CN106798948A - A kind of method of regulation and control biofilm surface topological structure to promote cell to creep - Google Patents
A kind of method of regulation and control biofilm surface topological structure to promote cell to creep Download PDFInfo
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- 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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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
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- 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
- A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
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- 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
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- 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
- A61L2300/416—Anti-neoplastic or anti-proliferative or anti-restenosis or anti-angiogenic agents, e.g. paclitaxel, sirolimus
Abstract
A kind of method the invention discloses regulation and control biofilm surface topological structure to promote cell to creep, belongs to high-molecular biologic medical material tech field.The inventive method is that, using polyelectrolyte self assembly target organism film layer by layer, regulation and control biofilm surface pore-size, interporal lacuna and nano surface are raised, specifically include following steps：Choose two kinds of polyelectrolyte with opposite charges, carrying opposite charges polyelectrolyte with it according to the selection of target organism film surface charge carries out self assembly, another kind is subsequently assembled with opposite charges polyelectrolyte, is so far one bilayer of assembling, by that analogy until obtaining the required number of plies.Layer-by-layer is applied to the present invention pore size and nano projection on biomembrane material surface, and assign biomembrane material extra performance, maximize its biomedical interest, many advantages, such as present invention has simple low raw-material cost, operating procedure, mild condition, it is adaptable to mass produce.
Description
Technical field
The invention belongs to high-molecular biologic medical material tech field, and in particular to one kind regulation and control biofilm surface topology knot
Method of the structure to promote cell to creep.
Background technology
The medical value of biomembrane material is not only embodied in its raw material biocompatibility good in itself, is also opened up with its surface
Flutter structural relation close.Used as the membrane material of cell growth support, for preferably analog cell epimatrix, they need conjunction
Suitable aperture, porosity high.Because the effect of cell and extracellular matrix is in Nano grade, people are to material surface nanometer
Rank projection and its it is also more and more to the research of cell adhesion, the influence creeped and break up.Because the reparation organized is new
The process of raw cell creeping substitution slough, only possesses the porous support of appropriate bore size, empty gap and nanometer projection
Can be the sticking of cell, grow, breed, breaking up and neoblastic generation provides good growing environment.Hole is excessive unfavorable
Creeped in cell, hole is too small, be unfavorable for that intercellular traffic and cell growth enter among timbering material.Additionally, working as material list
Face is not only beneficial to cell adhesion when possessing many nanoscales projections, can also promote creeping, breed and breaking up for cell.
According to the literature, by adjusting the parameter of solution concentration, proportioning, electrostatic spinning, including voltage strength, can receive
Distance is come the hole that adjusts the diameter of nanofiber so as to adjust fiber；By the concentration, the temperature of freeze-drying that adjust solution
To adjust the porosity of membrane material；The pore size of membrane material is adjusted by changing dissolution system.Also document points out biology
The roughness of material surface suitably increases and can promote sticking for cell, and the especially projection of material surface Nano grade can be effective
Promote sticking, stretch and creeping for cell.But so far it is not yet found that directly using the biological doctor of layer-by-layer regulation
Learn the research and application of material list face size, interporal lacuna and nano projection.The one of Patent No. " ZL 201310158591.0 "
Item invention being mentioned its aperture after be assembled to layer by layer polyelectrolyte nano fibrous membrane and being reduced；Number of patent application is
One of " 201510586136.X " invention prepares NF membrane using layer-by-layer, it is mentioned that by polyethyleneimine and
Graphene oxide is alternately assembled to the surface of polyacrylonitrile-radical film so that the hydrophily and pore size on film surface are obtained effectively
Regulation and control.Regrettably, this two patents belong to UF membrane field rather than biomedical materials field, and material is not discussed
Biocompatibility, material list face size, the raised influence to cell biological behavior of interporal lacuna and nano surface, therefore by membrane material
The purpose and polyelectrolyte species that material carries out LBL self-assembly are also far from each other with biomembrane material.
In recent years, layer-by-layer is widely applied in biomedical materials field.Early in, patent in 2010
Number the bio-medical material of static self-assembly modified nano fiber is just reported for a patent of " ZL 201010022450.2 "
Preparation method, its aim at the polyelectrolyte with other biological performance is assembled on nano fibrous membrane it is attached to assign its
Additivity can reach the purpose for increasing its biocompatibility, but this invention is not concerned about electrostatic self-assembled to nano fibrous membrane surface
These changes are not also studied the influence produced by cell behavior by the change of pore size and nano projection.Additionally, specially
Be assembled in for lysozyme and fibroin albumen for a patent utilization layer-by-layer of " ZL 201310456538.9 " by profit number
Cellulose nano-fibrous membrane surface, imparts nano fibrous membrane good anti-microbial property and cell adhesion, and manufacture the rat back of the body
Portion's wound model carries out experiment in vivo, it was demonstrated that modified cellulose nano-fibrous membrane can substantially shorten wound healing when
Between；One invention of Patent No. " ZL 201410423055.3 " describes a kind of for promoting regenerating heart tissue and doing thin
The preparation method of the Properties of Chitosan Fibroin Blend multi-functional sticking patch of albumen composite nano fiber of born of the same parents' monitoring, the method passes through LBL self-assembly
The shitosan of positively charged and electronegative fibroin albumen are successively alternately assembled into nanofiber surface by technology, then by between fat
Mesenchymal stem cells and cardiac progenitor cell seed cell are inoculated in nano fibrous membrane surface, and plan carries out cardiac muscle in this, as myocardium sticking patch
Repair.Two inventions of the above are using the modified main purpose of electrostatic self-chambering layer by layer still just with the three-dimensional branch of nano fibrous membrane
The polyelectrolyte with particular characteristic is simultaneously assembled in the surface of nano fibrous membrane for frame structure, and corresponding biomedicine is obtained with this
Performance.The shadow that their same changes for not inquiring into biomembrane material surface pore size, interporal lacuna and nano projection are creeped to cell
Ring, also do not have hole and surface nano-structure consciously to nano fibrous membrane and regulate and control.
The content of the invention
A kind of method it is an object of the invention to provide regulation and control biofilm surface topological structure to promote cell to creep, this
Inventive method is easy and effective, and being modified by surface makes biomembrane material obtain additional performance.
The object of the invention is achieved through the following technical solutions：
It is a kind of regulate and control method of the biofilm surface topological structure to promote cell to creep, be using polyelectrolyte self assembly mesh layer by layer
Mark biomembrane, regulates and controls biofilm surface topological structure；Described biofilm surface topological structure include pore-size, interporal lacuna and
Nano surface is raised.The method specifically includes following steps：Two kinds of polyelectrolyte with opposite charges are chosen, then according to mesh
The electric charge selection of mark biofilm surface carries opposite charges polyelectrolyte and is first begin to carry out self assembly with it, is subsequently assembled another kind
So far it is one bilayer of assembling, by that analogy until the number of plies needed for obtaining with opposite charges polyelectrolyte.Layer by layer from group
The method of dress can be fixed for solution infusion method, knife coating, spin-coating method, graft copolymerization, electrostatic spraying, electro-deposition, fluid.By anti-
The organizational project membrane material of surface topology improvement can be obtained after multiple multiple alternately assembling.Flied emission scanning electron is shot to show
Micro mirror observes the change of modified membrane material surface topography and hole.Finally by these biomembrane materials and modified membrane material
The surface of material carries out cell and is inoculated with and carries out MTT experiments or CCK-8 experiments and the shooting of ESEM after cultivating the different times
To assess whether modified membrane material possesses the ability for preferably promoting cell adherence, growth, propagation.If the poly- electrolysis of assembling
Matter is antibacterial substance, growth factor, antineoplastic, then such performance is estimated.Various experiments are commented more than
Estimate assembling which kind of number of plies effect it is the most excellent.
Described polyelectrolyte can be shitosan, fibroin albumen, collagen, lysozyme, polylysine, polypropylene ammonia
Base, polyethyleneimine, sodium alginate, gelatin, chondroitin sulfate, heparin sulfate, dextran sulfate, albumin, polyglutamic acid, thoroughly
One or more in the acid of bright matter.
The biomembrane material can be by the tape casting, electrostatic spinning, freeze-drying, electro-deposition method, spin-coating method, blade coating
Method, vacuum method, immersion precipitation phase inversion process are made, and its raw material can be one or more mixing in macromolecular material.
The inventive method is introduced as a example by using LBL self-assembly regulation fibroin protein film below, is comprised the following steps：
（1）Lysozyme and the I- collagen type aqueous solution are prepared respectively, and concentration is 1mg/mL, is slowly stirred until solute is complete
Dissolving；According to volume ratio 1:Above two solution is mixed to get lysozyme and collagen mixed solution by 1 ratio, and is adjusted
Its pH value is 6.3.
（2）Silk fibroin water solution is prepared, concentration is 1mg/mL, it is 8.0 to adjust its pH value.
（3）It is base plate film with fibroin protein film, the lysozyme-collagen of opposite charges will be carried using LBL self-assembly
Compound and fibroin albumen are alternately assembled to the surface of fibroin albumen base plate film, obtain different bimolecular number of plies lysozyme-collagens
Albumen/silk fibroin composite membrane.
Specifically, step（3）In described self assembling process be：By fibroin protein film in lysozyme-fibroin albumen
Soaked 20 minutes in mixed solution, then with milli-Q water three times, 3 minutes every time, so far to assemble 0.5 bilayer；
Then soaked 20 minutes in silk fibroin protein solution, it is same with milli-Q water three times, so far to assemble 1 bilayer；
Operation more than repeating obtains the composite protein film of the different bimolecular numbers of plies.
The invention provides using LBL self-assembly regulation and control biofilm surface topological structure promote cell on biomembrane
The application creeped.The present invention combines LBL self-assembly by adjusting biomembrane material surface topology, including hole size, Kong Jian
Gap and its nano surface are raised come the cell adhesion that promotes to be inoculated with thereon and to creep.By the method pair of LBL self-assembly
Biomembrane material carries out surface and is modified, and not only can also make its table by the size of surface laydown adjustment film surface pore
Face is roughened, and assigns the more nano projections in film surface, can more promote cell adhesion, propagation and break up.Although in life
Having a variety of methods in thing membrane material preparation process can carry out its surface pore regulation, but these methods still suffer from some to be lacked
Fall into, Yi Dan after shaping, hole maintains the original state material substantially.LBL self-assembly modification technology is simple, mild condition, with low cost,
Also the more additional properties in film surface can be assigned by assembling different polyelectrolyte, material surface is adjusted using the method
Control is a kind of to facilitate feasible approach.
The present invention, by layer-by-layer, preferably have adjusted membrane material surface holes with biomembrane material as carrier
The size of gap so that the size that larger gap shrinks to cell can be migrated across very well, and cause that the nanoscale on film surface is convex
Rise and greatly increase.Using mice embryonic bone precursor cells（MC3T3-E1）, human mouth mucosal epithelial cells（HIOEC）Or people is just
Normal dermal fibroblast（NHDFs）In vitro test is carried out, is as a result proved, the modified membrane material in surface can preferably promote
The growth of cell, propagation and break up.Additionally, this surface is modified also to impart biomembrane material others biological property, such as resist
Bacterium ability（Produced by the introducing of lysozyme）Deng.Compared with prior art, be applied to for layer-by-layer first by the present invention
The pore size and nano projection on regulation and control biomembrane material surface, and assign biomembrane material extra performance, make its biology doctor
Value maximization is learned, many advantages, such as the present invention has low raw-material cost, operating procedure simple, mild condition, it is adaptable to big
Large-scale production.
Brief description of the drawings
Fig. 1 is that fibroin protein film and the lysozyme-collagen/silk fibroin composite membrane of preparation are swept in embodiment 1
Retouch electron microscope.Lysozyme-collagen that a-f is successively fibroin protein film, the bimolecular number of plies is 0.5,5,5.5,10 and 10.5/
Silk fibroin composite membrane, multiplication factor be 1000 ×；A '-f ' be successively fibroin protein film, the bimolecular number of plies be 0.5,5,5.5,
10 and 10.5 lysozyme-collagen/silk fibroin composite membrane, multiplication factor be 5000 ×.
Fig. 2 is cell propagation and toxicity test result figure in embodiment 1, and subject cell is MC3T3-E1.A-g is successively silk
Fibroin film, the bimolecular number of plies are 0.5,1,5,5.5,10 and 10.5 lysozyme-collagen/silk fibroin composite membrane.
Fig. 3 is that MC3T3-E1 cells are inoculated in fibroin protein film and lysozyme-collagen/fibroin egg in embodiment 1
Later scanning electron microscope (SEM) photograph on tunica albuginea.Lysozyme-collagen that a-c is successively fibroin protein film, the bimolecular number of plies is 0.5 and 5.5
Albumen/silk fibroin composite membrane.
Fig. 4 is fibroin albumen/polycaprolactone composite nanometer fiber membrane and chitosan-collagen/fibroin in embodiment 3
The scanning electron microscope (SEM) photograph of albumen/polycaprolactone composite nanometer fiber membrane.A-e is successively fine fibroin albumen/polycaprolactone composite Nano
Dimension film, chitosan-collagen/fibroin albumen/polycaprolactone composite Nano that the bimolecular number of plies is 5,5.5,10 and 10.5 are fine
Dimension film.
Fig. 5 is people's normal dermal fibroblasts in embodiment 3（NHDFs）Fibroin albumen/polycaprolactone is inoculated in be combined
The scanning electron microscope (SEM) photograph of nano fibrous membrane and chitosan-collagen/fibroin albumen/polycaprolactone composite nanometer fiber membrane.a-d
Be successively fibroin albumen/polycaprolactone composite nanometer fiber membrane, the chitosan-collagen egg that the bimolecular number of plies is 0.5,10 and 10.5
In vain/fibroin albumen/polycaprolactone composite nanometer fiber membrane.
Specific embodiment
Technical scheme is described further below by specific embodiment, its object is to help preferably
Understand technical scheme, but these specific embodiments are not in any way limit the scope of the present invention.
Embodiment 1
（1）Lysozyme and the I- collagen type aqueous solution are prepared respectively, concentration is 1mg/mL, stirring is completely dissolved until solute；
According to volume ratio 1:Above two solution is mixed to get lysozyme and collagen mixed solution by 1 ratio, and uses ice vinegar
It is 6.3 that acid and ammoniacal liquor will adjust its pH value.
（2）Silk fibroin water solution is prepared, concentration is 1mg/mL, it is 8.0 that will adjust its pH value using glacial acetic acid and ammoniacal liquor.
（3）It is base plate film with the fibroin protein film that Hubei match los biosynthesis Science and Technology Ltd. provides.By fibroin protein film in
Soaked 20 minutes in lysozyme-fibroin albumen mixed solution, then with milli-Q water three times, 3 minutes every time, be so far considered as assembling
0.5 bilayer；Then soaked 20 minutes in silk fibroin protein solution, it is same to use milli-Q water three times, so far it is considered as
Assemble 1 bilayer；Operation more than repeating obtains the lysozyme-collagen/fibroin egg of bimolecular number of plies 0.5-10.5
White composite membrane, dries further vacuum drying naturally at room temperature.Then each group sample is shot into ESEM, is observed and comparison sheet
The face change of biofilm surface pattern afterwards before modified.
（4）Modified protein composite film is made the disk of a diameter of 6mm of card punch, ultraviolet irradiation 2h is sterilized
Afterwards, it is positioned in 96 orifice plates, and it is completely covered on bottom hole；And be divided into according to the difference of the bimolecular number of plies for being assembled
Corresponding group, 5 parallel controls of every group of setting.
（5）The mice embryonic bone precursor cells in exponential phase that will be cultivated（MC3T3-E1）It is 5 to be diluted to density
×103Individual/mL cell suspensions, take the cell suspension inoculation of 200 μ L in step respectively（4）In ready protein composite film sample
In this.It is put into culture in cell culture incubator（37 DEG C, 5% CO2）, change a cell culture medium within every 2 days.Culture is received after 5-7 days
Cell is obtained, then metal spraying after fixed, flushing, dehydration shoots scanning electric mirror observing cell adhesion situation and cell growth form.
（6）Such as step（4）The making sample is simultaneously grouped, and the group that protein composite film is not put in setting is blank
Group.3 × 10 are inoculated with per hole3Individual MC3T3-E1 cells, remove cell culture medium respectively at after 24h and 48h, replace with new
Culture medium containing cck-8 reagents, according to specification, lucifuge is inserted in incubator and taken out after culture 3h, is read in 450nm
Absorbance.Every group remove maximum and minimum value after average, be compared calculating cell proliferation rate with control group.
The pattern of gained lysozyme-collagen/silk fibroin composite membrane is shown in Fig. 1, it is seen that fibroin albumen surface exist compared with
More larger hole, and surface relative smooth.And after LBL self-assembly is modified, surface pore is obviously reduced, and first meeting
Tend to planarization, surface is also gradually coarse, possesses many nano projections.
Cell in vitro propagation and toxicity test are carried out using MC3T3-E1 cells, Fig. 2,3 are as a result seen.As seen from Figure 2
Self-chambering layer by layer be modified later composite membrane possess preferably promote cell propagation ability.Fig. 3 then intuitively illustrates fibroin egg
The larger gap on tunica albuginea surface is unfavorable for the migration of cell, and the promotion by the modified composite membrane in the method surface more preferably
The attaching of cell and propagation.But Fig. 2 also illustrate that surface biological is porous important, excessive assembling causes material surface
Tend to planarization will so that its promote growth and proliferation of cell ability decrease, therefore will according to target effect to assembling
The bimolecular number of plies is selected.
Embodiment 2
（1）Using 0.5% NaCO3Solution dissolves silkworm degumming of silk, concise rear calcium chloride/ethanol/water ternary solution, thoroughly
Obtain the silk fibroin protein solution of 2.5-3.5% after analysis, purifying, concentration, it is freeze-dried after obtain fibroin protein film.
（2）Gained fibroin protein film is crosslinked 1 hour with 75% ethanol, room temperature is further vacuum dried after drying.
（3）With 2% peracetic acid formulation chitosan solution（1mg/mL）, magnetic agitation adjusts pH to 5.0 to after being completely dissolved；Match somebody with somebody
The I- collagen types aqueous solution processed（1mg/mL）, magnetic agitation is to after being completely dissolved with glacial acetic acid and ammoniacal liquor regulation pH to 5.0.
（4）Chitosan solution is assembled in step using spin-coating method first（2）Gained fibroin albumen/polycaprolactone is compound to be received
Rice tunica fibrosa surface, dries naturally.Spin coating parameters are：0.3mL, spin speed is 1000rad, and spin-coating time is 3min, so far
It is depending on assembling 0.5 bilayer；Afterwards with same parameter spin coating collagen aqueous solution, dry naturally, be so far considered as assembling 1
Individual bilayer；Operation more than repeating obtains the surface modified fibroin protein film of bimolecular number of plies 5-20, dries naturally at room temperature
Further vacuum drying.Then each group sample is shot into ESEM, observe and comparison surface before modified after biofilm surface shape
The change of looks.
（5）Modified fibroin protein film is made the disk of a diameter of 16mm of card punch, ultraviolet irradiation 2h is gone out
After bacterium, it is positioned in 24 orifice plates, and it is completely covered on bottom hole；And according to the different by its point of the bimolecular number of plies for being assembled
Into corresponding group, 5 parallel controls of every group of setting.
（6）The mice embryonic bone precursor cells in exponential phase that will be cultivated（MC3T3-E1）It is 5 to be diluted to density
×104Individual/mL cell suspensions, take the cell suspension inoculation of 1mL in step respectively（4）In ready fibroin protein film and modified
In the sample of fibroin protein film.It is put into culture in cell culture incubator（37 DEG C, 5% CO2）, change a cell culture medium within every 2 days.
Harvesting after culture 5-7 days, it is fixed, rinse, metal spraying after dehydration, then shoot scanning electric mirror observing cell adhesion situation and carefully
Intracellular growth form.
ESEM result shows, shitosan and collagen are assembled in the surface of fibroin protein film using spin-coating method
Afterwards, its surface pore has reduced, and surface becomes gradually coarse by smooth, and nano surface is raised to be increased, and is inoculated with thereon
MC3T3-E1 cytochrome oxidase isozymes are more abundant, it is seen that more lamellipodiums and filopodia.
Embodiment 3
（1）Prepare 7wt% fibroin albumens/polycaprolactone composite nanometer fiber membrane using electrostatic spinning technique, wherein fibroin albumen with
The mass ratio of polycaprolactone is 5:1.Electrospinning parameters include：Rate of flooding is 1.0mL/h, and the voltage of high-voltage DC power supply is
16kV, the distance of syringe needle to receiver board is 15cm；Temperature is 27 DEG C, and humidity is 50%.
（2）Gained fibroin albumen/polycaprolactone composite nanometer fiber membrane is crosslinked 1 hour with 75% ethanol, after room temperature is dried
Further vacuum drying.
（3）With 2% peracetic acid formulation chitosan solution（1mg/mL）, magnetic agitation is to after being completely dissolved with glacial acetic acid and ammoniacal liquor
Regulation pH to 5.0；Prepare the I- collagen type aqueous solution（1mg/mL）, magnetic force be slowly stirred to after being completely dissolved with glacial acetic acid and
Ammoniacal liquor adjusts pH to 5.0.
（4）By step（2）Gained fibroin protein film soaks 20 minutes in being initially positioned at chitosan solution, then uses ultrapure washing
Wash three times, 3 minutes every time, be so far considered as and assemble 0.5 bilayer；Then 20 points are soaked in collagen aqueous solution
Clock, it is same to use milli-Q water three times, so far it is considered as and assembles 1 bilayer；Operation more than repeating obtains the bimolecular number of plies
The surface modified fibroin protein film of 0.5-10.5, dries further vacuum drying naturally at room temperature.Then each group sample is shot
ESEM, observes and the comparison surface change of biofilm surface pattern afterwards before modified.
（5）Modified fibroin protein film is made the disk of a diameter of 6mm of card punch, ultraviolet irradiation 2h is sterilized
Afterwards, it is positioned in 96 orifice plates, and it is completely covered on bottom hole；And be divided into according to the difference of the bimolecular number of plies for being assembled
Corresponding group, 5 parallel controls of every group of setting.
（6）The people's normal dermal fibroblasts in exponential phase that will be cultivated（NHDFs）Be diluted to density for 5 ×
103Individual/mL cell suspensions, take the cell suspension inoculation of 200 μ L in step respectively（4）In ready fibroin protein film and modified
In the sample of fibroin protein film.It is put into culture in cell culture incubator（37 DEG C, 5% CO2）, change a cell culture medium within every 2 days.
Harvesting after culture 5-7 days, it is fixed, rinse, metal spraying after dehydration, then shoot scanning electric mirror observing cell adhesion situation and carefully
Intracellular growth form.
The pattern of the present embodiment gained chitosan-collagen/fibroin albumen/polycaprolactone composite nanometer fiber membrane is shown in figure
4, it is seen that after LBL self-assembly is modified, nanofiber diameter becomes larger, and surface is gradually coarse, possesses many nanometer convexes
Rise.Cell adhesion experiment is carried out using NHDFs, Fig. 5 is as a result seen.The film surface that self-chambering layer by layer is modified later as seen from Figure 5
Cell volume is larger, extends more fully, shows its surface more conducively cell adherence, and modified composite nano-fiber membrane possesses
Preferably promote the ability of cell propagation.
Embodiment 4
（1）Compound concentration is 10wt% polycaprolactone solution, and solvent is hexafluoroisopropanol.Magnetic agitation 10h is complete to polycaprolactone
Dissolving.
（2）Appropriate polycaprolactone solution knifing on cleaned glass plate is taken, then film and glass plate are placed in fume hood
Until solvent volatilizees completely.24h is soaked after film is removed in distilled water, is then dried in atmosphere.
（3）Prepare Lysozyme in Aqueous Solution（3mg/mL）, pH to 5.0 is adjusted after being completely dissolved；Prepare I- collagen type water
Solution（2mg/mL）, magnetic agitation adjusts pH to 5.0 to after being completely dissolved.
（4）Appropriate Lysozyme in Aqueous Solution is taken first to scratch in step（1）The polycaprolactone film surface for obtaining final product, dries naturally
Afterwards, this is 0.5 bilayer of assembling；Appropriate collagen aqueous solution blade coating is then taken, is dried naturally, be so far considered as assembling 1
Individual bilayer.Operation more than repeating obtains the composite membrane of bimolecular number of plies 0.5-20, dries further vacuum naturally at room temperature
Dry.Then each group sample is shot into ESEM, is observed and the comparison surface change of biofilm surface pattern afterwards before modified.
（5）Modified fibroin protein film is made the disk of a diameter of 16mm of card punch, ultraviolet irradiation 2h is gone out
After bacterium, it is positioned in 24 orifice plates, and it is completely covered on bottom hole；And according to the different by its point of the bimolecular number of plies for being assembled
Into corresponding group, 5 parallel controls of every group of setting.
（6）The people's normal dermal fibroblasts in exponential phase that will be cultivated（NHDFs）Be diluted to density for 5 ×
103Individual/mL cell suspensions, take the cell suspension inoculation of 200 μ L in step respectively（4）In ready fibroin protein film and modified
In the sample of fibroin protein film.It is put into culture in cell culture incubator（37 DEG C, 5% CO2）, change a cell culture medium within every 2 days.
Harvesting after culture 5-7 days, it is fixed, rinse, metal spraying after dehydration, then shoot scanning electric mirror observing cell adhesion situation and carefully
Intracellular growth form.
ESEM result shows, lysozyme and collagen are assembled in the surface of polycaprolactone film using knife coating
Afterwards, its surface pore is reduced, and surface is gradually coarse with the increase of the assembling bimolecular number of plies, and nano surface is raised to be increased, and is connect
The NHDFs cytochrome oxidase isozymes planted thereon are more abundant, it is seen that more lamellipodiums and filopodia.
Embodiment 5
（1）Compound concentration is 10wt% polycaprolactone solution, and solvent is hexafluoroisopropanol.Magnetic agitation 10h is complete to polycaprolactone
Dissolving.
（2）Polycaprolactone nano fibrous membrane is prepared using electrostatic spinning technique.Electrospinning parameters include：Rate of flooding is
1.0mL/h, the voltage of high-voltage DC power supply is 16kV, and the distance of syringe needle to receiver board is 15cm；Temperature is 27 DEG C, and humidity is
50%。
（3）2wt% chitosan solutions are prepared, the glacial acetic acid of solvent 2%, magnetic force is slowly stirred to being completely dissolved；Prepare I-
The collagen type aqueous solution（2mg/mL）, magnetic force is slowly stirred to being completely dissolved.
（4）Chitosan solution is fitted into syringe, high voltage power supply is opened, predetermined voltage is adjusted to.Then electrostatic is passed through
Shitosan is fixed on polycaprolactone nano fibrous membrane surface by spraying technology, and spray time is to treat that it dries naturally after 30s, so far
It is 0.5 bilayer of assembling.EFI parameter includes：Rate of flooding is 1mL/h, and the voltage of high-voltage DC power supply is 20kV, pin
The distance that head arrives receiver board is 9cm；Environment temperature is 25 DEG C, and humidity is 50%.
（5）In the same way and collagen aqueous solution EFI is fixed on step by parameter（4）Gained composite Nano is fine
Dimension film surface, is so far considered as 1 bilayer of assembling.
（6）Repeat step（4）And step（5）The composite membrane of 0.5-20 layers of bilayer can be obtained, room temperature is dried naturally
Further vacuum drying afterwards.Then by each group sample shoot ESEM, observe and comparison surface before modified after biofilm surface
The change of pattern.
（7）Modified fibroin protein film is made the disk of a diameter of 6mm of card punch, ultraviolet irradiation 2h is sterilized
Afterwards, it is positioned in 96 orifice plates, and it is completely covered on bottom hole；And be divided into according to the difference of the bimolecular number of plies for being assembled
Corresponding group, 5 parallel controls of every group of setting.
（8）The human mouth mucosal epithelial cells in exponential phase that will be cultivated（HIOEC）It is 5 × 10 to be diluted to density3
Individual/mL cell suspensions, take the cell suspension inoculation of 200 μ L in step respectively（4）In ready fibroin protein film and modified silk
In the sample of fibroin film.It is put into culture in cell culture incubator（37 DEG C, 5% CO2）, change a cell culture medium within every 2 days.Training
Then harvesting after supporting 5-7 days, metal spraying after fixed, flushing, dehydration shoots scanning electric mirror observing cell adhesion situation and cell
Growthform.
ESEM result shows that after LBL self-assembly is modified, polycaprolactone film nanofiber diameter gradually becomes
Greatly, surface is gradually coarse, possesses many nano projections.It is seeded in the biofilm surface HIOEC volumes that self-chambering layer by layer is modified later
It is larger, extend more fully, show its surface more conducively cell adherence, modified composite membrane possesses and preferably promote cell increasing
The ability grown.
Above-described embodiment is the present invention preferably implementation method, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from Spirit Essence of the invention and the change, modification, replacement made under principle, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (6)
1. it is a kind of regulate and control method of the biofilm surface topological structure to promote cell to creep, it is characterised in that：Described method is
Using polyelectrolyte self assembly target organism film layer by layer, regulate and control biofilm surface topological structure；Described biofilm surface topology
Structure includes that pore-size, interporal lacuna and nano surface are raised.
2. method according to claim 1, it is characterised in that：Comprise the following steps：Choose two kinds and carry opposite charges
Polyelectrolyte, carrying opposite charges polyelectrolyte with it according to the selection of target organism film surface charge carries out self assembly, subsequent group
Dress is another to carry opposite charges polyelectrolyte, is so far one bilayer of assembling, by that analogy until the number of plies needed for obtaining.
3. method according to claim 1, it is characterised in that：The method of described LBL self-assembly be solution infusion method,
Knife coating, spin-coating method, graft copolymerization, electrostatic spraying, electro-deposition or fluid are fixed.
4. method according to claim 1, it is characterised in that：Described polyelectrolyte is shitosan, fibroin albumen, collagen
Albumen, lysozyme, polylysine, polypropylene amino, polyethyleneimine, sodium alginate, gelatin, chondroitin sulfate, heparin sulfate,
One or more in dextran sulfate, albumin, polyglutamic acid, hyaluronic acid.
5. method according to claim 1, it is characterised in that：Described biomembrane passes through the tape casting, electrostatic spinning, freezing
Drying, electro-deposition method, spin-coating method, knife coating, vacuum method or immersion precipitation phase inversion process are made.
6. using LBL self-assembly regulation and control biofilm surface topological structure in the application for promoting cell to be creeped on biomembrane.
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