CN1562391A - Mineralized composite material of gene recombination collagen and preparation method - Google Patents
Mineralized composite material of gene recombination collagen and preparation method Download PDFInfo
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- CN1562391A CN1562391A CN 200410033774 CN200410033774A CN1562391A CN 1562391 A CN1562391 A CN 1562391A CN 200410033774 CN200410033774 CN 200410033774 CN 200410033774 A CN200410033774 A CN 200410033774A CN 1562391 A CN1562391 A CN 1562391A
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Abstract
A mineralized genetically recombinant collagen composition for repairing bone is prepared through preparing the solution of genetically recombinant collagen in acid or water, dropping Ca ions contained solution in it while stirring, adding PO4 ions contained solution, regulating pH value, stirring, laying aside, centrifugal separation and washing several times, and freeze drying. It has high biocompatibility.
Description
Technical field
The present invention relates to a kind of mineralising gene recombinaton collagen composite materials and preparation method thereof, belong to technical field of bioengineering.
Background technology
Collagen protein is the extremely important structural protein of connective tissue, plays the function that supports organ, protection body.Collagen protein is the maximum protein of people's in-vivo content, accounts for the 25%-30% of body internal protein total amount, is equivalent to 6% of body weight.The collagen protein product is widely used in industries such as medical science, beauty treatment, cosmetics, but home and abroad no matter at present, it is produced and all adopts animal skins, heel string, cartilage and human placental extract, and all there are problems such as viral hidden danger, immunoreation or ethics in the collagen that extracts by these methods.Along with engineered development, the dna recombinant expression technology is applied to the production of recombinant collagen.The gene recombinaton collagen protein is a kind of high-molecular biologic albumen that adopts technique for gene engineering to produce, and its basic skills is to extract certain segment DNA of expressing human collagen, carry out certain modification after, in expression vector, express.A series of expression systems such as mammalian cell, silkworm, insect cell, yeast cells, escherichia coli all can be used for the production of serial recombinant collagen at present.The recombinant collagen of producing as Xi'an giant's biotechnology limited company is to become cDNA to be reconstituted in the escherichia coli after particular sequence repeats and modifies through the fragment gene behind the enzyme action the proteic mRNA reverse transcription of human collagen, and obtained high expressed, through a kind of gene recombinaton collagen of high density fermentation, separation, purifying process production.(Chinese patent application number 01106757.8 publication number CN1371919A)
Compare with the animal sources collagen protein, the gene recombinaton collagen protein not only has the inherent character of collagen protein, as: biocompatibility, biological degradability and absorbability, bioadhesive, short new cell form and urge epithelial cell and form, and have characteristics such as virus-free hidden danger, machinability and low rejection, can be widely used in a plurality of industries such as medical science, beauty treatment, health product and cosmetics.So adopt gene recombinaton collagen among the present invention.
Present clinical bone reparation is used to exist the danger of originating shortage, immunologic rejection and catching from body bone and allograph bone.The main component of biological osseous tissue is collagen protein and hydroxyapatite.Pure hydroxyapatite has excellent biocompatibility, bone conductibility and biological activity, has been widely used in the operation of plastic surgery operations and tooth section.But pure hydroxyapatite fragility is big, poor toughness, and easily come off from implant site, limited its use clinically.Chinese invention patent (application number 00107493.8, publication number CN 1272383A) discloses and has a kind ofly improved the character bionics method for preparation of hydroxyapatite with collagen protein, has obtained the good medical treatment effect.But because present used collagen protein is mainly derived from animal, have viral hidden danger, and the reorganization recombined collagen that adopts gene engineering method to produce can be avoided this problem.
Summary of the invention
One of purpose of the present invention provides a kind of mineralising gene recombinaton collagen composite materials that bone is repaired that can be used for, and another purpose provides the preparation method of mineralising gene recombinaton collagen composite materials.
The mineralising gene recombinaton collagen composite materials that the present invention proposes, it is characterized in that: contain organic composition and mineral in this material, described organic principle is a gene recombinaton collagen, described mineral is a calcium microcosmic salt crystal.
In above-mentioned mineralising gene recombinaton collagen composite materials, described gene recombinaton collagen, relative molecular weight are 10,000～400,000.
In above-mentioned mineralising gene recombinaton collagen composite materials, described gene recombinaton collagen is that the segment of choosing human I, II, III, IV, V, VI, XI Collagen Type VI gene is modified the back expression.。
In above-mentioned mineralising gene recombinaton collagen composite materials, described calcium microcosmic salt crystal comprises hydroxyapatite.
The preparation method of the mineralising gene recombinaton collagen composite materials that the present invention proposes, it is characterized in that: described method comprises the following steps:
(1) drip the solution that contains calcium ion in the gene recombinaton collagen solution, make the mass percent concentration of recombinant collagen in the solution finally be 0.3%-30%, the molar concentration of calcium ion finally is 0.01～2mol/L, needs continuous stirring in the dropping process; Can adopt acid or deionized water dissolving for water solublity gene recombinaton collagen, for non-water-soluble employing acid dissolving, used acid is hydrochloric acid, acetic acid, nitric acid, slowly adds the solution that contains calcium ion in this solution, and dripping quantity is that every grammes per square metre group collagen drips calcium ion 0.01～1mol;
(2) drip the aqueous solution contain phosphate anion while stirring slowly in the solution of step (1), the phosphate anion of adding and the mol ratio of calcium ion are Ca: P=1～2: 1;
(3) pH value of dripping alkali liquid adjusting while stirring is 7.4～13 in the solution of step (2);
(4) will leave standstill after the solution stirring, treat precipitation and supernatant layering after, remove supernatant, centrifugalize goes out precipitation, removes the solubility salinity with the deionized water cyclic washing, makes cleaning mixture be neutrality;
(5) precipitate is put into the freeze dryer lyophilization, subsequently dry thing is ground, promptly get described mineralising gene recombinaton collagen composite materials.
In above-mentioned preparation method, the solution of the described calcium ions of step (1) is any in calcium hydroxide and the calcium salt, and calcium salt is preferably lime nitrate, calcium chloride;
In above-mentioned preparation method, the solution of the described phosphorus-containing acid ion of step (2) is phosphoric acid and phosphatic any, and phosphate is preferably sodium dihydrogen phosphate, sodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, DAP, ammonium di-hydrogen phosphate.
In above-mentioned preparation method, the described alkali liquor of step (3) is alkali metal hydroxide or ammonium hydroxide, is preferably sodium hydroxide.
Use proof:, have the hidden danger of cross infection because the mineralising gene recombinaton collagen-based materials that the present invention adopts bone to repair has avoided collagen base bone material both domestic and external before this all with the collagen of animal extraction.Prepared material has the characteristics of virus-free hidden danger and low immunity rejection property, and good biocompatibility, but induction of bone growth, its calcium microcosmic salt crystalline size can be used for preparing the bone alternate material of biological property excellence in nanometer scale.In addition, this material combines this material from composition and the imitative nature bone of structure with other frame material, and can be used for preparing has the excellent biological property and the bone renovating material of mechanical property.
The specific embodiment
The present invention will be further described below in conjunction with embodiment:
Solid content 1%) and solid-state (purity 99%) recombinant collagen used gene recombinaton collagen is Xi'an giant's biotechnology limited company liquid state of producing (concentration: among the present invention.
The solution of used phosphorus-containing acid ion is phosphoric acid and phosphate among the present invention, and used phosphate can be sodium dihydrogen phosphate, sodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, DAP, ammonium di-hydrogen phosphate; The solution of used calcium ions can be calcium hydroxide and calcium salt, and calcium salt can be lime nitrate, calcium chloride; Used alkali liquor can be alkali metal hydroxide or ammonium hydroxide, selects sodium hydroxide in the test for use.Agents useful for same is analytical pure.
Embodiment 1
1) in the concentration that mixes up was 0.9% 500ml gene recombinaton collagen solution, Dropwise 5 00ml concentration was the Ca (OH) of 0.25mol/L
2Behind the solution, continuing slowly to add concentration is the NaH of 0.25mol/L
2PO
4Solution 300ml uses the magnetic stirring apparatus mix homogeneously in the time of dropping.
2) above-mentioned system is stirred, leaves standstill, remove supernatant, centrifugalize goes out precipitation, after removing salinity wherein and reach neutrality with the deionized water cyclic washing, puts into the freeze dryer lyophilization, dry thing is ground make dry powder subsequently.
Embodiment 2
1) in the concentration that mixes up is 33% 450ml gene recombinaton collagen solution, adding 25ml concentration is the CaCl of 0.2mol/L
2Behind the solution, continuing slowly to drip concentration is the Na of 0.2mol/L
2HPO
4Solution 25ml uses the magnetic stirring apparatus mix homogeneously in the time of dropping.
2) in the solution of the above-mentioned first step, slowly drip NaOH solution while stirring, stabilize to 7.4 until pH value.
3) above-mentioned system is stirred, leaves standstill, remove supernatant, centrifugalize goes out precipitation, after removing salinity wherein and reach neutrality with the deionized water cyclic washing, puts into the freeze dryer lyophilization, dry thing is ground make dry powder subsequently.
Embodiment 3
1) in the concentration that mixes up is 10% 100ml gene recombinaton collagen solution, adding concentration is the KH of 2mol/L
2PO
4Solution 500ml, continuing slowly to drip 400ml concentration is the Ca (NO of 5mol/L
3)
2Behind the solution, use the magnetic stirring apparatus mix homogeneously in the time of dropping.
2) slowly dripping KOH solution while stirring in the solution of the above-mentioned first step, is 8.0 until pH value.
3) above-mentioned system is stirred, leaves standstill, remove supernatant, centrifugalize goes out precipitation, after removing salinity wherein and reach neutrality with the deionized water cyclic washing, puts into the freeze dryer lyophilization, dry thing is ground make dry powder subsequently.
Embodiment 4
1) in the concentration that mixes up is 25% 450ml gene recombinaton collagen solution, adding 25ml concentration is the CaCl of 0.2mol/L
2Behind the solution, continuing slowly to drip concentration is the NH of 0.2mol/L
3H
2PO
4Solution 25ml uses the magnetic stirring apparatus mix homogeneously in the time of dropping.
2) in the solution of the above-mentioned first step, slowly drip NaOH solution while stirring, stabilize to 10.0 until pH value.
3) above-mentioned system is stirred, leaves standstill, remove supernatant, centrifugalize goes out precipitation, after removing salinity wherein and reach neutrality with the deionized water cyclic washing, puts into the freeze dryer lyophilization, dry thing is ground make dry powder subsequently.
Embodiment 5
1) concentration that mixes up is in 25% the 200ml gene recombinaton collagen solution, and adding concentration is the K of 2mol/L
2HPO
4Solution 500ml, continuing slowly to drip 300ml concentration is the Ca (NO of 5mol/L
3)
2Behind the solution, use the magnetic stirring apparatus mix homogeneously in the time of dropping.
2) slowly dripping KOH solution while stirring in the solution of the above-mentioned first step, is 13.0 until pH value.
3) above-mentioned system is stirred, leaves standstill, remove supernatant, centrifugalize goes out precipitation, after removing salinity wherein and reach neutrality with the deionized water cyclic washing, puts into the freeze dryer lyophilization, dry thing is ground make dry powder subsequently.
Embodiment 6
1) in the concentration that mixes up is 15% 200ml gene recombinaton collagen solution, adding 500ml concentration is the CaCl of 0.2mol/L
2Behind the solution, continuing slowly to drip concentration is the H of 0.2mol/L
3PO
4Solution 300ml uses the magnetic stirring apparatus mix homogeneously in the time of dropping.
2) in the solution of the above-mentioned first step, slowly drip NaOH solution while stirring, reach 8.5 until pH value.
3) above-mentioned system is stirred, leaves standstill, remove supernatant, centrifugalize goes out precipitation, after removing salinity wherein and reach neutrality with the deionized water cyclic washing, puts into the freeze dryer lyophilization, dry thing is ground make dry powder subsequently.
Embodiment 7
1) concentration that mixes up is in 30% the 200ml gene recombinaton collagen solution, and adding 500ml concentration is the CaCl of 1mol/L
2Behind the solution, continuing slowly to drip concentration is the NaH of 0.7mol/L
2PO
4Solution 300ml uses the magnetic stirring apparatus mix homogeneously in the time of dropping.
2) in the solution of the above-mentioned first step, slowly drip NaOH solution while stirring, reach 9.0 until pH value.
3) above-mentioned system is stirred, leaves standstill, remove supernatant, centrifugalize goes out precipitation, after removing salinity wherein and reach neutrality with the deionized water cyclic washing, puts into the freeze dryer lyophilization, dry thing is ground make dry powder subsequently.
Embodiment 8
1) in the concentration that mixes up is 25% 450ml gene recombinaton collagen solution, adding 25ml concentration is the Ca (NO of 1mol/L
3)
2Behind the solution, continuing slowly to drip concentration is the KH of 1mol/L
2PO
4Solution 25ml uses the magnetic stirring apparatus mix homogeneously in the time of dropping.
2) in the solution of the above-mentioned first step, slowly drip NaOH solution while stirring, stabilize to 9.5 until pH value.
3) above-mentioned system is stirred, leaves standstill, remove supernatant, centrifugalize goes out precipitation, after removing salinity wherein and reach neutrality with the deionized water cyclic washing, puts into the freeze dryer lyophilization, dry thing is ground make dry powder subsequently.
Claims (8)
1, mineralising gene recombinaton collagen composite materials is characterized in that: contain organic composition and mineral in this material, described organic principle is a gene recombinaton collagen, and described mineral is a calcium microcosmic salt crystal.
2, mineralising gene recombinaton collagen composite materials according to claim 1, it is characterized in that: described gene recombinaton collagen, relative molecular weight are 10,000～400,000.
3, according to claim 1,2 described mineralising gene recombinaton collagen composite materials, it is characterized in that: described gene recombinaton collagen is that the segment of choosing human I, II, III, IV, V, VI, XI Collagen Type VI gene is modified the back expression.
4, mineralising gene recombinaton collagen composite materials according to claim 1 is characterized in that: described calcium microcosmic salt crystal comprises hydroxyapatite.
5, the method for preparation mineralising gene recombinaton collagen composite materials as claimed in claim 1, it is characterized in that: described method comprises the following steps:
(1) drip the solution that contains calcium ion in the gene recombinaton collagen solution, make the mass percent concentration of recombinant collagen in the solution finally be 0.3%-30%, the molar concentration of calcium ion finally is 0.01～2mol/L, needs continuous stirring in the dropping process; Can adopt acid or deionized water dissolving for water solublity gene recombinaton collagen, for non-water-soluble employing acid dissolving, used acid is hydrochloric acid, acetic acid, nitric acid, slowly adds the solution that contains calcium ion in this solution, and dripping quantity is that every grammes per square metre group collagen drips calcium ion 0.01～1mol;
(2) drip the aqueous solution contain phosphate anion while stirring slowly in the solution of step (1), the phosphate anion of adding and the mol ratio of calcium ion are Ca: P=1～2: 1;
(3) pH value of dripping alkali liquid adjusting while stirring is 7.4～13 in the solution of step (2);
(4) will leave standstill after the solution stirring, treat precipitation and supernatant layering after, remove supernatant, centrifugalize goes out precipitation, removes the solubility salinity with the deionized water cyclic washing, makes cleaning mixture be neutrality;
(5) precipitate is put into the freeze dryer lyophilization, subsequently dry thing is ground, promptly get described mineralising gene recombinaton collagen composite materials.
6, preparation method according to claim 5 is characterized in that: the solution of the described calcium ions of step (1) is any in calcium hydroxide and the calcium salt, and calcium salt is preferably lime nitrate, calcium chloride;
7, preparation method according to claim 5, it is characterized in that: the solution of the described phosphorus-containing acid ion of step (2) is phosphoric acid and phosphatic any, and phosphate is preferably sodium dihydrogen phosphate, sodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, DAP, ammonium di-hydrogen phosphate.
8, preparation method according to claim 5 is characterized in that: the described alkali liquor of step (3) is alkali metal hydroxide or ammonium hydroxide, is preferably sodium hydroxide.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100425296C (en) * | 2006-01-05 | 2008-10-15 | 天津市赛宁生物工程技术有限公司 | Collagen base bionic bone matrix |
| CN102154786A (en) * | 2011-04-25 | 2011-08-17 | 东南大学 | Mineralized corn protein fibrous membrane and preparation method thereof |
| CN102190894A (en) * | 2011-03-25 | 2011-09-21 | 郑州大学 | Method for preparing collagen-based composite material |
| CN106492283A (en) * | 2016-11-22 | 2017-03-15 | 北京奥精医药科技有限公司 | A kind of mineralising guide tissue regeneration film and its preparation method and application |
| CN111375088A (en) * | 2020-04-29 | 2020-07-07 | 陕西巨子生物技术有限公司 | Double-layer osteochondral tissue repair scaffold and preparation method thereof |
| CN114425103A (en) * | 2022-04-06 | 2022-05-03 | 中国科学院苏州纳米技术与纳米仿生研究所 | Bionic biogel and preparation method and application thereof |
-
2004
- 2004-04-16 CN CN 200410033774 patent/CN1562391A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100425296C (en) * | 2006-01-05 | 2008-10-15 | 天津市赛宁生物工程技术有限公司 | Collagen base bionic bone matrix |
| CN102190894A (en) * | 2011-03-25 | 2011-09-21 | 郑州大学 | Method for preparing collagen-based composite material |
| CN102154786A (en) * | 2011-04-25 | 2011-08-17 | 东南大学 | Mineralized corn protein fibrous membrane and preparation method thereof |
| CN106492283A (en) * | 2016-11-22 | 2017-03-15 | 北京奥精医药科技有限公司 | A kind of mineralising guide tissue regeneration film and its preparation method and application |
| CN106492283B (en) * | 2016-11-22 | 2019-07-12 | 北京奥精医药科技有限公司 | A kind of mineralising guide tissue regeneration film and its preparation method and application |
| CN111375088A (en) * | 2020-04-29 | 2020-07-07 | 陕西巨子生物技术有限公司 | Double-layer osteochondral tissue repair scaffold and preparation method thereof |
| CN111375088B (en) * | 2020-04-29 | 2022-06-14 | 陕西巨子生物技术有限公司 | Double-layer osteochondral tissue repair scaffold and preparation method thereof |
| CN114425103A (en) * | 2022-04-06 | 2022-05-03 | 中国科学院苏州纳米技术与纳米仿生研究所 | Bionic biogel and preparation method and application thereof |
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