CN1763135A - A kind of preparation method of bone collagen gel liquid - Google Patents
A kind of preparation method of bone collagen gel liquid Download PDFInfo
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- CN1763135A CN1763135A CN 200510057329 CN200510057329A CN1763135A CN 1763135 A CN1763135 A CN 1763135A CN 200510057329 CN200510057329 CN 200510057329 CN 200510057329 A CN200510057329 A CN 200510057329A CN 1763135 A CN1763135 A CN 1763135A
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Abstract
The present invention relates to biomedical engineering field, be a kind ofly to make up the method that tissue engineered bone prepares osso-albumin for carrying out the double phase inoculation method, particularly a kind of preparation method of bone collagen gel liquid is characterized in that adopting following step of preparation process: the 1) preparation of decalcified bone matrix powder; 2) removal of the non-collagen impurity of decalcified bone matrix powder; 3) the collagen protein sex change of the decalcified bone matrix powder of the non-collagen impurity of removal; 4) the collagen protein enzymolysis of the decalcified bone matrix after the collagen protein sex change; 5) collagen protein purifying; 6) coagulant liquid preparation.The present invention has simple to operate, the extraction efficiency height, and collagen purity height becomes the good advantage of gelation.
Description
Technical field
The invention belongs to biomedical engineering field, is a kind ofly to make up the preparation method that tissue engineered bone prepares the method, particularly a kind of bone collagen gel liquid of osso-albumin for carrying out the double phase inoculation method.
Background technology
In the clinical position, multiple treatment of diseases as: nonunion, bone is damaged and various bone fusion all needs to use bone grafting material.At present, a lot of for the bone grafting material kind of clinical use, all there is defective in various degree, therefore, still there is not ideal bone grafting material source at present.The rise of organizational project and develop into the development bone grafting material novel, that have good osteogenic activity new thinking and technological method is provided.Through the development in surplus ten years, bone tissue engineer was obtained huge advance made in each key link.Yet at seed cell and timbering material is compound when carrying out tissue construction, seed cell inoculation inefficiency has had a strong impact on the quality of tissue engineered bone and has caused a large amount of wastes of seed cell, is the technical barrier that needs to be resolved hurrily at present.Have research use mouse tail collagen carry out seed cell double phase inoculation promptly earlier with seed cell under proper condition with collagen gel liquid uniform mixing, before the collagen cohesion that mixture and tissue engineering bracket is compound, thereby obtained the high seed cell rate of putting on the shelf, successfully constructed tissue engineering bone/cartilage with good properties.But mouse tail collagen source is limited, and has the ethics obstacle, be difficult to be applied to clinical, therefore need to seek a kind of source abundant, become gelation good, help the sophisticated collagen gel of tissue engineered bone source.
Collagen is the main structural protein of vertebrates and human body, accounts for the 25-30% of human body protein total amount, is distributed widely in various tissues such as bone, tendon, skin, blood vessel and the organ.The kind of collagen is a lot, and wherein I, II, III and IV type are four kinds of the most frequently used collagens.There are some researches show that type i collagen has promoter action to the expression of osteoblastic proliferation and phenotype, and that whether the collagen of other type has a negative impact to skeletonization is still unknown at present.The collagen of using in each field at present is mainly derived from tissues such as skin, tendon, but still contains other multiple collagen composition in these tissues except that containing a large amount of non-collagen compositions, is difficult for purifying, the extraction cost height.Osseous tissue is a huge collagen storehouse, and its collagen composition mainly is a type i collagen, and the content height, accounts for more than 90% of bone organic composition.Osso-albumin derives from bone simultaneously, and the source is abundant, and its composition should have no adverse effect to skeletonization in theory, so bone is to obtain to carry out the comparatively ideal source that tissue engineered bone makes up required collagen gel liquid.
But organizing different with other is that the crosslinking degree height of osso-albumin, acid-soluble method commonly used, neutral sulfity process are difficult to extract.Alkali solution technique can be used for extracting osso-albumin, document is arranged: Kemp, GD.Tristram, GR.The preparation of an alkali-collagen fromdemineralized bone.Biochem J, 1971,124 (5): its technical characterictic of 915-919. be will decalcified bone matrix with effect under the 5%NaOH20 ℃ of condition 48 hours, with hydrochloric acid the pH value of mixture is transferred to 4.0 then, have a large amount of flockss to produce, through centrifugal, dissolve, saltout, processes such as dialysis, freeze-drying obtain collagen.But the osso-albumin that alkali solution technique extracts, its soltion viscosity is low, becomes gelation poor, uses to be very limited in osseous tissue makes up.
Summary of the invention
The objective of the invention is to overcome aforementioned the deficiencies in the prior art, provide a kind of simple to operate, extraction efficiency height, collagen purity height, the preparation method of the bone collagen gel liquid that the one-tenth gelation is good.
The present invention has adopted such technical scheme: a kind of preparation method of bone collagen gel liquid is characterized in that adopting following step of preparation process:
1) preparation of decalcified bone matrix powder;
2) removal of the non-collagen impurity of decalcified bone matrix powder;
3) the collagen protein sex change of the decalcified bone matrix powder of the non-collagen impurity of removal;
4) the collagen protein enzymolysis of the decalcified bone matrix after the collagen protein sex change;
5) collagen protein purifying;
6) coagulant liquid preparation.
The preparation of decalcified bone matrix powder is to get the fresh animal long bone, remove surrounding soft tissue and periosteum, epiphysis spongy bone, strike off marrow, then cortex bone warded off into fragment, through clear water behind Xian, the methyl alcohol-chloroform degreasing 12-72 hour that adds 1: 1, add volumetric molar concentration again and be hydrochloric acid decalcification 3-14 days of 0.1-1.0M, after the tri-distilled water rinsing, once more through 1: 1 methyl alcohol-chloroform degreasing 12-72 hour, remollescent cortex bone piece blends into the decalcified bone matrix powder through organizing pulverizer to incite somebody to action, and is air-dry standby.
The removal of the non-collagen impurity of decalcified bone matrix powder is to get 5 parts of decalcified bone matrix powder, adding 150-250 part concentration is the NaOH solution of 0.01%-0.2%, place 4 ℃ of refrigerator effects 12-72 hour, take out then and filter, filter residuum tri-distilled water repetitive scrubbing.
The collagen protein sex change of removing the decalcified bone matrix powder of non-collagen impurity is to add 50-100 part to contain Guanidinium hydrochloride that volumetric molar concentration is 2.0-6.0M and pH value be in 7.4 the Tris-HCl solution removing decalcified bone matrix powder behind the non-collagen impurity, place 4 ℃ of refrigerator effects 12-72 hour, take out then and filter, filter residuum and remove Guanidinium hydrochloride with the tri-distilled water repetitive scrubbing.
The collagen protein enzymolysis of the decalcified bone matrix powder after the collagen protein sex change is that the decalcified bone matrix after the collagen protein sex change is added 200 parts of-800 parts of volumetric molar concentrations is in the acetum of 0.1-1.0M, add 0.05-2 part stomach en-again, place on the magnetic stirring apparatus, in 5 ℃ of-25 ℃ of environment continuously stirring 24-96 hour, obtain decalcified bone matrix liquid.
The purifying of collagen protein is to pass through the decalcified bone matrix liquid of collagen protein enzymolysis under 2-10 ℃ of temperature, be on the whizzer of 6000-20000rpm after centrifugal 10-60 minute at rotating speed, get its supernatant liquor, adding the NaCl crystal in supernatant liquor makes supernatant liquor concentration reach 2M, when having a large amount of purple shape precipitations to produce, centrifugal under aforementioned the same terms once more, taking precipitate, adding 100-200 part volumetric molar concentration is the Glacial acetic acid dissolving of 0.1-1.0M and the dialysis tubing of packing into, is the Na of 10-20mM in volumetric molar concentration
2HPO
42-24 hour inactivated pepsin of dialysis in the solution, the glacial acetic acid solution dialysis of putting into volumetric molar concentration again and be 0.1-1.0M is after 24-72 hour, repeat again above-mentionedly to saltout, centrifugal, dissolving, dialysis operation once, the viscosity collagen coagulant liquid that the obtains freeze-drying in the beaker of packing into obtains the freeze drying bone collagen of purifying.
The bone collagen gel liquid that concentration is 0.1-10% is made in coagulant liquid preparation and to solidify be to be that the Glacial acetic acid of 0.1-1.0M dissolves with freeze drying bone collagen volumetric molar concentration.
The present invention is directed to two key links that influence collagenolysis: crosslinked degree and position thereof; The existence of specificity noncollagen protein composition and designing, at first specificity noncollagen protein composition is removed with low concentration alkali, use the unspiralized region at the tropocollagen molecule two ends of the crosslinked concentrated distribution of stomach en-selective rhizotomy again, thus make osso-albumin by insoluble become solvable.Add this strong protein denaturant of Guanidinium hydrochloride in the scheme, destroy the hydrogen bond that forms between tropocollagen molecule by forming hydrogen bond between the amino acid side chain of itself and tropocollagen molecule, and the effect of hydrophobic bond between the blocking-up tropocollagen molecule, thereby the resolvability of enhancing osso-albumin, the extraction efficiency of raising osso-albumin.The present invention has easy and simple to handle, the extraction efficiency height, and collagen purity height becomes advantages such as gelation is good.
Embodiment
Apply the present invention to the extraction of pig osso-albumin below, embodiment of the present invention are described.A kind of preparation method of bone collagen gel liquid is characterized in that adopting following step of preparation process:
1) preparation of decalcified bone matrix powder;
2) removal of the non-collagen impurity of decalcified bone matrix powder;
3) the collagen protein sex change of the decalcified bone matrix powder of the non-collagen impurity of removal;
4) the collagen protein enzymolysis of the decalcified bone matrix after the collagen protein sex change;
5) collagen protein purifying;
6) coagulant liquid preparation.
The preparation of decalcified bone matrix powder
Get the fresh pig long bone, remove surrounding soft tissue and periosteum, epiphysis spongy bone, strike off marrow, then cortex bone is split into fragment, after a large amount of flushing with clean water, add in methyl alcohol-chloroform of 1: 1 and took off ester 24 hours, adding volumetric molar concentration again is the hydrochloric acid decalcification 8 days (changing decalcifying Fluid once in per 48 hours) of 0.6M, after a large amount of tri-distilled water rinsings, uses methyl alcohol-chloroform of 1: 1 to take off ester 24 hours once more, remollescent cortex bone piece blends into the decalcified bone matrix powder to organize pulverizer to incite somebody to action, and is air-dry standby.
The removal of the non-collagen impurity of decalcified bone matrix powder
Take by weighing 5.0 gram decalcified bone matrix powder, adding 150 gram concentration is 0.1%NaOH solution, places 4 ℃ of refrigerator effects 24 hours, filters then, filters residuum tri-distilled water repetitive scrubbing.
Remove the collagen protein sex change of the decalcified bone matrix powder of non-collagen impurity
The decalcified bone matrix powder of removing non-collagen impurity adds 100 grams, and to contain volumetric molar concentration be that 4.0M Guanidinium hydrochloride and pH value are in the solution of 7.4 Tris-HCl, place 4 ℃ of refrigerator continuation effects 24 hours, filter then, filter residuum and remove Guanidinium hydrochloride with the tri-distilled water repetitive scrubbing.
The collagen protein enzymolysis of the decalcified bone matrix after the collagen protein sex change
It is in the acetum of 0.5M that decalcified bone matrix after the collagen protein sex change adds 500 gram volumetric molar concentrations, add 0.5 gram stomach en-(Sigma company) again, place on the magnetic stirring apparatus, continuously stirring is 48 hours in 10 ℃ of-20 ℃ of environment, obtains decalcified bone matrix liquid.
The purifying of collagen protein
Through the decalcified bone matrix liquid behind the collagen protein enzymolysis under 4 ℃, 10000rpm condition centrifugal 20 minutes, getting supernatant liquor adding NaCl crystal makes its concentration reach 2M, there are this moment a large amount of purple shape precipitations to produce, centrifugal under aforementioned the same terms once more, taking precipitate, adding 100 gram concentration for the dissolving of 0.5M Glacial acetic acid and the dialysis tubing of packing into, is the Na of 20mM in concentration
2HPO
48 hours inactivated pepsins of dialysis in the solution, putting into concentration again is 0.175M glacial acetic acid solution dialysis 48 hours (dialyzates of replacing in per 8 hours), repeat above-mentionedly to saltout, centrifugal, dissolving, dialysis operation once, get the viscous adhesive original solution at last, with the collagen solution freeze-drying in the large beaker of packing into, at last the freeze drying bone collagen of purifying.
The coagulant liquid preparation
Freeze drying bone collagen volumetric molar concentration is the dissolving of 0.5M Glacial acetic acid, concentration is 4% bone collagen gel liquid.This collagen gel liquid is inserted in the ice bath, is that the NaOH of the NaOH of 1M and 0.1M is adjusted to 7.35-7.45 with the pH value of solution with concentration, joins rapidly in six orifice plates then, puts in 37 ℃ of water-baths and hatches 30 minutes, forms collagen gel.
Claims (7)
1, a kind of preparation method of bone collagen gel liquid is characterized in that adopting following step of preparation process:
1) preparation of decalcified bone matrix powder;
2) removal of the non-collagen impurity of decalcified bone matrix powder;
3) the collagen protein sex change of the decalcified bone matrix powder of the non-collagen impurity of removal;
4) the collagen protein enzymolysis of the decalcified bone matrix after the collagen protein sex change;
5) collagen protein purifying;
6) coagulant liquid preparation.
2, preparation method by the described bone collagen gel liquid of claim 1, the preparation that it is characterized in that said decalcified bone matrix powder is to get the fresh animal long bone, remove surrounding soft tissue and periosteum, the epiphysis spongy bone, strike off marrow, then cortex bone is warded off into fragment, through clear water behind Xian, the methyl alcohol-chloroform degreasing 12-72 hour that adds 1: 1, add volumetric molar concentration again and be hydrochloric acid decalcification 3-14 days of 0.1-1.0M, after the tri-distilled water rinsing, once more through 1: 1 methyl alcohol-chloroform degreasing 12-72 hour, through organize pulverizer remollescent cortex bone piece blend into the decalcified bone matrix powder, air-dry standby.
3, press the preparation method of the described bone collagen gel liquid of claim 1, the removal that it is characterized in that the non-collagen impurity of said decalcified bone matrix powder is to get 5 parts of decalcified bone matrix powder, adding 150-250 part concentration is the NaOH solution of 0.01%-0.2%, place 4 ℃ of refrigerator effects 12-72 hour, take out then and filter, filter residuum tri-distilled water repetitive scrubbing.
4, press the preparation method of the described bone collagen gel liquid of claim 1, the collagen protein sex change that it is characterized in that the decalcified bone matrix powder of the non-collagen impurity of said removal is to add 50-100 part to contain Guanidinium hydrochloride that volumetric molar concentration is 2.0-6.0M and pH value be in 7.4 the Tris-HCl solution removing decalcified bone matrix powder behind the non-collagen impurity, place 4 ℃ of refrigerator effects 12-72 hour, take out then and filter, filter residuum and remove Guanidinium hydrochloride with the tri-distilled water repetitive scrubbing.
5, press the preparation method of the described bone collagen gel liquid of claim 1, the collagen protein enzymolysis that it is characterized in that the decalcified bone matrix powder after the said collagen protein sex change is that the decalcified bone matrix after the collagen protein sex change is added 200 parts of-800 parts of volumetric molar concentrations is in the acetum of 0.1-1.0M, add 0.05-2 part stomach en-again, place on the magnetic stirring apparatus, in 5 ℃ of-25 ℃ of environment continuously stirring 24-96 hour, obtain decalcified bone matrix liquid.
6, preparation method by the described bone collagen gel liquid of claim 1, the purifying that it is characterized in that said collagen protein is to pass through the decalcified bone matrix liquid of collagen protein enzymolysis under 2-10 ℃ of temperature, be on the whizzer of 6000-20000rpm after centrifugal 10-60 minute at rotating speed, get its supernatant liquor, adding the NaCl crystal in supernatant liquor makes supernatant liquor concentration reach 2M, when having a large amount of purple shape precipitations to produce, centrifugal under aforementioned the same terms once more, taking precipitate, adding 100-200 part volumetric molar concentration is the Glacial acetic acid dissolving of 0.1-1.0M and the dialysis tubing of packing into, is the Na of 10-20mM in volumetric molar concentration
2HPO
42-24 hour inactivated pepsin of dialysis in the solution, the glacial acetic acid solution dialysis of putting into volumetric molar concentration again and be 0.1-1.0M is after 24-72 hour, repeat again above-mentionedly to saltout, centrifugal, dissolving, dialysis operation once, the viscosity collagen coagulant liquid that the obtains freeze-drying in the beaker of packing into obtains the freeze drying bone collagen of purifying.
7,, it is characterized in that the coagulant liquid preparation is is that the Glacial acetic acid of 0.1-1.0M dissolves with freeze drying bone collagen volumetric molar concentration, makes the bone collagen gel liquid that concentration is 0.1-10% by the preparation method of the described bone collagen gel liquid of claim 1.
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| CNB2005100573292A CN100485002C (en) | 2005-10-18 | 2005-10-18 | Bone collagen gel liquid preparation method |
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Cited By (7)
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| CN101570772B (en) * | 2008-05-04 | 2012-07-25 | 中国肉类食品综合研究中心 | Method for preparing natural ossein |
| CN104371910A (en) * | 2014-10-31 | 2015-02-25 | 王选明 | Method for extracting collagen from deer bones |
| CN104984410A (en) * | 2015-07-29 | 2015-10-21 | 陕西博与再生医学有限公司 | Controllable-degradation lacrimal passage suppository and preparation method thereof |
| CN105861608A (en) * | 2016-05-30 | 2016-08-17 | 深圳凯联龟业有限公司 | Method for preparing tortoise protein polypeptides |
| CN109464699A (en) * | 2018-12-28 | 2019-03-15 | 华中农业大学 | One kind being used for bone defect healing packing material and preparation method |
| CN113072834A (en) * | 2020-01-03 | 2021-07-06 | 兰州大学 | Collagen biological ink for 3D printing and 3D printing method |
| CN113633827A (en) * | 2021-08-16 | 2021-11-12 | 中国人民解放军总医院第四医学中心 | Silk woven meniscus implant and preparation method thereof |
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| CN1151222C (en) * | 2001-04-08 | 2004-05-26 | 内蒙古草原兴发股份有限公司 | Process for preparing bone collagen |
| CN1147553C (en) * | 2002-05-22 | 2004-04-28 | 赵成军 | Working technology for extracting ossein from animal cartilage |
| CN1214821C (en) * | 2003-05-27 | 2005-08-17 | 重庆大学 | Preparing method for heteroossein base materials |
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- 2005-10-18 CN CNB2005100573292A patent/CN100485002C/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101570772B (en) * | 2008-05-04 | 2012-07-25 | 中国肉类食品综合研究中心 | Method for preparing natural ossein |
| CN104371910A (en) * | 2014-10-31 | 2015-02-25 | 王选明 | Method for extracting collagen from deer bones |
| CN104371910B (en) * | 2014-10-31 | 2016-08-24 | 世茂（苏州）生物科技有限公司 | A kind of method extracting collagen from deer bone |
| CN104984410A (en) * | 2015-07-29 | 2015-10-21 | 陕西博与再生医学有限公司 | Controllable-degradation lacrimal passage suppository and preparation method thereof |
| CN105861608A (en) * | 2016-05-30 | 2016-08-17 | 深圳凯联龟业有限公司 | Method for preparing tortoise protein polypeptides |
| CN109464699A (en) * | 2018-12-28 | 2019-03-15 | 华中农业大学 | One kind being used for bone defect healing packing material and preparation method |
| CN113072834A (en) * | 2020-01-03 | 2021-07-06 | 兰州大学 | Collagen biological ink for 3D printing and 3D printing method |
| CN113072834B (en) * | 2020-01-03 | 2023-01-06 | 胶原蛋白(武汉)生物科技有限公司 | Collagen biological ink for 3D printing and 3D printing method |
| CN113633827A (en) * | 2021-08-16 | 2021-11-12 | 中国人民解放军总医院第四医学中心 | Silk woven meniscus implant and preparation method thereof |
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