CN106400312A - Method for preparing conductive composite nanofiber nervous tissue engineering scaffold based on graphene - Google Patents

Method for preparing conductive composite nanofiber nervous tissue engineering scaffold based on graphene Download PDF

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CN106400312A
CN106400312A CN201610806967.8A CN201610806967A CN106400312A CN 106400312 A CN106400312 A CN 106400312A CN 201610806967 A CN201610806967 A CN 201610806967A CN 106400312 A CN106400312 A CN 106400312A
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composite nano
graphene
poly
tissue engineering
silk fibroin
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莫秀梅
王娟
孙彬彬
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Donghua University
National Dong Hwa University
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Donghua University
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    • D01D5/0007—Electro-spinning
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Abstract

The invention provides a method for preparing a conductive composite nanofiber nervous tissue engineering scaffold based on graphene. The method includes the following steps: a step 1, dissolving tussur silk fibroin and poly (lactic acid)-poly caprolactone in a solvent with stirring till complete dissolution of the tussur silk fibroin and the poly (lactic acid)-poly caprolactone 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 tussur silk fibroin/ poly (lactic acid)-poly caprolactone composite nanofibers; and a step 3, dipping the tussur silk fibroin/ poly (lactic acid)-poly caprolactone composite nanofiber scaffold material obtained from the step 2 in a graphene oxide dispersion liquid, taking out the tussur silk fibroin/ poly (lactic acid)-poly caprolactone composite nanofiber scaffold material, cleaning the tussur silk fibroin/ poly (lactic acid)-poly caprolactone composite nanofiber scaffold material, soaking the tussur silk fibroin/ poly (lactic acid)-poly caprolactone composite nanofiber scaffold material in an ascorbic acid solution, taking out the tussur silk fibroin/ poly (lactic acid)-poly caprolactone composite nanofiber scaffold material, and cleaning the tussur silk fibroin/ poly (lactic acid)-poly caprolactone composite nanofiber scaffold material so as to acquiring the conductive composite nanofiber nervous tissue engineering scaffold based on the graphene. The method is simple to operate, is excellent in repeatability, and can provide a new thought for nerve defect repairing.

Description

Preparation based on graphene conductive type composite nano fiber neural tissue engineering support
Technical field
The present invention relates to a kind of preparation method based on graphene conductive type composite nano fiber neural tissue engineering support, Belong to biomedical material and be related to neural tissue engineering reparation field.
Background technology
In recent years, various nerve fiber defects bring great puzzlement and misery to patient.The regeneration of nerve fiber with Repair the focus being always international research, can be using nerve autograft, gene therapy, growth factor-induced and organizational project etc. Technology is treating neurologic defect and reparation.Wherein nerve fiber offer is rebuild or repaired to organizational project and developing into of regenerative medicine Effective treatment means, bring dawn for clinical neurologic defect reparation.
The design of Nerve Scaffold material not only will bionical human body cell epimatrix (ECM) structure and function solve in structure Timbering material and the biocompatibility issues of cell, but also should functionally guide neural tissue cell migration and promote to make With.The change being at all film potential on nerve fibre of nerve conduction, nerve conduction is the conduction of electric signal, therefore from knot Structure and functionally structure nerve regneration timbering material have wide DEVELOPMENT PROSPECT.
Tussah silk fibroin is a kind of natural activity albumen, has relatively low immunogenicity, good biocompatibility, its Itself and its catabolite are nontoxic to cell and body, or less will not cause inflammation and immunological rejection.It is except having Outside the advantage of common silk-fibroin, in molecular structure, also contain special arginine-glycine-aspartic acid acid (RGD) tripeptides sequence Row can promote cell to show the unrivaled advantage of other silk-fibroins for the identification of support and adhesion.Make it in biological doctor Of increased attention with the application study in field of tissue engineering technology with material.However, repairing with nerve fiber in application Again and during regeneration, its mechanical property need to improve further.
PLA-polycaprolactone is the copolymer of PLA and polycaprolactone, because it has good biodegradable Property and good mechanical properties, are widely used in field of tissue engineering technology.But it belongs to synthesis macromolecular material it is impossible to be nerve fiber Growth provides required bio signal, and hydrophily is poor simultaneously, is unfavorable for adhesion and the growth of cell.However, electrostatic spinning technique Timbering material can be made to have mechanical property that is higher and meeting nerve fiber in conjunction with the two advantage, and can be tissue life Long adhesion, increment and the differentiation providing bio signal, promoting nerve cell, thus meet the needs of nerve fiber.
Graphene is by sp2The two-dimensional layer Nanowire with cellular crystal lattice structure of hydbridized carbon atoms composition Dimension, as a kind of novel nano-material, has extremely extensive potential using value in biomedical sector.It has extremely excellent Different electric conductivity, mechanics and good biocompatibility, unique physicochemical properties can provide the extracellular of cell growth Matrix condition, this is its very big advantage as tissue engineering bracket material.At present, scholar is had to utilize Graphene Excellent performance prepares graphite alkene and its derivative strengthens three-dimensional compound rest and is used for bone tissue reparation and regeneration, but sharp With the conductive composite nano fiber neural tissue engineering support of the excellent electric conductivity preparation of Graphene also do not have pertinent literature and Patent is reported.
Content of the invention
It is an object of the invention to provide a kind of based on graphene conductive type composite nano fiber neural tissue engineering support Preparation method.
In order to achieve the above object, the invention provides a kind of be based on graphene conductive type composite nano fiber nerve fiber The preparation method of engineering rack is it is characterised in that include：
Step 1：Tussah silk fibroin and PLA-polycaprolactone are dissolved in solvent, stir to being completely dissolved, spun Silk liquid；
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 tussah silk fibroin/PLA-polycaprolactone composite nano fiber；
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, cleaning, be immersed in ascorbic acid solution, soak 1-3h at 50-70 DEG C, take out, be dried, Obtain based on graphene conductive type composite nano fiber neural tissue engineering support.
Preferably, the preparation method of described tussah silk fibroin includes：Natural tussah silk is carried out degumming, dissolving, thoroughly Analysis obtains tussah silk fibroin solution after processing, and carries out freeze-drying, obtains pure tussah silk fibroin.
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 24-48h.
Preferably, the tussah silk fibroin in described step 1 and the mass ratio of PLA-polycaprolactone are 25: 75-75 ∶25.
Preferably, the concentration of described spinning solution is 8%-10% (mass volume ratio).
Preferably, described electrospinning conditions are：Voltage is 8-15KV, the distance between spinning head and reception device (receiving range) is 6-12cm, and spinning speed is 0.8-1.5ml/h.
Preferably, the concentration of described graphene oxide dispersion is 0.02-10mg/ml.
Preferably, the fibroin albumen that described step 2 obtains/high molecular polymer composite nano-fiber support material dipping Dip time in graphene oxide dispersion is 0.3-12h.
Preferably, the concentration of described ascorbic acid solution is 10-100mM.
Preferably, by adjust the concentration of graphene oxide dispersion, dip time, at least one in dipping number of times Lai Adjust the hydrophilic and hydrophobic based on graphene conductive type composite nano fiber neural tissue engineering support, mechanical property and cell glue Attached property, is adjusted based on graphene conductive type composite nano fiber neural tissue engineering by the concentration adjusting ascorbic acid solution The electric conductivity of support.
Compared with prior art, the invention has the beneficial effects as follows：
1st, one kind of present invention preparation is based on graphene conductive type composite nano fiber Nerve Scaffold preparation method cleverly Prepare using electrostatic spinning technique that physical and chemical performance is excellent, the tussah silk-fibroin/PLA of good biocompatibility-poly- is in oneself Ester composite nano fiber scaffold, Graphene excellent for electric conductivity is restored method and tussah silk-fibroin using simple dipping Compound support frame material contacts, and forms effectively netted interconnection architecture on its timbering material surface, with the tussah silk rich in RGD sequence Albumen produces synergy, improves the biologically active of composite.
2nd, the composite nano-fiber support material tool compared with non-graphene-containing nano fiber scaffold material, based on Graphene There is mechanical property that is higher and meeting nerve fiber, additional electro photoluminescence more can promote differentiation and the propagation of nerve cell.
3rd, the physico of Nerve Scaffold material is controlled by regulation graphene oxide concentration, dip time, dipping number of times Learn performance, hydrophilic and hydrophobic, mechanical property and cell adhesion, control Graphene also by adjusting reducing agent ascorbic acid concentrations Former degree is thus adjust the electric conductivity of its timbering material.
4th, tussah silk-fibroin/PLA-polycaprolactone composite nano-fiber support material and Graphene pass through physics electrostatic Interact and hydrogen bond action occurs self assembly, do not destroy original nanofibrous structures so as to still have nanofiber Timbering material high-specific surface area, the advantage of porosity height etc..
5th, the inventive method is simple, materials safety reliable not using to any other harmful poisonous chemical reagent, Make it have application prospect well in terms of neural tissue engineering, easily realize industrialized production.
6th, the present invention will be compound using electrostatic spinning technique preparation to tussah silk fibroin and PLA-polycaprolactone blending Nano fiber scaffold can be to greatest extent from structurally and functionally bionical human body cell epimatrix, in addition, adopting immersion reduction method Coating graphite alkene has further the advantage that：(1) Graphene of coating has excellent electric conductivity, can be stimulated by additional band and promote Enter the differentiation of nerve cell；(2) Graphene has larger specific surface area, can be with tussah silk-fibroin composite nano-fiber material It is fully contacted, produce synergy, improve the biologically active of composite nano fiber nerve fiber timbering material；(3) low interpolation In the case of amount, it is remarkably improved the mechanical performance of timbering material；(4) Graphene has certain antibiotic property, advantageously reduces The postoperative infection rate of implant.In conjunction with the anti-technology of electrostatic and Graphene premium properties so as to be based on graphene conductive nanofiber Timbering material is expected to become the neural tissue engineering support in ideal.Before the present invention makes, also there is no document and patent report Cross relevant graphene oxide coating fibroin albumen/high molecular polymer complex nanometer fibrous tissue engineering rack preparation.This grinds Study carefully and bio-medical material and field of tissue engineering technology are had great importance.
7th, the conductive nanometer fibrous framework that the present invention prepares still keeps the feature of nano fiber scaffold, not only contains together The tussah silk fibroin of RGD sequence produces the biologically active of the timbering material that synergy improves, and leads from additional electro photoluminescence simultaneously Electric polymer graphite alkene promotes the reparation speed of nerve so as to have good application prospect in terms of neural tissue engineering.
Brief description
Fig. 1 is based on variable concentrations graphene conductive type composite nano fiber nerve group described in comparative example 1 and embodiment 1-3 Knit the optics picture of support：A () non-graphene-containing composite nano fiber scaffold (b) Graphene 0.5mg/ml composite nano fiber props up Frame, (c) Graphene 1.0mg/ml composite nano fiber scaffold, (d) Graphene 1.5mg/ml composite nano fiber scaffold；
Fig. 2 is based on variable concentrations graphene conductive type composite nano fiber nerve group described in comparative example 1 and embodiment 1-4 Knit the scanning electron microscopic picture of support：A () non-graphene-containing composite nano fiber scaffold (b) Graphene 0.5mg/ml composite Nano is fine Dimensional scaffold, (c) Graphene 1.0mg/ml composite nano fiber scaffold, (d) Graphene 1.5mg/ml composite nano fiber scaffold (e) Graphene 2.0mg/ml composite nano fiber scaffold.
Fig. 3 is to be based on graphene conductive type composite nano fiber nerve fiber support cyclic voltammetric matching described in embodiment 3 Curve synoptic diagram；
Fig. 4 is based on variable concentrations graphene conductive type composite nano fiber nerve group described in comparative example 1 and embodiment 1-4 Knit the Raman spectrum of support.A () non-graphene-containing composite nano fiber scaffold (b) Graphene 0.5mg/ml composite nano fiber props up Frame, (c) Graphene 1.0mg/ml composite nano fiber scaffold, (d) Graphene 1.5mg/ml composite nano fiber scaffold (e) graphite Alkene 2.0mg/ml composite nano fiber scaffold.
Fig. 5 is for comparative example 1 and schwann cell (SCs) described in embodiment 1-4 multiple based on variable concentrations graphene conductive type Close the scanning electron microscopic picture of nano-fiber nerve organization bracket growth adhesion.(a) non-graphene-containing composite nano fiber scaffold (b) Graphene 0.5mg/ml composite nano fiber scaffold, (c) Graphene 1.0mg/ml composite nano fiber scaffold, (d) Graphene 1.5mg/ml composite nano fiber scaffold (e) 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 conductive type composite nano fiber neural tissue engineering support, concrete steps For：
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 tussah silk fibroin 0.25g, PLA-polycaprolactone 0.75g, be dissolved in 10ml solvent hexafluoroisopropanol In, with certain speed magnetic agitation to being completely dissolved, obtain the spinning solution that concentration is 10% (w/v), spinning solution is carried out quiet Electrospun, with smooth looping 6x6cm of aluminium foil2Receiver board receives nanofiber, spinning condition：12 kilovolts of voltage；Receiving range 10cm, aluminium foil after spinning speed 1.0ml/h, about 4h receives certain thickness nano fibrous membrane.Put into airtight after taking off In container, it is 25 DEG C, carries out suffocating treatment 24h under conditions of standard atmospheric pressure and carry out in temperature with the ethanol of volume fraction 75% Crosslinking, after being disposed temperature be 25 DEG C, vacuum be -30KPa under conditions of be vacuum dried 48h, remove residual solvent, obtain To tussah silk fibroin/PLA-polycaprolactone composite nano fiber.
3rd, the fibroin albumen obtaining/high molecular polymer composite nano-fiber support material is immersed in 30min in pure water, Circulation dipping 5 times, takes out, and the phosphate buffer being 7.4 with pH (PBS) cleans for several times, and being immersed in concentration is the anti-bad of 20mM In hematic acid solution, soak 3h at 70 DEG C, take out, be dried, obtain composite nano fiber neural tissue engineering support.
Embodiment 1
A kind of preparation method based on graphene conductive type composite nano fiber neural tissue engineering support, concrete steps For：
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 tussah silk fibroin 0.25g, PLA-polycaprolactone 0.75g, be dissolved in 10ml hexafluoroisopropanol, with Certain speed magnetic agitation, to being completely dissolved, obtains the spinning solution that concentration is 10% (w/v).Spinning solution is carried out Static Spinning Silk, receives nanofiber, spinning condition with aluminium foil smooth looping 6x6cm2 receiver board：12 kilovolts of voltage, receiving range 10cm, spins Silk speed 1.0ml/h, aluminium foil after about 4h receives certain thickness nano fibrous membrane.Put into after taking off in closed container, It is 25 DEG C, carries out suffocating treatment 24h under conditions of standard atmospheric pressure and carry out crosslinking in temperature with the ethanol of volume fraction 75%, place Reason finish after temperature be 25 DEG C, vacuum be -30KPa under conditions of be vacuum dried 48h, remove residual solvent, obtain tussah Fibroin albumen/PLA-polycaprolactone composite nano fiber.
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 fibroin albumen obtaining/high molecular polymer composite nano-fiber support material is immersed in oxidation 30min in graphene dispersing solution, circulation dipping 5 times, takes out, the phosphate buffer being 7.4 with pH (PBS) cleans for several times, leaching Bubble, takes out for, in the ascorbic acid solution of 35mM, soaking 3h at 70 DEG C in concentration, is dried, and obtains multiple based on graphene conductive type Close nano-fiber nerve tissue engineering bracket.
Embodiment 2
A kind of preparation method based on graphene conductive type composite nano fiber neural tissue engineering support, concrete steps For：
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 tussah silk fibroin 0.25g, PLA-polycaprolactone 0.75g, be dissolved in 10ml hexafluoroisopropanol, with Certain speed magnetic agitation, to being completely dissolved, obtains the spinning solution that concentration is 10% (w/v).Spinning solution is carried out Static Spinning Silk, receives nanofiber, spinning condition with aluminium foil smooth looping 6x6cm2 receiver board：12 kilovolts of voltage, receiving range 10cm, spins Silk speed 1.0ml/h, aluminium foil after about 4h receives certain thickness nano fibrous membrane.Put into after taking off in closed container, It is 25 DEG C, carries out suffocating treatment 24h under conditions of standard atmospheric pressure and carry out crosslinking in temperature with the ethanol of volume fraction 75%, place Reason finish after temperature be 25 DEG C, vacuum be -30KPa under conditions of be vacuum dried 48h, remove residual solvent, obtain tussah Fibroin albumen/PLA-polycaprolactone composite nano fiber.
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 fibroin albumen obtaining/high molecular polymer composite nano-fiber support material is immersed in oxidation 30min in graphene dispersing solution, circulation dipping 5 times, takes out, the phosphate buffer being 7.4 with pH (PBS) cleans for several times, leaching Bubble, takes out for, in the ascorbic acid solution of 50mM, soaking 3h at 70 DEG C in concentration, is dried, and obtains multiple based on graphene conductive type Close nano-fiber nerve tissue engineering bracket.
Embodiment 3
A kind of preparation method based on graphene conductive type composite nano fiber neural tissue engineering support, concrete steps For：
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 tussah silk fibroin 0.25g, PLA-polycaprolactone 0.75g, be dissolved in 10ml hexafluoroisopropanol, with Certain speed magnetic agitation, to being completely dissolved, obtains the spinning solution that concentration is 10% (w/v).Spinning solution is carried out Static Spinning Silk, with smooth looping 6x6cm of aluminium foil2Receiver board receives nanofiber, spinning condition：12 kilovolts of voltage；Receiving range 10cm, spins Silk speed 1.0ml/h, aluminium foil after about 4h receives certain thickness nano fibrous membrane.Put into after taking off in closed container, It is 25 DEG C, carries out suffocating treatment 24h under conditions of standard atmospheric pressure and carry out crosslinking in temperature with the ethanol of volume fraction 75%, place Reason finish after temperature be 25 DEG C, vacuum be -30KPa under conditions of be vacuum dried 48h, remove residual solvent, obtain tussah Fibroin albumen/PLA-polycaprolactone composite nano fiber.
3rd, graphene oxide water solution is carried out ultrasonic disperse, vacuum filter goes the removal of impurity, is diluted to 1.5mg/ml, obtains Graphene oxide dispersion.The fibroin albumen obtaining/high molecular polymer composite nano-fiber support material is immersed in oxidation 30min in graphene dispersing solution, circulation dipping 5 times, takes out, the phosphate buffer being 7.4 with pH (PBS) cleans for several times, leaching Bubble, takes out for, in the ascorbic acid solution of 70mM, soaking 3h at 70 DEG C in concentration, is dried, and obtains multiple based on graphene conductive type Close nano-fiber nerve tissue engineering bracket.Described circulation based on graphene conductive type composite nano fiber nerve fiber support is lied prostrate Peace matched curve is as shown in Figure 4.
Embodiment 4
A kind of preparation method based on graphene conductive type composite nano fiber neural tissue engineering support, concrete steps For：
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 tussah silk fibroin 0.25g, PLA-polycaprolactone 0.75g, be dissolved in 10ml hexafluoroisopropanol, with Certain speed magnetic agitation, to being completely dissolved, obtains the spinning solution that concentration is 10% (w/v).Spinning solution is carried out Static Spinning Silk, receives nanofiber, spinning condition with aluminium foil smooth looping 6x6cm2 receiver board：12 kilovolts of voltage, receiving range 10cm, spins Silk speed 1.0ml/h, aluminium foil after about 4h receives certain thickness nano fibrous membrane.Put into after taking off in closed container, It is 25 DEG C, carries out suffocating treatment 24h under conditions of standard atmospheric pressure and carry out crosslinking in temperature with the ethanol of volume fraction 75%, place Reason finish after temperature be 25 DEG C, vacuum be -30KPa under conditions of be vacuum dried 48h, remove residual solvent, obtain tussah Fibroin albumen/PLA-polycaprolactone composite nano fiber.
3rd, graphene oxide water solution is carried out ultrasonic disperse, vacuum filter goes the removal of impurity, is diluted to 2mg/ml, obtains oxygen Graphite alkene dispersion liquid.The fibroin albumen obtaining/high molecular polymer composite nano-fiber support material is immersed in oxidation stone 30min in black alkene dispersion liquid, circulation dipping 5 times, takes out, the phosphate buffer being 7.4 with pH (PBS) cleans for several times, soaks In concentration for, in the ascorbic acid solution of 100mM, soaking 3h at 70 DEG C, take out, be dried, obtain being combined based on graphene conductive type Nano-fiber nerve tissue engineering bracket.
Graphene conductive type composite nano fiber scaffold optics picture such as Fig. 1 institute described in comparative example 1 and embodiment 1-3 Show, its result shows that the timbering material of non-graphene-containing is white, is black containing Graphene timbering material, it shows Graphene Existed with nano fiber scaffold material on.
Graphene conductive type composite nano fiber scaffold scanning electron microscopic picture 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 non-graphene-containing, has piece containing Graphene scaffold fibers rough surface Layer covers, its show Graphene existed with nano fiber scaffold material on and the structure of nanofiber will not be destroyed.
Fig. 3 is to be based on graphene conductive type composite nano fiber nerve fiber support cyclic voltammetric matching described in embodiment 3 Curve synoptic diagram；Electric conductivity using cyclic voltammetry nanofiber in electrochemical workstation (CHI660D).Sample is cut The sample of growth × wide=30mm × 10mm, tests its result as shown in figure 3, showing containing Graphene by cyclic voltammetry Nano fiber scaffold has good electric conductivity.
The electrical conductivity of the graphene conductive type composite nano fiber scaffold described in comparative example 1 and embodiment 1-4 such as following table institute Show：
Graphene concentration (mg/ml) Electrical conductivity (S/m)
0 0
0.5 1.96x 10-3±5.30x 10-4
1.0 1.35x 10-2±2.73x 10-3
1.5 3.32x10-2±1.31x 10-3
2.0 3.50x10-2±1.03x 10-3
Electric conductivity using cyclic voltammetry nanofiber in electrochemical workstation (CHI660D).Sample is cut into The sample of length × wide=3mm × 10mm, adopts micrometer caliper to measure the average thickness of nano-fiber film, every kind of sample simultaneously Produce 3 Duplicate Samples.According to formula σ=L/RS (σ, electrical conductivity S/cm；R, resistance Ω；S, the sectional area cm of sample2) calculate electricity Conductance simultaneously obtains the mean value of electrical conductivity.Its result shows：The nano fiber scaffold material containing Graphene does not have conduction Property, with the increase of Graphene concentration, nano fiber scaffold material conductivity increases.
Graphene conductive type composite nano fiber scaffold Raman spectrogram such as Fig. 4 institute described in comparative example 1 and embodiment 1-4 Show, different samples are adopted Raman spectrum analysis instrument to test, its result shows graphene oxide support D peak and the G peak oxygen of coating The ratio of graphite alkene is more than 1, shows that Graphene has existed on nano fiber scaffold material.
Schwann cell (SCs) described in comparative example 1 and embodiment 1-4 is given birth in graphene conductive type composite nano fiber scaffold The scanning electron microscopic picture of long adhesion, as shown in figure 5, carry out the preparation of each sample using 24 hole card punch, each sample is placed in 24 holes In culture plate, using cover glass as control sample, Jiang Geban is placed in the alcohol steam cylinder that volume fraction is 75% and carries out at sterilizing It is placed in superclean bench after reason 2h standby, clean support and blank culture plate 3 times successively using PBS, removed with this non-volatile Alcohol steam.The planting density of schwann cell is every hole 1 × 104Individual, then by the DMEM preparing culture medium (89%DMEM, 10% hyclone, 1% is dual anti-) sequentially add in each hole, put into 37 DEG C and 5%CO2Incubator in incubation, above operation step Suddenly all complete in super-clean bench, carry out dehydration of alcohol process, for the shooting of Electronic Speculum, its result shows the support containing Graphene Material 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 conductive type composite nano fiber neural tissue engineering support it is characterised in that Including：
Step 1：Tussah silk fibroin and PLA-polycaprolactone are dissolved in solvent, stir to being completely dissolved, obtain spinning Liquid；
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 tussah silk fibroin/PLA-polycaprolactone composite nano fiber；
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, cleaning, be immersed in ascorbic acid solution, soak 1-3h at 50-70 DEG C, take out, be dried, obtain Based on graphene conductive type composite nano fiber neural tissue engineering support.
2. the preparation side based on graphene conductive type composite nano fiber neural tissue engineering support as claimed in claim 1 Method is it is characterised in that the preparation method of described tussah silk fibroin includes：Natural tussah silk is carried out degumming, dissolving, dialysis Obtain tussah silk fibroin solution after process, carry out freeze-drying, obtain pure tussah silk fibroin.
3. the preparation side based on graphene conductive type composite nano fiber neural tissue engineering support as claimed in claim 1 Method is it is characterised in that the preparation method of described graphene oxide dispersion includes：Graphene oxide water solution is carried out ultrasonic Dispersion, vacuum filter goes the removal of impurity, dilution, obtains graphene oxide dispersion.
4. the preparation side based on graphene conductive type composite nano fiber neural tissue engineering support as claimed in claim 1 It is characterised in that the drying in described step 2 is vacuum drying, the time is 24-48h to method.
5. the preparation side based on graphene conductive type composite nano fiber neural tissue engineering support as claimed in claim 1 Method is it is characterised in that the tussah silk fibroin in described step 1 is 25: 75-75 with the mass ratio of PLA-polycaprolactone: 25.
6. the preparation side based on graphene conductive type composite nano fiber neural tissue engineering support as claimed in claim 1 Method is it is characterised in that the concentration of described spinning solution is 8%-10%.
7. the preparation side based on graphene conductive type composite nano fiber neural tissue engineering support as claimed in claim 1 Method is it is characterised in that described electrospinning conditions are：Voltage is 8-15KV, and the distance between spinning head and reception device are 6-12cm, spinning speed is 0.8-1.5ml/h.
8. the preparation side based on graphene conductive type composite nano fiber neural tissue engineering support as claimed in claim 1 Method is it is characterised in that the concentration of described graphene oxide dispersion is 0.02-10mg/ml.
9. the preparation side based on graphene conductive type composite nano fiber neural tissue engineering support as claimed in claim 1 Method is it is characterised in that fibroin albumen/high molecular polymer composite nano-fiber support material that described step 2 obtains impregnates Dip time in graphene oxide dispersion is 0.3-12h.
10. the preparation side based on graphene conductive type composite nano fiber neural tissue engineering support as claimed in claim 1 Method is it is characterised in that the concentration of described ascorbic acid solution is 10-100mM.
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