CN110510592A - The method that regulation preparation has the hydroxyapatite of superior cell compatibility - Google Patents
The method that regulation preparation has the hydroxyapatite of superior cell compatibility Download PDFInfo
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- CN110510592A CN110510592A CN201910840823.8A CN201910840823A CN110510592A CN 110510592 A CN110510592 A CN 110510592A CN 201910840823 A CN201910840823 A CN 201910840823A CN 110510592 A CN110510592 A CN 110510592A
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- hydroxyapatite
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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
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- 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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Abstract
The present invention provides a kind of methods that regulation preparation has the hydroxyapatite of superior cell compatibility, which comprises the following steps: step 1. is 1.5:1 according to calcium and phosphorus ions molar ratio, prepares Ca (NO respectively3)2·4H2O aqueous solution and (NH4)2HPO4Aqueous solution, and in Ca (NO3)2·4H2Small molecule amino acid is added in O aqueous solution, amino acid and calcium ion concentration ratio are 4:1 or 8:1；Parameter when mixing is added dropwise in step 2. setting, by (NH4)2HPO4Ca (the NO in stirring is slowly dropped to by peristaltic pump with the mixed aqueous solution of amino acid3)2·4H2In O aqueous solution；Obtained presoma is put into microwave dissolver by step 3., and setting microwave hydrothermal parameter is reacted；Obtained solid carries out centrifuge washing after step 4. will react, and then freeze-drying removes extra moisture, obtains hydroxyapatite.
Description
Technical field
The invention belongs to hydroxyapatite preparation technical fields, and in particular to regulation preparation has superior cell compatibility
The method of hydroxyapatite.
Technical background
Hydroxyapatite (Ca10(PO4)6(OH)2), as the main mine in a kind of bone for being widely present in animal, tooth
Substance is the preferred material at present in biomedical applications.Hydroxyapatite has good physicochemical property, and influences these
Performance be its crystal itself surface topography, size, crystallinity etc..These factors result in hydroxyapatite surface
Energy, mechanical property, the difference of biology performance, it is different so as to cause application aspect.
Traditional synthetic method has: the precipitation method, hydro-thermal method, sol-gel method etc..Wherein chemical precipitation method simple process,
The disadvantage is that synthesized grain crystalline degree is low, the simultaneously synthesizing period is long.The synthesis process of sol-gel method is simple, at low cost.
But influence synthesis of hydroxyapatite by sol-gel method factor it is very much, it is difficult to obtain well-crystallized, particle uniformly, purity is high and
The controllable particle of geomery.The grain crystalline degree of hydro-thermal method preparation is high and controllable, and required high-temperature and high-pressure conditions make
It is difficult to large-scale production.Also, the hydroxyapatite of these prior art preparations still has certain bio-toxicity, can be to people
Body causes additional side effect, and physico-chemical property and cell compatibility are to be improved.
Summary of the invention
The present invention is to carry out to solve the above-mentioned problems, and it is an object of the present invention to provide a kind of regulation preparation has superior cell
The method of the hydroxyapatite of compatibility.
The present invention to achieve the goals above, uses following scheme:
The present invention provides a kind of method of hydroxyapatite of the regulation preparation with superior cell compatibility, and feature exists
In, comprising the following steps: step 1. is 1.5:1 according to calcium and phosphorus ions molar ratio, prepares Ca (NO respectively3)2·4H2O aqueous solution and
(NH4)2HPO4Aqueous solution, and in Ca (NO3)2·4H2Small molecule amino acid is added in O aqueous solution, amino acid and calcium ion are dense
Degree is than being 4:1 or 8:1；Parameter when mixing is added dropwise in step 2. setting, by (NH4)2HPO4Pass through with the mixed aqueous solution of amino acid
Peristaltic pump is slowly dropped to the Ca (NO in stirring3)2·4H2In O aqueous solution；Obtained presoma is put into microwave and disappeared by step 3.
Xie Yizhong, setting microwave hydrothermal parameter are reacted；Obtained solid carries out centrifuge washing after step 4. will react, then cold
The extra moisture of dry removal is lyophilized, obtains hydroxyapatite.
Preferably, regulation preparation provided by the invention has the method for the hydroxyapatite of superior cell compatibility can be with
Have the feature that the small molecule amino acid in step 1, added in solution is glycine or serine, most preferably glycine.
Preferably, regulation preparation provided by the invention has the method for the hydroxyapatite of superior cell compatibility can be with
It has the feature that in step 1, amino acid and calcium ion concentration ratio are 8:1.
Preferably, regulation preparation provided by the invention has the method for the hydroxyapatite of superior cell compatibility can be with
It has the feature that in step 2, the rate of peristaltic pump is set as 20mL/min.
Preferably, regulation preparation provided by the invention has the method for the hydroxyapatite of superior cell compatibility can be with
It has the feature that during step 2 is added dropwise, adjusting the pH in reaction system with ammonium hydroxide is 9.
Preferably, regulation preparation provided by the invention has the method for the hydroxyapatite of superior cell compatibility can be with
It has the feature that in step 3, the response parameter of microwave hydrothermal reaction are as follows: microwave power 300W, 10 DEG C/min are from room temperature
160 DEG C are risen to, in 160 DEG C of reaction 40min, then furnace cooling.
Preferably, regulation preparation provided by the invention has the method for the hydroxyapatite of superior cell compatibility can be with
It has the feature that in step 4, centrifugal rotational speed is 8000rpm, and the time is 3min, with deionized water centrifuge washing 3 times.
Preferably, regulation preparation provided by the invention has the method for the hydroxyapatite of superior cell compatibility can be with
It has the feature that in step 4, freeze-drying process are as follows: freeze the product after centrifuge washing in -20 DEG C of refrigerator
12h, then dry 12h will be put into -45 DEG C of freeze drier.
Preferably, regulation preparation provided by the invention has the method for the hydroxyapatite of superior cell compatibility can be with
The length for having the feature that the hydroxyapatite being prepared is 50~130nm, and width is 20~30nm.
The action and effect of invention
Preparation method provided by the present invention is prepared into using microwave thermal water law, and using amino acid as adjusting control agent
To hydroxyapatite, whole process is easy to operate, short preparation period, and the phase purity and knot of resulting product hydroxy apatite
Brilliant degree is high, favorable dispersibility；And product hydroxy apatite powder cell proliferation and differentiation have positive effect, reach as high as
2.39 times of about pure HA have excellent cell compatibility and degradability.
Detailed description of the invention
Fig. 1 is XRD (X-ray diffraction analysis) figure of the method for hydroxyapatite obtained in the embodiment of the present invention；
Fig. 2 is the transmission electron microscope microscopic appearance figure of hydroxyapatite obtained in the embodiment of the present invention, wherein (a) is HAG4
Shape appearance figure, (b) be HAG8 shape appearance figure, (c) be HAS4 shape appearance figure, (d) be HAS8 shape appearance figure；
Fig. 3 is each group leaching liquor and the resulting cytotoxicity test figure of MC3T3-11 cell culture in the embodiment of the present invention；
Fig. 4 is each group leaching liquor and the cell morphology figure after MC3T3-11 cell culture 1,3,5 days in the embodiment of the present invention；
In above-mentioned figure, HAG4, HAG8, HAS4, HAS8 are corresponding in turn to product obtained by embodiment one to four,
Control group is that the blank group of material is not added, and HA is not by the pure hydroxyapatite group of regulation synthesis.
Specific embodiment
There is below in conjunction with attached drawing to regulation preparation of the present invention the side of the hydroxyapatite of superior cell compatibility
The specific embodiment of method is described in detail.
<embodiment one>
As shown in Figure 1, regulation preparation provided by the present embodiment one has the hydroxyapatite of superior cell compatibility
Method are as follows:
3.5423g four water-calcium nitrate and 4.5042g glycine are dissolved in 50ml deionized water, stirred 10 minutes；It will
1.3206g diammonium hydrogen phosphate is dissolved in 50ml deionized water, is stirred 10 minutes；With peristaltic pump by diammonium hydrogen phosphate with 20mL/
The speed of min is slowly dropped into the mixed solution of the four water-calcium nitrate into stirring and glycine, and keeping temperature is 25 DEG C, is used
Ammonium hydroxide adjusts the pH=9 for the process being entirely added dropwise, and drips off within 10min, and continues stirring 20min after dripping off and make solution
System is uniform.Obtained presoma is poured into microwave reaction kettle, the wattage that microwave dissolver is arranged is 300W, temperature 160
DEG C, time 40min.Microwave hydrothermal after reaction, pours into 50ml centrifuge tube, is carried out centrifuge washing 3 times with deionized water,
The revolving speed that centrifuge is arranged is 8000rpm, time 3min, washs extra ammonium ion and glycine.- 20 are put into after washing
DEG C refrigerator in freeze 12h；It then takes out, is put into freeze-drying 12h in -45 DEG C of freeze drying box and removes moisture removal, obtain product
Hydroxy apatite powder.It is HAG4 by the Product Labeling.
<embodiment two>
As shown in Figure 1, regulation preparation provided by the present embodiment two has the hydroxyapatite of superior cell compatibility
Method are as follows:
3.5423g four water-calcium nitrate and 9.0084g glycine are dissolved in 50ml deionized water, stirred 10 minutes；It will
1.3206g diammonium hydrogen phosphate is dissolved in 50ml deionized water, is stirred 10 minutes；With peristaltic pump by diammonium hydrogen phosphate with 20mL/
The speed of min is slowly dropped into the mixed solution of the four water-calcium nitrate into stirring and glycine, and keeping temperature is 25 DEG C, is used
Ammonium hydroxide adjusts the PH=9 for the process being entirely added dropwise, and drips off within 10min, and continues stirring 20min after dripping off and make solution
System is uniform.Obtained presoma is poured into microwave reaction kettle, the wattage that microwave dissolver is arranged is 300w, temperature 160
DEG C, time 40min.Microwave hydrothermal after reaction, pours into 50ml centrifuge tube, is carried out centrifuge washing 3 times with deionized water,
The revolving speed that centrifuge is arranged is 8000rpm, time 3min, washs extra ammonium ion and glycine.- 20 are put into after washing
DEG C refrigerator in freeze 12h；It then takes out, is put into freeze-drying 12h in -45 DEG C of freeze drying box and removes moisture removal, obtain product
Hydroxy apatite powder.It is HAG8 by the Product Labeling.
<embodiment three>
Regulation preparation provided by the present embodiment three has the method for the hydroxyapatite of superior cell compatibility are as follows:
3.5423g four water-calcium nitrate and 6.3054g serine are dissolved in 50ml deionized water, stirred 10 minutes；It will
1.3206g diammonium hydrogen phosphate is dissolved in 50ml deionized water, is stirred 10 minutes；With peristaltic pump by diammonium hydrogen phosphate with 20mL/
The speed of min is slowly dropped into the mixed solution of the four water-calcium nitrate into stirring and serine, and keeping temperature is 25 DEG C, is used
Ammonium hydroxide adjusts the PH=9 for the process being entirely added dropwise, and drips off within 10min, and continues stirring 20min after dripping off and make solution
System is uniform.Obtained presoma is poured into microwave reaction kettle, the wattage that microwave dissolver is arranged is 300w, temperature 160
DEG C, time 40min.Microwave hydrothermal after reaction, pours into 50ml centrifuge tube, is carried out centrifuge washing 3 times with deionized water,
The revolving speed that centrifuge is arranged is 8000rpm, time 3min, washs extra ammonium ion and serine.- 20 are put into after washing
DEG C refrigerator in freeze 12h；It then takes out, is put into freeze-drying 12h in -45 DEG C of freeze drying box and removes moisture removal, obtain product
Hydroxy apatite powder.It is HAS4 by the Product Labeling.
<example IV>
As shown in Figure 1, regulation preparation provided by the present embodiment four has the hydroxyapatite of superior cell compatibility
Method are as follows:
3.5423g four water-calcium nitrate and 12.6108g serine are dissolved in 50ml deionized water, stirred 10 minutes；It will
1.3206g diammonium hydrogen phosphate is dissolved in 50ml deionized water, is stirred 10 minutes；With peristaltic pump by diammonium hydrogen phosphate with 20mL/
The speed of min is slowly dropped into the mixed solution of the four water-calcium nitrate into stirring and serine, and keeping temperature is 25 DEG C, is used
Ammonium hydroxide adjusts the PH=9 for the process being entirely added dropwise, and drips off within 10min, and continues stirring 20min after dripping off and make solution
System is uniform.Obtained presoma is poured into microwave reaction kettle, the wattage that microwave dissolver is arranged is 300w, temperature 160
DEG C, time 40min.Microwave hydrothermal after reaction, pours into 50ml centrifuge tube, is carried out centrifuge washing 3 times with deionized water,
The revolving speed that centrifuge is arranged is 8000rpm, time 3min, washs extra ammonium ion and serine.- 20 are put into after washing
DEG C refrigerator in freeze 12h；It then takes out, is put into freeze-drying 12h in -45 DEG C of freeze drying box and removes moisture removal, obtain hydroxyl
Apatite powder.It is HAS8 by the Product Labeling.
Product made from Example one to four is successively used as sample HAG4, HAG8, HAS4, HAS8 to be tested for the property:
As shown in Figure 1, by X-ray diffractogram it is found that the diffraction maximum and hydroxyl of product obtained by embodiment one to four
Apatite PDF standard card JCPDS (09-0432) matches, and crystallinity and purity are good.After adding amino acid regulating and controlling, HAG8
It broadens with HAS8 relative to the diffraction maximum of HAG4 and HAS4, shows that the crystallinity of crystal becomes smaller.
Have as shown in Fig. 2, carrying out projection electron microscope from product and can be seen that product obtained by embodiment one to four all
The crystal grain of dispersibility is in apparent Rod-like shape.And be added presoma in amino acid substance amount increase, crystal grain by
The rodlike corynebacterium for being transformed into HAG8 and HAS8 of the length of HAG4 and HAS4 and rice-shaped.For two different amino acid, silk ammonia
Acid is more more obvious than the effect for the draw ratio that glycine shortens crystal.
The leaching liquor of product obtained by embodiment one to four and MC3T3-11 cell are co-cultured, test it with CCK8 method
The survival rate of 1,3,5 day absorbance, i.e. OD value, OD value and cell is positively correlated.As shown in figure 3, with pure ha phase
Than the hydroxyapatite cell survival rate of amino acid regulating and controlling is apparently higher than the group of the blank group of no HA (control group) and pure HA
Not, and the OD value of HAG8 group is about other 2.39 times of pure HA group, shows good cell compatibility.
As shown in figure 4, the pattern with fluorescence microscope cell at 1,3,5 day, as a result consistent with cytotoxicity test,
HAG4 and the cell density of HAG8 group are apparently higher than remaining group.
Above embodiments are only the illustration done to technical solution of the present invention.Regulation preparation according to the present invention
The method of hydroxyapatite with superior cell compatibility is not merely defined in described content in the embodiment above,
But it is defined by the scope defined by the claims..What those skilled in the art of the invention were done on the basis of the embodiment
Any modify or supplement or equivalence replacement, all in claim range claimed of the invention.
Claims (10)
1. a kind of method that regulation preparation has the hydroxyapatite of superior cell compatibility, which is characterized in that including walking as follows
It is rapid:
Step 1. is 1.5:1 according to calcium and phosphorus ions molar ratio, prepares Ca (NO respectively3)2·4H2O aqueous solution and (NH4)2HPO4Water
Solution, and in Ca (NO3)2·4H2Add small molecule amino acid in O aqueous solution, amino acid and calcium ion concentration ratio be 4:1 or
8:1；
Parameter when mixing is added dropwise in step 2. setting, by (NH4)2HPO4It is slow by peristaltic pump with the mixed aqueous solution of amino acid
Ca (the NO being added drop-wise in stirring3)2·4H2In O aqueous solution；
Obtained presoma is put into microwave dissolver by step 3., and setting microwave hydrothermal parameter is reacted；
Obtained solid carries out centrifuge washing after step 4. will react, and then freeze-drying removes extra moisture, obtains hydroxyl
Base apatite.
2. the method that regulation preparation according to claim 1 has the hydroxyapatite of superior cell compatibility, feature
It is:
Wherein, in step 1, the small molecule amino acid added in solution is glycine or serine.
3. the method that regulation preparation according to claim 1 has the hydroxyapatite of superior cell compatibility, feature
It is:
Wherein, in step 1, the small molecule amino acid added in solution is glycine.
4. the method that regulation preparation according to claim 1 has the hydroxyapatite of superior cell compatibility, feature
It is:
Wherein, in step 1, amino acid and calcium ion concentration ratio are 8:1.
5. the method that regulation preparation according to claim 1 has the hydroxyapatite of superior cell compatibility, feature
It is:
Wherein, in step 2, the rate of peristaltic pump is set as 20mL/min.
6. the method that regulation preparation according to claim 1 has the hydroxyapatite of superior cell compatibility, feature
It is:
Wherein, during step 2 is added dropwise, adjusting the pH in reaction system with ammonium hydroxide is 9.
7. the method that regulation preparation according to claim 1 has the hydroxyapatite of superior cell compatibility, feature
It is:
Wherein, in step 3, the response parameter of microwave hydrothermal reaction are as follows: microwave power 300W, 10 DEG C/min rise to from room temperature
160 DEG C, in 160 DEG C of reaction 40min, then furnace cooling.
8. the method that regulation preparation according to claim 1 has the hydroxyapatite of superior cell compatibility, feature
It is:
Wherein, in step 4, centrifugal rotational speed is 8000rpm, and the time is 3min, carries out centrifuge washing with deionized water.
9. the method that regulation preparation according to claim 1 has the hydroxyapatite of superior cell compatibility, feature
It is:
Wherein, in step 4, freeze-drying process are as follows: the product after centrifuge washing is freezed into 12h in -20 DEG C of refrigerator, then
Dry 12h will be put into -45 DEG C of freeze drier.
10. the method that regulation preparation according to claim 1 has the hydroxyapatite of superior cell compatibility, feature
It is:
Wherein, the length for the hydroxyapatite being prepared is 50~130nm, and width is 20~30nm.
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| CN111320470A (en) * | 2020-01-21 | 2020-06-23 | 武汉理工大学 | Method for preparing hydroxyapatite ceramic by combining microwave hydrothermal method and microwave sintering method |
| CN112978695A (en) * | 2021-03-16 | 2021-06-18 | 华东理工大学 | Preparation method of bionic hydroxyapatite nanoparticles with high osteogenic activity |
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| CN112978695A (en) * | 2021-03-16 | 2021-06-18 | 华东理工大学 | Preparation method of bionic hydroxyapatite nanoparticles with high osteogenic activity |
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