CN109602943A - A kind of preparation method of the high purity collagen sponge in beef tendon source - Google Patents
A kind of preparation method of the high purity collagen sponge in beef tendon source Download PDFInfo
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- CN109602943A CN109602943A CN201811567676.3A CN201811567676A CN109602943A CN 109602943 A CN109602943 A CN 109602943A CN 201811567676 A CN201811567676 A CN 201811567676A CN 109602943 A CN109602943 A CN 109602943A
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- collagen
- acetic acid
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- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 235000015278 beef Nutrition 0.000 title claims description 4
- 210000002435 tendon Anatomy 0.000 title claims description 4
- 239000000515 collagen sponge Substances 0.000 title abstract description 10
- 102000008186 Collagen Human genes 0.000 claims abstract description 60
- 108010035532 Collagen Proteins 0.000 claims abstract description 60
- 229920001436 collagen Polymers 0.000 claims abstract description 41
- 238000004132 cross linking Methods 0.000 claims abstract description 10
- 102000012422 Collagen Type I Human genes 0.000 claims abstract description 9
- 108010022452 Collagen Type I Proteins 0.000 claims abstract description 9
- 238000004108 freeze drying Methods 0.000 claims abstract description 9
- 230000001954 sterilising effect Effects 0.000 claims abstract description 9
- 238000000034 method Methods 0.000 claims abstract description 8
- 238000004140 cleaning Methods 0.000 claims abstract description 7
- 229940111202 pepsin Drugs 0.000 claims abstract description 7
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 4
- 239000002253 acid Substances 0.000 claims abstract description 3
- 238000000465 moulding Methods 0.000 claims abstract description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 69
- 239000000243 solution Substances 0.000 claims description 17
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 14
- 238000003756 stirring Methods 0.000 claims description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 238000000502 dialysis Methods 0.000 claims description 12
- 239000000047 product Substances 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 10
- 230000007071 enzymatic hydrolysis Effects 0.000 claims description 9
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims description 9
- 230000001376 precipitating effect Effects 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 8
- 230000008961 swelling Effects 0.000 claims description 8
- 239000012153 distilled water Substances 0.000 claims description 7
- 239000011780 sodium chloride Substances 0.000 claims description 7
- 210000001519 tissue Anatomy 0.000 claims description 7
- 102000057297 Pepsin A Human genes 0.000 claims description 6
- 108090000284 Pepsin A Proteins 0.000 claims description 6
- 239000002504 physiological saline solution Substances 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 5
- 101100366707 Arabidopsis thaliana SSL11 gene Proteins 0.000 claims description 4
- 101100366710 Arabidopsis thaliana SSL12 gene Proteins 0.000 claims description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 4
- KFSLWBXXFJQRDL-UHFFFAOYSA-N Peracetic acid Chemical compound CC(=O)OO KFSLWBXXFJQRDL-UHFFFAOYSA-N 0.000 claims description 4
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 4
- 210000003722 extracellular fluid Anatomy 0.000 claims description 4
- 238000007710 freezing Methods 0.000 claims description 4
- 230000008014 freezing Effects 0.000 claims description 4
- 238000004806 packaging method and process Methods 0.000 claims description 4
- 239000002244 precipitate Substances 0.000 claims description 4
- 102000002734 Collagen Type VI Human genes 0.000 claims description 3
- 108010043741 Collagen Type VI Proteins 0.000 claims description 3
- 239000004519 grease Substances 0.000 claims description 3
- 239000004615 ingredient Substances 0.000 claims description 3
- 238000002203 pretreatment Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 2
- 101100366711 Arabidopsis thaliana SSL13 gene Proteins 0.000 claims description 2
- 101100096719 Arabidopsis thaliana SSL2 gene Proteins 0.000 claims description 2
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 2
- 101100366560 Panax ginseng SS10 gene Proteins 0.000 claims description 2
- 101100366561 Panax ginseng SS11 gene Proteins 0.000 claims description 2
- 101100366562 Panax ginseng SS12 gene Proteins 0.000 claims description 2
- 101100366563 Panax ginseng SS13 gene Proteins 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims description 2
- 239000000284 extract Substances 0.000 claims description 2
- 210000003195 fascia Anatomy 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 230000007935 neutral effect Effects 0.000 claims description 2
- 238000005070 sampling Methods 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 238000005303 weighing Methods 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 claims 1
- 238000005516 engineering process Methods 0.000 abstract description 3
- 241001465754 Metazoa Species 0.000 abstract description 2
- 239000003814 drug Substances 0.000 abstract description 2
- 239000000463 material Substances 0.000 abstract description 2
- 230000008569 process Effects 0.000 abstract description 2
- 230000015556 catabolic process Effects 0.000 abstract 1
- 238000006731 degradation reaction Methods 0.000 abstract 1
- 230000005847 immunogenicity Effects 0.000 abstract 1
- 238000000746 purification Methods 0.000 abstract 1
- 239000002994 raw material Substances 0.000 abstract 1
- 230000017423 tissue regeneration Effects 0.000 abstract 1
- 150000001408 amides Chemical class 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 238000004566 IR spectroscopy Methods 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 239000011785 micronutrient Substances 0.000 description 2
- 235000013369 micronutrients Nutrition 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 230000008733 trauma Effects 0.000 description 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 238000005452 bending Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000033077 cellular process Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- 230000002439 hemostatic effect Effects 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000002715 modification method Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/42—Use of materials characterised by their function or physical properties
- A61L15/425—Porous materials, e.g. foams or sponges
-
- 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
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
- A61L15/32—Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
- A61L15/325—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
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/42—Use of materials characterised by their function or physical properties
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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
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/42—Use of materials characterised by their function or physical properties
- A61L15/44—Medicaments
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
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- 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
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- A61L2400/00—Materials characterised by their function or physical properties
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Abstract
The invention belongs to medical biotechnology field of material technology, and in particular to a method of Type I collagen sponge is made by raw material of animal heel string, is primarily characterized in that the collagen sponge of preparation is existed in the form of triple-helix structure.The collagen of low immunogenicity is extracted using acid plumping-Pepsin degradation new method, and the purification process such as saltout and dialyse is recycled to obtain the Type I collagen albumen of high protein content, high-purity.It finally using processes such as crosslinking, freeze-drying molding, for several times cleaning, secondary freeze-drying and irradiation sterilizations obtains that there is certain mechanical strength, have certain penetrability, the medical collagen sponge with suitable aperture and porosity and sterile three-dimensional structure.The high-purity I-type collagen protein sponge that the present invention obtains can be widely applied to field of medicaments (such as tissue repair).
Description
Technical field
The present invention relates to the purifying of collagen and the preparation methods of sponge.
Background technique
Collagen is the important component of extracellular matrix, is to form organ and organize essential ingredient, extensively
It is general to be distributed in each histoorgan of mammal.Collagen is made of two α 1 (I) chains and α 2 (I) chain, and sometimes two
α chain, which can overlap each other, to form a β chain or three α chains coincide with one another to form a γ chain, has triple-helix structure.Glue
Former protein sponge is a kind of physical form of collagen, is the three-dimensional sponge structure that the technology by freeze-drying obtains, cold
Freeze dry technology and maintains the intrinsic triple-helix structure of collagen well.Enzyme is carried out to animal heel string using pepsin
Solution, acts at one end collagen N 3/4, cuts away collagen end peptide and retain triple helix section and reduce the immunogene of collagen
Property.Collagen has many functions, and exogenous collagen tissue engineering material is filled out in tissue trauma hemostasis, reparation, defect
Fill etc. plays huge effect.
Up to the present, it has been found that 29 kinds of collagen-type, study more more is V Collagen Type VI of I-, wherein with I type
Collagen content is most, accounts for about the 90% of the total collagen of organism, using also the most extensive.Type I collagen is that fiber forms collagen, can be lived
Change epithelial cell, be promote rawhide hyperplasia, also can promote clostridiopetidase A generation, keep skin tensioned and elastic force, thus
Field of medicaments is increasingly attracted attention for repairing skin trauma and bone structure damage.
Summary of the invention
The purpose of the present invention is to provide a kind of Simple process to obtain high-purity, to body nonhazardous, hemostatic and tissue
The medical ox heel string Type I collagen protein sponge of repairing performance.
Institute of the invention is using technical solution:
A kind of preparation method of the Type I collagen protein sponge in beef tendon source comprising following steps:
S1, the grease removal of ox heel string, unless protein ingredient, takes 10g pre-treatment object, through rubbing, 3% acetic acid is swollen 48h, is added
1% pepsin extracts 48h, saltouts by NaCl, obtains collagen and slightly precipitate；It finally redissolves, then distinguishes through peracetic acid
It is dialysed with 0.02M disodium hydrogen phosphate, 1% acetic acid, 0.5% acetic acid, 0.1% acetic acid and distilled water, obtains high-purity I Collagen Type VI egg
It is white；
The molding crosslinking of S2, collagen protein sponge: crosslinking Treatment, freezing are carried out to collagen using chemical crosslink technique
At collagen protein sponge after drying.
The sterilizing of S3, collagen protein sponge: it will be used after the processed Type I collagen protein sponge basic products packaging of step 2
Co60 irradiation sterilization obtains collagen protein sponge product.
Wherein, high purity collagen sponge preparation method the following steps are included:
SS1, sampling: obtaining fresh ox heel string, rejects fascia, and tri-distilled water is cleaned up, dried, scalpel shave, group
Knit refiner rubbing；
SS2, remove non-collagen: the ox heel string for weighing 60g or so rubs object, after impregnating 4h with physiological saline, gauze mistake
Filter, then the sodium hydroxide for being 0.1M with concentration stir for 24 hours in 4 DEG C of refrigerators, and every 12h changes a not good liquor；
SS3, it removes fat: rubbing object filtered through gauze, it is neutrality that physiological saline, which is cleaned multiple times and filters to cleaning solution pH, is added
The isopropanol for entering 10% stirs for 24 hours at 4 DEG C, and every 12h is changed the liquid once；
SS4, it removes foreign protein: being reacted again with the Tris-HCl buffer solution of the 0.05mol/L of the sodium chloride containing 4.5M
24h；
SS5, washing: using filtered through gauze, and physiological saline cleans 6 times；
SS6, acid mention: rubbing the acetic acid solution that 3% a small amount of (0.5M) is added in object in cleaned ox heel string, use tissue
Refiner stirring is mixture of viscous form, and 3% acetic acid solution for adding 1000ml is swollen for 24 hours in 4 DEG C, and filtered through gauze takes
Precipitating is added the acetic acid that 1000ml concentration is 0.5M and continues swelling for 24 hours；
SS5, enzymatic hydrolysis: filtered through gauze takes precipitating, and the 0.5M acetic acid 1000ml containing 1% pepsin is added, stirs at 4 DEG C
It mixes for 24 hours.Filtered through gauze collects supernatant swelling solution, and precipitating is taken to continue to add enzymolysis liquid enzymatic hydrolysis for 24 hours,；
SS6, take enzymolysis liquid: enzymatic hydrolysis mixed liquor filtered through gauze is collected supernatant and is mixed with upper batch enzymolysis liquid.
SS7, adjust pH: adjusting enzymatic hydrolysis supernatant pH with 1M sodium hydroxide is 7.0；
SS8, it saltouts: being slowly added to the NaCl fine grained of final concentration of 2.7M into neutral swelling solution, it is stirring while adding, with
It is stood overnight afterwards in 4 DEG C；
SS9, collect collagen: filtered through gauze removes supernatant, obtains white precipitate；
SS10, dialysis: 0.5M acetic acid re-dissolves collagen deposit, be packed into bag filter first respectively with 1%, 0.5%,
0.01% acetic acid is that extracellular fluid dialysis is dialysed for 24 hours respectively, then is dialysed for 24 hours, most using the disodium hydrogen phosphate of 0.02M as extracellular fluid dialysis
Liquid is changed with tri-distilled water dialysis 72h, every 12h afterwards；
SS11, crosslinking: final concentration of 0.05% glutaraldehyde being added into the collagen solution after dialysis, stirs 10 points
Clock pours into the Tissue Culture Dish that diameter is 10cm, each mold 30g collagen, and 4 DEG C of planes stand crosslinking 4h, then turns
- 20 DEG C of refrigerators are moved to, 8-12h is freezed；
SS12, freeze-drying: the dry 36-48h in -58 DEG C of vacuum freeze driers being pre-chilled in advance；
SS13, cleaning: the collagen protein sponge after dry is cleaned 8-10 times with tri-distilled water, every all over 10min；
SS14, secondary freeze-drying: then the sponge after cleaning is freeze-dried 16-24h in -20 DEG C of freezing 4-6h；
SS15, sterilizing: Co60 irradiation sterilization is used after packaging.
Other feature and advantage of the disclosure will the following detailed description will be given in the detailed implementation section.
Detailed description of the invention
Attached drawing is and to constitute part of specification for providing further understanding of the disclosure, with following tool
Body embodiment is used to explain the disclosure together, but does not constitute the limitation to the disclosure.In the accompanying drawings:
Fig. 1 is collagen sponge finished product front, reverse side and side scanning electron microscopic picture.Fig. 2 is collagen protein sponge into magenta
External spectrum.Fig. 3 is collagen protein sponge finished product SDS-PAGE electrophoretic image.
Specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in
This:
Embodiment
It takes ox heel string grease removal except foreign protein pre-treatment product is appropriate, rubs spare.Sample that 10g is shredded is weighed in 1000ml
In beaker, 400ml0.5M acetic acid solution is added, is stirred for 24 hours under the conditions of 4 DEG C, filtered through gauze takes precipitating, continues in the same way
Swelling is for 24 hours.400ml1% pepsin acetic acid solution is added, wherein acetic acid is 0.5M, is digested for 24 hours, after the completion of enzymatic hydrolysis, by sample
In filtered through gauze.Take filtered fluid in clean 1000ml beaker.It is 7.0 with 0.1MNaOH tune pH, final concentration of 2.6M is added
NaCl pulverize after fine grained, be added while stirring.4 DEG C of standing 16-20h, filtered through gauze take precipitating, redissolve precipitating, dialysis.
35g swelling solution is weighed in 10cm culture dish aperture plate with electronic balance, and step-by-step freezing cooling: 4 DEG C of standing 0.5h, -25 DEG C quiet
Set 0.5h, -80 DEG C of standing 12h.Sample is put into freeze-drying 48h-72h in freeze dryer and obtains collagen sponge.
Test result is as follows for collagen sponge:
1, basic physical and chemical
1 collagen protein sponge finished product basic physical and chemical result of table
As shown in Table 1, collagen protein sponge basic physical and chemical meets national standard.
2, scanning electron microscope
Collagen sponge finished product front, reverse side and side scanning electron microscopic picture are as shown in Figure 1.
Surface, reverse side or the cross-section morphology of bracket all keep its three-dimensional porous structure, and pore size is 200-300 microns.
The inside aperture of bracket is big and mainly based on laminated structure, and the three-dimensional porous structure of collagen-based bracket can be the increasing of cell
It grows in functional expression and cellular process and passes through with the necessary physical support of extraneous mass exchange offer and space environment
The three-dimensional porous structure of collagen-based bracket must be kept while the cross-linking modified stability for improving collagen-based bracket, this is also to comment
A kind of important evidence of cross-linked modification method superiority and inferiority of valence.
3, infrared spectroscopy
Collagen protein sponge finished product infrared spectroscopy is as shown in Figure 2.
By the infrared spectrum spectrogram of collagen, collagen includes the characteristic absorption peak i.e. amide Ⅰ of three parts, II band of amide, acyl
III band of amine.Amide Ⅰ: 1651cm-1The stretching vibration peak position of C=O is in 1643cm in the collagen at place-1Place；II band of amide: amide
The bending vibration peak position of N-H is in 1538cm in key-1Place；Amide Ⅲ band: including the Change of absorption of two peak positions, the change of N-H in total
Shape vibrates peak position in 1237cm-1Nearby and the stretching vibration of C-N in 1538cm-1Near.At present it is believed that working as glue in document
1452cm in former infrared spectroscopy-1With 1237cm-1When the ratio for locating absorption peak is 1, it is believed that the triple helix knot of collagen
Structure still maintains completely, in this result, 1452cm-1With 1237cm-1Ratio is 1.023, it can be seen that, three strands of spiral shells of collagen
Rotation structure is to maintain completely.It can it therefore follows that combining the collagen in the method for extracting collagen extraction ox heel string to be from relieving haperacidity enzyme
It leans on.
4, gel electrophoresis
Collagen protein sponge finished product SDS-PAGE electrophoretic image is as shown in Figure 3.
From the figure 3, it may be seen that comparing with molecular weight standard (marker), there are two bands of a spectrum to be located near 1200kDa, one is located at
Near 200kDa, the α in collagen helix structure is corresponded respectively to1Chain, α2Chain and β chain.The triple-helix structure of natural collagen is
It being made of the α chain of three molecular weight 100kDa, the Type I collagen feature reported in bands of a spectrum position and document fits like a glove,
It there is no miscellaneous band in other positions, this can prove that self-carry collagen purity is very high.
5, microelement
Micronutrient levels in 2 collagen protein sponge finished product of table
| Type | As | Cd | Cr | Cu | Fe | Hg | Mo | Ni | Pb |
| Concentration (%) | <0.005 | <0.001 | <0.005 | <0.005 | <0.005 | <0.001 | <0.005 | <0.005 | <0.005 |
Make the compound national standard of 9 kinds of micronutrient levels contained by collagen protein sponge by oneself.
The above list is only a few specific embodiments of the present invention for finally, it should also be noted that.Obviously, this hair
Bright to be not limited to above embodiments, acceptable there are many deformations.Those skilled in the art can be from present disclosure
All deformations for directly exporting or associating, are considered as protection scope of the present invention.
Claims (2)
1. a kind of preparation method of the Type I collagen protein sponge in beef tendon source comprising following steps:
S1, the grease removal of ox heel string, unless protein ingredient, takes 10g pre-treatment object, through rubbing, 3% acetic acid is swollen 48h, is added 1%
Pepsin extracts 48h, saltouts by NaCl, obtains collagen and slightly precipitate；It finally redissolves through peracetic acid, then uses respectively
0.02M disodium hydrogen phosphate, 1% acetic acid, 0.5% acetic acid, 0.1% acetic acid and distilled water dialysis, obtain high-purity I Collagen Type VI egg
It is white；
The molding crosslinking of S2, collagen protein sponge: crosslinking Treatment, freeze-drying are carried out to collagen using chemical crosslink technique
Afterwards at collagen protein sponge；
The sterilizing of S3, collagen protein sponge: Co60 spoke will be used after the processed Type I collagen protein sponge basic products packaging of step 2
According to sterilizing, collagen protein sponge product is obtained.
2. preparation method according to claim 1 comprising following steps:
SS1, sampling: obtaining fresh ox heel string, rejects fascia, and tri-distilled water is cleaned up, dried, scalpel shave, organizes even
Pulp grinder rubs；
SS2, remove non-collagen: the ox heel string for weighing 60g or so rubs object, after impregnating 4h with physiological saline, filtered through gauze, then
For 24 hours in 4 DEG C of refrigerator stirrings, every 12h changes a not good liquor to the sodium hydroxide for being 0.1M with concentration；
SS3, it removes fat: rubbing object filtered through gauze, it is neutrality, addition that physiological saline, which is cleaned multiple times and filters to cleaning solution pH,
10% isopropanol stirs for 24 hours at 4 DEG C, and every 12h is changed the liquid once；
SS4, foreign protein is removed: again for 24 hours with the Tris-HCl buffer solution reaction of the 0.05mol/L of the sodium chloride containing 4.5M；
SS5, washing: using filtered through gauze, and physiological saline cleans 6 times；
SS6, acid mention: rubbing the acetic acid solution that 3% a small amount of (0.5M) is added in object in cleaned ox heel string, be homogenized with tissue
Machine stirring is mixture of viscous form, and 3% acetic acid solution for adding 1000ml is swollen for 24 hours in 4 DEG C, and filtered through gauze takes precipitating
The acetic acid that 1000ml concentration is 0.5M is added and continues swelling for 24 hours；
SS5, enzymatic hydrolysis: filtered through gauze takes precipitating, and the 0.5M acetic acid 1000ml containing 1% pepsin is added, stirs at 4 DEG C
24h.Filtered through gauze collects supernatant swelling solution, and precipitating is taken to continue to add enzymolysis liquid enzymatic hydrolysis for 24 hours,；
SS6, take enzymolysis liquid: enzymatic hydrolysis mixed liquor filtered through gauze is collected supernatant and is mixed with upper batch enzymolysis liquid.
SS7, adjust pH: adjusting enzymatic hydrolysis supernatant pH with 1M sodium hydroxide is 7.0；
SS8, saltout: being slowly added to the NaCl fine grained of final concentration of 2.7M into neutral swelling solution, it is stirring while adding, then in
4 DEG C stand overnight；
SS9, collect collagen: filtered through gauze removes supernatant, obtains white precipitate；
SS10, dialysis: 0.5M acetic acid re-dissolves collagen deposit, is packed into bag filter first respectively with 1%, 0.5%, 0.01%
Acetic acid be extracellular fluid dialysis dialyse for 24 hours respectively, then using the disodium hydrogen phosphate of 0.02M as extracellular fluid dialysis dialyse for 24 hours, finally with three
It steams water dialysis 72h, every 12h and changes liquid；
SS11, crosslinking: final concentration of 0.05% glutaraldehyde being added into the collagen solution after dialysis, stirs 10 minutes,
It pours into the Tissue Culture Dish that diameter is 10cm, each mold 30g collagen, 4 DEG C of planes stand crosslinking 4h, then shift
To -20 DEG C of refrigerators, 8-12h is freezed；
SS12, freeze-drying: the dry 36-48h in -58 DEG C of vacuum freeze driers being pre-chilled in advance；
SS13, cleaning: the collagen protein sponge after dry is cleaned 8-10 times with tri-distilled water, every all over 10min；
SS14, secondary freeze-drying: then the sponge after cleaning is freeze-dried 16-24h in -20 DEG C of freezing 4-6h；
SS15, sterilizing: Co60 irradiation sterilization is used after packaging.
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| CN114748679A (en) * | 2022-03-27 | 2022-07-15 | 卢玉华 | Preparation method of collagen sponge |
| CN115671365A (en) * | 2022-11-04 | 2023-02-03 | 浙江诸暨聚源生物技术有限公司 | Crosslinked recombinant collagen sponge and preparation method and application thereof |
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Application publication date: 20190412 |