CN108778353A - Artificial composite membrane, preparation method and applications - Google Patents

Artificial composite membrane, preparation method and applications Download PDF

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Publication number
CN108778353A
CN108778353A CN201680012639.9A CN201680012639A CN108778353A CN 108778353 A CN108778353 A CN 108778353A CN 201680012639 A CN201680012639 A CN 201680012639A CN 108778353 A CN108778353 A CN 108778353A
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preparation
collagen
added
composite membrane
type collagen
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Inventor
邢孟秋
商海涛
魏泓
蒋坤
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SHENZHEN QIANHAI JINZHUO BIOTECHNOLOGY Co.,Ltd.
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Shenzhen Chengnong Biomaterials Co ltd
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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
    • 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
    • A61L27/14—Macromolecular materials
    • A61L27/26—Mixtures of macromolecular compounds
    • 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
    • A61L27/58—Materials at least partially resorbable by the body

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Chemical & Material Sciences (AREA)
  • Dermatology (AREA)
  • Medicinal Chemistry (AREA)
  • Transplantation (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Biophysics (AREA)
  • Materials For Medical Uses (AREA)
  • Peptides Or Proteins (AREA)

Abstract

A kind of preparation method of artificial composite membrane, including fibroin albumen is mixed with collagen, and it is fabricated to composite membrane.A kind of artificial composite membrane is the artificial composite membrane made by the preparation method.The application of the reparation of spinal meninges after a kind of reparation or operation on vertebra by by the artificial composite membrane of the preparation method making for dura mater after operations on cranium and brain.The composite membrane is greatly improved film strength, to overcome disadvantage of the existing collagen film in graft application.

Description

Artificial composite membrane, preparation method and applications Technical field
The present invention relates to biomedical material, especially a kind of artificial composite membrane, preparation method and applications.
Background technique
Meningioma resection, craniocerebral injury and it is some other be related to endocranium damage in the case where, the transplanting of dura mater promotes its regeneration particularly important for substituting the dura mater of defect.Dura mater reparation not can lead to many complication, such as adhesion, leakage of cerebrospinal (CSF), infection entirely.Though some biomaterials can be obtained from androgynous or allosome, the propagation of disease may also result in, cause rejection, and scar, adhesion are still inevitable.The method that tradition repairs damage dura mater is directly to suture, but be not easy to suture after certain site tissue damages.Some synthetic polymers, as again expanded PTFE (ePTFE) can be expanded because of Fibrin Glue.
Ideal graft should meet the following conditions: (1) low (or nothing) immunogenicity, (2) good stretching, extension intensity, (3) pore size is suitable, it (4) can anti-leakage of cerebrospinal, (5) resist blocking and that, (6) suitable degradation rate, (7) are easy to surgical procedure.
Collagen film has apparent advantage in terms of transplanting, and has been successfully applied to clinic.Collagen is the main component of extracellular matrix, is the necessary material of cell survival and division.Since it is with good biocompatibility and biodegradability, therefore cell adherence can be promoted.Collagen is clinically applied to grafting of dura nearly century.However two big disadvantages limit its extensive use.Tensile capacity, shear resistance are poor to limit the application of collagen film during surgery.In addition, the purity of collagen also will affect therapeutic effect.
Summary of the invention
It is a primary object of the present invention to overcome the above-mentioned deficiency of the prior art, artificial composite membrane, the preparation method and applications of a kind of high intensity are provided.
To achieve the above object, the invention adopts the following technical scheme:
A kind of preparation method of artificial composite membrane, including fibroin albumen is mixed with collagen, and be fabricated to composite membrane.
Further, the collagen is I-type collagen.
Further, the fibroin albumen is extracted from silk cocoon.
Further, the I-type collagen is extracted from the knot hoof tissue such as ox tendon of animal.
Further, the method includes I-type collagen preparation step, the I-type collagen preparation step includes:
Sterilization treatment is carried out to ox tendon；
Tissue homogenate is made in ox tendon；
Remove the adipose tissue in ox tendon；
Remove the polysaccharide in ox tendon；
NaCl solution is added, taking precipitate obtains I-type collagen；
I-type collagen is purified through membrane filtration.
Further, the I-type collagen preparation step further include:
Purified I-type collagen is added in glacial acetic acid and is made it dissolve；
Centrifugal treating is carried out to solution, takes supernatant；
NaCl solution is added into supernatant, then carries out centrifugal treating, taking precipitate；
Sediment is added in glacial acetic acid, is purified through membrane filtration, filtrate adds in distilled water, then purifies through membrane filtration；
It is freeze-dried purified I-type collagen, obtains powder I-type collagen.
Further, after taking the sediment, sediment is successively added in the glacial acetic acid that concentration is 2~4%, 0.8~1.5%, 0.2~0.7% and is purified through filter membrane, added in distilled water and purified through filter membrane.
Further, the method includes fibroin albumen preparation step, the fibroin albumen preparation step includes:
By silk cocoon in Na2CO3It is boiled in solution to remove colloid；
The silk cocoon of degumming is dissolved in containing alcohol and CaCl2Mixed solution in；
Silk cocoon solution is filtered with aseptic filter paper；
Filtrate is added in distilled water, then purifies through membrane filtration；
It is freeze-dried purified fibroin albumen, obtains powder fibroin albumen.
Further, fibroin albumen is placed in acetum with I-type collagen and is mixed, then carry out homogenized, then make the composite membrane with resulting slurry.
Further, the pH value of the acetum is 2.5~3.5.
Further, homogenized 1~3 hour at 2~6 DEG C.
Further, resulting slurry is injected in mold, under the conditions of freeze-drying, the uniform film in aperture is made；Film is placed in vacuum oven baking, makes its crosslinking.
Further, the I-type collagen and the weight ratio of fibroin albumen are (3:1)~(5:1).
A kind of artificial composite membrane is the artificial composite membrane made by the preparation method.
A kind of application being used for the reparation of spinal meninges after the reparation of dura mater after operations on cranium and brain or operation on vertebra by the artificial composite membrane of the described preparation method production.
Beneficial effects of the present invention:
The present invention introduces fibroin albumen in collagen slurry for the first time to overcome the disadvantage of simple collagen protein film tension, shear resistance difference.Researches show that fibroin albumens to have good biocompatibility and biological degradability, and intensity is much higher than collagen.The composite membrane that both natural biopolymers are mixed with is greatly improved film strength, to overcome disadvantage of the collagen film in graft application.Technique of the invention can extract high purity collagen, and prepared composite membrane has good biocompatibility, in addition, it also has uniform microcellular structure and good toughness, be convenient for surgical procedure and suture.The dura mater that can be used to substitute excision in neurosurgery by artificial composite membrane prepared by method of the invention promotes dura mater regeneration for the reparation and regeneration of dura mater after operations on cranium and brain.
Specific embodiment
It elaborates below to embodiments of the present invention.It is emphasized that following the description is only exemplary, the range and its application being not intended to be limiting of the invention.
In one embodiment, a kind of preparation method of artificial composite membrane, including fibroin albumen is mixed with the preferred I-type collagen of collagen, and be fabricated to high-intensitive composite membrane.The artificial composite membrane is a kind of uniform cellular matrix, and thickness can be adjusted according to clinic needs, such as 6mm.High-intensitive artificial composite membrane includes I-type collagen and fibroin, and natural polymerization collagen and fibroin albumen can be extracted from animal tendon and silk cocoon respectively.Collagen and fibroin are natural protein, have good biocompatibility and degradation capability, have the characteristics that no antigen and low immunogenicity to human body.
Embodiment 1
Preparation flow is as follows:
One, the I-type collagen of high-purity is extracted
A, sterilization treatment is carried out to ox tendon with ultraviolet disinfection and 70% alcohol of concentration.
Ox is sent between clean operation as early as possible after putting to death 10 grams of ox tendons of acquirement, and disinfection by ultraviolet light or the sprinkling of 70% alcohol of concentration in case use in next step.
B, ox tendon is put into homogenate blender under the conditions of 4 DEG C and tissue homogenate is made.
Tissue is made in ox tendon using homogenate blender to be homogenized, dry ice, which is added, in this process prevents collagenous degeneration.
C, acetone (concentration > 98%) is added, sufficiently impregnates 3 days, to remove the adipose tissue in tendon.
10 grams of ox tendons are soaked into 50ml acetone (concentration > 98%), the acetone of replacement in every 24 hours.Whole operation carries out under the conditions of 4 DEG C.
D, concentration 0.3%NaH is added2PO4, impregnate 3 days, remove the polysaccharide in tendon.
Polysaccharide can increase the viscosity of collagen, cause organism immune response.4 DEG C, by the concentration 0.3%NaH of 100ml2PO4It is added in the ox tendon of removal adipose tissue, impregnates 3 days, the NaH of replacement in every 12 hours2PO4。
E, concentration 10%NaCl solution is added, obtains I-type collagen.
Collagen is slightly mentioned with concentration 10%NaCl solution first.NaCl solution is added in ox tendon, 4 DEG C overnight, and the white fiber of precipitation is collagen.
F, collagen is filtered and is purified 24 hours through filter membrane (trapped molecular weight 20,000D) in distilled water.
Collagen deposit is placed in the chimney filter that trapped molecular weight is 20,000D, is put into the beaker for filling distilled water, at 4 DEG C, is placed 24 hours, is replaced primary distilled water for every eight hours.
G, concentration 3% (v/v) glacial acetic acid is added in collagen, impregnates 3 days, sufficiently dissolves.
Under the conditions of 4 DEG C, in collagen be added concentration 3% (v/v) glacial acetic acid, impregnate 3 days sufficiently to dissolve, the glacial acetic acid solution of replacement in every 12 hours.It is glacial acetic acid solution used that 3ml glacial acetic acid, which is added in 997ml distilled water, and storage at 4 DEG C is in case use.
H, the centrifugation for carrying out 14,000g, takes supernatant.
Collagen solution moves into 50ml high speed centrifugation pipe, and at 4 DEG C, supernatant is collected in 14,000g centrifugations 45 minutes.
I, concentration 10%NaCl solution is added, 14,000g centrifugations take collagen deposit object.
Supernatant is moved into test tube, concentration 10%NaCl solution is added, ice bath is overnight, and the gummy white fiber of precipitation is collagen.
J, the gummy white fiber of precipitation is successively added in 3%, 1%, 0.5% glacial acetic acid of concentration, after through membrane filtration and respectively purifying 24 hours, is put into distilled water through membrane filtration and purifies 3 days, obtain the collagen of purifying.
K, freeze-drying obtains solid powder.
The collagen of purifying moves into test tube, moves into freeze dryer after placing 48 hours at -80 DEG C, can be obtained within about 4 days collagen freeze-dried powder, save under 4 DEG C of drying conditions.
Two, fibroin albumen is extracted from silk cocoon
A, in concentration 0.5%Na2CO3In boil silk cocoon to remove silk gum.
Concentration 0.5% (w/v) Na boiled is added in silk cocoon2CO3Solution distills water washing 3 times, removal sericin, 24 hours dry under the conditions of 65 DEG C in vacuum oven, spare.
B, under the conditions of 90 DEG C, the silk of degumming is dissolved in containing alcohol and CaCl2Mixed solution in obtain fibroin liquid.
100ml mixed solution C aCl2, distilled water, alcohol molar ratio be 1:8:2.
C, aseptic filter paper filters
Vacuum pump is evacuated under condition of negative pressure, and silk fibroin protein solution is filtered through filter paper.
D, silk fibroin protein solution is purified 72 hours through membrane filtration in distilled water
Filtrate moves into filtration pipe, places 3 days in distilled water to remove CaCl2And alcohol, every 12 hours replacement primary distilled waters.
E, freeze-drying obtains solid powder
The collagen of purifying is moved into test tube, freeze dryer is moved into after placing 48 hours at -80 DEG C, can be obtained within about 3~5 days collagen freeze-dried powder, saved under 4 DEG C of drying conditions.
Three, 0.8 gram of collagen and 0.25 gram of fibroin albumen is taken to be dissolved in 100ml acetic acid (PH=3) solution
Four, at 4 DEG C, homogenized 2 hours.
Collagen and fibroin albumen are added in homogenate blender, at 4 DEG C, homogenate is then moved into and is connected in the bottle of vacuum pump to remove bubble removing by 15,000rpm stirrings 2 hours.
Five, it is film-made
A, collagen fibroin albumen is homogenized in injection metal die.
75ml homogenate is poured into the metal die of 12cm × 12cm.
B, it freezes under (- 40 DEG C) drying condition, cellular film is made using controlled sequence.
Mold is put into freeze dryer by (20 DEG C) under room temperature, is cooled to cryogenic temperature (- 40 DEG C) with constant rate, and is placed 60 minutes at -40 DEG C；Vacuum environment (< 200mT) or 0 DEG C of 17 hour of placement are subsequently moved within, quality is uniform, cavernous solid composite membrane until being formed.
C, it is crosslinked film in 105 DEG C of vacuum ovens.
In Aluminium Foil Packing composite membrane, the sealing of three faces, recording quantity are crosslinked film in 105 DEG C of vacuum ovens, the thickness of most telolemma is about 6mm with evaporation water.
Six, film is cut into a certain size in case clinical application.
Seven, ethylene oxide sterilizing.
Eight, it packs to finished product.
Embodiment 2
Preparation flow is as follows:
One, the I-type collagen of high-purity is extracted
A, sterilization treatment is carried out to beef tendon with ultraviolet sterilization and 70% alcohol of concentration.
Ox, which is put to death, obtains ox tendon, takes 50 grams of ox tendons to be sent to clean operation room as early as possible, disinfection by ultraviolet light or the sprinkling of 70% alcohol of concentration in case use in next step.
B, fresh ox tendon is put into homogenate blender under the conditions of 4 DEG C and tissue homogenate is made.
Tissue homogenate is made in ox tendon using homogenate blender (dry ice, which is added, in this process prevents collagenous degeneration).
C, acetone (concentration > 98%) is added, sufficiently impregnates 3 days, to remove the adipose tissue in tendon.
500ml acetone (concentration > 98%) is added in 50 grams of ox tendons, impregnates 3 days, the acetone of replacement in every 24 hours, to remove the adipose tissue in ox tendon.Whole operation carries out under the conditions of 4 DEG C.
D, concentration 0.3%NaH is added2PO4, impregnate 3 days, remove the polysaccharide in tendon.
Polysaccharide can increase the viscosity of collagen, cause organism immune response.Under the conditions of 4 DEG C, the concentration 0.3%NaH that 1000ml is added in the ox tendon of adipose tissue is removed2PO4, impregnate 3 days, the NaH of replacement in every 12 hours2PO4。
E, concentration 10%NaCl solution is added, obtains I-type collagen.
Concentration 10%NaCl solution is added in ox tendon, slightly mentions collagen, and distilled water adjusts concentration, stays overnight at 4 DEG C, the White Flocculus being precipitated in solution is collagen.
F, collagen particle is filtered and is purified 24 hours through filter membrane (trapped molecular weight 20,000D) in distilled water.
Collagen deposit moves into the chimney filter that trapped molecular weight is 20,000D, is put into the beaker for filling distilled water, at 4 DEG C, places 24 hours, replaces primary distilled water for every eight hours.
G, concentration 3% (v/v) glacial acetic acid is added in collagen, impregnates 3 days, sufficiently dissolves.
Under the conditions of 4 DEG C, in collagen be added concentration 3% (v/v) glacial acetic acid, impregnate 3 days sufficiently to dissolve, the glacial acetic acid solution of replacement in every 12 hours.It is glacial acetic acid solution used that 9ml glacial acetic acid, which is added in 2991ml distilled water, and 4 DEG C of storages are in case use.
H, 14,000g centrifugation is carried out, supernatant is taken.
Collagen solution moves into 50ml high speed centrifugation pipe, and at 4 DEG C, supernatant is collected in 14,000g centrifugations 45 minutes.
I, concentration 10%NaCl solution, 14,000g centrifugations is added, taking precipitate obtains collagen.
Supernatant moves into test tube, concentration 10%NaCl solution is added, ice bath is overnight, and the gummy white fiber of precipitation is both collagen.
J, it is successively added in 3%, 1%, 0.5% glacial acetic acid of concentration, after through membrane filtration and respectively purifying 24 hours, is added in distilled water through membrane filtration and purifies 3 days, acquisition collagen.
K, freeze-drying obtains solid powder.
The collagen of purifying is moved into test tube, freeze dryer is moved into after placing 48 hours at -80 DEG C, can be obtained within about 4 days 45 grams of collagen freeze-dried powder, saved under drying condition at 4 DEG C.
Two, fibroin albumen is extracted from silk cocoon
A, in concentration 0.5%Na2CO3In boil silk cocoon to remove silk gum.
Concentration 0.5% (w/v) Na boiled is added in silk cocoon2CO3Solution distills water washing several times, removal sericin, 24 hours dry under the conditions of 65 DEG C in vacuum oven, spare.
B, under the conditions of 90 DEG C, the silk cocoon (10g) of degumming is dissolved in containing alcohol and CaCl2Mixed solution in.
Obtain fibroin liquid, 100mL mixed solution C aCl2, distilled water, alcohol molar ratio be 1:8:2.
C, aseptic filter paper filters.
Vacuum pump is evacuated under condition of negative pressure, and silk fibroin protein solution is filtered through filter paper.
D, silk fibroin protein solution is purified 72 hours after membrane filtration in distilled water.
Filtrate is moved into filtration pipe, places 3 days in distilled water to remove CaCl2And alcohol, replacement in distilled water every 12 hours are primary.
E, freeze-drying obtains solid powder.
The collagen of purifying is moved into test tube, freeze dryer is moved into after placing 48 hours at -80 DEG C, can be obtained within about 3~5 days collagen freeze-dried powder, saved under 4 DEG C of drying conditions.
Three, 0.8 gram of collagen and 0.25 gram of fibroin albumen is taken to be dissolved in 100ml acetic acid (PH=3) solution.
Four, it at 4 DEG C, is homogenized 2 hours.
Collagen and fibroin albumen are added in homogenate blender, at 4 DEG C, homogenate is then moved into and is connected in the bottle of vacuum pump to remove bubble removing by 15,000rpm stirrings 2 hours.
Five, it is film-made
A, in collagen fibroin albumen homogenate injection metal die.
300ml homogenate is poured into the metal die of 24cm × 24cm, totally 18 molds.
B, it freezes under (- 40 DEG C) drying condition, cellular film is made using controlled sequence.
Mold is put into freeze dryer by (20 DEG C) under room temperature, is cooled to cryogenic temperature (- 40 DEG C) with constant rate, and is placed 60 minutes at -40 DEG C；Vacuum environment (< 200mT) or 0 DEG C of 17 hour of placement are subsequently moved within, quality is uniform, cavernous solid composite membrane until being formed.
C, it is crosslinked film in 105 DEG C of vacuum ovens.
In Aluminium Foil Packing composite membrane, the sealing of three faces, recording quantity are crosslinked film in 105 DEG C of vacuum ovens, the thickness of most telolemma is about 6mm with evaporation water.
Six, film is cut into a certain size in case clinical application.
Seven, ethylene oxide sterilizing.
Eight, it packs to finished product.
Embodiment 3
Preparation process is as follows:
One, the I-type collagen of high-purity is extracted
A, sterilization treatment is carried out to ox tendon with ultraviolet sterilization and 70% alcohol of concentration
Ox takes 500 grams of ox tendons to be sent to clean operation room as early as possible after putting to death, disinfection by ultraviolet light or the sprinkling of 70% alcohol of concentration in case use in next step.
B, ox tendon is put into homogenate blender under the conditions of 4 DEG C and tissue homogenate is made.
Tissue homogenate is made in ox tendon using homogenate blender (dry ice, which is added, in this process prevents collagenous degeneration).
C, acetone (concentration > 98%) is added, sufficiently impregnates 3 days, to remove the adipose tissue in tendon.
5000ml acetone (concentration > 98%) is added in 500 grams of ox tendons, impregnates 3 days, the acetone of replacement in every 24 hours, to remove the adipose tissue in ox tendon.Whole operation carries out under the conditions of 4 DEG C.
D, concentration 0.3%NaH is added2PO4, impregnate 3 days, remove the polysaccharide in tendon.
Polysaccharide can increase collagen viscosity, cause organism immune response.Under the conditions of 4 DEG C, by 10 liters of concentration 0.3%NaH2PO4It is added in the tendon of removal adipose tissue, impregnates 3 days, the NaH of replacement in every 12 hours2PO4。
E, concentration 10%NaCl solution is added, obtains I-type collagen.
Concentration 10%NaCl solution is added in ox tendon, slightly mentions collagen, and distilled water adjusts concentration, stays overnight at 4 DEG C, the White Flocculus of precipitation is collagen.
F, in distilled water by collagen through filter membrane (trapped molecular weight 20,000D) filtered pure Change 24 hours.
Collagen deposit moves into the chimney filter that trapped molecular weight is 20,000D, is put into the beaker for filling distilled water, places 24 hours at 4 DEG C, replace primary distilled water for every eight hours.
G, concentration 3% (v/v) glacial acetic acid is added in collagen, impregnates 3 days, sufficiently dissolves.
At 4 DEG C, in collagen be added concentration 3% (v/v) glacial acetic acid, impregnate 3 days sufficiently to dissolve, the glacial acetic acid solution of replacement in every 12 hours.It is glacial acetic acid solution used that 90ml glacial acetic acid, which is added in 29910ml distilled water, and storage at 4 DEG C is in case use.
H, 14,000g is centrifuged, and takes supernatant.
Collagen solution moves into 50ml high speed centrifugation pipe, and at 4 DEG C, supernatant is collected in 14,000g centrifugations 45 minutes.
I, concentration 10%NaCl solution is added, 14,000g centrifugations take collagen deposit object.
Supernatant moves into test tube, concentration 10%NaCl solution is added, ice bath is overnight, and the gummy white fiber of precipitation is both collagen.
J, it successively in 3%, 1%, 0.5% glacial acetic acid of concentration, after through membrane filtration and respectively purifying 24 hours, through membrane filtration and is purified 3 days in distilled water, obtains collagen.
K, freeze-drying obtains solid powder.
The collagen of purifying is put into test tube, and freeze dryer is moved into after placing 48 hours at -80 DEG C, 45 grams of collagen freeze-dried powder is can be obtained within about 4 days, saves under drying condition at 4 DEG C.
Two, fibroin albumen is extracted from silk cocoon
A, in concentration 0.5%Na2CO3In boil silk cocoon to remove silk gum.
Concentration 0.5% (w/v) Na boiled is added in silk cocoon2CO3Solution distills water washing several times, removal sericin, 24 hours dry under the conditions of 65 DEG C in vacuum oven, spare.
B, at 90 DEG C, the silk (100g) of degumming is dissolved in containing alcohol and CaCl2Solution in obtain fibroin liquid.
1L mixed solution C aCl2, distilled water, alcohol molar ratio be 1:8:2.
C, aseptic filter paper filters.
Vacuum pump is evacuated under condition of negative pressure, and silk fibroin protein solution is filtered through filter paper.
D, silk fibroin protein solution through membrane filtration and is purified 72 hours in distilled water
Filtrate moves into filtration pipe, places 3 days in distilled water to remove CaCl2And alcohol, replacement in distilled water every 12 hours are primary.
E, freeze-drying obtains solid powder.
The collagen of purifying moves into test tube, moves into freeze dryer after placing 48 hours at -80 DEG C, about 3~ It can be obtained within 5 days collagen freeze-dried powder, saved under drying condition at 4 DEG C.
Three, 0.8 gram of collagen and 0.25 gram of fibroin albumen is taken to be dissolved in 100ml acetic acid (PH=3) solution.
Four, it at 4 DEG C, is homogenized 2 hours.
Collagen and fibroin albumen are added in homogenate blender, and at 4 DEG C, homogenate is then moved into and is connected in the bottle of vacuum pump to remove bubble removing by 15,000rpm stirrings 2 hours.
Five, it is film-made
A, collagen fibroin albumen is homogenized in injection metal die.
300ml homogenate is poured into the metal die of 24cm × 24cm, totally 180 molds.
B, it freezes under (- 40 DEG C) drying condition, cellular film is made using controlled sequence.
Mold is put into freeze dryer by (20 DEG C) under room temperature, is cooled to cryogenic temperature (- 40 DEG C) with constant rate, and is placed 60 minutes at -40 DEG C；Vacuum environment (< 200mT) or 0 DEG C of 17 hour of placement are subsequently moved within, quality is uniform, cavernous solid composite membrane until being formed.
C, it is crosslinked film in 105 DEG C of vacuum ovens.
In Aluminium Foil Packing composite membrane, the sealing of three faces, recording quantity are crosslinked film in 105 DEG C of vacuum ovens, the thickness of most telolemma is about 6mm with evaporation water.
Six, film is cut into a certain size in case clinical application.
Seven, ethylene oxide sterilizing.
Eight, it packs to finished product.
Embodiment 4
The collagen composite membrane of previous embodiment is used for the reparation of the dura defect of human or animal.In surgical procedure, film can be trimmed to a certain size, soak, and be attached at animal brain surface.The animal that the experimental animal of application composite membrane opens wide with dura mater in art or tightly sutures is compared.Crown middle line arc incision, right top is micro- to open cranium, avoids damage to Cerebral cortex and cuts off size about 15 × 15mm2Endocranium, the endocranium substitute for placing suitable size tightly sutures in brain surface and with dura mater around.
Embodiment 5
The collagen composite membrane of previous embodiment is used for the reparation of spinal meninges after human or animal's operation on vertebra.
The above content is combine specific/preferred embodiment made for the present invention further specifically It is bright, and it cannot be said that specific implementation of the invention is only limited to these instructions.For those of ordinary skill in the art to which the present invention belongs; without departing from the inventive concept of the premise; it can also make some replacements or modifications to the embodiment that these have been described, and these substitutions or variant all shall be regarded as belonging to protection scope of the present invention.

Claims (15)

  1. A kind of preparation method of artificial composite membrane, which is characterized in that including mixing fibroin albumen with collagen, and be fabricated to composite membrane.
  2. Preparation method as described in claim 1, which is characterized in that the collagen is I-type collagen.
  3. Preparation method as described in claim 1, which is characterized in that the fibroin albumen is extracted from silk cocoon.
  4. Preparation method as claimed in claim 2, which is characterized in that the I-type collagen is preferably extracted from ox tendon from the knot hoof tissue extraction of animal.
  5. Preparation method as claimed in claim 2, which is characterized in that including I-type collagen preparation step, the I-type collagen preparation step includes:
    Sterilization treatment is carried out to ox tendon；
    Tissue homogenate is made in ox tendon；
    Remove the adipose tissue in ox tendon；
    Remove the polysaccharide in ox tendon；
    NaCl solution is added, taking precipitate obtains I-type collagen；
    I-type collagen is purified through membrane filtration.
  6. Preparation method as claimed in claim 5, which is characterized in that the I-type collagen preparation step further include:
    Purified I-type collagen is added in glacial acetic acid and is made it dissolve；
    Centrifugal treating is carried out to solution, takes supernatant；
    NaCl solution is added into supernatant, then carries out centrifugal treating, taking precipitate；
    Sediment is added in glacial acetic acid, is purified through membrane filtration, filtrate adds in distilled water, then purifies through membrane filtration；
    It is freeze-dried purified I-type collagen, obtains powder I-type collagen.
  7. Preparation method as claimed in claim 6, which is characterized in that after taking the sediment, sediment is successively added in the glacial acetic acid that concentration is 2~4%, 0.8~1.5%, 0.2~0.7% and is purified through filter membrane, added in distilled water and purified through filter membrane.
  8. Such as the described in any item preparation methods of claim 2 to 7, which is characterized in that including fibroin albumen preparation step, the fibroin albumen preparation step includes:
    By silk cocoon in Na2CO3It is boiled in solution to remove colloid；
    The silk cocoon of degumming is dissolved in containing alcohol and CaCl2Mixed solution in；
    Silk cocoon solution is filtered with aseptic filter paper；
    Filtrate is added in distilled water, then purifies through membrane filtration；
    It is freeze-dried purified fibroin albumen, obtains powder fibroin albumen.
  9. Such as any one of claim 2 to 8 to the preparation method, which is characterized in that be placed in acetum fibroin albumen with I-type collagen and mix, then carry out homogenized, then make the composite membrane with resulting slurry.
  10. Such as claim 9 to the preparation method, which is characterized in that the pH value of the acetum is 2.5~3.5.
  11. Such as claim 9 to the preparation method, which is characterized in that homogenized 1~3 hour at 2~6 DEG C.
  12. Preparation method as claimed in claim 9, which is characterized in that resulting slurry is injected in mold, under the conditions of freeze-drying, the uniform film in aperture is made；Film is placed in vacuum oven baking, makes its crosslinking.
  13. Such as any one of claim 2 to 12 to the preparation method, which is characterized in that the I-type collagen and the weight ratio of fibroin albumen are (3:1)~(5:1).
  14. A kind of artificial composite membrane, which is characterized in that be by the artificial composite membrane of the described in any item preparation method production of such as claim 1 to 13.
  15. A kind of application being used for the reparation of spinal meninges after the reparation of dura mater after operations on cranium and brain or operation on vertebra by the artificial composite membrane of such as claim 1 to 13 described in any item preparation methods production.
CN201680012639.9A 2016-11-18 2016-11-18 Artificial composite membrane, preparation method and applications Pending CN108778353A (en)

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