CN110510592B - Method for regulating and preparing hydroxyapatite with excellent cell compatibility - Google Patents
Method for regulating and preparing hydroxyapatite with excellent cell compatibility Download PDFInfo
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- CN110510592B CN110510592B CN201910840823.8A CN201910840823A CN110510592B CN 110510592 B CN110510592 B CN 110510592B CN 201910840823 A CN201910840823 A CN 201910840823A CN 110510592 B CN110510592 B CN 110510592B
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- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 title claims abstract description 45
- 229910052588 hydroxylapatite Inorganic materials 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 40
- 230000001105 regulatory effect Effects 0.000 title claims abstract description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 23
- 150000001413 amino acids Chemical class 0.000 claims abstract description 18
- 238000006243 chemical reaction Methods 0.000 claims abstract description 17
- 239000007864 aqueous solution Substances 0.000 claims abstract description 12
- 239000011575 calcium Substances 0.000 claims abstract description 12
- 230000002572 peristaltic effect Effects 0.000 claims abstract description 9
- -1 phosphorus ions Chemical class 0.000 claims abstract description 8
- 239000002243 precursor Substances 0.000 claims abstract description 8
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 claims abstract description 7
- 229910001424 calcium ion Inorganic materials 0.000 claims abstract description 7
- 238000000120 microwave digestion Methods 0.000 claims abstract description 7
- 238000003756 stirring Methods 0.000 claims abstract description 6
- 238000002156 mixing Methods 0.000 claims abstract description 3
- 239000011574 phosphorus Substances 0.000 claims abstract description 3
- 229910052698 phosphorus Inorganic materials 0.000 claims abstract description 3
- 239000007787 solid Substances 0.000 claims abstract description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 22
- 238000005406 washing Methods 0.000 claims description 18
- 239000008367 deionised water Substances 0.000 claims description 14
- 229910021641 deionized water Inorganic materials 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 13
- 238000004108 freeze drying Methods 0.000 claims description 12
- 239000004471 Glycine Substances 0.000 claims description 11
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 9
- 230000008569 process Effects 0.000 claims description 9
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims description 7
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 6
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 6
- 238000007710 freezing Methods 0.000 claims description 6
- 230000008014 freezing Effects 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 abstract description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 abstract 1
- 229910052791 calcium Inorganic materials 0.000 abstract 1
- 239000000047 product Substances 0.000 description 16
- ZHJGWYRLJUCMRT-UHFFFAOYSA-N 5-[6-[(4-methylpiperazin-1-yl)methyl]benzimidazol-1-yl]-3-[1-[2-(trifluoromethyl)phenyl]ethoxy]thiophene-2-carboxamide Chemical compound C=1C=CC=C(C(F)(F)F)C=1C(C)OC(=C(S1)C(N)=O)C=C1N(C1=C2)C=NC1=CC=C2CN1CCN(C)CC1 ZHJGWYRLJUCMRT-UHFFFAOYSA-N 0.000 description 8
- 239000005696 Diammonium phosphate Substances 0.000 description 8
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 8
- 229910000388 diammonium phosphate Inorganic materials 0.000 description 8
- 235000019838 diammonium phosphate Nutrition 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 238000001027 hydrothermal synthesis Methods 0.000 description 7
- 101100515543 Arabidopsis thaliana HAM1 gene Proteins 0.000 description 6
- 239000013078 crystal Substances 0.000 description 6
- 235000013339 cereals Nutrition 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 5
- 239000011259 mixed solution Substances 0.000 description 4
- 238000002441 X-ray diffraction Methods 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000002386 leaching Methods 0.000 description 3
- 238000003980 solgel method Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 231100000263 cytotoxicity test Toxicity 0.000 description 2
- 229910052500 inorganic mineral Inorganic materials 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000011707 mineral Substances 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000009388 chemical precipitation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000001878 scanning electron micrograph Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
- C01B25/00—Phosphorus; Compounds thereof
- C01B25/16—Oxyacids of phosphorus; Salts thereof
- C01B25/26—Phosphates
- C01B25/32—Phosphates of magnesium, calcium, strontium, or barium
- C01B25/325—Preparation by double decomposition
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2002/00—Crystal-structural characteristics
- C01P2002/70—Crystal-structural characteristics defined by measured X-ray, neutron or electron diffraction data
- C01P2002/72—Crystal-structural characteristics defined by measured X-ray, neutron or electron diffraction data by d-values or two theta-values, e.g. as X-ray diagram
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2004/00—Particle morphology
- C01P2004/01—Particle morphology depicted by an image
- C01P2004/04—Particle morphology depicted by an image obtained by TEM, STEM, STM or AFM
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2004/00—Particle morphology
- C01P2004/60—Particles characterised by their size
- C01P2004/62—Submicrometer sized, i.e. from 0.1-1 micrometer
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2004/00—Particle morphology
- C01P2004/60—Particles characterised by their size
- C01P2004/64—Nanometer sized, i.e. from 1-100 nanometer
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- Materials For Medical Uses (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Abstract
本发明提供了一种调控制备具有优异细胞相容性的羟基磷灰石的方法，其特征在于，包括以下步骤：步骤1.按照钙磷离子摩尔比为1.5：1，分别配制Ca(NO3)2·4H2O水溶液和(NH4)2HPO4水溶液，并且在Ca(NO3)2·4H2O水溶液中添加小分子氨基酸，氨基酸和钙离子浓度比为4：1或8：1；步骤2.设定滴加混合时的参数，将(NH4)2HPO4和氨基酸的混合水溶液通过蠕动泵缓慢滴加到搅拌中的Ca(NO3)2·4H2O水溶液中；步骤3.将得到的前驱体放入微波消解仪中，设置微波水热参数进行反应；步骤4.将反应后所得到的固体进行离心洗涤，然后冷冻干燥去除多余的水分，得到羟基磷灰石。
The present invention provides a method for regulating and preparing hydroxyapatite with excellent cytocompatibility, which is characterized by comprising the following steps: Step 1. According to the molar ratio of calcium and phosphorus ions of 1.5:1, respectively prepare Ca(NO 3 ) ) 2 ·4H 2 O aqueous solution and (NH 4 ) 2 HPO 4 aqueous solution, and small molecular amino acids were added to the Ca(NO 3 ) 2 ·4H 2 O aqueous solution, and the concentration ratio of amino acid and calcium ion was 4:1 or 8:1 ; Step 2. Set the parameters during dropwise mixing, and slowly drop the mixed aqueous solution of (NH 4 ) 2 HPO 4 and amino acid into the stirring Ca(NO 3 ) 2 ·4H 2 O aqueous solution by peristaltic pump; step 3. Put the obtained precursor into a microwave digestion apparatus, and set microwave hydrothermal parameters to carry out the reaction; step 4. The solid obtained after the reaction is washed by centrifugation, and then freeze-dried to remove excess water to obtain hydroxyapatite.
Description
Technical Field
The invention belongs to the technical field of hydroxyapatite preparation, and particularly relates to a method for preparing hydroxyapatite with excellent cell compatibility by regulation.
Technical Field
Hydroxyapatite (Ca)10(PO4)6(OH)2) The mineral is a main mineral widely existing in bones and teeth of animals, and is the first choice material in the biomedical application at present. The hydroxyapatite has good physical and chemical properties, and the properties are influenced by the surface appearance, the size, the crystallinity and the like of the crystal. These factors lead to differences in the surface properties, mechanical properties and biological properties of hydroxyapatite, which leads to differences in application.
The traditional synthetic method comprises the following steps: precipitation methods, hydrothermal methods, sol-gel methods, and the like. The chemical precipitation method has simple process and has the defects of low crystallinity of the synthesized crystal grains and long synthesis period. The synthesis process of the sol-gel method is simple and the cost is low. However, many factors affect the preparation of hydroxyapatite by the sol-gel method, and it is difficult to obtain particles with good crystallization, uniform particles, high purity and controllable shape and size. The crystallinity of the crystal grains prepared by the hydrothermal method is high and controllable, and the required high-temperature and high-pressure conditions make the large-scale production difficult. Moreover, the hydroxyapatite prepared by the prior art still has certain biological toxicity, can cause additional side effects on human bodies, and the physicochemical property and the cell compatibility are required to be improved.
Disclosure of Invention
The present invention has been made to solve the above problems, and an object of the present invention is to provide a method for controlling the preparation of hydroxyapatite having excellent cell compatibility.
In order to achieve the purpose, the invention adopts the following scheme:
the invention provides a method for regulating and preparing hydroxyapatite with excellent cell compatibility, which is characterized by comprising the following steps: step 1, according to the molar ratio of calcium ions to phosphorus ions of 1.5: 1, respectively preparing Ca (NO)3)2·4H2Aqueous O solution and (NH)4)2HPO4In aqueous solution, and in Ca (NO)3)2·4H2Adding micromolecular amino acid into the O aqueous solution, wherein the concentration ratio of the amino acid to the calcium ion is 4: 1 or 8: 1; step 2, setting parameters during dropwise adding and mixing, and adding (NH)4)2HPO4The mixed aqueous solution of the amino acid and Ca (NO) is slowly dripped into the stirred Ca by a peristaltic pump3)2·4H2O in water solution; step 3, putting the obtained precursor into a microwave digestion instrument, and setting microwave hydrothermal parameters for reaction; and 4, centrifugally washing the solid obtained after the reaction, and then freeze-drying to remove redundant water to obtain the hydroxyapatite.
Preferably, the method for regulating and preparing hydroxyapatite with excellent cell compatibility provided by the invention can also have the following characteristics: in step 1, the small-molecule amino acid added to the solution is glycine or serine, preferably glycine.
Preferably, the method for regulating and preparing hydroxyapatite with excellent cell compatibility provided by the invention can also have the following characteristics: in step 1, the ratio of amino acid to calcium ion concentrations is 8: 1.
preferably, the method for regulating and preparing hydroxyapatite with excellent cell compatibility provided by the invention can also have the following characteristics: in step 2, the rate of the peristaltic pump was set to 20 mL/min.
Preferably, the method for regulating and preparing hydroxyapatite with excellent cell compatibility provided by the invention can also have the following characteristics: during the dropwise addition in step 2, the pH in the reaction system was adjusted to 9 with aqueous ammonia.
Preferably, the method for regulating and preparing hydroxyapatite with excellent cell compatibility provided by the invention can also have the following characteristics: in step 3, the reaction parameters of the microwave hydrothermal reaction are as follows: the microwave power is 300W, the temperature is increased from room temperature to 160 ℃ at the speed of 10 ℃/min, the reaction is carried out for 40min at the temperature of 160 ℃, and then the reaction is carried out with the furnace cooling.
Preferably, the method for regulating and preparing hydroxyapatite with excellent cell compatibility provided by the invention can also have the following characteristics: in step 4, the centrifugation speed is 8000rpm, the time is 3min, and the washing is carried out 3 times by centrifugation with deionized water.
Preferably, the method for regulating and preparing hydroxyapatite with excellent cell compatibility provided by the invention can also have the following characteristics: in step 4, the freeze-drying process is: freezing the product after centrifugal washing in a refrigerator at-20 deg.C for 12h, and drying in a freeze dryer at-45 deg.C for 12 h.
Preferably, the method for regulating and preparing hydroxyapatite with excellent cell compatibility provided by the invention can also have the following characteristics: the length of the prepared hydroxyapatite is 50-130 nm, and the width of the hydroxyapatite is 20-30 nm.
Action and Effect of the invention
The preparation method provided by the invention uses a microwave hot water method, and adopts amino acid as a regulating agent to prepare the hydroxyapatite, the whole process is simple to operate, the preparation period is short, and the obtained product hydroxyapatite has high phase purity and crystallinity and good dispersibility; the product hydroxyapatite powder HAs positive effect on cell proliferation and differentiation, can reach 2.39 times of pure HA at most, and HAs excellent cell compatibility and degradability.
Drawings
Fig. 1 is an XRD (X-ray diffraction analysis) pattern of the method of preparing hydroxyapatite according to an example of the present invention;
fig. 2 is a transmission electron microscope microscopic morphology image of hydroxyapatite prepared in the example of the present invention, wherein (a) is a morphology image of HAG4, (b) is a morphology image of HAG8, (c) is a morphology image of HAS4, and (d) is a morphology image of HAS 8;
FIG. 3 is a graph showing cytotoxicity tests of each group of the leaching solution and MC3T3-11 cell culture in the example of the present invention;
FIG. 4 is a cell morphology chart of each group of leaching solution and MC3T3-11 cells after 1, 3 and 5 days of culture in the embodiment of the invention;
in the above figures, HAG4, HAG8, HAS4 and HAS8 correspond to the products obtained in the first to fourth examples, respectively, the control group is a blank group without materials, and HA is a pure hydroxyapatite group without controlled synthesis.
Detailed Description
The following description will explain in detail specific embodiments of the method for regulating and preparing hydroxyapatite having excellent cell compatibility according to the present invention with reference to the accompanying drawings.
< example one >
As shown in fig. 1, the method for preparing hydroxyapatite with excellent cell compatibility according to the first embodiment of the present invention includes:
3.5423g of calcium nitrate tetrahydrate and 4.5042g of glycine are dissolved in 50ml of deionized water and stirred for 10 minutes; 1.3206g of diammonium phosphate is dissolved in 50ml of deionized water and stirred for 10 minutes; slowly dropping diammonium phosphate into a stirred mixed solution of calcium nitrate tetrahydrate and glycine at the speed of 20mL/min by using a peristaltic pump, keeping the temperature at 25 ℃, adjusting the pH value of the whole dropping process to be 9 by using ammonia water, completing dropping within 10min, and continuously stirring for 20min after completing dropping to ensure that a solution system is uniform. And pouring the obtained precursor into a microwave reaction kettle, and setting the wattage of a microwave digestion instrument to be 300W, the temperature to be 160 ℃ and the time to be 40 min. And after the microwave hydrothermal reaction is finished, pouring the mixture into a 50ml centrifugal tube, carrying out centrifugal washing for 3 times by using deionized water, setting the rotating speed of a centrifugal machine to be 8000rpm, and washing redundant ammonium ions and glycine for 3 min. After washing, putting the mixture into a refrigerator with the temperature of 20 ℃ below zero for freezing for 12 hours; then taking out, putting into a freeze drying oven with the temperature of minus 45 ℃ for freeze drying for 12h to remove water, and obtaining the product of the hydroxyapatite powder. This product was labeled HAG 4.
< example two >
As shown in fig. 1, the method for preparing hydroxyapatite with excellent cell compatibility according to the second embodiment of the present invention includes:
3.5423g of calcium nitrate tetrahydrate and 9.0084g of glycine are dissolved in 50ml of deionized water and stirred for 10 minutes; 1.3206g of diammonium phosphate is dissolved in 50ml of deionized water and stirred for 10 minutes; slowly dropping diammonium phosphate into a stirred mixed solution of calcium nitrate tetrahydrate and glycine at the speed of 20mL/min by using a peristaltic pump, keeping the temperature at 25 ℃, adjusting the pH value of the whole dropping process to be 9 by using ammonia water, completing dropping within 10min, and continuously stirring for 20min after completing dropping to ensure that a solution system is uniform. And pouring the obtained precursor into a microwave reaction kettle, and setting the wattage of a microwave digestion instrument to be 300w, the temperature to be 160 ℃ and the time to be 40 min. And after the microwave hydrothermal reaction is finished, pouring the mixture into a 50ml centrifugal tube, carrying out centrifugal washing for 3 times by using deionized water, setting the rotating speed of a centrifugal machine to be 8000rpm, and washing redundant ammonium ions and glycine for 3 min. After washing, putting the mixture into a refrigerator with the temperature of 20 ℃ below zero for freezing for 12 hours; then taking out, putting into a freeze drying oven with the temperature of minus 45 ℃ for freeze drying for 12h to remove water, and obtaining the product of the hydroxyapatite powder. This product was labeled HAG 8.
< example three >
The method for preparing hydroxyapatite with excellent cell compatibility by regulation and control provided by the third embodiment comprises the following steps:
3.5423g of calcium nitrate tetrahydrate and 6.3054g of serine are dissolved in 50ml of deionized water and stirred for 10 minutes; 1.3206g of diammonium phosphate is dissolved in 50ml of deionized water and stirred for 10 minutes; slowly dropping diammonium phosphate into a stirred mixed solution of calcium nitrate tetrahydrate and serine at the speed of 20mL/min by using a peristaltic pump, keeping the temperature at 25 ℃, adjusting the pH value of the whole dropping process to be 9 by using ammonia water, completing dropping within 10min, and continuously stirring for 20min after completing dropping to ensure that a solution system is uniform. And pouring the obtained precursor into a microwave reaction kettle, and setting the wattage of a microwave digestion instrument to be 300w, the temperature to be 160 ℃ and the time to be 40 min. And after the microwave hydrothermal reaction is finished, pouring the mixture into a 50ml centrifugal tube, carrying out centrifugal washing for 3 times by using deionized water, setting the rotating speed of a centrifugal machine to be 8000rpm and the time to be 3min, and washing redundant ammonium ions and serine. After washing, putting the mixture into a refrigerator with the temperature of 20 ℃ below zero for freezing for 12 hours; then taking out, putting into a freeze drying oven with the temperature of minus 45 ℃ for freeze drying for 12h to remove water, and obtaining the product of the hydroxyapatite powder. This product was labeled HAS 4.
< example four >
As shown in fig. 1, the method for preparing hydroxyapatite with excellent cell compatibility by regulation provided in the fourth embodiment is as follows:
3.5423g of calcium nitrate tetrahydrate and 12.6108g of serine are dissolved in 50ml of deionized water and stirred for 10 minutes; 1.3206g of diammonium phosphate is dissolved in 50ml of deionized water and stirred for 10 minutes; slowly dropping diammonium phosphate into a stirred mixed solution of calcium nitrate tetrahydrate and serine at the speed of 20mL/min by using a peristaltic pump, keeping the temperature at 25 ℃, adjusting the pH value of the whole dropping process to be 9 by using ammonia water, completing dropping within 10min, and continuously stirring for 20min after completing dropping to ensure that a solution system is uniform. And pouring the obtained precursor into a microwave reaction kettle, and setting the wattage of a microwave digestion instrument to be 300w, the temperature to be 160 ℃ and the time to be 40 min. And after the microwave hydrothermal reaction is finished, pouring the mixture into a 50ml centrifugal tube, carrying out centrifugal washing for 3 times by using deionized water, setting the rotating speed of a centrifugal machine to be 8000rpm and the time to be 3min, and washing redundant ammonium ions and serine. After washing, putting the mixture into a refrigerator with the temperature of 20 ℃ below zero for freezing for 12 hours; then taking out, putting into a freeze drying oven with the temperature of minus 45 ℃ for freeze drying for 12h to remove water, and obtaining the hydroxyapatite powder. This product was labeled HAS 8.
The products prepared in examples one to four were taken as samples HAG4, HAG8, HAS4, HAS8 in that order for performance testing:
as shown in FIG. 1, the diffraction peaks of the products obtained in examples one to four match with JCPDS (09-0432) which is the hydroxyapatite PDF standard card, and the crystallinity and purity are good as can be seen from the X-ray diffraction pattern. After the regulation of the added amino acids, diffraction peaks of HAG8 and HAS8 are widened relative to those of HAG4 and HAS4, and the crystallinity of the crystal is reduced.
As shown in FIG. 2, it can be seen from the SEM images of the products, the grains of the products prepared in examples one to four have dispersivity and are in obvious rod-like morphology. And as the amount of amino acid substances added into the precursor is increased, the crystal grains are changed from long rods of HAG4 and HAS4 into short rods of HAG8 and HAS8 and rice grains. For two different amino acids, serine has a more pronounced effect on shortening the aspect ratio of the crystals than glycine.
The leaching solution of the product prepared in the first to fourth examples is co-cultured with MC3T3-11 cells, and the absorbance, namely the OD value, of the cells in 1, 3 and 5 days is tested by a CCK8 method, and the OD value is in positive correlation with the survival rate of the cells. As shown in fig. 3, compared to pure hydroxyapatite, the cell survival rate of amino acid-regulated hydroxyapatite was significantly higher than that of the HA-free blank group (control group) and the pure HA group, and the OD value of HAG8 group was about 2.39 times that of the pure HA group, showing good cell compatibility.
As shown in fig. 4, the morphology of the cells was observed by a fluorescence microscope at 1, 3, and 5 days, and the cell density of the group of HAG4 and HAG8 was significantly higher than that of the other groups, in accordance with the cytotoxicity test.
The above embodiments are merely illustrative of the technical solutions of the present invention. The method for regulating and preparing hydroxyapatite with excellent cell compatibility according to the present invention is not limited to the contents described in the above embodiments, but is subject to the scope defined by the claims. Any modification or supplement or equivalent replacement made by a person skilled in the art on the basis of this embodiment is within the scope of the invention as claimed in the claims.
Claims (6)
1. A method for regulating and preparing hydroxyapatite with excellent cell compatibility is characterized by comprising the following steps:
step 1, according to the molar ratio of calcium ions to phosphorus ions of 1.5: 1, respectively preparing Ca (NO)3)2·4H2Aqueous O solution and (NH)4)2HPO4In aqueous solution, and in Ca (NO)3)2·4H2Adding micromolecular amino acid into the O aqueous solution, wherein the concentration ratio of the amino acid to the calcium ion is 4: 1 or 8: 1; the added small molecular amino acid is glycine or serine;
step 2, setting parameters during dropwise adding and mixing, and adding (NH)4)2HPO4The aqueous solution was slowly added dropwise to Ca (NO) under stirring by a peristaltic pump3)2·4H2In the mixed aqueous solution of O and amino acid, the speed of a peristaltic pump is set to be 20 mL/min;
step 3, putting the obtained precursor into a microwave digestion instrument, setting microwave hydrothermal parameters for reaction, wherein the microwave power is 300W, the temperature is increased from room temperature to 160 ℃ at the speed of 10 ℃/min, the reaction is carried out for 40min at the temperature of 160 ℃, and then the reaction is carried out along with furnace cooling;
and 4, centrifugally washing the solid obtained after the reaction, and then freeze-drying to remove redundant water to obtain hydroxyapatite with the length of 50-130 nm and the width of 20-30 nm.
2. The method for preparing hydroxyapatite with excellent cell compatibility according to claim 1, characterized in that:
wherein, in the step 1, the small molecular amino acid added into the solution is glycine.
3. The method for preparing hydroxyapatite with excellent cell compatibility according to claim 1, characterized in that:
wherein, in the step 1, the concentration ratio of the amino acid to the calcium ion is 8: 1.
4. the method for preparing hydroxyapatite with excellent cell compatibility according to claim 1, characterized in that:
wherein, in the dropping process of the step 2, the pH value in the reaction system is adjusted to 9 by ammonia water.
5. The method for preparing hydroxyapatite with excellent cell compatibility according to claim 1, characterized in that:
wherein, in the step 4, the centrifugal speed is 8000rpm, the time is 3min, and the centrifugal washing is carried out by deionized water.
6. The method for preparing hydroxyapatite with excellent cell compatibility according to claim 1, characterized in that:
wherein, in the step 4, the freeze drying treatment comprises the following steps: freezing the product after centrifugal washing in a refrigerator at-20 deg.C for 12h, and drying in a freeze dryer at-45 deg.C for 12 h.
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