CN105497984A - Preparation and preservation method of amniotic membrane biological agent - Google Patents
Preparation and preservation method of amniotic membrane biological agent Download PDFInfo
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- CN105497984A CN105497984A CN201510965775.7A CN201510965775A CN105497984A CN 105497984 A CN105497984 A CN 105497984A CN 201510965775 A CN201510965775 A CN 201510965775A CN 105497984 A CN105497984 A CN 105497984A
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- amniotic membrane
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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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/0231—Chemically defined matrices, e.g. alginate gels, for immobilising, holding or storing cells, tissue or organs for preservation purposes; Chemically altering or fixing cells, tissue or organs, e.g. by cross-linking, for preservation purposes
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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
- A61L27/3695—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 function or physical properties of the final product, where no specific conditions are defined to achieve this
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Health & Medical Sciences (AREA)
- Transplantation (AREA)
- Public Health (AREA)
- Dermatology (AREA)
- Medicinal Chemistry (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Molecular Biology (AREA)
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- Animal Behavior & Ethology (AREA)
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Abstract
The invention provides a preparation and preservation method of an amniotic membrane biological agent. The method includes the first step of raw material selection and pretreatment, the second step of inactivation treatment of viruses, the third step of preparation, the fourth step of dyeing and the fifth step of preservation. Compared with the prior art, the method has following advantages that the preparation method is simple, raw materials are wide in source, large-scale industrial production can be achieved, prepared decellularized amniotic membranes are low in treatment strength and good in decellularization effect, and dense collagenous structures of natural amniotic membranes can be fully maintained. Amniotic membrane features are optimized easily, and important technical support can be provided for tissue engineering amniotic membrane industrialization.
Description
Technical field
The present invention is a kind of preparation and store method thereof of amniotic membrane biological preparation, belongs to medical biomaterial technical field.
Background technology
Tissue adhesion is the anomalous structure that connective fiber band and adjacent tissue or organ combine and formed, various post-operative complication can be caused: such as, tendon repair tissue adhesion, the serious recovery affecting operative site function, even can cause the forfeiture of local function.
Amniotic membrane is the internal layer of people's fetal membrane, comprises basement membrane the thickest in human body and avascular interstitial, and amniotic membrane immunogenicity is low, has and alleviates the effect such as inflammatory reaction, suppression proliferation of fibrous tissue, can be used as a kind of desirable organizational project repair materials.
The TBS buffer that patent CN1369555A and CN102327640A uses trypsin respectively, TBS-SDS(contains sodium lauryl sulphate) etc. reagent de-cell is carried out to amniotic membrane, the amniotic material prepared is thinner, easily curling, adhesion is poor, easily sliding when using comes off.In order to overcome above-mentioned shortcoming, patent CN100368534C employing crosslinking technological is prepared compound bio and is derived amniotic membrane to improve its operability, but employ the multiple organic reagents such as chloroform, methanol, glutaraldehyde during process, process intensity is large, destroy very large after multiple steps such as defat to the natural structure of amniotic membrane, cause collagen bundle irregular arrangement, collagen fiber rupture, simultaneously to remain risk high for a large amount of bioactie agent inactivating agent, and.For the deficiency of biologically-derived amniotic membrane, patent CN1943796A discloses a kind of manufacture method of cross-linking radiation holey human acellular amniotic membrane hydrogel adjuvant, propose the concept of aerogel dressing, but adopt trypsin and ethylenediaminetetraacetic acid (EDTA) to carry out de-cell to amniotic membrane, very easily destroy the natural structure of amniotic membrane, collagen fiber are caused to rupture, reduce the mechanical property of material, and in this patent, employ cross-linking radiation and the irradiation sterilization step of high dose, generation often along with some side reactions in cross-linking radiation process reduces the safety of product, twice irradiation process can the space collagen structure of the natural amniotic membrane of large havoc.Obtained medicine amniotic membrane after amniotic membrane and somatomedin soak by patent CN101040616A, but do not improve thinner, the easily curling operational drawbacks of monolayer amniotic membrane yet, and also the somatomedin after soaking only is attached to amniotic membrane surface, and slow-releasing is poor.
Summary of the invention
For the deficiency that prior art exists, the object of the invention is to provide a kind of preparation and store method thereof of amniotic membrane biological preparation, and to solve the problem proposed in above-mentioned background technology, the present invention is easy to use, convenient operation, good stability, and reliability is high.
To achieve these goals, the present invention realizes by the following technical solutions: a kind of preparation of amniotic membrane biological preparation and store method thereof, comprise the following steps:
A) the choosing and pretreatment of raw material;
B) inactivation treatment is carried out to virus;
C) preparation process;
D) dye;
E) preserve.
Further, the mode of operation that described steps A is concrete is: after obtaining the informed consent of cesarean patient, obtain its postcesarean amniotic membrane, cesarean patient is feminine gender through hepatitis B virus, hepatitis C virus, chlamydia, HIV (human immunodeficiency virus) and syphilis inspection in advance, clean the bloodstain on its surface with physiological saline solution, clean multipass with physiological saline solution.
Further, the concrete mode of operation of described step B is: after being soaked with the PBS containing antibiotics by fresh amnion, soak time is 5-10min, PBS rinses 3 times × 5min, and antibiotics comprises 50,000 U/L penicillins, 80,000 U/L tobramycins, one or more combination in 100mg/L streptomycin, or be soaked in 1 ~ 3h in 75% alcoholic solution, or be soaked in 10 ~ 30min in 0.1% ~ 0.5% peracetic acid soln, then clean 3 ~ 5 times by purified water.
Further, the concrete mode of operation of described step C is: blunt separation between amniotic membrane and chorion, obtain smooth, after translucent amniotic membrane, sterile phosphate buffer is rinsed well, 0.25% trypsin, 37 ° of water bath with thermostatic control enzymolysis 30 minutes, fully clean up with sterile phosphate buffer, then amniotic membrane is laid on aseptic glass plate, its residual epithelial cell is scraped with cell sleaker, spongy layer, fiber mother layer cellular layer and serous exudate, amniotic membrane is laid on 0.45 μ nitrocellulose membrane, in hundred grades of sterile purifications, dry, taking-up epithelium posterius faces up, be laid on the nitrocellulose filter paper containing 0.45um micropore, and be cut into 3cm × 4cm size, be rolled into tubular and use silk thread tighten.
Further, the concrete mode of operation of described step C is: to remove human amnion membrane after digestion under 0.125% pancreatin 37 °, digestion one hour is continued with 0.01% collagenase and DNA enzymatic mixed liquor 37 °, put in agitator simultaneously and vibrate, the cell of centrifugal rear acquisition and human amnion mesenchymal stem cell.
Further, the dyeing pigment in described step D is Gentian Violet, and wherein temperature is 16 DEG C-37 DEG C, dyeing time 30s-5min.
Further, the mode of operation that described step e is concrete is: take off and the amniotic membrane handled well is placed in dmem culture medium, 4 DEG C of Refrigerator stores, uses in 24 hours.
Further, the mode of operation that described step e is concrete is: take off and the amniotic membrane handled well is put into 100% pure glycerin bottle, 4 DEG C of Refrigerator stores, after 24 hours, under sterile working, then moves in another 100% pure glycerin bottle, continues 4 DEG C of Refrigerator stores.
Beneficial effect of the present invention: the preparation of a kind of amniotic membrane biological preparation of the present invention and store method thereof, preparation method is simple, raw material sources are extensive, large-scale industrialization can be realized produce, low, the de-cell effect of prepared human acellular amniotic membrane process intensity is good, fully can retain the dense collagenous structure of natural amniotic membrane, the present invention is not only conducive to optimizing amniotic membrane characteristic, can provide important technical support for the industrialization of organizational project amniotic membrane simultaneously.
Detailed description of the invention
The technological means realized for making the present invention, creation characteristic, reaching object and effect is easy to understand, below in conjunction with detailed description of the invention, setting forth the present invention further.
The invention provides a kind of technical scheme: a kind of preparation of amniotic membrane biological preparation and store method thereof, comprise the following steps:
A) the choosing and pretreatment of raw material;
B) inactivation treatment is carried out to virus;
C) preparation process;
D) dye;
E) preserve.
The mode of operation that steps A is concrete is: after obtaining the informed consent of cesarean patient, obtain its postcesarean amniotic membrane, cesarean patient is feminine gender through hepatitis B virus, hepatitis C virus, chlamydia, HIV (human immunodeficiency virus) and syphilis inspection in advance, clean the bloodstain on its surface with physiological saline solution, clean multipass with physiological saline solution.
The concrete mode of operation of step B is: after being soaked with the PBS containing antibiotics by fresh amnion, soak time is 5-10min, PBS rinses 3 times × 5min, antibiotics comprises 50,000 U/L penicillins, 80000 U/L tobramycins, one or more combination in 100mg/L streptomycin, or be soaked in 1 ~ 3h in 75% alcoholic solution, or be soaked in 10 ~ 30min in 0.1% ~ 0.5% peracetic acid soln, then clean 3 ~ 5 times by purified water.
The concrete mode of operation of step C is: blunt separation between amniotic membrane and chorion, obtain smooth, after translucent amniotic membrane, sterile phosphate buffer is rinsed well, 0.25% trypsin, 37 ° of water bath with thermostatic control enzymolysis 30 minutes, fully clean up with sterile phosphate buffer, then amniotic membrane is laid on aseptic glass plate, its residual epithelial cell is scraped with cell sleaker, spongy layer, fiber mother layer cellular layer and serous exudate, amniotic membrane is laid on 0.45 μ nitrocellulose membrane, in hundred grades of sterile purifications, dry, taking-up epithelium posterius faces up, be laid on the nitrocellulose filter paper containing 0.45um micropore, and be cut into 3cm × 4cm size, be rolled into tubular and use silk thread tighten.
The concrete mode of operation of step C is: to remove human amnion membrane after digestion under 0.125% pancreatin 37 °, digestion one hour is continued with 0.01% collagenase and DNA enzymatic mixed liquor 37 °, put in agitator simultaneously and vibrate, the cell of centrifugal rear acquisition and human amnion mesenchymal stem cell.
Dyeing pigment in step D is Gentian Violet, and wherein temperature is 16 DEG C-37 DEG C, dyeing time 30s-5min.
The mode of operation that step e is concrete is: take off and the amniotic membrane handled well is placed in dmem culture medium, 4 DEG C of Refrigerator stores, uses in 24 hours.
The mode of operation that step e is concrete is: take off and the amniotic membrane handled well is put into 100% pure glycerin bottle, 4 DEG C of Refrigerator stores, after 24 hours, under sterile working, then moves in another 100% pure glycerin bottle, continues 4 DEG C of Refrigerator stores.
As a kind of embodiment of the present invention, comprise the following steps: after obtaining the informed consent of cesarean patient, obtain its postcesarean amniotic membrane, cesarean patient is in advance through hepatitis B virus, hepatitis C virus, chlamydia, HIV (human immunodeficiency virus) and syphilis inspection are feminine gender, the bloodstain on its surface is cleaned with physiological saline solution, multipass is cleaned with physiological saline solution, after fresh amnion is soaked with the PBS containing antibiotics, soak time is 5-10min, PBS rinses 3 times × 5min, antibiotics comprises 50,000 U/L penicillins, 80000 U/L tobramycins, one or more combination in 100mg/L streptomycin, blunt separation between amniotic membrane and chorion, obtain smooth, after translucent amniotic membrane, sterile phosphate buffer is rinsed well, 0.25% trypsin, 37 ° of water bath with thermostatic control enzymolysis 30 minutes, fully clean up with sterile phosphate buffer, then amniotic membrane is laid on aseptic glass plate, its residual epithelial cell is scraped with cell sleaker, spongy layer, fiber mother layer cellular layer and serous exudate, amniotic membrane is laid on 0.45 μ nitrocellulose membrane, in hundred grades of sterile purifications, dry, taking-up epithelium posterius faces up, be laid on the nitrocellulose filter paper containing 0.45um micropore, and be cut into 3cm × 4cm size, be rolled into tubular and use silk thread tighten, after dyeing process, take off and the amniotic membrane handled well is placed in dmem culture medium, 4 DEG C of Refrigerator stores, use in 24 hours, preparation method of the present invention is simple, raw material sources are extensive, large-scale industrialization can be realized produce, prepared human acellular amniotic membrane process intensity is low, de-cell effect is good, fully can retain the dense collagenous structure of natural amniotic membrane, the present invention is not only conducive to optimizing amniotic membrane characteristic, important technical support can be provided for the industrialization of organizational project amniotic membrane simultaneously.
More than show and describe ultimate principle of the present invention and principal character and advantage of the present invention, to those skilled in the art, obviously the invention is not restricted to the details of above-mentioned one exemplary embodiment, and when not deviating from spirit of the present invention or basic feature, the present invention can be realized in other specific forms.Therefore, no matter from which point, all should embodiment be regarded as exemplary, and be nonrestrictive, scope of the present invention is limited by claims instead of above-mentioned explanation, and all changes be therefore intended in the implication of the equivalency by dropping on claim and scope are included in the present invention.Any Reference numeral in claim should be considered as the claim involved by limiting.
In addition, be to be understood that, although this description is described according to embodiment, but not each embodiment only comprises an independently technical scheme, this narrating mode of description is only for clarity sake, those skilled in the art should by description integrally, and the technical scheme in each embodiment also through appropriately combined, can form other embodiments that it will be appreciated by those skilled in the art that.
Claims (8)
1. the preparation of amniotic membrane biological preparation and a store method thereof, is characterized in that: comprise the following steps:
A) the choosing and pretreatment of raw material;
B) inactivation treatment is carried out to virus;
C) preparation process;
D) dye;
E) preserve.
2. the preparation of a kind of amniotic membrane biological preparation according to claim 1 and store method thereof, is characterized in that: the mode of operation that described steps A is concrete is:
After obtaining the informed consent of cesarean patient, obtain its postcesarean amniotic membrane, cesarean patient is feminine gender through hepatitis B virus, hepatitis C virus, chlamydia, HIV (human immunodeficiency virus) and syphilis inspection in advance, clean the bloodstain on its surface with physiological saline solution, clean multipass with physiological saline solution.
3. the preparation of a kind of amniotic membrane biological preparation according to claim 1 and store method thereof, is characterized in that: the concrete mode of operation of described step B is:
After fresh amnion is soaked with the PBS containing antibiotics, soak time is 5-10min, PBS rinses 3 times × 5min, antibiotics comprises 50,000 U/L penicillins, 80000 U/L tobramycins, one or more combination in 100mg/L streptomycin, or be soaked in 1 ~ 3h in 75% alcoholic solution, or be soaked in 10 ~ 30min in 0.1% ~ 0.5% peracetic acid soln, then clean 3 ~ 5 times by purified water.
4. the preparation of a kind of amniotic membrane biological preparation according to claim 1 and store method thereof, is characterized in that: the concrete mode of operation of described step C is:
Blunt separation between amniotic membrane and chorion, obtain smooth, after translucent amniotic membrane, sterile phosphate buffer is rinsed well, 0.25% trypsin, 37 ° of water bath with thermostatic control enzymolysis 30 minutes, fully clean up with sterile phosphate buffer, then amniotic membrane is laid on aseptic glass plate, its residual epithelial cell is scraped with cell sleaker, spongy layer, fiber mother layer cellular layer and serous exudate, amniotic membrane is laid on 0.45 μ nitrocellulose membrane, in hundred grades of sterile purifications, dry, taking-up epithelium posterius faces up, be laid on the nitrocellulose filter paper containing 0.45um micropore, and be cut into 3cm × 4cm size, be rolled into tubular and use silk thread tighten.
5. the preparation of a kind of amniotic membrane biological preparation according to claim 1 and store method thereof, is characterized in that: the concrete mode of operation of described step C is:
To remove human amnion membrane after digestion under 0.125% pancreatin 37 °, continue digestion one hour with 0.01% collagenase and DNA enzymatic mixed liquor 37 °, put in agitator simultaneously and vibrate, the cell of centrifugal rear acquisition and human amnion mesenchymal stem cell.
6. the preparation of a kind of amniotic membrane biological preparation according to claim 1 and store method thereof, it is characterized in that: the dyeing pigment in described step D is Gentian Violet, wherein temperature is 16 DEG C-37 DEG C, dyeing time 30s-5min.
7. the preparation of a kind of amniotic membrane biological preparation according to claim 1 and store method thereof, is characterized in that: the mode of operation that described step e is concrete is:
Take off and the amniotic membrane handled well is placed in dmem culture medium, 4 DEG C of Refrigerator stores, using in 24 hours.
8. the preparation of a kind of amniotic membrane biological preparation according to claim 1 and store method thereof, is characterized in that: the mode of operation that described step e is concrete is:
Take off and the amniotic membrane handled well is put into 100% pure glycerin bottle, 4 DEG C of Refrigerator stores, after 24 hours, under sterile working, then moving in another 100% pure glycerin bottle, continue 4 DEG C of Refrigerator stores.
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Cited By (3)
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| CN107714609A (en) * | 2017-11-08 | 2018-02-23 | 郑楠 | A kind of amnion basement membrane, amnion facial mask based on amnion basement membrane and preparation method thereof |
| CN109182260A (en) * | 2018-09-11 | 2019-01-11 | 邵勇 | A kind of method of in vitro culture fetal membrane mescenchymal stem cell |
| CN113456893A (en) * | 2021-07-26 | 2021-10-01 | 温州医科大学附属眼视光医院 | Preparation method of fibrinogen-coated blue-dyed amnion basement membrane |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107714609A (en) * | 2017-11-08 | 2018-02-23 | 郑楠 | A kind of amnion basement membrane, amnion facial mask based on amnion basement membrane and preparation method thereof |
| CN109182260A (en) * | 2018-09-11 | 2019-01-11 | 邵勇 | A kind of method of in vitro culture fetal membrane mescenchymal stem cell |
| CN113456893A (en) * | 2021-07-26 | 2021-10-01 | 温州医科大学附属眼视光医院 | Preparation method of fibrinogen-coated blue-dyed amnion basement membrane |
| CN113456893B (en) * | 2021-07-26 | 2022-04-26 | 温州医科大学附属眼视光医院 | Preparation method of fibrinogen-coated blue-dyed amnion basement membrane |
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