CN106924807A - A kind of preparation method and applications for modifying nano-conductive polyaniline heart tissue engineering support - Google Patents
A kind of preparation method and applications for modifying nano-conductive polyaniline heart tissue engineering support Download PDFInfo
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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
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
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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
- A61L27/14—Macromolecular materials
- A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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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
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/225—Fibrin; Fibrinogen
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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
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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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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L89/00—Compositions of proteins; Compositions of derivatives thereof
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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
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
- A61L2300/414—Growth factors
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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
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/20—Materials or treatment for tissue regeneration for reconstruction of the heart, e.g. heart valves
Abstract
The invention discloses a kind of preparation method and application for modifying nano-conductive polyaniline heart tissue engineering support.Fibrin glue is obtained using the fibrin ferment of no cytotoxicity and fibrinogen crosslinked action synthesis first, then with chemical syntheses method, the electrically conductive polyaniline of nanoscale is directly synthesized on Fibrin glue（PANI）, upper angiogenic growth factor VEGF is finally grafted, obtain polyaniline fiber protein gel compound rest.The compound support frame material can promote the new life of growth and promotion body vessel of the cell on support, with good cell compatibility, to cytotoxic effect very little, with good application potential, the support decorative material in heart tissue engineering field is can be applied to, can further be researched and developed as the universality material of heart tissue engineering.
Description
Technical field
The invention belongs to biomedicine technical field.More particularly, to one kind modification nano-conductive polyaniline heart group
The preparation method and applications of weaver's engineering support.
Background technology
Angiocardiopathy is also called circulation system disease, be it is a series of be related to the disease of the circulatory system, mainly including blood high
Pressure, myocardial infarction, angina pectoris and heart failure etc., prolonged and repeated breaking-out are danger, urgency, the severe that can cause death.Painstaking effort
The harm of pipe disease is the present domestic or even world's all questions of common concern.WHO Report points out it now
Become the number one killer in the whole world, the treatment method on it is always just focus of concern.
Cardiovascular patient in most cases needs periodically and continuous contact detects health status, conventional treatment
Means include surgical intervention, medical treatment（Including stem cell transplantation and angiogenesis Therapy）, and meals physiotherapy and body
Educate health exercising.（That is drug therapy and supportive treatment, and its treatment cycle is life-long therapy, cure rate is 60% or so）.Face
On bed, the angiocardiopathy in whole latter stage is finally difficult to control to medicine, and heart transplant is solution best at present.But by
In donor's heart obtain be difficult and limit its extensive use.The reparation of injury of myocardium, it is necessary to suitable autograft tissue with
And suitable vascular canal replaces the blood vessel of inaccessible or lesion, due to the shortage of autograft, artificial graft's thing meet the tendency of and
Raw, however, comparing with natural tissues, also there are some defects in artificial graft's thing, such as easily form thrombus and calcification.For angiocarpy
For system, optimal transplanting tissue should have the ability of good biocompatibility and self growth.Although current stem cell
It is implanted in cardiac treatment and has achieved many infusive progress, but substantial amounts of research finds, simple stem cell transplantation
Improvement to cardiac function is not very good.It is severe mainly due to stem cell transplantation region microenvironment, such as ischemic, lack
Oxygen, inflammatory cell infiltration etc., after simple stem cell transplantation, transplanted cells are difficult in transplanting area stay and survival, therefore how
Promoting delay and survival of the stem cell in transplanting region becomes new study hotspot.
Support is the three-dimensional space environment based on simulation natural extracellular matrix, can provide suitable for the growth metabolism of cell
Suitable environment, while requirement will have enough mechanical strengths again, this is another important composition composition of cardiac muscle tissue engineering, is ground
Study carefully and show that timbering material can improve transplanting microenvironment, promote stem cell to be detained and survive, regulate and control stem cell etc..Can be used for cardiac muscle
The timbering material of organizational project has a lot, such as the common caprolactone of acellular matrix, PGA（PGCL）Deng.However, traditional branch
Frame material haves the shortcomings that certain, and such as mechanical property is weaker, lacks electrical conductivity.In recent years, with the development of nanometer technology,
Nano material is applied to field of tissue engineering technology and obtains obvious with the research that promotion organization is reproduced after being checked with conventional stent material
Progress.There is unique electrophysiological characteristics particularly in view of cardiac muscular tissue, be dispersed with cardiac muscle interweave it is webbed, with fax
Lead the Purkinje's fibers of function.Produce excited after the electricity impulsion that the conducting system of heart that Cardiomyocytes receives is transmitted and shrink
Coupling, myocardium room completes blood-pumping function as an entirety, synchronous.Therefore, preferable cardiac muscle tissue engineering material should
It is the extracellular microenvironment that can simulate cardiac muscle, with certain characteristics of electrical conductivity and mechanical strength.
The content of the invention
The technical problem to be solved in the present invention is the defect and technical deficiency for overcoming above-mentioned prior art, there is provided one kind has
Good cell compatibility, to cytotoxic effect very little, the heart that Myocyte growth can be promoted, promote new vessels regeneration
Dirty tissue engineering bracket.Nano material polyaniline PANI and Fibrin glue are combined to form compound rest material by the present invention
, then be grafted to angiogenic growth factor VEGF on timbering material by material, to promote growth and promotion body of the cell on support
The new life of interior blood vessel, is applied to growth and the cardiac repair of cardiac muscle cell, so that for heart tissue engineering establishes good application
Basis.
It is an object of the invention to provide a kind of preparation method for modifying nano-conductive polyaniline heart tissue engineering support.
The present invention is another object is that the polyaniline fiber protein gel compound rest prepared according to methods described.
Still a further object of the present invention is to provide the application of the polyaniline fiber protein gel compound rest.
Above-mentioned purpose of the present invention is achieved through the following technical solutions：
One kind modification nano-conductive polyaniline heart tissue engineering support（That is polyaniline fiber protein gel compound rest）System
Preparation Method, obtains fibrin gel branch using the fibrin ferment of no cytotoxicity and fibrinogen crosslinked action synthesis first
Frame, then with chemical syntheses method, directly synthesizes the electrically conductive polyaniline of nanoscale on Fibrin glue
（PANI）, upper angiogenic growth factor VEGF is finally grafted, obtain polyaniline fiber protein gel compound rest.
Specifically, the preparation method of the polyaniline fiber protein gel compound rest, is in fibrinogen in synthesis
On the basis of gel scaffold material, by fibrinogen gel scaffold infiltration in the reaction system solution of synthesis PANI, oxygen is allowed
The PANI being combined to " growth " in the middle of the space of support, so as to directly synthesize PANI on gel stent, is grafted
The fibrin prop composite of PANI materials（PANI-Scaffold）, upper angiogenic growth factor VEGF is finally grafted, obtain
Obtain polyaniline fiber protein gel compound rest.
In addition, particularly preferably, the preparation method of the polyaniline fiber protein gel compound rest comprises the following steps：
S1. fibrinogen gel support is prepared
S11. with sugar serum-free DMEM/F12 high（v/v=1:1, PH=7.4）Mixed culture medium solution fibrin is former（FG）It is lyophilized
Powder, is made the colloidal solution of 5~15 mg/ml；Clotting factor solution is added, colloidal mixture is mixed into；
S12. colloidal mixture is uniformly coated on culture plate（24 orifice plates, 500 μ L）It is interior, 35~38 DEG C of 25~35min of standing
（It is preferred that 37 DEG C of standing 30min）, fibrinogen is frozen into the fibrin of translucent gels shape in the presence of clotting factor
Former gel stent（Scaffold）；
S2. nano polyaniline（PANI）Synthesis on fibrinogen gel support
S21. aniline（An）Monomer solution mixes to obtain solution A, ammonium persulfate in adding HCl（APS）Dissolved in HCl, and added
Ethanol is stirred, ice-water bath cooling, obtains solution B；
S22. the fibrinogen gel scaffold infiltration for being prepared by step S1 is in solution B and in ice-water bath that solution A is slow
It is added in solution B（A small amount of lauryl sodium sulfate is properly added if necessary（SDS）, existed with the polyaniline product for increasing generation
Dissolubility in water）, mixing is stirred, fibrinogen gel support adheres to product；Reacted in ice-water bath after stopping stirring；
S23. fibrinogen gel support is taken out after reaction terminates, washs by product with HCl solution, dried, obtained
The fibrin prop composite of PANI materials is grafted, PANI-Scaffold is designated as；
S3. the graft growth factor：The amino of activated growth factor, the carboxyl on activation PANI-Scaffold, by suction-operated
With the effect of amino and carboxyl, growth factor VEGF is grafted on PANI-Scaffold.
Step S3 is specifically：DCC activated growth factors are used, the carboxyl on PANI-Scaffold is activated with EDC, by inhaling
Attached effect and chemical bond, growth factor VEGF is grafted on PANI-Scaffold, and the chemical bond refers to growth
The amino of factor Ⅴ EGF acts on forming amido link with the carboxyl of composite PANI-Scaffold.
Wherein it is preferred to, clotting factor described in step S11 is ox Thrombin plasma（PT）, dissolved with PBS when using, obtain
To clotting factor solution.
Preferably, the HCl in step S21 and S13 is the HCl of 0.05~0.15M.
It is highly preferred that the HCl in step S21 and S13 is the HCl of 0.1M.
Preferably, aniline solution and the volume ratio of HCl are 2 in solution A：45~55.
It is highly preferred that aniline solution and the volume ratio of HCl are 2 in solution A：50.
Preferably, the mass volume ratio of ammonium persulfate and HCl is 9~10g/L in solution B, and HCl and ethanol volume ratio
It is 5：2~4.
It is highly preferred that the mass volume ratio of ammonium persulfate and HCl is 9.6g/L in solution B, and HCl and ethanol volume ratio
It is 5：3.
Preferably, the time stirred described in step S21 is 5~15min.
It is highly preferred that the time stirred described in step S21 is 10min.
Preferably, the speed being slowly added to described in step S22 is 0.2~0.3ml/min.
It is highly preferred that the speed being slowly added to described in step S22 is 0.25ml/min（5ml solution As are added in 20min）.
Preferably, the volume ratio of solution A and solution B described in step S22 is 1：7~9.
It is highly preferred that the volume ratio of solution A and solution B described in step S22 is 1：8.
Preferably, the mixing time that mixing is stirred described in step S22 is 40~80min.
It is highly preferred that the mixing time that mixing is stirred described in step S22 is 60min.
Preferably, the time reacted described in step S22 is 10~15h.
It is highly preferred that the time reacted described in step S22 is 12h.
Furthermore it is preferred that the specific method of step S3 is：
S31. PANI-Scaffold is placed in EDC solution, slowly shakes 1~3h, the carboxyl on activation PANI-Scaffold；
S32. DCC solution is added in growth factor VEGF solution, with activated growth factor；
S33. by the product of step S31（PANI-Scaffold after activating）It is placed in the solution of step S32（After activating
Growth factor solution）In, 1~3h of concussion reaction；
S34. the product of step S33 in PBS lucifuge overnight, after taking-up again use PBS, wash away surface it is non-grafted on
Growth factor, dries and obtain polyaniline fiber protein gel compound rest, is designated as PANI-Scaffold-VEGF.
Wherein it is preferred to, the concentration of EDC solution described in step S31 is 3.5~4.0mg/ml.
It is highly preferred that the concentration of EDC solution described in step S31 is 3.8mg/ml.
Preferably, the time of slow concussion is 1~3h described in step S31 and S32.
It is highly preferred that the time of slow concussion described in step S31 and S32 is 2h.
Preferably, the concentration of growth factor VEGF solution described in step S32 is 90~110ng/ml.
It is highly preferred that the concentration of growth factor VEGF solution described in step S32 is 100ng/ml.
It is highly preferred that the solvent of growth factor VEGF solution described in step S32 is PBS, pH=7.4.
Preferably, the concentration of DCC solution described in step S32 is 20~30mg/ml.
It is highly preferred that the concentration of DCC solution described in step S32 is 24mg/ml.
In addition, the polyaniline fiber protein gel compound rest that the above method is prepared, and its as or prepare
Application in terms of heart tissue engineering timbering material, also all within protection scope of the present invention.
The present invention carries out the synthesis of conducting polyaniline material with the material to no toxic biological effect, and uses biomaterial
Carry out the synthesis of fibrin support.Both materials are combined, makes material both electric conductivity and nanometer with polyaniline material
Characteristic, has advantageous property of the support carriage in cell to embody the biocompatibility and functional characteristic of composite again.Herein
On the basis of promote the growth and the generation of blood vessel of cell in research with angiogenic growth factor VEGF, VEGF can be expressed as fibre
Dimension cell, vascular endothelial cell, macrophage etc., its main biological function is exactly the generation for promoting blood vessel.It can promote
Enter the growth of the endothelial cell in artery, vein and lymphatic vessel source.The VEGF of autogenous is that body injury recovers important
Angiogenesis factor.On this basis, VEGF can also increase vein and venular permeability etc. after capillary, with very
Good action effect.
Shown present invention uses analysis characterization methods such as droplet measurement, infrared spectrum, Raman surface analysis and Electronic Speculum,
The grafting compound action of the polyaniline of present invention synthesis, fibrin support and material, support and growth factor is all successfully
's.The result of transmission electron microscope can see polyaniline particle, and scanning electron microscope (SEM) photograph can see polyaniline material, fiber with let us
The surface topography of albumen support and the two composite.Show and confirm that the particle size of the polyaniline of synthesis exists in figure
50nm or so, the standard of the nano material needed for meeting us, from the point of view of support figure, support shows good loose structure,
It is easy to polyaniline material directly to adhere to or be bonded in the middle of synthesis, and from the electron microscope that the two is combined, polyaniline material
The good results are evident for attachment.And in quantitative experiment, by the measure of grafting rate also further demonstrated that polyaniline and growth because
Grafting of the son on support is effective.It is this to be grafted the porous speciality for being conceived to gel scaffold material, add polyaniline
The plasticity of material, the activation of growth factor, by many reattachments be bonded, and reach a certain amount of grafting materials, reach
Material requested is modified onto support carriage, so as to realize the action effect of multi-layer.
Found by thermal gravimetric analysis results, the stability of polyaniline material and simple fiber gel support is all not so good as support
The heat endurance of Polyaniline Grafted and growth factor is good on upper Polyaniline Grafted and support, and polyphenyl has especially been grafted on support
After two kinds of materials of amine and growth factor, the overall heat endurance of material is greatly enhanced.The content of polyaniline is 107.50
DEG C 98.01% is dropped down to, the content of simple fiber gel support is even more and drops down to 98.02% at 99.05 DEG C.And pass through
After polyaniline material and growth factor have been grafted on support, the temperature that the former content drops to 98.00% has brought up to 116.47
DEG C, the latter is even more and has brought up to 195.95 DEG C.It is therefore seen that, support is more simple with the composite of polyaniline and growth factor
The heat endurance of polyaniline and fiber gel support is obviously improved, and this also complies with our demands under study for action, obtains
The more preferable ideal material of stability.This is perhaps in the middle of the space that polyaniline and growth factor are grafted on support, to make whole
Material shows obvious gap, and then improves the heat endurance of material on the whole.
In addition, being processed with different materials H9c2 rat myocardial cells, material is respectively polyaniline (PANI), blank
Fibrin support (Scaffold), single growth factor VEGF, the polyaniline (PANI-Scaffold) synthesized on support,
Through the support that growth factor VEGF is modified, and six kinds of materials of whole compound support (PANI-Scaffold-VEGF), pass through
MTT oxicity analysis are detected, and the detection of fluidic cell apoptosis, have obtained influence effect of the material of each synthesis phase to cell
Really.Data result shows, compared with single polyaniline or blank fiber gel support, growth factor VEGF has promotion thin
The effect of intracellular growth.The polyaniline of " growth " is not changed significantly for the toxic effect of cell on fiber gel support, and
After growth factor VEGF has been modified on the fiber gel timbering material, the apoptosis rate of PANI-Scaffold-VEGF groups is then
Substantially reduce, it is shown that material can significantly suppress the apoptosis of cell after growth factor in grafting, illustrate PANI-
Good result of the Scaffold-VEGF materials to cell.In addition, after 72h is processed to cardiac muscle cell with various materials, material
Between the otherness of impact cell is significantly shown.By material process, the Apoptosis of independent polyaniline treatment group
Rate has approached 40%, and individually the apoptosis rate of bare stent group also has more than 20%, and by comparison, contains growth factor
The groups of cells of VEGF is reduced much on apoptosis rate.And most notably, overall compound rest PANI-Scaffold-
The apoptosis degree of the cell of VEGF group material process is substantially reduced, and only 3% or so, all have compared with other each groups obvious
Difference row, showing PANI-Scaffold-VEGF materials has the effect of obvious promotion cell growth.
Test result indicate that, the present invention successfully synthesizes heart tissue engineering timbering material, its effect in cellular level
Tested by the qualitative, quantitative of flow cytometry and confirmed, the PANI fibrin gels compound rest of acquisition can promote the heart
Myocyte preferably grows and breeds.Later heart tissue engineering art treatments means are entered one by the successful preparation of material
Step is expanded has directive significance.
The invention has the advantages that：
Present invention success has synthesized Nano particles of polyaniline on fibrinogen gel support, and rush blood vessel is grafted on support
Growth factor VEGF, synthesis obtains nano-conductive polyaniline nano-particle, makees with good cell compatibility, to cytotoxicity
With very little, growth and the propagation of cell can be promoted, and promote the regeneration of new vessels, with good application potential, can answered
For the support decorative material in heart tissue engineering field, can as the universality material of heart tissue engineering further research and
Exploitation.
The present invention uses chemical syntheses method, and with biomembrane and the similitude of biological support, first attempts in film
Upper synthesis conductive nano PANI, after by identical experimental technique be applied on biological support synthesize nanometer PANI so that reduce by
The modification of polyaniline sample simplifies experimental procedure to the link on support.And infrared spectrum is used, AFM and Electronic Speculum are swept
The characteristic manner such as retouch successfully to characterize it, it is thus identified that the structure of PANI.
Brief description of the drawings
Fig. 1 is the synthesis schematic diagram of polyaniline fiber protein gel compound rest.
Fig. 2 is the droplet measurement and its transmission electron microscope picture of PANI synthesis；The particle diameter distribution of electrically conductive polyaniline a.4nm；
The particle diameter distribution of the electrically conductive polyaniline under b.30nm；The transmission electron microscope picture of the electrically conductive polyaniline under c.4nm；D.30nm the conduction under
The transmission electron microscope picture of polyaniline.
Fig. 3 is polyaniline（PANI）, fibrinogen support（Scaffold）, the polyphenyl that synthesizes on fibrinogen support
Amine（PANI-Scaffold）And the two composite（PANI-Scaffold -VEGF）Infared spectrum.
Fig. 4 is polyaniline（PANI）, fibrinogen support（Scaffold）, the polyphenyl that synthesizes on fibrinogen support
Amine（PANI-Scaffold）And the two composite（PANI-Scaffold -VEGF）Raman collection of illustrative plates.
Fig. 5 is polyaniline（PANI）, fibrinogen support（Scaffold）, the polyphenyl that synthesizes on fibrinogen support
Amine（PANI-Scaffold）And the two composite（PANI-Scaffold -VEGF）Scanning electron microscope analysis.
Fig. 6 is the grafting rate of polyaniline and growth factor.
Fig. 7 is polyaniline（PANI）, fibrinogen support（Scaffold）, the polyphenyl that synthesizes on fibrinogen support
Amine（PANI-Scaffold）And the two composite（PANI-Scaffold -VEGF）Thermal stability analysis.
Fig. 8 is apoptosis rate（Material is detected to the toxic action of cardiac muscle cell）.
Fig. 9 is FCM analysis result.
Specific embodiment
The present invention, but embodiment are further illustrated below in conjunction with Figure of description and specific embodiment not to the present invention
Limit in any form.Unless stated otherwise, reagent, the method and apparatus that the present invention is used are for the art is routinely tried
Agent, method and apparatus.
Unless stated otherwise, agents useful for same of the present invention and material are purchased in market.
Main material, reagent and instrument used in following examples is as follows：
Aniline, ammonium persulfate, dichloroethanes, concentrated hydrochloric acid, NaOH, SDS, absolute ethyl alcohol, bioactive film.
Flask, beaker, six orifice plates, magnetic stirring apparatus, Rotary Evaporators, electronic analytical balance, vacuum drying chamber, ultraviolet point
Light photometer, infrared spectrum detector, Electronic Speculum etc..
DMEM/F12 culture mediums, phosphate buffer PBS（pH7.4）（Autogamy）, fibrinogen freeze-dried powder, fibrin ferment（Ox
Blood plasma）Freeze-dried powder, angiogenic growth factor VEGF, 24 orifice plates.
H9c2 cell lines, 0.25% trypsase, trypsase, 96 well culture plates, incubator, dimethyl sulfoxide (DMSO)（DMSO）、
CO2Incubator（5%CO2, 95% air, 100% humidity）, 0.8% low melting-point agarose（LMPA）, centrifuge, constant water bath box, suction
Pipe, vial（250ml、100ml）, waste liquid cylinder, pipette tips, plug, 1.5ml EP pipes, 15ml centrifuge tubes, cryopreservation tube（2ml）, fall
Put microscope, fluorescence microscope, constant temperature oscillator, ultrasonic disperse instrument, Rotary Evaporators, 1000ml volumetric flasks, 100ml capacity
Bottle, filter membrane of 0.20um etc..
The synthesis of the polyaniline fiber protein gel compound rest of embodiment 1
The synthesis schematic diagram of polyaniline fiber protein gel compound rest is as shown in Figure 1.
1st, nano-conductive polyaniline material（PANI）Synthesis
（1）Aniline（An）Solution A, ammonium persulfate (APS) 0.24g are mixed into the HCl of the μ L of monomer solution 200 addition 5ml 0.1M
Dissolved in the HCl of 25ml 0.1M, and add 15ml ethanol, after stirring 10min, cooled down in ice bucket, be mixed into solution B.
（2）After cooling is finished, solution A is slowly added into B solution in ice-water bath, the lower stirring of mixing, in 20min
Add 5ml solution As（A small amount of lauryl sodium sulfate is properly added if necessary（SDS）, existed with the polyaniline product for increasing generation
Dissolubility in water）；It is complete that aniline solution now is unmixed with HCl solution, and product is adhered to tunica fibrosa；Stop stirring after
12h is reacted in ice bucket.
（3）Reaction takes out tunica fibrosa after terminating, and washs by product with 0.1M HCl solutions, dries.
2nd, the synthesis of fibrinogen gel support
（1）With sugar serum-free DMEM/F12 high（v/v=1:1, PH=7.4）Mixed culture medium solution fibrin is former（FG）It is lyophilized
Powder, is made colloidal solution, and its concentration is 5-15 mg/ml.
（2）Add the ox Thrombin plasma dissolved with PBS（PT）The solution of freeze-dried powder, is mixed into colloidal mixture.
（3）Colloidal mixture is uniformly coated on 24 orifice plates（500μL）Interior, 37 DEG C stand 30min fibrinogens solidifying
Blood factor（PT）In the presence of be frozen into the fibrin support of translucent gels shape.
3rd, synthesis of the nano polyaniline on fibrinogen gel support
On the basis of the synthesis of fibrinogen gel timbering material, with the method similar to PANI synthesis, identical medicine is used
Addition, by fibrinogen gel scaffold infiltration in reaction system solution, allow oxidative synthesis PANI in the space of support
Central " growth ", so as to directly synthesize PANI on gel stent, the fibrin support for obtaining being grafted PANI materials is combined
Material PANI-Scaffold.
4th, grafting of the growth factor on material
DCC activated growth factors are used, the carboxyl on composite PANI-Scaffold is activated with EDC, by suction-operated and change
Learn key effect, growth factor VEGF be grafted on composite PANI-Scaffold, the chemical bond refer to growth because
The amino of sub- VEGF acts on forming amido link with the carboxyl of composite PANI-Scaffold.
Concrete operations scheme：3.8mg/ml EDC solutions are prepared, composite PANI-Scaffold EDC is placed on molten
In liquid, 2h, the carboxyl on activated fiber albumen support are slowly shaken.Growth factor VEGF is configured to PBS (pH=7.4)
The solution of 100ng/ml, takes 1-2ml or so, 24mg/ml DCC is added, with activated growth factor.Then by activation after it is compound
Material PANI-Scaffold is placed in concussion reaction 2h in growth factor solution.Reacted growth factor and timbering material are existed
Lucifuge overnight, uses PBS again in PBS after taking-up, wash away surface it is non-grafted on growth factor, to dry that obtain polyaniline fine
Fibrillarin gel compound rest PANI-Scaffold-VEGF.
The sign of the timbering material of embodiment 2
1st, granularity Detection instrument detection PANI particle diameters and transmission electron microscope detection
The droplet measurement and its transmission electron microscope picture of PANI synthesis are as shown in Figure 2.
In addition, being slightly different using the particle size that the polyaniline that different solvents synthesize has, but nanometer is all reached
Level, it is best in 4nm or so, illustrate to successfully synthesize nano level electrically conductive polyaniline, and by its shape of transmission electron microscope observing
State, it is also possible to know that it shows granular structure substantially, meet our demand.
2nd, infrared spectrum analysis：
The analysis and identification carried out to material molecule using infrared spectrum.The infrared-ray of a branch of different wave length is irradiated to material
Molecule on, the infrared-ray of some specific wavelengths is absorbed, and forms the infrared absorption spectroscopy of this molecule.Every kind of molecule has
The exclusive infrared absorption spectroscopy with structures shape is made from it, structural analysis and identification can be carried out to molecule accordingly.It is infrared
Absorption spectrum is ceaselessly made vibration and rotational motion by molecule and is produced, molecular vibration refer in molecule each atom in balance
Position nearby performs relative motion, and polyatomic molecule can constitute various vibration graphics.When in molecule each atom with same frequency, same
When phase makees simple harmonic oscillation near equilbrium position, this mode of vibration claims normal mode vibration（Such as stretching vibration and angle are shaken
It is dynamic）.The energy of molecular vibration is just corresponding with the light quantum energy of infrared-ray, therefore when the vibrational state of molecule changes,
Infrared spectrum can just be launched, it is also possible to vibrated because infra-red radiation excites molecule and produce infrared absorption spectroscopy.Molecule shakes
Energy that is dynamic and rotating is not continuous but quantized.But due to being also often associated with rotating during the vibrational transition of molecule
Transition, makes vibrational spectrum be in banding.So the infrared spectrum of molecule belongs to band spectrum.Molecule is bigger, and infrared band is also more.
With KBr pressed disc method sample preparations, determined on FT-IR Spectrometer.Structure according to polyaniline shows, due to
The presence of amino, imino group and part water in molecule, it is in 3400cm-1There is wide and strong absworption peak left and right；The presence of phenyl ring, makes
Obtain 1550cm-1To 1600cm-1There is the characteristic absorption peak of phenyl ring left and right；And the stretching vibration and flexural vibrations of some groups
Absworption peak.
Such as accompanying drawing 3, by infared spectrum, we can show that the polyaniline that the present invention synthesizes is successful, in 3467.89cm-1
Place is the peak of the N-H to be formed, and what 1553.99cm-1 embodied is the absorption vibration of quinoid structure N=Q=N, 1469.69cm-1And its saw
The other peak of dentation is the characteristic absorption vibration of the i.e. benzene formula structure N-B-N of characteristic peak of aromatic ring, all demonstrates the structure of polyaniline.
Fibrin support（Scaffold）In, carbonyl shows the framework of protein with the feature peak type of amino, and in composite
In, by comparing, the framework of protein is still present, but less than 1000cm-1In the range of show very big difference.
In infared spectrum after growth factor grafting, the peak type of hydroxyl is more obvious, and this should be the skew of poly hydroxyl peak, enter one
Step shows the successful of grafting, the trace analysis after fibrin support and composite and growth factor grafting,
Nano polyaniline has successfully been bonded to above fibrin support, and grafting of the growth factor on support is also successful, shape
Composite into needed for us.
3rd, Raman spectrum analysis：
Using Raman spectrum means, material molecule is analyzed.When light occurs frequency through transparent medium by the light of molecular scattering
Rate changes, and this phenomenon is referred to as Raman scattering；In the scattering spectrum of transparent medium, frequency and incident light frequency υ0Identical into
Divide and be referred to as Rayleigh scattering；Frequency is symmetrically distributed in υ0The spectral line or bands of a spectrum υ of both sides0±υ1As Raman spectrum, wherein frequency is smaller
Composition υ0- υ1It is also called stockes line, the larger composition υ of frequency0+ υ1It is also called anti-stockes line.Dissipated near Rayleigh
The spectral line of ray both sides is referred to as small Raman spectrum；The spectral line occurred away from the both sides of Rayleigh line is referred to as big Raman spectrum.Rayleigh dissipates
The intensity of ray only has the 10 of incident intensity-3, raman spectrum strength only about Rayleigh line 10-3.Small Raman spectrum with point
The rotational energy level of son is relevant, and big Raman spectrum is relevant with molecular vibration-rotational energy level.The theoretical explanation of Raman spectrum is, incident
There is inelastic scattering in photon, molecular absorption frequency is υ with molecule0Photon, launch υ0- υ1Photon, while molecule is from low
Energy state transitions are to upper state（Stockes line）；Molecular absorption frequency is υ0Photon, launch υ0+ υ1Photon, while molecule
Lower state is transitted to from upper state（Anti-stockes line）.The transition of molecular entergy level only relates to rotational energy level, and transmitting is small drawing
Graceful spectrum；It is related to vibration-rotational energy level, transmitting is big Raman spectrum.It is different from molecule infrared spectrum, polar molecule and non-
Polar molecule can produce Raman spectrum.
By polyaniline material, the material after timbering material and polyaniline and prop composite and the upper growth factor of grafting
Freeze-dried powder detected on Raman analyser, obtain the Raman spectrogram of each material surface.Due to amino in molecule, quinone ring and
The presence of phenyl ring, it is in 1590cm-1, there are the stretching vibration of C=C in quinone ring, 1500cm in left and right-1There is the spy of C=C in phenyl ring left and right
Levy stretching vibration, 1370cm-1There are the stretching vibration of aromatic amine Ar-N, 1200cm in left and right-1And 800cm-1Left and right be in phenyl ring the face in
The absworption peak of out-of-plane bending vibration.
Such as Fig. 4,1590cm-1Left and right is the stretching vibration of C=C in quinone ring, 1507cm-1Left and right is the flexible of C=C in phenyl ring
Vibration, 1371cm-1Left and right is the stretching vibration of aromatic amine Ar-N, 1166cm-1And 808cm-1Left and right is in phenyl ring face and face excurvation
Qu Zhendong, these all indicate the structure of polyaniline.In the composite, the Raman spectrum of polyaniline material has obvious
Change, illustrates in the middle of the process of Material cladding, the key of polyaniline material and gel stent all there occurs change, further illustrate
This bonding is successful.After being grafted growth factor on support, Raman collection of illustrative plates shows further difference, is demonstrated by whole
The composition contained individual material more.2400cm-1It is the peak of acyl group performance, 980cm-1What left and right was embodied is that amido link splits swarming,
On the basis of this, some peak types of the material before the graft growth factor are also maintained, the grafting for showing growth factor is successful.
4th, ESEM detection
ESEM is the various physical signallings that are ejected when sample surfaces are scanned using fine focusing electron beam to be modulated into
As it is a kind of new electronic optical instrument.It has that sample preparation is simple, multiplication factor adjustable extent is wide, image resolution
The features such as rate is high, the depth of field is big.Recent decades, ESEM has been widely used in the neck of the subjects such as biology, medical science, metallurgy
In domain, each development about subject is promoted.The sign of biological sample is detected frequently by ESEM, because of electron irradiation
There is damage and the pollution level very little of sample, the electron microscope with other modes compares, because electronics used during observation
Probe current is small（Typically about 10-10-10-12A）The beam spot size of electron probe is small（Typically 5nm to tens nanometers）, electricity
The energy of sub- probe is also smaller（Accelerating potential may diminish to 2kV）.And be not fixed any irradiation sample, but with grating
Shape scan mode irradiates sample.Therefore, because electron irradiation face occurs damage and the pollution level very little of sample, this point is to seeing
Examine some Biosamples especially important.Before electron microscopic observation is scanned, sample is correspondingly processed.Scanning electron microscope example
The major requirement of preparation is：As far as possible keep the surface texture of sample, without deformation and pollute, sample drying and have good
Good electric conductivity.
As shown in figure 5, electrically conductive polyanilines of a for the rod-like morphology of 50nm, b is the fibrinogen support of synthesis, and c is fibre
The polyaniline synthesized on fibrillarin original support, d is the polyaniline and VEGF on fibrinogen support.Can be with by scanning electron microscope (SEM) photograph
See the surface topography of polyaniline material, fibrin support and the two composite.
Show and confirm the particle size of polyaniline of synthesis in 50nm or so, the nanometer material needed for meeting us in figure
The standard of material, from the point of view of support figure, support shows good loose structure, it is easy to which polyaniline material is directly attached in the middle of synthesis
Or be bonded, and from the electron microscope that the two is combined, the good results are evident for polyaniline material attachment.
5th, PANI, the grafting rate of growth factor are determined（G250 Coomassie brilliant blue methods of testing）
Coomassie brilliant blue determination method is used, standard curve is made by corresponding protein of BSA, surveyed on the basis of this standard curve
The polyaniline of various concentrations and the UV absorption of growth factor concentration are determined, so as to the different polyaniline before and after being grafted and growth
The levels of the factor, obtain the grafting rate of polyaniline and growth factor.
Specific research approach：Two polyaniline reaction systems for starting simultaneously at reaction, one of them is left intact, separately
One is done support grafting, and reaction takes the μ L of polyaniline solutions 30 of same volume after terminating, and determines its suction respectively on standard curve
Luminosity, is calculated the concentration of the two respectively w1And w2.And the initial concentration of growth factor is 100ng/ml, activated by DCC
Afterwards, it is grafted on support, remaining growth factor solution determines its concentration by Coomassie brilliant blue ultraviolet spectroscopy, and then calculates
The grafting rate of growth factor, formula is as follows：
By the measure of grafting rate（As shown in Figure 6）, the grafting rate of polyaniline is in 20% or so, and the grafting rate of growth factor
Then 40% or so.It is about 0.5 × 0.5cm after a simple support is lyophilized from the point of view of the surface area that support has2's
Surface area, the amount of growth factor can reach 160ng/cm2, such grafting rate indicate polyaniline on support and growth because
The grafting of son is successful, and can successfully be grafted and be attached in the middle of the surface of support and brace aperture, as a result with scanning
The conclusion that other characterization methods such as Electronic Speculum, infrared spectrum are showed is consistent.
6th, heat stability test
Such as accompanying drawing 7, by the analysis of the thermal stability results to material, we are it can be found that polyaniline material and simple fibre
Tie up the stability of gel stent thermally-stabilised all not as Polyaniline Grafted on Polyaniline Grafted on support and support and growth factor
Property is good, and after being especially grafted two kinds of materials of polyaniline and growth factor on the support, the overall heat endurance of material is obtained
Greatly reinforce.The content of polyaniline drops down to 98.01% at 107.50 DEG C, and the content of simple fiber gel support is even more
99.05 DEG C drop down to 98.02%.And after being grafted polyaniline material and growth factor on support, the former declines content
Temperature to 98.00% has brought up to 116.47 DEG C, and the latter is even more and has brought up to 195.95 DEG C.
It is therefore seen that, the support polyaniline and fiber gel support more simple with the composite of polyaniline and growth factor
Heat endurance be obviously improved, this also complies with our demands under study for action, obtains the more preferable ideal material of stability.
The application study of the timbering material of embodiment 3
1st, H9C2 rat myocardial cells cell culture
（1）Cryopreservation tube is taken out from liquid nitrogen container, is directly immersed in 37 DEG C of warm water, and shake makes it melt as early as possible frequently.
（2）Cryopreservation tube is taken out from 37 DEG C of water-baths, lid is opened, cell suspension is suctioned out with suction pipe, be added to centrifuge tube and drip
Plus more than 10 times nutrient solutions, mix；
（3）Centrifugation, 1000rpm, 5min；
（4）Abandoning supernatant, is 95%, CO in relative humidity with the DMEM medium cultures containing 10% hyclone2Content is
5%, count, adjust cell density, inoculated and cultured bottle, 37 DEG C of incubator quiescent cultures.
（5）A not good liquor is changed within every two days, continues to cultivate, a second generation general passed per 3-4 days and passes three.
2nd, H9c2 (2-1) rat myocardial cells in exponential phase are processed with the different materials of synthesis, material
Material is respectively polyaniline（PANI）, blank fibrin support（Scaffold）, single growth factor VEGF, synthesis on support
Polyaniline（PANI-Scaffold）, through support and whole compound support that growth factor VEGF is modified（PANI-
Scaffold-VEGF）Six kinds of materials.
3rd, MTT cytotoxicity analysis
（1）Analysis method
1）Logarithmic phase cell is collected, concentration of cell suspension is adjusted, adds 100 μ l, bed board cell to be measured is adjusted density to 10 per hole4
Per hole（Edge hole is filled with aseptic PBS）.
2）5%CO2, 37 DEG C are incubated, and bottom hole is paved with to cell monolayer（96 hole flat undersides）, the medicine of concentration gradient is added,
Noon before that day bed board, the dosing of morning next day.General 5-7 gradient, per the μ l of hole 100, if 3-5 multiple holes.Suggestion sets 5, no
Then it is difficult to reflect truth.By the solution of best time insoluble drug release, thalline is filtered to remove through 0.22 μm of filter membrane.
3）5%CO2, 37 DEG C are incubated 24 hours, are observed under inverted microscope.
4）20 μ l MTT solution are added per hole（5mg/ml, i.e. 0.5%MTT）, continue to cultivate 4h.If medicine can be anti-with MTT
Should, can first be centrifuged and discard nutrient solution afterwards, carefully 2-3 is rushed after with PBS, add the nutrient solution containing MTT.
5）Terminate culture, carefully suck nutrient solution in hole.
6）150 μ l dimethyl sulfoxide (DMSO)s are added per hole, low-speed oscillation 10min on shaking table is put, crystal is fully dissolved.In enzyme
The light absorption value in each hole of measurement at connection immune detector OD 570nm.
7）Zeroing hole is set simultaneously（Culture medium, MTT, dimethyl sulfoxide (DMSO)）, control wells（Cell, same concentrations medicine it is molten
Solution medium, nutrient solution, MTT, dimethyl sulfoxide (DMSO)）.
（2）MTT toxicity detection analyses are carried out to cell, function influence effect of each synthetic material to cardiac muscle cell has been obtained
Really.
Such as accompanying drawing 8, as a result show, compared with single polyaniline or blank fiber gel support, growth factor VEGF
Effect with the apoptosis and promotion cell growth for suppressing cell.The polyaniline of " growth " on fiber gel support（PANI-
Scaffold）There is no obvious toxic effect for cardiac muscle cell, and growth factor has been modified on fiber gel timbering material
After VEGF, PANI-Scaffold-VEGF supports group is significantly improved with the compatibility of cardiac muscle cell, it is shown that grown in grafting
After the factor, timbering material can significantly facilitate the growth of cardiac muscle cell, illustrate PANI-Scaffold-VEGF timbering materials
To the good result of cardiac muscle cell, it was demonstrated that material to the toxic action very little of cardiac muscle cell, and after possessing growth factor,
There is facilitation to the growth of cardiac muscle cell.
4th, the double dyeing detection Apoptosis of streaming
By the H9c2 cells in exponential phase, with 5 × 105The cell density in individual/hole is inoculated into 6 well culture plates.Then
PBS, Spore-Curcumin-FA release liquid transfectional cell 12h are separately added into, the culture medium in culture plate is discarded.Collect thin
Born of the same parents, centrifugation removal culture medium.The Binding buffer for adding 200 μ l to each sample pipe the inside are mixed, and add 5 μ l
Annexin V-FITC, at room temperature lucifuge be incubated 10min；Centrifugation supernatant discarded, adds the Binding buffer of 200 μ l to mix
It is even, and add 5 μ l PI, directly with Flow cytometry check cell apoptosis situation.
Result shows（Accompanying drawing 9）, after 72h is processed to cardiac muscle cell with various materials, shadow of the material to cardiac muscle cell
Ringing has obvious otherness, and the material of each synthesis is all no very high to the degree of impairment of cell, and material is on the whole to thin
The toxic action of born of the same parents than relatively low, the apoptosis rate of the groups of cells of the single polyaniline treatment maximum to impact cell also only less than
40%, this shows that our material has good cell compatibility in terms of cytotoxicity.
Specifically, the apoptosis rate of independent polyaniline treatment group is close to 40%, and the individually cell of bare stent group
Apoptosis rate also has 20% or so, and by comparison, the groups of cells containing growth factor VEGF is reduced much on apoptosis rate,
This is attributed to the facilitation of angiogenic growth factor VEGF cell growths, reduces influence of the independent polyaniline to cell,
The facilitation to cell is shown on the whole.
Most notably, by overall compound rest PANI-Scaffold-VEGF group material process cell apoptosis
Degree is substantially reduced, and only 3% or so, almost can be ignored, there is the more prominent life to cell compared with other each groups
Facilitation long, showing PANI-Scaffold-VEGF timbering materials has extraordinary biocompatibility and cell is given birth to
Facilitation long, also demonstrates the polyaniline and the support by growth factor modified synthesized on Fibrin glue
Material and the prominent compatibility of cardiac muscle cell, and with the effect of apparent promotion cell growth.This for after material to the heart
Myocyte align and electrophysiologic study lays the first stone.
Claims (10)
1. a kind of preparation method of polyaniline fiber protein gel compound rest, it is characterised in that first using no cytotoxicity
Fibrin ferment and fibrinogen crosslinked action synthesis obtain fibrinogen gel support, then with chemical syntheses
Method, directly synthesizes the electrically conductive polyaniline of nanoscale on Fibrin glue, is finally grafted upper growth factor, is gathered
Aniline fibrin gel compound rest.
2. preparation method according to claim 1, it is characterised in that be in fibrinogen gel timbering material in synthesis
On the basis of, by fibrinogen gel scaffold infiltration in the reaction system solution of synthesis PANI, allow the PANI of oxidative synthesis
In the middle of the space of support " growth ", so as to directly synthesize PANI on gel stent, obtain being grafted the fiber of PANI materials
Albumen prop composite, is finally grafted upper angiogenic growth factor VEGF, obtains polyaniline fiber protein gel compound rest.
3. preparation method according to claim 1, it is characterised in that comprise the following steps：
S1. fibrinogen gel support is prepared
S11. with sugar serum-free DMEM/F12 mixed culture mediums solution fibrin original freeze-dried powder high, it is made the glue of 5~15mg/ml
Shape solution；Clotting factor solution is added, colloidal mixture is mixed into；
S12. by 35~38 DEG C of 25~35min of standing of colloidal mixture, fibrinogen gel support is obtained；
S2. synthesis of the nano polyaniline on fibrinogen gel support
S21. aniline solution mixes to obtain solution A in adding HCl；Ammonium persulfate dissolves in HCl, and adds ethanol to stir, frozen water
Bath cooling, obtains solution B；
S22. the fibrinogen gel scaffold infiltration for being prepared by step S1 is in solution B and in ice-water bath that solution A is slow
It is added in solution B, stirring mixing；Reacted in ice-water bath after stopping stirring；
S23. fibrinogen gel support is taken out after reaction terminates, the fibrin support for obtaining being grafted PANI materials is combined
Material, is designated as PANI-Scaffold；
S3. the graft growth factor：The amino of activated growth factor, the carboxyl on activation PANI-Scaffold, by suction-operated
With the effect of amino and carboxyl, growth factor VEGF is grafted on PANI-Scaffold.
4. preparation method according to claim 3, it is characterised in that clotting factor described in step S11 is that ox blood starches blood coagulation
Enzyme.
5. preparation method according to claim 3, it is characterised in that the HCl in step S21 and S13 is 0.05~0.15M
HCl；Aniline solution and the volume ratio of HCl are 2 in solution A：45~55；The mass volume ratio of ammonium persulfate and HCl in solution B
It is 9~10g/L, and HCl and the volume ratio of ethanol are 5：2~4.
6. preparation method according to claim 3, it is characterised in that the time stirred described in step S21 is 5~15min,
The speed being slowly added to described in step S22 is 0.2~0.3ml/min.
7. preparation method according to claim 3, it is characterised in that the volume ratio of solution A and solution B described in step S22
It is 1：The time reacted described in 7~9, step S22 is 10~15h.
8. preparation method according to claim 3, it is characterised in that the specific method of step S3 is：
S31. PANI-Scaffold is placed in EDC solution, slowly shakes 1~3h；
S32. DCC solution is added in growth factor VEGF solution, with activated growth factor；
S33. the product of step S31 is placed in the solution of step S32,1~3h of concussion reaction；
S34. the product of step S33 lucifuge in PBS overnight, uses PBS again after taking-up, dries and obtain polyaniline fibre
Fibrillarin gel compound rest.
9. the polyaniline fiber protein gel compound rest for being prepared according to any methods described of claim 1~8.
10. polyaniline fiber protein gel compound rest described in claim 9 as or prepare heart tissue engineering support material
Application in terms of material.
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