CN118121768A - High-strength extracellular matrix gel and preparation method thereof - Google Patents
High-strength extracellular matrix gel and preparation method thereof Download PDFInfo
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- CN118121768A CN118121768A CN202410552335.8A CN202410552335A CN118121768A CN 118121768 A CN118121768 A CN 118121768A CN 202410552335 A CN202410552335 A CN 202410552335A CN 118121768 A CN118121768 A CN 118121768A
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- 102000010834 Extracellular Matrix Proteins Human genes 0.000 title claims abstract description 89
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 title claims abstract description 89
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- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 238000001879 gelation Methods 0.000 title description 3
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- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
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- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 2
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- ZYFVNVRFVHJEIU-UHFFFAOYSA-N PicoGreen Chemical compound CN(C)CCCN(CCCN(C)C)C1=CC(=CC2=[N+](C3=CC=CC=C3S2)C)C2=CC=CC=C2N1C1=CC=CC=C1 ZYFVNVRFVHJEIU-UHFFFAOYSA-N 0.000 description 1
- 102000016611 Proteoglycans Human genes 0.000 description 1
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- 230000002500 effect on skin Effects 0.000 description 1
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- 239000002245 particle Substances 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
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- FHHPUSMSKHSNKW-SMOYURAASA-M sodium deoxycholate Chemical compound [Na+].C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 FHHPUSMSKHSNKW-SMOYURAASA-M 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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
- A61L27/3633—Extracellular matrix [ECM]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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
- A61L27/14—Macromolecular materials
- A61L27/20—Polysaccharides
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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
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/225—Fibrin; Fibrinogen
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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
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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
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- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/24—Collagen
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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
- A61L27/3687—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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/52—Hydrogels or hydrocolloids
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Transplantation (AREA)
- Epidemiology (AREA)
- Dermatology (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Molecular Biology (AREA)
- Botany (AREA)
- Biophysics (AREA)
- Dispersion Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Zoology (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention provides a high-strength extracellular matrix gel and a preparation method thereof, belonging to the technical field of biological materials. The mass fraction of the extracellular matrix in the high-strength extracellular matrix gel is 1.5% -6.0%; the mass of the insoluble matrix in the extracellular matrix accounts for 52% -72% of the mass of the extracellular matrix; the extracellular matrix includes one or more of collagen, elastin, fibronectin, laminin, and glycosaminoglycans. The compressive fracture stress of the high-strength extracellular matrix gel is 72.1-661.6kPa; compression fracture strain was 69.3% -83.1%. The high-strength extracellular matrix gel DNA and the alpha-gal antigen provided by the invention have low residual quantity, can greatly reduce the immunogenicity of animal-derived materials, and improve the biological safety.
Description
Technical Field
The invention belongs to the technical field of biological materials, and particularly relates to a high-strength extracellular matrix gel and a preparation method thereof.
Background
The extracellular matrix is a combination of structural and functional molecules secreted by cells in tissues and organs, and contains collagen, glycoprotein, proteoglycan, glycosaminoglycan, growth factor, etc. as the main component. It constitutes the ecological microenvironment of the cell, providing cellular structural support, signaling, etc., affecting various behaviors and functions of the cell. The extracellular matrix plays a very important role in maintaining cell homeostasis, injury repair, tissue reconstruction, etc.
The preparation method of the extracellular matrix mainly removes substances such as cells capable of causing immune rejection reaction in animal tissues by a decellularization technology, and leaves an external matrix with biological activity, so that the materials are also called as decellularized matrix materials. Materials prepared by decellularization technology of dermal tissue and Small Intestine Submucosa (SIS) are clinically applied at present, and have good effects in the aspects of treating burns, hernia repair and the like. However, the shape, thickness, size, etc. of such decellularized matrix materials are limited by the native state of the tissue and their application is also limited. To expand the application of extracellular matrix, it is an effective method to transform extracellular matrix into gel state by some technical treatments.
As patent CN104971380a discloses a decellularized matrix repair gel and a new method for preparing the same, which prepares a decellularized matrix repair gel by performing decellularization treatment and gelation treatment on tissue and organs taken from mammals. The acellular matrix repair gel provided by the invention eliminates immunogenicity of heterogeneous and allogenic tissues, retains activity of extracellular matrix components of tissues to the greatest extent, can specifically repair damaged tissues and organs of human bodies, is high in applicability, is suitable for various irregularly-shaped repair areas and environmental requirements of different parts in the bodies, has great clinical value, and adopts the main process of gelatification treatment in the method of enzyme digestion.
Patent CN105169483a discloses a method for preparing a acellular matrix gel with various shapes and properties by using a acellular matrix solution obtained by processing natural tissues or organs by using a decellularization technology, performing subsequent powdering and enzyme digestion treatment, and preparing the acellular matrix gel with various shapes and properties by using mild conditions. The acellular matrix solution is obtained through subsequent powdering and enzyme digestion treatment, and the acellular matrix gel with various shapes and properties can be prepared through mild conditions.
However, the acellular matrix gel prepared by the prior art has the defect of low mechanical strength, which limits some application scenes of the extracellular matrix gel.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide the extracellular matrix gel which has very good strength and toughness, well retains collagen, elastin, fibronectin, laminin, glycosaminoglycan and other biological macromolecules contained in extracellular matrix and has good biological activity.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
In one aspect, the present invention provides a high strength extracellular matrix gel consisting of an extracellular matrix and a salt solution;
The salt solution is sodium chloride solution or phosphate solution containing sodium chloride;
the mass fraction of the extracellular matrix in the high-strength extracellular matrix gel is 1.5% -6.0%;
the mass of the insoluble matrix in the extracellular matrix accounts for 52% -72% of the mass of the extracellular matrix;
the extracellular matrix comprises one or more of collagen, elastin, fibronectin, laminin, and glycosaminoglycan;
the insoluble matrix is the portion of the extracellular matrix that is not dissociated and is not soluble in water or hydrochloric acid solution.
The compressive fracture stress of the high-strength extracellular matrix gel is 72.1-661.6kPa; the compressive fracture strain of the high strength extracellular matrix gel is 69.3% -83.1%.
In another aspect, the present invention also provides a method for preparing the high-strength extracellular matrix gel, comprising the steps of:
(1) Taking the surrounding tissues of mammals, removing fat and adventitia tissues, and performing freeze thawing cycle once to obtain a tissue I;
(2) Cutting the tissue I obtained in the step (1), and then soaking the cut tissue I in a peracetic acid solution to obtain a tissue II;
(3) Soaking the tissue II obtained in the step (2) by using a sodium hydroxide solution, and then washing the tissue II by using flowing purified water until the pH value is less than 9.0 to obtain a tissue III;
(4) Cutting the tissue III obtained in the step (3), dispersing in purified water, adding organic acid to adjust pH to 2.5-3.5 to expand the tissue, and pulverizing into slurry;
(5) Adding sodium hydroxide solution into the slurry obtained in the step (4) to adjust the pH to 6.0-8.0, separating out floccules, and collecting floccules A;
(6) Soaking the floccule A obtained in the step (5) in an alkali solution, oscillating, filtering and collecting floccule B, and washing with flowing purified water until the pH value is less than 9.0 to obtain floccule C;
(7) Soaking the floccule C obtained in the step (6) in urea solution, and cleaning with purified water after the soaking is finished to obtain floccule D;
(8) Dispersing the floccule D obtained in the step (7) in an acid solution, and stirring to form a jelly;
(9) Filtering the jelly obtained in the step (8), and collecting a filtrate;
(10) Dialyzing the filtrate obtained in the step (9) by using purified water, and then freeze-drying to obtain a freeze-dried sample;
(11) Dispersing the freeze-dried sample obtained in the step (10) into jelly by using hydrochloric acid solution, regulating pH by using alkali liquor, and standing to obtain the high-strength extracellular matrix gel.
Wherein,
The surrounding tissue of the mammal in the step (1) is a porcine small intestine submucosa;
cutting, namely cutting the tissue I into fragments not larger than 20cm 2; the mass concentration of the peroxyacetic acid solution is 1% -2%; the soaking time is 1-2 hours;
The molar concentration of the sodium hydroxide solution in the step (3) is 0.05-0.5mol/L; the soaking time is 0.5-2 hours;
cutting in the step (4), namely cutting the tissue III into small pieces with the area not more than 4cm 2;
the mass ratio of the small pieces to the purified water is 1:2-1:10; the organic acid is one of acetic acid, citric acid or oxalic acid.
As some preferred embodiments, the slurry obtained in step (4) requires stirring for a period of time ranging from 2 to 12 hours.
The molar concentration of the sodium hydroxide solution in the step (5) is 1-5mol/L;
The alkali solution in the step (6) is sodium hydroxide solution or potassium hydroxide solution, and the pH value of the alkali solution is 11.0-13.0; the oscillation time is 6-24 hours;
The molar concentration of the urea solution in the step (7) is 4-6mol/L; the soaking time is 0.5-2 hours;
The acid solution in the step (8) is acetic acid solution, and the molar concentration of the acetic acid solution is 0.3-0.8mol/L; the mass ratio of the floccule D to the acid solution is 1:5-1:50; the stirring time is 24-72 hours;
The filtering in the step (9) is extrusion filtering by using a filter cloth, wherein the filter cloth is nylon filter cloth with 100-600 meshes;
The ratio of the freeze-dried sample to the hydrochloric acid solution in the step (11) is 1:60-1:15;
The molar concentration of the hydrochloric acid solution is 0.001-0.01mol/L;
The alkali liquor is sodium hydroxide solution or disodium hydrogen phosphate solution; the molar concentration of the sodium hydroxide solution is 0.1-1mol/L; the molar concentration of the disodium hydrogen phosphate solution is 0.02-0.2mol/L;
The pH is 6.0-10.0;
the standing temperature is 4-20 ℃; the standing time is 6-36 hours.
Compared with the prior art, the invention has the beneficial effects that:
(1) The high-strength extracellular matrix gel provided by the invention has higher compressive fracture stress and compressive fracture strain, shows very good strength and toughness, can be prepared into a sample with a certain three-dimensional shape, and overcomes the defect of poor mechanical property of the conventional extracellular matrix gel.
(2) The high-strength extracellular matrix gel provided by the invention well retains collagen, elastin, fibronectin, laminin, glycosaminoglycan and other biological macromolecules contained in the extracellular matrix, and has good biological activity.
(3) The high-strength extracellular matrix gel DNA and the alpha-gal antigen provided by the invention have low residual quantity, theoretically greatly reduce the immunogenicity of animal-derived materials and improve the biological safety.
(4) The high-strength extracellular matrix gel provided by the invention does not use any chemical crosslinking agent, so that adverse effects of the chemical crosslinking agent are avoided; the preparation method is relatively simple and the operation condition is mild.
Drawings
FIG. 1 shows the macroscopic morphology of the high strength extracellular matrix gel obtained in example 1 of the present invention.
FIG. 2 is a stress-strain curve of the high strength extracellular matrix gel obtained in accordance with various embodiments of the present invention.
FIG. 3 is a stress-strain curve of the high strength extracellular matrix gel obtained in each comparative example of the present invention.
FIG. 4 is a photograph showing the high-strength extracellular matrix gel-cultured rat chondrocytes obtained in example 1 of the present invention.
Detailed Description
The above-mentioned features of the invention, or of the embodiments, may be combined in any desired manner. All of the features explained in this specification can be used in combination with any form of method, and each feature disclosed in this specification can be replaced by any alternative feature serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the disclosed features are merely general examples of equivalent or similar features.
The invention will be further illustrated with reference to specific examples. These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. The following examples are presented to illustrate specific conditions, generally according to conventional conditions or according to manufacturer's recommended conditions. All percentages and fractions are by weight unless specifically indicated.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are exemplary only.
Example 1:
A high strength extracellular matrix gel was prepared as follows:
(1) Taking one small pig intestine, cleaning with tap water to remove residual blood and impurities, longitudinally cutting, and removing mucous membrane, serosa layer and myometrium by using a scraper to obtain submucosa of small pig intestine; washing with tap water for 1 time and then washing with purified water for 2 times; freezing in a refrigerator at-22deg.C for 12 hr, taking out, and thawing at room temperature;
(2) Cutting the submucosa of the small intestine of the pig treated in the step (1) into segments with the area of 10-20cm 2 by using scissors, soaking the segments in a 1% peracetic acid solution for 1 hour, and then fishing out and leaching out redundant solution;
(3) Immersing the submucosa section of the small intestine of the pig obtained in the step (2) into a sodium hydroxide solution with the concentration of 0.1mol/L for 2 hours, then washing with purified water until the pH value of the detected eluate is 8.6, and draining water;
(4) Cutting the porcine small intestine submucosa segment obtained in the step (3) into small pieces with the area of 2-4cm 2 by scissors, dispersing the small pieces in purified water with the mass 10 times of that of the small pieces, adding acetic acid to adjust the pH to 3.5, then crushing the small pieces into thick slurry by using a blade crusher, and continuously stirring the slurry for 12 hours by using mechanical stirring;
(5) Adding 1mol/L sodium hydroxide solution into the slurry obtained in the step (4) to adjust the pH to 6.8, completely separating out floccules, and filtering and collecting the floccules by using 200-mesh filter cloth;
(6) Dispersing the floccule obtained in the step (5) in a sodium hydroxide solution with the pH value of 13.0, placing in a shaker for continuous shaking for 6 hours, filtering and collecting the floccule by using 200-mesh filter cloth, and washing with purified water until the pH value of the detected eluate is 8.1;
(7) Soaking the floccule obtained in the step (6) into urea solution with the concentration of 4mol/L for 0.5 hour, and then washing with purified water;
(8) Adding 10 times of acetic acid solution with the mass and the concentration of 0.5mol/L into the floccule obtained in the step (7), uniformly dispersing, and continuously stirring for 24 hours after swelling to obtain jelly;
(9) Collecting the jelly obtained in the step (8), placing the jelly in a filter bag made of 200-mesh nylon filter cloth, extruding the filter bag to extrude the jelly from the pores, and collecting the extruded jelly;
(10) Placing the jelly obtained in the step (9) in a dialysis bag, dialyzing with purified water for 48 hours, and replacing the purified water every 8 hours; then the jelly is put into a freeze dryer for freeze drying;
(11) Dispersing the freeze-dried sample in the step (10) by using a hydrochloric acid solution with the mass of 60 times and the concentration of 0.01mol/L, uniformly mixing, adding a disodium hydrogen phosphate solution with the mass of 1/9 and the concentration of 0.02mol/L into the hydrochloric acid solution, regulating the pH value to 6.0, and placing the mixture at the temperature of 10 ℃ for 24 hours to obtain the high-strength extracellular matrix gel with the mass fraction of 1.5%.
Example 2:
A high strength extracellular matrix gel was prepared as follows:
(1) Taking one small pig intestine, cleaning with tap water to remove residual blood and impurities, longitudinally cutting, and removing mucous membrane, serosa layer and myometrium by using a scraper to obtain submucosa of small pig intestine; washing with tap water for 1 time and then washing with purified water for 2 times; freezing in a refrigerator at-22deg.C for 12 hr, taking out, and thawing at room temperature;
(2) Cutting the submucosa of the small intestine of the pig treated in the step (1) into fragments with the area of 10-20cm 2 by using scissors, soaking the fragments in 2% peracetic acid solution for 1 hour, and then fishing out and leaching out redundant solution;
(3) Immersing the submucosa sheet segment of the small intestine of the pig treated in the step (2) into a sodium hydroxide solution with the concentration of 0.5mol/L for 0.5 hour, then washing with purified water until the pH value of the detected eluate is 8.3, and draining water;
(4) Cutting the porcine small intestine submucosa segment obtained in the step (3) into small pieces with the area of 2-4cm 2 by scissors, dispersing the small pieces in purified water with the mass of 2 times of that of the small pieces of tissue, adding citric acid to adjust the pH value to 2.5, then crushing the small pieces of tissue into thick slurry by using a blade crusher, and continuously stirring the slurry for 6 hours by using mechanical stirring;
(5) Adding 5mol/L sodium hydroxide solution into the slurry obtained in the step (4) to adjust the pH to 7.9, completely separating out floccules, and filtering and collecting the floccules by using 200-mesh filter cloth;
(6) Dispersing the floccule obtained in the step (5) in a sodium hydroxide solution with the pH value of 11.0, placing in a shaker for continuous shaking for 24 hours, filtering and collecting the floccule by using 200-mesh filter cloth, and washing with purified water until the pH value of the detected eluate is 7.4;
(7) Soaking the floccule obtained in the step (6) into urea solution with the concentration of 6mol/L for 2 hours, and then washing with purified water;
(8) Adding acetic acid solution with the mass of 20 times and the concentration of 0.3mol/L into the floccule obtained in the step (7), uniformly dispersing, and continuously stirring for 72 hours after swelling to obtain jelly;
(9) Collecting the jelly obtained in the step (8), placing the jelly in a filter bag made of 200-mesh nylon filter cloth, extruding the filter bag to extrude the jelly from the pores, and collecting the extruded jelly;
(10) Placing the jelly obtained in the step (9) in a dialysis bag, dialyzing with purified water for 48 hours, and replacing the purified water every 8 hours; then the jelly is put into a freeze dryer for freeze drying;
(11) Dispersing the freeze-dried sample in the step (10) by using hydrochloric acid solution with the mass of the sample being 30 times that of the freeze-dried sample and the concentration being 0.01mol/L, uniformly mixing, adding sodium hydroxide solution with the mass of the sample being equal and the concentration being 1mol/L, adjusting the pH value to 7.6, and placing the mixture at the temperature of 20 ℃ for 6 hours to obtain the high-strength extracellular matrix gel with the mass fraction of 3.1%.
Example 3
A high strength extracellular matrix gel was prepared as follows:
(1) Taking one small pig intestine, cleaning with tap water to remove residual blood and impurities, longitudinally cutting, and removing mucous membrane, serosa layer and myometrium by using a scraper to obtain submucosa of small pig intestine; washing with tap water for 1 time and then washing with purified water for 2 times; freezing in a refrigerator at-22deg.C for 12 hr, taking out, and thawing at room temperature;
(2) Cutting the submucosa of the small intestine of the pig treated in the step (1) into fragments with the area of 10-20cm 2 by using scissors, soaking the fragments in 2% peracetic acid solution for 1 hour, and then fishing out and leaching out redundant solution;
(3) Immersing the submucosa section of the small intestine of the pig treated in the step (2) into a sodium hydroxide solution with the concentration of 0.25mol/L for 1 hour, then washing with purified water until the pH value of the detected eluate is 7.8, and draining water;
(4) Cutting the porcine small intestine submucosa segment obtained in the step (3) into small pieces with the area of 2-4cm 2 by scissors, dispersing the small pieces in purified water with the mass of 4 times of that of the small pieces, adding oxalic acid to adjust the pH to 2.5, then crushing the small pieces into thick slurry by using a blade crusher, and continuously stirring the slurry for 8 hours by using mechanical stirring;
(5) Adding 5mol/L sodium hydroxide solution into the slurry obtained in the step (4) to adjust the pH to 8.9, completely separating out floccules, and filtering and collecting the floccules by using 200-mesh filter cloth;
(6) Dispersing the floccule obtained in the step (5) in potassium hydroxide solution with the pH value of 12.0, placing in an oscillator for continuous oscillation for 24 hours, filtering and collecting the floccule by using 200-mesh filter cloth, and washing with purified water until the pH value of the detected eluate is 8.2;
(7) Soaking the floccule obtained in the step (6) into urea solution with the concentration of 6mol/L for 1 hour, and then washing with purified water;
(8) Adding the floccule obtained in the step (7) into acetic acid solution with the mass of 50 times and the concentration of 0.5mol/L, uniformly dispersing, and continuously stirring for 60 hours after swelling to obtain jelly;
(9) Collecting the jelly obtained in the step (8), placing the jelly in a filter bag made of 200-mesh nylon filter cloth, extruding the filter bag to extrude the jelly from the pores, and collecting the extruded jelly;
(10) Placing the jelly obtained in the step (9) in a dialysis bag, dialyzing with purified water for 48 hours, and replacing the purified water every 8 hours; then the jelly is put into a freeze dryer for freeze drying;
(11) Dispersing the freeze-dried sample obtained in the step (10) by using hydrochloric acid solution with the mass of the sample being 15 times and the concentration being 0.001mol/L, adding disodium hydrogen phosphate solution with the mass of 1/9 hydrochloric acid and the concentration being 0.2mol/L to adjust the pH value to 10.0 after uniformly mixing, and placing the mixture at the temperature of 4 ℃ for 36 hours to obtain the high-strength extracellular matrix gel with the mass fraction of 6.0%.
Example 4
A high strength extracellular matrix gel was prepared as follows:
(1) Taking one small pig intestine, cleaning with tap water to remove residual blood and impurities, longitudinally cutting, and removing mucous membrane, serosa layer and myometrium by using a scraper to obtain submucosa of small pig intestine; washing with tap water for 1 time and then washing with purified water for 2 times; freezing in a refrigerator at-22deg.C for 24 hr, taking out, and thawing at room temperature;
(2) Cutting the submucosa of the small intestine of the pig treated in the step (1) into fragments with the area of 10-20cm 2 by using scissors, soaking the fragments in a peracetic acid solution with the concentration of 1.5% for 1 hour, and then fishing out and leaching out redundant solution;
(3) Immersing the submucosa section of the small intestine of the pig treated in the step (2) into sodium hydroxide solution with the concentration of 0.05mol/L for 1 hour, then washing with purified water until the pH value of the detected eluate is 7.8, and draining water;
(4) Cutting the porcine small intestine submucosa segment obtained in the step (3) into small pieces with the area of 2-4cm 2 by using scissors, dispersing the small pieces in purified water with the mass of 4 times of that of the small pieces of tissue, adding oxalic acid to adjust the pH value to 3.0, then crushing the small pieces of tissue into thick slurry by using a blade crusher, and continuously stirring the slurry for 6 hours by using mechanical stirring;
(5) Adding 1mol/L sodium hydroxide solution into the slurry obtained in the step (4) to adjust the pH to 7.5, completely separating out floccules, and filtering and collecting the floccules by using 200-mesh filter cloth;
(6) Dispersing the floccule obtained in the step (5) in sodium hydroxide solution with the pH value of 12.0, placing in a shaker for continuous shaking for 16 hours, filtering and collecting the floccule by using 200-mesh filter cloth, and washing with purified water until the pH value of the detected eluate is 8.5;
(7) Soaking the floccule obtained in the step (6) into urea solution with the concentration of 5mol/L for 1 hour, and then washing with purified water;
(8) Adding the floccule obtained in the step (7) into acetic acid solution with the mass of 30 times and the concentration of 0.6mol/L, uniformly dispersing, and continuously stirring for 48 hours after swelling to obtain jelly;
(9) Collecting the jelly obtained in the step (8), placing the jelly in a filter bag made of 600-mesh nylon filter cloth, extruding the filter bag to extrude the jelly from the pores, and collecting the extruded jelly;
(10) Placing the jelly obtained in the step (9) in a dialysis bag, dialyzing with purified water for 48 hours, and replacing the purified water every 8 hours; then the jelly is put into a freeze dryer for freeze drying;
(11) Dispersing the freeze-dried sample in the step (10) by using a hydrochloric acid solution with the mass of 60 times and the concentration of 0.005mol/L, uniformly mixing, adding a disodium hydrogen phosphate solution with the mass of 1/9 hydrochloric acid and the concentration of 0.2mol/L, regulating the pH value to 8.1, and placing at 15 ℃ for 24 hours to obtain the high-strength extracellular matrix gel with the mass fraction of 1.5%.
Example 5
A high strength extracellular matrix gel was prepared as follows:
(1) Taking one small pig intestine, cleaning with tap water to remove residual blood and impurities, longitudinally cutting, and removing mucous membrane, serosa layer and myometrium by using a scraper to obtain submucosa of small pig intestine; washing with tap water for 1 time and then washing with purified water for 2 times; freezing in a refrigerator at-22deg.C for 24 hr, taking out, and thawing at room temperature;
(2) Cutting the submucosa of the small intestine of the pig treated in the step (1) into fragments with the area of 10-20cm 2 by using scissors, soaking the fragments in a peracetic acid solution with the concentration of 1.5% for 1 hour, and then fishing out and leaching out redundant solution;
(3) Immersing the submucosa section of the small intestine of the pig treated in the step (2) into a sodium hydroxide solution with the concentration of 0.2mol/L for 1 hour, then washing with purified water until the pH value of the detected eluate is 8.3, and draining water;
(4) Cutting the porcine small intestine submucosa segment obtained in the step (3) into small pieces with the area of 2-4cm 2 by scissors, dispersing the small pieces in purified water with the mass of 8 times of that of the small pieces, adding acetic acid to adjust the pH to 2.5, then crushing the small pieces into thick slurry by using a blade crusher, and continuously stirring the slurry for 4 hours by using mechanical stirring;
(5) Adding 1mol/L sodium hydroxide solution into the slurry obtained in the step (4) to adjust the pH to 7.5, completely separating out floccules, and filtering and collecting the floccules by using 200-mesh filter cloth;
(6) Dispersing the floccule obtained in the step (5) in potassium hydroxide solution with the pH value of 12.0, placing in an oscillator for continuously oscillating for 12 hours, filtering and collecting the floccule by using 200-mesh filter cloth, and washing with purified water until the pH value of the detected eluate is 8.3;
(7) Soaking the floccule obtained in the step (6) into urea solution with the concentration of 6mol/L for 1 hour, and then washing with purified water;
(8) Adding the floccule obtained in the step (7) into acetic acid solution with the mass of 20 times and the concentration of 0.5mol/L, uniformly dispersing, and continuously stirring for 48 hours after swelling to obtain jelly;
(9) Collecting the jelly obtained in the step (8), placing the jelly in a filter bag made of 100-mesh nylon filter cloth, extruding the filter bag to extrude the jelly from the pores, and collecting the extruded jelly;
(10) Placing the jelly obtained in the step (9) in a dialysis bag, dialyzing with purified water for 48 hours, and replacing the purified water every 8 hours; then the jelly is put into a freeze dryer for freeze drying;
(11) Dispersing the freeze-dried sample in the step (10) by using a hydrochloric acid solution with the mass of the sample being 30 times that of the sample and the concentration being 0.01mol/L, uniformly mixing, adding a sodium hydroxide solution with the mass of the sample being 2 times that of the sample and the concentration being 0.1mol/L, adjusting the pH value to 6.9, and placing at 8 ℃ for 36 hours to obtain the high-strength extracellular matrix gel with the mass fraction of 3.0%.
Example 6
A high strength extracellular matrix gel was prepared as follows:
(1) Taking one small pig intestine, cleaning with tap water to remove residual blood and impurities, longitudinally cutting, and removing mucous membrane, serosa layer and myometrium by using a scraper to obtain submucosa of small pig intestine; washing with tap water for 1 time and then washing with purified water for 2 times; freezing in a refrigerator at-22deg.C for 24 hr, taking out, and thawing at room temperature;
(2) Cutting the submucosa of the small intestine of the pig treated in the step (1) into segments with the area of 10-20cm 2 by using scissors, soaking the segments in a 1% peracetic acid solution for 1 hour, and then fishing out and leaching out redundant solution;
(3) Immersing the submucosa section of the small intestine of the pig treated in the step (2) into a sodium hydroxide solution with the concentration of 0.1mol/L for 1 hour, then washing with purified water until the pH value of the detected eluate is 7.8, and draining water;
(4) Cutting the porcine small intestine submucosa segment obtained in the step (3) into small pieces with the area of 2-4cm 2 by using scissors, dispersing the small pieces in purified water with the mass of 5 times of that of the small pieces of tissue, adding acetic acid to adjust the pH to 2.5, then crushing the small pieces of tissue into thick slurry by using a blade crusher, and continuously stirring the slurry for 6 hours by using mechanical stirring;
(5) Adding 1mol/L sodium hydroxide solution into the slurry obtained in the step (4) to adjust the pH to 7.0, completely separating out floccules, and filtering and collecting the floccules by using 200-mesh filter cloth;
(6) Dispersing the floccule obtained in the step (5) in sodium hydroxide solution with the pH value of 12.0, placing in a shaker for continuous shaking for 24 hours, filtering and collecting the floccule by using 200-mesh filter cloth, and washing with purified water until the pH value of the detected eluate is 8.8;
(7) Soaking the floccule obtained in the step (6) into urea solution with the concentration of 6mol/L for 1 hour, and then washing with purified water;
(8) Adding the floccule obtained in the step (7) into acetic acid solution with the mass of 20 times and the concentration of 0.5mol/L, uniformly dispersing, and continuously stirring for 72 hours after swelling to obtain jelly;
(9) Collecting the jelly obtained in the step (8), placing the jelly in a filter bag made of 400-mesh nylon filter cloth, extruding the filter bag to extrude the jelly from the pores, and collecting the extruded jelly;
(10) Placing the jelly obtained in the step (9) in a dialysis bag, dialyzing with purified water for 48 hours, and replacing the purified water every 8 hours; then the jelly is put into a freeze dryer for freeze drying;
(11) Dispersing the freeze-dried sample in the step (10) by using hydrochloric acid solution with the mass of 15 times and the concentration of 0.01mol/L, uniformly mixing, adding disodium hydrogen phosphate solution with the mass of 1/9 hydrochloric acid and the concentration of 0.2mol/L, adjusting the pH value to 7.2, and placing at the temperature of 10 ℃ for 24 hours to obtain the high-strength extracellular matrix gel with the mass fraction of 6.0%.
Comparative example 1
Referring to the procedure of example 4, the step (6) was replaced with "dispersing the floc obtained in the step (5) in a sodium hydroxide solution having a pH of 10.0, placing in a shaker for continuous shaking for 28 hours, filtering and collecting the floc using a 200 mesh filter cloth, and washing with purified water until the pH of the detected eluate was 7.6; the rest steps are unchanged, and the sample is prepared.
Comparative example 2
Referring to the operation of example 4, step (7) was replaced with "the floccule obtained in step (6) was immersed in a urea solution having a concentration of 2mol/L for 3 hours, followed by washing with purified water; the rest steps are unchanged, and the sample is prepared.
Comparative example 3
Referring to the procedure of example 4, the procedure (11) was replaced with "dispersing the lyophilized sample of step (10) with a hydrochloric acid solution having a concentration of 0.005mol/L and 60 times the mass of the sample, mixing uniformly, adding a disodium hydrogen phosphate solution having a concentration of 0.2mol/L and 1/9 mass of hydrochloric acid to adjust the pH to 8.0, and standing at 37℃for 24 hours; the rest steps are unchanged, and the sample is prepared.
Comparative example 4
Reference is made to the procedure of example 1 of patent CN105169483 a:
(1) Washing the small intestine of the pig with tap water to remove residual blood and impurities, longitudinally cutting, and removing mucous membrane, serosa layer and myometrium by using a scraper to obtain submucosa of the small intestine of the pig; washing with tap water for 1 time, washing with purified water for 2 times, shaking in distilled water, rinsing for 6 hours, putting the submucosa of small intestine into 3% Triton-X100 water solution for 12 hours during extraction, rinsing in distilled water for 3 times, putting into 4% deoxycholate sodium water solution for 24 hours at room temperature, and rinsing in distilled water for 3 times, wherein one cycle is 1 extraction time, and 2 cycles are carried out in total; the cell in the small intestine submucosa is removed through 2 times of cycle extraction, and substances such as DNA which can cause immune response are washed away, so that the decellularized pig small intestine submucosa is obtained.
(2) Freeze-drying the decellularized pig small intestine submucosa, and grinding the decellularized pig small intestine submucosa into powder with the diameter of about 200 mu m by using a grinder; adding 1 part of pepsin and 10 parts of decellularized pig small intestine submucosa into 100 parts of 0.01 mol/L HCl solution, keeping constant stirring at room temperature (25 ℃) for 24 hours, and removing undigested large particles by ultracentrifugation; taking supernatant fluid and storing the supernatant fluid at a low temperature for later use to obtain a pre-gel solution of the mucous membrane submucosa of the small intestine of the decellularized pig;
(3) Adding 0.1mol/L sodium hydroxide with the volume of 1/10 of the pre-gel solution into the pre-gel solution of the small intestine submucosa of the decellularized pig obtained in the step (2) at the temperature of 4 ℃ to adjust the pre-gel solution of the small intestine submucosa of the decellularized pig to be neutral (pH=7.4), adding 10 XPBS with the volume of 1/9 of the pre-gel solution, adjusting the mass fraction of the small intestine submucosa of the decellularized pig in the solution to be 1.5% by using PBS buffer solution with the pH value of 7.4 at the temperature of 4 ℃, and standing the solution at the temperature of 37 ℃ for 8 minutes to form gel.
Test example 1-determination of compressive stress at break and compressive strain at break of samples obtained in each example and comparative example
The compressive fracture stress and compressive fracture strain of the samples prepared in each example of the present invention and each comparative example were measured using a universal material tester.
The above samples were formed into a cylindrical shape having a diameter of 1.0cm and a height of 0.5cm using a die, and were axially compressed after being fixed on a jig, the compression speed was set at 30mm/min, the displacement and the pressure were recorded, the percentage of the displacement to the initial height of the sample was taken as strain, and the pressure divided by the initial cross-sectional area of the sample was taken as stress, and stress-strain graphs of the respective samples were shown in fig. 2 and 3. The compressive fracture stress and compressive fracture strain of each sample are shown in table 1. It can be seen that the samples prepared in the examples show higher compressive fracture stress and compressive fracture strain, and strong and tough properties in mechanical properties. The samples prepared in the comparative examples have the same mass fractions of the extracellular matrix and the compressive fracture stress is much smaller than that of example 4, and the compressive fracture strain is also lower.
Table 1, examples and comparative examples the compressive fracture stress and compressive fracture strain of the prepared samples.
Test example 2-determination of the composition of high-Strength extracellular matrix gel
The content of collagen in the high-strength extracellular matrix gel in the extracellular matrix is determined by a hydroxyproline method. The samples were treated with 6mol/L hydrochloric acid at 110℃for 24 hours to give a completely degraded amino acid solution, which was evaporated to dryness under reduced pressure using a rotary evaporator, and then the volume was determined with purified water, and the hydroxyproline content was measured with an amino acid analyzer, thereby converting the content of collagen in the samples prepared in each example to extracellular matrix.
The content of elastin, fibronectin, laminin and glycosaminoglycan in the high-strength extracellular matrix gel was determined by the Elisa method using a quantitative detection kit of the corresponding substances, respectively. After the samples are treated by the type I collagenase, experiments are carried out according to standard operation steps of corresponding substance kits respectively, and the content of each substance is detected.
The content of collagen, elastin, fibronectin, laminin and glycosaminoglycan in the extracellular matrix was experimentally determined as shown in table 2. It can be seen that the high strength extracellular matrix gel obtained according to the present invention retains several bio-macromolecular substances which are mainly present in the extracellular matrix.
TABLE 2 content of various biomacromolecule substances in the extracellular matrix in the high-strength extracellular matrix gel prepared in examples
The percentage of insoluble matrix in the high strength extracellular matrix gel to total extracellular matrix was determined as follows: the samples prepared in each example and each comparative example were diluted with hydrochloric acid at a concentration of 0.01mol/L to a content of extracellular matrix of 0.3mg/ml, and the above solutions were filtered through a filter membrane having a pore size of 0.22. Mu.m, and the viscous insoluble matter trapped on the filter membrane was collected. And freeze-drying the collected viscous insoluble substances, and weighing to obtain the mass of the insoluble matrix. The percentage of insoluble matrix in the samples of each example to extracellular matrix was calculated from the known amount of extracellular matrix and is shown in table 3. It can be seen that the insoluble matrix content of comparative examples 1,2 and 4 is lower than that of each example, and it is known that the change of each parameter in the step of preparing the jelly significantly affects the insoluble matrix content, and that there is a certain correlation between the insoluble matrix content and the detection result of the mechanical properties of the sample by comparison, whereas the insoluble matrix content of comparative example 3 is similar to that of each example because the parameter during gel formation is changed in comparative example 3, and the jelly generation is not affected and therefore the insoluble matrix content is not affected. It can be speculated from the above that the insoluble matrix content may be one of the factors determining the compressive fracture stress and compressive fracture strain of the sample.
Table 3, content of insoluble matrix in extracellular matrix gel of high strength prepared in each example.
Test example 3-determination of DNA residual amount and alpha-gal antigen residual amount of high-intensity extracellular matrix gel
The DNA residue of the high-strength extracellular matrix gel is determined by experiments according to the method of determining the DNA residue of animal-derived biological materials in the 25 th part of tissue engineering medical products of YY/T0606.25-2014, fluorescent staining method, using a Quant-IT PicoGreen dsDNA quantitative kit and standard operation procedures.
The residual quantity of the alpha-gal antigen of the high-strength extracellular matrix gel is determined by using an alpha-gal antigen quantitative detection kit according to the detection of the residual alpha-gal antigen of animal-derived scaffold materials of YY/T1561-2017 tissue engineering medical equipment products and according to standard operation steps.
The residual amounts of DNA and alpha-gal antigen in the samples of the examples obtained by the experiments are shown in Table 4. It can be seen that the sample DNA residual amounts are at very low levels, while the residual amounts of the alpha-gal antigen are below the detection limit (detection limit is 0.03 ng/g), DNA and alpha-gal antigen are the major sources of immunogenicity of animal-derived materials, and that the low levels of residual amounts indicate low immunogenicity of the high-strength extracellular matrix gel obtained according to the invention.
Table 4, residual amounts of DNA and alpha-gal in the high intensity extracellular matrix gel prepared in each example.
Test example 4 cell culture experiments
The high-strength extracellular matrix gel obtained in example 1 was placed in a cell culture plate well, DMEM medium was added thereto, rat chondrocytes were inoculated, and cell adhesion and proliferation were observed by staining after 12 hours and 48 hours of inoculation, respectively. As shown in FIG. 4, cells adhered to the gel surface completely after 12 hours and increased significantly in number after 48 hours, and it was found that the high-strength extracellular matrix gel was able to support cell adhesion and proliferation well.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (10)
1. A high strength extracellular matrix gel, characterized by: the high-strength extracellular matrix gel consists of an extracellular matrix and a salt solution; the mass fraction of the extracellular matrix is 1.5% -6.0%; the mass of the insoluble matrix in the extracellular matrix accounts for 52% -72% of the mass of the extracellular matrix; the extracellular matrix includes one or more of collagen, elastin, fibronectin, laminin, and glycosaminoglycans.
2. The high strength extracellular matrix gel of claim 1, wherein: the compressive fracture stress of the high-strength extracellular matrix gel is 72.1-661.6kPa; compression fracture strain was 69.3% -83.1%.
3. A method of preparing a high strength extracellular matrix gel according to claim 1 or 2, wherein: the method comprises the following steps:
(1) Taking the surrounding tissues of mammals, removing fat and adventitia tissues, and performing freeze thawing cycle once to obtain a tissue I;
(2) Cutting the tissue I obtained in the step (1), and then soaking the cut tissue I in a peracetic acid solution to obtain a tissue II;
(3) Soaking the tissue II obtained in the step (2) by using a sodium hydroxide solution, and then washing the tissue II by using flowing purified water until the pH value is less than 9.0 to obtain a tissue III;
(4) Cutting the tissue III obtained in the step (3), dispersing in purified water, adding organic acid to adjust pH to 2.5-3.5 to expand the tissue, and pulverizing into slurry;
(5) Adding sodium hydroxide solution into the slurry obtained in the step (4) to adjust the pH to 6.0-8.0, separating out floccules, and collecting floccules A;
(6) Soaking the floccule A obtained in the step (5) in an alkali solution, oscillating, filtering and collecting floccule B, and washing with flowing purified water until the pH value is less than 9.0 to obtain floccule C;
(7) Soaking the floccule C obtained in the step (6) in urea solution, and cleaning with purified water after the soaking is finished to obtain floccule D;
(8) Dispersing the floccule D obtained in the step (7) in an acid solution, and stirring to form a jelly;
(9) Filtering the jelly obtained in the step (8), and collecting a filtrate;
(10) Dialyzing the filtrate obtained in the step (9) by using purified water, and then freeze-drying to obtain a freeze-dried sample;
(11) Dispersing the freeze-dried sample obtained in the step (10) into jelly by using hydrochloric acid solution, regulating pH by using alkali liquor, and standing to obtain the high-strength extracellular matrix gel.
4. A method of preparation according to claim 3, characterized in that: the mass concentration of the peroxyacetic acid solution in the step (2) is 1% -2%; the soaking time is 1-2 hours.
5. A method of preparation according to claim 3, characterized in that: the molar concentration of the sodium hydroxide solution in the step (3) is 0.05-0.5mol/L; the soaking time is 0.5-2 hours.
6. A method of preparation according to claim 3, characterized in that: the molar concentration of the sodium hydroxide solution in the step (5) is 1-5mol/L; the alkali solution in the step (6) is sodium hydroxide solution or potassium hydroxide solution, and the pH value of the sodium hydroxide solution or the potassium hydroxide solution is 11.0-13.0; the shaking time is 6-24 hours.
7. A method of preparation according to claim 3, characterized in that: the molar concentration of the urea solution in the step (7) is 4-6mol/L; the soaking time is 0.5-2 hours.
8. A method of preparation according to claim 3, characterized in that: the acid solution in the step (8) is acetic acid solution, and the molar concentration of the acetic acid solution is 0.3-0.8mol/L; the mass ratio of the floccule D to the acid solution is 1:5-1:50; the stirring time is 24-72 hours.
9. A method of preparation according to claim 3, characterized in that: the ratio of the freeze-dried sample to the hydrochloric acid solution in the step (11) is 1:60-1:15; the molar concentration of the hydrochloric acid solution is 0.001-0.01mol/L.
10. A method of preparation according to claim 3, characterized in that: the alkali liquor in the step (11) is sodium hydroxide solution or disodium hydrogen phosphate solution; the molar concentration of the sodium hydroxide solution is 0.1-1mol/L; the molar concentration of the disodium hydrogen phosphate solution is 0.02-0.2mol/L; the pH is 6.0-10.0; the standing temperature is 4-20 ℃; the standing time is 6-36 hours.
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| CN104971380A (en) * | 2014-04-11 | 2015-10-14 | 烟台隽秀生物科技有限公司 | Acellular matrix repairing gel and new method for preparing the same |
| CN105169483A (en) * | 2015-10-20 | 2015-12-23 | 中山大学 | Preparation method of acellular matrix gels and acellular matrix gels |
| CN107320776A (en) * | 2017-07-06 | 2017-11-07 | 中山大学 | A kind of gel for promoting corium regeneration and preparation method thereof |
| CN108699522A (en) * | 2016-01-13 | 2018-10-23 | 高等教育联邦系统-匹兹堡大学 | Vascular extracellular matrix hydrogel |
| WO2024020635A1 (en) * | 2022-07-28 | 2024-02-01 | The University Of Newcastle | Extracellular matrix hydrogels |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104971380A (en) * | 2014-04-11 | 2015-10-14 | 烟台隽秀生物科技有限公司 | Acellular matrix repairing gel and new method for preparing the same |
| CN105169483A (en) * | 2015-10-20 | 2015-12-23 | 中山大学 | Preparation method of acellular matrix gels and acellular matrix gels |
| CN108699522A (en) * | 2016-01-13 | 2018-10-23 | 高等教育联邦系统-匹兹堡大学 | Vascular extracellular matrix hydrogel |
| CN107320776A (en) * | 2017-07-06 | 2017-11-07 | 中山大学 | A kind of gel for promoting corium regeneration and preparation method thereof |
| WO2024020635A1 (en) * | 2022-07-28 | 2024-02-01 | The University Of Newcastle | Extracellular matrix hydrogels |
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