CN118165099A - Composite collagen capable of recruiting stem cells and preparation method and application thereof - Google Patents
Composite collagen capable of recruiting stem cells and preparation method and application thereof Download PDFInfo
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- CN118165099A CN118165099A CN202410408895.6A CN202410408895A CN118165099A CN 118165099 A CN118165099 A CN 118165099A CN 202410408895 A CN202410408895 A CN 202410408895A CN 118165099 A CN118165099 A CN 118165099A
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- 102000008186 Collagen Human genes 0.000 title claims abstract description 62
- 108010035532 Collagen Proteins 0.000 title claims abstract description 62
- 229920001436 collagen Polymers 0.000 title claims abstract description 62
- 239000002131 composite material Substances 0.000 title claims abstract description 41
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 27
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 238000005406 washing Methods 0.000 claims abstract description 34
- 239000002244 precipitate Substances 0.000 claims abstract description 30
- 239000002608 ionic liquid Substances 0.000 claims abstract description 23
- 238000003756 stirring Methods 0.000 claims abstract description 23
- 239000012265 solid product Substances 0.000 claims abstract description 18
- 238000001914 filtration Methods 0.000 claims abstract description 9
- IBZJNLWLRUHZIX-UHFFFAOYSA-N 1-ethyl-3-methyl-2h-imidazole Chemical class CCN1CN(C)C=C1 IBZJNLWLRUHZIX-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000012620 biological material Substances 0.000 claims abstract description 4
- 238000001035 drying Methods 0.000 claims abstract description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 36
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 34
- 239000008367 deionised water Substances 0.000 claims description 33
- 229910021641 deionized water Inorganic materials 0.000 claims description 33
- 238000002791 soaking Methods 0.000 claims description 33
- 238000000034 method Methods 0.000 claims description 25
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 18
- 239000002245 particle Substances 0.000 claims description 14
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims description 10
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- 239000000463 material Substances 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- 238000004108 freeze drying Methods 0.000 claims description 8
- -1 1-ethyl-3-methylimidazole phosphate acetate Chemical compound 0.000 claims description 6
- 229910052786 argon Inorganic materials 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- 241000270295 Serpentes Species 0.000 claims description 4
- 239000003960 organic solvent Substances 0.000 claims description 4
- 230000008439 repair process Effects 0.000 claims description 4
- 241001494479 Pecora Species 0.000 claims description 3
- 239000003208 petroleum Substances 0.000 claims description 3
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 claims description 3
- 238000007634 remodeling Methods 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000000703 high-speed centrifugation Methods 0.000 claims description 2
- 239000002994 raw material Substances 0.000 claims description 2
- 230000008929 regeneration Effects 0.000 claims description 2
- 238000011069 regeneration method Methods 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 abstract description 12
- 241001465754 Metazoa Species 0.000 abstract description 4
- 239000000126 substance Substances 0.000 abstract description 4
- 210000000170 cell membrane Anatomy 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 3
- 210000000056 organ Anatomy 0.000 abstract description 3
- 230000003204 osmotic effect Effects 0.000 abstract description 3
- 102000004169 proteins and genes Human genes 0.000 abstract description 3
- 108090000623 proteins and genes Proteins 0.000 abstract description 3
- 239000011261 inert gas Substances 0.000 abstract 1
- 210000001519 tissue Anatomy 0.000 description 20
- 210000003491 skin Anatomy 0.000 description 12
- ZDIRKWICVFDSNX-UHFFFAOYSA-N diethyl phosphate 1-ethyl-3-methyl-1,2-dihydroimidazol-1-ium Chemical compound P(=O)(OCC)(OCC)O.C(C)N1CN(C=C1)C ZDIRKWICVFDSNX-UHFFFAOYSA-N 0.000 description 7
- 239000000706 filtrate Substances 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- VITQXNLPXZIPHQ-UHFFFAOYSA-N dimethyl phosphate;3-ethyl-1-methyl-1,2-dihydroimidazol-1-ium Chemical compound COP([O-])(=O)OC.CCN1C[NH+](C)C=C1 VITQXNLPXZIPHQ-UHFFFAOYSA-N 0.000 description 5
- 239000000835 fiber Substances 0.000 description 5
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 4
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 4
- 210000002744 extracellular matrix Anatomy 0.000 description 4
- 239000010985 leather Substances 0.000 description 4
- 230000004069 differentiation Effects 0.000 description 3
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- 102000004190 Enzymes Human genes 0.000 description 2
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- 238000005903 acid hydrolysis reaction Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
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- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 230000017423 tissue regeneration Effects 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 102000013373 fibrillar collagen Human genes 0.000 description 1
- 108060002894 fibrillar collagen Proteins 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000002439 hemostatic effect Effects 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
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- 210000000663 muscle cell Anatomy 0.000 description 1
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- 229920000642 polymer Polymers 0.000 description 1
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- 239000005017 polysaccharide Substances 0.000 description 1
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- 238000004064 recycling Methods 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 210000004927 skin cell Anatomy 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000007838 tissue remodeling Effects 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/60—Materials for use in artificial skin
-
- 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
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/24—Collagen
-
- 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
- A61L27/362—Skin, e.g. dermal papillae
-
- 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
- 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)
- Public Health (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Transplantation (AREA)
- Epidemiology (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
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- Dermatology (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
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Abstract
The invention provides a composite collagen capable of recruiting stem cells, a preparation method and application thereof, and belongs to the technical field of biological materials. The preparation method comprises the steps of immersing biological tissues containing collagen in 1-ethyl-3-methylimidazole salt ionic liquid after specific pretreatment, continuously stirring for 5-18 hours under the protection of inert gas, filtering to obtain a solid product, centrifuging the solid product at a high speed to obtain a precipitate, and washing and drying the precipitate in sequence to obtain the composite collagen. The invention creatively uses ionic liquid to dissolve cell membranes of animal-derived tissues and organs through osmotic effect and the like, separates DNA from protein, effectively removes cell substances in the tissues to realize the decellularization effect, and ensures that the prepared composite collagen has higher purity and bioactivity.
Description
Technical Field
The invention belongs to the technical field of biological materials, and particularly relates to a composite collagen capable of recruiting stem cells, and a preparation method and application thereof.
Background
Stem cells are cells that have not undergone differentiation, i.e., cells that have no specific function. Skin cells can protect the body, muscle cells can contract, and nerve cells can transmit information. Stem cells do not have any particular structure or function, but they have the potential to become any cell in the human body.
The stem cells differentiate without the "soil" environment, and the extracellular matrix acts as the "soil" environment. The extracellular matrix is a matrix of all components of the tissue except cells, including the matrix in a homogeneous state (collagen, polysaccharide and glycoprotein) and fibrillar collagen fibers, which have the function of linking and supporting cells, or a basic framework and metabolic site for cell attachment, the morphology and function of which directly affect the morphology and function of the formed tissue. The processed composite collagen material has good mechanical property, good tissue compatibility, no immunological rejection phenomenon when implanted into a human body, and plays roles of supporting and connecting cells in the human body, and simultaneously, the three-dimensional space structure and cell factors of the composite collagen material are beneficial to the adhesion and growth of stem cells.
The traditional preparation method of the composite collagen comprises an acid hydrolysis method and an enzyme hydrolysis method, and the methods have a plurality of treatment steps and serious pollution. The acid hydrolysis process has the problems of waste acid emission and the like, and the enzyme hydrolysis process has low efficiency, so that collagen resources are not fully utilized yet. Because the collagen is easy to combine with hydrogen bond and intertwine in wet state, the collagen is difficult to disperse by a simple mechanical or chemical method, and the collagen fiber is difficult to disperse by physical means such as freeze grinding and the like in dry state due to the characteristics of toughness and the like of the collagen fiber, and the collagen fiber is easy to be destroyed by excessive physical action.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide the composite collagen which can recruit stem cells, and the invention uses ionic liquid to dissolve cell membranes of animal-derived tissues and organs through osmotic effect and the like, separates DNA from protein, effectively removes cell substances in the tissues to realize the decellularization effect, so that the prepared composite collagen has higher purity and bioactivity.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for preparing composite collagen capable of recruiting stem cells, comprising the following steps:
(1) Pretreatment of raw materials: cutting biological tissues containing collagen into Huang Douli pieces, repeatedly washing the biological tissues with deionized water for 2-3 times, soaking the biological tissues with isopropanol, and washing the biological tissues with deionized water for 3-4 times after the soaking is finished;
(2) Immersing the ionic liquid: immersing the washed biological tissue particles containing collagen in 1-ethyl-3-methylimidazole salt ionic liquid, continuously stirring for 5-18 hours under the protection of argon or nitrogen, and filtering to obtain a solid product;
(3) And (3) centrifuging: carrying out high-speed centrifugation on the solid product obtained in the step (2) to obtain a precipitate;
(4) Washing: washing the precipitate in the step (3) for 3 times by adopting an organic solvent, and soaking the precipitate in deionized water for 3-5 hours;
(5) And (3) drying: and (3) freeze-drying the precipitate soaked in the step (4) in a sterile environment to obtain the composite collagen.
Specifically, the collagen-containing biological tissue in the step (1) is one of pigskin, cow skin, sheep skin or snake skin, and preferably pigskin.
Preferably, the soaking in isopropyl alcohol in the step (1) specifically comprises: the soaking time is 10-12 h, and the soaking is slowly stirred for 5min at intervals of 2h.
The step (1) is to cut the biological tissue containing collagen, wash with deionized water, soak with isopropyl alcohol and rewashing with deionized water, so that the collagen fiber structure is loose, the subsequent collagen extraction is easier, and the extraction time is shortened.
Preferably, the 1-ethyl-3-methylimidazole salt ionic liquid in the step (2) is any one of 1-ethyl-3-methylimidazole dimethyl phosphate, 1-ethyl-3-methylimidazole diethyl phosphate, 1-ethyl-3-methylimidazole phosphorus acetate or 1-ethyl-3-methylimidazole dicyan amine salt.
The ionic liquid has good solubility for various inorganic salts, organic matters, inorganic matters, polymers and other matters, and comprises weak polar matters and strong polar matters (such as carbohydrate). This allows some reactions to proceed in a homogeneous phase, increasing the reaction decellularization rate. On the other hand, the ionic liquid has better stability, and can form two phases after the reaction in a reaction system, so that the ionic liquid and the product are easy to separate, and the ionic liquid has higher recycling property than the traditional solvent.
Preferably, the rotation speed of the continuous stirring in the step (2) is 400-700 rpm.
Preferably, the rotational speed of the centrifugation in the step (3) is 8000-12000 rpm.
Preferably, the organic solvent in the step (4) is any one of ethanol, propanol, diethyl ether or petroleum ether. Washing the precipitate is favorable for improving the purity of the collagen and retaining the bioactivity of the collagen.
It is another object of the present invention to provide a composite collagen obtained by the above preparation method.
Another object of the present invention is to provide the use of the above-mentioned composite collagen for the preparation of biological tissue material.
Preferably, the biological tissue material is a biological material related to skin tissue regeneration, repair or remodeling.
According to the technical scheme, the invention discloses a composite collagen capable of recruiting stem cells, and a preparation method and application thereof. The invention adopts specific 1-ethyl-3-methylimidazole salt ionic liquid, utilizes the ionic liquid to dissolve cell membranes of animal-derived tissues and organs through osmotic effect and the like, separates DNA from protein, and effectively removes cell substances in the tissues to realize the decellularization effect. The process can remove antigen components capable of causing immune rejection reaction, simultaneously completely preserve the composition and three-dimensional space structure of collagen in extracellular matrix, and the 1-ethyl-3-methylimidazole salt ionic liquid can be recycled, thereby saving energy and protecting environment.
The composite collagen material obtained by the invention has good tissue compatibility, no immune rejection phenomenon when implanted into a human body, good biomechanical property, and can recruit and support human tissue stem cells in the human body, and meanwhile, the unique natural supermolecule three-dimensional space structure is particularly beneficial to the stem maintenance and differentiation of skin stem cells and the support of regeneration, repair and remodeling of skin tissues.
Drawings
FIG. 1 is a flow chart of a method for preparing composite collagen according to the present invention;
fig. 2 is a graph of differentiation of recruited stem cells after injection of complex collagen into the skin.
Detailed Description
The present invention will be further described with reference to examples, but the scope of the invention as claimed is not limited to the scope expressed by the examples.
Example 1
A method for preparing composite collagen capable of recruiting stem cells, comprising the following steps:
(1) Cutting 100g pigskin into Huang Douli pieces, repeatedly washing with deionized water for 2-3 times, transferring to isopropanol, soaking for 12 hr, and slowly stirring for 5min every 2 hr; repeatedly washing pigskin particles with deionized water for 3-4 times after isopropanol is removed;
(2) Transferring the pigskin particles into 1-ethyl-3-methylimidazole dimethyl phosphate, soaking and stirring for 15 hours under the protection of nitrogen, and filtering to obtain a solid product; wherein the stirring speed is 400rpm, and the mass concentration of the 1-ethyl-3-methylimidazole dimethyl phosphate solution is 60%;
(3) Transferring the solid product into a centrifuge, and centrifuging at a speed of 10000rpm to obtain a precipitate;
(4) Washing the precipitate with propanol for 3 times, washing with deionized water, and soaking in deionized water for 4 hr to remove residual ionic liquid;
(5) And (3) freeze-drying the precipitate washed in the step (4) in a sterile environment to obtain the composite collagen.
Example 2
A method for preparing composite collagen capable of recruiting stem cells, comprising the following steps:
(1) Cutting 100g of cowhide into Huang Douli pieces, repeatedly washing for 2-3 times by deionized water, transferring to isopropanol, soaking for 12 hours, and slowly stirring for 5 minutes every 2 hours in the soaking period; repeatedly washing the cow leather particles with deionized water for 3-4 times after removing isopropanol;
(2) Transferring the cow leather particles into 1-ethyl-3-methylimidazole diethyl phosphate, soaking and stirring for 15 hours under the protection of nitrogen, and filtering to obtain a solid product; wherein the stirring speed is 500rpm, and the mass concentration of the 1-ethyl-3-methylimidazole diethyl phosphate solution is 40%;
(3) Transferring the solid product into a centrifuge, and centrifuging at a high speed of 8000rpm to obtain a precipitate;
(4) Washing the precipitate with diethyl ether for 3 times, washing with deionized water, and soaking in deionized water for 4 hr to remove residual ionic liquid;
(5) And (3) freeze-drying the precipitate washed in the step (4) in a sterile environment to obtain the composite collagen.
Example 3
A method for preparing composite collagen capable of recruiting stem cells, comprising the following steps:
(1) Cutting 100g of sheep skin into Huang Douli pieces, repeatedly washing for 2-3 times by deionized water, transferring to isopropanol, soaking for 10 hours, and slowly stirring for 5 minutes every 2 hours in the soaking period; repeatedly washing the sheepskin particles with deionized water for 3-4 times after removing isopropanol;
(2) Transferring the sheepskin particles into 1-ethyl-3-methylimidazole dicyandiamide salt, soaking and stirring for 15 hours under the protection of argon, and filtering to obtain a solid product; wherein the stirring speed is 600rpm, and the mass concentration of the 1-ethyl-3-methylimidazole dicyandiamide salt solution is 60%;
(3) Transferring the solid product into a centrifuge, and centrifuging at a high speed of 12000rpm to obtain a precipitate;
(4) Washing the precipitate with petroleum ether for 3 times, washing with deionized water, and soaking in deionized water for 4 hr to remove residual ionic liquid;
(5) And (3) freeze-drying the precipitate washed in the step (4) in a sterile environment to obtain the composite collagen.
Example 4
A method for preparing composite collagen capable of recruiting stem cells, comprising the following steps:
(1) Cutting 100g of snake skin into Huang Douli pieces, repeatedly washing for 2-3 times by deionized water, transferring to isopropanol, soaking for 18 hours, and slowly stirring for 5 minutes every 2 hours in the soaking period; repeatedly washing the sheepskin particles with deionized water for 3-4 times after removing isopropanol;
(2) Transferring the snake skin particles into 1-ethyl-3-methylimidazole diethyl phosphate, soaking and stirring for 12 hours under the protection of argon, and filtering to obtain a solid product; wherein the stirring speed is 600rpm, and the mass concentration of the 1-ethyl-3-methylimidazole diethyl phosphate solution is 70%;
(3) Transferring the solid product into a centrifuge, and centrifuging at a speed of 10000rpm to obtain a precipitate;
(4) Washing the precipitate with ethanol for 3 times, washing with deionized water, and soaking in deionized water for 4 hr to remove residual ionic liquid;
(5) And (3) freeze-drying the precipitate washed in the step (4) in a sterile environment to obtain the composite collagen.
Example 5
A method for preparing composite collagen capable of recruiting stem cells, comprising the following steps:
(1) Cutting 100g pigskin into Huang Douli pieces, repeatedly washing with deionized water for 2-3 times, transferring to isopropanol, soaking for 12 hr, and slowly stirring for 5min every 2 hr; repeatedly washing pigskin particles with deionized water for 3-4 times after isopropanol is removed;
(2) Transferring the pigskin particles into 1-ethyl-3-methylimidazole dimethyl phosphate, soaking and stirring for 15 hours under the protection of argon, and filtering to obtain a solid product; wherein the stirring speed is 600rpm, and the mass concentration of the 1-ethyl-3-methylimidazole dimethyl phosphate solution is 80%;
(3) Transferring the solid product into a centrifuge, and centrifuging at a high speed of 8000rpm to obtain a precipitate;
(4) Washing the precipitate with ethanol for 3 times, washing with deionized water, and soaking in deionized water for 4 hr to remove residual ionic liquid;
(5) And (3) freeze-drying the precipitate washed in the step (4) in a sterile environment to obtain the composite collagen.
Example 6
A method for preparing composite collagen capable of recruiting stem cells, comprising the following steps:
(1) Cutting 100g of cowhide into Huang Douli pieces, repeatedly washing with deionized water for 2-3 times, transferring to isopropanol, soaking for 10 hr, and slowly stirring for 5min every 2 hr; repeatedly washing the cow leather particles with deionized water for 3-4 times after removing isopropanol;
(2) Transferring the cow leather particles into 1-ethyl-3-methylimidazole diethyl phosphate, soaking and stirring for 12 hours under the protection of nitrogen, and filtering to obtain a solid product; wherein the stirring speed is 700rpm, and the mass concentration of the 1-ethyl-3-methylimidazole diethyl phosphate solution is 50%;
(3) Transferring the solid product into a centrifugal machine, and centrifuging at a speed of 10000rpm at a high speed to obtain a precipitate;
(4) Washing the precipitate with diethyl ether for 3 times, washing with deionized water, and soaking in deionized water for 4 hr to remove residual ionic liquid;
(5) And (3) freeze-drying the precipitate washed in the step (4) in a sterile environment to obtain the composite collagen.
And (3) collecting the filtrate A in any embodiment step (2) and the filtrate B for washing the precipitate in the corresponding embodiment step (4), combining the filtrate A and the filtrate B as a unit volume, adding deionized water with a volume which is 1.2 times that of the filtrate A and the filtrate B, uniformly mixing, and removing water by a reduced pressure distillation method to obtain the corresponding 1-ethyl-3-methylimidazole salt ionic liquid which can be recycled.
The composite collagen obtained in any of the above examples, which recruits stem cells, was injected subcutaneously into animals (as shown in fig. 1), and after one month it was seen that mesenchymal stem cells appeared between the skin and the injected extracellular matrix (normal differentiated tissues did not have such cells); the composite collagen can also be used in biomedical fields such as cartilage tissue engineering scaffold materials, hemostatic materials, bone repair, skin repair and the like.
The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above examples, and all technical solutions belonging to the concept of the present invention belong to the protection scope of the present invention. It should be noted that modifications and adaptations to the present invention may occur to one skilled in the art without departing from the principles of the present invention and are intended to be within the scope of the present invention.
Claims (10)
1. A method for preparing composite collagen capable of recruiting stem cells, comprising the steps of:
(1) Pretreatment of raw materials: cutting biological tissues containing collagen into Huang Douli pieces, repeatedly washing the biological tissues with deionized water for 2-3 times, soaking the biological tissues with isopropanol, and rewashing the biological tissues with deionized water for 3-4 times after the soaking is finished;
(2) Immersing the ionic liquid: immersing the washed biological tissue particles containing collagen in 1-ethyl-3-methylimidazole salt ionic liquid, continuously stirring for 5-18 hours under the protection of argon or nitrogen, and filtering to obtain a solid product;
(3) And (3) centrifuging: carrying out high-speed centrifugation on the solid product obtained in the step (2) to obtain a precipitate;
(4) Washing: washing the precipitate in the step (3) for 3 times by adopting an organic solvent, and soaking the precipitate in deionized water for 3-5 hours;
(5) And (3) drying: and (3) freeze-drying the precipitate soaked in the step (4) in a sterile environment to obtain the composite collagen.
2. The method of claim 1, wherein the collagen-containing biological tissue of step (1) is any one of pigskin, cow hide, sheep skin or snake skin.
3. The method for preparing composite collagen capable of recruiting stem cells according to claim 1, wherein the isopropanol soaking in step (1) is specifically: the soaking time is 10-12 h, and the soaking is slowly stirred for 5min at intervals of 2 h.
4. The method for preparing composite collagen capable of recruiting stem cells according to claim 1, wherein the 1-ethyl-3-methylimidazole salt ionic liquid in the step (2) is any one of 1-ethyl-3-methylimidazole phosphate dimethyl salt, 1-ethyl-3-methylimidazole phosphate diethyl salt, 1-ethyl-3-methylimidazole phosphate acetate or 1-ethyl-3-methylimidazole dicyan salt.
5. The method for preparing composite collagen capable of recruiting stem cells according to claim 1, wherein the rotation speed of continuous stirring in the step (2) is 400-700 rpm.
6. The method for preparing composite collagen capable of recruiting stem cells according to claim 1, wherein the rotational speed of the centrifugation in the step (3) is 8000-12000 rpm.
7. The method for preparing composite collagen capable of recruiting stem cells according to claim 1, wherein the organic solvent in the step (4) is any one of ethanol, propanol, diethyl ether or petroleum ether.
8. A composite collagen obtainable by the method according to any one of claims 1 to 7.
9. Use of a composite collagen obtained according to the method of any one of claims 1 to 7 or according to claim 8 for the preparation of a biological tissue material.
10. The use according to claim 9, wherein the biological tissue material is a biological material associated with regeneration, repair or remodeling of skin tissue.
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