CN103820943B - Macropore three-dimensional order orientation silk fibroin nano-fiber support and preparation method thereof - Google Patents
Macropore three-dimensional order orientation silk fibroin nano-fiber support and preparation method thereof Download PDFInfo
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Abstract
The invention provides a kind of macropore three-dimensional order orientation silk fibroin nano-fiber support and preparation method thereof, the present invention mainly uses the method for electrostatic spinning, and the method for being collected by ethanol bath cylinder is realized.The feature of described ethanol bath drum process be the submergence of an electric conductivity cylinder part in ethanol, because ethanol has low surface tension, prepared silk fibroin nano-fiber can be made to infiltrate disperse wherein thus cause the gap enlargement between silk and silk, simultaneously cylinder can rotary motion, makes the silk collected can towards a direction ordered arrangement.Utilize the feature of the fibroin albumen Three Dimensional Fiber Scaffolds obtained by the present invention be in fibrous framework fibre diameter between 10nm is to 10 μm, fiber is the arrangement of orderly orientation, between fiber, average angle is at 0-30 °, fibrous framework has 3-D solid structure, thickness is 0.05-5mm, and between fiber, aperture is 0-30 μm.
Description
Technical field
The invention belongs to tissue engineering bracket material field, more specifically, relate to a kind of macropore three-dimensional order orientation silk fibroin nano-fiber support and preparation method thereof.
Background technology
Because disease wound causes the damage in various degree of tissue and organ, wherein some tissue damage is difficult to even cannot recover completely by body self-repairing capability.Organizational project is exactly how research promotes that injury repair even reaches the object of the regeneration of tissue and organ.Wherein, support is one of key factor in organizational project as the carrier of cell, growth factor and medicine.Optimal timbering material is extracellular matrix (ECM) composition and structure imitating tissue, thus analog cell external environment promotes the growth of cell.
Natural extracellular matrix composition is used for the making of tissue engineering bracket by a lot of people, comprises collagen, hyaluronic acid etc., although have good biologically active, machinery and processing characteristics are not as synthetic material, and cost is high, originate not easily.Though no cytotoxicity, degradable synthetic material have good mechanical and processing characteristics, its biologically active is poor; For taking into account both some advantages, Many researchers is used in combination by the two.Also some scholar develops other biological macromolecular material, these materials often have originates widely, low cost of manufacture, and there is good biologically active have preferably machinery and processing characteristics simultaneously, in the present invention, regenerated silk fibroin used is wherein a kind of, is widely used in various organizational project.
Different tissues has different extra-cellular matrix structure, and the aperture of timbering material, porosity, fibre diameter and distribution and fiber orientation should correspondingly change, thus affect the behavior of cell.Chen etc. are cultured cell in orderly or unordered two dimension or three-dimensional totally four kinds of supports, obtains having in cellular morphology, propagation, migration and differentiation etc. on the support of different structure form clearly distinguishing.(Chen,X.,X.Fu,etal."Regulationoftheosteogenesisofpre-osteoblastsbyspatialarrangementofelectrospunnanofibersintwo-andthree-dimensionalenvironments."Nanomedicine:Nanotechnology,BiologyandMedicine)。
In the ECM of mescenchymal tissue, collagen is topmost protein ingredient, accounts for 90% of protein ingredient.These collagens mainly exist with the form of nanofiber, and it is arranged in portion of tissue is orderly, as nerve, ligament, muscle, bone etc., at its hetero-organization then without particular orientation.Collagen fabric is not only different tissues and provides unique mechanical performance, has influence on the behavior of cell greatly yet.The support imitating collagen fabric making is a kind of well selection.
Researchers obtain fibrous framework with diverse ways, and find that cell can well grow on these fibers especially nano fiber scaffold.At present, the method obtaining nanofiber mainly contain be separated, self assembly and electrostatic spinning, in these methods, use the method for electrostatic spinning, 10nm can be obtained to micron-sized filament, compare with additive method, it is extensive that it has easy and simple to handle, that efficiency is high, available raw material, fiber and support performance adjustable, the advantage (K.Jayaraman such as sequence structure can be manufactured with, etal., Recentadvancesinpolymernanofibers, JournalofNanoscienceandNanotechnology4 (2004) 52 – 65.).
Use the nano fiber scaffold that conventional electrostatic spinning obtains, usually too fine and close, hinder the diffusion of cell, and along with the deposition of silk, change electric field, make support be difficult to do thick.In order to improve aperture, current researcher realizes mainly through following methods: percolation, photoetch method, micro-nano fiber composite method and ultrasonic method etc.But these methods destroy fiber original structure a bit, some makes material internal structure uneven.Other researcher improves aperture by the method changing receiving system, and the prerequisite of making every effort to not destroying spun structure is issued to object.Receiving system is made into the hollow foam hemisphere that one is plugged in stainless pin by Blakeney etc., obtains a kind of concentrated, low-density, not extruded electrospun scaffolds, likeness in form cotton mass.Cell experiment shows, and the fibrous framework that this method obtains and conventional method compare, and aperture and thickness all increase, and facilitate growth and the diffusion of cell.But this method can only obtain unordered fiber, orderly orientation is organized, more suitably support (Blakeney can not be provided, B.A., A.Tambralli, etal. (2011). " Cellinfiltrationandgrowthinalowdensity, uncompressedthree-dimensionalelectrospunnanofibrousscaff old. " Biomaterials32 (6): 1583-1590.).
Order fiber is obtained, mainly through parallel pole and cylinder two kinds of methods with method of electrostatic spinning.Wherein the fibrous material fiber-wall-element model that obtains of parallel pole is good, but size is little, limits its application.And the fibrous material size that cylinder is collected is large, but fiber-wall-element model is good not, and improves orientation and need to improve wire drawing strength, and the not good enough fiber of mechanical performance is easily pulled off.The same with traditional spinning in addition, it can only make fine and close, thin tunica fibrosa.The method that one layer of cells one deck support adds up such as Chen obtains a kind of three-dimensional, and cell is grown into orderly support wherein, but this rack making is complicated, the time that action need is a large amount of, and the operable time of cell is limited, improves thickness further more difficult.In addition, this support is the cumulative of very thin single-layer bracket, material continuity and homogeneity poor, external force must be used and could maintain form, limit in its body and use (Chen, X., X.Fu, etal. " Regulationoftheosteogenesisofpre-osteoblastsbyspatialarr angementofelectrospunnanofibersintwo-andthree-dimensiona lenvironments. " Nanomedicine:Nanotechnology, BiologyandMedicine).
In sum, there is the technological improvement requirement to preparing macropore three-dimensional order orientation nano fiber scaffold in this area.
Summary of the invention
For above defect or the technical need of prior art, the object of this invention is to provide a kind of preparation method of macropore three-dimensional order orientation silk fibroin nano-fiber support, possess feature easy and simple to handle, even structure, 3 D stereo, loose porous orderly orientation silk fibroin nano-fiber support can be obtained.
Its concrete technical scheme is as follows:
A preparation method for macropore three-dimensional order orientation silk fibroin nano-fiber support, is characterized in that: step is as follows:
(1) take silk cocoon as raw material, obtain regenerated silk fibroin through coming unstuck, dissolving, dialyse, after drying;
(2) with the mixed solution of carrene and trifluoroacetic acid for solvent, preparation mass fraction is the regenerated silk fibroin solution of 6-20%;
(3) upper step gained regenerated silk fibroin solution is carried out electrostatic spinning, then collect with cylinder collecting method in ethanolic solution, obtain the silk fibroin nano-fiber crude product of orderly orientation;
Described cylinder collecting method intermediate roll cylindrical shell horizontal positioned, the 1/3-1/2 of cylinder volume is immersed in ethanolic solution;
Described ethanol holds with circular flat bottom glass dish.
(4) by upper step gained silk fibroin nano-fiber crude product, be immersed in the ammoniacal liquor of 7% concentration and soak 20-30 minute, then rinsing 3-6 time in deionized water, finally carry out freeze drying and obtain macropore three-dimensional order orientation silk fibroin nano-fiber support.
In described step 3, the voltage of electrostatic spinning is 16-30kv, feeding speed is 0.0005-0.003mm/s; Collecting distance is 7-15cm; The diameter of cylinder is 2-10cm, is highly 5-15cm; Drum rotating frequency is 6-30Hz; The diameter of glass dish is 10-20cm, and height is 2-5cm.
Described cylinder is hollow cylinder, and its material is copper or iron or aluminium, or the macromolecule hollow cylinder of masking foil parcel.
In described step 2, the mixed solution mass ratio of carrene and trifluoroacetic acid is carrene: trifluoroacetic acid equals 3:7.
The present invention mainly relies on existing electrostatic spinning technique, this technology mainly volatilizable dissolution with solvents macromolecular material forms electrostatic spinning solution used, taylor cone is formed by the tip acting on injection apparatus of high voltage electric field to solution, under a suitable voltage, molecules in solution is finally broken through surface tension of liquid and is formed jet, rotate change such as whip dynamic grade aloft, along with solvent evaporates, finally remaining macromolecular fibre product is on gathering-device.The macromolecular fibre network formed can apply to multiple fields such as physics, chemistry, medical science.
The raw material regenerated silk fibroin that the present invention selects has originates widely, low cost of manufacture, good biologically active and good machinery and processing characteristics.Often be used to the research of tissue engineering bracket.
The present invention realizes mainly through a kind of ethanol bath cylinder collecting method.The feature of described ethanol bath drum process be the submergence of an electric conductivity cylinder part in ethanol, because ethanol has low surface tension, can make prepared silk fibroin nano-fiber infiltration spread wherein thus cause the gap enlargement between silk and silk, simultaneously cylinder can rotary motion under the drive of motor, makes the silk collected can towards a direction ordered arrangement.
The fibroin fiber part spraying to gathering-device is deposited on cylinder, is then taken in ethanol rapidly; A part is immersed in the ethanolic solution near cylinder, the upper cylinder surface of cylinder volume be scrolled.These have been understood because of adhesive tape ethanol by the fibroin fiber drenched and have become heavy, compare with under dry condition, cylinder need larger strength go tractive it, the tractive that this strength is larger is easier than same condition dry condition bottom roll collection is formed with sequence structure, it is a branch of that some fibre is adsorbed formation time close to each other, abdicate more wide arc gap between bundle and bundle, thus define the structure of macropore.
For conventional electrostatic spinning process, the fibroplastic barrier that the behavior Chang Yin that fiber is broken through surface tension and sprayed emitter has deposited has slackened electric field and has stopped, and finally causes the tunica fibrosa of collection very thin.And soak after fiber in ethanol rolled by cylinder, be gathered in narrower scope, and overall electric field is not affected greatly, fiber can be sprayed continuously, and the accumulation of fiber also by means of the intermolecular force of ethanol and can reach larger thickness.
Traditional cylinder is collected, in order to obtain more orderly fiber, usually need to improve drum speed to improve traction force, reach the object of drawing more straight, but for a lot of large biological molecule, be cross-linked before becoming more stable firmly structure, the usual non-constant of its mechanical performance, and the fiber of common electrostatic spinning ejection is usually formed by uncrosslinked molecule, when speed is brought up to certain time, these fibers are easy to be pulled off.Mainly there to be the form of random coil and α-helixstructure to exist time regenerated silk fibroin selected by us is dissolved in carrene/trifluoroacetic acid solution, this is a kind of unstable structure, electrostatic spinning is the process of the effect of a physical mechanics, so remain unstable structure when being ejected formation fibroin fiber, the fibroin fiber internal structure before that is entering into ethanol is insecure.Then, the alcoholic solution comprising ethanol can be cross-linked unstable fibroin albumen, make the beta sheet structure that random coil and α-helixstructure become stable, thus fibrous inside molecule is formed stable firmly to combine, improve the mechanical performance of fiber simultaneously.In the ethanol bath of collecting, fibroin albumen and occurred conformation change, and the comparatively tough fiber of formation within the specific limits, even if improve rotating speed to improve cylinder to its tractive strength, is also not easy to be pulled off.
Exception, because fibroin fiber forms rock-steady structure in collection process, does not need extra cross-linking step, it also avoid the extra contraction being cross-linked hole and the support entirety caused.
In sum, described ethanol bath cylinder collecting method can obtain possessing large aperture, 3-D solid structure and orderly nano fiber scaffold.That fibre diameter is between 10nm is to 10 μm by the feature of the Three Dimensional Fiber Scaffolds obtained by the present invention, fiber is the arrangement of orderly orientation, between fiber, average angle is at 0-30 °, and there is 3-D solid structure, thickness is 0.05-5mm, possess loose macroporous structure, between fiber, aperture is 0-30 μm.Aperture and backing thickness all solution concentration used and viscosities with electrostatic spinning between average angle, fiber between described fibre diameter, fiber, the speed of cylinder, the factor such as distance of shower nozzle and gathering-device is relevant, and these factors adjustable are to reach needs.
Pass through the present invention, can be able to a kind of easy and simple to handle, obtain even structure, 3 D stereo, loose porous orderly orientation silk fibroin nano-fiber support method, can be used in the organizational projects such as bone, nerve, tendon, muscle, fibrocartilage, blood vessel, the reparative regeneration of transmitting tissue.
Accompanying drawing explanation
Fig. 1 electrostatic spinning and collection process schematic diagram;
Fig. 2 nano fiber scaffold pictorial diagram;
Fig. 3 nano fiber scaffold scanning electron microscope (SEM) photograph;
In all of the figs, identical Reference numeral is used for representing identical element or structure, wherein:
1-electrospinning device 2-cylinder 3-ethanol.
Detailed description of the invention
In the present invention, electrospinning device and technology are prior art.
Below in conjunction with specific embodiment to technical scheme further instruction of the present invention.
Embodiment one:
1, silk cocoon is cut into 1cm
2fritter, boil 1.5 hours with the sodium carbonate liquor 500mL that mass fraction is 0.5%, clean by washed with de-ionized water, then boil 0.5h in 500mL deionized water, clean post-drying with deionized water.In the silk after process, add about 60mL calcium chloride/water/ethanol three-phase solution (mol ratio of three kinds of materials is 1/8/2), silk was dissolved completely in 1 hour 70 DEG C of stirring in water bath, then the solution of gained is dialysed 48 hours.Solution after dialysis filters after centrifugal 30min under the condition of 8000r/min, filters the solution that obtains and to carry out after liquid nitrogen frozen dry 48 hours, finally obtain regenerated silk fibroin.
2, regenerated silk fibroin being dissolved in mass ratio is in the carrene of 3:7 and the mixed solution of trifluoroacetic acid, and preparation regenerated silk fibroin mass fraction is the electrostatic spinning solution used of 6%.
3, spinning and collection:
Wherein the concrete operation method of ethanol bath cylinder collecting method is: by diameter 10cm, and the glass dish of high 2cm fills with ethanol, and bottom it, spread one deck tinfoil; The cylinder of cylinder used to be diameter be 2cm, high 5cm, its surface parcel one deck masking foil, utilizes motor to rotarily drive cylinder and rotates.Being positioned over by cylinder fills with in the culture dish of ethanol, and the submergence volume of cylinder is 1/2.
Electrostatic spinning use voltage is 16kv, feeding speed is 0.003mm/s, collection distance is 7cm, and cylinder rotates with the frequency of 30Hz in ethanol bath.Carry out the electrostatic spinning of different time under these conditions, the crude product of different-thickness can be collected on cylinder.
4, after electrostatic spinning completes, take off crude product, the ammonia spirit putting into 7% soaks 30min, then uses rinsed with deionized water 3-6 time, finally carries out freeze drying, obtains finished product.
5, the ordered three-dimensional material ultraviolet light, 75% ethanol or the oxirane disinfection that will obtain.Culture medium inoculates bone marrow stroma stem cell, IPS cell, osteocyte, nerve cell, Tenocyte cell, periodontal ligament stem cell, myocyte etc. after soaking, and carries out inducing, cultivating several weeks.By the long defect place such as stenter to implant bone, nerve, tendon, parodontium, muscle having cell.
Embodiment two
1, silk cocoon is cut into 1cm
2fritter, boil 1.5 hours with the sodium carbonate liquor 500mL that mass fraction is 0.5%, clean by washed with de-ionized water, then boil 0.5h in 500mL deionized water, clean post-drying with deionized water.In the silk after process, add about 60mL calcium chloride/water/ethanol three-phase solution (mol ratio of three kinds of materials is 1/8/2), silk was dissolved completely in 1 hour 70 DEG C of stirring in water bath, then the solution of gained is dialysed 48 hours.Solution after dialysis filters after centrifugal 30min under the condition of 8000r/min, filters the solution that obtains and to carry out after liquid nitrogen frozen dry 48 hours, finally obtain regenerated silk fibroin.
2, regenerated silk fibroin being dissolved in mass ratio is in the carrene of 3:7 and the mixed solution of trifluoroacetic acid, and preparation regenerated silk fibroin mass fraction is the electrostatic spinning solution used of 20%.
3, spinning and collection:
Wherein the concrete operation method of ethanol bath cylinder collecting method is: by diameter 20cm, and the glass dish of high 5cm fills with ethanol, and bottom it, spread one deck tinfoil; The cylinder of cylinder used to be diameter be 10cm, high 15cm, its surface parcel one deck masking foil, utilizes motor to rotarily drive cylinder and moves.Being positioned over by cylinder fills with in the culture dish of ethanol, and the submergence volume of cylinder is 1/3.
Electrostatic spinning use voltage is 30kv, feeding speed is 0.0005mm/s, collection distance is 15cm, and cylinder rotates with the speed of 6Hz in ethanol bath.Carry out the electrostatic spinning of different time under these conditions, the sample of different-thickness can be collected on cylinder.
4, after electrostatic spinning completes, take off sample, the ammonia spirit putting into 7% soaks 30min, then uses rinsed with deionized water 3-6 time, finally carries out freeze drying.
5, the ordered three-dimensional material ultraviolet light, 75% ethanol or the oxirane disinfection that will obtain.Culture medium inoculates bone marrow stroma stem cell, IPS cell, osteocyte, nerve cell, Tenocyte cell, periodontal ligament stem cell, myocyte etc. after soaking, and carries out inducing, cultivating several weeks.By the long defect place such as stenter to implant bone, nerve, tendon, parodontium, muscle having cell.
That fibre diameter is between 10nm is to 10 μm by the feature of the Three Dimensional Fiber Scaffolds obtained by the present invention, fiber is orientation ordered arrangement, between fiber, average angle is at 0-30 °, and there is 3-D solid structure, thickness is 0.05-5mm, possess loose macroporous structure, between fiber, aperture is 0-30 μm.Aperture and backing thickness all solution concentration used and viscosities with electrostatic spinning between average angle, fiber between described fibre diameter, fiber, the speed of cylinder, the factor such as distance of shower nozzle and gathering-device is relevant, and these factors adjustable are to reach needs.
Claims (5)
1. a preparation method for macropore three-dimensional order orientation silk fibroin nano-fiber support, is characterized in that: step is as follows:
(1) take silk cocoon as raw material, obtain regenerated silk fibroin through coming unstuck, dissolving, dialyse, after drying;
(2) with the mixed solution of carrene and trifluoroacetic acid for solvent, preparation mass fraction is the regenerated silk fibroin solution of 6-20%;
(3) upper step gained regenerated silk fibroin solution is carried out electrostatic spinning, collect with cylinder collecting method in ethanolic solution, obtain the silk fibroin nano-fiber crude product of orderly orientation;
Described cylinder collecting method intermediate roll cylindrical shell horizontal positioned, the 1/3-1/2 of cylinder volume is immersed in ethanolic solution;
(4) by upper step gained silk fibroin nano-fiber, be immersed in the ammoniacal liquor of 7% concentration and soak 20-30 minute, then rinsing 3-6 time in deionized water, finally carry out freeze drying and obtain macropore three-dimensional order orientation silk fibroin nano-fiber support.
2. the preparation method of macropore three-dimensional order orientation silk fibroin nano-fiber support as claimed in claim 1, is characterized in that: in described step 3, the voltage of electrostatic spinning is 16-30kv, feeding speed is 0.0005-0.003mm/s; Collecting distance is 7-15cm; The diameter of cylinder is 2-10cm, and drum rotating frequency is 6-30Hz.
3. the preparation method of macropore three-dimensional order orientation silk fibroin nano-fiber support as claimed in claim 1 or 2, is characterized in that: described cylinder is hollow cylinder, and its material is copper or iron or aluminium, or the macromolecule hollow cylinder of masking foil parcel.
4. the preparation method of macropore three-dimensional order orientation silk fibroin nano-fiber support as claimed in claim 1, is characterized in that: in described step 2, the mixed solution mass ratio of carrene and trifluoroacetic acid is carrene: trifluoroacetic acid equals 3:7.
5. the nano fiber scaffold utilizing preparation method described in claim 1 to prepare, it is characterized in that: in fibrous framework, fibre diameter is between 10nm is to 10 μm, fiber is the arrangement of orderly orientation, between fiber, average angle is at 0-30 °, fibrous framework has 3-D solid structure, thickness is 0.05-5mm, and between fiber, aperture is 0-30 μm.
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