CN100355405C - Production of porous stand for tissue engineering - Google Patents
Production of porous stand for tissue engineering Download PDFInfo
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- CN100355405C CN100355405C CNB200410025456XA CN200410025456A CN100355405C CN 100355405 C CN100355405 C CN 100355405C CN B200410025456X A CNB200410025456X A CN B200410025456XA CN 200410025456 A CN200410025456 A CN 200410025456A CN 100355405 C CN100355405 C CN 100355405C
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Abstract
The present invention relates to a method for preparing a porous support frame in tissue engineering, which comprises the following steps: polylactic acid copolymer particles are put in mould for heat compacting and shaping; then, the shaped polylactic acid copolymer is put in high-pressure CO2 at room temperature for mechanical saturation; the pressure of the high-pressure CO2 is from 3.0 to 30.0MPa, and simultaneously, polar solvents are sprayed from an inlet of liquid solution at the upper end of an expansion chamber; under enough the mechanical saturation time, the gas pressure is rapidly reduced to the atmospheric pressure level within 1 to 100 seconds; the solubility of the CO2 in the polylactic acid copolymer is rapidly reduced; a gas chamber for generating a great amount of CO2 is in a porous foam structure; the mechanical saturation time is from 1 to 120 hours. The technological process of a three-dimensional support frame prepared by the method of the present invention is simple. After a sample is taken out and simply treated, the sample can be used. The formed pores are mutually communicated, and most pores conform to the requirement of cell culture.
Description
Technical field
The present invention relates to macromolecular material and biomedical sector, be specifically related to a kind of preparation method of used in tissue engineering three-dimensional stent material.
Background technology
Tissue engineering bracket be meant can with organize active somatic cell combine and can implantable bioartificial intravital material, it is the basic boom of engineered tissue.Polyglycolic acid (PGA) and polylactic acid polylactic acid-based materials such as (PLA) are typical synthesized degradable polymer.Its general structure is [OCH (R) CO-], and the R in the formula is poly-through glycolic during for H, and R is CH
3The time be polylactic acid, because lactic acid and hydroxyacetic acid all are the tricarboxylic acid cycle intermediate metabolitess, and absorption and metabolic mechanism are clear and definite and have a reliable biological safety, thereby polylactic acid and polyglycolic acid be used for by drugs approved by FDA as first degradable absorbing material clinical, be study so far the most extensive, use maximum degradable biomaterials.As tissue engineering bracket material, PLA, PGA and copolymer biomaterial thereof not only have excellent biological compatibility, biodegradability and degraded adjustability, and can induce the rise of some gene to transcribe.At present, polylactic acid-based timbering material has been widely used in the timbering material of tissues such as bone, cartilage, blood vessel, nerve, skin, and shows its good prospects for application.
Should have big surface area and high porosity as the used in tissue engineering three-dimensional stent material, so that the exchange of the transmission of the adhesion of cell and nutrient substance and metabolite; And hole dimension should be enough big so that the abundant infiltration of cell.Above-mentioned factor often depends on the support preparation method.External at present existing people has carried out extensive studies to the preparation of used in tissue engineering porous support, specifically has following several:
1, fibrage method (Mikos A G, sarakinos G, Lyman M D, et al.Preparation ofpoly (glycolic acid) boned fiber structures for cell attachment and transplantation[J] .J Biomed Mater Res, 1993,27:183 ~ 189): the fibrage method is meant that with diameter be the PLLA of 10-16um or the fleece that PGA fibrage becomes three-dimensional communication.Zhi Bei biomaterial has higher porosity (70-80%) and big area/volume ratio in this way, can and breed the space that provides necessary for cell adhesion.But intensity, rigidity are lower. easily cause the repopulating cell damage.
2, solvent casting/particle leaching method (Mikos A G, Thorsen A J, Czerwonka L A, et al.Preparationand characterization of poly (L-lactide acid) foams[J] .Polymer, 1994,35 (5): 1068 ~ 1077): the advantage of solvent casting/particle leaching method is can prepare aperture and porosity according to the three-dimensional communication micropore support of design in advance.The porosity of its timbering material is by the volume fraction control of porogen, and pore size is determined by the porogen particle size.When the volume fraction percentage ratio of porogen meets or exceeds 70%, be interconnected between the micropore.(appoint the outstanding person, Wu Zhigang. polylactic acid-polyether block copolymer three-dimensional porous rack material and preparation method thereof [P]. China, the patent No.: CN 14446841A, 2003.10.8.)
3, gas foaming (Harris L D, Kim B S, Mooney D J.Open pore biodegrabable matrices formedwith gas foaming[J] .J Biomed Mater Res, 1998,42:396 ~ 402): the gas foaming technology adopts gas as porogen, in the drilling process not with an organic solvent.MMney adopts the gases at high pressure foaming technique, forms porous foam structure, the about 100um in aperture, and porosity reaches 93%.Utilize this method to prepare three-dimensional stent material and avoided the use of organic solvent and the influence of high-temperature process, help the growth factor-loaded repopulating cell that acts on.But this method is to the equipment requirements height, and microcellular structure is wayward and often form sealed porosity.Nam has reported method (the Nam Y that adopts a kind of gas foaming and particle leaching to combine, Yoon J, Park T.A novel fabrication method of macroporous biodegradable polymerscaffolds using gas foaming salt as a porogen additive[J] .J Biomed Mater Res (ApplBiomat), 2000, (53): 1-7).Be about to ammonium bicarbonate and join in PLLA or the PLCA solution, cast molding is treated the demoulding after the solvent evaporates, and puts into hot water that gaseous volatilization and particle leaching are carried out simultaneously.This method has not only kept the advantage of particle leaching method, and has solved the compact surfaces layer, the problem of the connective and salt residues of micropore, but that the problem of the The Long-term Effect of residual ammonium hydrogencarbonate pair cell has is to be solved.
4, (Lo H is separated/emulsifying, Ponticiello M S, Leong K W.Fabrication of controlled releasebiodegradable foams by phase separation[J] .Tissue Engineering, 1995,1:15-28): be separated/emulsifying comprises emulsifying/lyophilization and two kinds of methods of liquid-liquid phase separation.First method is after polymer is dissolved in dichloromethane, adds distilled water and forms emulsion, and it is poured in the mould, places liquid nitrogen acute cold.Then with support in-55 ℃ of following lyophilizations, dispersive moisture and polymer solvent are fallen in vacuum evaporation.Adopt this technology can make porosity, but the diameter in the hole that forms is less, is unfavorable for that the religion of cell echos propagation up to 95% support.
External report utilize supercritical CO
2During device preparation tissue engineered porous scaffold, adopt the method for direct PB, do not introduce organic solvent, the brace aperture rate that obtains can not be controlled preferably, and connective bad between the Kong Yukong, as depicted in figs. 1 and 2, can not well satisfy the requirement of cell culture.
Summary of the invention
The objective of the invention is to propose a kind of tissue engineered porous scaffold preparation method.
For achieving the above object, the present invention is achieved in that a kind of tissue engineered porous scaffold preparation method, and step is as follows: be placed in the mould copolymer of poly lactic acid granule hot-forming; At room temperature the copolymer of poly lactic acid of molding is placed on high pressure CO then
2Machinery is saturated in the gas, described high pressure CO
2The pressure of gas is 3.0-30.0Mpa, and the liquid solution import from the expanding chamber upper end simultaneously sprays into polar solvent; After enough mechanical saturation times, within 1～100 second, air pressure is reduced to the atmospheric pressure level rapidly, the CO in the described copolymer of poly lactic acid
2Gas Solubility descends rapidly, produces a large amount of CO
2Air cavity has just formed porous foam structure, and described mechanical saturation time is 1～120 hour.
Preferable, described copolymer of poly lactic acid is PLA-PEG, PLA-PEO, PLA-PolyTHF or PLGA.
Preferable, described polar solvent is chloroform, dichloromethane, acetone, ethyl acetate, oxolane.
Preferable, described high pressure CO
2The pressure of gas is 5～15MPa.
Preferable, the time that described machinery is saturated is 24～72 hours.
Preferable, in 10～50 seconds, air pressure is reduced to the atmospheric pressure level rapidly.
The invention has the beneficial effects as follows: it is simple relatively that employing this method prepares the three-dimensional stent material technological process, sample can use through simple processing after taking out, the hole that obtains when utilizing method provided by the invention to prepare three-dimensional stent material is interconnected, and the formed hole overwhelming majority meets the requirement of cell culture.
Description of drawings
Fig. 1 is the sem photograph (amplification is 100 times) that does not feed the porous support surface that micro-organic solvent prepares.
Fig. 2 is the sem photograph (amplification is 100 times) that does not feed the porous support cross section that micro-organic solvent prepares.
Fig. 3 is the sem photograph (amplification is 300 times) that feeds the porous support surface that micro-organic solvent prepares.
Fig. 4 is the sem photograph (amplification is 100 times) that feeds the porous support cross section that micro-organic solvent prepares.
Fig. 5 is the sem photograph (amplification is 1500 times) that feeds the porous support surface that micro-organic solvent prepares.
The specific embodiment
Below by further specifying the present invention in conjunction with the embodiments.
Embodiment 1:
Take by weighing the poly-lactic acid material of 0.8g, be ground into graininess, put into politef mould heating extrusion forming disk shape then.Above-mentioned polymer is put into supercritical CO
2In the device, be pressurized to 5.5Mpa under the room temperature, feed the acetone of 0.2ml/s simultaneously, machinery saturated 72 hours, then in 15 seconds rapid depressurization to atmospheric pressure.Take out sample and carry out sem test and porosity measurement, as shown in Figure 3, scanning electron microscopic observation is open to the hole of porous support, is interconnected between the Kong Yukong, and porosity is 89.2%.
Embodiment 2:
Take by weighing the PLA-PEG copolymer material of 0.8g, be ground into graininess, put into politef mould heating extrusion forming disk shape then.Above-mentioned polymer is put into supercritical CO
2In the device, be pressurized to 9.0Mpa under the room temperature, feed the ethyl acetate of 0.2ml/s simultaneously, machinery saturated 72 hours, then in 10 seconds rapid depressurization to atmospheric pressure.Take out sample and carry out sem test and porosity measurement, as shown in Figure 4 and Figure 5, scanning electron microscopic observation is open to the hole of porous support, is interconnected between the Kong Yukong, and porosity is 91.7%.
Embodiment 3:
Take by weighing the PLA-PEO copolymer material of 0.8g, be ground into graininess, put into politef mould heating extrusion forming disk shape then.Above-mentioned polymer is put into supercritical CO
2In the device, be pressurized to 9.0Mpa under the room temperature, feed the oxolane of 0.2ml/s simultaneously, machinery saturated 60 hours, then in 10 seconds rapid depressurization to atmospheric pressure.Take out sample and carry out sem test and porosity measurement, scanning electron microscopic observation is open to the hole of porous support, is interconnected between the Kong Yukong, and porosity is 86.2%.
Embodiment 4:
Take by weighing the PLGA material of 0.8g, be ground into graininess, put into politef mould heating extrusion forming disk shape then.Above-mentioned polymer is put into supercritical CO
2In the device, be pressurized to 15Mpa under the room temperature, feed the dichloromethane of 0.2ml/s simultaneously, machinery saturated 72 hours, then in 10 seconds rapid depressurization to atmospheric pressure.Take out sample and carry out sem test and porosity measurement, scanning electron microscopic observation is open to the hole of porous support, is interconnected between the Kong Yukong, and porosity is 83.9%.
Claims (5)
1. a tissue engineered porous scaffold preparation method is characterized in that may further comprise the steps: be placed in the mould copolymer of poly lactic acid granule hot-forming; At room temperature the copolymer of poly lactic acid of molding is placed on high pressure CO then
2Machinery is saturated in the gas, described high pressure CO
2The pressure of gas is 3.0-30.0Mpa, and the liquid solution import from the expanding chamber upper end simultaneously sprays into polar solvent chloroform, dichloromethane, acetone, ethyl acetate or oxolane; After enough mechanical saturation times, within 1～100 second, air pressure is reduced to the atmospheric pressure level rapidly, the CO in the described copolymer of poly lactic acid
2Gas Solubility descends rapidly, produces a large amount of CO
2Air cavity has just formed porous foam structure, and described mechanical saturation time is 1～120 hour.
2. according to the preparation method of tissue engineered porous scaffold described in the claim 1, it is characterized in that: described copolymer of poly lactic acid is PLA-PEG, PLA-PEO, PLA-PolyTHF or PLGA.
3. according to the preparation method of tissue engineered porous scaffold described in the claim 1, it is characterized in that: described high pressure CO
2The pressure of gas is 5～15MPa.
4. according to the preparation method of tissue engineered porous scaffold described in the claim 1, it is characterized in that: the time that described machinery is saturated is 24～72 hours.
5. according to the preparation method of tissue engineered porous scaffold described in the claim 1, it is characterized in that: in 10～50 seconds, air pressure is reduced to the atmospheric pressure level rapidly.
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| CNB200410025456XA CN100355405C (en) | 2004-06-24 | 2004-06-24 | Production of porous stand for tissue engineering |
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| CNB200410025456XA CN100355405C (en) | 2004-06-24 | 2004-06-24 | Production of porous stand for tissue engineering |
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| CN100355405C true CN100355405C (en) | 2007-12-19 |
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| CN100465209C (en) * | 2006-08-17 | 2009-03-04 | 同济大学 | Preparation method of degradable polymer material for stent in shape memory pipe cavity |
| CN105709323A (en) * | 2008-07-09 | 2016-06-29 | 科拉弗洛有限公司 | Methods, Apparatuses And Systems For Caval Stenting For Venous Drainage |
| CN106757772B (en) * | 2015-01-27 | 2018-10-12 | 浙江大学 | The preparation method of the three-dimensional porous unordered holder of fiber heat bonding solidification is spun in a kind of polylactic acid melting |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5584877A (en) * | 1993-06-25 | 1996-12-17 | Sumitomo Electric Industries, Ltd. | Antibacterial vascular prosthesis and surgical suture |
| CN1424115A (en) * | 2002-12-06 | 2003-06-18 | 暨南大学 | Tissue engineering stent material and preparation thereof |
| US6586246B1 (en) * | 1999-03-18 | 2003-07-01 | Innotech Medical, Inc. | Preparing porous biodegradable polymeric scaffolds for tissue engineering using effervescent salts |
| CN1446841A (en) * | 2003-01-16 | 2003-10-08 | 同济大学 | Porous frame material of poly-lactic acid-polyether block copolymer and its preparation method |
| CN1126515C (en) * | 2001-02-19 | 2003-11-05 | 南通医学院 | Medical artificial nerve graft and preparation process thereof |
| CN1476907A (en) * | 2003-06-30 | 2004-02-25 | 暨南大学 | Bio-active 3-D porous tissue engineering support material and its preparation method |
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5584877A (en) * | 1993-06-25 | 1996-12-17 | Sumitomo Electric Industries, Ltd. | Antibacterial vascular prosthesis and surgical suture |
| US6586246B1 (en) * | 1999-03-18 | 2003-07-01 | Innotech Medical, Inc. | Preparing porous biodegradable polymeric scaffolds for tissue engineering using effervescent salts |
| CN1126515C (en) * | 2001-02-19 | 2003-11-05 | 南通医学院 | Medical artificial nerve graft and preparation process thereof |
| CN1424115A (en) * | 2002-12-06 | 2003-06-18 | 暨南大学 | Tissue engineering stent material and preparation thereof |
| CN1446841A (en) * | 2003-01-16 | 2003-10-08 | 同济大学 | Porous frame material of poly-lactic acid-polyether block copolymer and its preparation method |
| CN1476907A (en) * | 2003-06-30 | 2004-02-25 | 暨南大学 | Bio-active 3-D porous tissue engineering support material and its preparation method |
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