CN106620847A - Collagen biological membrane and preparation method of collagen biological membrane - Google Patents
Collagen biological membrane and preparation method of collagen biological membrane Download PDFInfo
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- CN106620847A CN106620847A CN201611010805.XA CN201611010805A CN106620847A CN 106620847 A CN106620847 A CN 106620847A CN 201611010805 A CN201611010805 A CN 201611010805A CN 106620847 A CN106620847 A CN 106620847A
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- collagen
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- slurries
- biological film
- heel string
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- 108010035532 Collagen Proteins 0.000 title claims abstract description 111
- 102000008186 Collagen Human genes 0.000 title claims abstract description 111
- 229920001436 collagen Polymers 0.000 title claims abstract description 111
- 238000002360 preparation method Methods 0.000 title claims abstract description 26
- 239000012528 membrane Substances 0.000 title abstract 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 51
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 39
- 239000002002 slurry Substances 0.000 claims description 33
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 31
- 239000007788 liquid Substances 0.000 claims description 31
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 26
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 26
- 235000019836 ficin Nutrition 0.000 claims description 22
- 238000000034 method Methods 0.000 claims description 22
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 20
- 239000004365 Protease Substances 0.000 claims description 20
- 108090000270 Ficain Proteins 0.000 claims description 18
- POTUGHMKJGOKRI-UHFFFAOYSA-N ficin Chemical compound FI=CI=N POTUGHMKJGOKRI-UHFFFAOYSA-N 0.000 claims description 18
- 238000009938 salting Methods 0.000 claims description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 14
- 239000004310 lactic acid Substances 0.000 claims description 13
- 235000014655 lactic acid Nutrition 0.000 claims description 13
- 238000004108 freeze drying Methods 0.000 claims description 12
- 229910019142 PO4 Inorganic materials 0.000 claims description 11
- 239000007800 oxidant agent Substances 0.000 claims description 11
- 230000001590 oxidative effect Effects 0.000 claims description 11
- 239000010452 phosphate Substances 0.000 claims description 11
- 239000008363 phosphate buffer Substances 0.000 claims description 11
- 238000004132 cross linking Methods 0.000 claims description 9
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 8
- 150000003839 salts Chemical class 0.000 claims description 8
- 239000011780 sodium chloride Substances 0.000 claims description 8
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 6
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 6
- 239000007789 gas Substances 0.000 claims description 6
- 229910001919 chlorite Inorganic materials 0.000 claims description 4
- 229910052619 chlorite group Inorganic materials 0.000 claims description 4
- QBWCMBCROVPCKQ-UHFFFAOYSA-N chlorous acid Chemical compound OCl=O QBWCMBCROVPCKQ-UHFFFAOYSA-N 0.000 claims description 4
- 238000010382 chemical cross-linking Methods 0.000 claims description 3
- 230000018044 dehydration Effects 0.000 claims description 3
- 238000006297 dehydration reaction Methods 0.000 claims description 3
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 3
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 3
- 235000011152 sodium sulphate Nutrition 0.000 claims description 3
- 230000008901 benefit Effects 0.000 abstract description 5
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- 239000011148 porous material Substances 0.000 abstract 2
- 239000000243 solution Substances 0.000 description 34
- 239000008213 purified water Substances 0.000 description 21
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 19
- 239000000463 material Substances 0.000 description 16
- 241001465754 Metazoa Species 0.000 description 10
- 230000001954 sterilising effect Effects 0.000 description 9
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- 239000000523 sample Substances 0.000 description 7
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
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- 238000005406 washing Methods 0.000 description 5
- YCSMVPSDJIOXGN-UHFFFAOYSA-N CCCCCCCCCCCC[Na] Chemical compound CCCCCCCCCCCC[Na] YCSMVPSDJIOXGN-UHFFFAOYSA-N 0.000 description 4
- IAYPIBMASNFSPL-UHFFFAOYSA-N Ethylene oxide Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000004140 cleaning Methods 0.000 description 4
- 230000007547 defect Effects 0.000 description 4
- 210000001951 dura mater Anatomy 0.000 description 4
- 238000011010 flushing procedure Methods 0.000 description 4
- 238000007710 freezing Methods 0.000 description 4
- 230000008014 freezing Effects 0.000 description 4
- 239000001257 hydrogen Substances 0.000 description 4
- 229910052739 hydrogen Inorganic materials 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 229920002401 polyacrylamide Polymers 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 150000001408 amides Chemical class 0.000 description 3
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- 229910052760 oxygen Inorganic materials 0.000 description 3
- 230000002980 postoperative effect Effects 0.000 description 3
- UKLNMMHNWFDKNT-UHFFFAOYSA-M sodium chlorite Chemical compound [Na+].[O-]Cl=O UKLNMMHNWFDKNT-UHFFFAOYSA-M 0.000 description 3
- 229960002218 sodium chlorite Drugs 0.000 description 3
- 230000008467 tissue growth Effects 0.000 description 3
- 241001269524 Dura Species 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
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- 230000002378 acidificating effect Effects 0.000 description 2
- 210000000576 arachnoid Anatomy 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000002308 calcification Effects 0.000 description 2
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- 238000004925 denaturation Methods 0.000 description 2
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- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- WGRULTCAYDOGQK-UHFFFAOYSA-M sodium;sodium;hydroxide Chemical compound [OH-].[Na].[Na+] WGRULTCAYDOGQK-UHFFFAOYSA-M 0.000 description 2
- 238000002211 ultraviolet spectrum Methods 0.000 description 2
- 102000012422 Collagen Type I Human genes 0.000 description 1
- 108010022452 Collagen Type I Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000283977 Oryctolagus Species 0.000 description 1
- 208000035965 Postoperative Complications Diseases 0.000 description 1
- 102000029797 Prion Human genes 0.000 description 1
- 108091000054 Prion Proteins 0.000 description 1
- 101800000868 Tail peptide Proteins 0.000 description 1
- 102400001102 Tail peptide Human genes 0.000 description 1
- 208000031737 Tissue Adhesions Diseases 0.000 description 1
- 108010077465 Tropocollagen Proteins 0.000 description 1
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- 150000001413 amino acids Chemical class 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
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- 230000011382 collagen catabolic process Effects 0.000 description 1
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- ZZUFCTLCJUWOSV-UHFFFAOYSA-N furosemide Chemical compound C1=C(Cl)C(S(=O)(=O)N)=CC(C(O)=O)=C1NCC1=CC=CO1 ZZUFCTLCJUWOSV-UHFFFAOYSA-N 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
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- 238000007917 intracranial administration Methods 0.000 description 1
- 230000006651 lactation Effects 0.000 description 1
- 238000005461 lubrication Methods 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 210000002418 meninge Anatomy 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 239000005445 natural material Substances 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000003534 oscillatory effect Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 150000003016 phosphoric acids Chemical class 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- -1 salt ion Chemical class 0.000 description 1
- 210000002027 skeletal muscle Anatomy 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
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- 229920002994 synthetic fiber Polymers 0.000 description 1
- 230000009772 tissue formation Effects 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
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Classifications
-
- 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
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
-
- 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
-
- 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
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Epidemiology (AREA)
- Medicinal Chemistry (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Transplantation (AREA)
- Dermatology (AREA)
- Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Biophysics (AREA)
- Materials For Medical Uses (AREA)
Abstract
The invention discloses a collagen biological membrane and a preparation method of the collagen biological membrane. The collagen biological membrane is characterized in that collagen in the collagen biological membrane has a triple-helical structure; the thickness of the collagen biological membrane is 0.2cm to 0.9cm; the collagen biological membrane has a three-dimensional pore diameter structure and the size of a pore diameter is 60mm to 300mm. The collagen biological membrane provided by the invention not only has high mechanical strength, but also has the advantages of absorbability and low immunogenicity; the collagen biological membrane can be used as a substitute good of endocranium, spinal membranes and the like.
Description
Technical field
The present invention relates to biology medical material technical field, more particularly to a kind of collagenous biological film and preparation method thereof.
Background technology
Collagen (Collagen), as the main component that a kind of boiomacromolecule is in animal connective tissue, is also that lactation is moved
Thing in-vivo content is most, be distributed most wide functional protein, accounts for the 25%～30% of total protein.With tissue formation, into
Ripe, cell-tocell transmission, and joint lubrication, wound healing, calcification, blood clotting and aging etc. have close
Relation.Collagen is also one of most critical raw material of biotech industry, in medical material, cosmetics, food industry etc.
Equal extensive application.On space structure, collagen shows the structure of special triple helix winding, and three separate
Collagen peptide chain maintains the structure that triple helix mutually winds by the hydrogen bond formed between glycine.Collagen is this special
Triple helix structure ensure that its mechanical strength.Collagenous fibres are the grown forms that collagen exercises physiological action, in organism
Interior collagenous fibres are woven into the network structure for being rich in mechanical strength and elasticity becomes the most basic constituent of connective tissue.
Endocranium is the important barrier together for protecting brain tissue, for the structure and functional activity meaning of safeguarding nervous system
It is great.Operative repair dura mater, closing cavum subdurale, can obviously reduce or prevent leakage of cerebrospinal, intracranial infection, the insane symptom such as lame.
Therefore endocranium layer is kept to be completely necessary after neurosurgery.Wound, tumour corrode and surgical procedure endocranium shrinkage
Dural defect can be caused etc. factor so that original position is closed cavum subdurale and cannot be completed, additionally, the decompression of neurosurgery
Generally need to expand hypostegal cavity.Such situation generally needs to complete dura mater reparation using dural substitutes.
Natural material and synthetic material are divided into absorbable material and nonabsorable material as endocranium substitute, this
Class material is easily formed the shapes and sizes of needs, easily sterilization, is not concerned about transmitting the risk of disease.
Absorbable material can finally be degraded in vivo, absorbed, and substituted by the new meninx for generating and repaired defect of meninges.Collagen-based
Matter is generally extracted from the heel string or skin of the animals such as ox, pig, can be a kind of absorbable biological material as dural repairment material
Material, similar with people's dura mater main component, its main component is NTx albumen.Meninx substitute is at present with variform
As film, thin slice, sponge are tested and clinical practice.Have after the enzyme treated removal of tail peptide of the NTx molecule of animal
There are extremely low immunogenicity, and the vascularization of inducible fibroblast adhesion hyperplasia and film, so as to accelerate to be implanted into material certainly
Body process.Theoretically, collagen is optimal meninx alternative materials as a kind of absorbable material.
Accordingly, it would be desirable to substitute of the high-quality collagenous biological film as endocranium and backbone film etc..
The content of the invention
To solve above-mentioned technical problem, the invention provides a kind of collagenous biological film and preparation method thereof.Technical scheme is such as
Under：
In a first aspect, the invention provides a kind of collagenous biological film, the collagen in collagenous biological film has triple helix
Structure；The thickness of collagenous biological film is 0.2-0.9cm, and with three-dimensional aperture structure, the size in aperture is 60-300mm.
In second aspect, the invention provides the preparation method of above-mentioned collagenous biological film, comprises the following steps,
Collagen is mixed with water, lactic acid solution and/or hydrochloric acid solution is added thereto to, makes collagen dispersed, make slurry
Liquid；
The pH value of the slurries is adjusted to 5.5-6.5, afterwards to the slurries vacuum defoamation, freeze-drying is then carried out；
It is crosslinked using physically and/or chemically method, that is, obtains collagenous biological film.
In the preferred embodiment of the present invention, mass fraction of the collagen in the slurries is 0.3%-
1.5%.
In the preferred embodiment of the present invention, the pH value of the slurries is 2.8-3.8.
In the preferred embodiment of the present invention, the freeze-drying is the bar in vacuum for 120-250 millitorrs
Under part, 4-10 DEG C of holding 40min, -45--35 DEG C of holding 120-210min, 0 DEG C of holding 20-24h, 20-25 DEG C of holding 2-9h.
In the preferred embodiment of the present invention, the Physical cross linking methods are：Irradiated using the ultraviolet of 254nm
7-13h, or the Physical cross linking methods are：Under vacuum 100-120 DEG C of processed 2-3 days, is cooled to afterwards 50
Below DEG C；The Chemical Crosslinking Methods are：It is crosslinked using formaldehyde gas.
In the preferred embodiment of the present invention, the collagen is obtained by following steps：
First ox heel string is cut into slices；
The mass fraction that the ox heel string of section is immersed in again ficin delays for the phosphate of 0.005%-0.2%
In rushing liquid, at 4-15 DEG C 12-24h is kept；
Then oxidant is added in the phosphate buffer, with water the ox heel string of the section is rinsed；The oxidation
At least one of the agent in chlorite and hydrogen peroxide；
Finally the ox heel string of the section is immersed in the salting liquid that NaOH molar concentration is 1-2mol/L, in 4-
Kept for 1-4 days at 15 DEG C, the pH value that the salting liquid is adjusted afterwards is 4-7, collects product, and product is cleaned repeatedly, is taken off
Water process, obtains collagen.
In one kind more preferably embodiment of the present invention, the mass fraction of the ficin is 0.05%-
0.1%；The pH value of the PBS is 5-7, and the molar concentration of phosphate radical is 0.01-0.1mol/L.
In one kind more preferably embodiment of the present invention, the addition volume of the oxidant is the phosphate buffer
1 ‰ -5 ‰；The oxidant is hydrogen peroxide.
In one kind more preferably embodiment of the present invention, based on the gross mass of the salting liquid, the quality point of the salt
Number is 10%-30%；At least one of the salt in the salting liquid in sodium chloride, sodium sulphate, sodium carbonate.
In one kind more preferably embodiment of the present invention, before cutting into slices to ox heel string, first to the ox heel string
Pre-processed；The pretreatment is that ox heel string is rinsed repeatedly with water, is afterwards 20%-75% ethanol with mass fraction
Solution rinses 15-45min, is rinsed repeatedly with water.
The beneficial effect of technical scheme provided in an embodiment of the present invention is：The collagenous biological film that the present invention is provided not only keeps
The biology premium properties of collagen, i.e. triple helix structure, high mechanical strength, and also it is absorbable, immunogenicity is low, contributes to
Tissue growth, promote repair, degrade it is controllable the advantages of, can be used as the substitute of endocranium and backbone film etc..
Description of the drawings
Technical scheme in order to be illustrated more clearly that the embodiment of the present invention, below will be to making needed for embodiment description
Accompanying drawing is briefly described, it should be apparent that, drawings in the following description are only some embodiments of the present invention, for
For those of ordinary skill in the art, on the premise of not paying creative work, can be obtaining other according to these accompanying drawings
Accompanying drawing.
Fig. 1 is the scanning electron microscope (SEM) photograph of the collagenous biological film shown in an exemplary embodiment of the invention；
Fig. 2 be the collagenous biological film shown in an exemplary embodiment of the invention preparation method in collagen SDS-PAGE it is electric
Swimming figure；
Fig. 3 be the collagenous biological film shown in an exemplary embodiment of the invention preparation method in collagen infrared spectrum
Figure；
Fig. 4 be the collagenous biological film shown in an exemplary embodiment of the invention preparation method in collagen ultraviolet spectra
Figure.
Specific embodiment
To make technical scheme and advantage clearer, below in conjunction with accompanying drawing embodiment of the present invention is made into
One step ground is described in detail.
In a first aspect, as shown in figure 1, the invention provides a kind of collagenous biological film, the collagen tool in collagenous biological film
There is triple helix structure；The thickness of collagenous biological film is 0.2-0.9cm, it is preferable that the thickness of collagenous biological film is 0.5cm；Tool
There is three-dimensional aperture structure, the size in aperture is 60-300mm, it is preferable that the size in aperture is 120-250mm.
The collagenous biological film that the present invention is provided not only maintains the biology premium properties of collagen, i.e. triple helix structure,
High mechanical strength, and absorbable, immunogenicity is low, contributes to tissue growth, promotes to repair, and the advantages of degrade controllable, can make
For the substitute of endocranium and backbone film etc..
In second aspect, the invention provides a kind of preparation method of collagenous biological film, comprises the following steps,
Collagen is mixed with water, lactic acid solution and/or hydrochloric acid solution is added thereto to, makes collagen dispersed, make slurry
Liquid；
The pH value of the slurries is adjusted to 5.5-6.5, afterwards to the slurries vacuum defoamation, then freeze-drying；
It is crosslinked using physically and/or chemically method, that is, obtains collagenous biological film.
The preparation method of the collagenous biological film of the present invention, collagen is mixed with water, is added thereto to lactic acid solution and/or salt
Acid solution, is presented collagen elongated fibrous, and slurries are presented transparent state；It is to freeze that the pH of slurries is adjusted to into 5.5-6.5
The pH value weakly acidic pH of the collagen sponge formed after dry, close to the pH value of human body；Lactic acid and dissolving with hydrochloric acid collagen are selected, even if newborn
Acid and hydrochloric acid have residual on collagenous biological film, and because lactic acid is human body own metabolism material, hydrochloric acid can generate sodium chloride, so
The residual of the two is not hindered to human body.Therefore, the preparation method of collagenous biological film of the invention meets the needs of human body.Not only such as
This, the collagenous biological film of the preparation method preparation of the collagenous biological film provided using the present invention maintains collagen original three strands
Helical structure, with high mechanical strength and absorbable, immunogenicity is low, contributes to tissue growth, promotes to repair, and degraded can
The advantages of control, can be used as the substitute of endocranium and backbone film etc..Additionally, the preparation method process is simple.
In actual applications, the collagen of the preparation method of the collagenous biological film that the present invention is provided can be from ox, pig etc.
The heel string or skin of animal, preferably ox heel string.
Further, in actual applications, it is possible to use low-concentration sodium hydroxide solution, such as the NaOH of 0.5mol/L will be starched
The pH value of liquid is adjusted to 5.5-6.5, and in the range of the pH, the pH value weakly acidic pH of the collagen sponge formed after freeze-drying connects
The pH value of person of modern times's body.
Further, during collagen is evenly spread to into lactic acid solution and/or hydrochloric acid solution, if finding there is insoluble matter, can mistake
Filter is processed, specifically can be using the stainless filter-cloth filtering of 40 purposes twice.
Used as the embodiment of a modification of the present invention, mass fraction of the collagen in the slurries is 0.3%-
1.5%.The molar concentration of lactic acid is 0.01-0.05mol/L in the lactic acid solution；Hydrochloric acid is mole dense in the hydrochloric acid solution
Spend for 0.05-0.1mol/L.Preferably, the pH value of the slurries is 2.8-3.8, in this pH value range, slurries clear
Degree is high, and in subsequent adjustment pH value, reduces the consumption of alkali, the i.e. consumption and residual of salt ion, obtains highly purified immunity
The lower collagenous biological film of originality.
As the present invention another kind of improved embodiment, the freeze-drying be vacuum be 120-250 millitorrs
Under conditions of, 4-10 DEG C of holding 40min, -45--35 DEG C of holding 120-210min, 0 DEG C of holding 20-24h, 20-25 DEG C of holding 2-
9h.This freeze-drying condition is conducive to collagenous biological film to keep collagen to have triple helix structure, and preferably controls collagen
Biomembranous thickness and three-dimensional aperture structure.
In a particular embodiment, it is preferable that the Physical cross linking methods are：7- is irradiated using the ultraviolet of 254nm
13h, or the Physical cross linking methods are：Under vacuum 100-120 DEG C of processed 2-3 days, is cooled to afterwards 50 DEG C
Below.Being vacuum dried the method for this severe dehydration can improve the stability of collagen, prevent tissue collagen matrix from subsiding, and pass through
Dehydration, shortens the distance between Collage Activitv base, causes to be crosslinked between tropocollagen molecule, improves denaturation temperature, reduces
The content of its free amino acid, so as to improve the mechanical strength of collagen.
In a particular embodiment, it is preferable that the Chemical Crosslinking Methods are：It is crosslinked using formaldehyde gas.
In actual applications, the formaldehyde gas that mass fraction has been given play to for the formalin of 5%-30% can be adopted to freezing
Collagen after dry carries out cross-linking modified, and the usual time is 3-5h.
The physically or chemically cross-linking method provided using the present invention is modified, is conducive to improving the tensile strength of collagen-based materials
And anti-degradation capability, its expansion rate is reduced, improve water-resistance of collagen etc..
Used as another improved embodiment of the present invention, the collagen is obtained by following steps：
First ox heel string is cut into slices；
The mass fraction that the ox heel string of section is immersed in again ficin delays for the phosphate of 0.005%-0.2%
In rushing liquid, at 4-15 DEG C 12-24h is kept；
Then oxidant is added in the phosphate buffer, with water the ox heel string of the section is rinsed；The oxidation
At least one of the agent in chlorite and hydrogen peroxide；
Finally the ox heel string of the section is immersed in the salting liquid that NaOH molar concentration is 1-2mol/L, in 4-
Kept for 1-4 days at 15 DEG C, the pH value that the salting liquid is adjusted afterwards is 4-7, collects product, and product is cleaned repeatedly, is taken off
Water process, obtains collagen.
The present invention provide a kind of collagenous biological film preparation method, first ox heel string is thinly sliced, make collagen easily from
Discharge in ox heel string, then enzyme hydrolysis is carried out with ficin, making the ox heel string of the section compacted becomes loose, to it
In collagen tentatively extracted, ficin is that the protease of plant origin not only has the effect for extracting collagen well
Really, human body does not have rejection to it, and the protease relative to animal origin avoids immunogene interference.Phosphate-buffered
Solution provides suitable condition for the activity of ficin.Then basic hydrolysis, certain density hydrogen-oxygen are carried out with NaOH
Changing sodium can remove the paathogenic factors such as the self-contained prion of ox heel string, and collagen is dissociateed from ox heel string
Come, certain density salting liquid makes the collagen that dissociates in insoluble state, and easily separate with other impurity and solution, and
Collagen hydro is effectively prevented into collagen.It is to prevent collagen degradation during high temperature that temperature is strict controlled in into 4-15 DEG C.
It is to remove the various ions on collagen that product is cleaned repeatedly, then is dehydrated, and obtains the glue of highly purified triple helix structural integrity
It is former.
It should be noted that with slight oscillatory or can rock during the loose ox heel string of basic hydrolysis, but can not use
Power is stirred, and prevents mechanical external force from making the Fragmentation of collagen.
Need it is further noted that the pH value of salting liquid is adjusted to 4-7, preferably 6-7, concrete operations are molten to salt
Acid is added dropwise in liquid to adjust pH value, molar concentration is sulfuric acid solution, hydrochloric acid solution or acetic acid solution of 0.05-2mol/L etc..
In actual applications, first ox heel string cut into slices, then is rinsed repeatedly with water, remove moisture removal.By the section of ox heel string
It is to be easier to extract collagen therein.Rinsed repeatedly with water, be to further remove the impurity that section ox heel string contains.
Further, before cutting into slices to ox heel string, first ox heel string is pre-processed, specifically can be with the step of pretreatment
For：Ox heel string is rinsed repeatedly with water, is afterwards 20%-75% ethanol solutions rinsing 15-45min with mass fraction, used
Water is rinsed repeatedly.Removal of impurities is tentatively carried out to the ox heel string of whole, the impurity such as the blood stains dust on surface are washed away, mass fraction is
20%-75% ethanol solutions are relatively good to blood stains removal effect, and can also disinfection, preferably mass fraction be 65%-
75% ethanol solution, the more preferably ethanol solution of mass fraction 75%.Through rinsing, ox heel string can become whiter, subsequently
The collagen of acquisition also can more Bai Gengchun.
Further, section can be freezing microtome section, by ox heel string in -20 DEG C of freeze overnights, substantially 7-12h, it is easier to will
Ox heel string is thinly sliced, and specifically ox heel string can be cut into into the thin slice that thickness is 1-3mm using slicer.
Additionally, chlorite is preferably sodium chlorite.Sodium chlorite is easier to remove in follow-up water-washing process.
In order to strengthen the hydrolysis effect of ficin, while reducing reagent cost, it is preferable that ficin exists
Mass fraction in phosphate buffer is 0.05%-0.1%.
It is that ficin plays its enzyme hydrolysis effect and carries to further enhance the hydrolysis effect of ficin
For conditions and environment preferably, the pH value of PBS is 5-7, preferably pH value 5.8-6.5, more preferably pH value
6.4；The molar concentration of phosphate radical is 0.01-0.1mol/L, more preferably more preferably 0.02-0.06,0.03mol/L.Phosphoric acid
Salt buffer can be by least one preparation the in potassium dihydrogen phosphate, potassium phosphate,monobasic, sodium dihydrogen phosphate and disodium-hydrogen
Into.
Used as the embodiment of a modification of the present invention, oxidant is preferably hydrogen peroxide.Hydrogen peroxide is a kind of oxygen
The very strong oxidant of the property changed, can will not introduce other by ficin enzyme-deactivating, while resolving into oxygen and water in collagen
Ion.
Used as a kind of further improved embodiment of the present invention, the addition volume of oxidant is phosphate buffer
1‰-5‰.This addition can make ficin complete inactivation.
As another kind of improved embodiment of the present invention, based on the gross mass of the salting liquid, the quality of the salt
Fraction is 10%-30%.This concentration range, is more beneficial for collagen and separates out from salting liquid, prevents collagen from hydrolysis occurring and generates
Collagen.
Used as another improved embodiment of the present invention, the salt in salting liquid is selected from sodium chloride, sodium sulphate, sodium carbonate
In at least one.
Packaging sterilizing is carried out to the collagenous biological film that the present invention is provided.Low-temperature epoxy ethane sterilization is concrete：Freezing is dry
Double casing after the specification size cutting as requested of dry sponge, 100% ethylene oxide sterilization processes；Forevacuum is -50--
60Kpa；Sterilizing humidity is 55-74%RH；Sterilising temp is 35-39 DEG C；Rate of ventilation is 10 times, sterilization time 3-9h, optimization
Sterilization time 4-8h；Sterilizing determines aseptic and ethylene oxide residue after terminating, and confirmatory sample is aseptic and ethylene oxide residue
Meet regulation requirement.
It should be noted that water used in the present invention is the purified water used by pharmaceuticals industry, standards of pharmacopoeia is met.
Reagent and instrument
Reagent is commercially available.
Embodiment 1-4 extracts collagen
Embodiment 1
Fresh ox is chosen with tendinous tissue 1kg, water is rinsed 6 times, 75% alcohol rinsing 20min；With scissors reject manadesma and
Impurity, after washing, -20 DEG C of freeze overnights；Microtome is standby to about 1mm；
100g tendon pieces are weighed, purified water is rinsed 5 times；Purified water soaking at room temperature 5h of 5L；Purified water is rinsed 5 times, is filtered dry water
Divide standby；
It is 6 that tendon piece (the ox heel string cut into slices) after cleaning is immersed in the pH value containing 0.05% ficin
In the phosphate buffer of 0.05mol/L, after soaking 24 hours at 4 DEG C；Volume is added thereto to for phosphate-buffered liquid
The hydrogen peroxide of long-pending 3 ‰ stands 4h inactivation ficins after mixing, purified water is rinsed；Then under the conditions of 4 DEG C, 1M's
Soak 4 days in the sodium chloride solution of NaOH, gently rock in the middle of standing；The hydrochloric acid of 0.05mol/L is added dropwise, pH to 6 is adjusted；
Collagen precipitation is collected by filtration；Purified water flushing collagen 5 times, 4000 turns/min centrifugation 20min, obtains Collagen specimens.
As shown in Fig. 2 the molecular weight of lauryl sodium sulfate-polyacrylamide gel (SDS-PAGE) detection collagen is about
For 300,000 dalton.
Embodiment 2
Fresh ox is chosen with tendinous tissue 1kg, water is rinsed 9 times, 45% alcohol rinsing 30min；With scissors reject manadesma and
Impurity, after washing, -20 DEG C of freeze overnights；Microtome is standby to about 2mm；
100g tendon pieces are weighed, purified water is rinsed 5 times；Purified water soaking at room temperature 3h of 5L；Purified water is rinsed 5 times, is filtered dry water
Divide standby；
It is 5 that tendon piece (the ox heel string cut into slices) after cleaning is immersed in the pH value containing 0.005% ficin
In the phosphate buffer of 0.01mol/L, after soaking 24 hours at 4 DEG C；Volume is added thereto to for phosphate-buffered liquid
The sodium chlorite of long-pending 1 ‰ stands 4h inactivation ficins after mixing, purified water is rinsed；Then under the conditions of 4 DEG C, 1.5M
NaOH sodium chloride solution in soak 4 days, stand in the middle of gently rocks；The hydrochloric acid of 0.05mol/L is added dropwise, adjustment pH is extremely
5；Collagen precipitation is collected by filtration；Purified water flushing collagen 9 times, 4000 turns/min centrifugation 20min, obtains Collagen specimens.
The molecular weight of lauryl sodium sulfate-polyacrylamide gel (SDS-PAGE) detection collagen is about 300,000 dongles
.
Embodiment 3
Fresh ox is chosen with tendinous tissue 1kg, water is rinsed 3 times, 20% alcohol rinsing 40min；With scissors reject manadesma and
Impurity, after washing, -20 DEG C of freeze overnights；Microtome is standby to about 3mm；
100g tendon pieces are weighed, purified water is rinsed 5 times；Purified water soaking at room temperature 3h of 5L；Purified water is rinsed 5 times, is filtered dry water
Divide standby；
It is 5 that tendon piece (the ox heel string cut into slices) after cleaning is immersed in the pH value containing 0.1% ficin
In the phosphate buffer of 0.01mol/L, after soaking 24 hours at 15 DEG C；Volume is added thereto to for phosphate-buffered liquid
The hydrogen peroxide of long-pending 4 ‰ stands 5h inactivation ficins after mixing, purified water is rinsed；Then under the conditions of 15 DEG C,
Soak 4 days in the sodium chloride solution of the NaOH of 1.5M, gently rock in the middle of standing；The sulfuric acid of 0.05mol/L, adjustment is added dropwise
PH to 4；Collagen precipitation is collected by filtration；Purified water flushing collagen 10 times, 4000 turns/min centrifugation 20min, obtains Collagen specimens.
The molecular weight of lauryl sodium sulfate-polyacrylamide gel (SDS-PAGE) detection collagen is about 300,000 dongles
.
Embodiment 4
Fresh ox is chosen with tendinous tissue 1kg, water is rinsed 3 times, 65% alcohol rinsing 25min；With scissors reject manadesma and
Impurity, after washing, -20 DEG C of freeze overnights；Microtome is standby to about 3mm；
100g tendon pieces are weighed, purified water is rinsed 5 times；Purified water soaking at room temperature 3h of 5L；Purified water is rinsed 5 times, is filtered dry water
Divide standby；
It is 7 that tendon piece (the ox heel string cut into slices) after cleaning is immersed in the pH value containing 0.2% ficin
In the phosphate buffer of 0.1mol/L, after soaking 24 hours at 10 DEG C；Volume is added thereto to for phosphate-buffered liquid
The hydrogen peroxide of long-pending 5 ‰ stands 5h inactivation ficins after mixing, purified water is rinsed；Then under the conditions of 10 DEG C, 2M
NaOH sodium chloride solution in soak 4 days, stand in the middle of gently rocks；The sulfuric acid of 0.05mol/L is added dropwise, adjustment pH is extremely
7；Collagen precipitation is collected by filtration；Purified water flushing collagen 8 times, 4000 turns/min centrifugation 20min, obtains Collagen specimens.
The molecular weight of lauryl sodium sulfate-polyacrylamide gel (SDS-PAGE) detection collagen is about 300,000 dongles
.
The structure detection of the collagen of embodiment 5
Fig. 3 is the infrared spectrogram of the Collagen specimens that embodiment 1 is extracted.As can be seen from Figure 3,1653.9cm-1For amide I band
C=O stretching vibration absworption peaks, illustrate all to exist in sample the C=O for forming hydrogen bond in triple helix；1552.6cm-1For acid amides II
The N-H flexural vibrations absworption peaks of band；1239.1cm-1N-H for acid amides III bands deforms peak；1239.1cm-1～1452.2cm-1Model
The absworption peak for enclosing presence shows the integrality of collagen triple-helix structure；3422.8cm-1For the N-H stretching vibration peaks of collagen, explanation
The presence of peptide interchain hydrogen bond；1239.1cm-1With 1452.4cm-1Absorption peak strength ratio be 1.02, closely collagen features
Value 1.0.As can be seen here, the collagen prepared by the present invention is mainly NTx, the triple helix structure of NTx be to maintain compared with
Complete.The method of extraction collagen can keep the complete of the triple helix structure of collagen in a kind of heel string from ox that the present invention is provided
It is whole.
The purity detecting of the collagen of embodiment 6
Fig. 4 is the uv absorption spectra of the type i collagen sample that embodiment 1 is extracted.Figure 4, it is seen that purifying
Sample has ultraviolet spectra maximum absorption peak at wavelength 217nm, and peak area is 0.375.
From above-described embodiment, collagen helix structural intergrity is good, and purity is high, and the extracting method letter of collagen
Single, without the need for high-end devices, extraction cost is low.
The preparation of embodiment 7 to the collagenous biological film of embodiment 11
Embodiment 7
Collagen is added to the water, and 0.01M (mol/L) lactic acid solution is added dropwise, it is 3.0 to adjust pH, emulsification pump
6000rpm, mixes at a high speed 5min, makes slurries, and mass fraction of the collagen in slurries is 0.3%；40 mesh stainless steel filter cloth mistakes
Filter twice, removes insoluble material；The sodium hydroxide solution for being slowly added dropwise low concentration (0.5M) adjusts the pH to 5.5 of liquid；Cross
Filter and vacuum defoamation, pour the slurries for preparing into pallet, under conditions of vacuum is 120 millitorrs, 10 DEG C of holding 40min ,-
45 DEG C of holding 150min, 0 DEG C of holding 20h, 20 DEG C keep 5h to carry out freeze-drying, obtain collagen sponge.
Chemically it is crosslinked, in the formaldehyde that mass fraction is volatilized by 10% formalin 4h is kept, obtains finished product,
That is collagenous biological film.
Embodiment 8
Collagen is added to the water, and 0.03M (mol/L) lactic acid solution is added dropwise, it is 3.0 to adjust pH, emulsification pump
8000rpm, mixes at a high speed 5min, makes slurries, and mass fraction of the collagen in slurries is 0.5%；40 mesh stainless steel filter cloth mistakes
Filter twice, removes insoluble material；The sodium hydroxide solution for being slowly added dropwise low concentration (0.5M) adjusts the pH to 6 of liquid；Filter
And vacuum defoamation, by the slurries for preparing pour into pallet vacuum be 120 millitorrs under conditions of, 10 DEG C holding 40min, -35
DEG C keep 120min, 0 DEG C holding 22h, 25 DEG C keep 9h carry out freeze-drying, obtain collagen sponge.
Chemically it is crosslinked, in the formaldehyde that mass fraction is volatilized by 10% formalin 5h is kept, obtains finished product.
Embodiment 9
Collagen is added to the water, and 0.05M (mol/L) lactic acid solution is added dropwise, it is 3.8 to adjust pH, emulsification pump 8000rpm
5min is mixed at a high speed, slurries are made, and mass fraction of the collagen in slurries is 1.0%；40 mesh stainless steel filter-cloth filterings twice, go
Except insoluble material；The sodium hydroxide solution for being slowly added dropwise low concentration (0.5M) adjusts the pH to 6.5 of liquid；Filter and vacuum takes off
Bubble, pours the slurries for preparing into pallet, under conditions of vacuum is 220 millitorrs, 6 DEG C of holding 40min, and -38 DEG C of holdings
180min, 0 DEG C of holding 24h, 24 DEG C keep 7h to carry out freeze-drying, obtain collagen sponge.
Chemically it is crosslinked, gas crosslinking, mass fraction is to keep in the formaldehyde gas that 10% formalin is volatilized
5h, obtains finished product (as shown in Figure 1).
Embodiment 10
Collagen is added to the water, and 0.03M (mol/L) hydrochloric acid solution is added dropwise, it is 3.0 to adjust pH, emulsification pump
8000rpm, mixes at a high speed 5min, makes slurries, and mass fraction of the collagen in slurries is 0.5%；40 mesh stainless steel filter cloth mistakes
Filter twice, removes insoluble material；The sodium hydroxide solution for being slowly added dropwise low concentration (0.5M) adjusts the pH to 5.8 of liquid；Cross
Filter and vacuum defoamation, pour the slurries for preparing into pallet, under conditions of vacuum is 150 millitorrs, 10 DEG C of holding 40min ,-
45 DEG C of holding 150min, 0 DEG C of holding 20h, 20 DEG C keep 5h to carry out freeze-drying, obtain collagen sponge.
It is crosslinked with physical method, 120 DEG C, vacuum 48h obtains finished product.
Embodiment 11
Collagen is added to the water, and 0.05M (mol/L) lactic acid solution is added dropwise, it is 2.8 to adjust pH, emulsification pump
8000rpm, mixes at a high speed 5min, makes slurries, and mass fraction of the collagen in slurries is 1.0%；40 mesh stainless steel filter cloth mistakes
Filter twice, removes insoluble material；The sodium hydroxide solution for being slowly added dropwise low concentration (0.5M) adjusts the pH to 6.2 of liquid；Cross
Filter and vacuum defoamation, pour the slurries for preparing into pallet freezing, under conditions of vacuum is 150 millitorrs, 10 DEG C of holdings
40min, -45 DEG C of holding 150min, 0 DEG C of holding 24h, 25 DEG C of holding 8h are dried and obtain collagen sponge.
It is crosslinked with physical method, 100 DEG C, vacuum 72h obtains finished product.
Electron-microscope scanning detection is carried out to the collagenous biological film prepared by embodiment 7 to embodiment 11, as a result as shown in table 1.
The electron-microscope scanning result of the collagenous biological film of the embodiment 7 to 11 of table 1
From table 1 and Fig. 1, collagenous biological film thickness is about 0.2-0.9 centimetre, and with three-dimensional aperture structure, aperture is big
It is little for 60-300 microns.
The zoopery of embodiment 12
New zealand rabbit point is randomly divided into into a, b, c, 3 groups, 12 per group, after coronal suture, 1 bone is ground in center line right side with electric drill
Window, exposure and artificially causes dura defect at endocranium, a, b, c, and collagen prepared by 4 in three groups rabbits Application Example 9 is given birth to
Thing film, 4 rabbits carry out dura defect repairing using commercialized product (integra, as control), and 4 are not repaired.A, b, c tri-
Group was put to death respectively at postoperative 30 days, 90 days, 180 days.Collection each group venous blood lml carries out leucocyte, lymph before preoperative and execution
Cell count.It is postoperative that clinical observation is carried out to animal to target date execution, then extension windowing, together with endocranium, repair material
Material and brain tissue are removed, and carry out sample gross examination of skeletal muscle, and judge patching material inner surface and brain tissue adhesion degree.By sample one
Part is fixed on 1 week in the formalin that mass fraction is 10%, FFPE, slice row haematoxylin-eosin stains, row group
Knit to check, paired observation implantation material local cells Infiltrating observation local arachnoid, cavum subdurale and Cerebral cortex feelings
Condition.
Conclusion：Clinical observation each group animal occurs in addition to the sample repaired without leakage of cerebrospinal, do not it is found that animal goes out
Existing postoperative complications, our product has similar repairing performance to control sample.Each group animal is preoperative, put to death before leucocyte
Sum, LC are without significant difference (P>0.05).Postoperative 30 days, 180 days 90 days two kinds of meninx brain adhesion differences are without system
Meter learns meaning (P>0.05).Each group pathologic finding graft is without denaturation, parcel, calcification；Each group animal dura mater repairs local
See different degrees of cavum subdurale and arachnoid pathological change, each group implantation material (collagenous biological film) local inflammatory cells infiltration nothing
Significant difference (P>0.05).
The above is for only for ease of those skilled in the art and understands technical scheme, not to limit
The present invention.All any modification, equivalent substitution and improvements within the spirit and principles in the present invention, made etc., should be included in this
Within the protection domain of invention.
Claims (10)
1. a kind of collagenous biological film, it is characterised in that the collagen in collagenous biological film has triple helix structure；Collagenous biological film
Thickness be 0.2-0.9cm, with three-dimensional aperture structure, the size in aperture is 60-300mm.
2. the preparation method of the collagenous biological film of a kind of claim 1, it is characterised in that comprise the following steps,
Collagen is mixed with water, lactic acid solution and/or hydrochloric acid solution is added thereto to, makes collagen dispersed, make slurries；
The pH value of the slurries is adjusted to 5.5-6.5, afterwards to the slurries vacuum defoamation, freeze-drying is then carried out；
It is crosslinked using physically and/or chemically method, that is, obtains collagenous biological film.
3. preparation method according to claim 2, it is characterised in that mass fraction of the collagen in the slurries be
0.3%-1.5%.
4. preparation method according to claim 2, it is characterised in that the pH value of the slurries is 2.8-3.8.
5. preparation method according to claim 2, it is characterised in that it in vacuum is 120-250 that the freeze-drying is
Under conditions of millitorr, 4-10 DEG C of holding 40min, -45--35 DEG C of holding 120-210min, 0 DEG C of holding 20-24h, 20-25 DEG C of guarantor
Hold 2-9h.
6. preparation method according to claim 2, it is characterised in that the Physical cross linking methods are：Using the purple of 254nm
7-13h is irradiated in outside line, or the Physical cross linking methods are：100-120 DEG C of processed 2-3 days under vacuum, afterwards
It is cooled to less than 50 DEG C；The Chemical Crosslinking Methods are：It is crosslinked using formaldehyde gas.
7. preparation method according to claim 2, it is characterised in that the collagen is obtained by following steps：
First ox heel string is cut into slices；
The ox heel string of section is immersed in again the phosphate buffer of the mass fraction for 0.005%-0.2% of ficin
In, keep 12-24h at 4-15 DEG C；
Then oxidant is added in the phosphate buffer, with water the ox heel string of the section is rinsed；The oxidant choosing
At least one from chlorite and hydrogen peroxide；
Finally the ox heel string of the section is immersed in the salting liquid that NaOH molar concentration is 1-2mol/L, at 4-15 DEG C
Lower to be kept for 1-4 days, the pH value that the salting liquid is adjusted afterwards is 4-7, collects product, product is cleaned repeatedly, at dehydration
Reason, obtains collagen.
8. preparation method according to claim 7, it is characterised in that the mass fraction of the ficin is
0.05%-0.1%；The pH value of the PBS is 5-7, and the molar concentration of phosphate radical is 0.01-0.1mol/L.
9. method according to claim 7, it is characterised in that the addition volume of the oxidant is the phosphate-buffered
The 1 ‰ -5 ‰ of liquid；The oxidant is hydrogen peroxide.
10. method according to claim 7, it is characterised in that the gross mass based on the salting liquid, the quality of the salt
Fraction is 10%-30%；At least one of the salt in the salting liquid in sodium chloride, sodium sulphate, sodium carbonate.
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| CN107469145A (en) * | 2017-09-22 | 2017-12-15 | 北京华信佳音医疗科技发展有限责任公司 | A kind of preparation and its application of dura mater patching material |
| CN107551312A (en) * | 2017-10-19 | 2018-01-09 | 北京华信佳音医疗科技发展有限责任公司 | A kind of cotton-shaped collagen hemostasis fiber and preparation method thereof |
| CN107596428A (en) * | 2017-09-25 | 2018-01-19 | 北京华信佳音医疗科技发展有限责任公司 | A kind of collagen hemostasis sponge and preparation method thereof |
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| CN110893250A (en) * | 2019-10-11 | 2020-03-20 | 许和平 | Scar/adhesion barrier film and preparation method and use thereof |
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