CN1157232C - Prepn of osteological material containing nano phase calcium phosphate, collagen and alginate - Google Patents
Prepn of osteological material containing nano phase calcium phosphate, collagen and alginate Download PDFInfo
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- CN1157232C CN1157232C CNB011419016A CN01141901A CN1157232C CN 1157232 C CN1157232 C CN 1157232C CN B011419016 A CNB011419016 A CN B011419016A CN 01141901 A CN01141901 A CN 01141901A CN 1157232 C CN1157232 C CN 1157232C
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Abstract
The present invention relates to a method for preparing a bone material containing nano-phase calcium-phosphorus salt, collagen and alginate. Solution containing calcium ions is slowly and dropwisely added into acid soluble collagen solution at first, and then, water solution containing phosphate radical ions is slowly and dropwisely added while the solution is stirred. Alkaline liquor is slowly and dropwisely added to regulate the pH value while the solution is stirred, and then, the solution is set aside. Supernatant liquid is removed, precipitates are separated centrifugally and then are ground to prepare dry powder, and the dry powder is mixed with sodium alginate. Water is added for modification, products after modification are added into a mold to be molded and demolded, and then are soaked in distilled water, and after the products are dried, the bone material is obtained. The bone material prepared by the method has favorable biocompatibility and bone imitating structure. The crystal size of calcium-phosphorus salt is in the nanometer order of magnitude and is tightly combined with collagen as an organic constituent, and the arrangement is regular. Due to the adoption of natural raw material, the material has favorable biocompatibility. The material has reasonable material strength and porosity and can be used as implantation type degradable bone material.
Description
Technical field:
The present invention relates to a kind of preparation method that contains the bone material of nanometer phase calcium-phosphorus salt, collagen and alginate, belong to technical field of bioengineering.
Background technology:
The development of bone reparation is the direction that the biomedical engineering field scientist makes great efforts always.Developed now comprise metal, pottery, macromolecule with and the multiple bone renovating material made of composite, but because mechanical property, the deficiency of aspect such as biocompatibility and degradability, what the actual bone renovating material effect of using was best in the operation still is from body bone and allograph bone.But having from body bone and allograph bone has limited amount respectively and has immunologic rejection, the problem of the danger that catches.
Summary of the invention:
The objective of the invention is to propose a kind of preparation method that contains the bone material of nanometer phase calcium-phosphorus salt, collagen and alginate, this material is a raw material with bionical synthetic calcium microcosmic salt, protein and polysaccharide, make its microstructure have imitative bone characteristic, thereby have good biocompatibility and biological activity.
The preparation method of the bone material that contains nanometer phase calcium-phosphorus salt, collagen and alginate that the present invention proposes comprises following each step:
(1) in the molten collagen solution of acid, slowly drips the solution that contains calcium ion, the amount that adds calcium ion is that every gram collagen drips calcium ion 0.01～0.16mol, stirs in the time of dropping, wherein, acid in order to dissolving collagen is hydrochloric acid, nitric acid or acetic acid, and the concentration of collagen solution is 5.0 * 10
-5～5.0 * 10
-3G/ml.
(2) slowly drip the aqueous solution of phosphorus-containing acid ion while stirring in the solution in above-mentioned the 1st step, the mol ratio of the amount of the calcium ion of adding is Ca: P=1～2: 1 in the amount of the phosphate anion of adding and the 1st step.
(3) slowly to drip NaOH solution to pH value while stirring be 6～8 in the solution in above-mentioned the 2nd step, and pH value is measured with reagent paper or pH meter, is to begin to occur precipitation at 5～6 o'clock at pH value, and pH value is to occur white suspension at 7 o'clock.
(4) with solution left standstill 1～5 day, remove supernatant, centrifugalize goes out precipitation, clean three times repeatedly with deionized water after, put into the freeze dryer lyophilization, it is standby that grinding subsequently makes dry powder.
(5) be after 10～1: 1 the mixed with above-mentioned the 4th dry powder that makes of step and sodium alginate with mass ratio, adding low amounts of water fully is in harmonious proportion, the mass ratio of pressed powder and water is 1: 3～0.5, somatomedin such as recombinant human bone morphogenetic protein 2 (hereinafter to be referred as BMP) can the form with aqueous solution add in this step, and addition is to add 1～10 milligram in every gram pressed powder.
(6) product after will fully being in harmonious proportion is inserted in the mould of outer surface belt micropore, and placing weight percent concentration is that 0.5～10% calcium chloride solution soaked more than 8 hours, the demoulding to the forming materials.
(7) material after the demoulding was soaked in distilled water 12～48 hours, 30～50 ℃ of oven dry, or put into the lyophilization machine at-5～-20 ℃ after freezing and remove moisture, preceding a kind of method will obtain dense high-intensity bone material.Then a kind of method will obtain having the bone frame material of pore space structure.
The said goods is used oxirane steam disinfection 2～4 hours, or standby with preserving behind the Co60 illumination-based disinfection.
Method with biological self assembly in the invention has been synthesized nm-crysal collagen-based calcium phosphate composition, and this material is formed by the calcium microcosmic salt and the tropocollagen molecule self assembly of nanophase.It has the repetition lamellar structure on nanoscale, the cycle is 10-15nm, is alternately arranged by collagen layer and calcium microcosmic salt layer to form, because therefore it have good biocompatibility and very high biological activity from composition and the imitative nature bone of structure.Another kind of raw material sodium alginate in the invention is that to be used as cytoskeletal biomaterial at present in organizational project mainly be a kind of natural macromolecular material.It has nontoxic, hydrophilic, biocompatibility and the good characteristics of cellular affinity.The intensity of the material for preparing in the invention is mainly provided by the alginate of calcification, and its intensity level is all relevant with the addition and the calcification degree of sodium alginate with degradation property.This just can adjust the degradation speed of material to a certain extent.By this good biocompatibility, degradation speed is controlled, be easy to the natural macromolecular material of molding and the frame material that nm-crysal collagen-based calcium phosphate composition is integrated into high porosity, obtained excellent biocompatibility and bioactive degradable bone alternate material, can be used as the degradable bone alternate material and aspect medical, be used widely.Can also add an amount of somatomedin in the sample preparation process, improve the biological activity of material to greatest extent.Material imitative bone in microstructure is of the present invention two big characteristics with adopting natural material, and therefore material also has excellent biological property.
The prepared according to the methods of the invention bone material has excellent biocompatibility, and also has imitative bone on the structure.Its calcium microcosmic salt crystalline size is in nanometer scale, and is tight with combining of organic principle collagen, is arranged with certain rule.Because what adopt is that the biocompatibility of this material of natural material is also fine.And this strength of materials and porosity are reasonable, can be used as embedded type degradable bone material and are applied.
The specific embodiment:
The used collagen of the present invention is the type i collagen gel (concentration: solid content 1% of benefit Kanggong department, contained collagen is the Corii Bovis seu Bubali collagen of purification), the liquid type i collagen (concentration: 0.3% solution pH value is adjusted the Corii Bovis seu Bubali collagen that contained collagen is purification by HCl) that CELLON company buys, the type i collagen gel of Tianjin second Affiliated Hospital (concentration: solid content 1%, contained collagen are the cattle heel string collagen of purification); The used PO that contains
4 3-Solution be Na
2HPO
4, H
3PO
4, (NH
4)
2HPO
4Aqueous solution; The solution of used calcium ions is CaCl
26H
2O, Ca (NO
3)
2, CaCl
22H
2The aqueous solution of O.The sodium alginate that uses is from companies such as Acros Co., Jiangnan, Qingdao bright moon seaweed industry, Xisi, Beijing industrial chemicals.
Embodiment 1:
Material therefor is type i collagen gel (concentration: solid content 1%, contained collagen are the Corii Bovis seu Bubali collagen of purification), analytical pure CaCl from the purchase of benefit Kanggong department
26H
2O, analytical pure Na
2HPO
4, sodium alginate is from Acros Co..
(1) the 20g collagen gel is dissolved in the 300ml 0.5M acetic acid solution, slowly drips 18.3ml 1mol/l CaCl
2With 11ml 1mol/l Na
2HPO
4, use magnetic stirrer in the time of dropping;
(2) continue to stir, NaOH solution to the pH value that slowly drips 0.5mol/l simultaneously is 7;
(3) left standstill solution 1 day, remove supernatant, centrifugalize goes out to put into freeze dryer after precipitate with deionized water is cleaned three times repeatedly and carry out lyophilization, and it is standby to make dry powder after the grinding;
(4) adding 5 ml distilled waters after dry powder that (3) step of 2.4 grams is made and 0.6 gram sodium alginate mix fully is in harmonious proportion.
(5) product after will being in harmonious proportion is inserted in the mould of outer surface belt micropore, and micro-pore diameter is 0.45 micron, soaks the demoulding after 12 hours in the calcium chloride solution of 5% (w%).
(6) material after the demoulding in distilled water, soak after 12 hours in-20 ℃ freezing, put into the lyophilization machine subsequently and removed moisture in 32 hours.
(7) product in (6) step is preserved after 2 hours with the oxirane steam disinfection.
Embodiment 2:
Material therefor is type i collagen gel (concentration: solid content 1%, contained collagen are the cattle heel string collagen of purification), the analytical pure CaCl of Tianjin second Affiliated Hospital
22H
2O, analytical pure (NH
4)
2HPO
4, sodium alginate is from Xisi, Beijing Raw Materials Company of Chemical Industry.
(1) the 20g collagen gel is dissolved in 300ml 0.1M HNO
3In the solution, slowly drip 10ml 1mol/l CaCl
2With 10ml 1mol/l (NH
4)
2HPO
4, use magnetic stirrer in the time of dropping;
(2) continue to stir, NaOH solution to the pH value that slowly drips 0.5mol/l simultaneously is 7;
(3) left standstill solution 4 days, remove supernatant, centrifugalize goes out to put into freeze dryer after precipitate with deionized water is cleaned three times repeatedly and carry out lyophilization, and it is standby to make dry powder after the grinding;
(4) dry powder that (3) step of 1.8 grams is made and the mixed back of 0.6 gram sodium alginate fully are in harmonious proportion with the suspension of 3 milliliters of recombinant human bone morphogenetic protein 2BMP, and the content of BMP is 16.8 milligrams.
(5) product after will being in harmonious proportion is inserted in the mould of outer surface belt micropore, and micro-pore diameter is 0.45 micron, soaks the demoulding after 24 hours in the calcium chloride solution of 5% (w%).
(6) material after the demoulding soaks in distilled water after 48 hours in 30 ℃ of oven dry 24 hours.
(7) product in (6) step is preserved after 4 hours with the oxirane steam disinfection.
Embodiment 3:
Liquid type i collagen (concentration: 0.3% solution pH value is adjusted the Corii Bovis seu Bubali collagen that contained collagen is purification by HCl), analytically pure CaCl that material therefor is bought for CELLON company
26H
2O, analytical pure H
3PO
4(content＞=85% density 1.689g/ml), sodium alginate is from Acros Co..
(1) drips the 1.125ml H that is dissolved in the 10ml deionized water in the 100ml collagen solution
3PO
4, use magnetic stirrer in the time of dropping;
(2) take by weighing well-crystallized's 6.01g CaCl
26H
2O is dissolved in the 20ml deionized water, and it is dissolved fully, be added dropwise to it in solution that previous step makes after, continue to stir 1 hour;
(3) continue to stir, NaOH solution to the pH value that slowly drips 0.75mol/l simultaneously is 7;
(4) left standstill solution 1 day, remove supernatant, centrifugalize goes out to put into freeze dryer after precipitate with deionized water is cleaned three times repeatedly and carry out lyophilization, and it is standby to make dry powder after the grinding;
(5) dry powder that (4) step of 2.1 grams is made and the mixed back of 0.3 gram sodium alginate fully are in harmonious proportion with 5 milliliters of suspensions that are recombined into bone morphogenetic protein 2 (BMP), and the content of BMP is 7.2 milligrams.
(6) product after will being in harmonious proportion is inserted in the mould of outer surface belt micropore, and micro-pore diameter is 0.45 micron, soaks the demoulding after 32 hours in the calcium chloride solution of 3% (w%).
(7) material after the demoulding in distilled water, soak after 1 hour in-10 ℃ freezing, put into the lyophilization machine subsequently and removed moisture in 32 hours.
(8) product in (7) step is preserved after 2 hours with the oxirane steam disinfection.
Embodiment 4:
Material therefor is type i collagen gel (concentration: solid content 1%, contained collagen are the Corii Bovis seu Bubali collagen of purification), the analytical pure Ca (NO of the purchase of benefit Kanggong department
3)
2, analytical pure Na
2HPO
4, sodium alginate is from Acros Co..
(1) the 20g collagen gel is dissolved in the 300ml 0.5M acetic acid solution, slowly drips 16.5ml 1mol/lCa (NO
3)
2With 11ml 1mol/l H
3PO
4, use magnetic stirrer in the time of dropping;
(2) continue to stir, NaOH solution to the pH value that slowly drips 0.5mol/l simultaneously is 7;
(3) left standstill solution 1 day, remove supernatant, centrifugalize goes out to put into freeze dryer after precipitate with deionized water is cleaned three times repeatedly and carry out lyophilization, and it is standby to make dry powder after the grinding;
(4) directly insert in the mould of outer surface belt micropore after dry powder that (3) step of 2 grams is made and 1.5 gram sodium alginates mix, micro-pore diameter is 0.6 micron, soaks the demoulding after 12 hours in the calcium chloride solution of 8% (w%).
(5) material after the demoulding was soaked in distilled water 24 hours, dried 24 hours down at 45 ℃ subsequently
(6) preserve after with reference to GB with the Co60 illumination-based disinfection.
Embodiment 5:
Material therefor is type i collagen gel (concentration: solid content 1%, contained collagen are the Corii Bovis seu Bubali collagen of purification), the analytical pure CaCl of the purchase of benefit Kanggong department
26H
2O, analytical pure Na
2HPO
4, sodium alginate is from Jiangnan, Qingdao bright moon seaweed industry.
(1) the 30g collagen gel is dissolved in the 300ml 0.5M acetic acid solution, slowly drips 18.3ml 1mol/l CaCl
2With 11ml 1mol/l Na
2HPO
4, use magnetic stirrer in the time of dropping:
(2) continue to stir, NaOH solution to the pH value that slowly drips 0.5mol/l simultaneously is 7;
(3) left standstill solution 5 days, remove supernatant, centrifugalize goes out to put into freeze dryer after precipitate with deionized water is cleaned three times repeatedly and carry out lyophilization, and it is standby to make dry powder after the grinding;
(4) the 1.8 grams dry powder that makes of (3) step and 0.6 gram sodium alginate are mixed add 2 ml distilled waters and fully be in harmonious proportion.
(5) product after will being in harmonious proportion is inserted in the mould of outer surface belt micropore, and micro-pore diameter is 0.6 micron, soaks the demoulding after 8 hours in the calcium chloride solution of 10% (w%).
(6) material after the demoulding in distilled water, soak after 30 minutes in-20 ℃ freezing, put into the lyophilization machine subsequently and removed moisture in 24 hours.
(7) product in (6) step is preserved after 4 hours with the oxirane steam disinfection.
Claims (2)
1, a kind of preparation method that contains the bone material of nanometer phase calcium-phosphorus salt, collagen and alginate is characterized in that this method comprises following each step:
(1) in the molten collagen solution of acid, slowly drips the solution that contains calcium ion, the amount that adds calcium ion is that every gram collagen drips calcium ion 0.01～0.16mol, stirs in the time of dropping, wherein, acid in order to dissolving collagen is hydrochloric acid, nitric acid or acetic acid, and the concentration of collagen solution is 5.0 * 10
-5～5.0 * 10
-3G/ml;
(2) slowly drip the aqueous solution of phosphorus-containing acid ion while stirring in the solution in above-mentioned the 1st step, the mol ratio of the amount of the calcium ion of adding is Ca: P=1～2: 1 in the amount of the phosphate anion of adding and the 1st step;
(3) slowly to drip NaOH solution to pH value while stirring be 6～8 in the solution in above-mentioned the 2nd step, and pH value is measured with reagent paper or pH meter, is to begin to occur precipitation at 5～6 o'clock at pH value, and pH value is to occur white suspension at 7 o'clock;
(4) with solution left standstill 1～5 day, remove supernatant, centrifugalize goes out precipitation, clean three times repeatedly with deionized water after, put into the freeze dryer lyophilization, it is standby that grinding subsequently makes dry powder;
(5) above-mentioned the 4th dry powder that makes of step and sodium alginate are after 10～1: 1 the mixed, to add low amounts of water and fully be in harmonious proportion with mass ratio, and the mass ratio of pressed powder and water is 1: 3～0.5;
(6) product after fully being in harmonious proportion is inserted in the mould of outer surface belt micropore, and placing weight percent concentration is that 0.5～10% calcium chloride solution soaked more than 8 hours, the demoulding to the forming materials;
(7) material after the demoulding was soaked in distilled water 12～48 hours,, or put into the lyophilization machine at-5～-20 ℃ after freezing and remove moisture, obtain the bone frame material 30～50 ℃ of oven dry.
2, the method for claim 1 is characterized in that the form with aqueous solution adds the somatomedin rhBMP-2 in above-mentioned the 5th step, and addition is to add 1～10 milligram in every gram pressed powder.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011419016A CN1157232C (en) | 2001-09-21 | 2001-09-21 | Prepn of osteological material containing nano phase calcium phosphate, collagen and alginate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011419016A CN1157232C (en) | 2001-09-21 | 2001-09-21 | Prepn of osteological material containing nano phase calcium phosphate, collagen and alginate |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1337271A CN1337271A (en) | 2002-02-27 |
| CN1157232C true CN1157232C (en) | 2004-07-14 |
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ID=4676471
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB011419016A Expired - Fee Related CN1157232C (en) | 2001-09-21 | 2001-09-21 | Prepn of osteological material containing nano phase calcium phosphate, collagen and alginate |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108704165A (en) * | 2017-12-15 | 2018-10-26 | 中国科学院深圳先进技术研究院 | Alginate composite mortar, alginic acid hollow pipe and preparation method thereof |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1328322C (en) * | 2005-01-27 | 2007-07-25 | 浙江大学 | Biodegradable calcium phosphate/collagen composite materials for medical use and method for preparation thereof |
| CN100438927C (en) * | 2006-11-24 | 2008-12-03 | 清华大学 | Method for preparing injuctable material for repairing bones solidified in situ from calcium alginate |
| CN101628130B (en) * | 2009-08-20 | 2013-04-03 | 华中科技大学 | Nanometer bionic scaffold material and preparation method thereof |
| CN101773683B (en) * | 2010-03-03 | 2012-10-31 | 天津大学 | Chitosan modified alginate hydrogel three-dimensional porous bracket and preparation method thereof |
| CN102935246B (en) * | 2011-08-15 | 2015-02-04 | 国家纳米科学中心 | Three-dimensional cell culture scaffold, its preparation method and application |
| CN102406965B (en) * | 2011-12-01 | 2015-06-24 | 广西南宁博恩康生物科技有限公司 | Injectable gel material for treating bone defect and preparation method thereof |
-
2001
- 2001-09-21 CN CNB011419016A patent/CN1157232C/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108704165A (en) * | 2017-12-15 | 2018-10-26 | 中国科学院深圳先进技术研究院 | Alginate composite mortar, alginic acid hollow pipe and preparation method thereof |
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| Publication number | Publication date |
|---|---|
| CN1337271A (en) | 2002-02-27 |
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