CN102008747B - Foam carbon biological support, and preparation method and application thereof - Google Patents
Foam carbon biological support, and preparation method and application thereof Download PDFInfo
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- CN102008747B CN102008747B CN 201010586384 CN201010586384A CN102008747B CN 102008747 B CN102008747 B CN 102008747B CN 201010586384 CN201010586384 CN 201010586384 CN 201010586384 A CN201010586384 A CN 201010586384A CN 102008747 B CN102008747 B CN 102008747B
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Abstract
The invention discloses a foam carbon biological support, and a preparation method and an application thereof. The foam carbon biological support is prepared by carbonizing a precursor formed by the foaming and solidification of phenolic resin under ordinary pressure. The method comprises the following steps: when preparation, firstly preparing materials; and then arranging into water bath for foaming and solidifying; drying and solidifying to acquire the precursor; carbonizing the precursor to obtain the foam carbon biological support. When the foam carbon biological support is used for exo-cell culture, the foam carbon biological support planted with cells is arranged in a static or dynamic circulating culture system for culturing according to the demands in different cell types. The foam carbon biological support has good aperture ratio and biocompatibility and high mechanical strength, and can be widely used for exo-cell culture.
Description
Technical field
The present invention relates to a kind of Tissue Engineering Biomaterials and its preparation method and application, relate in particular to a kind of biological support and its preparation method and application.
Background technology
Extensive animal cell culture is organizational project, engineered basis, and a large amount of biomedicine experiments, external pharmacology test also be unable to do without cell in vitro and cultivate.Present general two dimensional surface cells in vitro training method in the world, obtaining cell is the plane cell monolayer, the cell life state is fully different from the 3-D solid structure in real human body, mutual locus between cell and cell, signal conduction and multiplication regulatory and internal milieu have very large difference, and vital movement will differ from normal cell state and may cause the regression of cell tissue.Therefore, for realizing having breakthrough highdensity cell culture mode future, make that cell can be in contact with one another with adjacent cells, conducted signal on three dimensions, simulate more truly the cell growth state in human body, the cell culture mode must adopt dimensional culture.But in default of mass transfer channel such as blood capillaries, the cell in vitro dimensional culture will inevitably run into the mass transfer difficult problem between multi-layer cellular---and oxygen, nutrient, somatomedin and metabolite between multi-layer cellular can't be carried, and Growth of Cells namely can't carry out mass exchange death with the external world because of it after certain thickness.Realize the extensive dimensional culture of cell for overcoming a mass transfer difficult problem, cell needs one " template "---three-dimensional stent material, should " template " can instruct cell adhesion propagation to form three-dimensional space and distribute, and utilize on timbering material the three-dimensional communication pore network to carry out the input of oxygen, nutrient, somatomedin and the output of metabolite.
The three-dimensional stent material that is widely studied at present, as poly-own lactide (PGA), Poly-L-lactic acid (PLLA), poly-L-lactic acid (PDLA), bioactive ceramics etc., possess high-specific surface area, three-dimensional communication pore network and biocompatibility, multiplex in body Repair of tissue defect among a small circle, but because degradability, absorbability and the comprcssive strength of these timbering materials are not high, be difficult to really as cell in vitro high density, the long-time timbering material of cultivating.And in extensive animal cell culture process, three-dimensional stent material not only needs suitable three dimensional structure and good biocompatibility, can make cell normally stick propagation, also must have good chemistry, mechanical stability, can keep in a long time normally to guarantee that cell large scale is cultivated.Therefore, the timbering material of cultivating as cell in vitro in the urgent need to researching and developing the good non-degraded porous material of a kind of biocompatibility.
Summary of the invention
The technical problem to be solved in the present invention is to overcome the deficiencies in the prior art, provide that a kind of percent opening is high, density is low, to organism avirulence, good biocompatibility and can keep the Carbon foam biological support of stable mechanical performance, the also corresponding preparation method and application that a kind of this Carbon foam biological support is provided in the cell in vitro long-term cultivation.
For solving the problems of the technologies described above, the technical scheme that the present invention proposes is a kind of Carbon foam biological support, and described Carbon foam biological support is to be formed through carbonization by the presoma that phenolic resins forms, and described presoma is that phenolic resins forms after foamed solidification under normal pressure.
In above-mentioned Carbon foam biological support, it preferably has three-dimensional intercommunicating pore structure.In this Carbon foam biological support with three-dimensional communication pore structure, the pore diameter range of described intercommunicating pore is preferably 50 microns～500 microns, and percent opening is preferably 70%～90% (percent opening refers to the ratio of the shared volume of bubble and the cumulative volume of Carbon foam biological support in the Carbon foam biological support).
As a total technical conceive, the present invention also provides a kind of preparation method of Carbon foam biological support, comprises the following steps:
(1) batching: take phenolic resins, and add respectively wherein foaming agent, 0.1%～0.5% emulsifying agent and 0.5%～0.9% the filler of phenolic resins quality 20%～30%, mix homogeneously gets resin compound;
(2) foamed solidification: add the firming agent of described phenolic resins quality 15%～20% in the described resin compound, be placed in immediately water-bath foaming (normal pressure foaming) after mix homogeneously, then carry out precuring in water-bath, obtain the precuring body;
(3) dry solidification: described precuring body is carried out drying with further curing, obtain the Carbon foam presoma;
(4) carbonization is burnt till: under isolated air condition, described Carbon foam presoma is carried out carbonization burn till, obtain the Carbon foam biological support.
In the preparation method of above-mentioned Carbon foam biological support, described foaming agent is preferably pentane; Described emulsifying agent is preferably cetyl benzene sulphur sodium sulfate; Described filler is preferably graphite powder; Described firming agent is preferably the dilute sulfuric acid of mass concentration 15%～25%.
In the preparation method of above-mentioned Carbon foam biological support, the temperature of described water-bath preferably is controlled at 60 ℃～70 ℃, the general stirring 5～10 minutes when foaming in described water-bath, and the time of described precuring is more than 30 minutes.
In the preparation method of above-mentioned Carbon foam biological support, the temperature during described drying preferably is controlled at 100 ℃～150 ℃, and drying time is preferably more than 24 hours.
In the preparation method of above-mentioned Carbon foam biological support, the temperature when described carbonization is burnt till preferably is controlled at 800 ℃～1000 ℃, and temperature retention time is not preferably (heating-up time calculates in described temperature retention time) more than 3 hours.
As a total technical conceive, the present invention also provides the application of a kind of above-mentioned Carbon foam biological support on cell in vitro is cultivated, and the Carbon foam biological support that will be implanted with cell for the demand of different cell categories during application is placed in static state or dynamic Cyclic culture system is cultivated.
Compared with prior art, the invention has the advantages that: 1) utilize the Carbon foam of non-degraded three-dimensional communication pore network structure as the cell culture biologic bracket material, not only can realize the cells in vitro High Density Cultivation, reduce the cell culture cost, functionalization in the time of more keeping the cells in vitro cultivation realizes the cultivation of high activity 3 D stereo; 2) add filler graphite in the foaming process of Carbon foam presoma of the present invention, be used for to improve foaming efficient and stability, greatly improved the percent opening that foaming prepares the Carbon foam biological support.
Carbon foam biological support of the present invention not only has mechanical strength steady in a long-term, and percent opening is high, density is low, and selected material with carbon element is due to the chemical inertness of itself, and is nontoxic to organism, good biocompatibility; And the three-D space structure of Carbon foam biological support of the present invention is conducive to cell and connects each other in solid space, thereby more intends very presenting cell upgrowth situation in vivo, is the cell in vitro cultivation support that a class has application prospect.
Description of drawings
Fig. 1 is the low power pattern microphotograph of the Carbon foam biologic bracket material that makes of the embodiment of the present invention.
Fig. 2 is the high power pattern microphotograph of the Carbon foam biologic bracket material that makes of the embodiment of the present invention.
Fig. 3 is the cell attachment microphotograph of the Carbon foam biologic bracket material that makes of the embodiment of the present invention, and after wherein a figure～e figure is respectively cell seeding, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours five time point cell attachment are in the microphotograph on biologic bracket material surface.
The specific embodiment
Embodiment
A kind of this Carbon foam biological support is to be formed through carbonization by the presoma that phenolic resins forms as Fig. 1～Carbon foam biological support of the present invention shown in Figure 2, forms after foamed solidification under normal pressure and presoma is phenolic resins.The Carbon foam biological support of the present embodiment has three-dimensional intercommunicating pore structure, and the aperture of its intercommunicating pore is 220 microns left and right, and percent opening is 80 ± 2%.
The preparation method of the Carbon foam biological support of above-mentioned the present embodiment mainly comprises the following steps:
(1) batching: the phenolic resins (what adopt in the present embodiment is 2130 trade mark phenolic resins) of taking 100 parts, the foaming agent pentane that first adds 30 parts, then add respectively the emulsifying agent cetyl benzene sulphur sodium sulfate of 0.5 part and the filler graphite powder (graphite powder is crossed 200 mesh sieves) of 0.9 part, mix to get resin compound;
(2) foamed solidification: add 20 parts of firming agent dilute sulfuric acids (mass concentration is 20%) in the resin compound that obtains to step (1), being placed in immediately 60 ℃ of water-baths after mix homogeneously foams, stirred 5～10 minutes during foaming, then precuring 30 minutes in water-bath obtains the precuring body;
(3) dry solidification: the precuring body that step (2) is obtained is placed in the air dry oven of 150 ℃ and further solidified 24 hours, obtains the Carbon foam presoma;
(4) carbonization is burnt till: under the state of isolated air, the Carbon foam presoma that obtains after dry solidification is put in ceramic crucible, toner seal completely cuts off air, ceramic crucible being placed in resistance furnace carries out carbonization and burns till again, what carbonization was burnt till is machine-processed as shown in table 1 below, burns till to obtain the Carbon foam biological support after completing.
Mechanism is burnt till in the carbonization of table 1: embodiment
The Carbon foam biological support that the present embodiment makes is a kind of three-dimensional communication pore structure that is interconnected and forms, the pore diameter range of intercommunicating pore is between 50 microns～500 microns, percent opening reaches more than 70% (as the biological culture support, the optimum opening porosity of the present embodiment can reach 80 ± 2%), this Carbon foam biological support has the adjustable characteristics in aperture.The Carbon foam biological support that the present embodiment is made soaks a period of time in simulated body fluid after, its surface and hole wall thereof namely have the inorganic salt coating formation of calcic P elements, and cell can be effectively on this biologic bracket material adherent and propagation, it has good biological activity this sufficient proof Carbon foam biologic bracket material of the present invention, has broad prospects in the application of cell in vitro cultivation support.
The Carbon foam biologic bracket material cell in vitro of the present embodiment adheres to microphotograph as shown in a figure～e figure in Fig. 3, and in 5 hours after repopulating cell, cellular morphology is normal, adheres to good.For the specific demand of different cell categories, the long-term cultivation of cell in vitro can be selected dynamic cultivation or static culture.Table 2 is Alamar Blue detected value contrasts of using Carbon foam biologic bracket material cultured cell under dynamic circulation cultivating system and static culture system of the present embodiment.The progressively increase of Alamar Blue detected value can be found out from following table 2, and with the prolongation of incubation time, propagation all appears in cell on the Carbon foam biologic bracket material under two kinds of different culture environment.
Table 2: the Alamar Blue detected value of Carbon foam biologic bracket material under different cultivating systems
Above Alamar Blue detection method is a kind of comparatively desirable method of cell proliferation situation on the multiporous biological timbering material that detects, its ultimate principle is: the Alamar Blue of oxidized form can be reduced by the cyclophorase in living cells, after reduction, dyestuff generation color and fluorescence change, its depth is directly proportional to living cells quantity, available spectrophotometer or fluorescence detector detect, and the Alamar Blue numerical value of measuring at last is relevant with biocompatibility.The method is not only simple to operate, and controllability is strong, and this dyestuff is to cell nonhazardous effect, and the specified operational procedure of measuring Alamar Blue numerical value in the present embodiment is:
1, cell culture: use the RPMI1640 culture medium, add 10% hyclone, suitable antibiotic;
When 2, the cell coverage rate reaches 80%, with trypsinization and diluting cells density at 50000/ml;
3, above-mentioned celliferous culture fluid is added drop-wise on the Carbon foam biologic bracket material of the present embodiment, additional culture fluid is immersed under liquid level material monolithic;
4, the Carbon foam biologic bracket material with repopulating cell is transferred to 37 ℃, 5%CO
2Incubator in cultivate a period of time;
5, detect the cytoactive time point at need, press fresh medium: Alamar Blue=20: 1 preparation detects uses culture fluid, 5 minutes mixings of sonic oscillation;
6, Carbon foam biologic bracket material original fluid is sucked, add to contain Alamar Blue and detect and to use culture fluid, again put back to 37 ℃, 5%CO
2Cultivate reaction 6 hours in incubator;
7, to the rear reduction situation that detects Alamar Blue of predetermined time point, the reaction wavelength is 570nm, and reference wavelength 600nm calculates activity value according to absorbance reading;
8, will contain Alamar Blue detection and suck with culture fluid, add fresh medium, put back to 37 ℃, 5%CO
2Cultivate a period of time in incubator, next time point is active to be detected to do.
Claims (6)
1. the preparation method of a Carbon foam biological support, is characterized in that, comprises the following steps:
(1) batching: take phenolic resins, and add respectively wherein foaming agent, 0.1%～0.5% emulsifying agent and 0.5%～0.9% the filler of phenolic resins quality 20%～30%, mix homogeneously gets resin compound; Described foaming agent is pentane; Described filler is graphite powder;
(2) foamed solidification: add the firming agent of described phenolic resins quality 15%～20% in the described resin compound, be placed in immediately water-bath after mix homogeneously and foam, then carry out precuring in water-bath, obtain the precuring body; Described firming agent is the dilute sulfuric acid of mass concentration 15%～25%;
(3) dry solidification: described precuring body is carried out drying with further curing, obtain the Carbon foam presoma; Temperature during described drying is controlled at 100 ℃～150 ℃, and drying time is more than 24 hours;
(4) carbonization is burnt till: under isolated air condition, described Carbon foam presoma is carried out carbonization burn till, obtain the Carbon foam biological support.
2. the preparation method of Carbon foam biological support according to claim 1, it is characterized in that: the temperature of described water-bath is controlled at 60 ℃～70 ℃, stirs 5～10 minutes during foaming in described water-bath, and the described precuring time is more than 30 minutes.
3. the preparation method of Carbon foam biological support according to claim 1, it is characterized in that: the temperature when described carbonization is burnt till is controlled at 800 ℃～1000 ℃, and temperature retention time is more than 3 hours.
4. Carbon foam biological support that makes as any one preparation method in claim 1～3, it is characterized in that: described Carbon foam biological support is to be formed through carbonization by the presoma that phenolic resins forms, and described presoma is that phenolic resins forms after foamed solidification under normal pressure; Described Carbon foam biological support has three-dimensional intercommunicating pore structure.
5. Carbon foam biological support according to claim 4, it is characterized in that: in described Carbon foam biological support, the pore diameter range of intercommunicating pore is 50 microns～500 microns, and percent opening is 70%～90%.
6. a Carbon foam biological support as described in claim 4 or 5 application on cell in vitro is cultivated, the Carbon foam biological support that will be implanted with cell for the demand of different cell categories during application is placed in static state or dynamic Cyclic culture system is cultivated.
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| CN101066756A (en) * | 2007-06-11 | 2007-11-07 | 湖南大学 | Process of preparing carbon naotube foam |
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| CN101066756A (en) * | 2007-06-11 | 2007-11-07 | 湖南大学 | Process of preparing carbon naotube foam |
Non-Patent Citations (4)
| Title |
|---|
| 曹敏等.炭素泡沫材料的制备和应用.《材料科学与工程学报》.2004,第22卷(第4期),第613-616页. |
| 正戊烷发泡法制备多孔碳泡沫材料;甘礼华等;《同济大学学报(自然科学版)》;20081130;第36 卷(第11 期);第1552-1555页 * |
| 炭素泡沫材料的制备和应用;曹敏等;《材料科学与工程学报》;20040831;第22卷(第4期);第613-616页 * |
| 甘礼华等.正戊烷发泡法制备多孔碳泡沫材料.《同济大学学报(自然科学版)》.2008,第36 卷(第11 期),第1552-1555页. |
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