CN1407002A - Collagen fibre extracting method and manufacture of tissue filling material therefrom - Google Patents
Collagen fibre extracting method and manufacture of tissue filling material therefrom Download PDFInfo
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- CN1407002A CN1407002A CN 01131437 CN01131437A CN1407002A CN 1407002 A CN1407002 A CN 1407002A CN 01131437 CN01131437 CN 01131437 CN 01131437 A CN01131437 A CN 01131437A CN 1407002 A CN1407002 A CN 1407002A
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- collegen filament
- collagen
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Abstract
A process for extracting collagen fibres includes such steps as immersing animal tissue in high-penetrability acid, washing to remove acid, enzymolyzing and grinding. A process for preparing tissue filler includes such steps as diluting said collagen fibres, packing, sealing and sterilizing to obtain the filler for soft tissue, or die pressing said collagen fibres, drying and sterilizing to obtain the filler for hard dissue. Its advantages are high output rate up to more than 80% and quality, and long storage period.
Description
Technical field
The present invention relates to a kind of extracting method and a kind of method and a kind of method of making sclerous tissues's weighting agent with collegen filament of making soft tissue filler with collegen filament of collegen filament.
Background technology
At present, tissue filling agent is divided into soft tissue filler and sclerous tissues's weighting agent.As the animal-origin injectivity collagen of soft tissue filler for many years, generally be difficult to from the collagen extracting solution, produce as the collagem membrane of sclerous tissues's weighting agent in clinical application.
Collegen filament are harder, and higher tension force is arranged.A tropocollagen molecule diameter is 1.5 μ m, and length is 280 μ m, is made up of the polypeptide chain of three α configurations; Every chain contains 1052 amino-acid residues, and wherein glycine accounts for 1/3rd, and proline(Pro) and oxyproline respectively account for 1/4th, and every next but two amino acid has a glycine, and glycine and proline(Pro), oxyproline or other amino acid are formed triamino acid association.The N-terminal of polypeptide chain and C-terminal all do not have triamino acid association, leave acidic amino acid and basic aminoacids, and these zones of tropocollagen molecule are called the end peptide, do not form triple helix; The crosslinked structural relation that is meant between tropocollagen molecule inside and tropocollagen molecule; for the fiber maturation provides more tension force and stability; crosslinkedly in organizing ripening process, carry out; lysyl or hydroxyprolyl-change corresponding aldehyde under the effect of enzyme; form Schiff alkali or aidimines type compound, the spontaneous generation of this reaction with aidimines or hydroxyl then.
Collagen is a kind of inflexible scleroproein that has, and general proteolytic enzyme is difficult to its degraded, and its physiological transforms also well below other protein.The collagen transformation period is longer, Chang Yiyue or year calculate, but when organizing when rebuilding rapidly, and the degraded of collagen or change in quality relatively will be hurry up, its transformation period is then with sky or hour calculating.The problem that xenogenesis or collagen of the same race are transplanted most critical is the antigenicity problem of material, and verified from molecular level, injectable collagen is a kind of poor antigen material.The major antigen site of collagen is positioned at the N-terminal and the C-terminal zone of molecule.These two peptide ends have very big species variation, play main immunization.These two zones are by optionally hydrolysis or removal and inactivation when extracting collagen, and only the triple helix inside configuration in minute daughter keeps faint antigenicity, and is not enough to cause tangible rejection, thereby greatly reduces the antigenicity of collagen.Animal collagen may excite certain immune response, with clinical dosage syringeability collagen treatment patient the time, can detect increasing of anticol original antibody, even in the patient of no skin reaction, also can detect, but these antibody and people's collagen no cross reaction contacts harmless concerning most patient with collagen biological.
Collagen is divided into soluble collagen and insoluble collagen.Insoluble definition is relative, and its insoluble reason is relevant with the interaction of intercellular substance composition, in addition with advancing age, between tropocollagen molecule and other intermolecularly also can form bridge formation---crosslinked.These insoluble collagen molecules and other non-collagen tissues mix, and have caused certain difficulty to extract collagen from tissue.The soluble collagen extraction yield has 5% in the human fetal skin, enter juvenile period after extraction yield have only 1%.The formation of extracting and adding inhibitor inhibition collegen filament repeatedly can make calf skin skin collagen have 50% to become soluble collagen under certain condition.
At present, the method for extracting collagen normally: with new calves skin or human placenta degrease, chopping acid is molten, enzymolysis, acid is carried, high speed centrifugation repeats above-mentioned acid and lifts from heart step, the dialysis of saltouing, crosslinked reorganization, sterilization precipitates lyophilize.
Summary of the invention
The extract of existing collagen extracting method is a soluble collagen, because animal material source limitation is strong, the leaching process complexity needs to add linking agent, and the product viscosity is big, and extraction yield is not high, can not make collagem membrane, so clinical application is very restricted.
The objective of the invention is to: a kind of extracting method of collegen filament is provided, and method is simple, and product is stable, and the collegen filament that this method obtains can arrive widespread use clinically.
The present invention is achieved in that a kind of extracting method of collegen filament, and this method comprises the steps:
(1) selects to scrub unhairing behind the healthy slaughtering animal, get back degrease holostrome skin and/or tendon and/or plate muscle and/or bone, oozed acid soak 24-120 hour with height under the normal temperature;
(2) stand-by tissue is taken out, clean deacidification, it being soaked in temperature is 4 ℃ again--in-20 ℃ the 0.1%---2% pepsin solution, and 12---24 hour;
(3) stand-by tissue is taken out cleaning, obtain acellular collagen framework, acellular collagen framework is cut into the tissue block of 0.05cm---0.5cm size;
(4) repeat above-mentioned steps (1) and step (2)
(5) tissue block being ground to form diameter is 10 μ m---50 μ m, and length is the collegen filament silk of 300 μ m---500 μ m.
According to aforesaid method, wherein step (1) adopts acetic acid or the hydrochloric acid of 3---10N.
According to aforesaid method, wherein step (2) adopts the alkali neutralizing acid of 0.5---6N.
According to aforesaid method, wherein step (5) adopts the polarizing microscope monitoring.
A kind of collegen filament that make with aforesaid method are made the method for soft tissue filler, this method comprises the step of said extracted collegen filament, also comprises: the collegen filament silk is diluted to 80---120mg/ml with physiological saline or phosphate buffer solution, the syringe of packing into, seal, use Co
60Irradiation.
A kind of collegen filament that make with aforesaid method are made the method for sclerous tissues's weighting agent, and this method comprises the step of said extracted collegen filament, also comprises: the collegen filament silk is inserted in the mould of definite shape, and extrusion forming, at 40 ℃---90 ℃ are dry down, use Co
60Irradiation.
The extracting method of collegen filament is different from existing soluble collagen extraction process fully among the present invention.The tissues such as skin that raw material comprises animal, tendon, cartilage, bone of producing of the present invention, productive rate can reach more than 80%.Destroyed the structure of cell and dissolved the extracellular soluble components by adopting height to ooze acid, species specificity is eliminated in enzymatic reaction, produce acellular collagen framework, do not need to add linking agent, do not need high speed centrifugation, saltout, step such as dialysis, therefore the degraded product of collagen-free molecule and soluble collagen in the product of acquisition are not high adhesive materials, extracted amount is brought up to 400mg/ml from traditional 35mg/ml, and product uses 80---120mg/ml usually.
The manufacture method of soft tissue of the present invention and sclerous tissues's weighting agent is the result of the further in-depth on the extracting method of above-mentioned collegen filament.The soft tissue filler long preservative period that it obtained, the productive rate height, product is stable; Sclerous tissues's weighting agent has been filled up the blank that present this product directly obtains from animal tissues.
Embodiment
The present invention will be further described below in conjunction with specific embodiments of the invention.
Ox after embodiment 1 will newly butcher is scrubbed peeling, gets tendon 500 grams, the salt acid soak of the following usefulness of normal temperature 2N 48 hours; Stand-by tissue is taken out, clean with distilled water, 0.5N alkali neutralizing acid, it being soaked in temperature is 20 ℃ again, in 0.1% pepsin solution 12 hours; Stand-by tissue is taken out water clean, obtain acellular collagen framework.Acellular collagen framework is cut into the tissue block of 0.05cm---0.5cm size; Repeat above-mentioned acidleach and enzymolysis step once; Under the polarizing microscope monitoring tissue block being ground to form diameter is 10 μ m---50 μ m, and length is the collegen filament silk of 300 μ m---500 μ m.Obtain product 2000 grams altogether.
The collagen filament product of getting certain volume is at 50 ℃--and be dried to constant temperature between-90 ℃, calculate the back of weighing, and content is 400mg/ml.
The collegen filament that obtain with present embodiment carry out the following compositions identification experiment.[purpose]:,, determine the quality of the injectivity collagen that the present invention obtains with the amino acid kind and the degree of infrared absorption spectrum (molecular spectrum), high pressure liquid phase analysis degraded with standard substance, positive control contrast.[sample source and measuring unit]: standard substance---collagen protein, Sigma company produces.The injectivity collagen that positive control---people placenta is made, medical collagen company of China prevention academy of sciences produces.Measuring unit---Tsing-Hua University carries out the infrared absorption spectrum experiment.
High pressure liquid phase analysis amino acid percentage composition, chemical method survey sugar are carried out in analysis testing research center, Liaoning Province.
[result] product that the present invention obtained is a collagen protein, is not general protein;
All kinds of collagen amino acid content of product that the present invention obtained and reference substance do not have significant difference (p＜0.05);
The product that the present invention obtained is pure collagen protein, the sugar-free prothetic group.
Pig after embodiment 2 will newly butcher is scrubbed unhairing, gets back degrease holostrome skin 500 grams, and normal temperature soaked 68 hours with the acetic acid of 10N down; Stand-by tissue is taken out, clean with distilled water, the neutralization of 0.5N alkali, it being soaked in temperature is 4 ℃ again, in 0.2% pepsin solution 24 hours; Stand-by tissue is taken out water clean, obtain acellular collagen framework.Acellular collagen framework is cut into the tissue block of 0.05cm---0.5cm size; Repeat above-mentioned acidleach and enzymolysis step once; Under the polarizing microscope monitoring, it is 10 μ m---50 μ m that tissue block is ground to form diameter, and length is the collegen filament silk of 300 μ m---500 μ m, obtains product 2000 grams altogether.
Embodiment 3 is according to embodiment 1 described method, and wherein the salt acid soak 72 hours of 3N is adopted in acidleach, and it is that 1.5% pepsin solution soaked 18 hours that enzymolysis adopts concentration.
Embodiment 4 is diluted to 120mg/ml with the collegen filament silk that obtains with physiological saline, and the syringe of packing into seals, and uses Co
60Irradiation obtains soft tissue filler---injectable collagen.Carry out microbial culture with the syringeability collagen that present embodiment obtained, no bacterial growth; Carry out the antigenicity experiment, the result is as follows: with the syringeability collagen injection in the rat tails subcutis, through 1.5,3.0 and 6.0 months, kill three groups of rats respectively, get the section of afterbody injection subcutis, H.E dyeing, the characteristics of different time behind the observation injection collagen under polarizing microscope, the syringeability collagen that proof makes with method of the present invention does not have obvious inflammatory reaction, no antigen reaction, with tissue compatible, do not have the parcel phenomenon, still have the residual collagen of part after 6.0 months in muscular tissue gap and fatty tissue.
Embodiment 5 is diluted to 80mg/ml with the collagen filament with phosphate buffer solution according to embodiment 4 described methods.
Embodiment 6 inserts the collegen filament silk that obtains in the mould of definite shape according to embodiment 1 described method, extrusion forming, at 40 ℃---90 ℃ are dry down, use Co
60Irradiation obtains sclerous tissues's weighting agent.
Claims (6)
1. the extracting method of collegen filament, this method comprises the steps:
(1) selects to scrub unhairing behind the healthy slaughtering animal, get back degrease holostrome skin and/or tendon and/or plate muscle and/or bone, oozed acid soak 24-120 hour with height under the normal temperature;
(2) stand-by tissue is taken out, clean deacidification, it being soaked in temperature is 4 ℃ again--in-20 ℃ the 0.1%---2% pepsin solution, and 12---24 hour;
(3) stand-by tissue is taken out cleaning, obtain acellular collagen framework, acellular collagen framework is cut into the tissue block of 0.05cm---0.5cm size;
(4) repeat above-mentioned steps (1) and step (2);
(5) tissue block being ground to form diameter is 10 μ m---50 μ m, and length is the collegen filament silk of 300 μ m---500 μ m.
2. the extracting method of collegen filament according to claim 1 is characterized in that: acetic acid or the hydrochloric acid of described step (1) employing 3---10N.
3. the extracting method of collegen filament according to claim 1 and 2 is characterized in that: the alkali neutralizing acid of described step (2) employing 0.5---6N.
4. the extracting method of collegen filament according to claim 1 and 2 is characterized in that: the monitoring of described step (5) employing polarizing microscope.
5. method of making soft tissue filler with the described collegen filament of claim 1, this method comprises the step of said extracted collegen filament, also comprises: the collegen filament silk is diluted to 80---120mg/ml with physiological saline or phosphate buffer solution, the syringe of packing into, seal, use Co
60Irradiation.
6. method of making sclerous tissues's weighting agent with the described collegen filament of claim 1, this method comprises the step of said extracted collegen filament, also comprises: the collegen filament silk is inserted in the mould of definite shape extrusion forming, at 40 ℃---90 ℃ are dry down, use Co
60Irradiation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01131437 CN1235947C (en) | 2001-09-10 | 2001-09-10 | Collagen fibre extracting method and manufacture of tissue filling material therefrom |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01131437 CN1235947C (en) | 2001-09-10 | 2001-09-10 | Collagen fibre extracting method and manufacture of tissue filling material therefrom |
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| CN1407002A true CN1407002A (en) | 2003-04-02 |
| CN1235947C CN1235947C (en) | 2006-01-11 |
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| CN 01131437 Expired - Fee Related CN1235947C (en) | 2001-09-10 | 2001-09-10 | Collagen fibre extracting method and manufacture of tissue filling material therefrom |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100351167C (en) * | 2005-08-26 | 2007-11-28 | 张立文 | Collagen fiber stuffing product and processing method thereof |
| CN101831724A (en) * | 2010-04-16 | 2010-09-15 | 陈雄生 | Acellular tendon or ligament collagenous fiber material and preparation method thereof |
| WO2017107996A1 (en) * | 2015-12-25 | 2017-06-29 | 北京瑞健高科生物科技有限公司 | Cell-growing scaffold having structure memory property |
| WO2017107997A1 (en) * | 2015-12-25 | 2017-06-29 | 北京瑞健高科生物科技有限公司 | Method for preparing cell growth scaffold having structural memory properties |
| CN108245711A (en) * | 2018-02-26 | 2018-07-06 | 王由 | A kind of collagen compound 45S5 bioactivity glass and its preparation method and application |
| CN108383904A (en) * | 2018-02-26 | 2018-08-10 | 王由 | Application of the extracting method, collagenous fibres of collagenous fibres on manufacture tissue filling agent |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101173486B (en) * | 2007-10-09 | 2011-04-06 | 李瑜 | Method for producing corium fabric leather by continuous wet method |
-
2001
- 2001-09-10 CN CN 01131437 patent/CN1235947C/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100351167C (en) * | 2005-08-26 | 2007-11-28 | 张立文 | Collagen fiber stuffing product and processing method thereof |
| CN101831724A (en) * | 2010-04-16 | 2010-09-15 | 陈雄生 | Acellular tendon or ligament collagenous fiber material and preparation method thereof |
| CN101831724B (en) * | 2010-04-16 | 2012-12-12 | 陈雄生 | Acellular tendon or ligament collagenous fiber material and preparation method thereof |
| WO2017107996A1 (en) * | 2015-12-25 | 2017-06-29 | 北京瑞健高科生物科技有限公司 | Cell-growing scaffold having structure memory property |
| WO2017107997A1 (en) * | 2015-12-25 | 2017-06-29 | 北京瑞健高科生物科技有限公司 | Method for preparing cell growth scaffold having structural memory properties |
| US10814037B2 (en) | 2015-12-25 | 2020-10-27 | Beijing Ruijian Gaoke Biotechnology Co., Ltd. | Method for preparing cell growth scaffold having structural memory properties |
| CN108245711A (en) * | 2018-02-26 | 2018-07-06 | 王由 | A kind of collagen compound 45S5 bioactivity glass and its preparation method and application |
| CN108383904A (en) * | 2018-02-26 | 2018-08-10 | 王由 | Application of the extracting method, collagenous fibres of collagenous fibres on manufacture tissue filling agent |
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| CN1235947C (en) | 2006-01-11 |
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