can be reduced by adopting a reducing agent, and the effect of stabilizing chemical bonds is achieved. The reducing agent is not limited, and reducing agents commonly used in the art may be used, and for example, the reducing agent may 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 may include one or more of an amino acid, an amino amide, an alkyl amide, a saturated alkyl amine, an unsaturated alkyl amine, an amino alcohol, a polyether amine, an amino alkyl acid, an amino polysaccharide, an amino alkyl diacid, an amino surfactant, an alkyl diamine, and a fatty diamine.
In some embodiments, the concentration of the capping agent in the capping agent solution may be 1mM to 50mM.
In some embodiments, the drying mode is not limited, and any drying method commonly used in the art can be selected, for example, freeze drying can be adopted, wherein the pressure of freeze drying is less than 100Pa, the temperature can be between-60 ℃ and 25 ℃, the drying time can be between 2 hours and 48 hours, and the freeze drying time can be between 1 and 10 times.
In some embodiments, the degreasing treatment method may specifically be solvent soaking; alternatively, the solvent is selected mainly from low toxicity, nonpolar and volatile solvents, and exemplary solvents may include one or more of dichloromethane, n-hexane, cyclohexane and petroleum ether. Wherein, the soaking time can be 12-48 h, the temperature is 15-37 ℃, and the soaking times can be 1-20 times.
It will be appreciated that the biological tissue may also be subjected to a sealing and/or sterilization step after degreasing, wherein the sterilization process is selected from the processes commonly used in the art, and may be Ethylene Oxide (EO) sterilization, for example.
In some embodiments, the method of preparing a biological tissue material may specifically include the steps of:
Soaking biological tissue in a protective agent solution, and then performing cell removal treatment;
Placing the biological tissue subjected to cell removal treatment into a fixing solution for fixing and crosslinking;
Drying the biological tissue after the fixation and crosslinking; and
Immersing the dried biological tissue in a solvent for degreasing.
The second object of the present application is also to provide a biological tissue material prepared by the preparation method as described above.
The third object of the present application is further to provide the use of a biological tissue material as described above as a prosthetic valve or biological patch or in the preparation of a prosthetic valve or biological patch.
The present application will be described in further detail with reference to specific examples.
Example 1
1) The cleaned bovine pericardium tissue was placed in a neomycin sulfate solution at 1mM concentration in PBS buffer at room temperature and shaken for 2h. Subsequently, the bovine pericardial tissue is soaked in hypotonic PBS buffer (the concentration is 0.1M and the pH is 7.4), and then 0.5wt% Triton X-100, 20 mu g/mL RNase A and 0.2mg/mL DNase are added to be mixed and vibrated for 24 hours, and the bovine pericardial tissue is taken out and washed 8 times by using sterile physiological saline;
2) An aqueous glutaraldehyde solution of 0.625wt% and pH7.4 was prepared as a fixation solution, and bovine pericardial tissue washed in step 1) was placed in the fixation solution for fixation for 24 hours. Subsequently, the bovine pericardium tissue was washed 3 times in PBS buffer at a concentration of 0.1M and a pH of 7.4 for 5min each time;
3) Preparing 1L of PBS buffer solution (with the concentration of 0.1M and the pH of 7.4) containing 10mM lysine, soaking the bovine pericardium tissue washed in the step 2) in the PBS buffer solution, placing the soaked bovine pericardium tissue on a constant temperature shaking table, vibrating the soaked bovine pericardium tissue at a constant temperature of 25 ℃ and at a constant speed of 90rpm for 48 hours, and then washing the bovine pericardium tissue with a sterile physiological saline solution for 30 minutes;
4) Lyophilizing the cleaned bovine pericardium tissue in step 3) at-20deg.C under 100Pa for 4 hr, and then heating to 20deg.C for analysis to obtain dry bovine pericardium tissue. Soaking the dried bovine pericardium tissue in cyclohexane at room temperature, standing for 12h each time, repeating for 6 times, taking out the bovine pericardium tissue, standing in a fume hood for 48h to volatilize the cyclohexane, and obtaining the treated bovine pericardium tissue. The bovine pericardial tissue may then be sealed and EO sterilized.
Example 2
1) The cleaned bovine pericardium tissue was placed into a quercetin solution with a concentration of 1mM in Tris buffer at room temperature and shaken for 1h. Subsequently, the bovine pericardium tissue is soaked in hypotonic PBS buffer (the concentration is 0.1M, the pH is 7.4), then 0.5 weight percent of sodium deoxycholate and 2 weight percent of Sodium Dodecyl Sulfate (SDS) are added to be mixed and oscillated for 12 hours, the mixture is taken out, then 0.5 weight percent of trypsin is used for processing for 2 hours, and the bovine pericardium tissue is washed 8 times by using sterile physiological saline;
2) The bovine pericardial tissue obtained in step 1) was placed in carbodiimide (EDC) and N-hydroxysuccinimide (NHS) (concentration 15mM:15 mM) was immersed in the mixed solution for 24 hours. Then 0.625wt% glutaraldehyde solution with pH of 7.4 is prepared as a fixing solution, and the bovine pericardium tissue soaked in the step 1) is placed into the fixing solution to be fixed for 24 hours. Subsequently, the bovine pericardium tissue was washed 3 times in PBS buffer at a concentration of 0.1M and a pH of 7.4 for 5min each time;
3) Lyophilizing the cleaned bovine pericardium tissue in step 2) at-20deg.C under 100Pa for 4 hr, and then heating to 20deg.C for analysis to obtain dry bovine pericardium tissue. Soaking the dried bovine pericardium tissue in dichloromethane at room temperature, standing for 12h each time, repeating for 6 times, taking out the bovine pericardium tissue, standing in a fume hood for 48h to volatilize the dichloromethane, and obtaining the treated bovine pericardium tissue. The bovine pericardial tissue may then be sealed and EO sterilized.
Example 3
1) The cleaned bovine pericardium tissue was placed in a curcumin solution at 1mM concentration in PBS buffer at room temperature and shaken for 1h. Subsequently, the bovine pericardium tissue is soaked in hypotonic PBS buffer (the concentration is 0.1M and the pH is 8), 1wt percent of alkyl glycoside, 20 mu g/mL of RNase A and 0.2mg/mL of DNase are added, mixed and vibrated for 48 hours, taken out, and the bovine pericardium tissue is washed 8 times by using sterile physiological saline;
2) Preparing 0.625wt% glutaraldehyde solution with pH of 7.4 as a fixing solution, and fixing the bovine pericardium tissue washed in the step 1) in the fixing solution for 4 hours, and then fixing in 5wt% procyanidine solution (PBS buffer solution) for 72 hours. Subsequently, the bovine pericardium tissue was washed 3 times in PBS buffer at a concentration of 0.1M and a pH of 7.4 for 5min each time;
3) Preparing 1L of PBS buffer solution (with the concentration of 0.1M and the pH of 7.4) containing 10mM lysine, soaking the bovine pericardium tissue washed in the step 2) in the PBS buffer solution, placing the soaked bovine pericardium tissue on a constant temperature shaking table, vibrating the soaked bovine pericardium tissue at a constant temperature of 25 ℃ and at a constant speed of 90rpm for 48 hours, and then washing the bovine pericardium tissue with a sterile physiological saline solution for 30 minutes;
4) Lyophilizing the cleaned bovine pericardium tissue in step 3) at-40deg.C under 60Pa for 2 hr, and then heating to 10deg.C for analysis to obtain dry bovine pericardium tissue. Soaking the dried bovine pericardium tissue in n-hexane at room temperature, standing for 12h each time, repeating for 6 times, taking out the bovine pericardium tissue, standing in a fume hood for 48h to volatilize n-hexane, and obtaining the treated bovine pericardium tissue. The bovine pericardial tissue may then be sealed and EO sterilized.
Example 4
The present embodiment is basically the same as the processing method of embodiment 1, except that: the biological tissue material is a porcine aortic valve, and comprises the following specific steps:
1) The cleaned aortic valve tissue of the pig was placed in a neomycin sulfate solution in PBS buffer at a concentration of 5mM at room temperature and shaken for 2h. Subsequently, the tissue was immersed in hypotonic PBS buffer (concentration: 0.1M, pH: 7.4), and 1wt% Triton X-100, 20. Mu.g/mL RNase A and 0.2mg/mL DNase were added thereto, mixed and shaken for 24 hours, taken out, and washed 8 times with sterile physiological saline;
2) A glutaraldehyde solution of 0.625wt% and pH 7.4 was prepared as a fixation solution, and the porcine aortic valve tissue washed in step 1) was placed in the fixation solution for fixation for 24 hours. Subsequently, the tissue was washed 3 times in PBS buffer at a concentration of 0.1M and pH 7.4 for 5min each;
3) Preparing 1L of PBS buffer solution (with the concentration of 0.1M and the pH of 7.4) containing 10mM lysine, soaking the tissue washed in the step 2) in the PBS buffer solution, placing the tissue on a constant temperature shaking table, oscillating the tissue at the constant temperature of 37 ℃ and 90rpm for 24 hours, and then washing the tissue with sterile physiological saline solution for 30 minutes;
4) Lyophilizing the tissue washed in step 3) at-20deg.C under 100Pa for 4 hr, and then heating to 20deg.C for analysis to obtain dry porcine aortic tissue. Soaking the dried tissue in cyclohexane at room temperature, standing for 12h each time, repeating for 3 times, taking out the tissue, standing in a fume hood for 48h to volatilize the cyclohexane, and obtaining the treated pig aorta tissue. The porcine aortic tissue may then be sealed and EO sterilized.
Example 5
The present embodiment is basically the same as the processing method of embodiment 1, except that: the biological tissue material is jugular vein, and the specific steps are as follows:
1) The cleaned jugular vein tissue was placed in a neomycin sulfate solution at a concentration of 3mM in PBS buffer at room temperature and shaken for 12h. Subsequently, the tissue was immersed in hypotonic PBS buffer (concentration: 0.1M, pH: 7.4), 3wt% Triton X-100, 20. Mu.g/mL RNase A and 0.2mg/mL DNase were added thereto, mixed and shaken for 24 hours, taken out, and washed 8 times with sterile physiological saline;
2) A glutaraldehyde solution was prepared as a fixation solution at 0.625wt% and pH 7.4, and the jugular vein tissue washed in step 1) was placed in the fixation solution for fixation for 24 hours. Subsequently, the tissue was washed 3 times in PBS buffer at a concentration of 0.1M and pH 7.4 for 5min each;
3) Preparing 1L of PBS buffer solution (with the concentration of 0.1M and the pH of 7.4) containing 10mM lysine, soaking the tissue washed in the step 2) in the PBS buffer solution, placing the tissue on a constant temperature shaking table, oscillating the tissue at the constant temperature of 25 ℃ and at the speed of 90rpm for 24 hours, and washing the tissue for 30 minutes by using a sterile physiological saline solution;
4) Lyophilizing the tissue washed in step 3) at-20deg.C under 100Pa for 4 hr, and then heating to 20deg.C for analysis to obtain dry jugular vein tissue. Soaking the dried tissue in cyclohexane at room temperature, standing for 12h each time, repeating for 3 times, taking out the tissue, standing in a fume hood for 48h to volatilize the cyclohexane, and obtaining the treated jugular vein tissue. The jugular vein tissue may then be sealed and EO sterilized.
Example 6
The present embodiment is basically the same as the processing method of embodiment 1, except that: the decellularization treatment technology is different, and comprises the following specific steps:
1) The cleaned bovine pericardium tissue was placed in a neomycin sulfate solution at 1mM concentration in PBS buffer at room temperature and shaken for 2h. Freezing bovine pericardial tissue at-20deg.C for 4 hr, thawing at room temperature for 4 hr, circulating for 3 times, adding 1wt% CHAPS, mixing and shaking for 24 hr, taking out, ultrasonic washing with physiological saline after ultrasonic treatment with trypsin solution (PBS buffer, pH=7.4, 1 mM) for 2 hr, ultrasonic washing with EDTA solution (Tris buffer, pH=7.4, 1 mM) for 2 hr, and washing bovine pericardial tissue with sterile physiological saline for 8 times;
2) A glutaraldehyde solution of 0.625wt% and pH 7.4 was prepared as a fixation solution, and bovine pericardial tissue washed in step 1) was placed in the fixation solution for fixation for 24 hours. Subsequently, the bovine pericardium tissue was washed 3 times in PBS buffer at a concentration of 0.1M and a pH of 7.4 for 5min each time;
3) Preparing 1L of PBS buffer solution (with the concentration of 0.1M and the pH of 7.4) containing 10mM lysine, soaking the bovine pericardium tissue washed in the step 2) in the PBS buffer solution, placing the soaked bovine pericardium tissue on a constant temperature shaking table, vibrating the soaked bovine pericardium tissue at a constant temperature of 25 ℃ and at a constant speed of 90rpm for 48 hours, and then washing the bovine pericardium tissue with a sterile physiological saline solution for 30 minutes;
4) Lyophilizing the cleaned bovine pericardium tissue in step 3) at-20deg.C under 100Pa for 4 hr, and then heating to 20deg.C for analysis to obtain dry bovine pericardium tissue. Soaking the dried bovine pericardium tissue in cyclohexane at room temperature, standing for 12h each time, repeating for 6 times, taking out the bovine pericardium tissue, standing in a fume hood for 48h to volatilize the cyclohexane, and obtaining the treated bovine pericardium tissue. The bovine pericardial tissue may then be sealed and EO sterilized.
Example 7
The present embodiment is basically the same as the processing method of embodiment 1, except that: the decellularization treatment technology is different, and comprises the following specific steps:
1) The cleaned bovine pericardium tissue was placed in a 1mM neomycin sulfate solution at room temperature and shaken for 2 hours. Then, the bovine pericardium tissue is soaked in hypotonic Tris solution, 1wt% tributyl phosphate and 0.5wt% sodium cholate are added, and the mixture is vibrated for 24 hours, washed and soaked in hypertonic PBS solution, 1wt% glucoside is added, and the mixture is vibrated for 24 hours. Washing bovine pericardium tissue 8 times by using sterile physiological saline after the reaction is completed;
2) A glutaraldehyde solution of 0.625wt% and pH 7.4 was prepared as a fixation solution, and bovine pericardial tissue washed in step 1) was placed in the fixation solution for fixation for 24 hours. Subsequently, the bovine pericardium tissue was washed 3 times in PBS buffer at a concentration of 0.1M and a pH of 7.4 for 5min each time;
3) Preparing 1L of PBS buffer solution (with the concentration of 0.1M and the pH of 7.4) containing 10mM lysine, soaking the bovine pericardium tissue washed in the step 2) in the PBS buffer solution, placing the soaked bovine pericardium tissue on a constant temperature shaking table, vibrating the soaked bovine pericardium tissue at a constant temperature of 25 ℃ and at a constant speed of 90rpm for 48 hours, and then washing the bovine pericardium tissue with a sterile physiological saline solution for 30 minutes;
3) Lyophilizing the cleaned bovine pericardium tissue in step 2) at-20deg.C under 100Pa for 4 hr, and then heating to 20deg.C for analysis to obtain dry bovine pericardium tissue. Soaking the dried bovine pericardium tissue in cyclohexane at room temperature, standing for 12h each time, repeating for 6 times, taking out the bovine pericardium tissue, standing in a fume hood for 48h to volatilize the cyclohexane, and obtaining the treated bovine pericardium tissue. The bovine pericardial tissue may then be sealed and EO sterilized.
Comparative example 1
The bovine pericardial tissue was treated in this comparative example without cell removal and degreasing. The method comprises the following specific steps:
1) A glutaraldehyde solution was prepared as a fixation solution at 0.625wt% and pH7.4, and the cleaned bovine pericardium tissue was placed in the fixation solution at room temperature and fixed for 24 hours. Subsequently, the bovine pericardium tissue is soaked in PBS buffer with the concentration of 0.1M and the pH of 7.4 for 3 times of washing for 5min each time;
2) 1L of PBS buffer containing 10mM lysine (concentration: 0.1M, pH: 7.4) was prepared, and the bovine pericardium tissue washed in step 1) was immersed in the above PBS buffer and placed on a constant temperature shaking table, and subjected to constant temperature shaking at 25℃and 90rpm for 48 hours, and then washed with a sterile physiological saline solution for 30 minutes, followed by storage in glutaraldehyde solution.
Comparative example 2
This comparative example was substantially identical to the treatment of example 2, except that: no protective agent was added prior to the decellularization treatment.
1) Immersing the cleaned bovine pericardium tissue in hypotonic PBS buffer (with the concentration of 0.1M and the pH of 7.4) at room temperature, adding 0.5wt% of sodium deoxycholate and 2wt% of Sodium Dodecyl Sulfate (SDS), mixing and oscillating for 12 hours, taking out, treating for 2 hours by using 0.5wt% of trypsin, and cleaning the bovine pericardium tissue by using sterile normal saline for 8 times;
2) Bovine pericardial tissue was placed in carbodiimide (EDC) and N-hydroxysuccinimide (NHS) (concentration 15mM:15 mM) was immersed in the mixed solution for 24 hours. Then 0.625wt% glutaraldehyde solution with pH of 7.4 is prepared as a fixing solution, and the bovine pericardium tissue soaked in the step 1) is placed into the fixing solution to be fixed for 24 hours. Subsequently, the bovine pericardium tissue was washed 3 times in PBS buffer at a concentration of 0.1M and a pH of 7.4 for 5min each time;
3) Lyophilizing the cleaned bovine pericardium tissue in step 2) at-20deg.C under 100Pa for 4 hr, and then heating to 20deg.C for analysis to obtain dry bovine pericardium tissue. Soaking the dried bovine pericardium tissue in dichloromethane at room temperature, standing for 12h each time, repeating for 6 times, taking out the bovine pericardium tissue, standing in a fume hood for 48h to volatilize the dichloromethane, and obtaining the treated bovine pericardium tissue. The bovine pericardial tissue may then be sealed and EO sterilized.
Comparative example 3
This comparative example was substantially identical to the treatment of example 1, except that: no degreasing treatment was performed.
1) The cleaned bovine pericardium tissue was placed in a 1mM neomycin sulfate solution (neomycin sulfate in PBS buffer) at room temperature and shaken for 2 hours. Subsequently, the bovine pericardial tissue is soaked in hypotonic PBS buffer (the concentration is 0.1M and the pH is 7.4), and then 0.5wt% Triton X-100, 20 mu g/mL RNase A and 0.2mg/mL DNase are added to be mixed and vibrated for 24 hours, and the bovine pericardial tissue is taken out and washed 8 times by using sterile physiological saline;
2) An aqueous glutaraldehyde solution of 0.625wt% and pH7.4 was prepared as a fixation solution, and bovine pericardial tissue washed in step 1) was placed in the fixation solution for fixation for 24 hours. Subsequently, the bovine pericardium tissue was washed 3 times in PBS buffer at a concentration of 0.1M and a pH of 7.4 for 5min each time;
3) Preparing 1L of PBS buffer solution (with the concentration of 0.1M and the pH of 7.4) containing 10mM lysine, soaking the bovine pericardium tissue washed in the step 2) in the PBS buffer solution, placing the soaked bovine pericardium tissue on a constant temperature shaking table, vibrating the soaked bovine pericardium tissue at a constant temperature of 25 ℃ and at a constant speed of 90rpm for 48 hours, and then washing the bovine pericardium tissue with a sterile physiological saline solution for 30 minutes;
4) Lyophilizing the cleaned bovine pericardium tissue in step 3) at-20deg.C under 100Pa for 4 hr, and then heating to 20deg.C for analysis to obtain dry bovine pericardium tissue. The bovine pericardial tissue may then be sealed and EO sterilized.
Comparative example 4
This comparative example is essentially the same as example 1, except that crosslinking is performed prior to decellularization, and the specific steps are as follows:
1) The cleaned bovine pericardium was placed in a 1mM neomycin sulfate solution (neomycin sulfate in PBS buffer) and shaken for 2h. Subsequently, an aqueous glutaraldehyde solution of 0.625wt% and pH 7.4 was prepared as a fixation solution, and the cleaned bovine pericardium tissue was placed in the fixation solution at room temperature for fixation for 24 hours. Subsequently, the bovine pericardium tissue was washed 3 times in PBS buffer at a concentration of 0.1M and a pH of 7.4 for 5min each time;
2) Subsequently, the fixed and crosslinked bovine pericardium tissue in the step 1) is soaked in hypotonic PBS buffer (the concentration is 0.1M and the pH is 7.4), and then 0.5wt% Triton X-100, 20 mug/mL RNase A and 0.2mg/mL DNase are added to be mixed and vibrated for 24 hours, taken out, and the bovine pericardium tissue is washed 8 times by using sterile physiological saline;
3) Placing the cleaned bovine pericardium tissue in the step 2) into a PBS buffer solution (the concentration is 0.1M and the pH is 7.4) containing 10mM lysine, soaking the cleaned bovine pericardium tissue in the PBS buffer solution and placing the immersed bovine pericardium tissue on a constant-temperature shaking table, vibrating the immersed bovine pericardium tissue at a constant temperature of 25 ℃ and 90rpm for 48 hours, and cleaning the bovine pericardium tissue for 30 minutes by using a sterile physiological saline solution;
4) Lyophilizing the cleaned bovine pericardium tissue in step 3) at-20deg.C under 100Pa for 4 hr, and then heating to 20deg.C for analysis to obtain dry bovine pericardium tissue. Soaking the dried bovine pericardium tissue in cyclohexane at room temperature, standing for 12h each time, repeating for 6 times, taking out the bovine pericardium tissue, standing in a fume hood for 48h to volatilize the cyclohexane, and obtaining the treated bovine pericardium tissue. The bovine pericardial tissue may then be sealed and EO sterilized.
The mechanical properties of the bovine pericardial tissues treated in examples 1 to 7 and comparative examples 1 to 2 were evaluated as follows:
Rehydrating the processed bovine pericardium tissue for 5min, cutting the bovine pericardium tissue into rectangles of 5mm multiplied by 50mm, and testing the mechanical properties of the bovine pericardium tissue on a universal material testing machine. The test gauge length was set at 25mm and the tensile rate at 20mm/min, and tensile test was performed until the test specimen was broken, and the test results were recorded as shown in table 1.
TABLE 1 results of mechanical Properties of bovine pericardium tissue
As can be seen from the test results of the table, compared with comparative example 1, the treatment method used in the application does not affect the mechanical properties of biological tissues; compared with comparative example 2, the protective agent is used, and the decellularized tissue does not damage the mechanical properties of the tissue.
Calcification performance evaluation was performed on bovine pericardial tissues treated in example 1 and comparative examples 1 and 3, and the specific procedure was as follows:
the samples of example 1 and comparative examples 1 and 3 were cut into wafers having a diameter of 10mm, and each group of samples was washed with anhydrous physiological saline for 2 times, respectively, for use.
9 Wistar rats of good health and substantially uniform body type, 3 weeks old, were selected as subcutaneously implanted animal models (named respectively 1,2, 3, … …,8 and 9), two samples of different compositions were implanted in their back surgical areas after anesthesia, 6 replicates were implanted in each group, and sample laying was ensured. The subcutaneous implantation profile of rats is shown below:
TABLE 2 subcutaneous implantation schematic of rats
The implantation time was 60 days, after 60 days the bovine pericardium tissue was removed and dried at a constant temperature of 60 ℃ for 48h. The calcium content of bovine pericardial tissue was then measured using inductively coupled plasma spectroscopy and the test results are shown in figure 1. As can be seen from FIG. 1, the calcium content in the bovine pericardium tissue in example 1 was only 1.03. Mu.g/mg, the calcium content in the bovine pericardium tissue in comparative example 1 was as high as 19.94. Mu.g/mg, and the calcium content in the bovine pericardium tissue in comparative example 3 was 7.86. Mu.g/mg. As is clear from example 1 and comparative example 3, in the case where the remaining conditions are substantially identical, comparative example 3 is not subjected to degreasing treatment, and the calcium content in bovine pericarp tissue obtained by the treatment is higher than that of example 1, which means that the treatment method provided in comparative example 3 cannot effectively reduce the lipid content in biological tissue material, resulting in higher calcium content and poor calcification resistance. Therefore, the treatment method provided by the application can obviously remove or reduce the lipid content in the biological tissue material, reduce the calcium content in the biological tissue material and obviously improve the calcification resistance.
The bovine pericardial tissues obtained in example 1 and comparative example 4 were subjected to HE section staining, and the procedure was as follows: bovine pericardium tissues obtained in example 1 and comparative example 4 were respectively selected, embedded in paraffin blocks, the cross-sectional directions of the tissues were selected using a soft tissue slicer, 3 μm thick sheets were cut out, and stained with hematoxylin and eosin stain, and the staining results are shown in fig. 2; wherein a is a graph of the staining result of the HE section of bovine pericardium tissue obtained in example 1, and b is a graph of the staining result of the HE section of bovine pericardium tissue obtained in comparative example 4. As can be seen from FIG. 2, the bovine pericardium tissue obtained by the treatment process of example 1 can be decellularized, whereas the bovine pericardium tissue of comparative example 4 has no decellularized effect.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. The scope of the invention is therefore intended to be covered by the appended claims, and the description and drawings should be construed in view of the scope of the appended claims.
Claims (14)
1. A method for preparing a biological tissue material, comprising the steps of:
soaking biological tissue in a protective agent solution, and then performing cell removal treatment; and
And (3) performing fixed cross-linking, drying and degreasing on the biological tissue subjected to the decellularization treatment.
2. The method of claim 1, wherein the biological tissue comprises one or more of pericardium, valve, blood vessel, jugular vein, skin, small intestinal submucosa, meninges, bladder intima, ligament, tendon, and swim bladder.
3. The method for preparing a biological tissue material according to claim 1, wherein the protective agent in the protective agent solution comprises one or more of neomycin sulfate, tannic acid, quercetin, curcumin and polyethylene glycol;
The concentration of the protective agent in the protective agent solution is 0.1 mM-1 mM.
4. The method of claim 1, wherein the decellularized treatment is performed by one or more of physical, chemical, and biological methods;
the physical method comprises one or more of ultrasonic treatment, mechanical stirring, perfusion, pressure treatment, supercritical fluid, repeated freezing and thawing and osmotic pressure;
The chemical method is that a chemical reagent is adopted for cell removal treatment, wherein the chemical reagent comprises one or more of chaotropic agent, detergent, acid, alkali, chelating agent, protease inhibitor, tributyl phosphate and antibiotics;
The biological method is to adopt enzyme to carry out cell removal treatment.
5. The method of preparing biological tissue material according to claim 4, wherein the chemical agent satisfies at least one of the following characteristics:
(1) The chaotropic agent comprises one or two of urea and thiourea;
(2) The detergent comprises one or more of an ionic detergent, a nonionic detergent and a zwitterionic detergent;
(3) The acid comprises one or more of acetic acid, peracetic acid, hydrochloric acid and sulfuric acid;
(4) The alkali comprises one or more of ammonium hydroxide, sodium hydroxide and ammonia water;
(5) The chelating agent comprises one or two of ethylenediamine tetraacetic acid and ethylene glycol bis (2-aminoethyl ether) tetraacetic acid.
6. The method of preparing a biological tissue material according to claim 5, wherein the detergent satisfies at least one of the following characteristics:
(1) The ionic detergent comprises one or more of sodium dodecyl sulfate, sodium deoxycholate, sodium cholate, sarcosine, sodium dodecyl polyoxyethylene ether sulfate and glucose ester quaternary ammonium salt;
(2) The nonionic detergent comprises one or more of triton, tween, glycocholate, glycosyl ferment, digitonin, glucosides, alkyl glycosides and cocoyl monoethanolamide;
(3) The zwitterionic detergent comprises one or more of 3- (3- (cholamidopropyl) dimethylamino) propane sulfonic acid inner salt, sulfobetaines, carboxylic acid betaines, oleic sulfuric acid ester salt type imidazolines, dodecylaminopropionic acid, amino acids and cocamidopropylamine oxides.
7. The method for producing a biological tissue material according to claim 5, wherein the chemical agent is a solvent containing a detergent, and the concentration of the detergent in the solvent is 0.01wt% to 1wt%.
8. The method of claim 1, wherein the fixation cross-linking is performed in a fixation solution containing a cross-linking agent comprising a chemical cross-linking agent comprising one or more of glutaraldehyde, formaldehyde, carbodiimide, and polyepoxide, and/or a natural cross-linking agent comprising one or both of genipin and procyanidins.
9. The method of preparing a biological tissue material according to claim 8, wherein the concentration of the cross-linking agent in the fixing solution is 0.05wt% to 10wt%.
10. The method for preparing a biological tissue material according to any one of claims 1 to 9, wherein the degreasing treatment method is solvent soaking; the solvent comprises one or more of dichloromethane, n-hexane, cyclohexane and petroleum ether.
11. The method of any one of claims 1 to 9, further comprising the step of capping with a capping agent solution after the immobilized cross-links.
12. The method of claim 11, wherein the capping agent in the capping agent solution comprises one or more of an amino acid, an amino amide, an alkyl amide, a saturated alkyl amine, an unsaturated alkyl amine, an amino alcohol, a polyether amine, an amino alkyl acid, an amino polysaccharide, an amino alkyl diacid, an amino surfactant, and a fatty diamine.
13. A biological tissue material produced by the production method according to any one of claims 1 to 12.
14. Use of the biological tissue material according to claim 13 as a prosthetic valve or biological patch or in the preparation of a prosthetic valve or biological patch.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211520655.2A CN118105544A (en) | 2022-11-30 | 2022-11-30 | Biological tissue material and preparation method and application thereof |
| PCT/CN2023/119105 WO2024114050A1 (en) | 2022-11-30 | 2023-09-15 | Biological tissue material, preparation method therefor, and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN202211520655.2A CN118105544A (en) | 2022-11-30 | 2022-11-30 | Biological tissue material and preparation method and application thereof |
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| CN118105544A true CN118105544A (en) | 2024-05-31 |
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| CN202211520655.2A Pending CN118105544A (en) | 2022-11-30 | 2022-11-30 | Biological tissue material and preparation method and application thereof |
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| Country | Link |
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| CN (1) | CN118105544A (en) |
| WO (1) | WO2024114050A1 (en) |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE69912998T2 (en) * | 1999-09-22 | 2004-09-02 | Baxter International Inc., Deerfield | HEART VALVE AND METHOD FOR PRODUCING A BIOLOGICAL TISSUE |
| CN101224313B (en) * | 2008-02-04 | 2012-02-15 | 中国科学院上海硅酸盐研究所 | Quercetin crosslinking method for preparing artificial bioprosthesis heart valve materials |
| KR101075399B1 (en) * | 2008-12-31 | 2011-10-25 | 서울대학교산학협력단 | Production method of heterograft |
| CN104888274A (en) * | 2015-05-19 | 2015-09-09 | 暨南大学 | Acellular matrix with natural level of glycosaminoglycan and preparation and application thereof |
| CN109125810A (en) * | 2017-06-19 | 2019-01-04 | 上海微创心通医疗科技有限公司 | A kind of preparation method of biovalve |
| CA3138296A1 (en) * | 2019-05-22 | 2020-11-26 | Filippo Naso | Method for preventing the formation of calcified deposits and for inactivating xenoantigens in biological matrices |
-
2022
- 2022-11-30 CN CN202211520655.2A patent/CN118105544A/en active Pending
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| WO2024114050A1 (en) | 2024-06-06 |
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