CN103394125A - Tissue engineering double-layered tubular support and preparation method thereof - Google Patents
Tissue engineering double-layered tubular support and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a tissue engineering double-layered tubular support and a preparation method thereof. The support is composed of two polymer layers with different pore structures, the inner layer is a compact nanofiber structure, and the outer layer is of a porous nanofiber structure. The preparation method comprises the following steps of: dissolving a polymer material in a solvent, pouring in a mould, performing phase separation overnight, demoulding to obtain a polymer gel, performing solvent replacement, and freeze drying to obtain a nanofiber tubular support with a compact structure; sleeving the nanofiber tubular support on a mould stem to obtain a casting mould; and dissolving a polymer material in a solvent, casing the polymer solution in the casing mould by combing an electroporation technology, performing phase separation overnight, demoulding, performing solvent replacement, removing the solvent and/or porogen, and freeze drying to obtain the tissue engineering double-layered tubular support. The preparation method disclosed by the invention is simple in operation, free of complicated equipment, suitable for large-scale production and low in preparation cost; the whole mechanical property of the support is improved by introducing other polymers in the phase separation system.
Description
Technical field
The invention belongs to tissue engineering tubular scaffold and preparation field thereof, particularly a kind of organizational project double layered tubular support and preparation method thereof.
Background technology
The higher mammal body contains abundant tubular tissue, and as trachea, blood vessel, lymphatic vessel and intestinal etc., these tubular tissues have obvious characteristics, and its tube wall is comprised of dissimilar cellular layer.When damaged and serious pathological changes appear in these tissues, generally all implement to transplant and reproduce.In the pathological changes of these tubular tissues, cardiovascular disease is to threaten one of serious disease of human beings.The grafts such as autologous, the allosome that uses clinically exist many defects, and are as not enough as donor, easily cause immunological rejection etc., limited its application.Develop into external structure 26S Proteasome Structure and Function and the similar graft of natural tubular tissue of organizational project provide new approach, and the layering cellularity feature of bionical natural tubular tissue is the precondition of external structure functionalization tubular bracket.Support is important ingredient in organizational project, plays effects such as supporting Growth of Cells, guide tissue regeneration.And cell can promote the regeneration of cambium to the growth in support.Therefore, the 26S Proteasome Structure and Function that desirable tubular bracket should bionical natural tubular tissue, and should have the inside growth that suitable aperture is beneficial to cell.On microcosmic, the extracellular matrix (ECM) in body shows as nanometer fibrous network structure, thereby prepares the physical arrangement that nano fiber scaffold can be good at bionical ECM.Electrostatic spinning technique is to apply at present more nanofiber preparation method.Scholar (Biomaterials is arranged, 2010,31:4313-4321) by changing the electrostatic spinning parameter, adopt the double-deck intravascular stent that made of two step electrostatic spinning technique successes, internal layer is nanofibrous structures, aperture is less, is suitable for adhesion and the propagation of vascular endothelial cell (ECs) at tube chamber, is conducive to the endothelialization of tube chamber; Skin is the micrometer fibers structure, and aperture is larger, is suitable for vascular smooth muscle (SMCs) to the growth in support, can be used for the blood vessel in vitro construction support.But support layer structure prepared by the method does not have the nanofibrous structures of good bionical ECM.Thermic be separated (TIPS) be considered to a kind of nano fiber scaffold technology of preparing that has application prospect most, be mainly by the polymer solution quick freezing by higher temperature, by temperature change, drive and be separated to prepare the nano fiber scaffold material.The support that adopts TIPS to prepare, fibre diameter surpasses 90% at 50-500nm, porosity.In conjunction with hole forming technology, TIPS can also be for the preparation of the porous nano-fibre support.Have the scholar (Adv.Funct.Mater.2010,20,2833-2841) utilize the die assembly with different heat conductivitys, adopt TIPS to prepare the nano-fiber tubular scaffold of the inner aperture of tube wall Gradient distribution.Wherein a kind of support tube wall endothecium structure aperture is little, is suitable for adhesion and the propagation of ECs, is beneficial to the endothelialization of ECs at tube chamber; Tube wall layer structure aperture is larger, is suitable for SMCs and grows in support, can be used for external structure functionalization intravascular stent.But support composition prepared by the method is single, and mechanical property is difficult to meet the requirement of tubular tissue.
The improvement of mechanical property can reach by introducing other polymer, when the polymer of introducing is compatible with original copolymer, can make the compact nanometer fibrous framework that mechanical property is improved, and sustenticular cell is in adhesion and the propagation on surface.And when the polymer of introducing is incompatible with original copolymer (Chinese patent, application number: CN201210384648.4, publication number: CN102908208A), can make the porous nano-fibre support, the method both had been compounded with other polymer to improve the mechanical property of support, realize simultaneously again the macroporous structure of support, promoted cell to grow in support.With porogen pore method commonly used, compare, thisly from the pore-forming technology, saved porogen and removed this link, preparation is more simple, quick, and avoided the residual risk of poisonous porogen (as paraffin), more safe and reliable, in organizational project porous nano-fibre support Application and preparation, have application prospect preferably.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of organizational project double layered tubular support and preparation method thereof, and the method is simple to operate, do not need complex device, can be mass-produced, preparation cost is cheap; The present invention can be by introducing the whole mechanical property that other polymer improves support in phase-separated system.
A kind of organizational project double layered tubular support of the present invention, described support is comprised of the polymeric layer of two-layer different pore structures, and internal layer is the compact nanometer fibre structure, and aperture is less than 1-5 μ m; Skin is the porous nano-fibre structure, and aperture is 10-500 μ m.
The preparation method of a kind of organizational project double layered tubular support of the present invention comprises:
(1) under 40-80 ℃ of condition, polymeric material is dissolved in solvent, obtains uniform solution, then be cast in mould, at-20 ℃～-80 ℃, be separated and spend the night, the mould of decorporating, obtain polymer gel, then carry out solvent exchange, lyophilization, obtain the compact texture nano-fiber tubular scaffold;
(2) above-mentioned nano-fiber tubular scaffold is enclosed within on the mould stem stem, obtains casting die mould;
(3) under 40-80 ℃ of condition, polymeric material is dissolved in solvent, obtain polymer solution, then in conjunction with following hole forming technology: porogen pore method, namely first porogen is filled in step (2) gained casting die mould, then be cast into wherein polymer solution, or by polymer solution be cast in step (2) gained casting die mould after porogen mixes; Wherein to account for the polymer quality ratio be 10%-90% to porogen; Perhaps the gas foaming method, be cast in step (2) gained casting die mould after being about to polymer solution and gas foaming agent mixing, and under 50-100 ℃ of condition, allows gas foaming agent decompose; Wherein to account for the polymer quality ratio be 10%-50% to gas foaming agent; Perhaps, from the pore-forming technology, be about to have of one's own the pore system polymer solution and directly be cast in step (2) gained casting die mould;
Finally at-20 ℃～-80 ℃, be separated and spend the night, the mould of decorporating, then carry out solvent exchange, and except desolventizing and/or porogen, lyophilization, obtain organizational project double layered tubular support.
in described step (1) and (3), polymeric material is polylactic acid, poly-epsilon-caprolactone, polyglycolic acid, poly-β-hydroxybutyric acid, polyhydroxy-alkanoate, sebacic acid and propyl tri-alcohol ester, polycaprolactone-D-lactic acid copolymer, Poly(D,L-lactide-co-glycolide, polyurethane, polyvinylpyrrolidone, polyethylene, polypropylene, polystyrene, nylon, polyethylene terephthalate, collagen, gelatin, fibroin albumen, Fibrinogen, cellulose, one or more in chitosan.
In described step (1) and (3), solvent is water, methanol, ethanol, hexane, cyclohexane extraction, the tert-butyl alcohol, hexafluoroisopropanol, trifluoroethanol, 1,4-dioxane, oxolane, N, one or more in N-dimethylformamide, chloroform, acetone, dichloromethane.In described step (3), porogen is one or more in paraffin, sodium chloride, gelatine microsphere, sugared ball.
In the middle gas foaming method of described step (3), gas foaming agent is one or more in carbonate, bicarbonate, azo-compound, sulfonyl hydrazines compound, nitroso compound.
In described step (1) and (3), solvent exchange solvent used is one or more in water, ethanol, methanol, hexane, cyclohexane extraction, and temperature is 0 ℃ of left and right, and exchange solvent 2-3 days, change solvent every day 3-5 time.
Organizational project double layered tubular support prepared by the method is comprised of two-layer polymeric layer with different pore structures, and internal layer is the compact nanometer fibre structure, can promote adhesion, the propagation of cell; Skin is the porous nano-fibre structure, can promote adhesion, the propagation of cell and grow in support; Different layers can be cultivated dissimilar cell, bionical natural tubular tissue on 26S Proteasome Structure and Function.
Cell can relate to endotheliocyte, smooth muscle cell and/or the fibroblast of vascular applications, also can relate to other tubular tissues (as esophagus, trachea etc.) and regenerates or repair required cell etc.
The present invention takes full advantage of the advantage that the thermic phase detachment technique can prepare nano fiber scaffold, adopt medically acceptable polymeric material as substrate, reach and promote adhesion, propagation and the differentiation etc. of cell on support also to promote the transportation of nutrient substance in support and the discharge of metabolic waste simultaneously; Introduce other medically acceptable polymeric material, improve the mechanical property of support, and in conjunction with macropore support technology of preparing, realize the layered characteristic of macroporous structure on the tubular bracket tube wall, for managing on inside and outside wall, cultivate different cells, the 26S Proteasome Structure and Function of bionical natural tubular tissue, can be used for the repair and reconstruction of damaged tubular tissue clinically.
Beneficial effect
(1) the present invention is simple to operate, does not need complex device, can be mass-produced, and preparation cost is cheap;
(2) the present invention can be by introducing the whole mechanical property that other polymer improves support in phase-separated system;
(3) the organizational project double layered tubular support for preparing of the present invention is comprised of two-layer nanofibre polymer layer with different aperture, the inner compact layer structure is suitable for adhesion and the propagation of cell, outer loose structure is conducive to growing into of cell, cell layered characteristic that can bionical natural tubular tissue, be conducive to the tissue engineering tubular scaffold of external structure functionalization.
The accompanying drawing explanation
Fig. 1 is the be separated picture of PLLA/PLCL compact nanometer fiber tubular bracket of preparation of the first step, and wherein A is digital photograph, and B is the SEM picture of PLLA/PLCL tubular bracket cross section, and C is the SEM picture after B amplifies;
Fig. 2 is enclosed within the digital pictures of the new die that forms on the casting die mould stem stem by the be separated PLLA/PLCL tubular bracket of preparation of the first step, and it is left be tubular die, in for overlapping the stem stem that compact nanometer fiber tubular bracket is arranged, the right side is plug;
Fig. 3 is the be separated picture of double layered tubular support of preparation of second step, wherein A is digital photograph, B is the SEM picture of double layered tubular support cross section, and C is the SEM picture after outer PLLA/PCL macroporous structure amplifies, and D is the SEM picture after internal layer PLLA/PLCL compact texture is amplified.
The specific embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only are not used in and limit the scope of the invention be used to the present invention is described.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims limited range equally.
Embodiment 1
Endothecium structure is PLLA/PLCL compact nanometer fibre structure, and layer structure is the double layered tubular support that PLLA/PCL has pore system of one's own.
(1) PLLA and PLCL are dissolved in oxolane in 55 ℃ with mass ratio 40:60, stir and be mixed with 10%(w/v) uniform solution;
(2) the PLLA/PLCL mixed solution is cast in the politef mould, is placed in rapidly under-80 ℃ of conditions it is separated, spend the night;
(3) from low temperature, taking out mould, and the mould of decorporating, gelatinous polymer is immersed in the deionization mixture of ice and water and exchanges solvent 2 days, change the deionization frozen water every day 3 times;
(4) from deionized water, taking out tubular bracket, lyophilization, the PLLA/PLCL nano-fiber tubular scaffold that namely obtains having compact texture;
(5) by the compact nanometer fiber tubulose support casing that makes in (4) on the stem stem of politef mould, be assembled into the new casting die mould of a cover;
(6) PLLA and PCL are dissolved in oxolane in 50 ℃ with mass ratio 70:30, stir and be mixed with 10%(w/v) uniform solution;
(7) the PLLA/PCL mixed solution is cast in step (5) gained casting die mould, is placed in rapidly under-80 ℃ of conditions it is separated, spend the night;
(8) from low temperature, taking out mould, and the mould of decorporating, support is immersed in the deionization mixture of ice and water and exchanges solvent 2 days, change the deionization frozen water every day 3 times;
(9) from deionized water, taking out support, lyophilization, namely obtain the double layered tubular support.
Embodiment 2
Endothecium structure is PLLA/PLCL compact nanometer fibre structure, and layer structure is the double layered tubular support of the PLLA porous nano-fibre structure of sugared ball pore.
(1) PLLA and PLCL are dissolved in oxolane in 55 ℃ with mass ratio 70:30, stir and be mixed with 10%(w/v) uniform solution;
(2) the PLLA/PLCL mixed solution is cast in the politef mould, is placed in rapidly under-20 ℃ of conditions it is separated, spend the night;
(3) from low temperature, taking out mould, and the mould of decorporating, gelatinous polymer is immersed in the deionization frozen water and exchanges solvent 2 days, change the deionization frozen water every day 3 times;
(4) from deionized water, taking out tubular bracket, lyophilization, the PLLA/PLCL nano-fiber tubular scaffold that namely obtains having compact texture;
(5) by the compact nanometer fiber tubulose support casing that makes in (4) on the stem stem of politef mould, be assembled into the new casting die mould of a cover;
(6) the sugared ball of diameter 200-500 μ m is filled in step (5) gained casting die mould, standing 30min under 37 ℃ of conditions, allow part adhesion between sugared ball;
(7), PLLA is dissolved in to oxolane under 50 ℃ of conditions in, stirs and to be mixed with 10%(w/v) uniform solution;
(8) PLLA solution is cast in the casting die mould that is filled with sugared ball of step (6) gained, is placed in rapidly under-80 ℃ of conditions it is separated, spend the night;
(9) from low temperature, taking out mould, and the mould of decorporating, support is immersed in cyclohexane extraction and exchanges solvent 2 days, change cyclohexane extraction every day 3 times;
(10) from cyclohexane extraction, taking out support, after lyophilization, support is immersed in deionized water and soaks 2 days except the desaccharide ball, change deionized water every day 3 times.
(11) from deionized water, taking out support, lyophilization, namely obtain the double layered tubular support.
Embodiment 3
Endothecium structure is PLLA/PLCL compact nanometer fibre structure, and layer structure is the double layered tubular support of the standby PLLA porous nano-fibre structure of gas foaming legal system.
(1) PLLA and PLCL are dissolved in oxolane in 55 ℃ with mass ratio 50:50, stir and be mixed with 10%(w/v) uniform solution;
(2) the PLLA/PLCL mixed solution is cast in the politef mould, is placed in rapidly under-80 ℃ of conditions it is separated, spend the night;
(3) from low temperature, taking out mould, and the mould of decorporating, gelatinous polymer is immersed in the deionization frozen water and exchanges solvent 2 days, change the deionization frozen water every day 3 times;
(4) from deionized water, taking out tubular bracket, lyophilization, the PLLA/PLCL nano-fiber tubular scaffold that namely obtains having compact texture;
(5) by the compact nanometer fiber tubulose support casing that makes in (4) on the stem stem of politef mould, be assembled into the new casting die mould of a cover;
(6), PLLA is dissolved in to oxolane under 50 ℃ of conditions in, stirs and to be mixed with 10%(w/v) uniform solution;
Under (7) 50 ℃ of conditions, the sodium bicarbonate (NaHCO of PLLA quality 20% will be accounted for
3) after powder adds in PLLA solution and fully to stir 2min, be cast into rapidly in step (5) gained casting die mould, after standing 5min under 60 ℃ of conditions, be placed in rapidly under-80 ℃ of conditions it is separated, spend the night;
(8) from low temperature, taking out mould, and the mould of decorporating, support is immersed in the deionization mixture of ice and water and exchanges solvent 2 days, change the deionization frozen water every day 3 times;
(9) from deionized water, taking out support, lyophilization, namely obtain the double layered tubular support.
Claims (7)
1. organizational project double layered tubular support, it is characterized in that: described support is comprised of the polymeric layer of two-layer different pore structures, and internal layer is the compact nanometer fibre structure, and aperture is less than 1-5 μ m; Skin is the porous nano-fibre structure, and aperture is 10-500 μ m.
2. the preparation method of an organizational project double layered tubular support comprises:
(1) under 40-80 ℃ of condition, polymeric material is dissolved in solvent, obtains uniform solution, then be cast in mould, at-20 ℃～-80 ℃, be separated and spend the night, the mould of decorporating, obtain polymer gel, then carry out solvent exchange, lyophilization, obtain the compact texture nano-fiber tubular scaffold;
(2) above-mentioned nano-fiber tubular scaffold is enclosed within on the mould stem stem, obtains casting die mould;
(3) under 40-80 ℃ of condition, polymeric material is dissolved in solvent, obtain polymer solution, then in conjunction with following hole forming technology: porogen pore method, namely first porogen is filled in step (2) gained casting die mould, then be cast into wherein polymer solution, or by polymer solution be cast in step (2) gained casting die mould after porogen mixes; Wherein to account for the polymer quality ratio be 10%-90% to porogen; Perhaps the gas foaming method, be cast in step (2) gained casting die mould after being about to polymer solution and gas foaming agent mixing, and under 50-100 ℃ of condition, allows gas foaming agent decompose; Wherein to account for the polymer quality ratio be 10%-50% to gas foaming agent; Perhaps, from the pore-forming technology, be about to have of one's own the pore system polymer solution and directly be cast in step (2) gained casting die mould;
Finally at-20 ℃～-80 ℃, be separated and spend the night, the mould of decorporating, then carry out solvent exchange, and except desolventizing and/or porogen, lyophilization, obtain organizational project double layered tubular support.
3. the preparation method of a kind of organizational project double layered tubular support according to claim 2, it is characterized in that: in described step (1) and (3), polymeric material is polylactic acid, poly-epsilon-caprolactone, polyglycolic acid, poly-β-hydroxybutyric acid, polyhydroxy-alkanoate, sebacic acid and propyl tri-alcohol ester, polycaprolactone-D-lactic acid copolymer, Poly(D,L-lactide-co-glycolide, polyurethane, polyvinylpyrrolidone, polyethylene, polypropylene, polystyrene, nylon, polyethylene terephthalate, collagen, gelatin, fibroin albumen, Fibrinogen, cellulose, one or more in chitosan.
4. the preparation method of a kind of organizational project double layered tubular support according to claim 2, it is characterized in that: in described step (1) and (3), solvent is water, methanol, ethanol, hexane, cyclohexane extraction, the tert-butyl alcohol, hexafluoroisopropanol, trifluoroethanol, 1,4-dioxane, oxolane, N, one or more in N-dimethylformamide, chloroform, acetone, dichloromethane.
5. the preparation method of a kind of organizational project double layered tubular support according to claim 2 is characterized in that: in described step (3), porogen is one or more in paraffin, sodium chloride, gelatine microsphere, sugared ball.
6. the preparation method of a kind of organizational project double layered tubular support according to claim 2 is characterized in that: in described step (3) in the gas foaming method gas foaming agent be one or more in carbonate, bicarbonate, azo-compound, sulfonyl hydrazines compound, nitroso compound.
7. the preparation method of a kind of organizational project double layered tubular support according to claim 2, it is characterized in that: in described step (1) and (3), solvent exchange solvent used is one or more in water, ethanol, methanol, hexane, cyclohexane extraction, temperature is 0 ℃ of left and right, exchange solvent 2-3 days, change solvent every day 3-5 time.
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| CN109568661A (en) * | 2018-12-27 | 2019-04-05 | 郑州大学 | A kind of high tenacity scaffold for vascular tissue engineering and preparation method thereof |
| CN110215540A (en) * | 2019-04-09 | 2019-09-10 | 盐城工业职业技术学院 | A kind of fibroin with three-dimensional order and unordered dual network structure/polymer matrix tubular bracket and its preparation and application |
| CN110215540B (en) * | 2019-04-09 | 2021-07-27 | 盐城工业职业技术学院 | Silk fibroin/polymer based tubular stent with three-dimensional ordered and disordered double-network structure and preparation and use methods thereof |
| CN112807491A (en) * | 2020-12-31 | 2021-05-18 | 东华大学 | Elastic tissue engineering scaffold with communicated macroporous structure and preparation method thereof |
| WO2023138593A1 (en) * | 2022-01-21 | 2023-07-27 | 北京大学口腔医学院 | Antibacterial stent having micro-nano double-layer structure, and preparation method therefor and use thereof |
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