CN103566409A - Preparation method and application of medical collagen retaining biological activity - Google Patents
Preparation method and application of medical collagen retaining biological activity Download PDFInfo
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- CN103566409A CN103566409A CN201210262253.7A CN201210262253A CN103566409A CN 103566409 A CN103566409 A CN 103566409A CN 201210262253 A CN201210262253 A CN 201210262253A CN 103566409 A CN103566409 A CN 103566409A
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Abstract
The invention relates to an extraction method for a type I medical collagen raw material retaining biological activity. The method is characterized in that an acid process, an alkali process and an enzyme process are comprehensively utilized to produce the high-purity medical collagen raw material retaining unique biological activity, low immunogenicity and high biocompatibility of collagen under the conditions that a temperature, a pH value and reaction time are strictly controlled and the usage amount of protease is controlled, and the medical collagen raw material extracted by using the method has yield of more than 90% and purity of more than 99% and is suitable for industrial large scale production. The material can be used for production of high-grade hemostatic materials, bone fusion materials, tissue regeneration membranes, active collagen solutions for cosmetic Injection, etc.
Description
Technical field
The invention belongs to biological medicine technology field, be specifically related to a kind of preparation technology and application thereof that keeps bioactive medical collagen.
Background technology
Collagen is a kind of natural biomacromolecule, and source is abundant, removes after the end peptide at its antigen place and can process and be prepared into the biomaterial with good biocompatibility, has all dropped into a large amount of human and material resources both at home and abroad and has carried out the raw-material research of medical adhesive.
Collagen is the main component of extracellular matrix (ECMs); there is unique triple-helix structure; it not only plays support and protective effect to tissue and organ; and a series of bioprocesss of participation mediation; comprise adhesion, migration and differentiation and the tissue regeneration of cell, these special biological functions of collagen are to realize by the specific binding of the bioactive molecules such as membrane receptor, soluble factor and other ECM component and collagen triple-helix structure.Therefore, for the research of collagen triple-helix structure and function, oneself becomes the focus of current technical field of biological material.The thermal denaturation process of collagen is the process that triple-helix structure recurs variation, and along with the rising of temperature, triple helical can untwist, and finally forms random wall scroll polypeptide chain, and this process can be summarized as the thermal denaturation process of " triple helical one monomer ".In this process, the Hyarogen-bonding of stablizing triple-helix structure is destroyed gradually, and after collagen thermal denaturation, triple-helix structure is destroyed completely, and this process is irreversible, removes after the end peptide at its antigen place and can process and be prepared into the biomaterial with good biocompatibility.
Collagen utilizes its unique triple-helix structure not only can form a surface, allow cell and other enzyme or bioactie agent combined with it, and this spiral helicine structure comprising information, make the physiological environment of organism be able to move without any confusion.
The triple helix of collagen is a kind of structure that can hold the distinctive aminoacid composition of collagen protein and sequence.The studies and clinical application of bio-medical material is from first generation bio-inert material, second filial generation bioactive materials and Biodegradable material develop into the third generation biomaterial based on cell and molecular level, an important research direction of third generation biomaterial is analog cell epimatrix environment, on material, introduce special protein, polypeptide and other biological molecule, make material irritation cell on molecular level produce special responsing reaction, cause the interaction of cellular integration element, inducing cell propagation, synthetic and the assembling of differentiation and extracellular matrix, thereby start the regenerative system of body, and bringing into play important regulating action for the thermal balance of animal body.Collagen has good biocompatibility in addition, research and widely used bio-medical material, as tissue engineering bracket, absorbable suture, guided tissue regeneration's barrier film etc., but how to obtain the collagen of the activated triple-helix structure of this tool of maintenance and to utilize this collagen to prepare biocompatibility medical material by conventional means, be the target that people pursue.
Summary of the invention
The inventor is through repetition test, and through ultraviolet spectra, infrared spectrum and Electronic Speculum test, the preservation that type i collagen prepared by result proof preparation method of the present invention is complete the triple-helix structure of collagen, and effectively remove and hold peptide, reduce the immunogenicity of collagen, made it have better biocompatibility.
The object of the present invention is to provide the preparation method that a kind of antigenicity is low, maintenance biological activity possesses the I type medical collagen of triple-helix structure.
Another object of the present invention is to provide a kind of take bone that I type medical collagen of the present invention is prepared from as raw material and merges the preparation method that biomaterial and this bone merge biomaterial, and prepared bone merges the reparation that biomaterial can be used for the damaged damage location of bone.
Another object of the present invention is to provide a kind of biological tissue's regeneration membrane that I type medical collagen of the present invention is raw material and preparation method thereof of take, prepared biological diaphragm not only can be used for high-grade hemostatic material, also can be used for skin and meninges regeneration field simultaneously.
The present invention keeps the bioactive preparation method of collagen to have the advantages that extract yield is high, antigenicity is low, the pure collagen yield of solubility that common extracting method obtains can only reach 10-20%, generally can only be for laboratory research, and the yield of active collagen that the present invention extracts can reach more than 90%, improve greatly the utilization rate of animal tissue, be applicable to large-scale industrial production.
Maintenance biological activity of the present invention possesses the I type biological collagen of triple-helix structure can be prepared by following method step:
1), raw material pre-treatment: choose the cattle heel string of newly slaughtering cattle, transportation is preserved in frozen water, arrive and accelerate to process behind ground, processed, removes unnecessary tissue with shears immediately, is positioned over-20 degree refrigerator and cooled and freezes, with blade, scrape off the unnecessary fascia in surface afterwards, with aluminium foil, be bundled into 10mm size cylindric, be positioned over that refrigerator and cooled is frozen puts, standby.
2), collagen extraction:
A, get the cattle heel string after freezing, cut into after the thin slice ，Yong historrhexis machine coarse crushing of 0.5 millimeter, with the NaCl solution soaking 15-30min of 5%-20%, then with the KH that adds 0.5%
2pO
4, at the solution soaking 1-3h of 0-5 ℃, it is neutral by purified water, rinsing to pH value, after add ethanol and trichloroethane mixed liquor (1: 1) to soak, defat 1-3 hour under slow stirring condition, obtains pretreated feedstock.
B, raw material add containing 23%NaCl-0.05mol/L Tris-HCl (pH value 7.5) buffer and soak 3h after rinsing by purified water, during draw at any time the oils and fats on liquid level, to floating over liquid level without lipid.
C ,Hou Yong historrhexis machine carries out in small, broken bits, and regulator solution pH value to 6.8, adds 1: 100 ficin, under 4 ℃ of conditions, within 20 minutes, stirs once, process 5 hours, after repeatedly rinsing with 1% H
2o
2after processing 4h, clean.
D, above gained collagen is added in saturated NaCl solution (PH7.0-7.2), standing 6-24 hour under 4 ℃ of conditions, after add the NaOH solution of 1M and 0.5M ammonium chloride solution to soak, under 15-20 ℃ of condition standing 36 hours, finally with HCl, neutralize, regulate pH value to 7.2, under stirring condition, by purified water, rinse 3 times, then the water for injection that is 6.8 with pH soaks 3 hours at 5 ℃ of temperature, filter, remove excessive moisture, freezing preservation, obtains.
I type medical collagen prepared by the preparation method of the present invention of take is prepared biological haemostatic membrane as raw material and can be made by following methods:
1, lyophilizing: gained collagen is joined in 0.5M glacial acetic acid solution by 3% (mass fraction) proportion, with online high-shear homogenizer, disperse 2-3 time, its temperature is controlled at below 10 ℃, and the liquid vacuum after dispersion is removed bubble, puts into freezer dryer lyophilizing pallet, make thickness at 4-6mm, at subzero 5 ℃ (5 ℃), to-15 ℃ of pre-freezes 2 hours, then-45 ℃ to-55 ℃ lyophilizing 30 hours, at-10 ℃ of degree, distil 15 hours, 15 ℃ of secondaries distil 3 hours, obtain biomembrane of the present invention.
2, strengthen: diaphragm is soaked in 3% genipin solution to 6 hours, carries out, after chemical crosslinking, putting into vacuum drying oven 90-110 ℃ of vacuum drying 15-20 hour.
3, sterilizing: gained diaphragm is carried out to double casing with aluminum-plastic composite membrane, the product after packing is carried out to sterilization treatment with gamma ray, obtain.
Gained diaphragm not only can be used for high-grade hemostatic material, also can be used for skin and meninges regeneration field simultaneously.
I type medical collagen prepared by the preparation method of the present invention of take is prepared bone fusion medical material as raw material and can be prepared with following methods: collagen is dissolved in organic acid (citric acid or the glacial acetic acid) aqueous solution containing 0.1%-1%, regulate pH value to 2.0-5.0, adopt high-speed shearing machine to disperse, temperature is controlled at 0 ℃-5 ℃ during this time, it is scattered that rear to add respectively with collagen weight ratio be 1: the tricalcium phosphate of 50-100: 50-100 and hydroxyapatite, disperse again, mix homogeneously, fill, sterilizing, obtains.
I type medical collagen prepared by the preparation method of the present invention of take is prepared external skin anti-decrepit beauty frost as raw material and can be made by following methods: I type medical collagen of the present invention is dissolved in citric acid, through high-speed shearing machine, disperse, with superhigh-voltage homogenizing machine, the collagen solution after disperseing is carried out to nanoscale fragmentation again, obtain can Transdermal absorption active nano collagen, mix with other adjuvants, skin protection is made skin care item and get final product with Emulsion routinely.
The specific embodiment:
Embodiment 1
The preparation method of type i collagen of the present invention.
1, raw material pre-treatment: choose the cattle heel string of newly slaughtering cattle, transportation is preserved in frozen water, after reaching the spot, with shears, remove unnecessary tissue, be positioned over-20 degree refrigerator and cooled and freeze, with blade, scrape off the unnecessary fascia in surface afterwards, with aluminium foil, be bundled into 10mm size cylindric, be positioned over that refrigerator and cooled is frozen puts, stand-by.
2, collagen extraction:
1), by the cattle heel string 1000g after freezing, cut into the thin slice of 1 millimeters thick, the NaCl solution soaking 20min with 5%, the KH with 0.5%
2p
4, 5 ℃ of temperature are soaked 2h, it is neutral by purified water, rinsing to pH value, after add ethanol and trichloroethane mixed liquor (1: 1) to soak, under slow stirring condition, defat is 1 hour, obtains pretreated feedstock 950g.
2) add containing 23%NaCl-0.05mol/L Tris-HCl (pH value 7.5) buffer after rinsing by purified water and soak 3h, during draw at any time the oils and fats on liquid level, to floating over liquid level without lipid.
3) Hou Yong historrhexis machine carries out fragmentation by lamellar heel string, and regulator solution pH value to 6.8 adds 1: 100 ficoin, under 4 ℃ of conditions, slowly stirs 5 hours, repeatedly rinses after the rear H2O2 with 1% processes 4h and cleans.
4) above gained collagen is added in saturated NaCl solution (PH7.2), under 4 degree conditions standing 12 hours, after add the NaOH solution of 1M and 0.5M ammonium chloride solution to soak, under 15 degree conditions standing 36 hours, finally with HCl, neutralize, regulate pH value to 7.2, under stirring condition, by purified water, rinse 3 times, then the water for injection that is 6.8 with PH, 5 ℃ are soaked 3 hours, remove excessive moisture, obtain 910 grams of pure collagens.
Embodiment 2
Biomembranous preparation of the present invention
By I type medical collagen of the present invention lyophilizing after dispersion and emulsion, then high-temperature physics is crosslinked, and packing, sterilizing, obtain.Specific as follows:
1, lyophilizing: gained collagen is joined in 0.5M glacial acetic acid solution by 3% (mass fraction) proportion, with online high-shear homogenizer, disperse 2-3 time, its temperature is controlled at below 10 ℃, and the liquid vacuum after dispersion is removed bubble, puts into freezer dryer lyophilizing pallet, make thickness at 4-6mm, at subzero 5 ℃ (5 ℃), to-15 ℃ of pre-freezes 2 hours, then-45 ℃ to-55 ℃ lyophilizing 30 hours, at-10 ℃ of degree, distil 15 hours, 15 ℃ of secondaries distil 3 hours, obtain biomembrane of the present invention.
2, strengthen: diaphragm is soaked in 3% genipin solution to 6 hours, carries out, after chemical crosslinking, putting into vacuum drying oven 90-110 ℃ of vacuum drying 15-20 hour.
3, sterilizing: the dried diaphragm of gained is carried out to double casing with aluminum-plastic composite membrane, the product after packing is carried out to sterilization treatment with gamma ray, obtain biomembrane of the present invention.
Embodiment 3
The preparation of injection bone integrating materials of the present invention
I type medical collagen of the present invention is dissolved in to (citric acid or glacial acetic acid) in organic acid, after high shear disperses, evenly mixes with a certain proportion of tricalcium phosphate and hydroxyapatite, packing, sterilizing, obtains.Specific as follows:
1, disperse: get 1000ml water for injection, add 0.5% glacial acetic acid, regulate pH value to 3.2, after add 6g collagen, with high speed dispersor, under the condition of 21000 revs/min, stir 4 hours, during temperature be controlled at 3-5 ℃.
2, mix: get tricalcium phosphate 400g, hydroxyapatite 600g after sterilizing, join in above-mentioned scattered collagen solution, use dispersion machine mix homogeneously.
3, packing: gained mixture is encapsulated in 10ml syringe, packs.
4, sterilizing: the product after packing carries out sterilization treatment with gamma ray, obtains injectivity bone integrating materials of the present invention.
Embodiment 4
The preparation of skin anti-aging frost of the present invention
I type medical collagen of the present invention is dissolved in citric acid, through high shear, disperse, then with TIS superhigh-voltage homogenizing machine, the collagen solution after dispersion carried out to nanoscale fragmentation, obtain can Transdermal absorption active nano collagen, mix and make the anti-ageing frost of reduce wrinkle with other adjuvants, specific as follows:
Formula:
Batching title: content (%Wt)
1, vitamin A palmitic acid lipid 1.00
2, vitamin E acetic acid fat 0.5
3, (mountain Yu acyl dilactic acid sodium) 1.5
4, cetostearyl alcohol (50/50) 3.00
5, monoglyceride 2.60
6, isopropyl palmitate 4.00
7, senior olive oil 5.00
8, deionized water 69.20
9, glycerol 3.00
10, DMDM Hydantoin 0.20
11, magnesium ascorbyl phosphate and lecithin 6.00
12, active nano collagen 3.00
13, palmityl hydroxylation wheat protein 1.00
Technique:
1, by formula ratio, take raw material 1-7, add in stainless steel cask and stir, mix homogeneously, is heated to 80C, makes oil phase.
2, get formula ratio glycerol again, join in deionized water, mix and blend in stainless steel cask, is heated to 80C, makes water.
3, the water making is poured in high speed homogenization tank, again oil phase is added, start twice of high-shear homogenizer homogenizing, be cooled to 25 ℃, add again DMDM Hydantoin, magnesium ascorbyl phosphate and lecithin, nanometre collagen and palmityl hydroxylation wheat protein, under 15 ℃ of cryogenic conditions, with high shear, carry out homogenizing and stir 120 minutes, obtain pure white paste anti-aging cream.
4, gained cream is added to filling machine under 15-25 ℃ of condition, carry out fill, sealing.
5, the good cream liquid bottle of fill is delivered between outer package, labeling, mounted box, get product.
Through experiment test, the collagen cosmetics of making by the method are because containing active collagen composition, after Transdermal absorption, can help new subcutaneous cell regeneration at the new netted collagen scaffold of subcutaneous formation, can effectively remove the wrinkle producing because of skin aging, through clinical verification, effect is very obvious.
Claims (7)
1. keep the raw-material preparation method of biological activity I type medical adhesive, it is characterized in that:
1), raw material pre-treatment: choose the cattle heel string of newly slaughtering cattle, transportation is preserved in frozen water, arrive and accelerate to process behind ground, processed, removes unnecessary tissue with shears immediately, is positioned over-20 degree refrigerator and cooled and freezes, with blade, scrape off the unnecessary fascia in surface afterwards, with aluminium foil, be bundled into 10mm size cylindric, be positioned over that refrigerator and cooled is frozen puts, standby.
2), collagen extraction:
A, get the cattle heel string after freezing, cut into the thin slice of 0.5-2 millimeters thick, with the NaCl solution soaking 15-30min of 5%-20%, then use 0.5% KH
2p
4temperature is at the solution soaking 1-3h of 0-5 ℃, and it is neutral by purified water, rinsing to pH value, after add ethanol and trichloroethane mixed liquor (1: 1) to soak, defat 1-3 hour under slow stirring condition, obtains pretreated feedstock.
B, raw material add containing 23%NaCl-0.05mol/L Tris-HCl (pH value 7.5) buffer and soak 3h after rinsing by purified water, during draw at any time the oils and fats on liquid level, to floating over liquid level without lipid.
C ,Hou Yong historrhexis machine carries out fragmentation by lamellar heel string, and regulator solution pH value to 6.8, adds 1: 100 ficoin, under 4 ℃ of conditions, slowly stir 5 hours, after repeatedly rinsing with 1% H
2o
2after processing 4h, clean.
D, above gained collagen is added in saturated NaCl solution (PH7.0-7.2), standing 6-24 hour under 4 ℃ of conditions, after add the NaOH solution of 1M and 0.5M ammonium chloride solution to soak, under 15-20 ℃ of condition standing 36 hours, finally with HCl, neutralize, regulate pH value to 7.2, under stirring condition, by purified water, rinse 3 times, then the water for injection that is 6.8 with pH soaks 3 hours at 5 ℃ of temperature, filter, remove excessive moisture, freezing preservation, obtains.
2. a biological haemostatic membrane, is characterized in that: it is that the collagen with biological activity maintenance collagen triple helical knot being made by claim 1 is that raw material is prepared from.
3. a preparation method for biological haemostatic membrane, is characterized in that: it is that to keep the collagen of collagen triple helical knot be that raw material is prepared from by following method to the biological activity that has being made by claim 1:
1), lyophilizing: collagen is joined in 0.5M glacial acetic acid solution by 3% (mass fraction) proportion, with online high-shear homogenizer, disperse 2-3 time, temperature is controlled at below 10 ℃, and the liquid vacuum after dispersion is removed bubble, puts into freezer dryer lyophilizing pallet, make thickness at 4-6mm, subzero 5 ℃ to subzero 15 ℃ of pre-freezes 2 hours, then-45 ℃ to-55 ℃ lyophilizing 30 hours, at-10 ℃ of degree, distil 15 hours, 15 ℃ of secondaries distil 3 hours, standby;
2), strengthen: diaphragm is soaked in 3% genipin solution to 6 hours, carries out, after chemical crosslinking, putting into vacuum drying oven 90-110 ℃ of vacuum drying 15-20 hour.
3), sterilizing: gained diaphragm is carried out to double casing with aluminum-plastic composite membrane, the product after packing is carried out to sterilization treatment with gamma ray, obtain.
4. biological bone merges a medical material, it is characterized in that: it is that the collagen with biological activity maintenance collagen triple helical knot being made by claim 1 is that raw material is prepared from.
5. a biological bone merges the preparation method of medical material, it is characterized in that: it is that the collagen with biological activity maintenance collagen triple helical knot being made by claim 1 is that raw material is prepared from by following method: collagen is dissolved in organic acid (citric acid or the glacial acetic acid) aqueous solution containing 0.1%-1%, regulate pH value to 2.0-5.0, adopt high-speed shearing machine to disperse, temperature is controlled at 0 ℃-5 ℃ during this time, it is scattered that rear to add respectively with collagen weight ratio be 1: the tricalcium phosphate of 50-100: 50-100 and hydroxyapatite, disperse again, mix homogeneously, fill, sterilizing, obtain.
6. a skin anti-decrepit beauty frost, is characterized in that: it is that the collagen with biological activity maintenance collagen triple helical knot being made by claim 1 is that raw material is prepared from.
7. the preparation method of a skin anti-decrepit beauty frost, it is characterized in that: it is that the collagen with biological activity maintenance collagen triple helical knot being made by claim 1 is that raw material is prepared from by following method: I type medical collagen of the present invention is dissolved in citric acid, through high-speed shearing machine, disperse, with superhigh-voltage homogenizing machine, the collagen solution after disperseing is carried out to nanoscale fragmentation again, obtain the active nano collagen of maintenance triple-helix structure that can Transdermal absorption, mix with other adjuvants, skin protection is made skin care item and get final product with Emulsion routinely.
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| CN110025820A (en) * | 2019-05-30 | 2019-07-19 | 福建吉特瑞生物科技有限公司 | A kind of absorbable collagen sutures and preparation method thereof |
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Application publication date: 20140212 |