CN102908208A - Preparation method of porous nano-fiber tubular scaffold - Google Patents
Preparation method of porous nano-fiber tubular scaffold Download PDFInfo
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Abstract
The invention relates to a preparation method of a porous nano-fiber tubular scaffold, which comprises the steps that (1) PLLA (Poly L Lactic Acid) and other polymers are dissolved in a solvent to get a polymer solution; (2) the polymer solution is injected into a tubular mold and rapidly placed at a low temperature for phase separation, then the polymer solution is taken out, a tubular mold housing is removed, polymer gel after the phase separation and a core mold are immersed in ice water together, then the core mold is taken out, the polymer gel is immersed in deionized ice water to exchange the solvent, and the tubular scaffold is obtained; and (3) finally, the tubular scaffold obtained is frozen and dried for 48-120 hours, and then the porous nano-fiber tubular scaffold is obtained. The preparation method is simple to operate, and requires no additional hole-foaming agent, is suitable for volume production, and is lower in preparation cost; the prepared tubular scaffold has a nano-fiber structure similar to an extracellular matrix of a human tissue, and a porous structure with the diameter and porosity capable of being adjusted, and facilitates growth of cells and reconstruction of a cambium.
Description
Technical field
The invention belongs to the preparation field of tubular tissue engineering rack, particularly a kind of preparation method of porous nano-fibre tubular bracket.
Background technology
Contain a lot of tube chamber shape tissues in the human body, such as cardiovascular, peripheral blood vessel, nerve trachea, esophagus, intestinal, bile duct, urethra and bone etc., repair the damaged organization bracket that often will use tubulose of these tissues.In the pathological changes of these tubular tissues, cardiovascular disease is to threaten one of the most serious disease of human health, usually need implement transplant operation to the blood vessel of damaged and serious pathological changes.At present, clinically commonly used from the body blood vessel such as hidden greatly, IMA as graft.But limited from body blood vessel source, and autotransplantation easily causes secondary damage to human body.Use the support of heteroplastic transplantation easily to cause immunological rejection.At present the method for comparative maturity is that the synthetic blood vessel prosthesis of using artificial such as expanded polytetrafluoroethylsealing (ePTFE) and terylene (Dacron) are as tremulous pulse substitute reconstruction tremulous pulse.These artificial blood vessels are used for, and substitution effects large, medium-sized artery is satisfactory, but be used for small-bore (<when 6mm) blood vessel substitutes, because artificial blood vessel's tube wall lacks endotheliocyte, small-bore blood flow rate slowly causes transplanting early stage Acute thrombosis, transplanting later stage anastomotic hyperplasia causes luminal stenosis until inaccessible, and patency rate is difficult to reach requirement.Cell-biomaterial composites based on tissue engineering technique makes up implants after external structure, and with reinventing that the host blood vessel tissue merges and organizes, it is unobstructed to recover blood vessel seriality and blood, is considered to desirable replacement vessels.
Intravascular tissue engineering comprises three parts of somatomedin of three-dimensional rack, seed cell, necessity.Wherein, intravascular stent plays effects such as supporting Growth of Cells, guide tissue regeneration, control organizational structure and release active factors, is one of key factor that determines the organizational project success or failure.Desirable intravascular stent should possess following characteristics: the 26S Proteasome Structure and Function of (1) bionic extracellular matrix (ECM); (2) the good compatibility and controlled degradation rate; (3) has certain pore structure; (4) have the mechanical property that is complementary with natural blood vessel, be convenient to blood flow; (5) release of active growth factor is arranged.Each tool pluses and minuses of intravascular stent of using clinically at present, do not support adhesion and the growth of endotheliocyte such as synthetic non-degradable material, transplanting after-poppet can't be absorbed in vivo, degrade, the blood vessel of its structure is not engineering blood vessel truly, and limited owing to its source from the blood vessel of body heteroplastic transplantation, can't satisfy the demands.
Desirable tissue blood vessel support should be able to be reinvented along with the degraded of support in agglutination, thereby form one and have the neovascularity of same mechanical performance and biological property with host's aorta, except structure and function bionical, also to be conducive to the differentiation of cell, identify, play the remodeling process of damaged blood vessel.For structure and the function of bionical natural blood vessel, support, structure depends on preparation method.Therefore from microcosmic, extracellular matrix forms (wherein the diameter of collagen fiber is 50-500nm) by fibrous reticular structure, with the physical arrangement of ECM in the at utmost bionical human body of cytoskeleton of submicron or nanofiber preparation.The structural approach of preparation nanofiber has a lot, present topmost have electrostatic spinning and phase separation method.Method with electrostatic spinning can form long nanofiber.The method and apparatus that a kind of electrostatic spinning prepares tissue engineering bracket material is disclosed such as Chinese patent (application number 03137309.7), this method has material source extensively and is easy to the characteristics of molding, but usually be difficult to control its pore structure, utilize phase disengagement method having certain advantage aspect the preparation three-dimensional porous rack.Phase detachment technique is the important method of preparation polymer porous material, its process be under certain condition with polymer dissolution in suitable solvent, keep solution temperature to be higher than and be separated a little, macromolecular chain is fully unfolded in solvent; When drop in temperature to the thermodynamic instability district or when having non-solvent to exist, solution is separated, it is stingy and mutually rich to form polymer; Under cooling conditions, the rich phase region of polymer removes the mode of solvent with lyophilizing or displacement again, obtains porous polymer material.For semi-crystalline polymer such as PLLA, can obtain the nanofiber porous support by conditions such as the combination of control solvent, polymer concentration, thermal annealing, solvent exchange and cryogenic temperatures.
In the process of reconstruction of tubular tissue, support must provide larger aperture to utilize growing into of cell, and particle leaching is the most frequently used pore means.Such as (the Biomaterials of Ma seminar, 2010,31:7971-7977) prepare the nano fiber scaffold with macropore connection with sugared ball as porogen and be used for revascularization, but this full communicating macroporous structure has not only been sacrificed the mechanical strength of support, also has the risk of blood leakage.(the Polymer such as S.Ramakrishna, 2009,50:4128-4138) adopting dioxane and water is mixed solvent dissolving PLLA, be separated by thermic and prepared the large hole nano fibrous framework, but simple PLLA support fragility is larger, is difficult to provide mechanical strengths such as commonly using toughness essential in the tubular tissue repair process and elasticity.
Summary of the invention
Technical problem to be solved by this invention provides a kind of preparation method of porous nano-fibre tubular bracket, the method is simple to operate, need not add porogen, can be mass-produced, preparation cost is lower, obtain tubular bracket and had aperture and the adjustable loose structure of porosity, be conducive to growing into and the reconstruction of cambium of cell.
The preparation method of a kind of porous nano-fibre tubular bracket of the present invention comprises:
(1) Poly-L-lactic acid PLLA and other polymer are dissolved in the solvent in 50-70 ℃, obtain the polymer solution of homogeneous;
(2) above-mentioned polymer solution is injected in the tubular die, places rapidly-4 ℃ to-196 ℃ low temperature, it to be separated; Then take out, and the tubular die shell of decorporating, polymer gel after being separated is soaked in the deionization frozen water 10-30 minute with core mold, take out again core mold, and continuing polymer gel was soaked in the deionization frozen water exchange solvent 48-96 hour, change the deionization frozen water every day 2-5 time, obtain tubular bracket;
(3) at last with the tubular bracket lyophilization that obtains 48-120 hour, namely obtain having the nano-fiber tubular scaffold of loose structure.
Other polymer described in the step (1) are poly-epsilon-caprolactone (PCL), polyglycolic acid (PGA), poly-β-hydroxybutyric acid (PHB), polyhydroxy-alkanoate (PHA), sebacic acid and propyl tri-alcohol ester (PGS), polycaprolactone-Poly-L-lactic acid copolymer (PCL-b-PLLA), Poly(D,L-lactide-co-glycolide (PLGA), polyurethane (PU), polyvinylpyrrolidone (PVP), polyethylene (PE), polypropylene (PP), polystyrene (PS), nylon (Nylon), polyethylene terephthalate (PET), collagen, gelatin, fibroin, Fibrinogen, cellulose, a kind of in the chitosan etc.
The mass ratio of the Poly-L-lactic acid described in the step (1) and other polymer is 6-8:4-2.
Solvent described in the step (1) is water, methanol, ethanol, hexane, cyclohexane extraction, the tert-butyl alcohol, hexafluoroisopropanol, trifluoroethanol, 1,4-dioxane, oxolane, N, one or more in N-METHYLFORMAMIDE, chloroform, acetone, the dichloromethane etc.
The total concentration of polymer is 0.02-0.2g/mL in the polymer solution described in the step (1).
Tubular die described in the step (2) is made of the cylindrical shell of end sealing and shaft-like core with cover, and the diameter of cylindrical shell and shaft-like core is adjustable, thereby thickness and the inside and outside footpath of the tubular bracket of preparation are controlled.
The size range of the resulting porous nano-fibre tubular bracket of the present invention is: internal diameter 2-6cm, thickness 0.5-5mm.
Tubular bracket of the present invention is take PLLA as material of main part, and the support that obtains has loose structure and nanofibrous structures simultaneously.
The invention discloses a kind of porous nano-fibre support technology of preparing, with some elastomeric polymers or biocompatibility preferably polymer add among the PLLA, prepared porous PLLA based composite nano fiber tubular bracket, the adding of these compositions has not only increased the elasticity of support, also can form to regulate by changing material biocompatibility and the degradation property of support.Because the difference of two kinds of Gelation temperature and times, the polymer of part low content forms microsphere because being separated, can obtain the porous nano-fibre structure through suitable processing, this loose structure can be cell and grows into sufficient space is provided, but can not cause owing to the complete UNICOM in duct the seepage of blood, urine etc., its mechanical property and micro structure are controlled.Be more suitable for the structure of engineered luminal structure.
Tubular bracket of the present invention is take biodegradable Poly-L-lactic acid as material of main part, add with it inconsistent another kind of polymer, the control phase separation makes two kinds of incompatible materials form " island " shape structure, and the aperture of support and porosity can be regulated according to ratio and the phase separation of two kinds of polymer.
Tubular tissue engineering rack of the present invention can be used for the structure of the tubular tissues such as organizational project cardiovascular, peripheral blood vessel, nerve trachea, esophagus, intestinal, bile duct, urethra and bone.Adopt the porous nano-fibre tubular bracket of thermally induced phase separation preparation, the method has that equipment requirements is low, preparation process simply and not need add the characteristics such as porogen.The tubular bracket of preparation not only has the nanofibrous structures that is similar to human tissue cell's epimatrix, also has the adjustable loose structure of aperture and porosity, is conducive to growing into and the reconstruction of cambium of cell.In phase-separated system, add toughness and the elasticity that the incompatible polymer with better mechanical strength can improve support, be conducive to make up tubular tissue through engineering approaches Organ and tissue.
Beneficial effect
(1) the present invention simple to operate, need not add porogen, can be mass-produced, preparation cost is lower;
(2) the present invention adds toughness and the elasticity that the incompatible polymer with better mechanical strength can improve support in phase-separated system, is conducive to make up tubular tissue through engineering approaches Organ and tissue;
(2) tubular bracket of the present invention preparation not only has the nanofibrous structures that is similar to human tissue cell's epimatrix, also has the adjustable loose structure of aperture and porosity, is conducive to growing into and the reconstruction of cambium of cell.
Description of drawings
Fig. 1 is the mould photo of the porous nano-fibre tubular bracket of preparation;
Fig. 2 is the optical photograph figure of the tubular bracket of preparation;
Fig. 3 is the electromicroscopic photograph of tubular bracket, and (A) figure is the cross-sectional view of tubular bracket, and (B) figure is the local SEM figure that amplifies;
Fig. 4 is the infrared spectrogram of tubular bracket, and wherein a is the infrared spectrogram of pure PLLA, and b is that PLLA and PCL ratio are the infrared spectrogram of 70:30, and c is the infrared spectrogram of pure PCL;
Pore size and pore volume that the bi-material of Fig. 5 reality different mixing proportion is made support change.
The specific embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.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.
With mass ratio PLLA:PCL=70:30, the polymer total concentration is the nano-fiber tubular scaffold that preparation has loose structure under the 0.05g/mL condition.
(1) PLLA and PCL are dissolved in the oxolane formation uniform solution with the 70:30 ratio in 55 ℃;
(2) polymer solution is injected in the politef mould, places rapidly-80 ℃ to make it that phase separation occur, spend the night;
(3) from low temperature, take out, and the mold shell of decorporating, place the polymer of gel the deionization frozen water to soak 30 minutes with core mold, take out the mould of core, and polymer is soaked in the deionization frozen water exchange solvent 2 days, change the deionization frozen water every day three times;
(4) take out tubular bracket from deionized water, lyophilization namely obtains having the nano-fiber tubular scaffold of loose structure.
With the PLLA:PU=70:30 of mass ratio, total polymer concentration is preparation porous nano-fibre tubular bracket under the 0.1g/mL condition.
(1) PLLA and PU are dissolved in the oxolane formation uniform solution with the 70:30 ratio in 60 ℃;
(2) polymer solution is injected in the politef mould, places rapidly-100 ℃ to make it that phase separation occur, spend the night;
(3) from low temperature, take out, and the mold shell of decorporating, place the polymer of gel the deionization frozen water to soak 30 minutes with core mold, take out the mould of core, and polymer is soaked in the deionization frozen water exchange solvent 3 days, change the deionization frozen water every day three times;
(4) take out tubular bracket from deionized water, lyophilization namely obtains having the nano-fiber tubular scaffold of loose structure.
With the PLLA:PLGA=60:40 of mass ratio, total polymer concentration is preparation porous nano-fibre tubular bracket under the 0.15g/mL condition.
(1) PLLA and PLGA are dissolved in the oxolane in 70 ℃ with the ratio of 60:40, form uniform solution;
(2) polymer solution is injected in the politef mould, places rapidly-50 ℃ to make it that phase separation occur, spend the night;
(3) from low temperature, take out, and the mold shell of decorporating, place the polymer of gel the deionization frozen water to soak 30 minutes with core mold, take out the mould of core, and polymer is soaked in the deionization frozen water exchange solvent 3 days, change the deionization frozen water every day three times;
(4) take out tubular bracket from deionized water, lyophilization namely obtains having the nano-fiber tubular scaffold of loose structure.
Claims (6)
1. the preparation method of a porous nano-fibre tubular bracket comprises:
(1) Poly-L-lactic acid PLLA and polymer are dissolved in the solvent in 50-70 ℃, obtain the polymer solution of homogeneous;
(2) above-mentioned polymer solution is injected in the tubular die, places rapidly under-4 ℃ to-196 ℃ it is separated; Then take out, and the tubular die shell of decorporating, polymer gel after being separated is soaked in the deionization frozen water 10-30 minute with core mold, take out again core mold, and continuing polymer gel was soaked in the deionization frozen water exchange solvent 48-96 hour, change the deionization frozen water every day 2-5 time, obtain tubular bracket;
(3) at last with the tubular bracket lyophilization that obtains 48-120 hour, namely obtain having the nano-fiber tubular scaffold of loose structure.
2. the preparation method of a kind of porous nano-fibre tubular bracket according to claim 1, it is characterized in that: the polymer described in the step (1) is poly-epsilon-caprolactone, polyglycolic acid, poly-β-hydroxybutyric acid, polyhydroxy-alkanoate, sebacic acid and propyl tri-alcohol ester, polycaprolactone-Poly-L-lactic acid copolymer, Poly(D,L-lactide-co-glycolide, polyurethane, polyvinylpyrrolidone, polyethylene, polypropylene, polystyrene, nylon, polyethylene terephthalate, collagen, gelatin, fibroin, Fibrinogen, cellulose, a kind of in the chitosan.
3. the preparation method of a kind of porous nano-fibre tubular bracket according to claim 1, it is characterized in that: the mass ratio of the Poly-L-lactic acid described in the step (1) and polymer is 6-8:4-2.
4. the preparation method of a kind of porous nano-fibre tubular bracket according to claim 1, it is characterized in that: the solvent described in the step (1) is water, methanol, ethanol, hexane, cyclohexane extraction, the tert-butyl alcohol, hexafluoroisopropanol, trifluoroethanol, 1, in 4-dioxane, oxolane, DMF, chloroform, acetone, the dichloromethane one or more.
5. the preparation method of a kind of porous nano-fibre tubular bracket according to claim 1, it is characterized in that: the total concentration of polymer is 0.02-0.2g/mL in the polymer solution described in the step (1).
6. the preparation method of a kind of porous nano-fibre tubular bracket according to claim 1, it is characterized in that: the tubular die described in the step (2) is made of the cylindrical shell of end sealing and shaft-like core with cover, and the diameter of cylindrical shell and shaft-like core is adjustable.
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