CN102491299A - Preparation method of nano hydroxyapatite - Google Patents
Preparation method of nano hydroxyapatite Download PDFInfo
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- CN102491299A CN102491299A CN2011103595814A CN201110359581A CN102491299A CN 102491299 A CN102491299 A CN 102491299A CN 2011103595814 A CN2011103595814 A CN 2011103595814A CN 201110359581 A CN201110359581 A CN 201110359581A CN 102491299 A CN102491299 A CN 102491299A
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Abstract
The invention provides a preparation method of nano hydroxyapatite. The method comprises the steps that after silk fibroin water solution is conducted to sealing incubation, silk fibroin nanosphere water solution is obtained; and the silk fibroin nanosphere water solution is mixed with a phosphate group source and a calcium source, and then nano hydroxyapatite is obtained after reaction. The invention provides another preparation method of nano hydroxyapatite. The method comprises the steps that firstly, silk fibroin water solution is treated under electrostatic field, and silk fibroin nanosphere is obtained; secondly, the silk fibroin nanosphere obtained in the first step is mixed with a phosphate group source and a calcium source, and then nano hydroxyapatite is obtained after reaction. The preparation method has the advantages that noxious solvent is not required to be used, the preparation method is simple, the condition is mild and controllable, the secondary structure of silk fibroin can not be destroyed, and silk fibroin nanosphere with controllable size and appearance can be obtained, so that nano hydroxyapatite with controllable size and appearance can be obtained.
Description
Technical field
The invention belongs to technical field of nano material, relate in particular to a kind of preparation method of nanometer hydroxyapatite.
Background technology
Silk fibroin is the structural protein that a kind of occurring in nature extensively exists; Can from the domestic silkworm silk fiber, extract; Not only the source is sufficient, process for extracting is simple; And have adhesion, amplification and the Differentiation of excellent biological compatibility, biodegradability, good mechanical performance and good pair cell, be the good organic substrate material of a kind of development prospect.Silk fibroin can be prepared into various ways such as solution, film, porous support, gel, has a good application prospect at biomedical sector.
Win 40350 (Hydroxyapatite, HA, Ca
10(PO
4)
6(OH)
2) be the weakly alkaline calcium phosphate salt that are slightly soluble in water, belong to hexagonal system, be one type of inorganic materials with good biological activity, biocompatibility and absorption property.In vivo; Win 40350 since with organic substrate generation special combination such as a small amount of collagen, chitosan; Formed better, the different inorganic/organic composite material of inducibility with medium property; Biological intravital Win 40350 not only has special mechanical property, optical property and complicated pattern, and in the biomineralization process, and biological physical efficiency is controlled the mineralisation process of Win 40350 on from nanometer to micron-sized yardstick accurately.In recent years; The research of relevant biomimetic mineralization is very noticeable, and its major cause is that not only this field is in the joint of Life Science and Inorganic Chemistry, biophysics and Materials science, has the characteristics of tangible subject crossing and infiltration; It provides new thinking for crystalline material and biological intelligence material that synthetic has unique exquisite pattern what is more important; And energy consumption is lower in the building-up process, meets the Materials science requirements of green environmental protection, be present materialogy research focus it
Hydroxyapatite nano grade particles or microballoon have extensive and important purposes in a plurality of fields such as medical science, pharmacy, coating.At present existing multiple mature technology can prepare the hydroxyapatite powder of producing different structure and pattern, but all there are problems such as energy consumption is high, preparation condition hydroxyapatite powder distribution of sizes harsh, that prepare is big, homogeneity is relatively poor in most of technology.The development of bionic technology has brought new opportunity to develop for the preparation of hydroxy apatite powder; Utilize the bionic technology; Can in normal temperature, normal pressure, water solution system, obtain the Win 40350 product of size pattern homogeneous, thereby effectively improve the quality of products, cut down the consumption of energy.
Prior art discloses the multiple report that utilizes bionics techniques to prepare hydroxyapatite nanoparticle; Be published in 2010 like investigators such as Thachepan S in the document " Mesoscale crystallization of calcium phosphate nanostructures in protein (casein) micelles " of " NANOSCALE " and point out, utilize protein micelle to do nanostructure and pattern that template can be regulated and control Win 40350; Rivera-Munoz; Investigators such as EM were published in the document " Growth of Hydroxyapatite Nanoparticles on Silica Gels " of " JOURNAL OF NANOSCIENCE AND NANOTECHNOLOGY " and point out in 2011; Utilize silica gel to do template; Can control the pattern of Win 40350, but the aforesaid method desired raw material costs an arm and a leg and is not suitable for large-scale production.
Application number is that the Chinese patent document of CN201010618923.5 discloses a kind of Win 40350/fibroin albumen composite material, at first with ca nitrate soln and sodium silicate solution hybrid reaction, with obtaining wollastonite after the reactant calcining; Again silk fibroin is dissolved in the fused nitrocalcite, forms SF-Ca (NO
3)
2Solution; With dripping SF-Ca (NO behind the wollastonite powder formation suspension-s soluble in water
3)
2Solution obtains Win 40350/fibroin albumen composite material, and still, the matrix material dispersiveness that this method obtains is relatively poor; Investigators such as Lin Niu were published in the document " A Novel Nanocomposite Particle of Hydroxyapatite and Silk Fibroin:Biomimetic Synthesis and Its Biocompatibility " of Journal of Nanomaterials in 2010; It is the method that template prepares Win 40350 that the document " Facile fabrication of nano-hydroxyapatite/silk fibroin composite via a simplified coprecipitation route " that Chunquan Fan etc. was published in " J Mater Sci " in 2010 all discloses with the silk fibroin; But the structure and the pattern of the Win 40350 that it obtains are uncontrollable; And it is dispersed relatively poor; Very easily reunite; Can't be dispersed in the aqueous solution, seriously hinder the further application of Win 40350.
Summary of the invention
In view of this, the technical problem that the present invention will solve is to provide a kind of preparation method of nanometer hydroxyapatite, and preparation method provided by the invention can obtain size homogeneous, dispersiveness nanometer hydroxyapatite preferably.
The invention provides a kind of preparation method of nanometer hydroxyapatite, may further comprise the steps:
After silk fibroin water solution sealing cultivation, obtain the silk fibroin nanometer ball aqueous solution;
The said silk fibroin nanometer ball aqueous solution is mixed with phosphoric acid root and calcium source, obtain nanometer hydroxyapatite after the reaction.
Preferably, the mass concentration of said silk fibroin water solution is 0.001%～20%.
Preferably, the temperature that said sealing is cultivated is 0 ℃～90 ℃, and the time that said sealing is cultivated is 0.5h～96h.
Preferably, the mass concentration of the said silk fibroin nanometer ball aqueous solution is 1%～20%.
Preferably, said phosphoric acid root is phosphoric acid, sodium phosphate or calcium phosphate.
Preferably, said calcium source is calcium hydroxide or nitrocalcite.
Preferably, the temperature of said reaction is 50 ℃～80 ℃.
The present invention also provides a kind of preparation method of nanometer hydroxyapatite, may further comprise the steps:
A) silk fibroin water solution is handled under electrostatic field, obtained silk fibroin nanometer ball gel;
B) the silk fibroin nanometer ball gel that said step a) is obtained mixes with phosphoric acid root and calcium source, obtains nanometer hydroxyapatite after the reaction.
Preferably, the strength of electric field of said electrostatic field is 1.0 * 10
2V/m～1.0 * 10
5V/m.
Preferably, said step a) specifically comprises:
A1) the silk fibroin water solution sealing is cultivated, obtain the silk fibroin nanometer ball aqueous solution;
A2) the said silk fibroin nanometer ball aqueous solution is handled under electrostatic field, obtained silk fibroin nanometer ball gel.
Compared with prior art, the present invention at first cultivates the silk fibroin water solution sealing, obtains the silk fibroin nanometer ball aqueous solution; Be template with this silk fibroin nanometer ball aqueous solution then; The synthesis of nano Win 40350 on silk fibroin nanometer ball surface; Silk fibroin and Win 40350 interact; The reunion of Win 40350 be not only can suppress, and can size and the controlled nanometer hydroxyapatite of pattern be obtained through the concentration of control silk fibroin water solution.Preparing method provided by the invention need not to use noxious solvent, the preparation method is simple, mild condition is controlled; Can not destroy the secondary structure of silk fibroin; The controlled silk fibroin nanometer ball of size and pattern be can obtain, thereby size and the controlled nanometer hydroxyapatite of pattern obtained; Silk fibroin and nanometer hydroxyapatite interact, and can realize the hydroxyapatite crystal growth and assemble the control of behavior, the nanometer hydroxyapatite emulsion that acquisition can stable existence; The nano-hydroapatite particles that obtains after said emulsion is centrifugal, washing, the drying can be scattered in the water once more, can not form reunion, helps the further utilization of nanometer hydroxyapatite.
Description of drawings
The AFM analysis of spectra of the silk fibroin nanometer ball solution that Fig. 1 provides for the embodiment of the invention;
The infrared spectrogram of the silk fibroin nanometer ball solution that Fig. 2 provides for the embodiment of the invention;
The stereoscan photograph of the nanometer hydroxyapatite that Fig. 3 provides for the embodiment of the invention;
The infrared spectrum of the nanometer hydroxyapatite that Fig. 4 provides for the embodiment of the invention;
The size distribution figure of the nanometer hydroxyapatite that Fig. 5 provides for the embodiment of the invention.
Embodiment
The invention provides a kind of preparation method of nanometer hydroxyapatite, may further comprise the steps:
After silk fibroin water solution sealing cultivation, obtain the silk fibroin nanometer ball aqueous solution;
The said silk fibroin nanometer ball aqueous solution is mixed with phosphoric acid root and calcium source, obtain nanometer hydroxyapatite after the reaction.
The present invention at first cultivates the silk fibroin water solution sealing, obtains the silk fibroin nanometer ball aqueous solution; Be template with this silk fibroin nanometer ball aqueous solution then; The synthesis of nano Win 40350 on silk fibroin nanometer ball surface; Silk fibroin and Win 40350 interact; The reunion of Win 40350 be not only can suppress, and can size and the controlled nanometer hydroxyapatite of pattern be obtained through the concentration of control silk fibroin water solution.
The present invention at first cultivates the silk fibroin water solution sealing, obtains the silk fibroin nanometer ball aqueous solution.Said silk fibroin water solution is preferably according to following method preparation:
Silk is handled in sodium carbonate solution, obtained silk fibroin;
Said silk fibroin is dissolved in the solvent, in the aqueous solution, dialyses then, obtain silk fibroin water solution.
Silk is handled in sodium carbonate solution, and the sericin that silk is outside is removed, and obtains silk fibroin.The mass concentration of said sodium carbonate solution is preferably 0.1%～1%, and more preferably 0.3%～0.8%; Said treatment temperature is preferably 80 ℃～120 ℃, more preferably 90 ℃～110 ℃; The time of said processing is preferably 40min～80min, more preferably 50min～70min; Said processing is specially boiling, and in boiling process, outside sericin and the yellow soda ash of silk reacts, thereby separates with silk fibroin, obtains silk fibroin.
After disposing, use deionized water to wash the product that obtains repeatedly, obtain silk fibroin 60 ℃ of following dryings then.After obtaining silk fibroin, said silk fibroin is dissolved in the solvent, is contained in the dialysis tubing then and in deionized water, dialyses, obtain silk fibroin water solution.The solvent that said solvent is preferably lithium-bromide solution or is made up of calcium chloride, water and ethanol.Dialysis time is preferably 3 days～and 4 days, whenever between dialysis period change primary water at a distance from 2h, obtain silk fibroin water solution.
In the present invention, the mass concentration of said silk fibroin water solution is preferably 0.001%～20%, and more preferably 0.01%～15%, most preferably be 0.1%～10%.
When said silk fibroin water solution sealing was cultivated, silk fibroin was self-assembled into and is nanometer ball, obtains the silk fibroin nanometer ball aqueous solution, and the temperature that said sealing is cultivated is preferably 0 ℃～90 ℃, more preferably 5 ℃～85 ℃, most preferably is 10 ℃～80 ℃; The time that said sealing is cultivated is preferably 0.5h～96h, and more preferably 1h～90h most preferably is 2h～80h.
In the present invention; Can seal the temperature and time of cultivating according to the concentration decision of silk fibroin water solution; Thereby the diameter of the silk fibroin nanometer ball that control obtains as when silk fibroin water solution concentration is 1%～20%, is cultivated temperature and is preferably 10 ℃～90 ℃; The cultivation time is preferably 2h～96h, and can obtain diameter is the silk fibroin nanometer ball of 20nm～60nm; When silk fibroin water solution concentration is 0.001%～1%, to cultivate temperature and be preferably 0 ℃～60 ℃, the cultivation time is preferably 0.5h～80h, and can obtain diameter is the silk fibroin nanometer ball of 10nm～40nm.
After obtaining the silk fibroin nanometer ball aqueous solution; With this aqueous solution as template solution synthesis of nano Win 40350; Be about to the said silk fibroin nanometer ball aqueous solution and mix, obtain nanometer hydroxyapatite after the reaction, specifically may further comprise the steps with phosphoric acid root and calcium source:
The said silk fibroin nanometer ball aqueous solution is scattered in the phosphoric acid root aqueous solution, obtains mixing solutions, said mixing solutions is joined in the aqueous solution of calcium source, obtain the Win 40350 emulsion after the reaction;
With obtaining nanometer hydroxyapatite after said Win 40350 separation of emulsions, washing, the drying.
In the present invention, the mass concentration of the said silk fibroin nanometer ball aqueous solution is preferably 1%～20%, and more preferably 2%～15%, most preferably be 3%～10%; When the silk fibroin nanometer ball concentration of aqueous solution that obtains is low, when the synthesis of nano Win 40350, can the said silk fibroin nanometer ball aqueous solution be adjusted into above-mentioned concentration.
The said silk fibroin nanometer ball aqueous solution is scattered in the phosphoric acid root aqueous solution, obtains mixing solutions; Said phosphoric acid root is preferably phosphoric acid, sodium phosphate or calcium phosphate, more preferably phosphoric acid; The volumetric molar concentration of phosphorus is preferably 0.01mol/L～0.1mol/L in the said phosphoric acid root, more preferably 0.02mol/L～0.08mol/L.
With said mixing solutions and calcium source aqueous solution, said phosphoric acid root and said calcium source react, and form hydroxyapatite crystal at silk fibroin nanometer ball particle surface, thereby obtain the Win 40350 emulsion; Said calcium source is preferably calcium hydroxide or nitrocalcite, more preferably calcium hydroxide; The temperature of said reaction is preferably 50 ℃～80 ℃, more preferably 60 ℃～70 ℃.
In the present invention, said silk fibroin is preferably 3: 7 with the mass ratio of the Win 40350 that obtains～and 9: 1, more preferably 4: 6～8: 2, most preferably be 4: 5～7: 3.
In said Win 40350 emulsion; Silk fibroin can interact with its surperficial hydroxyapatite crystal; Win 40350 can be dispersed in the silk fibroin macromolecule network, thereby suppresses the reunion of Win 40350, make said Win 40350 emulsion can be in water stable existence; Help the homodisperse of Win 40350 in other emulsions, obtain stay-in-grade matrix material.
After obtaining the Win 40350 emulsion, with obtaining nanometer hydroxyapatite after said separation of emulsions, washing, the drying.Said separation is preferably spinning, and the rotating speed during spinning is preferably more than the 10000rpm, more preferably more than the 11000rpm; Said washing is preferably water and washs, and washing times is preferably more than 3 times; Said drying is preferably vacuum-drying.
Obtain nanometer hydroxyapatite, said nanometer hydroxyapatite is carried out electron-microscope scanning, the result shows its uniform particles, size homogeneous.In addition, control the mass concentration of said silk fibroin water solution, can obtain the nanometer hydroxyapatite of controllable size.
After obtaining nanometer hydroxyapatite, it is scattered in the water once more, good dispersion property can not form reunion.
The nano-hydroapatite particles that the present invention prepares is even, can be used as the weighting material of coating, also can be used as tissue renovation material or pharmaceutical carrier etc. and is used for biomedical sector.The nanometer hydroxyapatite that the present invention is obtained is dispersed in the matrix, can obtain stay-in-grade bone or teeth repairing material.
The present invention at first cultivates the silk fibroin water solution sealing, obtains the silk fibroin nanometer ball aqueous solution; Be template with this silk fibroin nanometer ball aqueous solution then; The synthesis of nano Win 40350 on silk fibroin nanometer ball surface; Silk fibroin and Win 40350 interact; The reunion of Win 40350 be not only can suppress, and can size and the controlled nanometer hydroxyapatite of pattern be obtained through the concentration of control silk fibroin water solution.Preparing method provided by the invention need not to use noxious solvent, the preparation method is simple, mild condition is controlled; Can not destroy the secondary structure of silk fibroin; The controlled silk fibroin nanometer ball of size and pattern be can obtain, thereby size and the controlled nanometer hydroxyapatite of pattern obtained; Silk fibroin and nanometer hydroxyapatite interact, and can realize the hydroxyapatite crystal growth and assemble the control of behavior, the nanometer hydroxyapatite emulsion that acquisition can stable existence; The nano-hydroapatite particles that obtains after said emulsion is centrifugal, washing, the drying can be scattered in the water once more, can not form reunion, helps the further utilization of nanometer hydroxyapatite.
The present invention also provides a kind of preparation method of nanometer hydroxyapatite, may further comprise the steps:
A) silk fibroin water solution is handled under electrostatic field, obtained silk fibroin nanometer ball gel;
B) the silk fibroin nanometer ball gel that said step a) is obtained mixes with phosphoric acid root and calcium source, obtains nanometer hydroxyapatite after the reaction.
In this technical scheme, silk fibroin water solution is issued in electrostatic field be conigenous assembling, obtain silk fibroin nanometer ball gel, prepare the controlled hydroxyl nano-apatite of good dispersion property, size and pattern with this silk fibroin nanometer ball gel as template.In this technical scheme, handle except silk fibroin water solution being adopted electrostatic field, obtain outside the silk fibroin nanometer ball gel, other, like the concentration of silk fibroin water solution, phosphoric acid root, calcium source etc., all identical with technique scheme.
The present invention places electrostatic field to handle silk fibroin water solution, and silk fibroin self-assembly under electrostatic field forms silk fibroin nanometer ball gel.In the present invention, the strength of electric field of said electrostatic field is preferably 1.0 * 10
2V/m～1.0 * 10
5V/m, more preferably 1.5 * 10
2V/m～0.5 * 10
5V/m most preferably is 2.0 * 10
2V/m～0.1 * 10
5V/m; The said treatment time is preferably 10min～30min, more preferably 15min～20min.
In the said silk fibroin nanometer ball gel, the diameter of said silk fibroin nanometer ball and the concentration of silk fibroin water solution, relevant through electric field strength and treatment time, can obtain diameter is the silk fibroin nanometer ball gel of 50nm～2000nm.
In the present invention; In order to make the silk fibroin nanometer ball particle diameter that obtains more even; Before silk fibroin being carried out the electrostatic field processing, preferably said silk fibroin water solution is sealed cultivation, the step that promptly prepares the silk fibroin template preferably includes following steps:
A1) the silk fibroin water solution sealing is cultivated, obtain the silk fibroin nanometer ball aqueous solution;
A2) the said silk fibroin nanometer ball aqueous solution is handled under electrostatic field, obtained silk fibroin nanometer ball gel.
The mass concentration of said silk fibroin water solution is preferably 0.1%～20%, and more preferably 0.5%～15%, most preferably be 1%～10%; The temperature that said sealing is cultivated is preferably 10 ℃～90 ℃, more preferably 20 ℃～80 ℃, most preferably is 30 ℃～70 ℃; The time that said sealing is cultivated is preferably 0.5h～48h, more preferably 1h～45h.
The present invention at first carries out electrostatic field with silk fibroin water solution and handles, and obtains silk fibroin nanometer ball gel; Be template with this silk fibroin nanometer ball gel then; The synthesis of nano Win 40350 on silk fibroin nanometer ball surface; Silk fibroin and Win 40350 interact; The reunion of Win 40350 be not only can suppress, and can size and the controlled nanometer hydroxyapatite of pattern be obtained through the concentration of control silk fibroin water solution.Preparing method provided by the invention need not to use noxious solvent, the preparation method is simple, mild condition is controlled; Can not destroy the secondary structure of silk fibroin; The controlled silk fibroin nanometer ball of size and pattern be can obtain, thereby size and the controlled nanometer hydroxyapatite of pattern obtained; Silk fibroin and nanometer hydroxyapatite interact, and can realize the hydroxyapatite crystal growth and assemble the control of behavior, the nanometer hydroxyapatite emulsion that acquisition can stable existence; The nano-hydroapatite particles that obtains after said emulsion is centrifugal, washing, the drying can be scattered in the water once more, can not form reunion, helps the further utilization of nanometer hydroxyapatite.
In order to further specify the present invention, the preparation method of nanometer hydroxyapatite provided by the invention is described in detail below in conjunction with embodiment.
Embodiment 1
0.5% Na with silk in mass concentration
2CO
3100 ℃ are boiled 60min in the solution, remove the outside sericin of silk; The product that uses deionized water rinsing to obtain repeats 3 times, obtains silk fibroin after 60 ℃ of dryings;
27 gram silk fibroins are dissolved in the 100mL lithium-bromide solution, the silk fibroin protein solution that obtains are contained in dialysis tubing are immersed in the deionized water dialysis 3 days, during every two hours change primary water, obtain purified silk fibroin protein solution, its mass concentration is 5.9%; Said silk fibroin protein solution was cultivated 12 hours at 60 ℃ of lower seals, obtained silk fibroin nanometer ball solution, the diameter of said silk fibroin nanometer ball is 30～50 nanometers;
Said silk fibroin nanometer ball solution is carried out AFM analysis and IR spectroscopy; The result is referring to Fig. 1 and Fig. 2; The AFM analysis of spectra of the silk fibroin nanometer ball solution that Fig. 1 provides for the embodiment of the invention; The infrared spectrogram of the silk fibroin nanometer ball solution that Fig. 2 provides for the embodiment of the invention can know that by Fig. 1 and Fig. 2 the present invention has obtained the silk fibroin nanometer ball.
Said silk fibroin nanometer ball solution is dispersed in the phosphate aqueous solution that phosphorus acid ion concentration is 0.06mol/L obtains mixing solutions, and the adjustment silk fibroin with synthetic after the Win 40350 mass ratio be 6: 4; Measure the said mixing solutions of 20mL and be contained in the beaker, said mixing solutions is added drop-wise in the calcium hydroxide aqueous solution, obtain the Win 40350 emulsion with the speed of 180mL/60min; Said Win 40350 emulsion is at room temperature placed, but its stable existence; After said emulsion is centrifugal, washing, the drying, obtain nano-hydroapatite particles.
Said hydroapatite particles is carried out electron-microscope scanning, IR spectroscopy and particle size analysis; The result is referring to Fig. 3, Fig. 4 and Fig. 5; The stereoscan photograph of the nanometer hydroxyapatite that Fig. 3 provides for the embodiment of the invention; The infrared spectrum of the nanometer hydroxyapatite that Fig. 4 provides for the embodiment of the invention; The size distribution figure of the nanometer hydroxyapatite that Fig. 5 provides for the embodiment of the invention can be known that by Fig. 3, Fig. 4 and Fig. 5 the present invention has prepared uniform particles, size homogeneous, dispersive nanometer hydroxyapatite.
Said nano-hydroapatite particles is scattered in the water once more, and good dispersion property can not form reunion.
Embodiment 2
0.5% Na with silk in mass concentration
2CO
3100 ℃ are boiled 60min in the solution, remove the outside sericin of silk; The product that uses deionized water rinsing to obtain repeats 3 times, obtains silk fibroin after 60 ℃ of dryings;
27 gram silk fibroins are dissolved in the 100mL lithium-bromide solution, the silk fibroin protein solution that obtains are contained in dialysis tubing are immersed in the deionized water dialysis 3 days, during every two hours change primary water, obtain purified silk fibroin protein solution, its mass concentration is 5.9%;
Said silk fibroin protein solution was cultivated 4 hours at 90 ℃ of lower seals, obtained silk fibroin nanometer ball solution, the diameter of said silk fibroin nanometer ball is 20～40 nanometers;
Said silk fibroin nanometer ball solution is carried out AFM analysis and IR spectroscopy, and the result shows that the present invention has obtained the silk fibroin nanometer ball.
Said silk fibroin nanometer ball solution is dispersed in the phosphate aqueous solution that phosphorus acid ion concentration is 0.06mol/L obtains mixing solutions, and the adjustment silk fibroin with synthetic after the Win 40350 mass ratio be 8: 2; Measure the said mixing solutions of 20mL and be contained in the beaker, said mixing solutions is added drop-wise in the calcium hydroxide aqueous solution, obtain the Win 40350 emulsion, said Win 40350 emulsion is at room temperature placed with the speed of 180mL/60min, but its stable existence; After said emulsion is centrifugal, washing, the drying, obtain nano-hydroapatite particles.
Said hydroapatite particles is carried out electron-microscope scanning, IR spectroscopy and particle size analysis, and the result shows that the present invention has prepared uniform particles, size homogeneous, dispersive nanometer hydroxyapatite.
Said nano-hydroapatite particles is scattered in the water once more, and good dispersion property can not form reunion.
Embodiment 3
0.5% Na with silk in mass concentration
2CO
3100 ℃ are boiled 60min in the solution, remove the outside sericin of silk; The product that uses deionized water rinsing to obtain repeats 3 times, obtains silk fibroin after 60 ℃ of dryings;
27 gram silk fibroins are dissolved in the 100mL lithium-bromide solution; The silk fibroin protein solution that obtains is contained in dialysis tubing is immersed in the deionized water dialysis 3 days; Every two hours change primary water during this time; Obtain purified silk fibroin protein solution, its mass concentration is 5.9%, and the concentration of said silk fibroin protein solution is adjusted into 2%;
Said silk fibroin protein solution was cultivated 86 hours at 20 ℃ of lower seals, obtained silk fibroin nanometer ball solution, the diameter of said silk fibroin nanometer ball is 40～60 nanometers;
Said silk fibroin nanometer ball solution is carried out AFM analysis and IR spectroscopy, and the result shows that the present invention has obtained the silk fibroin nanometer ball.
Said silk fibroin nanometer ball solution is dispersed in the phosphate aqueous solution that phosphorus acid ion concentration is 0.06mol/L obtains mixing solutions, and the adjustment silk fibroin with synthetic after the Win 40350 mass ratio be 4: 6; Measure the said mixing solutions of 20mL and be contained in the beaker, said mixing solutions is added drop-wise in the calcium hydroxide aqueous solution, obtain the Win 40350 emulsion, said Win 40350 emulsion is at room temperature placed with the speed of 180mL/60min, but its stable existence; After said emulsion is centrifugal, washing, the drying, obtain nano-hydroapatite particles.
Said hydroapatite particles is carried out electron-microscope scanning, IR spectroscopy and particle size analysis, and the result shows that the present invention has prepared uniform particles, size homogeneous, dispersive nanometer hydroxyapatite.
Said nano-hydroapatite particles is scattered in the water once more, and good dispersion property can not form reunion.
Embodiment 4
0.5% Na with silk in mass concentration
2CO
3100 ℃ are boiled 60min in the solution, remove the outside sericin of silk; The product that uses deionized water rinsing to obtain repeats 3 times, obtains silk fibroin after 60 ℃ of dryings;
0.3 gram silk fibroin is dissolved in the 100mL lithium-bromide solution, the silk fibroin protein solution that obtains is contained in dialysis tubing is immersed in the deionized water dialysis 3 days, during every two hours change primary water, obtain purified silk fibroin protein solution, its mass concentration is about 0.05%;
Said silk fibroin protein solution was cultivated 48 hours at 10 ℃ of lower seals, obtained silk fibroin nanometer ball solution, the diameter of said silk fibroin nanometer ball is 20～40 nanometers;
Said silk fibroin nanometer ball solution is carried out AFM analysis and IR spectroscopy, and the result shows that the present invention has obtained the silk fibroin nanometer ball.
Said silk fibroin nanometer ball solution is dispersed in the phosphate aqueous solution that phosphorus acid ion concentration is 0.06mol/L obtains mixing solutions, and the adjustment silk fibroin with synthetic after the Win 40350 mass ratio be 6: 4; Measure the said mixing solutions of 20mL and be contained in the beaker, said mixing solutions is added drop-wise in the calcium hydroxide aqueous solution, obtain the Win 40350 emulsion, said Win 40350 emulsion is at room temperature placed with the speed of 180mL/60min, but its stable existence; After said emulsion is centrifugal, washing, the drying, obtain nano-hydroapatite particles.
Said hydroapatite particles is carried out electron-microscope scanning, IR spectroscopy and particle size analysis, and the result shows that the present invention has prepared uniform particles, size homogeneous, dispersive nanometer hydroxyapatite.
Said nano-hydroapatite particles is scattered in the water once more, and good dispersion property can not form reunion.
Embodiment 5
0.5% Na with silk in mass concentration
2CO
3100 ℃ are boiled 60min in the solution, remove the outside sericin of silk; The product that uses deionized water rinsing to obtain repeats 3 times, obtains silk fibroin after 60 ℃ of dryings;
3 gram silk fibroins are dissolved in the 100mL lithium-bromide solution, the silk fibroin protein solution that obtains are contained in dialysis tubing are immersed in the deionized water dialysis 3 days, during every two hours change primary water, obtain purified silk fibroin protein solution, its mass concentration is about 0.6%;
Said silk fibroin protein solution was cultivated 2 hours at 5 ℃ of lower seals, obtained silk fibroin nanometer ball solution, the diameter of said silk fibroin nanometer ball is 10～40 nanometers;
Said silk fibroin nanometer ball solution is carried out AFM analysis and IR spectroscopy, and the result shows that the present invention has obtained the silk fibroin nanometer ball.
Said silk fibroin nanometer ball solution is dispersed in the phosphate aqueous solution that phosphorus acid ion concentration is 0.06mol/L obtains mixing solutions, and the adjustment silk fibroin with synthetic after the Win 40350 mass ratio be 8: 2; Measure the said mixing solutions of 20mL and be contained in the beaker, said mixing solutions is added drop-wise in the calcium hydroxide aqueous solution, obtain the Win 40350 emulsion, said Win 40350 emulsion is at room temperature placed with the speed of 180mL/60min, but its stable existence; After said emulsion is centrifugal, washing, the drying, obtain nano-hydroapatite particles.
Said hydroapatite particles is carried out electron-microscope scanning, IR spectroscopy and particle size analysis, and the result shows that the present invention has prepared uniform particles, size homogeneous, dispersive nanometer hydroxyapatite.
Said nano-hydroapatite particles is scattered in the water once more, and good dispersion property can not form reunion.
Embodiment 6
0.5% Na with silk in mass concentration
2CO
3100 ℃ are boiled 60min in the solution, remove the outside sericin of silk; The product that uses deionized water rinsing to obtain repeats 3 times, obtains silk fibroin after 60 ℃ of dryings;
It is in the solution that forms of 1: 6: 2 calcium chloride, water and ethanol by mol ratio that 1 gram silk fibroin is dissolved in 10mL; The silk fibroin protein solution that obtains is contained in dialysis tubing is immersed in the deionized water dialysis 4 days; Every two hours change primary water during this time; Obtain purified silk fibroin protein solution, its mass concentration is 2%;
The said silk fibroin protein solution of 15mL is contained in the container that electrode is installed, the voltage of the logical 30V of energized, making up field intensity is 1 * 10
3The directed electric field of V/m was switched on 15 minutes, formed the silk fibroin gellike, and it is made up of at the silk fibroin nanometer ball of 50～500 nanometers diameter;
Said silk fibroin gellike is dispersed in the phosphate aqueous solution that phosphorus acid ion concentration is 0.06mol/L obtains mixing solutions, and the adjustment silk fibroin with synthetic after the Win 40350 mass ratio be 3: 7; Measure the said mixing solutions of 20mL and be contained in the beaker, said mixing solutions is added drop-wise in the calcium hydroxide aqueous solution, obtain the Win 40350 emulsion, said Win 40350 emulsion is at room temperature placed with the speed of 180mL/60min, but its stable existence; After said emulsion is centrifugal, washing, the drying, obtain nano-hydroapatite particles.
Said hydroapatite particles is carried out electron-microscope scanning, IR spectroscopy and particle size analysis, and the result shows that the present invention has prepared uniform particles, size homogeneous, dispersive nanometer hydroxyapatite.
Said nano-hydroapatite particles is scattered in the water once more, and good dispersion property can not form reunion.
Embodiment 7
0.5% Na with silk in mass concentration
2CO
3100 ℃ are boiled 60min in the solution, remove the outside sericin of silk; The product that uses deionized water rinsing to obtain repeats 3 times, obtains silk fibroin after 60 ℃ of dryings;
27 gram silk fibroins are dissolved in the 100mL lithium-bromide solution, the silk fibroin protein solution that obtains are contained in dialysis tubing are immersed in the deionized water dialysis 3 days, during every two hours change primary water, obtain purified silk fibroin protein solution, its mass concentration is about 5.9%;
Said silk fibroin protein solution was cultivated 24 hours at 60 ℃ of lower seals, the silk fibroin protein solution after the said cultivation of 15mL is contained in the container that electrode is installed, the voltage of the logical 20V of energized, making up field intensity is 5 * 10
2The directed electric field of V/m was switched on 10 minutes, formed the silk fibroin gellike, and it is made up of at the silk fibroin nanometer ball of 100～2000 nanometers diameter;
Said silk fibroin gellike is dispersed in the phosphate aqueous solution that phosphorus acid ion concentration is 0.06mol/L obtains mixing solutions, and the adjustment silk fibroin with synthetic after the Win 40350 mass ratio be 7: 3; Measure the said mixing solutions of 20mL and be contained in the beaker, said mixing solutions is added drop-wise in the calcium hydroxide aqueous solution, obtain the Win 40350 emulsion, said Win 40350 emulsion is at room temperature placed with the speed of 180mL/60min, but its stable existence; After said emulsion is centrifugal, washing, the drying, obtain nano-hydroapatite particles.
Said hydroapatite particles is carried out electron-microscope scanning, IR spectroscopy and particle size analysis, and the result shows that the present invention has prepared uniform particles, size homogeneous, dispersive nanometer hydroxyapatite.
Said nano-hydroapatite particles is scattered in the water once more, and good dispersion property can not form reunion.
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; Can also make some improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.
Claims (10)
1. the preparation method of a nanometer hydroxyapatite may further comprise the steps:
After silk fibroin water solution sealing cultivation, obtain the silk fibroin nanometer ball aqueous solution;
The said silk fibroin nanometer ball aqueous solution is mixed with phosphoric acid root and calcium source, obtain nanometer hydroxyapatite after the reaction.
2. preparation method according to claim 1 is characterized in that, the mass concentration of said silk fibroin water solution is 0.001%～20%.
3. preparation method according to claim 1 is characterized in that, the temperature that said sealing is cultivated is 0 ℃～90 ℃, and the time that said sealing is cultivated is 0.5h～96h.
4. preparation method according to claim 1 is characterized in that, the mass concentration of the said silk fibroin nanometer ball aqueous solution is 1%～20%.
5. preparation method according to claim 1 is characterized in that, said phosphoric acid root is phosphoric acid, sodium phosphate or calcium phosphate.
6. preparation method according to claim 1 is characterized in that, said calcium source is calcium hydroxide or nitrocalcite.
7. preparation method according to claim 1 is characterized in that, the temperature of said reaction is 50 ℃～80 ℃.
8. the preparation method of a nanometer hydroxyapatite may further comprise the steps:
A) silk fibroin water solution is handled under electrostatic field, obtained silk fibroin nanometer ball gel;
B) the silk fibroin nanometer ball gel that said step a) is obtained mixes with phosphoric acid root and calcium source, obtains nanometer hydroxyapatite after the reaction.
9. preparation method according to claim 8 is characterized in that, the strength of electric field of said electrostatic field is 1.0 * 10
2V/m～1.0 * 10
5V/m.
10. preparation method according to claim 8 is characterized in that, said step a) specifically comprises:
A1) the silk fibroin water solution sealing is cultivated, obtain the silk fibroin nanometer ball aqueous solution;
A2) the said silk fibroin nanometer ball aqueous solution is handled under electrostatic field, obtained silk fibroin nanometer ball gel.
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| CN107374980A (en) * | 2017-09-04 | 2017-11-24 | 苏州博瑞华仿生材料科技有限公司 | Controllable calcium phosphate gelatin nano composite material of a kind of component content and crystal structure and preparation method thereof |
| CN111849013A (en) * | 2020-07-14 | 2020-10-30 | 重庆医科大学附属口腔医院 | Nano-hydroxyapatite-silk fibroin mineralized scaffold and preparation method and application thereof |
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| CN111849013A (en) * | 2020-07-14 | 2020-10-30 | 重庆医科大学附属口腔医院 | Nano-hydroxyapatite-silk fibroin mineralized scaffold and preparation method and application thereof |
| CN115054733A (en) * | 2022-07-05 | 2022-09-16 | 武汉纺织大学 | Preparation method and application of silk fibroin/hydroxyapatite composite micro-nano particle material |
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