CN102935246A - 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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- CN102935246A CN102935246A CN2011102332154A CN201110233215A CN102935246A CN 102935246 A CN102935246 A CN 102935246A CN 2011102332154 A CN2011102332154 A CN 2011102332154A CN 201110233215 A CN201110233215 A CN 201110233215A CN 102935246 A CN102935246 A CN 102935246A
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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 the bioengineered tissue field, particularly a kind of preparation method and application that are applied to three-dimensional cell cultivation support.The present invention is take nano tricalcium phosphate as initial feed, by the synthesizing porous calcium phosphate support of polymethyl methacrylate template.The calcium phosphate support is immersed the complex that forms tricalcium phosphate/sodium alginate thin film in the acid sodium alginate soln, finally prepared three-dimensional cell and cultivated support.
Background technology
Two dimension (2D) cell culture be a kind ofly prepare, observation of cell and in vitro study cell and the interactional technology of medicine, biotic factor and biomaterial.But it and cell growth pattern in vivo is not similar.In animal body, cell is three-dimensional (3D) growth and makes up formation living tissue or organ.Therefore, cell three-dimensional is cultivated to cultivate with two dimension and is compared, more the cell micro-environment in the animal body.Have report to point out, cultivate by external three-dimensional cell, we can understand the structure-function under the organization condition that is organized in normal and pathology.Therefore, three-dimensional cell cultivation support has very high using value.
What adopt in traditional three-dimensional cell cultivation support preparation is the organic polymer support, and such as gelatin-glutaraldehyde, but its restricted standby process technology limit often adopts specific organic principle, and forming machine complexity or intensity are low, and inconvenience is as product.
Summary of the invention
Therefore, the object of the invention is to overcome above-mentioned preparation technology's deficiency, provide a kind of novel three-dimensional cell to cultivate support.
Another object of the present invention provides the preparation method that described three-dimensional cell is cultivated support, and this preparation process is simple and easy to do.And provide above-mentioned three-dimensional cell to cultivate the application of support in the preparation cell culture system.
Be used for realizing that the technical scheme of above-mentioned purpose is as follows:
On the one hand, the invention provides a kind of three-dimensional cell for cell culture and cultivate support, it comprises porous calcium phosphate support and the sodium alginate layer that is coated on the 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 that above-mentioned three-dimensional cell is cultivated support, this preparation method may further comprise the steps:
Step 1: the solid phase calcium phosphate content is the preparation of the slip of 50～55% (w/vs);
Step 2: the preparation of poly (methyl methacrylate) micro-sphere;
Step 3: the preparation of porous calcium phosphate support;
Step 4: it is 4～7% sodium alginate hydrochloric acid solution that the porous calcium phosphate support of step 3 preparation is put into the quality percentage composition, and vacuum is soaked 1-2h, with ultra-pure water cyclic washing three times, and get final product.
Preferably, in step 1, described calcium phosphate is nano-calcium phosphate.
Preferably, in step 1, the preparation of described slip may further comprise the steps:
Step 1.1: the preparation of nanometer tricalcium phosphate powder;
Step 1.2: with nanometer tricalcium phosphate powder, dispersant and ultra-pure water ball milling 0.5-2h, and get final product.
Preferably, the preparation of described nanometer tricalcium phosphate powder may further comprise the steps:
Step 1.1.1: lime nitrate, diammonium hydrogen phosphate and carbamide are dissolved in the ultra-pure water, and preferably, the concentration of each raw material is in the described ultra-pure water solution: lime nitrate 0.5-0.6 mole/L, diammonium hydrogen phosphate 0.4-0.5 mole/L and carbamide 0.56-0.6 mole/L;
Step 1.1.2: the solution that step 1.1.1 is made is positioned in the baking oven, in 70-80 ℃ of dry 3-7h;
Step 1.1.3: dried material is put in the Muffle furnace, and in 700-800 ℃ of temperature, calcining 1-2h obtains white nanometer tricalcium phosphate powder.
Preferably, in step 2, the preparation of described poly (methyl methacrylate) micro-sphere may further comprise the steps:
Step 2.1: be that the 5-6% sodium hydroxide solution slowly cleans methyl methacrylate twice with mass fraction, again with the methyl methacrylate 12-24h after the dry washing of anhydrous calcium chloride, preferably, be that the 5-6% sodium hydroxide solution slowly cleans methyl methacrylate twice with the 25mL mass fraction;
Step 2.2: it is in 0.3% the poly-vinyl alcohol solution that basic magnesium carbonate is dissolved in the quality percentage composition, adds methyl methacrylate solution, initiator, Ethylene glycol dimethacrylate, at N again
2Protection is lower, and as for 66-70 ℃ of lower stirring 5-6h, mixing speed is 240-440r/min with reaction system, 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 poly-vinyl alcohol solution is 1: 12.5-1: 13, and more preferably, described methyl methacrylate liquid density 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 poly-vinyl alcohol solution is 1: 625-1: 667, and more preferably, the density of described Ethylene glycol dimethacrylate is 1.0151g/mL.
Step 2.3: reaction is removed residual solution with the filter paper filtering crude product after finishing, and washs 5 times with ultrapure ultra-pure water;
Step 2.4: in 60-65 ℃ of baking oven, vacuum drying obtains the poly (methyl methacrylate) micro-sphere that diameter is 200～300 μ m.
Preferably, in step 3, the preparation of described porous calcium phosphate support may further comprise the steps:
Step 3.1: in aluminium sulfite, carbamide, polyvinyl pyrrolidone solution ultra-pure water, preparation obtains the nano aluminium oxide suspension, preferably, the concentration that is dissolved in the ultra-pure water of described each raw material is respectively: aluminium sulfite 0.05-0.06 mole/L, carbamide 0.56-0.6 mole/L, polyvinyl pyrrolidone 6.5x10
-6Mole/L;
Step 3.2: will gather methyl star diluted acid methyl ester microsphere and be positioned in the beaker, in drying baker, under 180-200 ℃, dry 1-2h;
Step 3.3: under the room temperature, the nano aluminium oxide suspension slowly is paved with poly (methyl methacrylate) micro-sphere, dry in vacuum drying oven, obtain the poly (methyl methacrylate) micro-sphere support.
Step 3.4: polymethyl methacrylate/support that step 3.3 is made carries out application of vacuum, prepared tricalcium phosphate slip in the implantation step 1, and mix homogeneously obtains slip/polymethyl methacrylate/scaffold complex.
Step 3.5: slip/polymethyl methacrylate/scaffold complex is put in sintering formation porous calcium phosphate support in the Muffle furnace.
Preferably, described sintering step is: place successively room temperature to 200 ℃ evenly to heat up and sintering 50 minutes, 200-400 ℃ evenly heats up and sintering 600 minutes, and 400-1300 ℃ evenly heats up and sintering 400 minutes last 1250 ℃ of sintering 120 minutes.
Another aspect the invention provides above-mentioned three-dimensional cell and cultivates the application of support in the preparation cell culture system.
The invention provides a kind of novel cell three-dimensional and cultivate support, it comprises porous calcium phosphate support and the sodium alginate that is coated on the described porous calcium phosphate support, and compared with prior art, dimensional culture support intensity of the present invention is high, convenient operation and transportation; And preparation technology is simple, and cost is low, need not special installation.The sodium alginate support of the present invention preparation is a kind of growth masterplate for cell culture, the regeneration that it can transmitting tissue, the simultaneously shape of control tissue.Support of the present invention, is had and makes the advantage simple, that anti-pressure ability is strong as main body by polymethyl methacrylate; And polymethyl methacrylate has nontoxic, and the advantage of non-immunogenicity is widely used at biomedicine fields such as blood storages.Three-dimensional cell culturing rack with appropriate channel size more is conducive to the formation of blood vessel and tissue.
Description of drawings
Below, describe by reference to the accompanying drawings embodiment of the present invention in detail, wherein:
Fig. 1 is the stereoscan photograph of injecting in the poly (methyl methacrylate) micro-sphere template before the slip sintering;
Fig. 2 is the microphotograph of cultivating support for the three-dimensional cell of cell culture of the present invention.
The specific embodiment
Below in conjunction with specific embodiment, and comparable data describes in further detail the present invention.Should be understood that these embodiment just in order to demonstrate the invention, but not limit by any way scope of invention.
In following embodiment, various processes and the method do not described in detail are conventional methods as known in the art.
Embodiment 1
1) the solid phase tricalcium phosphate is that 50% slip preparation steps is as follows:
(a) solid phase tricalcium phosphate powder preparation: lime nitrate 9.8412g, diammonium hydrogen phosphate 5.2836g, carbamide 3.3665g are dissolved in the 100mL ultra-pure water.
(b) above-mentioned aqueous solution is positioned over dry 3h in 70 ℃ of baking ovens.
(c) after the drying, solid is put in the Muffle furnace.Under 700 ℃ of temperature, calcining 1h obtains white nanometer tricalcium phosphate powder.
(d) nanometer tricalcium phosphate powder 3.0106g and dispersant ammonium polyacrylate 1.2mL are dissolved in the 3mL ultra-pure water, with planetary ball mill ball milling 2h, prepare the slip of solid phase DFP.
2) the poly (methyl methacrylate) micro-sphere preparation process is as follows:
(a) the preparation mass fraction is 5% sodium hydrate aqueous solution.
(b) at first, get above-mentioned sodium hydroxide solution 25mL and slowly clean methyl methacrylate twice.Then, clean methyl methacrylate twice with above-mentioned sodium hydroxide solution 25mL.At last, with the methyl methacrylate 12h after the dry washing of anhydrous calcium chloride.
(c) the preparation mass fraction is 0.3% polyvinyl alcohol water solution.
(d) in straight conical flask, it is 0.3% poly-vinyl alcohol solution that basic magnesium carbonate 0.5182g is dissolved in the 100mL mass fraction.Then, add 8mL methyl methacrylate solution (density p=0.9410g/mL), initiator benzoyl peroxide 0.0282g, 0.16mL Ethylene glycol dimethacrylate (density p=1.051g/mL).At N
2Protection is lower, and as for 66 ℃ of lower stirring 5h, mixing speed is 240r/min with reaction system.
(d) after reaction finishes, remove residual solution with the filter paper filtering crude product, and wash 5 times with ultra-pure water.
(e) in 60 ℃ of baking ovens, vacuum drying obtains the poly (methyl methacrylate) micro-sphere that diameter is 200～300 μ m.
3) preparation process of polymethyl methacrylate/support is as follows:
(a) aluminium sulfite 3.6752g, carbamide 8.4084g, polyvinyl pyrrolidone 0.0462g are dissolved in the 250mL ultra-pure water, preparation obtains the nano aluminium oxide suspension.
(b) will gather methyl star diluted acid methyl ester microsphere and be positioned in the beaker, in drying baker.Under 180 ℃, dry 1h.
(c) under the room temperature, with the nano aluminium oxide suspension slowly be paved with poly (methyl methacrylate) micro-sphere, dry in vacuum drying oven, obtain the poly (methyl methacrylate) micro-sphere support.
4) with 3) polymethyl methacrylate/support of making in the step carries out application of vacuum, injects the 1st) prepared tricalcium phosphate slip of step, mix homogeneously obtains slip/polymethyl methacrylate/scaffold complex.
5) slip/polymethyl methacrylate/scaffold complex is put in the Muffle furnace, room temperature to 200 ℃ even intensification and sintering 50 minutes, 200-400 ℃ evenly heats up and sintering 600 minutes, 400-1300 ℃ evenly heats up and sintering 400 minutes, last 1250 ℃ of sintering 120 minutes obtain hole calcium phosphate support.
6) the dimensional culture support that is used for cell culture is that the preparation process of sodium alginate support is as follows:
(a) the preparation mass concentration is 4% sodium alginate gel;
(b) with the 5th) porous calcium phosphate of preparation props up and is placed in the 4% sodium alginate hydrochloric acid solution vacuum and soaks 1h in the step, with ultra-pure water flushing 3 times, prepares the sodium alginate support.
Embodiment 2
1) the solid phase tricalcium phosphate is that 55% slip preparation steps is as follows:
(a) solid phase tricalcium phosphate powder preparation: lime nitrate 12.0222g, diammonium hydrogen phosphate 6.8940g, carbamide 3.7730g are dissolved in the 100mL ultra-pure water.
(b) above-mentioned aqueous solution is positioned over dry 7h in 80 ℃ of baking ovens.
(c) after the drying, solid is put in the Muffle furnace.Under 800 ℃ of temperature, calcining 2h obtains white nanometer tricalcium phosphate powder.
(d) nanometer tricalcium phosphate powder 3.0259g and dispersant ammonium polyacrylate 1.2mL are dissolved in the 3mL ultra-pure water, with planetary ball mill ball milling 0.5h, prepare the slip of solid phase DFP.
2) the poly (methyl methacrylate) micro-sphere preparation process is as follows:
(a) the preparation mass fraction is 6% sodium hydrate aqueous solution.
(b) at first, get above-mentioned sodium hydroxide solution 40mL and slowly clean methyl methacrylate twice.At last, with the methyl methacrylate 24h after the dry washing of anhydrous calcium chloride.
(c) the preparation mass fraction is 0.4% polyvinyl alcohol water solution.
(d) in straight conical flask, it is 0.4% poly-vinyl alcohol solution that basic magnesium carbonate 0.5482g is dissolved in the 100mL mass fraction.Then, add 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).At N
2Protection is lower, and as for 70 ℃ of lower stirring 6h, mixing speed is 440r/min with reaction system.
(d) after reaction finishes, remove residual solution with the filter paper filtering crude product, and wash 5 times with ultra-pure water.
(e) in 65 ℃ of baking ovens, vacuum drying obtains the poly (methyl methacrylate) micro-sphere that diameter is 200～300 μ m.
3) preparation process of polymethyl methacrylate/support is as follows:
(a) aluminium sulfite 4.4109g, carbamide 9.0010g, polyvinyl pyrrolidone 0.0475g are dissolved in the 250mL ultra-pure water, preparation obtains the nano aluminium oxide suspension.
(b) will gather methyl star diluted acid methyl ester microsphere and be positioned in the beaker, in drying baker.Under 200 ℃, dry 2h.
(c) under the room temperature, with the nano aluminium oxide suspension slowly be paved with poly (methyl methacrylate) micro-sphere, dry in vacuum drying oven, obtain the poly (methyl methacrylate) micro-sphere support.
4) with the 3rd) polymethyl methacrylate/support of making of step carries out application of vacuum, injects the 1st) prepared tricalcium phosphate slip of step, mix homogeneously obtains slip/polymethyl methacrylate/scaffold complex.
5) slip/polymethyl methacrylate/scaffold complex is put in the Muffle furnace, room temperature to 200 ℃ even intensification and sintering 50 minutes, 200-400 ℃ evenly heats up and sintering 600 minutes, 400-1300 ℃ evenly heats up and sintering 400 minutes, last 1250 ℃ of sintering 120 minutes obtain hole calcium phosphate support.
6) the dimensional culture support that is used for cell culture is that the preparation process of sodium alginate support is as follows:
(a) the preparation mass concentration is 7% sodium alginate gel;
(b) with the 5th) porous calcium phosphate of preparation props up to be placed in the 7% sodium alginate hydrochloric acid solution and soaks 1h in the step, with ultra-pure water flushing 3 times, prepares the sodium alginate support.
Embodiment 3
1) the solid phase tricalcium phosphate is that 52% slip preparation steps is as follows:
(a) solid phase tricalcium phosphate powder preparation: lime nitrate 10.6191g, diammonium hydrogen phosphate 6.0986g, carbamide 3.6099g are dissolved in the 100mL ultra-pure water.
(b) above-mentioned aqueous solution is positioned over dry 5h in 75 ℃ of baking ovens.
(c) after the drying, solid is put in the Muffle furnace.Under 780 ℃ of temperature, calcining 1.5h obtains white nanometer tricalcium phosphate powder.
(d) nanometer tricalcium phosphate powder 3.0119g and dispersant ammonium polyacrylate 1.2mL are dissolved in the 3mL ultra-pure water, with planetary ball mill ball milling 0.5h, prepare the slip of solid phase DFP.
2) the poly (methyl methacrylate) micro-sphere preparation process is as follows:
(a) the preparation mass fraction is 5.5% sodium hydrate aqueous solution.
(b) at first, get above-mentioned sodium hydroxide solution 40mL and slowly clean methyl methacrylate twice.At last, with the methyl methacrylate 20h after the dry washing of anhydrous calcium chloride.
(c) the preparation mass fraction is 0.36% polyvinyl alcohol water solution.
(d) in straight conical flask, it is 0.3% poly-vinyl alcohol solution that basic magnesium carbonate 0.5295g is dissolved in the 100mL mass fraction.Then, add 8mL methyl methacrylate solution (density p=0.9410g/mL), initiator benzoyl peroxide 0.0282g, 0.16mL Ethylene glycol dimethacrylate (density p=1.051g/mL).At N
2Protection is lower, and as for 68 ℃ of lower stirring 5.5h, mixing speed is 300r/min with reaction system.
(d) after reaction finishes, remove residual solution with the filter paper filtering crude product, and wash 5 times with ultra-pure water.
(e) in 65 ℃ of baking ovens, vacuum drying obtains the poly (methyl methacrylate) micro-sphere that diameter is 200～300 μ m.
3) preparation process of polymethyl methacrylate/support is as follows:
(a) aluminium sulfite 4.1452g, carbamide 8.8762g, polyvinyl pyrrolidone 0.0456g are dissolved in the 250mL ultra-pure water, preparation obtains the nano aluminium oxide suspension.
(b) will gather methyl star diluted acid methyl ester microsphere and be positioned in the beaker, in drying baker.Under 190 ℃, dry 1.5h.
(c) under the room temperature, with the nano aluminium oxide suspension slowly be paved with poly (methyl methacrylate) micro-sphere, dry in vacuum drying oven, obtain the poly (methyl methacrylate) micro-sphere support.
4) with the 3rd) polymethyl methacrylate/support of making of step carries out application of vacuum, injects the 1st) prepared tricalcium phosphate slip of step, mix homogeneously obtains slip/polymethyl methacrylate/scaffold complex.
5) slip/polymethyl methacrylate/scaffold complex is put in the Muffle furnace, room temperature to 200 ℃ even intensification and sintering 50 minutes, 200-400 ℃ evenly heats up and sintering 600 minutes, 400-1300 ℃ evenly heats up and sintering 400 minutes, last 1250 ℃ of sintering 120 minutes obtain hole calcium phosphate support.
6) the dimensional culture support that is used for cell culture is that the preparation process of sodium alginate support is as follows:
(a) the preparation mass concentration is 5% sodium alginate gel;
(b) with the 5th) porous calcium phosphate of preparation props up to be placed in the 5% sodium alginate hydrochloric acid solution and soaks 1h in the step, with ultra-pure water flushing 3 times, prepares the sodium alginate support.
Embodiment 4
1) the solid phase tricalcium phosphate is that 54% slip preparation steps is with embodiment 1
2) the poly (methyl methacrylate) micro-sphere preparation process is with embodiment 1:
3) preparation process of polymethyl methacrylate/support is with embodiment 1:
4) slip/polymethyl methacrylate/scaffold complex preparation process is with embodiment 1.
5) slip/polymethyl methacrylate/scaffold complex is put in the Muffle furnace, room temperature to 200 ℃ even intensification and sintering 50 minutes, 200-400 ℃ evenly heats up and sintering 600 minutes, 400-1300 ℃ evenly heats up and sintering 400 minutes, last 1250 ℃ of sintering 120 minutes obtain hole calcium phosphate support.
6) the dimensional culture support that is used for cell culture is that the preparation process of sodium alginate support is with embodiment 1.
Embodiment 5
1) the solid phase tricalcium phosphate is that 55% slip preparation steps is with embodiment 1
2) the poly (methyl methacrylate) micro-sphere preparation process is with embodiment 1:
3) preparation process of polymethyl methacrylate/support is with embodiment 1:
4) slip/polymethyl methacrylate/scaffold complex preparation process is with embodiment 1.
5) slip/polymethyl methacrylate/scaffold complex is put in the Muffle furnace, 1250 ℃ of lower sintering 120 minutes, obtains the porous calcium phosphate support.
6) the dimensional culture support that is used for cell culture is that the preparation process of sodium alginate support is with embodiment 1.
Be used for the cell culture three-dimensional cell and cultivate the application of support: the dimensional culture that is used for cell culture of the present invention's preparation is that the sodium alginate support is a kind of growth masterplate for cell culture, the regeneration that it can transmitting tissue, the simultaneously shape of control tissue.Support of the present invention, is had and makes the advantage simple, that anti-pressure ability is strong as main body by polymethyl methacrylate; And polymethyl methacrylate has nontoxic, and the advantage of non-immunogenicity is widely used at biomedicine fields such as blood storages.Three-dimensional cell culturing rack with appropriate channel size more is conducive to the formation of blood vessel and tissue.
Experiment shows: the homemade polymethyl methacrylate of the present invention has good 3 D tropism.The structure of this polymer before sintering characterizes by scanning electron 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, the three-dimensional cell support demonstrates porous and 3 D tropism.
Claims (9)
1. a dimensional culture support that is used for cell culture is characterized in that, described dimensional culture support comprises porous calcium phosphate support and the sodium alginate layer that is coated on the described porous calcium phosphate support; Described porous calcium phosphate support is provided with equally distributed micropore.
2. the preparation method of dimensional culture support according to claim 1 is characterized in that, described preparation method comprises:
Step 1: the solid phase calcium phosphate content is the preparation of the slip of 50～55% (w/vs);
Step 2: the preparation of poly (methyl methacrylate) micro-sphere;
Step 3: the preparation of porous calcium phosphate support;
Step 4: it is 4～7% sodium alginate hydrochloric acid solution that the porous calcium phosphate support of step 3 preparation is put into the quality percentage composition, and vacuum is soaked 1-2h, with ultra-pure water cyclic washing three times, and get final product.
3. preparation method according to claim 2 is characterized in that, in step 1, described calcium phosphate is nano-calcium phosphate.
4. according to claim 2 or 3 described preparation methoies, it is characterized in that in step 1, the preparation of described slip may further comprise the steps:
Step 1.1: the preparation of nanometer tricalcium phosphate powder;
Step 1.2: with nanometer tricalcium phosphate powder, dispersant and ultra-pure water ball milling 0.5-2h, and get final product.
5. preparation method according to claim 4 is characterized in that, the preparation of described nanometer tricalcium phosphate powder may further comprise the steps:
Step 1.1.1: lime nitrate, diammonium hydrogen phosphate and carbamide are dissolved in the ultra-pure water, and preferably, the concentration of each raw material is in the described ultra-pure water solution: lime nitrate 0.5-0.6 mole/L, diammonium hydrogen phosphate 0.4-0.5 mole/L and carbamide 0.56-0.6 mole/L;
Step 1.1.2: the solution that step 1.1.1 is made is positioned in the baking oven, in 70-80 ℃ of dry 3-7h;
Step 1.1.3: dried material is put in the Muffle furnace, and in 700-800 ℃ of temperature, calcining 1-2h obtains white nanometer tricalcium phosphate powder.
6. according to claim 2 to 5 each described preparation methoies, it is characterized in that in step 2, the preparation of described poly (methyl methacrylate) micro-sphere may further comprise the steps:
Step 2.1: be that the 5-6% sodium hydroxide solution slowly cleans methyl methacrylate twice with mass fraction, again with the methyl methacrylate 12-24h after the dry washing of anhydrous calcium chloride, preferably, be that the 5-6% sodium hydroxide solution slowly cleans methyl methacrylate twice with the 25mL mass fraction;
Step 2.2: it is in 0.3% the poly-vinyl alcohol solution that basic magnesium carbonate is dissolved in the quality percentage composition, add again methyl methacrylate solution, initiator, Ethylene glycol dimethacrylate, under the N2 protection, reaction system is stirred 6h under 66-70 ℃, mixing speed is 240-440r/min, 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 poly-vinyl alcohol solution is 1: 12.5-1: 13, and more preferably, described methyl methacrylate liquid density 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 poly-vinyl alcohol solution is 1: 625-1: 667, and more preferably, the density of described Ethylene glycol dimethacrylate is 1.0151g/mL;
Step 2.3: reaction is removed residual solution with the filter paper filtering crude product after finishing, and washs 5 times with ultra-pure water;
Step 2.4: in 60-65 ℃ of baking oven, vacuum drying obtains the poly (methyl methacrylate) micro-sphere that diameter is 200～300 μ m.
7. each described preparation method is characterized in that according to claim 2-5, and in step 3, the preparation of described porous calcium phosphate support may further comprise the steps:
Step 3.1: in aluminium sulfite, carbamide, polyvinyl pyrrolidone ultra-pure water, preparation obtains the nano aluminium oxide suspension, preferably, the concentration that is dissolved in the ultra-pure water of described each raw material is respectively: aluminium sulfite 0.05-0.06 mole/L, carbamide 0.56-0.6 mole/L, polyvinyl pyrrolidone 6.5x10-6 mole/L;
Step 3.2: the polymethyl acrylate microsphere is positioned in the beaker, in drying baker, under 180-200 ℃, dry 1-2h;
Step 3.3: under the room temperature, the nano aluminium oxide suspension slowly is paved with poly (methyl methacrylate) micro-sphere, dry in vacuum drying oven, obtain the poly (methyl methacrylate) micro-sphere support.
Step 3.4: polymethyl methacrylate/support that step 3.3 is made carries out application of vacuum, prepared tricalcium phosphate slip in the implantation step 1, and mix homogeneously obtains slip/polymethyl methacrylate/scaffold complex.
Step 3.5: slip/polymethyl methacrylate/scaffold complex is put in sintering formation porous calcium phosphate support in the Muffle furnace.
8. preparation method according to claim 7, it is characterized in that, described sintering step is: placed successively room temperature to 200 ℃ even intensification and sintering 50 minutes, 200-400 ℃ evenly heats up and sintering 600 minutes, 400-1300 ℃ evenly heats up and sintering 400 minutes last 1250 ℃ of sintering 120 minutes.
9. the application of dimensional culture support claimed in claim 1 in the preparation cell culture system.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108686270A (en) * | 2018-06-04 | 2018-10-23 | 杭州电子科技大学 | A kind of high porosity tissue engineering bracket and preparation method thereof |
| CN112587731A (en) * | 2020-12-03 | 2021-04-02 | 广东省医疗器械研究所 | Composite stent and preparation method and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1337271A (en) * | 2001-09-21 | 2002-02-27 | 清华大学 | Prepn of osteological material containing nano phase calcium phosphate, collagen and alginate |
| US20030082808A1 (en) * | 1998-11-13 | 2003-05-01 | Limin Guan | Composite biodegradable polymer scaffold |
| CN1528471A (en) * | 2003-10-15 | 2004-09-15 | 浙江大学 | Method for preparing tissue engineered porous composite scaffold material |
| CN1644221A (en) * | 2005-01-26 | 2005-07-27 | 徐小良 | Composite material for porous material and gel use thereof |
| CN1736928A (en) * | 2005-08-26 | 2006-02-22 | 清华大学 | Preparation method of calcium orthophosphate bone cement degradable to pore in human body |
| CN101766843A (en) * | 2010-02-05 | 2010-07-07 | 清华大学 | Artificial bone with porous laminated structure and passages and preparation method thereof |
-
2011
- 2011-08-15 CN CN201110233215.4A patent/CN102935246B/en not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030082808A1 (en) * | 1998-11-13 | 2003-05-01 | Limin Guan | Composite biodegradable polymer scaffold |
| CN1337271A (en) * | 2001-09-21 | 2002-02-27 | 清华大学 | Prepn of osteological material containing nano phase calcium phosphate, collagen and alginate |
| CN1528471A (en) * | 2003-10-15 | 2004-09-15 | 浙江大学 | Method for preparing tissue engineered porous composite scaffold material |
| CN1644221A (en) * | 2005-01-26 | 2005-07-27 | 徐小良 | Composite material for porous material and gel use thereof |
| CN1736928A (en) * | 2005-08-26 | 2006-02-22 | 清华大学 | Preparation method of calcium orthophosphate bone cement degradable to pore in human body |
| CN101766843A (en) * | 2010-02-05 | 2010-07-07 | 清华大学 | Artificial bone with porous laminated structure and passages and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 王嫣: "海藻酸钠凝胶对骨髓间充质干细胞生物学效应的初步研究", 《重庆医科大学学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108686270A (en) * | 2018-06-04 | 2018-10-23 | 杭州电子科技大学 | A kind of high porosity tissue engineering bracket and preparation method thereof |
| CN112587731A (en) * | 2020-12-03 | 2021-04-02 | 广东省医疗器械研究所 | Composite stent and preparation method and application thereof |
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