CN108159493A - A kind of preparation method of alginate-hydrogel nano fiber scaffold - Google Patents
A kind of preparation method of alginate-hydrogel nano fiber scaffold Download PDFInfo
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- CN108159493A CN108159493A CN201711457839.8A CN201711457839A CN108159493A CN 108159493 A CN108159493 A CN 108159493A CN 201711457839 A CN201711457839 A CN 201711457839A CN 108159493 A CN108159493 A CN 108159493A
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/52—Hydrogels or hydrocolloids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/56—Porous materials, e.g. foams or sponges
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
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- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
- D01D5/00—Formation of filaments, threads, or the like
- D01D5/0007—Electro-spinning
- D01D5/0015—Electro-spinning characterised by the initial state of the material
- D01D5/003—Electro-spinning characterised by the initial state of the material the material being a polymer solution or dispersion
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- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
- D01D5/00—Formation of filaments, threads, or the like
- D01D5/0007—Electro-spinning
- D01D5/0061—Electro-spinning characterised by the electro-spinning apparatus
- D01D5/0076—Electro-spinning characterised by the electro-spinning apparatus characterised by the collecting device, e.g. drum, wheel, endless belt, plate or grid
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- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
- D01D5/00—Formation of filaments, threads, or the like
- D01D5/0007—Electro-spinning
- D01D5/0061—Electro-spinning characterised by the electro-spinning apparatus
- D01D5/0092—Electro-spinning characterised by the electro-spinning apparatus characterised by the electrical field, e.g. combined with a magnetic fields, using biased or alternating fields
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- D—TEXTILES; PAPER
- D04—BRAIDING; LACE-MAKING; KNITTING; TRIMMINGS; NON-WOVEN FABRICS
- D04H—MAKING TEXTILE FABRICS, e.g. FROM FIBRES OR FILAMENTARY MATERIAL; FABRICS MADE BY SUCH PROCESSES OR APPARATUS, e.g. FELTS, NON-WOVEN FABRICS; COTTON-WOOL; WADDING ; NON-WOVEN FABRICS FROM STAPLE FIBRES, FILAMENTS OR YARNS, BONDED WITH AT LEAST ONE WEB-LIKE MATERIAL DURING THEIR CONSOLIDATION
- D04H1/00—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres
- D04H1/40—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres from fleeces or layers composed of fibres without existing or potential cohesive properties
- D04H1/42—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres from fleeces or layers composed of fibres without existing or potential cohesive properties characterised by the use of certain kinds of fibres insofar as this use has no preponderant influence on the consolidation of the fleece
- D04H1/4382—Stretched reticular film fibres; Composite fibres; Mixed fibres; Ultrafine fibres; Fibres for artificial leather
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/08—Methods for forming porous structures using a negative form which is filled and then removed by pyrolysis or dissolution
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
Abstract
The present invention relates to a kind of preparation methods of alginate-hydrogel nano fiber scaffold, include the following steps:Prepare polycaprolactone solution;Prepare sodium alginate soln;Electrostatic spinning is carried out using twin-jet nozzle electrostatic spinning system, obtains polycaprolactone sodium alginate nano fiber film;Polycaprolactone-sodium alginate nano fiber film is completely soaked in calcium chloride solution, is then fully washed with ethanol water, is freeze-dried, then chloroform washing by soaking is used, it is finally washed, is freeze-dried to get the alginate-hydrogel nano fiber scaffold successively with ethyl alcohol and distilled water.The alginate-hydrogel nano fiber scaffold has stable three-dimensional structure, has preferable biocompatibility, can be applied to 3D cell culture, and can optimize the penetration depth of cell.
Description
Technical field
The invention belongs to nano fiber scaffold fields, and in particular to a kind of alginate-hydrogel nano fiber scaffold
Preparation method.
Background technology
Electro spinning nano fiber is the common timbering material that extra-cellular matrix structure is simulated in organizational project.But nanometer fiber net
Cell culture environment whether to represent true vivo environment still unclear.Because cell itself mixes and electrospun nanofiber fibre
The porous property of dimension, enables cell lateral transfer on grid, three-D displacement of the interference cell on nano fiber scaffold.By
Difference between two and three dimensions cell culture environment has a significant impact cell metabolism, then Three-dimensional cell culture into
A hot spot for tumor tissues modeling and stem cell culture application.Such as Chinese patent " is based on collagen sponge-nano-cellulose
Three-dimensional cell stent preparation method " in (201710515612.8) report by preparing collagen sponge and nanofiber respectively
Then element is mixed and three-dimensional cell stent is prepared.Its advantage is that result is reliable, easy to operate, repeatable strong, can
The three dimensional growth and proliferation of cell are effectively facilitated for a long time, while to the generation of all kinds of human body three-dimensional micro-assembly robots, especially to liver
The formation of cancerous tissue function has certain effect, but its shortcoming is with regard to apparent, it does not overcome cell surface culture this
One problem needs that cell is placed in the autonomous expansion on stent rather than by cell on stent by biometric print technology
It dissipates;One kind is reported in Chinese patent " two-phase porous three-dimensional cell culturing bracket " (CN102719391A) and has both two-dimentional cell training
The dimensional culture system of the convenient part of whole for the system of supporting, this ideal Three-dimensional cell culture system will need have energy first
User is enough allowed as easily observing life of the cell in Three-dimensional cell culture stent using plane tissue culture plate
Long situation, the preparation method first step:First prepare the semi-finished product porous three-dimensional stent that crude fibre is mutually formed.Second step:Half into
Product porous three-dimensional rack outer surface sprays fine fibre using electrostatic spinning technique.Because electrostatic spinning technique is more in this semi-finished product
Spraying on the three-dimensional rack of hole is influenced by the depth of three-dimensional rack in itself, and its spinning density drops therewith with the intensification of depth
It is low, and easily form porous three-dimensional rack outer surface and be capped, the situation for causing lining electrostatic spinning few.Therefore second step just limits
The thickness of semi-finished product porous three-dimensional stent that crude fibre in the first step is mutually formed, and the electrostatic spinning of the stent lining formed are made
Density is extremely unstable.
Current research hot spot in being simulated as organizational project, compared with traditional two-dimentional cell culture, three-dimensional cell branch
The advantages of Three-dimensional cell culture that frame is brought, is:1) 2) being more nearly with the growth pattern of cell in vivo can deepen to just
The understanding of the structure-function relationship of normal and pathology tissue;3) direction of research is more extensive;4) result of study is more reliable.It is near
Nian Lai, three-dimensional cell stent have good application potential in field in organizational project simulation, can provide with internal cell more
The near growing environment of adjunction, and the idea of more organizational project cell simulations is realized on this basis, including cell and growth
Interaction, intensification between the factor, cell and drug is to understanding of structure-function relationship of normal and pathology tissue etc.
Deng.And electrostatic spinning technique is also a kind of method popular at present for preparing three-dimensional manometer cytoskeleton, the cell prepared
Stent specific surface area height, tridimensional network, gap are continuous and porosity is high etc. perfect meets our requirement, but also can be with
Subsidiary antibacterial properties.Electrospun material with biocompatibility, degradability and good mechanical performance, such as:Polylactic acid
(PLA), the synthetic macromolecular compounds such as polylactic-co-glycolic acid (PLGA), polycaprolactone (PCL), polyurethane (PU).
However, three-dimensional cell stent is prepared at present and the technology of approximate cell growth environment and immature is provided, due to thin
The being not fixed property and fibre porosity of born of the same parents position so that cell generates horizontal displacement and is easy to three-D displacement, real in three-dimensional
Existing displacement is not easy to.From solid it is intuitive the result is that:The cell of three-dimensional cell rack surface is not easy to permeate to stent depths.
Therefore, it is badly in need of preparing a kind of three-dimensional manometer fibrocyte stent that can optimize Premeabilisation of cells depth.
Invention content
The present invention is described above in order to solve the problems, such as, provides a kind of alginate-hydrogel nano fiber scaffold
Preparation method prepares alginate-hydrogel nano fiber scaffold using Dispersion relation and removal technology, wherein sodium alginate and
Polycaprolactone is injected jointly by internal and external pin, and polycaprolactone can support the fibre morphology of sodium alginate, until sodium alginate
It is crosslinked in calcium chloride solution.Polycaprolactone layer then in chloroform impregnates and removes removing repeatedly, and crosslinked alginate-
Hydrogel nano fiber scaffold keeps complete in inside.Thus alginate-the water-setting generated using polycaprolactone nanofiber
Glue nano-bracket can be used as 3D cell culture because of its preferable biocompatibility.
To achieve the above object, the present invention adopts the following technical scheme that:
A kind of preparation method of alginate-hydrogel nano fiber scaffold, the preparation method include the following steps:
1) polycaprolactone is dissolved in chloroform/methanol solvent, obtains polycaprolactone solution;
2) sodium alginate and Triton X-100 are dissolved in solvent, prepare sodium alginate soln;
3) using twin-jet nozzle electrostatic spinning system, weighing the polycaprolactone solution of 18g steps 1) preparation, to be placed in twin-jet nozzle quiet
In the outer nozzle of electrospinning system, the sodium alginate soln for weighing the preparation of 20g steps 2) adds in twin-jet nozzle electrostatic spinning system
In inner nozzle, electrostatic spinning is carried out, obtains polycaprolactone-sodium alginate nano fiber film;
4) calcium chloride is dissolved in ethanol water solvent, obtains calcium chloride solution, then by the polycaprolactone described in step 3)-
Sodium alginate nano fiber film is completely soaked in the calcium chloride solution of above-mentioned configuration, uses ethyl alcohol water-soluble after impregnating 6h at room temperature
Liquid fully washs, and is then freeze-dried;
5) the nano fibrous membrane chloroform washing by soaking after step 4) is freeze-dried, then with ethyl alcohol and distilled water successively
Washing removes chloroform to complete, is freeze-dried to get the alginate-hydrogel nano fiber scaffold.
Preferably, in the polycaprolactone solution that the step 1) is prepared, the volume ratio of the chloroform and methanol is 3:1,
The content of the polycaprolactone in the solution is 16wt%.
Preferably, in the sodium alginate soln that the step 2) is prepared, the solvent is distilled water and dimethyl sulfoxide (DMSO)
Mixture, the volume ratio of the distilled water and dimethyl sulfoxide (DMSO) is 19:1.
Preferably, in the sodium alginate soln that the step 2) is prepared, the content of the sodium alginate in the solution is
The content of 2wt%, the Triton X-100 in the solution is 0.5wt%.
Preferably, the spinning condition in the step 3) is that the flow velocity of the solution in control outer nozzle is 0.5ml/h,
The flow velocity of solution in inner nozzle be 2.5ml/h, collection cylinder diameter 100mm, length 200mm, rotating speed 100rpm, nozzle away from
Roller distance is 12cm, spinning voltage 20kV.
Preferably, in the step 3), controlled at 23-26 DEG C during electrostatic spinning, humidity 25-
35%.
Preferably, in the calcium chloride solution that the step 4) is prepared, volume of the ethanol water solvent for ethyl alcohol and distilled water
Than being 3:7 mixed solution, the mass fraction of calcium chloride is 0.002-2%.
Preferably, in the step 5), the nano fibrous membrane chloroform washing by soaking 10 after step 4) is freeze-dried
It is secondary, 1h is impregnated every time.The polycaprolactone in nano fibrous membrane can gradually be removed with chloroform multiple washing by soaking, so that completely
Remove polycaprolactone.
In order to which sodium alginate is made further to be crosslinked in the nanofiber of electrostatic spinning, by polycaprolactone-alginic acid
Sodium nano fibrous membrane impregnates in the calcium chloride solution of various concentration, make its it is spontaneous crosslink behavior, calcium chloride is dissolved in second
Alcohol is 3 with distilled water volume ratio:In 7 ethanol water, the mass fraction of calcium chloride solution is controlled in 0.002-2% ranges
It is interior, polycaprolactone-sodium alginate nano fiber film is completely soaked in calcium chloride solution, and incubates 6h at room temperature, is incubated
After fully washed using ethanol water, calcium chloride is removed from nano fibrous membrane, is then freeze-dried, to remove nanometer
The ingredients such as ethyl alcohol, water in tunica fibrosa, and maintain its original shape.Nano fibrous membrane after freeze-drying is soaked in chloroform
Bubble 10 times, impregnates 1h, with phased separation polycaprolactone every time.Then it is washed successively with ethyl alcohol and distilled water to remove chloroform, most
It is freeze-dried the shape to keep nano fibrous membrane afterwards.
A kind of alginate-hydrogel nanometer prepared by the preparation method of alginate-hydrogel nano fiber scaffold
Fibrous framework.
Further, the fibre diameter of the alginate-hydrogel nano fiber scaffold is 88 ± 47nm
After removing polycaprolactone from crosslinked alginate-hydrogel nano fiber scaffold, nano fiber scaffold retains
Fibre morphology is good, and with the concentration for gradually decreasing calcium chloride, the fibre density of nano fiber scaffold also gradually decreases.Research hair
Existing, before removing polycaprolactone, the average diameter of the fiber of nano fiber scaffold is 555 ± 173nm, however, no matter calcium ion reaches
To what concentration, after removing polycaprolactone, the fibre diameter of alginate-hydrogel nano fiber scaffold can all be reduced to 88 ±
47nm.The fibre diameter of alginate-hydrogel nano fiber scaffold reduces the polycaprolactone for confirming covering sodium alginate by chlorine
It is imitative to remove completely, and due to the package of polycaprolactone, crosslinked alginate-hydrogel nano fiber scaffold keeps fiber
Form.However, after washing, alginate-hydrogel nanofiber branch in, under the conditions of the calcium ion of low concentration after crosslinking
Fiber mechanical performance in frame is low, and most of fiber is all rinsed, and only remains the fiber with more preferable mechanical performance.
Beneficial effects of the present invention:
The present invention uses a kind of coaxial electrostatic spinning technology, forms a kind of sodium alginate Nanowire wrapped up by polycaprolactone
Then dimensional scaffold makes sodium alginate further be crosslinked in calcium chloride solution, while the method for removing polycaprolactone is finally made
The alginate-hydrogel nano fiber scaffold.
(1) the application is noted jointly by Dispersion relation and removal technology, sodium alginate and polycaprolactone by internal and external pin
Enter to carry out electrostatic spinning, collect the non-woven fabrics for forming three-dimensional structure receiving, then remove polycaprolactone, polycaprolactone can be supported
The fibre morphology of the three-dimensional structure of sodium alginate is until sodium alginate is crosslinked in calcium chloride, and immersion removing is poly- repeatedly in chloroform
After caprolactone, crosslinked alginate-hydrogel nano fiber scaffold can also keep complete, and there is no because of polycaprolactone
The fibre morphology of three-dimensional structure is removed and changes, and the alginate-hydrogel nano fiber scaffold has preferable biofacies
Capacitive can be applied to 3D cell culture.
(2) alginate-hydrogel nano fiber scaffold optimizes the penetration depth of cell, especially using hydrogel as
The nano fiber scaffold of raw material, this, which allows, needs the research using hydrogel as timbering material that can really be given birth to being more nearly cell
It is cultivated to obtain more true and reliable achievement in research in the stent of condition living.
(3) during sodium alginate cross-linking and removal polycaprolactone, the concentration pair of the calcium ion in calcium chloride solution
The regularity of the influence of stent can control the parameter of stent, ensure that the controllability of stent, can be needed with experimental difference
It asks, makes the alginate-hydrogel nano fiber scaffold for meeting demand accordingly.
Description of the drawings
Fig. 1 is the TEM pictures of alginate-hydrogel nano fiber scaffold in embodiment 1.
Fig. 2 is the picture of alginate-hydrogel nano fiber scaffold prepared by embodiment 1-3.
Fig. 3 is the cell migration picture under tracer technique.
Fig. 4 is the cell migration data of alginate-hydrogel nano fiber scaffold prepared by embodiment 1.
Fig. 5 is the cell migration data of alginate-hydrogel nano fiber scaffold prepared by embodiment 2.
Fig. 6 is the cell migration data in Gel2.
Specific embodiment
For the present invention is better described, be easy to understand technical scheme of the present invention, below by embodiment to the present invention into
One step is described in detail.But following embodiments is only the simple example of the present invention, does not represent or limit the right of the present invention
Protection domain, protection scope of the present invention are subject to claims.
Embodiment 1
A kind of preparation method of alginate-hydrogel nano fiber scaffold, the preparation method include the following steps:
1) it is 3 by volume by chloroform and methanol:1 ratio mixing, is dissolved in chloroform/methanol mixed liquor by polycaprolactone
In, prepare the polycaprolactone solution that polycaprolactone content is 16wt%;
2) it is 19 by volume by distilled water and dimethyl sulfoxide (DMSO):1 ratio mixing, by sodium alginate and TritonX-
100 are dissolved in distilled water and dimethyl sulfoxide (DMSO) mixed liquor, preparation obtains sodium alginate soln, and the sodium alginate is in the solution
Content is 2wt%, and the contents of the Triton X-100 in the solution are 0.5wt%;
3) using twin-jet nozzle electrostatic spinning system, weighing the polycaprolactone solution of 18g steps 1) preparation, to be placed in twin-jet nozzle quiet
In the outer nozzle of electrospinning system, the sodium alginate soln for weighing the preparation of 20g steps 2) adds in twin-jet nozzle electrostatic spinning system
In inner nozzle, electrostatic spinning is carried out, obtains polycaprolactone-sodium alginate nano fiber film, spinning condition is:It controls in outer nozzle
The flow velocity of solution is 0.5ml/h, and the flow velocity of the solution in inner nozzle is 2.5ml/h, collection cylinder diameter 100mm, length
200mm, rotating speed 100rpm, nozzle away from roller distance for 12cm, spinning voltage 20kV, controlled at 23-26 DEG C, humidity
25-35%;
4) calcium chloride is dissolved in ethanol water solvent, obtains calcium chloride solution, the ethanol water solvent is ethyl alcohol and distillation
The volume ratio of water is 3:7 mixed solution, the content of the calcium chloride are 2wt%, then by the polycaprolactone described in step 3)-
Sodium alginate nano fiber film is completely soaked in the calcium chloride solution of above-mentioned configuration, is impregnated 6h at room temperature, is then used ethyl alcohol
Aqueous solution fully washs, and is then freeze-dried;
5) the nano fibrous membrane chloroform washing by soaking 10 times after step 4) is freeze-dried, impregnates 1h, Ran Houyong every time
Ethyl alcohol and distilled water wash successively to complete and remove chloroform, are freeze-dried to get the alginate-hydrogel nanofiber
Stent.
A kind of alginate-hydrogel nano fiber scaffold by prepared by above-mentioned preparation method.
The fibre diameter of the alginate-hydrogel nano fiber scaffold is 88 ± 47nm.
As shown in Figure 1, Fig. 1 a are to impregnate polycaprolactone-sodium alginate nanometer after 6h in step 4) in calcium chloride solution
Tunica fibrosa scanning electron microscopic picture, Fig. 1 b are alginate-hydrogel nanometer after fully being washed with ethanol water in step 4)
The scanning electron microscopic picture of tunica fibrosa, it can be seen that removed from the calcium cross-linked alginate of chlorination-hydrogel nano fibrous membrane poly-
After caprolactone, it is good that alginate-hydrogel nano fibrous membrane retains fibre morphology.
Embodiment 2
A kind of preparation method of alginate-hydrogel nano fiber scaffold, the preparation method include the following steps:
1) it is 3 by volume by chloroform and methanol:1 ratio mixing, is dissolved in chloroform/methanol mixed liquor by polycaprolactone
In, prepare the polycaprolactone solution that polycaprolactone content is 16wt%;
2) it is 19 by volume by distilled water and dimethyl sulfoxide (DMSO):1 ratio mixing, by sodium alginate and TritonX-
100 are dissolved in distilled water and dimethyl sulfoxide (DMSO) mixed liquor, preparation obtains sodium alginate soln, and the sodium alginate is in the solution
Content is 2wt%, and the contents of the Triton X-100 in the solution are 0.5wt%;
3) using twin-jet nozzle electrostatic spinning system, weighing the polycaprolactone solution of 18g steps 1) preparation, to be placed in twin-jet nozzle quiet
In the outer nozzle of electrospinning system, the sodium alginate soln for weighing the preparation of 20g steps 2) adds in twin-jet nozzle electrostatic spinning system
In inner nozzle, electrostatic spinning is carried out, obtains polycaprolactone-sodium alginate nano fiber film, spinning condition is:It controls in outer nozzle
The flow velocity of solution is 0.5ml/h, and the flow velocity of the solution in inner nozzle is 2.5ml/h, collection cylinder diameter 100mm, length
200mm, rotating speed 100rpm, nozzle away from roller distance for 12cm, spinning voltage 20kV, controlled at 23-26 DEG C, humidity
25-35%;
4) calcium chloride is dissolved in ethanol water solvent, obtains calcium chloride solution, the ethanol water solvent is ethyl alcohol and distillation
The volume ratio of water is 3:7 mixed solution, the content of the calcium chloride is 0.02wt%, then by gathering in oneself described in step 3)
Ester-sodium alginate nano fiber film is completely soaked in the calcium chloride solution of above-mentioned configuration, and ethyl alcohol is used after impregnating 6h at room temperature
Aqueous solution fully washs, and is then freeze-dried;
5) the nano fibrous membrane chloroform washing by soaking 10 times after step 4) is freeze-dried, impregnates 1h, Ran Houyong every time
Ethyl alcohol and distilled water wash successively to complete and remove chloroform, are freeze-dried to get the alginate-hydrogel nanofiber
Stent.
A kind of alginate-hydrogel nano fiber scaffold by prepared by above-mentioned preparation method.
The fibre diameter of the alginate-hydrogel nano fiber scaffold is 88 ± 47nm.
Embodiment 3
A kind of preparation method of alginate-hydrogel nano fiber scaffold, the preparation method include the following steps:
1) it is 3 by volume by chloroform and methanol:1 ratio mixing, is dissolved in chloroform/methanol mixed liquor by polycaprolactone
In, prepare the polycaprolactone solution that polycaprolactone content is 16wt%;
2) it is 19 by volume by distilled water and dimethyl sulfoxide (DMSO):1 ratio mixing, by sodium alginate and TritonX-
100 are dissolved in distilled water and dimethyl sulfoxide (DMSO) mixed liquor, preparation obtains sodium alginate soln, and the sodium alginate is in the solution
Content is 2wt%, and the contents of the Triton X-100 in the solution are 0.5wt%;
3) using twin-jet nozzle electrostatic spinning system, weighing the polycaprolactone solution of 18g steps 1) preparation, to be placed in twin-jet nozzle quiet
In the outer nozzle of electrospinning system, the sodium alginate soln for weighing the preparation of 20g steps 2) adds in twin-jet nozzle electrostatic spinning system
In inner nozzle, electrostatic spinning is carried out, obtains polycaprolactone-sodium alginate nano fiber film, spinning condition is:It controls in outer nozzle
The flow velocity of solution is 0.5ml/h, and the flow velocity of the solution in inner nozzle is 2.5ml/h, collection cylinder diameter 100mm, length
200mm, rotating speed 100rpm, nozzle away from roller distance for 12cm, spinning voltage 20kV, controlled at 23-26 DEG C, humidity
25-35%;
4) calcium chloride is dissolved in ethanol water solvent, obtains calcium chloride solution, the ethanol water solvent is ethyl alcohol and distillation
The volume ratio of water is 3:7 mixed solution, the content of the calcium chloride is 0.006wt%, then will gather oneself described in step 3)
Lactone-sodium alginate nano fiber film is completely soaked in the calcium chloride solution of above-mentioned configuration, and second is used after impregnating 6h at room temperature
Alcohol solution fully washs, and is then freeze-dried;
5) the nano fibrous membrane chloroform washing by soaking 10 times after step 4) is freeze-dried, impregnates 1h, Ran Houyong every time
Ethyl alcohol and distilled water wash successively to complete and remove chloroform, are freeze-dried to get the alginate-hydrogel nanofiber
Stent.
A kind of alginate-hydrogel nano fiber scaffold by prepared by above-mentioned preparation method.
The fibre diameter of the alginate-hydrogel nano fiber scaffold is 88 ± 47nm.
As shown in Fig. 2, Fig. 2 a are obtained polycaprolactone-sodium alginate nano fiber film scanning electron microscope (SEM) photograph after electrostatic spinning
Piece, Fig. 2 b, Fig. 2 c, Fig. 2 d are respectively the scanning electricity of alginate-hydrogel nano fiber scaffold made from embodiment 1-3 difference
Mirror picture, it is recognised that removing polycaprolactone on alginate-hydrogel nano fiber scaffold after being crosslinked by calcium chloride
Afterwards, nano fiber scaffold reservation fibre morphology is good, with the concentration for gradually decreasing calcium chloride, alginate-hydrogel nanometer
Fibre density in fibrous framework also gradually decreases.Due to the package of polycaprolactone, alginate-hydrogel nano fiber scaffold
The form of fiber is kept, however, after washing, alginate-hydrogel in, under the conditions of the calcium ion of low concentration after crosslinking
Fiber mechanical performance in nano fiber scaffold is low, and most of fiber is all rinsed, and only remains with more preferable mechanicalness
The fiber of energy.
Embodiment 4
A kind of preparation method of alginate-hydrogel nano fiber scaffold, the preparation method include the following steps:
1) it is 3 by volume by chloroform and methanol:1 ratio mixing, is dissolved in chloroform/methanol mixed liquor by polycaprolactone
In, prepare the polycaprolactone solution that polycaprolactone content is 16wt%;
2) it is 19 by volume by distilled water and dimethyl sulfoxide (DMSO):1 ratio mixing, by sodium alginate and TritonX-
100 are dissolved in distilled water and dimethyl sulfoxide (DMSO) mixed liquor, preparation obtains sodium alginate soln, and the sodium alginate is in the solution
Content is 2wt%, and the contents of the Triton X-100 in the solution are 0.5wt%;
3) using twin-jet nozzle electrostatic spinning system, weighing the polycaprolactone solution of 18g steps 1) preparation, to be placed in twin-jet nozzle quiet
In the outer nozzle of electrospinning system, the sodium alginate soln for weighing the preparation of 20g steps 2) adds in twin-jet nozzle electrostatic spinning system
In inner nozzle, electrostatic spinning is carried out, obtains polycaprolactone-sodium alginate nano fiber film, spinning condition is:It controls in outer nozzle
The flow velocity of solution is 0.5ml/h, and the flow velocity of the solution in inner nozzle is 2.5ml/h, collection cylinder diameter 100mm, length
200mm, rotating speed 100rpm, nozzle away from roller distance for 12cm, spinning voltage 20kV, controlled at 23-26 DEG C, humidity
25-35%;
4) calcium chloride is dissolved in ethanol water solvent, obtains calcium chloride solution, the ethanol water solvent is ethyl alcohol and distillation
The volume ratio of water is 3:7 mixed solution, the content of the calcium chloride is 0.002wt%, then will gather oneself described in step 3)
Lactone-sodium alginate nano fiber film is completely soaked in the calcium chloride solution of above-mentioned configuration, and second is used after impregnating 6h at room temperature
Alcohol solution fully washs, and is then freeze-dried;
5) the nano fibrous membrane chloroform washing by soaking 10 times after step 4) is freeze-dried, impregnates 1h, Ran Houyong every time
Ethyl alcohol and distilled water wash successively to complete and remove chloroform, are freeze-dried to get the alginate-hydrogel nanofiber
Stent.
A kind of alginate-hydrogel nano fiber scaffold by prepared by above-mentioned preparation method.
The fibre diameter of the alginate-hydrogel nano fiber scaffold is 88 ± 47nm.
Embodiment 5
A kind of preparation method of alginate-hydrogel nano fiber scaffold, the preparation method include the following steps:
1) it is 3 by volume by chloroform and methanol:1 ratio mixing, is dissolved in chloroform/methanol mixed liquor by polycaprolactone
In, prepare the polycaprolactone solution that polycaprolactone content is 16wt%;
2) it is 19 by volume by distilled water and dimethyl sulfoxide (DMSO):1 ratio mixing, by sodium alginate and TritonX-
100 are dissolved in distilled water and dimethyl sulfoxide (DMSO) mixed liquor, preparation obtains sodium alginate soln, and the sodium alginate is in the solution
Content is 2wt%, and the contents of the Triton X-100 in the solution are 0.5wt%;
3) using twin-jet nozzle electrostatic spinning system, weighing the polycaprolactone solution of 18g steps 1) preparation, to be placed in twin-jet nozzle quiet
In the outer nozzle of electrospinning system, the sodium alginate soln for weighing the preparation of 20g steps 2) adds in twin-jet nozzle electrostatic spinning system
In inner nozzle, electrostatic spinning is carried out, obtains polycaprolactone-sodium alginate nano fiber film, spinning condition is:It controls in outer nozzle
The flow velocity of solution is 0.5ml/h, and the flow velocity of the solution in inner nozzle is 2.5ml/h, collection cylinder diameter 100mm, length
200mm, rotating speed 100rpm, nozzle away from roller distance for 12cm, spinning voltage 20kV, controlled at 23-26 DEG C, humidity
25-35%;
4) calcium chloride is dissolved in ethanol water solvent, obtains calcium chloride solution, the ethanol water solvent is ethyl alcohol and distillation
The volume ratio of water is 3:7 mixed solution, the content of the calcium chloride is 1.00wt%, then by gathering in oneself described in step 3)
Ester-sodium alginate nano fiber film is completely soaked in the calcium chloride solution of above-mentioned configuration, and ethyl alcohol is used after impregnating 6h at room temperature
Aqueous solution fully washs, and is then freeze-dried;
5) the nano fibrous membrane chloroform washing by soaking 10 times after step 4) is freeze-dried, impregnates 1h, Ran Houyong every time
Ethyl alcohol and distilled water wash successively to complete and remove chloroform, are freeze-dried to get the alginate-hydrogel nanofiber
Stent.
A kind of alginate-hydrogel nano fiber scaffold by prepared by above-mentioned preparation method.
The fibre diameter of the alginate-hydrogel nano fiber scaffold is 88 ± 47nm.
In order to determine the influence of the concentration versus cell penetration depth of calcium ion, in alginate-water made from embodiment 1,2
Using fluorescence labeled cell in gel nano fiber scaffold, then measure under the microscope cell vertically pass through the alginate-
The length of hydrogel nano fiber scaffold.Concrete operations are as follows:By alginate-water-setting of 3mg in the environment for being 4 DEG C in temperature
Glue nano fiber scaffold, which is put into, fills 0.1ml, 50 μ g mL-1The depth of protein solution is 2mm, the round PDMS of a diameter of 8mm
In (dimethyl silicone polymer) lattice.After 12h, the orifice plate full of alginate-hydrogel nano fiber scaffold is just obtained and has used
In cell culture, while one group of control group is set, is 2% sodium alginate gel and 100 microlitres by 100 microlitres of mass fractions
Calcium chloride crosslinking after a kind of substance (Gel2) for generating.By 1*105The culture plate that NIH3T3 cell inoculations are coated to company's albumen
In, then put it into the DMEM medium cultures 12h containing 10%FBS.Then 25 μm of addition is wine-colored in the medium
Cell tracker solution is to fluorescent staining cell.The culture plate of cell covering is inverted, is placed on alginate-hydrogel nanometer
The top of fibrous framework.The migration of the cell of fluorescent marker is monitored by laser scanning co-focusing microscope, preceding 12h is every
3h monitorings are primary, behind 5 days per being monitored for 24 hours.Using He-Ne laser excitation 633nm laser, emission filter and 640-
The bandpass filter of 700nm is respectively perpendicular scanning alginate-hydrogel nano fiber scaffold.As seen in figures 3-6, this is observed
The penetration depth of alginate-hydrogel nano fiber scaffold prepared by inventive method is good, especially under high-calcium ionic concentration
Carry out showing very superior Premeabilisation of cells ability in crosslinked alginate-hydrogel nano fiber scaffold, by its with
The comparison of cell migration can intuitively obtain this conclusion very much in Gel2.And the alginic acid under the processing of different calcium concentrations
The permeability of salt-hydrogel nano fiber scaffold is different, and due to the direct shadow of the concentration of a certain range of calcium ion
Rung the crosslinking degree of alginate-hydrogel nano fiber scaffold, thus permeability and seepage velocity all in a certain range
Calcium ion concentration it is directly proportional.
The above is the preferred embodiment of the present invention, and its object is to person skilled in the art is allowed to understand the present invention
Content is simultaneously implemented, it is noted that for those skilled in the art, is not departing from the principle of the invention
Under the premise of, the addition to the equivalence replacement of raw material selected by the present invention and auxiliary element, the selection of concrete mode can also be made,
Or to each particular technique feature described in above-mentioned specific embodiment, in the case of no contradiction, pass through any conjunction
Suitable mode is combined, and the addition of these equivalence replacements and auxiliary element, combination of particular technique feature etc. are also considered as this
The protection domain of invention.
Claims (10)
1. a kind of preparation method of alginate-hydrogel nano fiber scaffold, which is characterized in that the preparation method include with
Lower step:
1) polycaprolactone is dissolved in chloroform/methanol solvent, obtains polycaprolactone solution;
2) sodium alginate and Triton X-100 are dissolved in solvent, prepare sodium alginate soln;
3) using twin-jet nozzle electrostatic spinning system, the polycaprolactone solution for weighing the preparation of 18g steps 1) is placed in twin-jet nozzle Static Spinning
In the outer nozzle of silk system, the sodium alginate soln for weighing the preparation of 20g steps 2) adds in the interior spray of twin-jet nozzle electrostatic spinning system
In mouth, electrostatic spinning is carried out, obtains polycaprolactone-sodium alginate nano fiber film;
4) calcium chloride is dissolved in ethanol water solvent, obtains calcium chloride solution, then by polycaprolactone-seaweed described in step 3)
Sour sodium nano fibrous membrane is completely soaked in the calcium chloride solution of above-mentioned configuration, is filled after impregnating 6h at room temperature with ethanol water
Divide washing, be then freeze-dried;
5) the nano fibrous membrane chloroform washing by soaking after step 4) is freeze-dried, is then washed successively with ethyl alcohol and distilled water
Chloroform is removed to complete, is freeze-dried to get the alginate-hydrogel nano fiber scaffold.
2. a kind of preparation method of alginate-hydrogel nano fiber scaffold as described in claim 1, which is characterized in that
In the polycaprolactone solution that the step 1) is prepared, the volume ratio of the chloroform and methanol is 3:1, the polycaprolactone is molten
Content in liquid is 16wt%.
3. a kind of preparation method of alginate-hydrogel nano fiber scaffold as described in claim 1, which is characterized in that
In the sodium alginate soln that the step 2) is prepared, the solvent is distilled water and the mixture of dimethyl sulfoxide (DMSO), the steaming
Distilled water and the volume ratio of dimethyl sulfoxide (DMSO) are 19:1.
4. a kind of preparation method of alginate-hydrogel nano fiber scaffold as described in claim 1, which is characterized in that
In the sodium alginate soln that the step 2) is prepared, the content of the sodium alginate in the solution is 2wt%, the Triton
The contents of X-100 in the solution are 0.5wt%.
5. a kind of preparation method of alginate-hydrogel nano fiber scaffold as described in claim 1, which is characterized in that
Spinning condition in the step 3) is, the flow velocity for controlling the solution in outer nozzle is 0.5ml/h, the solution in inner nozzle
Flow velocity is 2.5ml/h, collection cylinder diameter 100mm, length 200mm, rotating speed 100rpm, and nozzle is 12cm away from roller distance,
Spinning voltage is 20kV.
6. a kind of preparation method of alginate-hydrogel nano fiber scaffold as described in claim 1, which is characterized in that
In the step 3), controlled at 23-26 DEG C during electrostatic spinning, humidity 25-35%.
7. a kind of preparation method of alginate-hydrogel nano fiber scaffold as described in claim 1, which is characterized in that
In the calcium chloride solution that the step 4) is prepared, ethanol water solvent is that the volume ratio of ethyl alcohol and distilled water is 3:7 mixing is molten
Liquid, the mass fraction of calcium chloride is 0.002-2%.
8. a kind of preparation method of alginate-hydrogel nano fiber scaffold as described in claim 1, which is characterized in that
In the step 5), the nano fibrous membrane chloroform washing by soaking 10 times after step 4) is freeze-dried impregnates 1h every time.
9. a kind of a kind of preparation method institute of alginate of claim 1-8 any one of them-hydrogel nano fiber scaffold
The alginate of preparation-hydrogel nano fiber scaffold.
A kind of 10. alginate-hydrogel nano fiber scaffold as claimed in claim 9, which is characterized in that the alginic acid
The fibre diameter of salt-hydrogel nano fiber scaffold is 88 ± 47nm.
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