CN115300674A - Preparation method and application of composite collagen capable of recruiting stem cells - Google Patents
Preparation method and application of composite collagen capable of recruiting stem cells Download PDFInfo
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- CN115300674A CN115300674A CN202210969335.9A CN202210969335A CN115300674A CN 115300674 A CN115300674 A CN 115300674A CN 202210969335 A CN202210969335 A CN 202210969335A CN 115300674 A CN115300674 A CN 115300674A
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- 238000000034 method Methods 0.000 claims abstract description 15
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
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Images
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
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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
- A61L27/14—Macromolecular materials
- 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
- A61L27/3604—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
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- A—HUMAN NECESSITIES
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—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
Abstract
The invention relates to a method for preparing composite collagen capable of recruiting stem cells, which comprises the steps of immersing biological tissues containing collagen in 1-ethyl-3-methylimidazolium salt ionic liquid, continuously stirring for 5-18 hours under the protection of inert gas, filtering to obtain solid products, carrying out high-speed centrifugation on the solid products to obtain precipitates, and sequentially washing and drying the precipitates to obtain the composite collagen capable of recruiting stem cells.
Description
Technical Field
The invention relates to the technical field of biological materials, in particular to a preparation method of composite collagen capable of recruiting stem cells.
Background
Stem cells are cells that have not been differentiated, i.e., cells that have no specific function. Skin cells can protect the body, muscle cells can contract, and nerve cells can transmit information. The stem cells do not have any special structure or function, but have the potential to become any cell in the human body.
Stem cells do not differentiate from the "soil" environment, and the extracellular matrix serves as the "soil" environment. The extracellular matrix is all components except cells in tissues, including a matrix (collagen, polysaccharide and glycoprotein) in a homogeneous state and filamentous collagen fibers, has the function of connecting and supporting cells, is a basic framework and a metabolic site for cell attachment, and has the shape and the function of directly influencing the shape and the function of the formed tissues. The processed composite glue material has good mechanical property, good histocompatibility, no immunological rejection phenomenon when implanted into human body, and functions of supporting and connecting cells in human body, and the three-dimensional space structure and cell factor are favorable for 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 multiple treatment steps and serious pollution. The problems of waste acid discharge and the like exist in the acid hydrolysis process, and the efficiency of the enzyme hydrolysis process is low, so that the collagen resource is not fully utilized. The collagen is easy to be combined by hydrogen bonds and intertwined with each other in a wet state, so that the collagen is difficult to disperse by a simple mechanical or chemical method, and the collagen is difficult to disperse by physical means such as freeze grinding and the like in a dry state due to the characteristics of the toughness and the like of the collagen fibers, so that the collagen fibers are easy to damage due to excessive physical action.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a preparation method of composite collagen capable of recruiting stem cells, which creatively uses ionic liquid to dissolve cell membranes of animal-derived tissues and organs through osmosis effect and the like, separates DNA from protein, and effectively removes cell substances in the tissues to realize the effect of decellularization.
In order to achieve the purpose, the invention is realized by the following technical scheme.
A preparation method of composite collagen capable of recruiting stem cells comprises the steps of immersing biological tissues containing collagen in 1-ethyl-3-methylimidazolium salt ionic liquid, continuously stirring for 5-18 hours under the protection of inert gas, filtering to obtain a solid product, carrying out high-speed centrifugation on the solid product to obtain a precipitate, and sequentially washing and drying the precipitate to obtain the composite collagen capable of recruiting stem cells.
Optionally, the 1-ethyl-3-methylimidazole salt ionic liquid comprises one or more of 1-ethyl-3-methylimidazole dimethyl phosphate, 1-ethyl-3-methylimidazole diethyl phosphate, 1-ethyl-3-methylimidazole phosphorus acetate, and 1-ethyl-3-methylimidazole dicyandiamide salt. The ionic liquid has good solubility for various substances such as inorganic salts, organic substances, inorganic substances, polymers and the like, and comprises substances with weak polarity and substances with strong polarity (such as carbohydrate). This enables some reactions to be carried out in homogeneous phase, increasing the reaction decellularisation rate. On the other hand, the catalyst has better stability, and can form two phases after reaction in a reaction system, so that the ionic liquid and a product are easy to separate, and the catalyst has higher recoverability and reusability compared with the traditional solvent.
Optionally, the collagen-containing biological tissue is one of pigskin, cow skin, sheep skin and snake skin, and is preferably pigskin.
Optionally, the collagen-containing biological tissue needs to be pretreated before being immersed in the 1-ethyl-3-methylimidazolium salt ionic liquid, wherein the pretreatment is to cut the collagen-containing biological tissue into particles, and sequentially carry out initial washing with deionized water, immersion in isopropanol, and rewashing with deionized water.
Optionally, the inert gas is argon or nitrogen.
Optionally, the precipitate is washed with any one of ethanol, propanol, diethyl ether and petroleum ether, then soaked and washed with deionized water for 3-5 hours, and then dried.
Optionally, the precipitate is dried in a sterile environment.
The application has the following beneficial effects: creatively uses the ionic liquid to dissolve cell membranes of animal-derived tissues and organs through osmosis effect and the like, separates DNA from protein, and effectively removes cell substances in the tissues to realize the effect of decellularization. The process can remove antigen components capable of causing immunological rejection, while completely retaining the composition and three-dimensional spatial structure of collagen in extracellular matrix. The composite gel raw material obtained by the invention has good histocompatibility, no immune rejection phenomenon when being implanted into a body, and good biomechanical property, can recruit and support human tissue stem cells in the body, has a specific natural supermolecule three-dimensional space structure, and is particularly beneficial to the dryness maintenance and differentiation of the skin stem cells and the support of the regeneration, repair and remodeling of the skin tissue.
Drawings
FIG. 1 is a flow diagram of a method for producing complex collagen that recruits stem cells;
fig. 2 is a graph of the differentiation of recruited stem cells after injection of complex collagen into the skin.
Detailed Description
The invention is further described below with reference to examples, but the scope of the invention as claimed is not limited to the examples.
Example 1
A preparation method of composite collagen capable of recruiting stem cells comprises the following steps:
(1) Cutting 100g of pigskin into soybean grains, repeatedly washing with deionized water for 2-3 times, then transferring to isopropanol to soak for 12 hours, and slowly stirring for 5 minutes every 2 hours during the soaking period; repeatedly washing the pigskin particles for 3-4 times by using deionized water after removing the isopropanol;
(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 percent;
(3) Transferring the solid product into a centrifuge, and centrifuging at a high 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 (4) freeze-drying the precipitate washed in the step (4) in a sterile environment to obtain the compound collagen capable of recruiting stem cells.
Example 2
A preparation method of composite collagen capable of recruiting stem cells comprises the following steps:
(1) Cutting 100g of cow leather into soybean particles, repeatedly washing with deionized water for 2-3 times, transferring into isopropanol, soaking for 12 hours, and slowly stirring for 5 minutes every 2 hours during soaking; after removing isopropanol, repeatedly washing the cow leather particles for 3-4 times by using deionized water;
(2) Transferring the cowhide 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 percent;
(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 ether for 3 times, washing with deionized water, and soaking in deionized water for 4 hr to remove residual ionic liquid;
(5) And (4) freeze-drying the precipitate washed in the step (4) in a sterile environment to obtain the compound collagen capable of recruiting stem cells.
Example 3
A preparation method of composite collagen capable of recruiting stem cells comprises the following steps:
(1) Cutting 100g of sheepskin into soybean grains, repeatedly washing with deionized water for 2-3 times, then transferring to isopropanol to soak for 10 hours, and slowly stirring for 5 minutes every 2 hours during the soaking period; after removing isopropanol, repeatedly washing the sheepskin particles for 3-4 times by using deionized water;
(2) Transferring the sheepskin particles into 1-ethyl-3-methylimidazole dicyandiamide salt, soaking and stirring for 15 hours under the protection of argon gas, 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 percent;
(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 (4) freeze-drying the precipitate washed in the step (4) in a sterile environment to obtain the compound collagen capable of recruiting stem cells.
Example 4
A preparation method of composite collagen capable of recruiting stem cells comprises the following steps:
(1) Cutting 100g of snake skin into soybean particles, repeatedly washing with deionized water for 2-3 times, then transferring the snake skin into isopropanol to soak for 18 hours, and slowly stirring for 5 minutes every 2 hours during the soaking period; after removing isopropanol, repeatedly washing sheepskin particles for 3-4 times by using deionized water;
(2) Transferring the snakeskin particles into 1-ethyl-3-methylimidazole diethyl phosphate, soaking and stirring for 12 hours under the protection of argon gas, 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 salt solution is 70 percent;
(3) Transferring the solid product into a centrifuge, and centrifuging at a high 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 (4) freeze-drying the precipitate washed in the step (4) in a sterile environment to obtain the compound collagen capable of recruiting stem cells.
Example 5
A preparation method of composite collagen capable of recruiting stem cells comprises the following steps:
(1) Cutting 100g of pigskin into soybean grains, repeatedly washing the pigskin for 2 to 3 times by using deionized water, then transferring the pigskin into isopropanol to soak for 12 hours, and slowly stirring the pigskin for 5 minutes every 2 hours during the soaking period; repeatedly washing the pigskin particles for 3-4 times by using deionized water after removing the isopropanol;
(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 percent;
(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 (5) freeze-drying the precipitate washed in the step (4) in a sterile environment to obtain the compound collagen capable of recruiting stem cells.
Example 6
A preparation method of composite collagen capable of recruiting stem cells comprises the following steps:
(1) Cutting 100g of cow leather into soybean particles, repeatedly washing with deionized water for 2-3 times, transferring into isopropanol, soaking for 10 hours, and slowly stirring for 5 minutes every 2 hours during soaking; after removing isopropanol, repeatedly washing the cow leather particles for 3-4 times by using deionized water;
(2) Transferring the cowhide 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 percent;
(3) Transferring the solid product into a centrifuge, and centrifuging at a high speed of 10000rpm 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 (4) freeze-drying the precipitate washed in the step (4) in a sterile environment to obtain the compound collagen capable of recruiting stem cells.
And (3) collecting the filtrate A in the step (2) of any embodiment and the filtrate B for washing the precipitate in the step (4) of the corresponding embodiment, combining the filtrate A and the filtrate B to be a unit volume, adding 1.2 times of volume of deionized water into the unit volume, uniformly mixing, and removing water by using a reduced pressure distillation method to obtain the corresponding 1-ethyl-3-methylimidazolium salt ionic liquid which can be recycled.
The compound collagen capable of recruiting stem cells obtained in any of the above examples was injected subcutaneously into animals (as shown in figure 1), and after one month, mesenchymal stem cells appeared between the skin and the injected extracellular matrix (normal differentiated tissues do 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 embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and adaptations to those skilled in the art without departing from the principles of the present invention should also be considered as within the scope of the present invention.
Claims (8)
1. A preparation method of composite collagen capable of recruiting stem cells is characterized in that biological tissues containing collagen are immersed in 1-ethyl-3-methylimidazolium salt ionic liquid, continuously stirred for 5-18 hours under the protection of inert gas, then filtered to obtain solid products, the solid products are subjected to high-speed centrifugation to obtain precipitates, and the precipitates are sequentially washed and dried to obtain the composite collagen capable of recruiting stem cells.
2. The method for preparing complex collagen capable of recruiting stem cells according to claim 1, wherein the ionic liquid of 1-ethyl-3-methylimidazole salt comprises one or more of dimethyl 1-ethyl-3-methylimidazole phosphate, diethyl 1-ethyl-3-methylimidazole phosphate, 1-ethyl-3-methylimidazole phosphorus acetate, and 1-ethyl-3-methylimidazole dicyandiamide salt.
3. The method for preparing composite collagen capable of recruiting stem cells according to claim 1, wherein the biological tissue containing collagen is selected from pigskin, cow skin, sheep skin, snake skin, and preferably pigskin.
4. The method for preparing composite collagen capable of recruiting stem cells according to claim 1, wherein the collagen-containing biological tissue is pre-treated before being immersed in the 1-ethyl-3-methylimidazolium salt ionic liquid, and the pre-treatment comprises cutting the collagen-containing biological tissue into particles, and sequentially carrying out initial washing with deionized water, soaking with isopropanol, and re-washing with deionized water.
5. The method for preparing composite collagen for recruiting stem cells according to claim 1, wherein the inert gas is argon or nitrogen.
6. The method for preparing composite collagen for recruiting stem cells according to claim 1, wherein said precipitate is washed with any one of ethanol, propanol, ether and petroleum ether, then soaked in deionized water for 3-5 hours, and then dried.
7. The method for preparing a complex collagen for recruiting stem cells according to claim 1, wherein the precipitate is dried in a sterile environment.
8. Use of a method for producing composite collagen according to any one of claims 1 to 7 for recruiting stem cells to produce a biological tissue material.
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| CN103554247A (en) * | 2013-10-28 | 2014-02-05 | 大连工业大学 | Method for preparing collagen microfiber by use of ionic liquid mixing solvent |
| US20170006858A1 (en) * | 2014-03-24 | 2017-01-12 | Kingstronbio (Changshu) Co., Ltd. | Acellular collagenous tissue and a processing method of artificial heart valve comprising the acellular collagenous tissue |
| CN108640987A (en) * | 2018-06-26 | 2018-10-12 | 四川大学 | A method of undenatured natural collagen is extracted with ionic liquid pretreatment |
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| CN103554247A (en) * | 2013-10-28 | 2014-02-05 | 大连工业大学 | Method for preparing collagen microfiber by use of ionic liquid mixing solvent |
| US20170006858A1 (en) * | 2014-03-24 | 2017-01-12 | Kingstronbio (Changshu) Co., Ltd. | Acellular collagenous tissue and a processing method of artificial heart valve comprising the acellular collagenous tissue |
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