CN106400311A - Method for preparing composite nanofiber tissue engineering scaffold based on graphene oxide - Google Patents
Method for preparing composite nanofiber tissue engineering scaffold based on graphene oxide Download PDFInfo
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- CN106400311A CN106400311A CN201610806923.5A CN201610806923A CN106400311A CN 106400311 A CN106400311 A CN 106400311A CN 201610806923 A CN201610806923 A CN 201610806923A CN 106400311 A CN106400311 A CN 106400311A
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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
- D04H1/70—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres
- D04H1/72—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres the fibres being randomly arranged
- D04H1/728—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres the fibres being randomly arranged by electro-spinning
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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
-
- 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
- D01D5/0069—Electro-spinning characterised by the electro-spinning apparatus characterised by the spinning section, e.g. capillary tube, protrusion or pin
-
- 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
- D04H1/4266—Natural fibres not provided for in group D04H1/425
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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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- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
- D06M11/00—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with inorganic substances or complexes thereof; Such treatment combined with mechanical treatment, e.g. mercerising
- D06M11/73—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with inorganic substances or complexes thereof; Such treatment combined with mechanical treatment, e.g. mercerising with carbon or compounds thereof
- D06M11/74—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with inorganic substances or complexes thereof; Such treatment combined with mechanical treatment, e.g. mercerising with carbon or compounds thereof with carbon or graphite; with carbides; with graphitic acids or their salts
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- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
- D06M2101/00—Chemical constitution of the fibres, threads, yarns, fabrics or fibrous goods made from such materials, to be treated
- D06M2101/02—Natural fibres, other than mineral fibres
- D06M2101/10—Animal fibres
- D06M2101/12—Keratin fibres or silk
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- Engineering & Computer Science (AREA)
- Textile Engineering (AREA)
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- Chemical & Material Sciences (AREA)
- Dispersion Chemistry (AREA)
- Inorganic Fibers (AREA)
- Materials For Medical Uses (AREA)
- Artificial Filaments (AREA)
Abstract
The invention provides a method for preparing a composite nanofiber tissue engineering scaffold based on graphene oxide. The method includes the following steps: a step 1, dissolving silk fibroin and high-molecular polymer in a solvent with stirring till complete dissolution of the silk fibroin and the high-molecular polymer so as to acquiring a spinning solution; a step 2, performing electrostatic spinning on the spinning solution obtained from the step 1 so as to acquire a nanofiber membrane, performing steam fumigation treatment by using ethyl alcohol, and performing drying so as to acquire a silk fibroin/ high-molecular polymer composite nanofiber scaffold material; and a step 3, dipping the silk fibroin/ high-molecular polymer composite nanofiber scaffold material obtained from the step 2 in a graphene oxide dispersion liquid, taking out the silk fibroin/ high-molecular polymer composite nanofiber scaffold material, and drying the silk fibroin/ high-molecular polymer composite nanofiber scaffold material so as to acquire the composite nanofiber tissue engineering scaffold. The composite nanofiber tissue engineering scaffold is high in mechanical property, can provide biological signals for tissue growth, can promote adherence, propagation and differentiation of cells.
Description
Technical field
The present invention relates to a kind of preparation method based on graphene oxide complex nanometer fibrous tissue engineering rack.
Background technology
In recent years, increasing with aging population and injury gained in sports, the defect of organ and tissue or exhaustion are to occur
For problem that is frequent, most destructive and spending costliness the most.The recovery of regeneration and function is always perplexs clinic
One difficult problem.
The developing into reconstruction or repair mankind's disease damage tissue and organ provides and effectively controls of organizational project and regenerative medicine
Treatment means.One of key factor being related to decision organizational project success or failure of its timbering material.Maximum limit is answered in the design of timbering material
Spend bionical human body cell epimatrix (ECM) structure and functional characteristics, the biocompatibility issues of ability effectively solving material.Preferable
Timbering material not only need the mechanics of appropriateness be cell growth provide three-dimensional support, also need to be conducive to cell to glue on its surface
Attached, propagation and migrate, promote body regeneration with repair.Therefore, from the desired tissue of structurally and functionally bionic extracellular matrix
Engineering rack has wide DEVELOPMENT PROSPECT.Silk-fibroin is a kind of natural copolymerization extracted from silkworm, wild silkworm and spider silk
Thing, it is a kind of natural activity egg as a kind of natural fiber albumen containing 18 kinds of amino acid to the great nutritive value of human body
In vain, there is relatively low immunogenicity, good biocompatibility, itself and its catabolite are nontoxic to cell and body, no
Meeting or less cause inflammation and immunological rejection.Meanwhile, contain special smart ammonia in the Tussah Silk Protein Molecules in tussah
Acid-glycine-asparatate (RGD) tripeptide sequence.RGD sequence is tied with extracellular ligand phase as cell integral membrane element acceptor
The recognition site closing, the interaction between mediated cell and extracellular matrix and cell, cell can be promoted for support
Identification and adhesion.Silk fibroin bracket can be by providing one properly with reference to cell or growth factor for repairing impaired tissue
Microenvironment, serve as interim tissue substitute timbering material.Biologic bracket material therefore based on silk-fibroin is in bio-medical group
There is good application prospect in the field of knitting.But due to the poor needs that cannot meet function of organization of native protein mechanical property, institute
Applied in biomedical sector with limiting it.
High molecular polymer, due to good mechanical properties, is widely used in organizational project and field of medicine release.But its
Belong to synthesis macromolecular material it is impossible to for tissue growth silk-fibroin and high molecular polymer blending can in conjunction with the advantage of the two,
Timbering material can be made to have mechanical property that is higher and meeting nerve fiber, bio signal can be provided for tissue growth again, promote
Enter adhesion, increment and the differentiation of cell, thus meeting the needs of various tissues.
Graphene oxide is a kind of derivative of Graphene, and microstructure can expand to tens of at any time on lateral dimension
Micron, its lamella contains a lot of oxy radicals such as carboxyl, hydroxyl and epoxy radicals etc., has amphipathic.Graphene oxide as
Interfacial agent generally there are interface, reduces the energy between interface, has superior dispersiveness in water.Especially, aoxidize
The oxygen-content active group chemistry of Graphene is different, is that the interaction and multiple fibroin albumen between provides substantial amounts of knot
Close site.There is also the combination of the non-covalent bonds such as ionic bond and hydrogen bond simultaneously.In addition, graphene oxide has excellent power
Learn performance and good biocompatibility, the mechanics that can be used as Biocomposite material strengthens phase, improves biologic bracket material
Mechanical property.At present, graphene oxide is studied in terms of organizational project and is concentrated mainly on two-dimensional film, nanometer sheet increases as performance
Strong additive and Graphene three-dimensional foam is prepared using chemical gaseous phase deposition technology.Will be multiple based on coating for graphene oxide preparation
The research closing nano fibrous tissue engineering timbering material is rarely reported.
Content of the invention
It is an object of the invention to provide a kind of physical and chemical performance is excellent, the complex nanometer fibrous tissue of good biocompatibility
The preparation method of engineering rack.
In order to achieve the above object, the present invention provides one kind to be based on graphene oxide complex nanometer fibrous tissue engineering rack
Preparation method it is characterised in that include：
Step 1：Fibroin albumen and high molecular polymer are dissolved in solvent, stir to being completely dissolved, obtain spinning solution；
Step 2：The spinning solution that step 1 is obtained carries out electrostatic spinning and obtains nano fibrous membrane, using ethanol or its solution
Carry out suffocating treatment, be dried to obtain fibroin albumen/high molecular polymer composite nano-fiber support material；
Step 3：The fibroin albumen that step 2 is obtained/high molecular polymer composite nano-fiber support material is immersed in oxygen
In graphite alkene dispersion liquid, take out, be dried to obtain complex nanometer fibrous tissue engineering rack.
Preferably, the preparation method of described graphene oxide dispersion includes：Graphene oxide water solution is surpassed
Sound disperses, and vacuum filter goes the removal of impurity, and dilution obtains graphene oxide dispersion.
Preferably, the drying in described step 2 is vacuum drying, and the time is 24 72h.
Preferably, the preparation method of the fibroin albumen in described step 1 includes：By fibroin albumen raw material carry out degumming,
Obtain silk fibroin protein solution after dissolving, dialysis treatment, carry out freeze-drying, obtain pure fibroin albumen.
It is highly preferred that described fibroin albumen raw material is domestic silkworm silk, tussah, spider silk or transgenosis silk, wherein wild
Silk is tussah silk.
Preferably, described fibroin albumen and the mass ratio of high molecular polymer are 1: 99-99: 1.
Preferably, the concentration of described spinning solution is 4-12% (mass volume ratio).
Preferably, the technological parameter of described electrostatic spinning is：Regulation voltage is 8-15KV, spinning head and receiving device it
Between distance (receiving range) be 6-15cm, spinning speed be 0.8-1.2ml/h.
Preferably, the concentration of described graphene oxide dispersion is 0.05-10mg/ml.
Preferably, the dip time in described step 3 is 20min-12h.
Preferably, described high molecular polymer be PCL, PLA, PGA, PLLA, P (LLA-CL), PLGA, PEO, PEUU,
At least one in PVA or PU.
Present invention also offers above-mentioned complex nanometer fibrous tissue engineering rack is manufacturing blood vessel, nerve trachea, bone group
Knit, the application in tendon tissue and skin.
The present invention is blended and adopts composite Nano prepared by electrostatic spinning technique fine by fibroin albumen with high molecular polymer
Dimensional scaffold can be to greatest extent from structurally and functionally bionical human body cell epimatrix, in addition, in its rack surface coating oxidation
Graphene not only makes composite Nano timbering material have large specific surface area, can be combined with fibroin albumen/high molecular polymer
Nano-fiber material is fully contacted, and improves its mechanical performance, is conducive to the conduction of interfacial stress, and can produce with fibroin albumen
Raw synergy, improves the biologically active of composite nano-fiber support material.Its organizational project being expected to become in ideal is made to prop up
Frame.Before the present invention makes, document and the excessively relevant graphene oxide of patent report is not also had to coat fibroin albumen/polyphosphazene polymer
Prepared by compound complex nanometer fibrous tissue engineering rack.The present invention is for bio-medical material and biomedical sector nerve fiber
Engineering field has great importance.
The present invention is excellent with physical and chemical performance, and the silk-fibroin/high molecular polymer composite Nano of good biocompatibility is fine
Tie up as matrix, by physics electrostatic interaction and hydrogen bond action, graphene oxide realize on composite nano fiber matrix from
Assembling, forms, on its surface, the sheet coating that piece footpath is 50nm-200nm, through drying stifling wait process obtain having different dense
The tissue engineering bracket of the fibroin albumen/high molecular polymer of graphene oxide coating of degree.
Compared with prior art, the invention has the beneficial effects as follows：
(1) it is an advantage of the current invention that physical and chemical performance is prepared using electrostatic spinning technique excellent, biocompatibility
Good silk-fibroin/high molecular polymer composite nano fiber scaffold, can in the graphene oxide of its face coat variable concentrations
Make timbering material have mechanical property that is higher and meeting tissue, bio signal can be provided for tissue growth again, promote cell
Adhesion, propagation and differentiation.
(2) there is self assembly in the contact between composite nano-fiber support material and graphene oxide, need not add and appoint
What crosslinking agent and regulation PH, simple to operate, reproducible.
(3) thing of tissue engineering bracket can be controlled by adjusting graphene oxide concentration and dip time, cycle-index
Physicochemical performance.The composite nano-fiber support material of the graphene oxide coating of preparation does not only destroy original nanometer
Fibre structure is so as to still have nano fiber scaffold material high-specific surface area, the advantage of porosity height etc., simultaneously also with fibroin egg
White generation acts synergistically, and improves the biologically active of timbering material on the basis of original.Make it in terms of neural tissue engineering
There is application prospect well；
(4) have broad application prospects：Can apply to Various Tissues reparation：Blood vessel, nerve trachea, bone tissue, flesh
Tendinous tissue, skin etc.
(5) present invention is simple to operate, is capable of industrialized production, and materials safety is reliable not to be harmful to using to any other
Poisonous chemical reagent, easily realizes industrialized production.
Brief description
Fig. 1 is the graphite oxide ene coatings tussah silk fibroin/PLA of variable concentrations in comparative example 1 and embodiment 1-3
Polycaprolactone composite nano fiber scaffold optics picture：Composite nano fiber scaffold (b) the coating oxidation Graphene of (a) non-coating
0.5mg/ml composite nano fiber scaffold, (c) coating oxidation Graphene 1.0mg/ml composite nano fiber scaffold, (d) coating oxygen
Graphite alkene 1.5mg/ml composite nano fiber scaffold；
Fig. 2 is the graphite oxide ene coatings tussah silk fibroin/PLA of variable concentrations in comparative example 1 and embodiment 1-4
Polycaprolactone composite nano fiber scaffold scanning electron microscopic picture：Composite nano fiber scaffold (b) the coating oxidation stone of (a) non-coating
Black alkene 0.5mg/ml composite nano fiber scaffold, (c) coating oxidation Graphene 1.0mg/ml composite nano fiber scaffold, (d) applies
Layer graphene oxide 1.5mg/ml composite nano fiber scaffold (e) coating oxidation Graphene 2.0mg/ml composite nano fiber props up
Frame.
Fig. 3 is the graphite oxide ene coatings tussah silk fibroin/polylactic acid poly of comparative example 1 and embodiment 1-4 variable concentrations
Caprolactone composite nano fiber scaffold mechanical property；
Fig. 4 is the graphite oxide ene coatings tussah silk fibroin/polylactic acid poly of comparative example 1 and embodiment 1-4 variable concentrations
The Raman spectrum of caprolactone composite nano fiber scaffold.Composite nano fiber scaffold (b) the coating oxidation Graphene of (a) non-coating
0.5mg/ml composite nano fiber scaffold, (c) coating oxidation Graphene 1.0mg/ml composite nano fiber scaffold, (d) coating oxygen
Graphite alkene 1.5mg/ml composite nano fiber scaffold (e) coating oxidation Graphene 2.0mg/ml composite nano fiber scaffold.
Fig. 5 is that the graphene oxide of variable concentrations described in comparative example 1 and embodiment 1-4 for the schwann cell (SCs) applies
The scanning electron microscopic picture of layer tussah silk fibroin/polylactic acid poly caprolactone composite nano fiber scaffold growth adhesion.(a) non-coating
Composite nano fiber scaffold (b) coating oxidation Graphene 0.5mg/ml composite nano fiber scaffold, (c) coating oxidation Graphene
1.0mg/ml composite nano fiber scaffold, (d) coating oxidation Graphene 1.5mg/ml composite nano fiber scaffold (e) coating oxidation
Graphene 2.0mg/ml composite nano fiber scaffold.
Specific embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than restriction the scope of the present invention.In addition, it is to be understood that after having read the content of present invention instruction, people in the art
Member can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited
Scope.
In various embodiments of the present invention, high molecular polymer used is PLA-polycaprolactone, and it is commercially available prod, its point
Son amount Mn is 300,000, and wherein, the mol ratio of lactic acid units and caprolactone units is 50：50.
Graphene oxide water solution in various embodiments of the present invention is commercially available prod, and concentration is 2mg/ml.
Comparative example 1
A kind of preparation method based on graphene oxide complex nanometer fibrous tissue engineering rack, concretely comprises the following steps：
1st, by tussah silk under 95～100 DEG C of environment, it is placed in Na containing 5g/L2CO3Solution in degumming 3 times, every time
30min, bath raio 1: 50.Tussah silk peptide fiber, 60 DEG C of drying are obtained after degumming.Tussah silk peptide fiber is placed in full by bath raio 1: 10
In the LiSCN solution of sum, at 50 DEG C ± 2 DEG C, dissolve 70min, the tussah silk fibroin solution of acquisition loads trapped molecular weight and is
In the bag filter of 8-10KDa, deionized water is dialysed 3d, freeze-dried obtains tussah silk fibroin.
2nd, weigh fibroin albumen 0.125g, high molecular polymer 0.375g, be dissolved in 5ml hexafluoroisopropanol, with certain
Speed magnetic agitation, to being completely dissolved, obtains the spinning solution that concentration is 10% (w/v), carries out electrostatic spinning, with the smooth bag of aluminium foil
Twine 5x5cm2Receiver board receives nanofiber, spinning condition：12 kilovolts of voltage, receiving range 8cm, spinning speed 1.2ml/h, greatly
Certain thickness nano fibrous membrane is received on aluminium foil after about 4h.Put into after taking off in closed container, with volume fraction 75%
Ethanol is 25 DEG C, carries out suffocating treatment 24h under conditions of standard atmospheric pressure and carry out crosslinking in temperature, after being disposed in temperature is
25 DEG C, vacuum be -30KPa under conditions of be vacuum dried 48h, remove residual solvent, obtain fibroin albumen/high molecular polymer
Composite nano-fiber support material.
3rd, the composite nano fiber scaffold obtaining is immersed in 3min in pure water, circulation dipping 5 times, the phosphorus being 7.4 with pH
Phthalate buffer (PBS) cleans for several times, takes out, is finally dried to obtain complex nanometer fibrous tissue engineering rack.
Embodiment 1
A kind of preparation method based on graphene oxide complex nanometer fibrous tissue engineering rack, concretely comprises the following steps：
1st, by tussah silk under 95～100 DEG C of environment, it is placed in Na containing 5g/L2CO3Solution in degumming 3 times, every time
30min, bath raio 1: 50.Tussah silk peptide fiber, 60 DEG C of drying are obtained after degumming.Tussah silk peptide fiber is placed in full by bath raio 1: 10
In the LiSCN solution of sum, at 50 DEG C ± 2 DEG C, dissolve 70min, the tussah silk fibroin solution of acquisition loads trapped molecular weight and is
In the bag filter of 8-10KDa, deionized water is dialysed 3d, freeze-dried obtains tussah silk fibroin.
2nd, weigh fibroin albumen 0.125g, high molecular polymer 0.375g, be dissolved in 5ml hexafluoroisopropanol, with certain
Speed magnetic agitation, to being completely dissolved, obtains the spinning solution that concentration is 10% (w/v).Carry out electrostatic spinning, with the smooth bag of aluminium foil
Twine 5x5cm2Receiver board receives nanofiber, spinning condition：12 kilovolts of voltage；Receiving range 8em, spinning speed 1.2ml/h, greatly
Certain thickness nano fibrous membrane is received on aluminium foil after about 4h.Put into after taking off in closed container, with volume fraction 75%
Ethanol is 25 DEG C, carries out suffocating treatment 24h under conditions of standard atmospheric pressure and carry out crosslinking in temperature, after being disposed in temperature is
25 DEG C, vacuum be -30KPa under conditions of be vacuum dried 48h, remove residual solvent, obtain fibroin albumen/high molecular polymer
Composite nano-fiber support material.
3rd, graphene oxide water solution is carried out ultrasonic disperse, vacuum filter goes the removal of impurity, is diluted to 0.5mg/ml, obtains
Graphene oxide dispersion；The composite nano fiber scaffold obtaining is immersed in 30min in graphene oxide dispersion, circulation leaching
The phosphate buffer (PBS) that stain 5 times is 7.4 with pH cleans for several times, takes out, is finally dried to obtain complex nanometer fibrous tissue
Engineering rack, its optics picture is as shown in figure 1, scanning electron microscopic picture is as shown in Figure 2.
Embodiment 2
A kind of preparation method based on graphene oxide complex nanometer fibrous tissue engineering rack, concretely comprises the following steps：
1st, by tussah silk under 95～100 DEG C of environment, it is placed in containing 5g/LNa2CO3Solution in degumming 3 times, each 30min,
Bath raio 1: 50.Tussah silk peptide fiber, 60 DEG C of drying are obtained after degumming.Tussah silk peptide fiber is placed in saturation by bath raio 1: 10
In LiSCN solution, dissolve 70min at 50 DEG C ± 2 DEG C, it is 8- that the tussah silk fibroin solution of acquisition loads trapped molecular weight
In the bag filter of 10KDa, deionized water is dialysed 3d, freeze-dried obtains tussah silk fibroin.
2nd, weigh fibroin albumen 0.125g, high molecular polymer 0.375g, be dissolved in 5ml hexafluoroisopropanol, with certain
Speed magnetic agitation, to being completely dissolved, obtains the spinning solution that concentration is 10% (w/v).Carry out electrostatic spinning, with the smooth bag of aluminium foil
Twine 5x5cm2Receiver board receives nanofiber, spinning condition：12 kilovolts of voltage；Receiving range 8cm, spinning speed 1.2ml/h, greatly
Certain thickness nano fibrous membrane is received on aluminium foil after about 4h.Put into after taking off in closed container, with volume fraction 75%
Ethanol is 25 DEG C, carries out suffocating treatment 24h under conditions of standard atmospheric pressure and carry out crosslinking in temperature, after being disposed in temperature is
25 DEG C, vacuum be -30KPa under conditions of be vacuum dried 48h, remove residual solvent, obtain fibroin albumen/high molecular polymer
Composite nano-fiber support material.
3rd, graphene oxide water solution is carried out ultrasonic disperse, vacuum filter goes the removal of impurity, is diluted to 1.0mg/ml, obtains
Graphene oxide dispersion；The composite nano fiber scaffold obtaining is immersed in 30min in graphene oxide dispersion, circulation leaching
Stain 5 times.The phosphate buffer being 7.4 with pH (PBS) cleans for several times, takes out, is finally dried to obtain complex nanometer fibrous tissue
Engineering rack, its optics picture is as shown in figure 1, scanning electron microscopic picture is as shown in Figure 2.
Embodiment 3
A kind of preparation method based on graphene oxide complex nanometer fibrous tissue engineering rack, concretely comprises the following steps：
1st, by tussah silk under 95～100 DEG C of environment, it is placed in Na containing 5g/L2CO3Solution in degumming 3 times, every time
30min, bath raio 1: 50.Tussah silk peptide fiber, 60 DEG C of drying are obtained after degumming.Tussah silk peptide fiber is placed in full by bath raio 1: 10
In the LiSCN solution of sum, at 50 DEG C ± 2 DEG C, dissolve 70min, the tussah silk fibroin solution of acquisition loads trapped molecular weight and is
In the bag filter of 8-10KDa, deionized water is dialysed 3d, freeze-dried obtains tussah silk fibroin.
2nd, weigh fibroin albumen 0.125g, high molecular polymer 0.375g, be dissolved in 5ml hexafluoroisopropanol, with certain
Speed magnetic agitation, to being completely dissolved, obtains the spinning solution that concentration is 10% (w/v).Carry out electrostatic spinning, with the smooth bag of aluminium foil
Twine 5x5cm2Receiver board receives nanofiber, spinning condition：12 kilovolts of voltage；Receiving range 8cm, spinning speed 1.2ml/h, greatly
Certain thickness nano fibrous membrane is received on aluminium foil after about 4h.Put into after taking off in closed container, with volume fraction 75%
Ethanol is 25 DEG C, carries out suffocating treatment 24h under conditions of standard atmospheric pressure and carry out crosslinking in temperature, after being disposed in temperature is
25 DEG C, vacuum be -30KPa under conditions of be vacuum dried 48h, remove residual solvent, obtain fibroin albumen/high molecular polymer
Composite nano-fiber support material.
3rd, the graphene oxide water solution of gained is carried out ultrasonic disperse, vacuum filter goes the removal of impurity, is diluted to 1.5mg/
Ml, obtains graphene oxide dispersion；The composite nano fiber scaffold obtaining is immersed in graphene oxide dispersion
30min, circulation dipping 5 times, the phosphate buffer being 7.4 with pH (PBS) cleans for several times, takes out, is finally dried to obtain compound
Nano fibrous tissue engineering support, its optics picture is as shown in figure 1, scanning electron microscopic picture is as shown in Figure 2.
Embodiment 4
A kind of preparation method based on graphene oxide complex nanometer fibrous tissue engineering rack, concretely comprises the following steps：
1st, by tussah silk under 95～100 DEG C of environment, it is placed in Na containing 5g/L2CO3Solution in degumming 3 times, every time
30min, bath raio 1: 50.Tussah silk peptide fiber, 60 DEG C of drying are obtained after degumming.Tussah silk peptide fiber is placed in full by bath raio 1: 10
In the LiSCN solution of sum, at 50 DEG C ± 2 DEG C, dissolve 70min, the tussah silk fibroin solution of acquisition loads trapped molecular weight and is
In the bag filter of 8-10KDa, deionized water is dialysed 3d, freeze-dried obtains tussah silk fibroin.
2nd, weigh fibroin albumen 0.125g, high molecular polymer 0.375g, be dissolved in 5ml hexafluoroisopropanol, with certain
Speed magnetic agitation, to being completely dissolved, obtains the spinning solution that concentration is 10% (w/v).Carry out electrostatic spinning, with the smooth bag of aluminium foil
Twine 5x5cm2Receiver board receives nanofiber, spinning condition：12 kilovolts of voltage；Receiving range 8cm, spinning speed 1.2ml/h, greatly
Certain thickness nano fibrous membrane is received on aluminium foil after about 4h.Put into after taking off in closed container, with volume fraction 75%
Ethanol is 25 DEG C, carries out suffocating treatment 24h under conditions of standard atmospheric pressure and carry out crosslinking in temperature, after being disposed in temperature is
25 DEG C, vacuum be -30KPa under conditions of be vacuum dried 48h, remove residual solvent.Obtain fibroin albumen/high molecular polymer
Composite nano-fiber support material
3rd, graphene oxide water solution is carried out ultrasonic disperse, vacuum filter goes the removal of impurity, is diluted to 2.0mg/ml, obtains
Graphene oxide dispersion；The composite nano fiber scaffold obtaining is immersed in 30min in graphene oxide dispersion, circulation leaching
The phosphate buffer (PBS) that stain 5 times is 7.4 with pH cleans for several times, takes out, is finally dried to obtain complex nanometer fibrous tissue
Engineering rack.
The nano fiber scaffold of the graphite oxide ene coatings described in comparative example 1 and embodiment 1-3 its optics picture such as Fig. 1 institute
Show, its result shows that the timbering material of non-coating graphene oxide is white, and coating oxidation Graphene timbering material is brown, its
Show that graphene oxide is present on nano fiber scaffold material.
Its scanning electron microscopic picture of nano fiber scaffold of graphite oxide ene coatings described in comparative example 1 and embodiment 1-4 is such as
Shown in Fig. 2.Its result shows the scaffold fibers flat smooth of coating oxidation Graphene, containing graphene oxide scaffold fibers surface
Coarse have lamella to cover, and its result display graphene oxide coating and will not destroy Nanowire on nano fiber scaffold material
The structure of dimension.
Nano fiber scaffold mechanical property such as Fig. 3 institute of the graphite oxide ene coatings described in comparative example 1 and embodiment 1-4
Show, the rectangle sample that its timbering material is cut into 3cm x1cm is detected on pulling force tester for elongation, the display of its result applies
After layer graphene oxide, the mechanical property of composite nano fiber scaffold is substantially better than the nano-composite fiber support of non-coating, fracture
Percentage elongation and fracture strength all increase.
Nano fiber scaffold Raman spectroscopy such as Fig. 4 of graphite oxide ene coatings described in comparative example 1 and embodiment 1-4
Shown, different samples are adopted Raman spectrum analysis instrument to test, its result shows graphene oxide support D peak and the G peak of coating
The ratio of graphene oxide is less than 1, shows graphene oxide coating to nano fiber scaffold material.
Schwann cell (SCs) described in comparative example 1 and embodiment 1-4 is in the graphite oxide ene coatings tussah silk of variable concentrations
The scanning electron microscopic picture of fibroin/polylactic acid poly caprolactone composite nano fiber scaffold growth adhesion will be as shown in figure 5, will not be same
Product carry out sterilization treatment for the culture of schwann cell, specially carry out the preparation of each sample using 24 hole card punch, by individual sample
Product are placed in 24 well culture plates, and using cover glass as control sample, Jiang Geban is placed in the alcohol steam cylinder that volume fraction is 75%
It is placed in superclean bench after carrying out sterilization treatment 2h standby, clean support and blank culture plate 3 times successively using PBS, with this
Remove non-volatile alcohol steam.The planting density of schwann cell is every hole 1 × 104Individual, then by the DMEM preparing culture medium
(1% is dual anti-for 89%DMEM, 10% hyclone) sequentially adds in each hole, puts into 37 DEG C and 5%CO2Incubator in incubation,
Above operating procedure all completes in super-clean bench.Carry out dehydration of alcohol process, for the shooting of Electronic Speculum, its result shows coating oxygen
The timbering material of graphite alkene is more beneficial for cell growth adhesion with the nano fiber scaffold material of non-coating.
Claims (10)
1. a kind of preparation method based on graphene oxide complex nanometer fibrous tissue engineering rack is it is characterised in that include：
Step 1：Fibroin albumen and high molecular polymer are dissolved in solvent, stir to being completely dissolved, obtain spinning solution；
Step 2：The spinning solution that step 1 is obtained carries out electrostatic spinning and obtains nano fibrous membrane, is carried out using ethanol or its solution
Suffocating treatment, is dried to obtain fibroin albumen/high molecular polymer composite nano-fiber support material；
Step 3：The fibroin albumen that step 2 is obtained/high molecular polymer composite nano-fiber support material is immersed in oxidation stone
In black alkene dispersion liquid, take out, be dried to obtain complex nanometer fibrous tissue engineering rack.
2. the preparation method of complex nanometer fibrous tissue engineering rack as claimed in claim 1 is it is characterised in that described oxygen
The preparation method of graphite alkene dispersion liquid includes：Graphene oxide water solution is carried out ultrasonic disperse, vacuum filter goes the removal of impurity,
Dilution, obtains graphene oxide dispersion.
3. the preparation method of complex nanometer fibrous tissue engineering rack as claimed in claim 1 is it is characterised in that described step
Drying in rapid 2 is vacuum drying, and the time is 24-72h.
4. the preparation method of complex nanometer fibrous tissue engineering rack as claimed in claim 1 is it is characterised in that described step
The preparation method of the fibroin albumen in rapid 1 includes：Fibroin albumen raw material is carried out degumming, dissolving, obtains fibroin after dialysis treatment
Protein solution, carries out freeze-drying, obtains pure fibroin albumen.
5. the preparation method of complex nanometer fibrous tissue engineering rack as claimed in claim 4 is it is characterised in that described silk
Fibroin raw material is domestic silkworm silk, tussah, spider silk or transgenosis silk.
6. the preparation method of complex nanometer fibrous tissue engineering rack as claimed in claim 1 is it is characterised in that described silk
Fibroin is 1: 99-99: 1 with the mass ratio of high molecular polymer.
7. the preparation method of complex nanometer fibrous tissue engineering rack as claimed in claim 1 is it is characterised in that described spinning
The concentration of silk liquid is 4-12%.
8. the preparation method of complex nanometer fibrous tissue engineering rack as claimed in claim 1 is it is characterised in that described is quiet
The technological parameter of Electrospun is：Regulation voltage is 8-15KV, and the distance between spinning head and receiving device are 6-15cm, spinning speed
Rate is 0.8-1.2ml/h.
9. the preparation method of complex nanometer fibrous tissue engineering rack as claimed in claim 1 is it is characterised in that described oxygen
The concentration of graphite alkene dispersion liquid is 0.05-10mg/ml.
10. the complex nanometer fibrous tissue engineering rack any one of claim 1-9 manufacture blood vessel, nerve trachea,
Application in bone tissue, tendon tissue and skin.
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