CN102935246B - Three-dimensional cell culture scaffold, its preparation method and application - Google Patents
Three-dimensional cell culture scaffold, its preparation method and application Download PDFInfo
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- CN102935246B CN102935246B CN201110233215.4A CN201110233215A CN102935246B CN 102935246 B CN102935246 B CN 102935246B CN 201110233215 A CN201110233215 A CN 201110233215A CN 102935246 B CN102935246 B CN 102935246B
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Abstract
The invention provides a three-dimensional culture scaffold for cell culture. The three-dimensional culture scaffold comprises a porous calcium phosphate stand and sodium alginate coated on the porous calcium phosphate stand. The scaffold has the advantages of simple preparation, and convenient operation and transportation, etc. The invention also provides a preparation method and application of the three-dimensional scaffold.
Description
Technical field
The invention belongs to bioengineered tissue field, particularly a kind of preparation method and application being applied to Three-dimensional cell culture support.The present invention take nano tricalcium phosphate as initial feed, by polymethylmethacrylate templated synthesis porous calcium phosphate support.Calcium phosphate support is immersed the mixture forming tricalcium phosphate/alginate films in acid sodium alginate soln, finally prepared Three-dimensional cell culture support.
Background technology
Two dimension (2D) cell cultures is prepared by one, observation of cell in vitro study cell and the interactional technology of medicine, biotic factor and biomaterial.But it and cell growth pattern is in vivo not similar.In animal body, cell is that three-dimensional (3D) grows and build formation living tissue or organ.Therefore, compared with three-dimensional cell cultivation is cultivated with two dimension, more similar in appearance to the cell micro-environment in animal body.Have report to point out, by external Three-dimensional cell culture, we can understand the structure-function under the organization condition being organized in normal and pathology.Therefore, Three-dimensional cell culture support has very high using value.
What adopt in traditional Three-dimensional cell culture support preparation is organic polymer support, and as gelatin-glutaraldehyde, but it often adopts specific organic composition by preparation process technology limit, and forming machine is complicated or intensity is low, and inconvenience is as product.
Summary of the invention
Therefore, the object of the invention is to the deficiency overcoming above-mentioned preparation technology, a kind of novel Three-dimensional cell culture support is provided.
Another object of the present invention is to provide the preparation method of described Three-dimensional cell culture support, and this preparation process is simple and easy to do.And provide above-mentioned Three-dimensional cell culture support preparing the application in cell culture apparatus.
Technical scheme for realizing above-mentioned purpose is as follows:
On the one hand, the invention provides a kind of Three-dimensional cell culture support for cell cultures, it comprises porous calcium phosphate support and is coated in the sodium alginate layer on porous calcium phosphate support, and described porous calcium phosphate support is provided with equally distributed micropore.
On the other hand, the invention provides the preparation method of above-mentioned Three-dimensional cell culture support, this preparation method comprises the following steps:
Step 1: solid-phase calcium phosphate content is the preparation of the slip of 50 ~ 55% (weightmeasurement ratios);
Step 2: the preparation of poly (methyl methacrylate) micro-sphere;
Step 3: the preparation of porous calcium phosphate support;
Step 4: the sodium alginate hydrochloric acid soln that mass percentage is 4 ~ 7% put into by porous calcium phosphate support step 3 prepared, vacuum soaks 1-2h, with ultrapure water repetitive scrubbing three times, to obtain final product.
Preferably, in step 1, described calcium phosphate is nano-calcium phosphate.
Preferably, in step 1, the preparation of described slip comprises the following steps:
Step 1.1: the preparation of nanometer tricalcium phosphate powder;
Step 1.2: by nanometer tricalcium phosphate powder, dispersion agent and ultrapure water ball milling 0.5-2h, to obtain final product.
Preferably, the preparation of described nanometer tricalcium phosphate powder comprises the following steps:
Step 1.1.1: nitrocalcite, diammonium hydrogen phosphate and urea are dissolved in ultrapure water, preferably, in described ultrapure water solution, the concentration of each raw material is: nitrocalcite 0.5-0.6 mole/L, diammonium hydrogen phosphate 0.4-0.5 mole/L and urea 0.56-0.6 mole/L;
Step 1.1.2: the solution that step 1.1.1 is obtained is positioned in baking oven, in 70-80 DEG C of dry 3-7h;
Step 1.1.3: be put in retort furnace by dried material, in 700-800 DEG C of temperature, calcining 1-2h, obtains the nanometer tricalcium phosphate powder of white.
Preferably, in step 2, the preparation of described poly (methyl methacrylate) micro-sphere comprises the following steps:
Step 2.1: be that 5-6% sodium hydroxide solution slowly cleans methyl methacrylate twice with massfraction, again with the methyl methacrylate 12-24h after Calcium Chloride Powder Anhydrous drying washing, preferably, be that 5-6% sodium hydroxide solution slowly cleans methyl methacrylate twice with 25mL massfraction;
Step 2.2: magnesium basic carbonate being dissolved in mass percentage is in the polyvinyl alcohol solution of 0.3%, then adds methyl methacrylate solution, initiator, Ethylene glycol dimethacrylate, at N
2under protection, reaction system is stirred 5-6h at 66-70 DEG C, stirring velocity is 240-440r/min, and preferably, the concentration of described basic carbonate magnesium solution is 0.0142-0.015 mole/L; Preferably, the volume ratio of described methyl methacrylate solution and polyvinyl alcohol solution is 1: 12.5-1: 13, and more preferably, the density of described methyl methacrylate solution is 0.9410g/mL; Preferably, described initiator is benzoyl peroxide, and the mass concentration of described benzoyl peroxide is 0.3%; Preferably, the volume ratio of described Ethylene glycol dimethacrylate and polyvinyl alcohol solution is 1: 625-1: 667, and more preferably, the density of described Ethylene glycol dimethacrylate is 1.0151g/mL.
Step 2.3: after reaction terminates, by filter paper filtering crude product removing residual solution, and with ultrapure milli-Q water 5 times;
Step 2.4: in 60-65 DEG C of baking oven, vacuum-drying obtains the poly (methyl methacrylate) micro-sphere that diameter is 200 ~ 300 μm.
Preferably, in step 3, the preparation of described porous calcium phosphate support comprises the following steps:
Step 3.1: by aluminium sulfite, urea, polyvinyl pyrrolidone solution ultrapure water, preparation obtains nano aluminium oxide suspension liquid, preferably, the concentration that described each raw material is dissolved in ultrapure water is respectively: aluminium sulfite 0.05-0.06 mole/L, urea 0.56-0.6 mole/L, polyvinyl pyrrolidone 6.5x10
-6mole/L;
Step 3.2: poly-methyl star diluted acid methyl esters microballoon is positioned in beaker, in loft drier, at 180-200 DEG C, dry 1-2h;
Step 3.3: under room temperature, is slowly paved with poly (methyl methacrylate) micro-sphere by nano aluminium oxide suspension liquid, dry in vacuum drying oven, obtains poly (methyl methacrylate) micro-sphere support.
Step 3.4: polymethylmethacrylate/support that step 3.3 is obtained carries out vacuum-treat, tricalcium phosphate slip prepared in implantation step 1, mixes and obtains slip/polymethylmethacrylate/scaffold complex.
Step 3.5: slip/polymethylmethacrylate/scaffold complex is put in sintering in retort furnace and forms porous calcium phosphate support.
Preferably, described sintering step is: be placed in room temperature successively and evenly heat up to 200 DEG C and sinter 50 minutes, and 200-400 DEG C evenly heats up and sinter 600 minutes, and 400-1300 DEG C evenly heats up and sinter 400 minutes, last 1250 DEG C of sintering 120 minutes.
Another aspect, the invention provides above-mentioned Three-dimensional cell culture support and is preparing the application in cell culture apparatus.
The invention provides a kind of novel three-dimensional cell cultivation support, it comprises porous calcium phosphate support and is coated in the sodium alginate on described porous calcium phosphate support, and compared with prior art, dimensional culture support intensity of the present invention is high, convenient operation and transport; And preparation technology is simple, and cost is low, without the need to specific installation.Sodium alginate support prepared by the present invention is a kind of growth masterplate for cell cultures, and it can the regeneration of transmitting tissue, controls the shape of tissue simultaneously.Support of the present invention as main body by polymethylmethacrylate, is had and makes the advantage simple, anti-pressure ability is strong; And polymethylmethacrylate has nontoxic, the advantage of non-immunogenicity, is widely used at biomedicine fields such as blood storage.The Three-dimensional cell culture frame with appropriate channel size is more conducive to the formation of blood vessel and tissue.
Accompanying drawing explanation
Below, describe embodiment of the present invention in detail by reference to the accompanying drawings, wherein:
Fig. 1 injects the stereoscan photograph before fused slurry in poly (methyl methacrylate) micro-sphere template;
Fig. 2 is the microphotograph of the Three-dimensional cell culture support for cell cultures of the present invention.
Embodiment
Below in conjunction with specific embodiment, and comparable data describes in further detail the present invention.Should be understood that these embodiments just in order to demonstrate the invention, but not limit scope of invention by any way.
Below in an example, the various process do not described in detail and method are ordinary methods as known in the art.
embodiment 1
1) solid phase tricalcium phosphate be 50% slurry preparation step as follows:
A prepared by () solid phase tricalcium phosphate powder: nitrocalcite 9.8412g, diammonium hydrogen phosphate 5.2836g, urea 3.3665g are dissolved in 100mL ultrapure water.
B the above-mentioned aqueous solution is positioned over dry 3h in 70 DEG C of baking ovens by ().
After (c) drying, solid is put in retort furnace.At 700 DEG C of temperature, calcining 1h, obtains the nanometer tricalcium phosphate powder of white.
D nanometer tricalcium phosphate powder 3.0106g and dispersants ammonium polyacrylate 1.2mL is dissolved in 3mL ultrapure water by (), with planetary ball mill ball milling 2h, prepare the slip of solid phase DFP.
2) poly (methyl methacrylate) micro-sphere preparation process is as follows:
A () preparation massfraction is the aqueous sodium hydroxide solution of 5%.
B () first, is got above-mentioned sodium hydroxide solution 25mL and is slowly cleaned methyl methacrylate twice.Then, methyl methacrylate is cleaned twice with above-mentioned sodium hydroxide solution 25mL.Finally, with the methyl methacrylate 12h after Calcium Chloride Powder Anhydrous drying washing.
C () preparation massfraction is the polyvinyl alcohol water solution of 0.3%.
D magnesium basic carbonate 0.5182g, in straight Erlenmeyer flask, is dissolved in the polyvinyl alcohol solution that 100mL massfraction is 0.3% by ().Then, 8mL methyl methacrylate solution (density p=0.9410g/mL), initiator benzoyl peroxide 0.0282g, 0.16mL Ethylene glycol dimethacrylate (density p=1.051g/mL) is added.At N
2under protection, reaction system is stirred 5h at 66 DEG C, stirring velocity is 240r/min.
D () reaction terminates after, by filter paper filtering crude product removing residual solution, and with milli-Q water 5 times.
E (), in 60 DEG C of baking ovens, vacuum-drying obtains the poly (methyl methacrylate) micro-sphere that diameter is 200 ~ 300 μm.
3) preparation process of polymethylmethacrylate/support is as follows:
A aluminium sulfite 3.6752g, urea 8.4084g, polyvinyl pyrrolidone 0.0462g are dissolved in 250mL ultrapure water by (), preparation obtains nano aluminium oxide suspension liquid.
B poly-methyl star diluted acid methyl esters microballoon is positioned in beaker, in loft drier by ().At 180 DEG C, dry 1h.
Under (c) room temperature, by nano aluminium oxide suspension liquid slowly be paved with poly (methyl methacrylate) micro-sphere, dry in vacuum drying oven, obtain poly (methyl methacrylate) micro-sphere support.
4) by 3) polymethylmethacrylate/support obtained in step carries out vacuum-treat, injects the 1st) the prepared tricalcium phosphate slip of step, mix and obtain slip/polymethylmethacrylate/scaffold complex.
5) slip/polymethylmethacrylate/scaffold complex is put in retort furnace, room temperature evenly heats up to 200 DEG C and sinters 50 minutes, 200-400 DEG C evenly heats up and sinters 600 minutes, 400-1300 DEG C evenly heats up and sinters 400 minutes, last 1250 DEG C of sintering 120 minutes, obtain hole calcium phosphate support.
6) for the dimensional culture support of cell cultures and the preparation process of sodium alginate support as follows:
A () preparation mass concentration is the sodium alginate gel of 4%;
B () is by the 5th) porous calcium phosphate prepared in step props up and is placed on vacuum in 4% sodium alginate hydrochloric acid soln and soaks 1h, with ultrapure water 3 times, prepares sodium alginate support.
embodiment 2
1) solid phase tricalcium phosphate be 55% slurry preparation step as follows:
A prepared by () solid phase tricalcium phosphate powder: nitrocalcite 12.0222g, diammonium hydrogen phosphate 6.8940g, urea 3.7730g are dissolved in 100mL ultrapure water.
B the above-mentioned aqueous solution is positioned over dry 7h in 80 DEG C of baking ovens by ().
After (c) drying, solid is put in retort furnace.At 800 DEG C of temperature, calcining 2h, obtains the nanometer tricalcium phosphate powder of white.
D nanometer tricalcium phosphate powder 3.0259g and dispersants ammonium polyacrylate 1.2mL is dissolved in 3mL ultrapure water by (), with planetary ball mill ball milling 0.5h, prepare the slip of solid phase DFP.
2) poly (methyl methacrylate) micro-sphere preparation process is as follows:
A () preparation massfraction is the aqueous sodium hydroxide solution of 6%.
B () first, is got above-mentioned sodium hydroxide solution 40mL and is slowly cleaned methyl methacrylate twice.Finally, with the methyl methacrylate 24h after Calcium Chloride Powder Anhydrous drying washing.
C () preparation massfraction is the polyvinyl alcohol water solution of 0.4%.
D magnesium basic carbonate 0.5482g, in straight Erlenmeyer flask, is dissolved in the polyvinyl alcohol solution that 100mL massfraction is 0.4% by ().Then, 7.7mL methyl methacrylate solution (density p=0.9410g/mL), initiator benzoyl peroxide 0.0282g, 0.15mL Ethylene glycol dimethacrylate (density p=1.051g/mL) is added.At N
2under protection, reaction system is stirred 6h at 70 DEG C, stirring velocity is 440r/min.
D () reaction terminates after, by filter paper filtering crude product removing residual solution, and with milli-Q water 5 times.
E (), in 65 DEG C of baking ovens, vacuum-drying obtains the poly (methyl methacrylate) micro-sphere that diameter is 200 ~ 300 μm.
3) preparation process of polymethylmethacrylate/support is as follows:
A aluminium sulfite 4.4109g, urea 9.0010g, polyvinyl pyrrolidone 0.0475g are dissolved in 250mL ultrapure water by (), preparation obtains nano aluminium oxide suspension liquid.
B poly-methyl star diluted acid methyl esters microballoon is positioned in beaker, in loft drier by ().At 200 DEG C, dry 2h.
Under (c) room temperature, by nano aluminium oxide suspension liquid slowly be paved with poly (methyl methacrylate) micro-sphere, dry in vacuum drying oven, obtain poly (methyl methacrylate) micro-sphere support.
4) by the 3rd) the obtained polymethylmethacrylate/support of step carries out vacuum-treat, injects the 1st) the prepared tricalcium phosphate slip of step, mix and obtain slip/polymethylmethacrylate/scaffold complex.
5) slip/polymethylmethacrylate/scaffold complex is put in retort furnace, room temperature evenly heats up to 200 DEG C and sinters 50 minutes, 200-400 DEG C evenly heats up and sinters 600 minutes, 400-1300 DEG C evenly heats up and sinters 400 minutes, last 1250 DEG C of sintering 120 minutes, obtain hole calcium phosphate support.
6) for the dimensional culture support of cell cultures and the preparation process of sodium alginate support as follows:
A () preparation mass concentration is the sodium alginate gel of 7%;
B () is by the 5th) porous calcium phosphate prepared in step props up to be placed in 7% sodium alginate hydrochloric acid soln and soaks 1h, with ultrapure water 3 times, prepares sodium alginate support.
Embodiment 3
1) solid phase tricalcium phosphate be 52% slurry preparation step as follows:
A prepared by () solid phase tricalcium phosphate powder: nitrocalcite 10.6191g, diammonium hydrogen phosphate 6.0986g, urea 3.6099g are dissolved in 100mL ultrapure water.
B the above-mentioned aqueous solution is positioned over dry 5h in 75 DEG C of baking ovens by ().
After (c) drying, solid is put in retort furnace.At 780 DEG C of temperature, calcining 1.5h, obtains the nanometer tricalcium phosphate powder of white.
D nanometer tricalcium phosphate powder 3.0119g and dispersants ammonium polyacrylate 1.2mL is dissolved in 3mL ultrapure water by (), with planetary ball mill ball milling 0.5h, prepare the slip of solid phase DFP.
2) poly (methyl methacrylate) micro-sphere preparation process is as follows:
A () preparation massfraction is the aqueous sodium hydroxide solution of 5.5%.
B () first, is got above-mentioned sodium hydroxide solution 40mL and is slowly cleaned methyl methacrylate twice.Finally, with the methyl methacrylate 20h after Calcium Chloride Powder Anhydrous drying washing.
C () preparation massfraction is the polyvinyl alcohol water solution of 0.36%.
D magnesium basic carbonate 0.5295g, in straight Erlenmeyer flask, is dissolved in the polyvinyl alcohol solution that 100mL massfraction is 0.3% by ().Then, 8mL methyl methacrylate solution (density p=0.9410g/mL), initiator benzoyl peroxide 0.0282g, 0.16mL Ethylene glycol dimethacrylate (density p=1.051g/mL) is added.At N
2under protection, reaction system is stirred 5.5h at 68 DEG C, stirring velocity is 300r/min.
D () reaction terminates after, by filter paper filtering crude product removing residual solution, and with milli-Q water 5 times.
E (), in 65 DEG C of baking ovens, vacuum-drying obtains the poly (methyl methacrylate) micro-sphere that diameter is 200 ~ 300 μm.
3) preparation process of polymethylmethacrylate/support is as follows:
A aluminium sulfite 4.1452g, urea 8.8762g, polyvinyl pyrrolidone 0.0456g are dissolved in 250mL ultrapure water by (), preparation obtains nano aluminium oxide suspension liquid.
B poly-methyl star diluted acid methyl esters microballoon is positioned in beaker, in loft drier by ().At 190 DEG C, dry 1.5h.
Under (c) room temperature, by nano aluminium oxide suspension liquid slowly be paved with poly (methyl methacrylate) micro-sphere, dry in vacuum drying oven, obtain poly (methyl methacrylate) micro-sphere support.
4) by the 3rd) the obtained polymethylmethacrylate/support of step carries out vacuum-treat, injects the 1st) the prepared tricalcium phosphate slip of step, mix and obtain slip/polymethylmethacrylate/scaffold complex.
5) slip/polymethylmethacrylate/scaffold complex is put in retort furnace, room temperature evenly heats up to 200 DEG C and sinters 50 minutes, 200-400 DEG C evenly heats up and sinters 600 minutes, 400-1300 DEG C evenly heats up and sinters 400 minutes, last 1250 DEG C of sintering 120 minutes, obtain hole calcium phosphate support.
6) for the dimensional culture support of cell cultures and the preparation process of sodium alginate support as follows:
A () preparation mass concentration is the sodium alginate gel of 5%;
B () is by the 5th) porous calcium phosphate prepared in step props up to be placed in 5% sodium alginate hydrochloric acid soln and soaks 1h, with ultrapure water 3 times, prepares sodium alginate support.
embodiment 4
1) solid phase tricalcium phosphate is that the slurry preparation step of 54% is with embodiment 1
2) poly (methyl methacrylate) micro-sphere preparation process is with embodiment 1:
3) preparation process of polymethylmethacrylate/support is with embodiment 1:
4) slip/polymethylmethacrylate/scaffold complex preparation process is with embodiment 1.
5) slip/polymethylmethacrylate/scaffold complex is put in retort furnace, room temperature evenly heats up to 200 DEG C and sinters 50 minutes, 200-400 DEG C evenly heats up and sinters 600 minutes, 400-1300 DEG C evenly heats up and sinters 400 minutes, last 1250 DEG C of sintering 120 minutes, obtain hole calcium phosphate support.
6) for the dimensional culture support of cell cultures and the preparation process of sodium alginate support with embodiment 1.
embodiment 5
1) solid phase tricalcium phosphate is that the slurry preparation step of 55% is with embodiment 1
2) poly (methyl methacrylate) micro-sphere preparation process is with embodiment 1:
3) preparation process of polymethylmethacrylate/support is with embodiment 1:
4) slip/polymethylmethacrylate/scaffold complex preparation process is with embodiment 1.
5) slip/polymethylmethacrylate/scaffold complex is put in retort furnace, at 1250 DEG C, sinters 120 minutes, obtain porous calcium phosphate support.
6) for the dimensional culture support of cell cultures and the preparation process of sodium alginate support with embodiment 1.
Application for cell cultures Three-dimensional cell culture support: the dimensional culture for cell cultures prepared by the present invention and sodium alginate support are a kind of growth masterplates for cell cultures, it can the regeneration of transmitting tissue, controls the shape of tissue simultaneously.Support of the present invention as main body by polymethylmethacrylate, is had and makes the advantage simple, anti-pressure ability is strong; And polymethylmethacrylate has nontoxic, the advantage of non-immunogenicity, is widely used at biomedicine fields such as blood storage.The Three-dimensional cell culture frame with appropriate channel size is more conducive to the formation of blood vessel and tissue.
Experiment shows: the homemade polymethylmethacrylate of the present invention has good 3 D tropism.This polymkeric substance structure is before sintering characterized by scanning electronic microscope (accompanying drawing 1), and the structure of this three-dimensional rack is observed by microscope (accompanying drawing 2).As can be seen from the figure, three-dimensional cell support demonstrates porousness and 3 D tropism.
Claims (15)
1. a preparation method for dimensional culture support, is characterized in that, described preparation method comprises:
Step 1: solid-phase calcium phosphate content is the preparation of the slip of 50 ~ 55% (weightmeasurement ratios);
Step 2: the preparation of poly (methyl methacrylate) micro-sphere;
Step 3: the preparation of porous calcium phosphate support;
Step 4: the sodium alginate hydrochloric acid soln that mass percentage is 4 ~ 7% put into by porous calcium phosphate support step 3 prepared, vacuum soaks 1-2h, with ultrapure water repetitive scrubbing three times, to obtain final product,
Wherein, in step 3, the preparation of described porous calcium phosphate support comprises the following steps:
Step 3.1: by aluminium sulfite, urea, polyvinyl pyrrolidone ultrapure water, preparation obtains nano aluminium oxide suspension liquid;
Step 3.2: polymethyl acrylate microballoon is positioned in beaker, in loft drier, at 180-200 DEG C, dry 1-2h;
Step 3.3: under room temperature, is slowly paved with poly (methyl methacrylate) micro-sphere by nano aluminium oxide suspension liquid, dry in vacuum drying oven, obtains poly (methyl methacrylate) micro-sphere support;
Step 3.4: the poly (methyl methacrylate) micro-sphere support that step 3.3 is obtained carries out vacuum-treat, tricalcium phosphate slip prepared in implantation step 1, mixes and obtains slip/polymethylmethacrylate/scaffold complex;
Step 3.5: slip/polymethylmethacrylate/scaffold complex is put in sintering in retort furnace and forms porous calcium phosphate support.
2. preparation method according to claim 1, is characterized in that, in step 1, described calcium phosphate is nano-calcium phosphate.
3. preparation method according to claim 2, is characterized in that, in step 1, the preparation of described slip comprises the following steps:
Step 1.1: the preparation of nanometer tricalcium phosphate powder;
Step 1.2: by nanometer tricalcium phosphate powder, dispersion agent and ultrapure water ball milling 0.5-2h, to obtain final product.
4. preparation method according to claim 3, is characterized in that, the preparation of described nanometer tricalcium phosphate powder comprises the following steps:
Step 1.1.1: nitrocalcite, diammonium hydrogen phosphate and urea are dissolved in ultrapure water;
Step 1.1.2: the solution that step 1.1.1 is obtained is positioned in baking oven, in 70-80 DEG C of dry 3-7h;
Step 1.1.3: be put in retort furnace by dried material, in 700-800 DEG C of temperature, calcining 1-2h, obtains the nanometer tricalcium phosphate powder of white.
5. preparation method according to claim 4, is characterized in that, in step 1.1.1, in described ultrapure water solution, the concentration of each raw material is: nitrocalcite 0.5-0.6 mole/L, diammonium hydrogen phosphate 0.4-0.5 mole/L and urea 0.56-0.6 mole/L.
6. the preparation method according to any one of claim 1 to 5, is characterized in that, in step 2, the preparation of described poly (methyl methacrylate) micro-sphere comprises the following steps:
Step 2.1: be that 5-6% sodium hydroxide solution slowly cleans methyl methacrylate twice with massfraction, then with the methyl methacrylate 12-24h after Calcium Chloride Powder Anhydrous drying washing,
Step 2.2: magnesium basic carbonate being dissolved in mass percentage is in the polyvinyl alcohol solution of 0.3%, add methyl methacrylate solution, initiator, Ethylene glycol dimethacrylate again, under N2 protection, 6h is stirred at reaction system being placed in 66-70 DEG C, stirring velocity is 240-440r/min, step 2.3: after reaction terminates, by filter paper filtering crude product removing residual solution, and with milli-Q water 5 times;
Step 2.4: in 60-65 DEG C of baking oven, vacuum-drying obtains the poly (methyl methacrylate) micro-sphere that diameter is 200 ~ 300 μm.
7. preparation method according to claim 6, is characterized in that, in step 2.1, is that 5-6% sodium hydroxide solution slowly cleans methyl methacrylate twice with 25mL massfraction.
8. preparation method according to claim 6, is characterized in that, in step 2.2, the concentration of described basic carbonate magnesium solution is 0.0142-0.015 mole/L.
9. preparation method according to claim 6, is characterized in that, in step 2.2, the volume ratio of described methyl methacrylate solution and polyvinyl alcohol solution is 1:12.5-1:13.
10. preparation method according to claim 6, is characterized in that, in step 2.2, the density of described methyl methacrylate solution is 0.9410g/mL.
11. preparation methods according to claim 6, is characterized in that, in step 2.2, described initiator is benzoyl peroxide, and the mass concentration of described benzoyl peroxide is 0.3%.
12. want the preparation method described in 6 according to right, it is characterized in that, in step 2.2, the volume ratio of described Ethylene glycol dimethacrylate and polyvinyl alcohol solution is 1:625-1:667.
13. preparation methods according to claim 6, is characterized in that, in step 2.2, the density of described Ethylene glycol dimethacrylate is 1.0151g/mL.
14. preparation methods according to claim 1, is characterized in that, in step 3.1, the concentration that described each raw material is dissolved in ultrapure water is respectively: aluminium sulfite 0.05-0.06 mole/L, urea 0.56-0.6 mole/L, polyvinyl pyrrolidone 6.5x10
-6mole/L.
15. preparation methods according to claim 1, it is characterized in that, described sintering step is: be placed in room temperature successively and evenly heat up to 200 DEG C and sinter 50 minutes, 200-400 DEG C evenly heats up and sinters 600 minutes, 400-1300 DEG C evenly heats up and sinters 400 minutes, last 1250 DEG C of sintering 120 minutes.
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