CN103820943A - Macropore three-dimensional ordered-orientation silk fibroin nano-fiber support and preparation method thereof - Google Patents
Macropore three-dimensional ordered-orientation silk fibroin nano-fiber support and preparation method thereof Download PDFInfo
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Abstract
The invention provides a macropore three-dimensional ordered-orientation silk fibroin nano-fiber support and a preparation method thereof. The macropore three-dimensional ordered-orientation silk fibroin nano-fiber support is realized through an ethyl alcohol bath roller collecting method by mainly applying an electrostatic spinning method. The ethyl alcohol bath roller collecting method is characterized in that a part of a conducting roller is immersed in ethyl alcohol, and the ethyl alcohol has low surface tension, so that the prepared silk fibroin nano-fiber can be soaked and dispersed in the ethyl alcohol, and gaps between filaments are enlarged, and meanwhile, the roller can make rotational motion, so that the collected filaments can be configured in sequence toward one direction. The silk fibroin nano-fiber support prepared through the preparation method provided by the invention is characterized in that the diameter of each fiber in the fiber support is 10nm-10[mu]m, the fibers are arranged in an ordered-orientation manner, the average angle between every fibers is 0-30 degrees, the fiber support has a three-dimensional structure and is 0.05-5mm in thickness, and the bore diameter between every two fibers is 0-30[mu]m.
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 injury repair even to reach 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 that imitates tissue, thereby analog cell external environment promotes the growth of cell.
N cell epimatrix composition is the making for 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, source is difficult for.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 mixed the two to use.Also some scholar has developed other biological macromolecular material, these materials often have source widely, low cost of manufacture, and there is good biologically active and have good machinery and processing characteristics simultaneously, in the present invention, regenerated silk fibroin used is wherein one, is widely used in various organizational projects.
Different tissues has different extracellular matrix structures, and aperture, porosity, fibre diameter and the distribution of timbering material and fiber orientation are answered corresponding changing, thereby affects the behavior of cell.Chen etc. are at orderly or unordered two dimension or three-dimensional cultured cell on totally four kinds of supports, and obtaining on the support of different structure form the aspects such as cellular morphology, propagation, migration and differentiation has clearly difference.（Chen,X.,X.Fu,et?al."Regulation?of?the?osteogenesis?of?pre-osteoblasts?by?spatial?arrangement?of?electrospunnanofibers?in?two-and?three-dimensional?environments."Nanomedicine:Nanotechnology,Biology?and?Medicine）。
In the ECM of mescenchymal tissue, collagen is topmost protein ingredient, accounts for 90% of protein ingredient.These collagens are mainly to 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 without particular orientation.Collagen fabric not only provides unique mechanical performance for different tissues, also has influence on greatly the behavior of cell.The support that imitates collagen fabric making is a kind of well selection.
Researchers have obtained fibrous framework with diverse ways, and find that cell can well grow on these fibers especially nano fiber scaffold.At present, the method that obtains nanofiber mainly contains and is separated, self assembly and electrostatic spinning, in these methods, use the method for electrostatic spinning, can obtain 10nm to micron-sized filament, with additive method comparison, it is extensive that it has raw material easy and simple to handle, efficiency is high, available, fiber and support performance are adjustable, can be manufactured with the advantage (K.Jayaraman such as sequence structure, et al., Recent advances in polymer nanofibers, Journal of Nanoscience and Nanotechnology4 (2004) 52 – 65.).
The nano fiber scaffold that uses conventional electrostatic spinning to obtain, conventionally too fine and close, hinder the diffusion of cell, and along with the deposition of silk, changed electric field, make support be difficult to do thick.In order to improve aperture, researcher mainly realizes by the following method at present: percolation, photoetch method, micro-nano fiber composite method and ultrasonic method etc.But some has destroyed fiber original structure these methods, some makes material internal structure inhomogeneous.Other researcher improves aperture by the method that changes receiving system, makes every effort to be issued to object in the prerequisite of not destroying spinning structure.Blakeney etc. make receiving system into a hollow foam hemisphere of plugging in stainless pin, have obtained a kind of concentrated, low-density, not extruded electrostatic spinning support, likeness in form cotton mass.Cell experiment shows, the fibrous framework that this method obtains and conventional method comparison, and aperture and thickness all increase, and have promoted growth and the diffusion of cell.But this method can only obtain unordered fiber, organize more suitably support (Blakeney can not be provided for orderly orientation, B.A., A.Tambralli, et al. (2011). " Cell infiltration and growth in a low density, uncompressed three-dimensional electrospunnanofibrous scaffold. " Biomaterials32 (6): 1583-1590.).
Obtain order fiber with method of electrostatic spinning, mainly by parallel pole and two kinds of methods of cylinder.The fibrous material fiber favorable orientation that wherein parallel pole obtains, but size is little, has limited its application.And the fibrous material size that cylinder is collected is large, but fiber orientation is good not, need to improve wire drawing strength and improve orientation, 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 cumulative methods of use one layer of cells one deck support such as Chen obtain a kind of three-dimensional, the cell orderly support wherein of growing into, but this rack making complexity, and the time that action need is a large amount of, and the operable time of cell is limited, further improves thickness more difficult.In addition, this support is the cumulative of very thin single-layer bracket, material continuity and homogeneity are poor, must use external force and just can maintain form, limit utilization (Chen in its body, X., X.Fu, et al. " Regulation of the osteogenesis of pre-osteoblasts by spatial arrangement of electrospunnanofibers in two-and three-dimensional environments. " Nanomedicine:Nanotechnology, Biology and Medicine).
In sum, in this area, exist the technological improvement requirement to preparing macropore three-dimensional order orientation nano fiber scaffold.
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, can to obtain even structure, 3 D stereo, loose porous orderly orientation silk fibroin nano-fiber support.
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, after coming unstuck, dissolve, dialyse, being dried, obtain regenerated silk fibroin;
(2) take the mixed solution of carrene and trifluoroacetic acid as solvent, the regenerated silk fibroin solution that preparation mass fraction is 6-20%;
(3) upper step gained regenerated silk fibroin solution is carried out to electrostatic spinning, then in ethanolic solution, collect with cylinder collecting method, obtain the silk fibroin nano-fiber crude product of orientation in order;
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 in deionized water rinsing 3-6 time, 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 that 16-30kv, feeding speed are 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 is mainly that volatilizable dissolution with solvents macromolecular material forms electrostatic spinning solution used, by high voltage electric field, the tip that acts on injection apparatus of solution is formed to taylor cone, under a suitable voltage, molecules in solution is finally broken through surface tension of liquid and is formed jet, rotate the variation of the moving grade of whip aloft, be accompanied by solvent evaporates, finally remaining macromolecular fibre product is on gathering-device.The macromolecular fibre network forming can apply to multiple fields such as physics, chemistry, medical science.
The raw material regenerated silk fibroin that the present invention selects has widely source, low cost of manufacture, good biologically active and preferably machinery and processing characteristics.Often be used to the research of tissue engineering bracket.
The present invention mainly realizes by a kind of ethanol bath cylinder collecting method.The feature of described ethanol bath drum process is that an electric conductivity cylinder part is immersed in ethanol, because ethanol has low surface tension, thereby can make prepared silk fibroin nano-fiber invasion wherein cause the gap enlargement between silk and silk, simultaneously cylinder can rotatablely move under the drive of motor, makes the silk of collecting can be towards a direction ordered arrangement.
A fibroin fiber part that sprays to gathering-device is deposited on cylinder, is then taken to rapidly in ethanol; A part is immersed near ethanolic solution cylinder, the upper cylinder surface of cylinder volume being scrolled.The fibroin fiber that these are drenched has been understood because of adhesive tape ethanol and has been become heavy, with comparison under dry condition, cylinder need larger strength go tractive it, the tractive ratio that this strength is larger is collected and is more easily formed ordered structure with condition dry condition bottom roll, some fibre mutually near time be adsorbed form a branch of, between bundle and bundle, abdicate larger gap, thereby formed the structure of macropore.
For conventional electrostatic spinning process, fiber is broken through surface tension and is sprayed the fibroplastic barrier that the behavior Chang Yin of emitter deposited and slackened electric field and stopped, and finally causes the tunica fibrosa collected very thin.And after being immersed in fiber in ethanol and being rolled by cylinder, be gathered in narrower scope, and overall electric field do not affected greatly, fiber can be sprayed continuously, and the accumulation of fiber also by ethanol intermolecular force and can reach larger thickness.
Collect for traditional cylinder, in order to obtain more orderly fiber, conventionally need to improve drum speed and improve traction force, reach the object of drawing more directly, but for a lot of large biological molecules, crosslinked become more stable firmly structure before, the common non-constant of its mechanical performance, and the fiber of common electrostatic spinning ejection is usually formed by uncrosslinked molecule, to bring up to certain time when speed, these fibers are easy to be pulled off.When being dissolved in carrene/trifluoroacetic acid solution, our selected regenerated silk fibroin mainly exists with the form that has random coil and α-helixstructure, this is a kind of unstable structure, electrostatic spinning is the process of the effect of a physical mechanics, so remain unstable structure being ejected to form in fibroin fiber, that is to say that the fibroin fiber internal structure entering into before ethanol is insecure.Then, alcoholic solution including ethanol can be cross-linked unstable fibroin albumen, make random coil and α-helixstructure become stable beta sheet structure, thereby make fibrous inside molecule form stable firmly combination, improve the mechanical performance of fiber simultaneously.In the ethanol bath of collecting, fibroin albumen is that occurred conformation changes, and the comparatively tough fiber of formation within the specific limits, improves the tractive strength of cylinder to it even if improve rotating speed, is also not easy to be pulled off.
Exception, because fibroin fiber has formed rock-steady structure in collection process, does not need extra cross-linking step, has avoided the extra crosslinked hole causing and the contraction of support entirety yet.
In sum, described ethanol bath cylinder collecting method can obtain possessing large aperture, 3-D solid structure and orderly nano fiber scaffold.By the feature of the prepared three-dimensional fiber support of the present invention be fibre diameter at 10nm between 10 μ m, fiber is orderly orientation and arranges, 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 μ m.Between described fibre diameter, fiber between average angle, fiber aperture and backing thickness all with electrostatic spinning solution concentration used and viscosity, the speed of cylinder, the factors such as the distance of shower nozzle and gathering-device are relevant, adjustable these factors are to reach needs.
By the present invention, can be able to a kind of easy and simple to handle, method of obtaining even structure, 3 D stereo, loose porous orderly orientation silk fibroin nano-fiber support, 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 institute's drawings attached, identical Reference numeral is used for representing identical element or structure, wherein:
1-electrospinning device 2-cylinder 3-ethanol.
The specific embodiment
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 mono-:
1, silk cocoon is cut into 1cm
2fritter, the sodium carbonate liquor 500mL that is 0.5% with mass fraction boils 1.5 hours, cleans up with deionized water, then boil 0.5h in 500mL deionized water, cleans post-drying with deionized water.In silk after treatment, 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 ℃ 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 obtaining and carries out being dried 48 hours after liquid nitrogen frozen, finally obtains regenerated silk fibroin.
2, regenerated silk fibroin being dissolved in to mass ratio is in the carrene of 3:7 and the mixed solution of trifluoroacetic acid, the electrostatic spinning solution used that preparation regenerated silk fibroin mass fraction is 6%.
3, spinning and collection:
Wherein the concrete operation method of ethanol bath cylinder collecting method is: by diameter 10cm, the glass dish of high 2cm fills with ethanol, and at its bottom paving one deck tinfoil; Cylinder used is that diameter is 2cm, the cylinder of high 5cm, and its surface parcel one deck masking foil, utilizes motor to rotarily drive cylinder and rotates.Cylinder is positioned in the culture dish that fills with ethanol, the submergence volume of cylinder is 1/2.
Electrostatic spinning uses voltage for 16kv, feeding speed are for 0.003mm/s, collection are apart from being 7cm, and cylinder frequency with 30Hz in ethanol bath is rotated.Carry out under these conditions the electrostatic spinning of different time, can on cylinder, collect the crude product of different-thickness.
4, after electrostatic spinning completes, take off crude product, put into 7% ammonia spirit and soak 30min, then use rinsed with deionized water 3-6 time, finally carry out freeze drying, obtain finished product.
5, by orderly ultraviolet light, 75% ethanol or the oxirane disinfection for three-dimensional material that obtain.After culture medium soaks, inoculation bone marrow stroma stem cell, IPS cell, osteocyte, nerve cell, Tenocyte cell, periodontal ligament stem cell, myocyte etc., induce, cultivate several weeks.Damaged places such as having the support implantable bone of cell, nerve, tendon, parodontium, muscle will be grown.
Embodiment bis-
1, silk cocoon is cut into 1cm
2fritter, the sodium carbonate liquor 500mL that is 0.5% with mass fraction boils 1.5 hours, cleans up with deionized water, then boil 0.5h in 500mL deionized water, cleans post-drying with deionized water.In silk after treatment, 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 ℃ 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 obtaining and carries out being dried 48 hours after liquid nitrogen frozen, finally obtains regenerated silk fibroin.
2, regenerated silk fibroin being dissolved in to mass ratio is in the carrene of 3:7 and the mixed solution of trifluoroacetic acid, the electrostatic spinning solution used that preparation regenerated silk fibroin mass fraction is 20%.
3, spinning and collection:
Wherein the concrete operation method of ethanol bath cylinder collecting method is: by diameter 20cm, the glass dish of high 5cm fills with ethanol, and at its bottom paving one deck tinfoil; Cylinder used is that diameter is 10cm, the cylinder of high 15cm, and its surface parcel one deck masking foil, utilizes motor to rotarily drive cylinder and moves.Cylinder is positioned in the culture dish that fills with ethanol, the submergence volume of cylinder is 1/3.
Electrostatic spinning uses voltage for 30kv, feeding speed are for 0.0005mm/s, collection are apart from being 15cm, and cylinder speed with 6Hz in ethanol bath is rotated.Carry out under these conditions the electrostatic spinning of different time, can on cylinder, collect the sample of different-thickness.
4, after electrostatic spinning completes, take off sample, put into 7% ammonia spirit and soak 30min, then use rinsed with deionized water 3-6 time, finally carry out freeze drying.
5, by orderly ultraviolet light, 75% ethanol or the oxirane disinfection for three-dimensional material that obtain.After culture medium soaks, inoculation bone marrow stroma stem cell, IPS cell, osteocyte, nerve cell, Tenocyte cell, periodontal ligament stem cell, myocyte etc., induce, cultivate several weeks.Damaged places such as having the support implantable bone of cell, nerve, tendon, parodontium, muscle will be grown.
By the feature of the prepared three-dimensional fiber support of the present invention be fibre diameter at 10nm between 10 μ m, 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 μ m.Between described fibre diameter, fiber between average angle, fiber aperture and backing thickness all with electrostatic spinning solution concentration used and viscosity, the speed of cylinder, the factors such as the distance of shower nozzle and gathering-device are relevant, adjustable these factors 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, after coming unstuck, dissolve, dialyse, being dried, obtain regenerated silk fibroin;
(2) take the mixed solution of carrene and trifluoroacetic acid as solvent, the regenerated silk fibroin solution that preparation mass fraction is 6-20%;
(3) upper step gained regenerated silk fibroin solution is carried out to electrostatic spinning, in ethanolic solution, collect with cylinder collecting method, obtain the silk fibroin nano-fiber crude product of orientation in order;
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 in deionized water rinsing 3-6 time, 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 that 16-30kv, feeding speed are 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. one kind is utilized the nano fiber scaffold that prepared by preparation method described in claim 1, it is characterized in that: in fibrous framework fibre diameter at 10nm between 10 μ m, fiber is orderly orientation and arranges, 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 μ m.
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