Cell shielding membrane and its manufacturing method

Abstract

To provide a cell shielding film that can inhibit the development of infection and has the function of promoting tissue formation such as bone tissue.SOLUTION: A cell shielding film has a carbon nanotube film, and a tissue regeneration promoter carried on the carbon nanotube film. The carbon nanotube film can include a carbon nanotube bundle with a diameter of 10 nm or more and less than 60 nm, and the carbon nanotube bundle may form a network structure. The carbon nanotube bundle can include a plurality of single-wall carbon nanotubes.SELECTED DRAWING: Figure 6

Description

[Technical field]

[0001] One embodiment of the present invention relates to a cell-shielding membrane that can be used in guiding tissue regeneration and a method for producing the same. For example, one embodiment of the present invention relates to a carbon nanotube membrane that functions as a cell-shielding membrane and is loaded with a drug that promotes tissue formation, and a method for producing the same. [Background technology]

[0002] Guided tissue regeneration (GTR) and guided bone regeneration (GBR) are known as methods for regenerating tissues in dental treatment. In these regeneration methods, a non-resorbable or resorbable cell-shielding membrane is placed in the defective area of ​​the periodontal ligament or bone, providing a space for the periodontal ligament tissue or bone tissue to regenerate, while preventing the invasion and growth of tissue cells other than the tissue to be regenerated. This makes it possible to selectively promote the regeneration of the periodontal ligament or bone tissue in the defective area (see Non-Patent Documents 1 to 3). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Lim, Glendale et al., "International Journal of Oral & Maxillofacial Implants", 2018, Vol. 33, pp. 41-50 [Non-Patent Document 2] Kucherer, Urique et al., "International Journal of Oral and Maxillofacial Implants", 2014, Vol. 29, pp. 14-24 [Non-Patent Document 3] Mendes, M. Renato et al., "Life Sciences", 2010, Vol. 87, pp. 215-222 Summary of the Invention [Problem to be solved by the invention]

[0004] An object of one embodiment of the present invention is to provide a cell-shielding membrane that can suppress the onset of infectious diseases and has a function of promoting the formation of tissues such as periodontal ligament, bone tissue, etc., and a method for producing the same. For example, an object of one embodiment of the present invention is to provide a carbon nanotube-based cell-shielding membrane that can be used in guiding the regeneration of bone and tissues, and a method for producing the same. [Means for solving the problem]

[0005] One embodiment of the present invention is a cell barrier membrane, which includes a carbon nanotube membrane and a tissue regeneration-promoting agent supported on the carbon nanotube membrane.

[0006] One embodiment of the present invention is a method for producing a cell-shielding membrane, which includes preparing a dispersion containing carbon nanotubes and a tissue regeneration-promoting agent, and forming a membrane using the filtrate obtained by filtering the dispersion.

[0007] One embodiment of the present invention is a method for regenerating tissue. The regeneration method includes covering a tissue defect with a cell-blocking membrane. The cell-blocking membrane includes a carbon nanotube membrane and a tissue regeneration-promoting agent supported on the carbon nanotube membrane. [Brief description of the drawings]

[0008] [Figure 1] 1 is a flowchart showing a method for producing a cell-shielding membrane according to one embodiment of the present invention. [Diagram 2] 4 shows ultraviolet absorption spectra of the filtrate obtained by filtering the standard aqueous solution of hyaluronic acid and the dispersion of hyaluronic acid and carbon nanotubes in Example 1. [Diagram 3] 1 is a scanning electron microscope (SEM) image of the cell-shielding membrane of Example 1. [Figure 4] 1 is an SEM image of the cell-shielding membrane of Example 1. [Diagram 5] 1 shows a histogram and Pareto curve of the diameters of the bundles of carbon nanotubes contained in the cell-shielding film of Example 1. [Figure 6]1 is a micro-computed tomography (micro-CT) image of a bone defect formed in a rat calvarium covered with the cell-occluding membrane of Example 1, taken 28 days after surgery. [Figure 7] 1 is an optical microscope image of the anterior cross section of a bone defect formed in a rat calvarium covered with the cell-occluding membrane of Example 1, taken 28 days after surgery. [Figure 8] 4 shows ultraviolet absorption spectra of a standard aqueous solution of epigallocatechin (EGCG) and a filtrate obtained by filtering a dispersion of EGCG and carbon nanotubes in Example 2. [Figure 9] 1 is a plot of the amount of EGCG supported on the carbon nanotube film versus the amount of EGCG added in Example 2. [Figure 10] 1 is an SEM image of the cell-shielding membrane of Example 2. [Figure 11] 1 shows a histogram and Pareto curve of the diameters of the bundles of carbon nanotubes contained in the cell-shielding film of Example 2. [Figure 12] 13 is a micro-CT image of a bone defect formed in a rat calvarium covered with the cell-shielding membrane of Example 2, taken 28 days after surgery. [Figure 13] 13 is an optical microscope image of the subcutaneous tissue of a rat 56 days after surgery, into which the cell barrier membrane of Example 2 was embedded. [Figure 14] 1 is an SEM image of a carbon nanotube film of a comparative example. [Figure 15] 1 shows a histogram and Pareto curve of the diameters of carbon nanotube bundles contained in a carbon nanotube film of a comparative example. [Figure 16] 13 is a soft X-ray photograph of a bone defect formed in a rat calvarium, covered with a carbon nanotube film of a comparative example, taken 14 days after surgery. [Figure 17] 13 is an optical microscope image of a rat subcutaneous tissue 14 days after surgery, into which a carbon nanotube film of the comparative example was embedded. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Hereinafter, each embodiment of the present invention will be described with reference to the drawings, etc. However, the present invention can be embodied in various forms without departing from the gist of the present invention, and should not be construed as being limited to the description of the embodiments and examples exemplified below.

[0010] Even if there are other effects and advantages different from those brought about by the aspects of the following embodiments, those that are clear from the description in this specification or that can be easily predicted by a person skilled in the art are naturally understood to be brought about by the present invention.

[0011] One embodiment of the present invention is a carbon nanotube-based cell-shielding membrane (hereinafter also referred to as a CNT-based cell-shielding membrane or a cell-shielding membrane), which has the function of shielding cells that inhibit tissue regeneration in tissue regeneration using, for example, the GBR method or the GTR method, and promoting the regeneration of the tissue to be regenerated. The configuration and manufacturing method of the CNT-based cell-shielding membrane, and the tissue regeneration method using the CNT-based cell-shielding membrane are described below.

[0012] 1. Configuration The CNT-based cell-shielding film includes a CNT film containing carbon nanotubes (hereinafter, also simply referred to as CNT), and a tissue regeneration promoter supported on the CNT film. As an optional configuration, the CNT-based cell-shielding film may include an additive.

[0013] Each of the CNTs contained in the CNT film is substantially sp 2It is a rod-shaped molecule having a cylindrical main skeleton composed of carbon atoms, and its diameter and length can be selected arbitrarily. For example, the diameter is appropriately selected from the range of 0.60 nm to 10 nm, 0.60 nm to 5.0 nm, or 0.60 nm to 3.0 nm, and the length is appropriately selected from the range of 0.1 μm to 5 mm, 10 μm to 1 mm, or 10 μm to 100 μm. In addition, the CNT may be a CNT containing other molecules or ions such as peapots, or the surface may be molecular-modified. Alternatively, it may be a CNT with both ends capped, or a CNT with a structure in which one or both ends are open. Furthermore, the CNT may be a single-walled CNT (SWCNT) or a multi-walled CNT (MWCNT).

[0014] CNTs easily associate with each other due to their high intermolecular force, forming bundles composed of multiple CNT molecules. In the CNT-based cell-shielding membrane, multiple bundles form a dense mesh structure. More specifically, the mesh structure is formed by randomly entangling multiple relatively thin bundles. Because of this dense mesh structure, the CNT-based cell-shielding membrane can not only exist as a free-standing membrane with high strength, but also maintain its function as a cell-shielding membrane, that is, the function of securing a space for selective growth of bone cells and tissue cells in the defective area of ​​the bone or tissue to be healed, without the collapse of the membrane structure even after being embedded in the living body for a long time. Here, a free-standing membrane means a membrane that can maintain its shape without using other supporting members such as a substrate.

[0015] The mesh structure can be evaluated, for example, using the diameter of the bundles observed in the SEM image of the CNT-based cell shielding film. The CNTs contained in the CNT-based cell shielding film mainly include bundles with a diameter of 10 nm or more and 50 nm or less, and the probability of existence of bundles with a diameter of 60 nm or more is 10% or less, or 5% or less. The probability of existence may be higher than 0%. Here, the probability of existence of bundles with a diameter of 60 nm or more is the ratio of bundles having a diameter of 60 nm or more out of a total of 300 bundles including 10 bundles crossing the central transverse line of the image in each image obtained by acquiring 30 SEM images of the CNT-based cell shielding film at 200,000 times magnification. When there are more than 10 bundles crossing the central transverse line of the image, 10 bundles are selected in order from the surface observed in the SEM image.

[0016] The size of the CNT-based cell-shielding membrane can be determined as desired, and can be adjusted to fit the tissue defect where it is placed. The thickness of the CNT-based cell-shielding membrane can also be determined as desired, and can be appropriately selected from the ranges of, for example, 100 nm to 500 μm, 1 μm to 300 μm, or 10 μm to 200 μm. By selecting a thickness within this range, the membrane can be secured to have a strength sufficient to function as a cell-shielding membrane, and can be deformed into any shape according to the shape of the object to be healed.

[0017] There is no restriction on the tissue regeneration promoter, and a compound having a function of promoting the regeneration of cells constituting periodontal ligament tissue, bone tissue, etc. can be used. For example, hyaluronic acid, catechin and its isomers or derivatives, basic fibroblast growth factor (bFGF), bone morphogenetic protein (BMP), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), etc. can be used. Examples of isomers and derivatives of catechin include epicatechin, epigallocatechin (EGCG), gallocatechin, etc. Preferably, the CNT-based cell-occluding membrane contains a bone regeneration promoter capable of promoting the dispersion of CNT in water. Examples of such bone regeneration promoters include the above-mentioned hyaluronic acid, or catechin and its isomers or derivatives.

[0018] The composition ratio of the tissue regeneration promoter to the CNT membrane may be determined arbitrarily depending on the adsorption capacity of the CNT membrane for the tissue regeneration promoter. The weight ratio of the tissue regeneration composition to the CNT membrane may be selected from the range of, for example, 3.0% by weight to 75% by weight, 5.0% by weight to 60% by weight, or 10% by weight to 50% by weight.

[0019] The additives, which are optional components, can be used to adjust the properties of the CNT-based cell shielding membrane, such as its strength, in vivo resistance, hydrophilicity, gas permeability, water permeability, etc. Specifically, they may be synthetic polymers having biocompatibility, such as regenerated cellulose, polyvinyl alcohol, ethylene-vinyl alcohol copolymer, polymethacrylate, polyacrylonitrile, polystyrene, polypropylene, polyethylene terephthalate, nylon, polytetrafluoroethylene, polysiloxane, polylactic acid, and lactic acid-glycolic acid copolymer, or biopolymers exemplified by collagen derived from animals such as cows or pigs, or decellularized tissues such as decellularized human dermis.

[0020] The additive may be supported on the CNT film. In this case, for example, the weight ratio of the additive to the CNT film may be 5% or more and 200% or less, 30% or more and 100% or less, or 50% or more and 80% or less. Alternatively, the additive may be used as one film and laminated with the CNT film. For example, the CNT-based cell-shielding film may have a structure in which a film containing an additive is sandwiched between two CNT films. Alternatively, the CNT film may be sandwiched between films containing an additive. Even when a film containing an additive and a CNT film are laminated, the weight ratio of these films may be selected from the above-mentioned range.

[0021] 2. Manufacturing method of CNT-based cell-shielding membrane

[0022] An example of a manufacturing method for a CNT-based cell-shielding film is shown in Figure 1. One of the manufacturing methods for a CNT-based cell-shielding film involves the synthesis of CNTs, loading a tissue regeneration-promoting agent onto the CNTs, and recovering and forming a film of the CNTs with the tissue regeneration-promoting agent adsorbed thereon.

[0023] The CNTs constituting the CNT film can be synthesized by using various methods such as the arc discharge method, the laser ablation method, and the chemical vapor synthesis method. For example, in the chemical vapor synthesis method, a substrate carrying metal catalyst particles such as iron, nickel, or cobalt may be used, and CNTs may be grown from the surface of the substrate or powder. More specifically, the substrate carrying the metal catalyst particles is placed in a heating furnace such as an electric furnace. Then, a CNT growth gas (carbon source) such as benzene, acetylene, ethylene, carbon monoxide, methane, or ethanol, and a catalyst activation material such as water are supplied into the heating furnace, and the furnace is heated at a temperature range of 600°C to 1200°C, so that the chemical growth of CNTs can be started from the surface of the metal catalyst particles. An inert gas such as argon may be supplied into the heating furnace to dilute the CNT growth gas. This method allows the synthesis of SWCNTs that are long, exceeding 100 μm or 1 mm, called super-growth CNTs.

[0024] Alternatively, CNTs can be grown from the surface of metal catalyst particles suspended in the gas phase (gas-phase flow method). In this case, the reaction field can be optimized by using CNT growth gases with different decomposition characteristics. This method is called the improved direct injection pyrolysis synthesis method (eDIPS method). By applying the eDIPS method, it is possible to selectively synthesize single-walled CNTs with precisely controlled diameters at high purity.

[0025] The tissue regeneration promoter is supported by treating the CNT and the tissue regeneration promoter in a solvent (hereinafter, dispersion solvent) and adsorbing the tissue regeneration promoter onto the surface of the CNT dispersed in the dispersion solvent. Examples of the dispersion solvent include water, alcohols such as ethanol and isopropanol, ketones such as methyl ethyl ketone, esters such as ethyl acetate, carbonates such as ethylene carbonate and propylene carbonate, amide solvents such as N,N-dimethylacetamide and N,N-dimethylformamide, and aromatic hydrocarbons such as toluene and xylene. Among these, water is preferred because it is non-toxic and inexpensive. In this case, ultrasonic waves may be irradiated onto the dispersion liquid containing the CNT and the tissue regeneration promoter to accelerate the dispersion of the CNT.

[0026] The CNTs synthesized by the above-mentioned method are strongly aggregated, but in the dispersion solvent, the aggregated state of the CNTs is partially dissolved and bundles are formed. In this case, by using a tissue regeneration promoter that also functions as a dispersant to promote the dispersion of CNTs, it is possible to obtain an assembly of bundles with a smaller width. As mentioned above, the dispersion of CNTs can be effectively promoted by using hyaluronic acid, catechin, and its isomers or derivatives.

[0027] The dispersion obtained by dispersing CNTs in a dispersion solvent in the presence of a tissue regeneration promoter is filtered by suction or natural filtration to separate the filtrate and the filtrate. Filter paper or glass filters, or filters containing polyethylene called membrane filters, may be used as the filters used for filtration. The filtrate is then collected, formed into a membrane, and dried (i.e., the dispersion solvent is distilled off) to produce a CNT-based cell-shielding membrane. For example, the filter may be removed from the filtrate deposited on the filter and used as a CNT-based cell-shielding membrane.

[0028] In addition, when the solubility of the tissue regeneration promoter in the dispersion solvent is low, CNTs can be dispersed using the dispersion solvent, filtered, and formed into a CNT film, and then the tissue regeneration promoter can be dissolved in a solvent that dissolves the tissue regeneration promoter, and the CNT film can be immersed in this solution to support the tissue regeneration promoter.

[0029] When an additive is used, the additive may be dissolved or dispersed in the above-mentioned dispersion liquid and then filtered. Alternatively, a separately prepared film containing the additive and the CNT-based cell-shielding film may be laminated.

[0030] In the above-mentioned manufacturing method, the tissue regeneration promoter is adsorbed on the CNT surface in the dispersion liquid of CNT and supported on the CNT film, but the tissue regeneration promoter may be supported after the CNT film is prepared using a dispersant. For example, a dispersant is added to a dispersion solvent containing CNT to promote dispersion of CNT, and then the CNT is collected by filtration and formed into a film. The dispersant is then removed by treating the CNT film with a solvent in which the dispersant is soluble. Furthermore, the CNT film may be immersed in the tissue regeneration promoter or a solution containing the tissue regeneration promoter to support the tissue regeneration promoter.

[0031] Examples of dispersants used in this case include ionic or nonionic surfactants. Examples of ionic surfactants include cationic surfactants such as quaternary ammonium salts, such as alkylammonium salts; amphoteric surfactants such as betaine and amine oxide having an ammonium cation and a carboxyl anion; and anionic surfactants such as alkylbenzenesulfonates, alkyl sulfates, alkyl phosphates, and carboxylates. Examples of nonionic surfactants include sugar ester surfactants such as sorbitan fatty acid esters and polyoxyethylene sorbitan fatty acid esters; fatty acid ester surfactants such as polyoxyethylene resin acid esters and polyoxyethylene fatty acid diethyl; ether surfactants such as polyoxyethylene alkyl ethers, polyoxyethylene alkyl phenyl ethers, and polyoxyethylene polypropylene glycols; and aromatic surfactants such as polyoxyalkylene octyl phenyl ethers, polyoxyalkylene nonyl phenyl ethers, polyoxyalkyl dibutyl phenyl ethers, polyoxyalkyl styryl phenyl ethers, polyoxyalkyl benzyl phenyl ethers, polyoxyalkyl bisphenyl ethers, and polyoxyalkyl cumyl phenyl ethers.

[0032] As an optional step, the CNT-based cell-shielding membrane may be subjected to a hydrophilization treatment, for example, by plasma treatment in the presence of argon or air, or by UV irradiation in the presence of ozone.

[0033] 3.Tissue regeneration method Tissue regeneration can be achieved by placing and fixing a CNT cell shielding membrane so as to cover the tissue defect. A known method can be used for fixing, for example, by forming the CNT cell shielding membrane to fit the shape of the target to be healed, attaching absorbable or non-absorbable sutures to the edges of the CNT cell shielding membrane, and ligating and fixing it to the target to be healed. After tissue regeneration is complete, the CNT cell shielding membrane is removed (re-entry).

[0034] Since CNTs are not absorbed or decomposed in vivo unless they are detached from the membrane, the CNT-based cell-shielding membrane according to one embodiment of the present invention is classified as a non-absorbable cell-shielding membrane. Therefore, it is possible to suppress the occurrence of inflammation, which is a problem when performing tissue regeneration using an absorbable cell-shielding membrane, and there is no need to worry about decomposition or collapse before tissue regeneration. For example, when polylactic acid is used as a cell-shielding membrane, it may cause strong inflammation because it dissolves rapidly in the body, but it is possible to suppress inflammation by using a CNT-based cell-shielding membrane.

[0035] On the other hand, when a cell-shielding membrane containing polytetrafluoroethylene or titanium is used as a non-absorbable cell-shielding membrane, sufficient tissue regeneration cannot be achieved unless it is tightly sutured to the healing target, which can lead to the onset of infection. However, since CNTs are unlikely to cause foreign body reactions or rejection reactions in the body, as shown in the examples, the use of a CNT-based cell-shielding membrane according to one of the embodiments of the present invention can suppress inflammatory reactions and the onset of infection. In addition, due to the high molecular weight and rigidity of CNTs and the dense network structure of the CNT bundles, the CNT-based cell-shielding membrane can be easily manufactured as a self-supporting membrane. Therefore, it is possible to secure a space for bone cell growth for a long period of time (space making) without decomposing or collapsing in the body.

[0036] Furthermore, whether absorbable or non-absorbable cell shielding membranes are used, tissue regeneration takes a very long time (e.g., six months or more), whereas the CNT-based cell shielding membrane according to one embodiment of the present invention is capable of carrying a relatively large amount of tissue regeneration-promoting agent, allowing the tissue regeneration-promoting agent to act effectively, enabling rapid tissue regeneration. EXAMPLES

[0037] An example of regeneration of bone tissue using a CNT-based cell shielding film according to one embodiment of the present invention will be described below.

[0038] 1. Example 1: CNT-based cell-blocking membrane carrying hyaluronic acid 1-1. Preparation of CNT-based cell-shielding membrane High-purity SWCNT (10 mg) synthesized by the eDIPS method was added to a hyaluronic acid aqueous solution (20 mL) adjusted to a concentration of 0.1 wt%, and the resulting dispersion was subjected to ultrasonic treatment at room temperature for about 30 minutes. The solution was further treated with a tip-type ultrasonic treatment machine (manufactured by SONICS, model number VCX500, the same below) for 10 minutes. Thereafter, the dispersion was suction-filtered using a membrane filter (manufactured by Merck, model number JGWP04700, the same below). The filtrate collected on the membrane filter was dried in a dryer at 60°C for 24 hours, then peeled off from the membrane filter, and the surface was hydrophilized with a UV ozone cleaner (manufactured by Meiwafosis, model number PC450, the same below) to obtain a CNT-based cell-shielding membrane carrying hyaluronic acid. 1-2.Evaluation of CNT-based cell shielding membrane

[0039] Figure 2 shows the ultraviolet absorption spectra of the filtrate and the standard aqueous solution of hyaluronic acid (0.1 mg / mL, i.e., 0.01 wt%). The ultraviolet absorption spectrum was obtained using a Shimadzu ultraviolet-visible-near infrared spectrophotometer (model number UV-3100, the same applies below). The amount of hyaluronic acid supported on the CNT membrane was quantified from the difference in absorption intensity between the filtrate and the standard aqueous solution, and it was found that 1.2 mg of hyaluronic acid was supported per 10 mg of CNT.

[0040] Figure 3 is a 100,000x SEM image (Hitachi High-Technologies Corporation, scanning electron microscope, model SU8200) of the obtained hyaluronic acid-loaded CNT-based cell-shielding membrane. This SEM image confirmed that bundles of thin CNTs with diameters of about 5 nm to 50 nm formed a mesh structure.

[0041] Quantitative evaluation of the mesh structure was performed as follows. First, 30 SEM images were taken at 200,000 times magnification at 30 randomly selected locations on the CNT-based cell shielding membrane. An example of an SEM image is shown in Figure 4. As shown in Figure 4, the diameters (lengths of the straight lines numbered in the figure) of 10 bundles crossing the central transverse line of the image (horizontal dotted line in Figure 4) in each SEM image were measured. If there were more than 10 bundles crossing the central transverse line of the image, 10 bundles were selected from the surface side of the CNT-based cell shielding membrane. Similar measurements were performed on 30 SEM images, and the results obtained for a total of 300 bundles are shown in Figure 5 using histograms and Pareto curves. As shown in Figure 5, it can be seen that the CNTs contained in the CNT-based cell shielding membrane are mostly composed of bundles with a diameter of 50 nm or less. The average bundle diameter was 25.6 nm, the standard deviation was 16.5 nm, and 95% of the bundles had a diameter of less than 60 nm. From this, it was found that the probability of existence of bundles with a diameter of 60 nm or more in the CNT-based cell shielding membrane is 5%. It is believed that such a dense mesh structure gives the CNT membrane the ability to form a self-supporting membrane, suppresses the collapse of the membrane structure in the body, and as a result, suppresses the induction of an inflammatory response. In this example, no surfactant was added in the preparation of the CNT dispersion. However, the dense mesh structure suggests that CNTs exhibit good dispersibility in water even in the absence of a surfactant. This means that hyaluronic acid also functions as a dispersant.

[0042] 1-3. Bone formation using CNT-based cell-shielding membrane A bone defect (approximately 7 mm in diameter) was created in the rat calvaria, and the defect was covered with a hyaluronic acid-loaded CNT-based cell-shielding membrane, and bone formation was observed. Figures 6(A) to 6(D) are micro-CT images of the bone defect 28 days after surgery. Figures 6(A) and 6(B) are horizontal plane images, and Figures 6(C) and 6(D) are cross-sectional images. Compared to when the cell-shielding membrane was not applied (Figures 6(A) and 6(C)), bone regeneration was clearly confirmed when the cell-shielding membrane was applied (Figures 6(B) and 6(D)).

[0043] Figure 7 shows an optical micrograph of the anterior cross section of the center of the bone defect 28 days after surgery. As shown in Figure 7, active new bone formation (*) was observed on the underside of the cellular barrier membrane (↑), and thin fibrous connective tissue (△) was observed on the upper surface of the cellular barrier membrane, but almost no inflammatory cells were observed within the connective tissue.

[0044] 2. Example 2: EGCG-loaded CNT-based cell-blocking membrane 2-1. Preparation of CNT-based cell-shielding membrane High-purity SWCNTs (10 mg) synthesized by the eDIPS method were added to an EGCG aqueous solution (20 mL) adjusted to a concentration of 0.2 wt%, and the resulting dispersion was subjected to ultrasonic treatment at room temperature for about 30 minutes. The solution was further treated with a tip-type ultrasonic treatment machine for 10 minutes. The dispersion was then suction-filtered using a membrane filter with a diameter of 47 mm. The residue collected on the membrane filter was dried in a dryer at 60°C for 24 hours, and then peeled off from the membrane filter. The surface was further hydrophilized with a UV ozone cleaner to obtain a CNT-based cell-shielding membrane carrying EGCG.

[0045] 2-2. Evaluation of CNT-based cell-shielding membrane Figure 8 shows the ultraviolet absorption spectra of the filtrate and the standard aqueous solution of EGCG (0.1 mg / mL, i.e., 0.01 wt%). The amount of EGCG supported on the CNT membrane was quantified from the difference in absorption intensity between the filtrate and the standard aqueous solution. Almost no EGCG was detected in the filtrate, and it was found that 4 mg of EGCG was supported per 10 mg of CNT. In addition, curve fitting was performed on the plot of the amount of EGCG supported against the amount of EGCG added (Figure 9), and it was estimated that the maximum amount of EGCG supported per 10 mg of SWCNT was 7.52 mg. The reason that such a large amount of EGCG can be supported on the CNT membrane is probably due to the sp of the CNT membrane. 2 This is believed to be due to strong intermolecular forces based on π-π stacking interactions between the carbon and the aromatic rings in EGCG.

[0046] Figure 10 shows an SEM image (100,000x magnification) of the obtained EGCG-loaded CNT-based cell-shielding membrane. This SEM image shows that thin bundles with diameters of about 10 nm to 100 nm form a mesh structure.

[0047] Quantitative evaluation of the mesh structure was performed in the same manner as in Example 1. FIG. 11 shows a histogram and Pareto curve of the diameters of a total of 300 bundles obtained from 30 SEM images. As can be seen from FIG. 11, like the CNT-based cell shielding film of Example 1, the CNTs contained in the CNT-based cell shielding film of this example are mostly composed of bundles with a diameter of 50 nm or less. The average diameter of the bundles was 30.7 nm, the standard deviation was 21.6 nm, and 90% of the bundles had a diameter of less than 60 nm. From this, it was found that the probability of bundles with a diameter of 60 nm or more existing in the CNT-based cell shielding film was 10%. In this example, no surfactant was added in the preparation of the CNT dispersion. However, from the dense mesh structure, it can be seen that the CNTs show good dispersibility even in the absence of a surfactant. This means that EGCG also functions as a dispersant for dispersing CNTs.

[0048] 2-3. Bone formation using CNT-based cell-shielding membrane A bone defect (approximately 7 mm in diameter) was created in the rat calvaria and covered with an EGCG-loaded CNT-based cell-shielding membrane, and bone formation was observed. Figures 12(A) to 12(D) are micro-CT images of the bone defect 28 days after surgery. Figures 12(A) and 12(B) are horizontal plane images, and Figures 12(C) and 12(D) are cross-sectional images. Compared to when the cell-shielding membrane was not applied (Figures 12(A) and 12(C)), bone regeneration was clearly confirmed when the cell-shielding membrane was applied (Figures 12(B) and 12(D)).

[0049] Figure 13 shows an optical microscope image of the EGCG-loaded CNT cell-shielding membrane 56 days after it was embedded subcutaneously in a rat. From Figure 13, it was observed that the CNT cell-shielding membrane (↑) was covered by fibrous connective tissue (△). Fibroblasts were observed within the fibrous connective tissue, but almost no inflammatory cells were observed, and inflammation was mild.

[0050] 3. Comparative Example: CNT membrane not carrying bone regeneration promoter As a comparative example, an example will be described in which a CNT film prepared by dispersing CNTs in chloroform was used without using a bone regeneration promoter.

[0051] 3-1. Preparation of CNT film High-purity SWCNTs (10 mg) synthesized by the eDIPS method were added to chloroform (20 mL), and the resulting dispersion was subjected to ultrasonic treatment at room temperature for about 30 minutes. The solution was further treated with a tip-type ultrasonic treatment device for 10 minutes. The dispersion was then suction filtered using a membrane filter with a diameter of 47 mm. The residue collected on the membrane filter was dried in a dryer at 60°C for 24 hours, and then peeled off from the membrane filter. The surface was further hydrophilized with a UV ozone cleaner to obtain a CNT film.

[0052] 3-2. Evaluation of CNT films Figure 14 shows an SEM image of the CNT film of the comparative example. It can be seen that thick bundles with diameters of about 10 nm to 200 nm form a coarse mesh structure, unlike Examples 1 and 2. Note that, since CNTs hardly disperse in water when no dispersant is used, thicker bundles are formed when water is used as the dispersion solvent instead of chloroform.

[0053] Quantitative evaluation of the mesh structure was performed in the same manner as in Examples 1 and 2. FIG. 15 shows a histogram and Pareto curve of the diameters of a total of 300 bundles obtained from 30 SEM images. The data width in this histogram is the same as that in Examples 1 and 2. As can be seen by comparing FIG. 15 with FIG. 5 and FIG. 10, unlike the CNT-based cell shielding films of Examples 1 and 2, the bundle diameters and their distribution are large, and bundles with diameters exceeding 150 nm exist, with the maximum bundle diameter being 521 nm. The average bundle diameter was 63.5 nm, and the standard deviation was 64.5 nm. It was also confirmed that the CNT bundle-like structures with diameters less than 60 nm accounted for 67%, and the probability of bundles with diameters of 60 nm or more in the CNT film was 37%.

[0054] 3-3. Bone formation using CNT membrane A bone defect (diameter approximately 7 mm) was created in the rat calvaria, and the bone defect was covered with a comparative example of a CNT membrane not carrying a bone regeneration promoter, and bone formation was observed. Figure 16 shows a soft X-ray image of the bone defect 14 days after surgery. When the CNT membrane was not loaded with a bone regeneration promoter as in the comparative example, almost no opacity was observed even when the CNT membrane was coated on the bone defect, making it clear that bone regeneration had not progressed.

[0055] Figure 17 shows optical microscope images of subcutaneous tissue 14 days after surgery. The CNT membrane (left image, ↑) is covered with fibrous connective tissue containing many mesenchymal cells, but moderate inflammatory cell infiltration (*) was observed, and many foreign body giant cells (△) were also observed. It was also confirmed that the structure of the CNT membrane had significantly collapsed within the tissue (left image, circled), and that some of the SWCNTs had fallen off the membrane and become mixed into the cells as foreign bodies (right image, ↑).

[0056] As shown in the above examples, it was confirmed that the use of a CNT-based cell-shielding membrane according to one of the embodiments of the present invention suppresses the breakdown of the membrane and the occurrence of inflammation, and enables the regeneration of tissue cells in a short period of time. This clearly indicates that the CNT-based cell-shielding membrane according to one of the embodiments of the present invention can suppress the onset of infectious diseases and also functions as a cell-shielding membrane that promotes tissue formation.

Claims

1. A carbon nanotube freestanding film; A cell-shielding membrane consisting of only a tissue regeneration promoter supported on the carbon nanotube freestanding membrane.

2. The cell-shielding membrane according to claim 1 , wherein the carbon nanotube free-standing membrane comprises bundles of carbon nanotubes having a diameter of 10 nm or more and less than 60 nm that form a mesh structure.

3. The cell-shielding membrane described in claim 1, wherein, among a total of 300 bundles including 10 carbon nanotube bundles crossing the central transverse line of the image observed in each of 30 images of the carbon nanotube freestanding membrane at 200,000 magnification scanning electron microscope images, the probability of the existence of bundles having a diameter of 60 nm or more is 10% or less.

4. The cell-shielding membrane of claim 2 , wherein the bundle comprises a plurality of single-walled carbon nanotubes.

5. The tissue regeneration promoter is a bone regeneration promoter, The cell-occlusion membrane of claim 1 , wherein the bone regeneration promoter is selected from hyaluronic acid, catechin, epicatechin, epigallocatechin, gallocatechin, basic fibroblast growth factor, bone morphogenetic protein, vascular endothelial growth factor, and platelet-derived growth factor.

6. The cell barrier membrane according to claim 1 , further comprising an additive selected from polytetrafluoroethylene, polylactic acid, a copolymer of lactic acid and glycolic acid, collagen derived from bovine or porcine origin, and decellularized human dermis.

7. A method for producing a cell-shielding membrane, comprising: Preparing a dispersion containing carbon nanotubes and a tissue regeneration promoter; forming a carbon nanotube free-standing membrane using a residue obtained by filtering the dispersion; The tissue regeneration promoter is supported on the carbon nanotube freestanding film, A method for producing a cell-shielding film, wherein the cell-shielding film consists only of the carbon nanotube freestanding film and the tissue regeneration promoter.

8. The method according to claim 7 , further comprising subjecting the carbon nanotube freestanding film to a hydrophilic treatment.

9. The method according to claim 7 , wherein the carbon nanotubes are synthesized by a gas-phase flow method using multiple carbon sources.

10. The process of claim 9, wherein the carbon source is selected from methane, ethylene, and acetylene.

11. The tissue regeneration promoter is a bone regeneration promoter, The method according to claim 7, wherein the bone regeneration promoter is selected from hyaluronic acid, catechin, epicatechin, epigallocatechin, gallocatechin, basic fibroblast growth factor, bone morphogenetic protein, vascular endothelial growth factor, and platelet-derived growth factor.

12. The method of claim 7 , wherein the dispersion further comprises an additive selected from polytetrafluoroethylene, polylactic acid, a copolymer of lactic acid and glycolic acid, collagen derived from bovine or porcine origin, and decellularized human dermis.

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