WO2016013848A1 - Polyvinylidene fluoride nanocomposite scaffold for cell culture, and method for producing same - Google Patents

Polyvinylidene fluoride nanocomposite scaffold for cell culture, and method for producing same Download PDF

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WO2016013848A1
WO2016013848A1 PCT/KR2015/007556 KR2015007556W WO2016013848A1 WO 2016013848 A1 WO2016013848 A1 WO 2016013848A1 KR 2015007556 W KR2015007556 W KR 2015007556W WO 2016013848 A1 WO2016013848 A1 WO 2016013848A1
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polyvinylidene fluoride
cell culture
scaffold
composite membrane
carbon nanotubes
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French (fr)
Korean (ko)
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서용석
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서울대학교 산학협력단
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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
    • 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
    • A61L27/02—Inorganic materials
    • A61L27/08—Carbon ; Graphite
    • 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
    • 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
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C12M3/006—Cell injection or fusion devices

Definitions

  • the present invention relates to a polyvinylidene fluoride nanocomposite scaffold for cell culture prepared by mixing polyvinylidene fluoride, a polymer material showing piezoelectric properties, with carbon nanotubes, and more specifically, polyvinylidene A mixed solution preparation step of mixing the fluoride solution and the carbon nanotube suspension; A composite membrane manufacturing step of preparing a composite membrane using the mixed solution; A composite membrane stretching step of stretching the composite membrane; And a method of preparing a polyvinylidene fluoride nanocomposite scaffold for cell culture, characterized in that it comprises a composite membrane polarization step of applying an electric field to the composite membrane.
  • the present invention relates to a polyvinylidene fluoride nanocomposite scaffold for cell culture, which has excellent cell deposition and proliferation ability as well as excellent physical performance.
  • Scaffolds refer to physical scaffolds and adhesive substrates that are made for in vitro culture and transplantation of tissue cells. Such scaffolds are used for cell transplantation for regeneration of human tissues. It is related to the adhesion and the migration and proliferation of epithelial cells, which is important for the mass culture and proliferation of cells. That is, most biologically active cells have a basic step that must pass through to survive in contact with substances in or outside the body. The first step is cell adhesion. In particular, when looking at the survival stage of fibroblasts and tissue cells, the cells preferentially adhere to the substrate, and after adhesion, the metabolism of organelles in the cytoplasm becomes active, and a new site is used to facilitate the proliferation and supply of nutrients. Will move.
  • the surface that activates the deposition of cells is the most basic means of doubling cell proliferation.
  • the adhesion of these cells to the substrate can be artificially controlled by the components of the substrate.
  • Scaffolds are carriers that support scaffolds as they regenerate and grow, that is, the basis of artificial substrates. Recently, scaffolds have been used in mass culture and proliferation vessels or flasks of cells. Regarding such substrates, the Korean Patent Application No. 1991-0005802 of the present invention relates to a cell culture semipermeable membrane and a method for producing the same, and discloses a cell culture semipermeable membrane prepared as a matrix. In Korea Patent Registration No.
  • Polyvinyl alcohol-collagen hydrogel scaffold for cell culture and its manufacturing method are blended polyvinyl alcohol and collagen at a constant ratio to hydro Cell culture scaffolds prepared by gelation and radiation solidification have excellent cell deposition and proliferation ability, and also have excellent physical properties of scaffolds such as gel strength, swelling and elongation, It is reported that it can be easily coated, but by preparing a mixed cross-linking by irradiation The problem of cell deformity following irradiation in culture is not considered.
  • the present inventors have solved the above-mentioned problems, the cells can be easily adhered, the physical properties are excellent, suitable for mass culture of cells, the purification and sterilization process does not require a new cell culture for easy manufacturing
  • the scaffold and its manufacturing method were invented.
  • an object of the present invention is to provide a cell culture scaffold that can easily adhere to cells so as to enable mass culture and proliferation of cells, and which has excellent physical properties of the support surface.
  • Another object of the present invention is to provide a method which can more easily produce a scaffold having the above characteristics without the purification and sterilization process.
  • the object of the present invention described above is to disperse carbon nanotubes in polyvinylidene fluoride showing piezoelectric properties, to prepare nanocomposites, and to maximize the content of the beta phase showing piezoelectric properties through the stretching step and the polarization step. And it was intended to prepare a polyvinylidene fluoride nanocomposite scaffold for cell culture as well as excellent proliferation ability.
  • Cell culture polyvinylidene fluoride scaffold of the present invention for achieving the above object is a polyvinylidene fluoride (Polyvinylidene fluoride) as a main component in the solvent and the carbon nanotubes dispersing the solution to the functional group on the surface After mixing with the solution and uniformly dispersing with homogenizer, spinning the nanofibers by electrospinning process to obtain the film made of nanofibers after stretching the nanofibers, and applying the stretching and polling to the film to improve the piezoelectric properties of the film.
  • Polyvinylidene fluoride nanocomposite scaffolds for maximizing the beta phase content exhibiting piezoelectric properties were prepared by using the membrane obtained by maximizing the scaffold or by directly applying the solution onto a flat surface.
  • the carbon nanotubes dispersed in the polyvinylidene fluoride are contained in less than 2% by weight.
  • the piezoelectric properties of the nanocomposites are found to decrease, and thus, the proliferation of the cells is reduced, and the physical properties of the nanocomposites may be partially broken during stretching. This becomes undesirable.
  • An important factor in preparing the nanocomposite for the scaffold of the present invention is that the polarizations in the molecules are oriented in a certain direction to indicate the piezoelectric properties of the polyvinylidene fluoride, which is most effective when the carbon nanotubes contain about 0.5 to 1% by weight.
  • Cell culture scaffold of the present invention configured as described above is polyvinylidene fluoride, unlike the general fluorine-containing polymers, excellent cell deposition and proliferation ability, and also contains carbon nanotubes to improve the strength and piezoelectric properties of the nanocomposite Increasing, the physical performance as a scaffold is complemented and can be easily coated on the inner wall of the cell culture vessel.
  • the carbon nanotube-containing polyvinylidene fluoride prepared according to the present invention maximizes the content of the beta phase exhibiting piezoelectric properties, and is excellent in cell deposition and proliferation ability as well as excellent physical performance, making it useful as a scaffold for cell culture. Very good
  • the polyvinylidene fluoride nanocomposite according to the present invention promotes cell activity and improves cell adhesion, and thus can be utilized as biomaterials such as scaffolds for culture of cells and cells, culture assays, culture tubes, and neural junctions. Do.
  • FIG. 1 is a schematic diagram of an electrospinning apparatus used in the present invention.
  • FIG. 2 is a graph showing changes in beta-phase content in the crystal phase in the polyvinylidene fluoride nanocomposite sample by electrospinning method according to an embodiment of the present invention with respect to carbon nanotube content.
  • FIG. 3 is a graph showing the change in beta phase content in the crystal phase in the polyvinylidene fluoride nanocomposite sample by the solution casting method according to an embodiment of the present invention with respect to the carbon nanotube content.
  • Figure 4 shows the results when applying the polyvinylidene fluoride nanocomposites prepared according to an embodiment of the present invention as a cell culture scaffold.
  • Figure 5 shows the results of immunofluorescence microscopy 7 days after cell seeding according to an embodiment of the present invention
  • A RT4-D6P2T
  • B U87-MG
  • C as F-actin of the cytoplasm nucleus, respectively
  • SH-SY5Y cell is a pure polyvinylidene fluoride scaffold
  • b is a scaffold containing 0.05% carbon nanotubes
  • c is a membrane containing 0.5% carbon nanotubes 7 days after cell seeding.
  • Method for producing a polyvinylidene fluoride nanocomposite scaffold for cell culture comprises the steps of: (i) preparing a mixed solution for mixing a polyvinylidene fluoride solution and a carbon nanotube suspension; (ii) a composite membrane manufacturing step of preparing a composite membrane using the mixed solution; (iii) a composite membrane stretching step of stretching the composite membrane; And (iv) polarizing the composite membrane by applying an electric field to the composite membrane.
  • the content of carbon nanotubes dispersed in the composite film is preferably 0.5 to 1% by weight based on polyvinylidene fluoride.
  • a composite membrane may be prepared by electrospinning the mixed solution, or a composite membrane may be prepared by solution casting the mixed solution.
  • the carbon nanotubes are preferably subjected to a functionalization step using a mixed acid of sulfuric acid and nitric acid before preparing the suspension.
  • Cell culture polyvinylidene fluoride nanocomposite scaffold according to an aspect of the present invention is prepared by the above method is the content of carbon nanotubes dispersed in the composite membrane is 0.5 to 1% by weight compared to polyvinylidene fluoride It is characterized by.
  • the polyvinylidene fluoride nanocomposite scaffold for cell culture has a beta phase composition of more than 80% of polyvinylidene fluoride, which maximizes the content of the beta phase, which exhibits piezoelectric properties, and provides excellent cell deposition and proliferation ability. Its excellent performance makes it an excellent scaffold for cell culture.
  • the polyvinylidene fluoride nanocomposite according to the present invention promotes cell activity and improves cell adhesion, and thus can be utilized as biomaterials such as scaffolds for culture of cells and cells, culture assays, culture tubes, and neural junctions. Do.
  • Polyvinylidene fluoride is a semicrystalline polymer whose molecular structure is in the form of- (CH 2 CF 2 ) ⁇ -in which units of vinylidene fluoride are linearly bonded.
  • the electronegativity of fluorine (3.98) is very high compared to hydrogen (2.20), so that the electrons in the molecule tend to be biased in the direction in which fluorine is located, creating a dipole moment in the direction perpendicular to the chain.
  • the polarization and piezoelectric properties of the TTTT type beta phase have a tetragonal unit cell.
  • the gamma phase is also a tetragonal crystal structure, but shows a TTTGTTTG array structure, and in the case of other delta and epsilon phases, it is similar to the alpha and beta phases.
  • Alpha and beta phases are the most common polyvinylidene fluoride phases.
  • the alpha phase is non-piezoelectric, whereas all dipoles in the beta phase (TTTT) are piezoelectric with moments in the same direction perpendicular to the chain.
  • Electrospinning process of the polymer solution has been widely used in the manufacture of nanofibers as an effective method for producing ultra-fine fibers up to several micrometers in diameter.
  • the resulting film is stretched or polarized to maximize the beta phase content exhibiting piezoelectric properties, or made of polyvinylidene fluoride nanofibers containing carbon nanotubes using an electrospinning process.
  • the beta phase-containing membrane was first prepared by the stretching of the electrospinning process and the orientation of the CF 2 group due to the interaction between the surface functional group of the carbon nanotubes and the fluorine atoms of the polyvinylidene fluoride.
  • the polyvinylidene fluoride nanocomposite is used as a cell culture scaffold to clearly show cell culture activity. Can be improved. Scaffolds prepared in the present invention can be modified in various forms and used in various cell multipliers.
  • Semi-crystalline polyvinylidene fluoride (average molecular weight 5.210 5 ) was dried for 24 hours under vacuum at 80 °C to remove moisture and impurities, dimethyl acetamide and acetone were mixed in a ratio of 50:50 and dried poly Vinylidene fluoride is added to the combined solvents in an appropriate ratio and stirred at 60 ° C. until a homogeneous solution is formed. At this time, the concentration of polyvinylidene fluoride was fixed at 15% by weight, which is a value corresponding to an appropriate viscosity for obtaining fine and uniform fibers.
  • the carbon nanotubes are subjected to surface acid treatment for functionalization.
  • the solution was added to a sulfuric acid / nitric acid (3: 1 volume ratio) solution, stirred for 48 hours, and then subjected to strong ultrasonic waves for 2 hours.
  • the multilayer carbon nanotubes were filtered through a 0.4 ⁇ m polyvinylidene fluoride filter membrane and washed with pure distilled water until the pH value reached 7. After washing, the multilayered carbon nanotubes are dried in half day at 80 ° C. in a vacuum.
  • the multi-layered carbon nanotubes to which the functional groups are attached are dispersed in a dimethylacetamide solvent for 30 minutes using an ultrasonic bath.
  • Polyvinylidene fluoride is quantitatively dissolved in acetone and stirred for 30 minutes using a magnetic stirrer. Thereafter, the acetone / polyvinylidene fluoride solution and the multilayer carbon nanotube / dimethylacetamide suspension are mixed and stirred at 40 ° C. After loading the solution into a 10 CC syringe and using an electrospinning process to obtain a nanocomposite consisting of nanofibers.
  • the alpha phase of polyvinylidene fluoride is first converted into the beta phase by the stretching action of the electrospinning process.
  • the added carbon nanotubes cause and help the alpha phase to be converted to the beta phase due to the surface polar group.
  • the appearance looks to increase with the content of the multi-layer carbon nanotubes in a 840cm -1 and 1270cm -1.
  • the alpha phase composition corresponding to 2 ⁇ 18.6 and 20.2 degrees decreases and the beta phase composition of 20.9 ° increases as the multilayer carbon nanotube content increases. This is due to the nucleation of the multi-walled carbon nanotubes.
  • the carboxyl group is attached to the surface of the multi-walled carbon nanotubes and dispersed in a polyvinylidene fluoride solution, an electrostatic interaction occurs between the carboxyl group and CF 2 . This interaction not only contributes to the formation of higher crystallinity, but also facilitates the phase-to-beta phase transition because the TTTT sequence of the beta phase exhibits a more stable energy state than the TGTG array of the alpha phase composition.
  • the multi-walled carbon nanotubes reach a concentration higher than the critical point, the viscoelastic force becomes stronger than the electrostatic force by the electrospinning process, and the variation of the beta phase composition is insignificant.
  • the multi-layered carbon nanotubes are more than 0.5%, the beta phase is found to be reduced slightly because depolarization occurs due to charge accumulation at the interface.
  • the combination of the stretching or polarization process of the nanofiber film greatly affects the beta phase composition, and the alpha phase composition decreases in the analysis using Fourier transform infrared spectroscopy and wide-angle X-ray diffraction analyzer. At the same time an increase in the composition of the beta phase is observed.
  • the stretching process exerts a mechanical external force to induce the polymer chains in the crystal to be arranged in a row, and to form a beta phase of the longest form of the crystal phase.
  • the polarization process creates an electric field in a direction perpendicular to the polyvinylidene fluoride fiber, elicits a dipole moment in that direction and increases the beta phase composition as in the stretching process.
  • the beta phase composition change when the stretching and polarization process is applied at various multilayer carbon nanotube contents is shown in FIG. 2.
  • Specimens containing multilayered carbon nanotubes have a beta-phase composition of at least 91%, while those that do not have only about 80% of the composition, and the bonding between the multilayer carbon nanotubes and polyvinylidene fluoride makes the stretching process more efficient. Because of making. The polarization process does not have a large phase shift effect compared to the stretching process, but similarly, there is an influence by multilayer carbon nanotubes. Accumulation of charge on both sides of the polyvinylidene fluoride specimens results in polarization of the current, which does not pass through, resulting in nonuniform dipoles. However, when the multi-walled carbon nanotubes having high capacitance are dispersed and bonded to the surface of the alpha phase crystal, the dipole moment direction is more efficiently induced and the phase conversion to the beta phase is promoted.
  • the polyvinylidene fluoride nanocomposite prepared by the electrospinning method was placed on the bottom, and two kinds of cells (RT4-D6P2T (schwannoma cell line in mouse) and U87-MG (human glioblastoma cell line) in volume ratio were 10%.
  • DMEM Dulbecco® Modified Eagle® medium
  • DMEM Dulbecco® Modified Eagle® medium
  • bobbin serum containing bobbin serum and 1% antibiotic / antifungal solution at a temperature of 37 ° C. and 5% CO 2
  • Cells were cultured in a culture medium with 0.25% trypsin / ethylenediaminetetra It was isolated with acid solution and incubated directly on the nanocomposite (40000 cells / cm 2 ).
  • MTT Metalthiazolyldiphenyl-tetrazolium bromide
  • the nanocomposites containing 0.5% carbon nanotubes in the 1 week culture scaffold were more than doubled than the pure polyvinylidene fluoride. It can be seen growing (FIG. 4).
  • U87-MG cells increased slowly with increasing carbon nanotubes and showed the highest growth rate when 1% of carbon nanotubes were contained.
  • the results of direct observation of the growth pattern of cells after one week with an immunofluorescence microscope are shown in FIG. 5. The more carbon tubes it contains (0.5%), the more the cells grew most.
  • the above results suggest that the more nanocomposites with better piezoelectric properties (including carbon nanotubes), the more polarized CF 2 groups on the surface and the more cells are stimulated by these polarities and grown.
  • Semi-crystalline polyvinylidene fluoride (average molecular weight 5.210 5 ) was dried for 24 hours under vacuum at 80 °C to remove moisture and impurities, dimethyl acetamide and acetone were mixed in a ratio of 50:50 and dried poly Vinylidene fluoride is added to the combined solvents in an appropriate ratio and stirred at 60 ° C. until a homogeneous solution is formed. At this time, the concentration of polyvinylidene fluoride was fixed at 15% by weight, which is a value corresponding to an appropriate viscosity for obtaining fine and uniform fibers.
  • the surface of the carbon nanotubes is subjected to acid treatment for functionalization.
  • the solution was added to a sulfuric acid / nitric acid (3: 1 volume ratio) solution, stirred for 48 hours, and then subjected to strong ultrasonic waves for 2 hours.
  • the multilayer carbon nanotubes were filtered through a 0.4 ⁇ m polyvinylidene fluoride filter membrane and washed with pure distilled water until the pH value reached 7. After washing, the multilayered carbon nanotubes are dried in half day at 80 ° C. in a vacuum.
  • the multi-layered carbon nanotubes to which the functional groups are attached are dispersed in a dimethylacetamide solvent for 30 minutes using an ultrasonic bath.
  • Polyvinylidene fluoride is quantitatively dissolved in acetone and stirred for 30 minutes using a magnetic stirrer.
  • the acetone / polyvinylidene fluoride solution and the multilayer carbon nanotube / dimethylacetamide suspension are mixed and stirred at 40 ° C.
  • the mixed solution is applied on a petri dish and left in the air for 24 hours to wait until the solvent evaporates completely, and then the dried membrane is stretched with a biaxial stretching machine and polarized again.
  • the content of the beta phase was measured using a Fourier transform infrared spectrometer and a wide-angle X-ray spectrometer as in Example 1 (FIG. 3). What is unusual is that the beta phase reaches a maximum when 0.5% of carbon nanotubes are contained, unlike the membrane prepared by the electrospinning described above, and then depolarization phenomenon occurs and decreases.
  • Dulbecco containing polyvinylidene fluoride nanocomposite prepared by the solution casting method at the bottom of the container and containing 10% petalbobin serum and 1% antibiotic / antifungal solution in volume ratio of SH-SY5Y cells (human neuroblastoma cell line).
  • SH-SY5Y cells human neuroblastoma cell line.
  • DMEM Modified Eagle® medium
  • Cells were separated from the culture medium with 0.25% trypsin / ethylenediaminetetraacid solution and cultured directly onto the composite membrane (40000 cells / cm 2 ).
  • Cell adhesion and growth were measured by MTT (Methylthiazolyldiphenyl-tetrazolium bromide) assay. Morphology of the cells was observed by immunofluorescence.
  • the growth rate of the cells increases with time, and in the case of the culture scaffold of one week, the growth of the cells is more than doubled than that of the pure polyvinylidene fluoride membrane. . It can be directly seen that the cells grew most at the carbon nanotube concentration with the highest beta phase (0.5%). The better the piezoelectric properties (containing carbon nanotubes), the more polarized CF 2 groups on the surface. The cells appear to have grown more stimulated by these polarities.
  • Polyvinylidene fluoride nanocomposites which are mostly in the beta phase showing high piezoelectric properties according to the present invention, promote cell activity and improve cell adhesion, so that cells and cell culture scaffolds, culture assays, culture tubes, and neural junction tubes It can be used as a bio material.

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Abstract

The present invention relates to a polyvinylidene fluoride nanocomposite scaffold for cell culture, and a method for producing same, the polyvinylidene fluoride nanocomposite scaffold for cell culture being produced by mixing carbon nanotubes with polyvinylidene fluoride which is a polymer substance showing piezoelectric properties. More specifically, the present invention relates to a method for producing a polyvinylidene fluoride nanocomposite scaffold for cell culture, the method comprising: a mixed solution preparing step for mixing a polyvinylidene fluoride solution and a carbon nanotube suspension; a composite film producing step for producing a composite film by using the mixed solution; a composite film stretching step for stretching the composite film; and a composite film polarizing step for applying an electric field to the composite film, and the present invention also relates to a polyvinylidene fluoride nanocomposite scaffold for cell culture produced by the method, the scaffold having a maximized content of beta-phase showing piezoelectric properties, thereby having not only excellent cell adhesion and proliferation capabilities but also excellent physical performance.

Description

세포배양용 폴리비닐리덴플루오라이드 나노복합체 스캐폴드 및 그 제조방법Polyvinylidene fluoride nanocomposite scaffolds for cell culture and preparation method thereof
본 발명은 압전특성을 보이는 고분자 물질인 폴리비닐리덴플루오라이드를 탄소나노튜브와 혼합하여 제조되는 세포배양용 폴리비닐리덴플루오라이드 나노복합체 스캐폴드 및 그의 제조방법에 대한 것으로 보다 상세하게는 폴리비닐리덴플루오라이드 용액과 탄소나노튜브 현탁액을 혼합하는 혼합용액 준비단계; 상기 혼합용액을 이용하여 복합막을 제조하는 복합막 제조단계; 상기 복합막을 연신하는 복합막 연신단계; 및 상기 복합막에 전기장을 가하는 복합막 분극화 단계를 포함하는 것을 특징으로 하는 세포배양용 폴리비닐리덴플루오라이드 나노복합체 스캐폴드의 제조방법 및 이러한 방법으로 제조되어 압전특성을 보이는 베타상의 함유율을 극대화 되어 세포의 증착 및 증식능력이 뛰어날 뿐 아니라 물리적 성능이 우수한 세포배양용 폴리비닐리덴플루오라이드 나노복합체 스캐폴드에 관한 것이다. The present invention relates to a polyvinylidene fluoride nanocomposite scaffold for cell culture prepared by mixing polyvinylidene fluoride, a polymer material showing piezoelectric properties, with carbon nanotubes, and more specifically, polyvinylidene A mixed solution preparation step of mixing the fluoride solution and the carbon nanotube suspension; A composite membrane manufacturing step of preparing a composite membrane using the mixed solution; A composite membrane stretching step of stretching the composite membrane; And a method of preparing a polyvinylidene fluoride nanocomposite scaffold for cell culture, characterized in that it comprises a composite membrane polarization step of applying an electric field to the composite membrane. The present invention relates to a polyvinylidene fluoride nanocomposite scaffold for cell culture, which has excellent cell deposition and proliferation ability as well as excellent physical performance.
본 출원은 2014년 7월 21일에 출원된 한국특허출원 제10-2014-0091898호에 기초한 우선권을 주장하며, 해당 출원의 명세서 및 도면에 개시된 모든 내용은 본 출원에 원용된다.This application claims priority based on Korean Patent Application No. 10-2014-0091898 filed on July 21, 2014, and all the contents disclosed in the specification and drawings of the application are incorporated in this application.
스캐폴드(Scaffold)는 조직 세포의 체외 배양과 체내 이식이 가능하도록 만들어진 물리적 지지체 및 점착 기질을 칭하는 것으로, 이러한 스캐폴드는 인체조직 재생을 위한 세포 이식에 사용되고 있으며, 세포와 기질 간의 접촉부분에서 세포의 점착 및 이에 수반되는 상피세포의 이동과 증식과 관련되어 있어 세포의 대량배양 및 증식에 있어서 그 중요성은 매우 높다. 즉, 생물학적으로 활성을 지니고 있는 대부분의 세포는 체내 혹은 체외의 물질과 접촉 시 생존하기 위하여 반드시 거쳐야 하는 기본단계가 있으며, 그 첫 단계는 세포의 점착이다. 특히, 섬유아세포 및 조직세포의 생존단계를 살펴보면, 세포는 우선적으로 기질에 점착을 하며 점착 후 세포질에서의 세포기관(organelle)의 대사가 활발해지고, 증식 및 양분의 공급을 원활히 하기 위하여 새로운 부위로 이동하게 된다. 따라서, 세포의 증착을 활성화시키는 표면은 세포의 증식을 배가하는데 가장 기본이 되는 수단이다. 이러한 세포의 기질에 대한 점착능은 기질의 성분에 의하여 인위적으로 조절될 수 있다. 스캐폴드는 세포의 재생과 이들이 성장할 때 지지체가 되어주는 담체, 즉 인공기질의 근간이 되는 물질로서, 최근 세포의 대량배양 및 증식용기 혹은 플라스크에 코팅되어 사용된다. 이러한 기질에 대해, 종래의 대한민국 특허출원 제1991-0005802호의 "세포배양용 반투막 겔과 그의 제조방법"은 세포 배양용 반투막 겔과 그의 제조 방법에 관한 것으로 매트릭스로 제조된 세포배양용 반투막겔을 개시하고 있으며, 대한민국 특허등록 제10-0783228호의 "세포배양용 폴리비닐알코올-콜라겐 하이드로젤 스캐폴드 및그 제조방법은 폴리비닐알코올(Polyvinyl alcohol)과 콜라겐(Collagen) 을 일정비율로 블렌드(blend)하여 하이드로젤화하고 방사선을 조사하여 고형화 시킨 것으로서 제조된 세포배양용 스캐폴드는 세포의 증착 및 증식능력이 뛰어나면서도, 겔강도, 팽윤도 및 연신율과 같은 스캐폴드의 물리적 성능 또한 우수하여, 세포배양용기의 내벽에 용이하게 코팅될 수 있다고 보고하고 있으나, 방사선조사에 의하여 혼합가교체를 제조함으로서 세포배양시 방사선조사에 따른 세포변형문제는 고려하지 않고 있다.Scaffolds refer to physical scaffolds and adhesive substrates that are made for in vitro culture and transplantation of tissue cells. Such scaffolds are used for cell transplantation for regeneration of human tissues. It is related to the adhesion and the migration and proliferation of epithelial cells, which is important for the mass culture and proliferation of cells. That is, most biologically active cells have a basic step that must pass through to survive in contact with substances in or outside the body. The first step is cell adhesion. In particular, when looking at the survival stage of fibroblasts and tissue cells, the cells preferentially adhere to the substrate, and after adhesion, the metabolism of organelles in the cytoplasm becomes active, and a new site is used to facilitate the proliferation and supply of nutrients. Will move. Thus, the surface that activates the deposition of cells is the most basic means of doubling cell proliferation. The adhesion of these cells to the substrate can be artificially controlled by the components of the substrate. Scaffolds are carriers that support scaffolds as they regenerate and grow, that is, the basis of artificial substrates. Recently, scaffolds have been used in mass culture and proliferation vessels or flasks of cells. Regarding such substrates, the Korean Patent Application No. 1991-0005802 of the present invention relates to a cell culture semipermeable membrane and a method for producing the same, and discloses a cell culture semipermeable membrane prepared as a matrix. In Korea Patent Registration No. 10-0783228, "Polyvinyl alcohol-collagen hydrogel scaffold for cell culture and its manufacturing method are blended polyvinyl alcohol and collagen at a constant ratio to hydro Cell culture scaffolds prepared by gelation and radiation solidification have excellent cell deposition and proliferation ability, and also have excellent physical properties of scaffolds such as gel strength, swelling and elongation, It is reported that it can be easily coated, but by preparing a mixed cross-linking by irradiation The problem of cell deformity following irradiation in culture is not considered.
본 발명자 등은 상기한 종래의 문제점을 해결하여 세포가 용이하게 점착할 수 있고, 물리적 특성이 우수하여 세포의 대량배양에 적합하고, 정제 및 멸균 공정이 필요하지 않아 제조가 용이한 새로운 세포배양용 스캐폴드 및 그 제조방법을 발명하였다. The present inventors have solved the above-mentioned problems, the cells can be easily adhered, the physical properties are excellent, suitable for mass culture of cells, the purification and sterilization process does not require a new cell culture for easy manufacturing The scaffold and its manufacturing method were invented.
따라서, 본 발명의 목적은 세포의 대량배양 및 증식이 가능하도록 세포가 용이하게 점착할 수 있고, 지지면의 물리적 특성이 우수한 세포배양용 스캐폴드를 제공하기 위한 것이다. 본 발명의 다른 목적은 상기의 특성을 갖는 스캐폴드를 정제 및 멸균 공정을 요하지 않으면서 보다 용이하게 제조할 수 있는 방법을 제공하기 위한 것이다. 상기한 본 발명의 목적은 압전특성을 보이는 폴리비닐리덴플루오라이드에 탄소나노튜브를 분산시킨 후 나노복합체를 제조하고 이를 연신단계와 분극단계를 거쳐서 압전특성을 보이는 베타상의 함유율을 극대화 되어 세포의 증착 및 증식능력이 뛰어날 뿐 아니라 물리적 성능이 우수한 세포배양용 폴리비닐리덴플루오라이드 나노복합체 스캐폴드를 제조하고자 하였다.Accordingly, an object of the present invention is to provide a cell culture scaffold that can easily adhere to cells so as to enable mass culture and proliferation of cells, and which has excellent physical properties of the support surface. Another object of the present invention is to provide a method which can more easily produce a scaffold having the above characteristics without the purification and sterilization process. The object of the present invention described above is to disperse carbon nanotubes in polyvinylidene fluoride showing piezoelectric properties, to prepare nanocomposites, and to maximize the content of the beta phase showing piezoelectric properties through the stretching step and the polarization step. And it was intended to prepare a polyvinylidene fluoride nanocomposite scaffold for cell culture as well as excellent proliferation ability.
상기 목적을 달성하기 위한 본 발명의 세포배양용 폴리비닐리덴플루오라이드 스캐폴드는 주성분으로 폴리비닐리덴플루오라이드(Polyvinylidene fluoride)를 용매에 녹이고 그 용액을 표면에 관능기를 부착시킨 탄소나노튜브를 분산시킨 용액과 혼합하여 호모게나이저로 균일하게 분산시킨 후 전기방사 공정을 이용하여 나노화이버를 방사하여 연신공정을 일차적으로 거친 후 나노화이버로 이루어진 막을 얻고 이 막에 연신과 폴링을 적용하여 막의 압전특성을 극대화 시킴으로써 얻어지는 막을 스캐폴드로 사용하거나 용액을 직접 평면 위에 도포한 후 생성된 막을 연신하거나 분극화시켜 압전특성을 보이는 베타상 함유량을 극대화 시킨 세포배양용 폴리비닐리덴플루오라이드 나노복합체 스캐폴드를 제조하였다.Cell culture polyvinylidene fluoride scaffold of the present invention for achieving the above object is a polyvinylidene fluoride (Polyvinylidene fluoride) as a main component in the solvent and the carbon nanotubes dispersing the solution to the functional group on the surface After mixing with the solution and uniformly dispersing with homogenizer, spinning the nanofibers by electrospinning process to obtain the film made of nanofibers after stretching the nanofibers, and applying the stretching and polling to the film to improve the piezoelectric properties of the film. Polyvinylidene fluoride nanocomposite scaffolds for maximizing the beta phase content exhibiting piezoelectric properties were prepared by using the membrane obtained by maximizing the scaffold or by directly applying the solution onto a flat surface.
본 발명 일 구현예에 따르면, 상기 폴리비닐리덴플루오라이드 내에 분산된 탄소나노튜브는 2중량% 미만으로 함유함을 특징으로 한다. 상기 탄소나노튜브함량이 2중량%를 넘을 경우 나노복합체의 압전특성은 오히려 감소하는 것으로 나타났으며 이렇게 되면 세포의 증식이 감소하게 되고 더욱이 연신 시에 나노복합체가 부분적으로 파단될 수가 있기 때문에 물리적 물성이 바람직하지 못하게 된다. 본 발명의 스케폴드용 나노복합체 제조시 중요한 인자는 폴리비닐리덴플루오라이드의 압전특성을 나타내기 위한 분자 내의 분극들이 일정한 방향으로 배향되는 것인데 이는 탄소 나노튜브가 0.5~1 중량% 정도 함유되었을 때 가장 큰 분극을 이룰 수 있으며 가장 좋은 압전 특성을 보이고 또 이때 가장 많은 세포증식을 이루게 된다. 상기와 같이 구성되는 본 발명의 세포배양용 스캐폴드는 폴리비닐리덴플루오라이드로 일반적인 불소함유 고분자들과는 달리 세포의 증착 및 증식능력이 뛰어나며, 또한 탄소나노튜브가 함유됨으로써 나노복합체의 강도와 압전특성을 증가시키게 되어 스캐폴드로서의 물리적 성능이 보완되었으며, 세포배양용기의 내벽에 용이하게 코팅될 수 있다.According to an embodiment of the present invention, the carbon nanotubes dispersed in the polyvinylidene fluoride are contained in less than 2% by weight. When the carbon nanotube content is more than 2% by weight, the piezoelectric properties of the nanocomposites are found to decrease, and thus, the proliferation of the cells is reduced, and the physical properties of the nanocomposites may be partially broken during stretching. This becomes undesirable. An important factor in preparing the nanocomposite for the scaffold of the present invention is that the polarizations in the molecules are oriented in a certain direction to indicate the piezoelectric properties of the polyvinylidene fluoride, which is most effective when the carbon nanotubes contain about 0.5 to 1% by weight. Large polarization can be achieved, showing the best piezoelectric properties, and at this time, the most cell proliferation. Cell culture scaffold of the present invention configured as described above is polyvinylidene fluoride, unlike the general fluorine-containing polymers, excellent cell deposition and proliferation ability, and also contains carbon nanotubes to improve the strength and piezoelectric properties of the nanocomposite Increasing, the physical performance as a scaffold is complemented and can be easily coated on the inner wall of the cell culture vessel.
본 발명에 따라 제조된 탄소나노튜브 함유 폴리비닐리덴플루오라이드는 압전특성을 보이는 베타상의 함유율이 극대화되어 세포의 증착 및 증식능력이 뛰어날 뿐 아니라 물리적 성능이 우수하여 세포배양용 스캐폴드로 활용성이 매우 뛰어나다.The carbon nanotube-containing polyvinylidene fluoride prepared according to the present invention maximizes the content of the beta phase exhibiting piezoelectric properties, and is excellent in cell deposition and proliferation ability as well as excellent physical performance, making it useful as a scaffold for cell culture. Very good
또한, 탄소나노튜브가 함유됨으로써 나노복합체의 강도와 압전특성을 증가시키게 되어 스캐폴드로서의 물리적 성능이 보완되었으며, 세포배양용기의 내벽에 용이하게 코팅될 수 있다.In addition, by containing the carbon nanotubes to increase the strength and piezoelectric properties of the nanocomposite to compensate for the physical performance as a scaffold, can be easily coated on the inner wall of the cell culture vessel.
본 발명에 따른 폴리비닐리덴프루오라이드 나노복합체는 세포활성을 촉진시키고 셀의 접착을 좋게 하므로 셀 및 세포의 배양용 스캐폴드, 배양에세이, 배양관, 신경접합관 등의 바이오재료로써 활용이 가능하다.The polyvinylidene fluoride nanocomposite according to the present invention promotes cell activity and improves cell adhesion, and thus can be utilized as biomaterials such as scaffolds for culture of cells and cells, culture assays, culture tubes, and neural junctions. Do.
도 1은 본 발명에 사용한 전기방사장치의 모식도이다.1 is a schematic diagram of an electrospinning apparatus used in the present invention.
도 2는 본 발명의 일 실시 예인 전기방사법에 의한 폴리비닐리덴플루오라이드 나노복합체 시료내 결정상 중 베타상 함유량의 변화를 탄소나노튜브 함유량에 대하여 각 공정단계별 변화를 나타낸 그래프이다.FIG. 2 is a graph showing changes in beta-phase content in the crystal phase in the polyvinylidene fluoride nanocomposite sample by electrospinning method according to an embodiment of the present invention with respect to carbon nanotube content.
도 3은 본 발명의 일 실시 예인 용액캐스팅법에 의한 폴리비닐리덴플루오라이드 나노복합체 시료내 결정상 중에서 베타상 함유량의 변화를 탄소나노튜브 함유량에 대하여 나타낸 그래프이다.3 is a graph showing the change in beta phase content in the crystal phase in the polyvinylidene fluoride nanocomposite sample by the solution casting method according to an embodiment of the present invention with respect to the carbon nanotube content.
도 4는 본 발명의 일 실시예에 따라 제조한 폴리비닐리덴플루오라이드 나노복합체를 셀 배양 스캐폴드로 적용하였을 때 결과를 나타낸 것이다.Figure 4 shows the results when applying the polyvinylidene fluoride nanocomposites prepared according to an embodiment of the present invention as a cell culture scaffold.
도 5는 본 발명의 일 실시예에 따른 셀 파종 후 7일 뒤의 면역형광현미경 결과를 나타낸 것으로, 세포질 핵의 F-actin 으로서 각각 (A)RT4-D6P2T, (B)U87-MG, (C)SH-SY5Y 셀 이고, (a)는 순수 폴리비닐리덴플루오라이드 스캐폴드이며 (b)는 0.05%의 탄소나노튜브가 함유된 스캐폴드 (c)는 0.5%의 탄소나노튜브가 함유된 막으로서 셀 파종 후 7일 뒤의 형태이다.Figure 5 shows the results of immunofluorescence microscopy 7 days after cell seeding according to an embodiment of the present invention, (A) RT4-D6P2T, (B) U87-MG, (C) as F-actin of the cytoplasm nucleus, respectively ) SH-SY5Y cell, (a) is a pure polyvinylidene fluoride scaffold (b) is a scaffold containing 0.05% carbon nanotubes (c) is a membrane containing 0.5% carbon nanotubes 7 days after cell seeding.
이하 본 발명의 일 구현 예에 따른 폴리비닐리덴플루오라이드 나노복합체 스캐폴드 및 그 제조방법에 대하여 설명한다.Hereinafter, a polyvinylidene fluoride nanocomposite scaffold and a manufacturing method thereof according to an embodiment of the present invention will be described.
본 발명의 일 측면에 따른 세포배양용 폴리비닐리덴플루오라이드 나노복합체 스캐폴드의 제조방법은 (i) 폴리비닐리덴플루오라이드 용액과 탄소나노튜브 현탁액을 혼합하는 혼합용액 준비단계; (ii) 상기 혼합용액을 이용하여 복합막을 제조하는 복합막 제조단계; (iii) 상기 복합막을 연신하는 복합막 연신단계; 및 (iv) 상기 복합막에 전기장을 가하는 복합막 분극화 단계를 포함한다.Method for producing a polyvinylidene fluoride nanocomposite scaffold for cell culture according to an aspect of the present invention comprises the steps of: (i) preparing a mixed solution for mixing a polyvinylidene fluoride solution and a carbon nanotube suspension; (ii) a composite membrane manufacturing step of preparing a composite membrane using the mixed solution; (iii) a composite membrane stretching step of stretching the composite membrane; And (iv) polarizing the composite membrane by applying an electric field to the composite membrane.
상기 복합막 내에 분산된 탄소나노튜브의 함량은 폴리비닐리덴플루오라이드 대비 0.5 내지 1 중량%인 것이 바람직하다.The content of carbon nanotubes dispersed in the composite film is preferably 0.5 to 1% by weight based on polyvinylidene fluoride.
상기 복합막 제조단계는 혼합용액을 전기방사하여 복합막을 제조하거나 또는 혼합용액을 용액캐스팅하여 복합막을 제조할 수 있다.In the composite membrane manufacturing step, a composite membrane may be prepared by electrospinning the mixed solution, or a composite membrane may be prepared by solution casting the mixed solution.
상기 탄소나노튜브는 현탁액을 제조하기 이전에 황산과 질산의 혼합산을 이용하여 기능화하는 단계를 거치는 것이 바람직하다. The carbon nanotubes are preferably subjected to a functionalization step using a mixed acid of sulfuric acid and nitric acid before preparing the suspension.
본 발명의 일 측면에 따른 세포배양용 폴리비닐리덴플루오라이드 나노복합체 스캐폴드는 상기의 방법에 의하여 제조되어 복합막 내에 분산된 탄소나노튜브의 함량이 폴리비닐리덴플루오라이드 대비 0.5 내지 1 중량%인 것을 특징으로 한다.Cell culture polyvinylidene fluoride nanocomposite scaffold according to an aspect of the present invention is prepared by the above method is the content of carbon nanotubes dispersed in the composite membrane is 0.5 to 1% by weight compared to polyvinylidene fluoride It is characterized by.
또한, 세포배양용 폴리비닐리덴플루오라이드 나노복합체 스캐폴드는 폴리비닐리덴플루오라이드의 베타상 조성이 80% 이상으로 압전특성을 보이는 베타상의 함유율이 극대화되어 세포의 증착 및 증식능력이 뛰어날 뿐 아니라 물리적 성능이 우수하여 세포배양용 스캐폴드로 활용성이 매우 뛰어나다.In addition, the polyvinylidene fluoride nanocomposite scaffold for cell culture has a beta phase composition of more than 80% of polyvinylidene fluoride, which maximizes the content of the beta phase, which exhibits piezoelectric properties, and provides excellent cell deposition and proliferation ability. Its excellent performance makes it an excellent scaffold for cell culture.
또한, 탄소나노튜브가 함유됨으로써 나노복합체의 강도와 압전특성을 증가시키게 되어 스캐폴드로서의 물리적 성능이 보완되었으며, 세포배양용기의 내벽에 용이하게 코팅될 수 있다.In addition, by containing the carbon nanotubes to increase the strength and piezoelectric properties of the nanocomposite to compensate for the physical performance as a scaffold, can be easily coated on the inner wall of the cell culture vessel.
본 발명에 따른 폴리비닐리덴프루오라이드 나노복합체는 세포활성을 촉진시키고 셀의 접착을 좋게 하므로 셀 및 세포의 배양용 스캐폴드, 배양에세이, 배양관, 신경접합관 등의 바이오재료로써 활용이 가능하다.The polyvinylidene fluoride nanocomposite according to the present invention promotes cell activity and improves cell adhesion, and thus can be utilized as biomaterials such as scaffolds for culture of cells and cells, culture assays, culture tubes, and neural junctions. Do.
폴리비닐리덴플루오라이드는 반결정성 고분자로서 분자 구조는 비닐리덴플루오라이드의 단위체가 선형적으로 결합된 -(CH2CF2)ξ- 의 형태이다. 플루오르의 전기음성도(3.98)는 수소(2.20)에 비해 매우 높은 값을 가지며, 때문에 분자 내의 전자들은 플루오르가 위치한 방향에 편중되는 경향을 보이고 곧 사슬에 수직한 방향으로의 쌍극자모멘트를 생성한다. 폴리비닐리덴플루오라이드는 다섯가지 형태의 결정을 갖고 있으며 그 중 가장 일반적인 형태는 알파상으로서, TGTG (T = trans, G+ = gauche +, G- =gauche -)의 단사정계 단위셀을 갖는다. 분극 및 압전성의 특성을 보이는 것은 TTTT 형태의 베타상이고 이는 사방정계의 단위셀를 갖는다. 감마상 역시 사방정계 결정구조에 해당하지만 TTTGTTTG 배열 구조를 보이며, 다른 델타- 와 입실론상의 경우에는 알파, 베타상의 유사 형태에 해당한다. 알파와 베타상이 가장 일반적 폴리비닐리덴플루오라이드 상에 해당한다. 알파상의 단위셀은 TGTG 배열을 갖는 두 사슬이 역 평행하게 배치되어 있기에 전체 쌍극자모멘트는 0의 값을 갖게 된다. 따라서 알파상은 비 압전성, 을 보이는 반면 베타상 (TTTT) 에 존재하는 모든 쌍극자는 사슬에 수직한 같은 방향의 모멘트를 압전성을 보이게 된다. Polyvinylidene fluoride is a semicrystalline polymer whose molecular structure is in the form of- (CH 2 CF 2 ) ξ -in which units of vinylidene fluoride are linearly bonded. The electronegativity of fluorine (3.98) is very high compared to hydrogen (2.20), so that the electrons in the molecule tend to be biased in the direction in which fluorine is located, creating a dipole moment in the direction perpendicular to the chain. Polyvinylidene fluoride has five types of crystals, the most common of which is the alpha phase, having a monoclinic unit cell of TGTG (T = trans, G + = gauche +, G- = gauche−). The polarization and piezoelectric properties of the TTTT type beta phase have a tetragonal unit cell. The gamma phase is also a tetragonal crystal structure, but shows a TTTGTTTG array structure, and in the case of other delta and epsilon phases, it is similar to the alpha and beta phases. Alpha and beta phases are the most common polyvinylidene fluoride phases. In the unit cell of the alpha phase, since the two chains having the TGTG array are arranged in parallel in parallel, the total dipole moment has a value of zero. Thus, the alpha phase is non-piezoelectric, whereas all dipoles in the beta phase (TTTT) are piezoelectric with moments in the same direction perpendicular to the chain.
모든 트랜스-폴리비닐리덴플루오라이드에서는 이웃하는 플루오르 원자 간에 반발력이 존재하는데, 이는 플루오르 원자의 직경(0.270nm)이 트랜스 탄소 사슬에 의해 생기는 공간(0.256nm)에 비해 크기 때문에 발생한다. 이러한 반발력을 제거하기 위해 CF2그룹은 대부분 알파상 혹은 감마상의 형태를 가지며상대적으로 베타상의 조성 비율이 줄어든다. 베타상의 조성을 높이는 가장 일반적인 방법은 연신, 그리고 분극화다. 베타상 함량을 증가시키기 위한 방법 중 하나로 무기 물질(세라믹, 금속, 자기입자, 나노클레이) 혹은 유기 물질을 첨가재로서 사용하는 방법이 있다.이러한 방식으로 복합재료를 형성하면 압전, 초전율이 크게 증가하는 경향을 보이며, 이는 폴리비닐리덴플루오라이드와 첨가물 사이의 어떠한 상호작용에 의해 발생하는 효과이다. In all trans-polyvinylidene fluorides there is a repulsive force between neighboring fluorine atoms because the diameter of the fluorine atoms (0.270 nm) is large compared to the space created by the trans carbon chain (0.256 nm). In order to eliminate this repulsion, the CF 2 group is mostly in the form of alpha or gamma phase, and the proportion of the beta phase is relatively decreased. The most common ways to increase the composition of the beta phase are stretching and polarization. One way to increase the beta-phase content is to use inorganic materials (ceramic, metal, magnetic particles, nanoclays) or organic materials as additives. Forming composites in this way greatly increases piezoelectric and pyroelectricity. This is an effect caused by any interaction between polyvinylidene fluoride and additives.
고분자 용액의 전기방사 공정은 직경이 수 마이크로미터에 이르는 초 미세 섬유를 제작하기 위한 효과적인 방법으로서 나노화이버 제조에 많이 사용되고 있다. 본 발명에서는 혼합용액을 직접 도포한 후 생성된 막을 연신하거나 분극화시켜서 압전특성을 보이는 베타상 함유량을 극대화 시키거나, 전기방사공정을 이용하여 탄소나노튜브를 함유하는 폴리비닐리덴플루오라이드 나노화이버로 이루어진 막을 제조하여 일차적으로 전기방상공정의 연신과정 및 탄소나노튜브의 표면기능기와 폴리비닐리덴플루오라이드의 불소 원자들과의 상호작용으로 인한 CF2 그룹의 배향화로 일차적으로 베타상이 함유된 막을 제조한 후 이 막을 연신과 분극화 (폴링) 공정을 거침으로써 결정상이 거의 베타상으로 존재하게되어 압전특성이 향상된 막을 제조하고, 이 폴리비닐리덴플루오라이드 나노복합체를 세포배양 스캐폴드로 사용함으로써 세포배양활성을 뚜렷이 향상시킬 수 있다. 본 발명에서 제조된 스캐폴드는 다양한 형태로 변형 가능하며 여러 세포증식기에 사용될 수 있다. Electrospinning process of the polymer solution has been widely used in the manufacture of nanofibers as an effective method for producing ultra-fine fibers up to several micrometers in diameter. In the present invention, by applying the mixed solution directly, the resulting film is stretched or polarized to maximize the beta phase content exhibiting piezoelectric properties, or made of polyvinylidene fluoride nanofibers containing carbon nanotubes using an electrospinning process. After the membrane was prepared, the beta phase-containing membrane was first prepared by the stretching of the electrospinning process and the orientation of the CF 2 group due to the interaction between the surface functional group of the carbon nanotubes and the fluorine atoms of the polyvinylidene fluoride. By stretching and polarizing (polling) the membrane, the crystal phase is almost in the beta phase to produce a membrane having improved piezoelectric properties. The polyvinylidene fluoride nanocomposite is used as a cell culture scaffold to clearly show cell culture activity. Can be improved. Scaffolds prepared in the present invention can be modified in various forms and used in various cell multipliers.
이하, 실시예를 통하여 본 발명을 보다 구체적으로 설명하고자 한다. 본 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것이며, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. This embodiment is intended to illustrate the present invention in more detail, and the scope of the present invention is not limited to these examples.
실시예Example 1: 전기방사법 1: electrospinning
반결정성의 폴리비닐리덴플루오라이드(평균 분자량은 5.2105)를 습기 및 불순물 제거를 위해 80℃ 진공 하에서 24시간 동안 건조시키고, 디메틸아세트마이드와 아세톤은 50:50의 비율로 혼합한 후 건조시킨 폴리비닐리덴플루오라이드를 적정 비율로 배합된 용매에 첨가하고 균질의 용액이 형성될 때까지 60℃에서 교반시킨다. 이때 폴리비닐리덴플루오라이드의 농도는 15중량%로 고정하였는데, 미세하고 균일한 섬유를 얻기 위한 적정 점도에 해당하는 값이다. 다층탄소나노튜브가 폴리비닐리덴플루오라이드 용액에 성공적으로 분산되도록 하기 위해서 탄소나노튜브를 표면산처리하여 기능화를 시킨다. 황산/질산(3:1 부피비) 용액에 넣고 48시간 동안 저은 뒤 2시간 동안 강한 초음파를 가한다. 그 뒤 0.4㎛ 폴리비닐리덴플루오라이드 거름막에 다층 탄소나노튜브를 걸러내고 순수한 증류수를 사용하여 pH 값이 7에 도달할 때까지 세척한다. 세척이 완료된 다층 탄소나노튜브는 80℃ 진공에서 반나절 건조시킨다. 작용기가 부착된 다층 탄소나노튜브는 초음파 수조를 이용, 디메틸아세트마이드 용매에 30분간 분산시킨다. 폴리비닐리덴플루오라이드는 정량하여 아세톤에 용해시키고 자기 교반기를 이용하여 30분간 젓는다. 이후 아세톤/폴리비닐리덴플루오라이드 용액과 다층탄소나노튜브/디메틸아세트마이드 현탁액을 혼합하고 40℃에서 교반한다. 이후 이 용액을 10 CC 주사기에 장전한 후 전기방사공정을 이용하여 나노화이버로 이루어진 나노복합체를 얻게 된다. 전기방사공정을 적용함으로써 전기방사공정의 연신작용에 의하여 일차적으로 폴리비닐리덴플루오라이드의 알파상들을 베타상으로 변환시킨다. 이때 첨가된 탄소나노튜브는 그 표면극성기로 인하여 알파상이 베타상으로 전환되는 것을 유발하고 도와주게 된다. 푸리에 변환 적외선 스펙트럼을 877cm-1 에서 관찰되는 값(시편의 두께에 비례)을 기준으로 하여 비교 분석하였을 때, 840cm-1 및 1270cm-1에서 다층 탄소나노튜브의 함량에 따라 증가하는 모습을 보인다. 광각 X선 분석에 의하면 다층 탄소나노튜브 함량이 증가할 수록 2θ= 18.6, 20.2 도에 해당하는 알파상 조성이 감소하고, 20.9°의 베타상 조성이 크게 증가하는 것이 관측된다. 이것은 다층 탄소나노튜브의 기핵 작용에 의한 것으로서, 다층 탄소나노튜브의 표면에 카르복실 그룹이 붙어 폴리비닐리덴플루오라이드 용액에 분산되었을 때 카르복실 그룹과 CF2간에 정전기적 상호작용이 발생한다. 이 상호작용은 더욱 높은 결정화도의 형성에 기여할 뿐 아니라, 베타상의 TTTT 배열이 알파상 조성의 TGTG 배열에 비해 더욱 안정적인 에너지 상태를 보이기 때문에 알파에서 베타상으로의 상 변환을 더욱 촉진시키는 기능을 한다. 실제 다층 탄소나노튜브를 0.05중량% 첨가하였을 때 결정 구조에 큰 변화가 생겨나는 것이 확인된다. 다층 탄소나노튜브가 증가할수록 폴리비닐리덴플루오라이드와의 접촉 면적이 증가하며, 이로부터 더욱 많은 양의 베타상을 얻을 수 있다. 이러한 현상은 다층 탄소나노튜브의 농도가 1중량%에 이를 때까지 지속된다. 다층 탄소나노튜브와 폴리비닐리덴플루오라이드 간의 상호작용이 강할수록 다층 탄소나노튜브가 균일하게 분산되어 용액의 점도는 증가한다. 따라서 다층 탄소나노튜브가 임계점 이상의 농도에 이르면 전기 방사 공정에 의한 정전기력 이상으로 점탄성력이 강해지고 베타상 조성의 변화 폭이 미미해진다. 다층탄소나노튜브가 0.5% 이상이 되면 오히려 미미하게나마 베타상이 감소되는 것이 발견되었으며 이는 계면에서의 전하축적으로 인하여 탈분극화 현상이 일어나기 때문이다. 방사공정이후 나노화이버로 이루어진 막의연신 혹은 분극화 공정의 조합이 베타상 조성에 크게 영향을 미치며, 해당 공정을 거쳤을 때 푸리에 변환 적외선 분광기 및 광각 X선 회절 분석기를 이용한 분석에서 알파상의 조성이 감소하는 동시에 베타상의 조성이 늘어나는 것이 관측된다. 연신 공정은 기계적인 외력을 가하여 결정 내에서의 고분자 사슬이 일렬로 배열되도록 유도하고, 결정상 중 가장 긴 형태를 갖는 베타상의 배좌가 형성되도록 한다. 그리고 분극화 공정은 폴리비닐리덴플루오라이드 섬유에 수직한 방향으로 전기장을 형성하여 그 방향으로의 쌍극자 모멘트를 이끌어내고 연신 공정과 마찬가지로 베타상 조성을 증가시킨다. 먼저, 다양한 다층 탄소나노튜브 함량에서 연신 및 분극화 공정을 가했을 때의 베타상 조성 변화가 도2에 나타나 있다. 다층 탄소나노튜브를 함유하는 시편은 91% 이상의 베타상 조성을 갖는 반면 그렇지 않은 시편은 약 80% 근처의 조성에 그치는데, 다층 탄소나노튜브와 폴리비닐리덴플루오라이드 간의 접합이 연신 공정을 더욱 효율적으로 만들기 때문이다. 분극화 공정은 연신 공정에 비해 상 변이 효과가 크지는 않지만 마찬가지로 다층 탄소나노튜브에 의한 영향이 존재한다. 폴리비닐리덴플루오라이드 시편의 양면에 전하가 축적되면 전류가 투과되지 못하고 분극화 되며 결과적으로 불균일한 쌍극자를 형성하게 된다. 그러나 높은 전기 용량을 갖는 다층탄소나노튜브가 분산되어 알파상 결정의 표면에 접합되어 있으면 더욱 효율적으로 쌍극자 모멘트의 방향 유도가 이루어지고 베타상으로의 상 변환이 촉진된다. Semi-crystalline polyvinylidene fluoride (average molecular weight 5.210 5 ) was dried for 24 hours under vacuum at 80 ℃ to remove moisture and impurities, dimethyl acetamide and acetone were mixed in a ratio of 50:50 and dried poly Vinylidene fluoride is added to the combined solvents in an appropriate ratio and stirred at 60 ° C. until a homogeneous solution is formed. At this time, the concentration of polyvinylidene fluoride was fixed at 15% by weight, which is a value corresponding to an appropriate viscosity for obtaining fine and uniform fibers. In order to successfully disperse the multilayer carbon nanotubes in the polyvinylidene fluoride solution, the carbon nanotubes are subjected to surface acid treatment for functionalization. The solution was added to a sulfuric acid / nitric acid (3: 1 volume ratio) solution, stirred for 48 hours, and then subjected to strong ultrasonic waves for 2 hours. Thereafter, the multilayer carbon nanotubes were filtered through a 0.4 μm polyvinylidene fluoride filter membrane and washed with pure distilled water until the pH value reached 7. After washing, the multilayered carbon nanotubes are dried in half day at 80 ° C. in a vacuum. The multi-layered carbon nanotubes to which the functional groups are attached are dispersed in a dimethylacetamide solvent for 30 minutes using an ultrasonic bath. Polyvinylidene fluoride is quantitatively dissolved in acetone and stirred for 30 minutes using a magnetic stirrer. Thereafter, the acetone / polyvinylidene fluoride solution and the multilayer carbon nanotube / dimethylacetamide suspension are mixed and stirred at 40 ° C. After loading the solution into a 10 CC syringe and using an electrospinning process to obtain a nanocomposite consisting of nanofibers. By applying the electrospinning process, the alpha phase of polyvinylidene fluoride is first converted into the beta phase by the stretching action of the electrospinning process. At this time, the added carbon nanotubes cause and help the alpha phase to be converted to the beta phase due to the surface polar group. When compared on the basis of the value (in proportion to the thickness of the specimen) is observed in the Fourier transform infrared spectrum 877cm -1, the appearance looks to increase with the content of the multi-layer carbon nanotubes in a 840cm -1 and 1270cm -1. According to the wide-angle X-ray analysis, the alpha phase composition corresponding to 2θ = 18.6 and 20.2 degrees decreases and the beta phase composition of 20.9 ° increases as the multilayer carbon nanotube content increases. This is due to the nucleation of the multi-walled carbon nanotubes. When the carboxyl group is attached to the surface of the multi-walled carbon nanotubes and dispersed in a polyvinylidene fluoride solution, an electrostatic interaction occurs between the carboxyl group and CF 2 . This interaction not only contributes to the formation of higher crystallinity, but also facilitates the phase-to-beta phase transition because the TTTT sequence of the beta phase exhibits a more stable energy state than the TGTG array of the alpha phase composition. It is confirmed that a significant change in crystal structure occurs when 0.05 wt% of the multi-layered carbon nanotubes are actually added. As the multi-walled carbon nanotubes increase, the contact area with the polyvinylidene fluoride increases, from which a larger amount of beta phase can be obtained. This phenomenon continues until the concentration of the multilayer carbon nanotubes reaches 1% by weight. The stronger the interaction between the multilayer carbon nanotubes and the polyvinylidene fluoride, the more uniformly the multilayer carbon nanotubes are dispersed and the viscosity of the solution increases. Therefore, when the multi-walled carbon nanotubes reach a concentration higher than the critical point, the viscoelastic force becomes stronger than the electrostatic force by the electrospinning process, and the variation of the beta phase composition is insignificant. When the multi-layered carbon nanotubes are more than 0.5%, the beta phase is found to be reduced slightly because depolarization occurs due to charge accumulation at the interface. After the spinning process, the combination of the stretching or polarization process of the nanofiber film greatly affects the beta phase composition, and the alpha phase composition decreases in the analysis using Fourier transform infrared spectroscopy and wide-angle X-ray diffraction analyzer. At the same time an increase in the composition of the beta phase is observed. The stretching process exerts a mechanical external force to induce the polymer chains in the crystal to be arranged in a row, and to form a beta phase of the longest form of the crystal phase. And The polarization process creates an electric field in a direction perpendicular to the polyvinylidene fluoride fiber, elicits a dipole moment in that direction and increases the beta phase composition as in the stretching process. First, the beta phase composition change when the stretching and polarization process is applied at various multilayer carbon nanotube contents is shown in FIG. 2. Specimens containing multilayered carbon nanotubes have a beta-phase composition of at least 91%, while those that do not have only about 80% of the composition, and the bonding between the multilayer carbon nanotubes and polyvinylidene fluoride makes the stretching process more efficient. Because of making. The polarization process does not have a large phase shift effect compared to the stretching process, but similarly, there is an influence by multilayer carbon nanotubes. Accumulation of charge on both sides of the polyvinylidene fluoride specimens results in polarization of the current, which does not pass through, resulting in nonuniform dipoles. However, when the multi-walled carbon nanotubes having high capacitance are dispersed and bonded to the surface of the alpha phase crystal, the dipole moment direction is more efficiently induced and the phase conversion to the beta phase is promoted.
세포배양실험 1 Cell culture experiment 1
바닥에 상기 전기방사법으로 제조된 폴리비닐리덴플루오라이드 나노복합체를 깔고 두 종류의 세포들 (RT4-D6P2T (쥐의 Schwannoma cell line), U87-MG (사람의 glioblastoma cell line)을 부피비로 10% 페탈보빈세럼과 1%의 항생/항진균용액을 함유하는 Dulbecco® Modified Eagle® medium (DMEM)내에서 37℃의 온도와 5% CO2 분위기에서 배양시켰다. 셀들은 배양지에서 0.25% 트립신/에틸렌다이아민테트라액시드용약으로 분리시켰으며 나노복합체 위에 직접 배양시켰다 (40000 cells/cm2). 셀의 접착과 성장은 MTT (Methylthiazolyldiphenyl -tetrazolium bromide)시금평가방법으로 측정하였다. 셀의 형태학은 면역형광검사법으로 관찰하였다.The polyvinylidene fluoride nanocomposite prepared by the electrospinning method was placed on the bottom, and two kinds of cells (RT4-D6P2T (schwannoma cell line in mouse) and U87-MG (human glioblastoma cell line) in volume ratio were 10%. Incubated in a Dulbecco® Modified Eagle® medium (DMEM) containing bobbin serum and 1% antibiotic / antifungal solution at a temperature of 37 ° C. and 5% CO 2 Cells were cultured in a culture medium with 0.25% trypsin / ethylenediaminetetra It was isolated with acid solution and incubated directly on the nanocomposite (40000 cells / cm 2 ). Cell adhesion and growth were measured by MTT (Methylthiazolyldiphenyl-tetrazolium bromide) assay. Morphology of the cells was observed by immunofluorescence.
셀이 파종된뒤 12시간 후에 폴리비닐리덴플루오라이드 나노복합체 위에 접합된 비율을 살펴보았다. 각 셀에 대하여 최적의 접합율을 보이는 탄소나노튜브 농도가 존재하는데 이는 전술한 압전효과와 일치하는 결과를 보인다. 전반적으로 압전현상이 높은 복합체일수록 셀 수는 증가하였으며 탄소나노튜브를 함유하는 나노복합체의 경우 순수 폴리비닐리덴플루오라이드보다 20% 이상 증가된 셀 접착율을 보이고 있다. 셀 접착율의 최대치를 보이는 농도는 조금씩 틀리지만 전반적으로 압전특성의 변화와 상관관계를 보이는 것을 알 수 있다(도 4). 셀들의 증가율은 시간이 지날수록 늘어나는 것을 볼 수 있으며 RT4-D6P2T 셀의 경우 배양 1주일 된 스캐폴드의 경우 0.5% 탄소나노튜브를 함유한 나노복합체는 순수 폴리비닐리덴플루오라이드 보다 배 이상으로 세포가 성장하는 것을 볼 수 있다(도 4). U87-MG 셀은 탄소나노튜브가 증가함에 따라 서서히 증가하였으며 1%의 탄소나노튜브가 함유된 경우 가장 높은 성장율을 나타내었다. 일주일 후의 셀의 성장형태를 면역형광현미경으로 직접 관찰한 결과가 도5 에 도시되어 있다. 탄소튜브가 더 많이 함유될수록 (0.5%)에서 셀이 가장 많이 자랐음을 직접 볼 수 있다. 이상의 결과로 볼 때 압전특성이 좋은 나노복합체 일수록(탄소나노튜브함유) 표면에 극화된 CF2 그룹이 많고 셀들이 이들 극성에 자극을 받아서 더 성장한 것으로 보인다.12 hours after the cells were seeded, the proportions of the polyvinylidene fluoride nanocomposites were examined. For each cell, there is a concentration of carbon nanotubes showing an optimum bonding rate, which is consistent with the piezoelectric effect described above. In general, the higher the piezoelectric composite, the higher the number of cells, and the nanocomposite containing carbon nanotubes showed more than 20% increase in cell adhesion rate compared to pure polyvinylidene fluoride. The concentration showing the maximum cell adhesion rate is slightly different but it can be seen that the correlation with the change in piezoelectric properties as a whole (Fig. 4). The growth rate of the cells can be seen to increase over time. For the RT4-D6P2T cells, the nanocomposites containing 0.5% carbon nanotubes in the 1 week culture scaffold were more than doubled than the pure polyvinylidene fluoride. It can be seen growing (FIG. 4). U87-MG cells increased slowly with increasing carbon nanotubes and showed the highest growth rate when 1% of carbon nanotubes were contained. The results of direct observation of the growth pattern of cells after one week with an immunofluorescence microscope are shown in FIG. 5. The more carbon tubes it contains (0.5%), the more the cells grew most. The above results suggest that the more nanocomposites with better piezoelectric properties (including carbon nanotubes), the more polarized CF 2 groups on the surface and the more cells are stimulated by these polarities and grown.
실시예Example 2: 용액캐스팅법 2: solution casting method
반결정성의 폴리비닐리덴플루오라이드(평균 분자량은 5.2105)를 습기 및 불순물 제거를 위해 80℃ 진공 하에서 24시간 동안 건조시키고, 디메틸아세트마이드와 아세톤은 50:50의 비율로 혼합한 후 건조시킨 폴리비닐리덴플루오라이드를 적정 비율로 배합된 용매에 첨가하고 균질의 용액이 형성될 때까지 60℃에서 교반시킨다. 이때 폴리비닐리덴플루오라이드의 농도는 15중량%로 고정하였는데, 미세하고 균일한 섬유를 얻기 위한 적정 점도에 해당하는 값이다. 다층탄소나노튜브가 폴리비닐리덴플루오라이드 용액에 성공적으로 분산되도록 하기 위해서 탄소나노튜브를 표면을 산처리하여 기능화를 시킨다. 황산/질산(3:1 부피비) 용액에 넣고 48시간 동안 저은 뒤 2시간 동안 강한 초음파를 가한다. 그 뒤 0.4㎛ 폴리비닐리덴플루오라이드 거름막에 다층 탄소나노튜브를 걸러내고 순수한 증류수를 사용하여 pH 값이 7에 도달할 때까지 세척한다. 세척이 완료된 다층 탄소나노튜브는 80℃ 진공에서 반나절 건조시킨다. 작용기가 부착된 다층 탄소나노튜브는 초음파 수조를 이용, 디메틸아세트마이드 용매에 30분간 분산시킨다. 폴리비닐리덴플루오라이드는 정량하여 아세톤에 용해시키고 자기 교반기를 이용하여 30분간 젓는다. 이후 아세톤/폴리비닐리덴플루오라이드 용액과 다층탄소나노튜브/디메틸아세트마이드 현탁액을 혼합하고 40℃에서 교반한다. 이후 혼합용액을 페트리디쉬위에 도포한 뒤 대기중에서 24시간 방치하여 용매가 완전증발할 때까지 기다린 후 건조된 막을 이축연신기로 연신하고 이를 다시 분극화공정을 거친다. 실시예 1과 같이 푸리에변환적외선 분광기와 광각X선 분광기를 이용하여 베타상의 함량을 측정하였다(도 3). 특이한 것은 전술한 전기방사로 제조된 막과 달리 탄소나노튜브가 0.5% 함유되었을 때 베타상이 최고치에 이르며 이후는 탈분극화현상이 일어나서 오히려 감소한다는 것이다.Semi-crystalline polyvinylidene fluoride (average molecular weight 5.210 5 ) was dried for 24 hours under vacuum at 80 ℃ to remove moisture and impurities, dimethyl acetamide and acetone were mixed in a ratio of 50:50 and dried poly Vinylidene fluoride is added to the combined solvents in an appropriate ratio and stirred at 60 ° C. until a homogeneous solution is formed. At this time, the concentration of polyvinylidene fluoride was fixed at 15% by weight, which is a value corresponding to an appropriate viscosity for obtaining fine and uniform fibers. In order to successfully disperse the multi-layered carbon nanotubes in the polyvinylidene fluoride solution, the surface of the carbon nanotubes is subjected to acid treatment for functionalization. The solution was added to a sulfuric acid / nitric acid (3: 1 volume ratio) solution, stirred for 48 hours, and then subjected to strong ultrasonic waves for 2 hours. Thereafter, the multilayer carbon nanotubes were filtered through a 0.4 μm polyvinylidene fluoride filter membrane and washed with pure distilled water until the pH value reached 7. After washing, the multilayered carbon nanotubes are dried in half day at 80 ° C. in a vacuum. The multi-layered carbon nanotubes to which the functional groups are attached are dispersed in a dimethylacetamide solvent for 30 minutes using an ultrasonic bath. Polyvinylidene fluoride is quantitatively dissolved in acetone and stirred for 30 minutes using a magnetic stirrer. Thereafter, the acetone / polyvinylidene fluoride solution and the multilayer carbon nanotube / dimethylacetamide suspension are mixed and stirred at 40 ° C. Thereafter, the mixed solution is applied on a petri dish and left in the air for 24 hours to wait until the solvent evaporates completely, and then the dried membrane is stretched with a biaxial stretching machine and polarized again. The content of the beta phase was measured using a Fourier transform infrared spectrometer and a wide-angle X-ray spectrometer as in Example 1 (FIG. 3). What is unusual is that the beta phase reaches a maximum when 0.5% of carbon nanotubes are contained, unlike the membrane prepared by the electrospinning described above, and then depolarization phenomenon occurs and decreases.
세포배양실험 2Cell Culture Experiment 2
용기 바닥에 상기 용액캐스팅법으로 제조된 폴리비닐리덴플루오라이드 나노복합체를 깔고 SH-SY5Y 셀(사람의 neuroblastoma cell line )을 부피비로 10% 페탈보빈세럼과 1%의 항생/항진균용액을 함유하는 Dulbecco® Modified Eagle® medium (DMEM)내에서 37℃의 온도와 5% CO2 분위기에서 배양시켰다. 셀은 배양지에서 0.25% 트립신/에틸렌다이아민테트라액시드용약으로 분리시켰으며 복합막위에 직접 배양시켰다 (40000 cells/cm2). 셀의 접착과 성장은 MTT(Methylthiazolyldiphenyl -tetrazolium bromide)시금평가방법으로 측정하였다. 셀의 형태학은 면역형광검사법으로 관찰하였다.Dulbecco containing polyvinylidene fluoride nanocomposite prepared by the solution casting method at the bottom of the container and containing 10% petalbobin serum and 1% antibiotic / antifungal solution in volume ratio of SH-SY5Y cells (human neuroblastoma cell line). Incubated in a Modified Eagle® medium (DMEM) at a temperature of 37 ° C. and 5% CO 2 atmosphere. Cells were separated from the culture medium with 0.25% trypsin / ethylenediaminetetraacid solution and cultured directly onto the composite membrane (40000 cells / cm 2 ). Cell adhesion and growth were measured by MTT (Methylthiazolyldiphenyl-tetrazolium bromide) assay. Morphology of the cells was observed by immunofluorescence.
셀이 파종된뒤 12시간 후에 폴리비닐리덴플루오라이드 나노복합체 위에 접합된 비율을 살펴보았다. 각 셀에 대하여 최적의 접합율을 보이는 탄소나노튜브 농도가 존재하는데 이는 앞서의 압전효과와 일치하는 결과를 보인다. 실시예 1의 전기방사법에 의한 나노복합체의 경우와 같이 전반적으로 압전현상이 높을 수록 셀 수는 증가하였으며 탄소나노튜브를 함유하는 경우 순수 폴리비닐리덴플루오라이드 보다 20% 이상 증가된 셀 접착율을 보이고 있다. 셀 접착율의 최대치를 보이는 농도는 조금씩 틀리지만 전반적으로 압전특성의 변화와 상관관계를 보이는 것을 알 수 있다. 셀들의 증가율은 시간이 지날수록 늘어나는 것을 볼 수 있으며 배양 1주일 된 스캐폴드의 경우 0.5% 탄소나노튜브를 함유한 경우는 순수 폴리비닐리덴플루오라이드막보다 배 이상으로 세포가 성장하는 것을 볼 수 있다. 베타상이 가장 많은 탄소나노튜브 농도에서(0.5%) 셀이 가장 많이 자랐음을 직접 볼 수 있다.이상의 결과로 볼 때 압전특성이 좋을수록(탄소나토튜브함유) 표면에 극화된 CF2 그룹이 많고 셀들이 이들 극성에 자극을 받아서 더 성장한 것으로 보인다.12 hours after the cells were seeded, the proportions of the polyvinylidene fluoride nanocomposites were examined. For each cell, there is a carbon nanotube concentration that shows the optimum bonding rate, which is consistent with the piezoelectric effect. As in the case of the nanocomposite by the electrospinning method of Example 1, the overall number of cells increased as the piezoelectric phenomenon was increased, and the cell adhesion rate was increased by 20% or more than that of pure polyvinylidene fluoride. have. It can be seen that the concentration showing the maximum cell adhesion rate is slightly different but correlates with the change in piezoelectric properties. It can be seen that the growth rate of the cells increases with time, and in the case of the culture scaffold of one week, the growth of the cells is more than doubled than that of the pure polyvinylidene fluoride membrane. . It can be directly seen that the cells grew most at the carbon nanotube concentration with the highest beta phase (0.5%). The better the piezoelectric properties (containing carbon nanotubes), the more polarized CF 2 groups on the surface. The cells appear to have grown more stimulated by these polarities.
본 발명에 따른 높은 압전특성을 보이는 베타상이 대부분인 폴리비닐리덴프루오라이드 나노복합체는 세포활성을 촉진시키고 셀의 접착을 좋게 하므로 셀 및 세포의 배양용 스캐폴드, 배양에세이, 배양관, 신경접합관 등의 바이오재료로써 활용이 가능하다.Polyvinylidene fluoride nanocomposites, which are mostly in the beta phase showing high piezoelectric properties according to the present invention, promote cell activity and improve cell adhesion, so that cells and cell culture scaffolds, culture assays, culture tubes, and neural junction tubes It can be used as a bio material.

Claims (8)

  1. (i) 폴리비닐리덴플루오라이드 용액과 탄소나노튜브 현탁액을 혼합하는 혼합용액 준비단계;(i) a mixed solution preparation step of mixing a polyvinylidene fluoride solution and a carbon nanotube suspension;
    (ii) 상기 혼합용액을 이용하여 복합막을 제조하는 복합막 제조단계;(ii) a composite membrane manufacturing step of preparing a composite membrane using the mixed solution;
    (iii) 상기 복합막을 연신하는 복합막 연신단계; 및 (iii) a composite membrane stretching step of stretching the composite membrane; And
    (iv) 상기 복합막에 전기장을 가하는 복합막 분극화 단계를 포함하는 것을 특징으로 하는 세포배양용 폴리비닐리덴플루오라이드 나노복합체 스캐폴드의 제조방법.(iv) a method for producing a polyvinylidene fluoride nanocomposite scaffold for cell culture, comprising the step of polarizing a composite membrane applying an electric field to the composite membrane.
  2. 청구항 1에 있어서,The method according to claim 1,
    상기 복합막 내에 분산된 탄소나노튜브의 함량은 폴리비닐리덴플루오라이드 대비 0.5 내지 1 중량%인 것을 특징으로 하는 세포배양용 폴리비닐리덴플루오라이드 나노복합체 스캐폴드의 제조방법.The content of carbon nanotubes dispersed in the composite membrane is a method for producing a polyvinylidene fluoride nanocomposite scaffold for cell culture, characterized in that 0.5 to 1% by weight compared to polyvinylidene fluoride.
  3. 청구항 1에 있어서,The method according to claim 1,
    상기 복합막 제조단계는 혼합용액을 전기방사하여 복합막을 제조하는 것을 특징으로 하는 세포배양용 폴리비닐리덴플루오라이드 나노복합체 스캐폴드의 제조방법.The composite membrane manufacturing step of producing a polyvinylidene fluoride nanocomposite scaffold for cell culture, characterized in that for producing a composite membrane by electrospinning the mixed solution.
  4. 청구항 1에 있어서,The method according to claim 1,
    상기 복합막 제조단계는 혼합용액을 용액캐스팅하여 복합막을 제조하는 것을 특징으로 하는 세포배양용 폴리비닐리덴플루오라이드 나노복합체 스캐폴드의 제조방법.The composite membrane manufacturing step is a method for producing a polyvinylidene fluoride nanocomposite scaffold for cell culture, characterized in that the composite membrane is prepared by solution casting the mixed solution.
  5. 청구항 1에 있어서,The method according to claim 1,
    상기 탄소나노튜브는 현탁액을 제조하기 이전에 황산과 질산의 혼합산을 이용하여 기능화하는 단계를 거치는 것을 특징으로 하는 세포배양용 폴리비닐리덴플루오라이드 나노복합체 스캐폴드의 제조방법.The carbon nanotube is a method for producing a polyvinylidene fluoride nanocomposite scaffold for cell culture, characterized in that the step of functionalizing using a mixed acid of sulfuric acid and nitric acid before preparing the suspension.
  6. 청구항 1 내지 청구항 5의 어느 한 항의 방법에 의하여 제조되고,Prepared by the method of any one of claims 1 to 5,
    복합막 내에 분산된 탄소나노튜브의 함량이 폴리비닐리덴플루오라이드 대비 0.5 내지 1 중량%인 것을 특징으로 하는 세포배양용 폴리비닐리덴플루오라이드 나노복합체 스캐폴드.Cell culture polyvinylidene fluoride nanocomposite scaffold, characterized in that the content of the carbon nanotubes dispersed in the composite membrane is 0.5 to 1% by weight compared to the polyvinylidene fluoride.
  7. 청구항 1 내지 청구항 5 중 어느 한 항의 방법에 의하여 제조되어,Prepared by the method of any one of claims 1 to 5,
    폴리비닐리덴플루오라이드의 베타상 조성이 80% 이상인 것을 특징으로 하는 세포배양용 폴리비닐리덴플루오라이드 나노복합체 스캐폴드.A polyvinylidene fluoride nanocomposite scaffold for cell culture, characterized in that the beta phase composition of polyvinylidene fluoride is 80% or more.
  8. 청구항 7의 나노복합체 스캐폴드가 표면에 코팅된 것을 특징으로 하는 생체배양용기.The biocomposite vessel characterized in that the nanocomposite scaffold of claim 7 is coated on the surface.
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