JP2011167237A - Bio-applicable material - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a bio-applicable material without mixing acid, alkaline or harmful solvents, and without danger to the living body at all. <P>SOLUTION: Nanofiber whose average fiber diameter is approximately 20 nm including chitin or chitosan or their composite is prepared without using acid, alkaline, organic solvents but by using water only. As a result, thus obtained nanofiber has no danger to the living body, and has excellent cell adhesiveness because of the nano-fiberization. The nanofiber is useful as a medical material or bio-applicable material for artificial skin, a wound dressing agent, a scaffold for adhesion of cells required for regeneration of various organs, or the like. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a biomaterial.

  In recent years, in the field of regenerative medicine, research and development of artificial skin using materials excellent in biocompatibility centering on natural polymers have been promoted. In addition, since tissue regeneration requires a three-dimensional structure as a scaffold, materials for artificial skin contribute to appropriate mechanical strength and cell adhesion in addition to biocompatibility and safety. It is required that a sufficient surface area or a porous structure that can supply oxygen and nutrients and become an invasion path for cells and regenerative tissues can be formed.

  As a three-dimensional structure that functions as a scaffold, a sponge body, a honeycomb body, or a nonwoven fabric layered with fibers is known. Among them, as a function of constituting a nonwoven fabric, a conventionally known fiber width (short diameter) is known. ) Is being developed and put to practical use in addition to micrometer-order ultrafine fibers and even finer nanometer-order fibers (nanofibers). Nanofibers are defined as having a fiber width of 100 nm or less, and have a much larger specific surface area than conventional ultrafine fibers, which is advantageous in terms of cell adhesion efficiency.

  Nonwoven fabrics of fibers with a short diameter of several nanometers to several tens of micrometers using gelatin, collagen, etc. as natural polymers, and polysaccharides such as chitosan, chondroitin sulfate, pectin, and hyaluronic acid have a short diameter of about 500 nm. A certain nonwoven fabric is applied as a medical material. In particular, chitin and chitosan, which are biomass abundant in nature, are attracting attention in the field of regenerative medicine because they have excellent biocompatibility, but they are decomposed and refined because they have a strong crystal structure. It is difficult and its use is stagnant.

  Acid-treated chitin / chitosan and chitin / chitosan paste (Patent Document 1), production method of ultrafine nanofiber (Patent Document 2), functional wound dressing (Patent Document 3), bone filling material, and production method thereof (Patent Document) Although the literature regarding 4) is known, a special reagent having extremely high toxicity is used for the production thereof, which is problematic in terms of safety to living bodies. Moreover, in the case of artificial skin made of chitin fibers, the fiber diameter is on the order of micrometers, so the engraftment with cells is not sufficient.

JP2008-1829 JP2009-270210 JP 10-151184 A JP 10-309287 A

  Therefore, the problem to be solved by the present invention is a medical material, a biocompatible material, and a cell culture substrate that are safe as artificial skin and wound dressing materials and have excellent cell adhesion as a scaffold for tissue regeneration. And providing cosmetics. Therefore, attention is focused on chitin and chitosan having excellent biocompatibility, and these are processed into various forms by efficiently converting them into nanofibers and used as biomaterials.

  When the present inventors applied materials such as chitin nanofibers and, if necessary, films, slurries, pastes, and emulsions further containing chitosan nanofibers to the living body, they have superior effects compared to materials in which they are dissolved. It was found to have

The present invention provides the following biomaterials.
Item 1. Biomaterials containing chitin nanofibers and / or chitosan nanofibers.
Item 2. Item 2. The biocompatible material according to Item 1, which is in the form of a paste, gel, slurry, film, sheet, porous body, dispersion, suspension or emulsion.
Item 3. Item 2. The biomaterial according to Item 1, comprising chitin nanofibers and chitosan nanofibers.
Item 4. Item 4. The biomaterial according to any one of Items 1 to 3, which is used as a medical culture material such as artificial skin and a wound dressing or a cell culture substrate that is a scaffold for regeneration of various organs.
Item 5. Item 5. The biomaterial according to any one of Items 1 to 4, wherein an average fiber width (minor axis) of the nanofiber is 4 to 100 nm, preferably 4 to 20 nm.
Item 6. A film, sheet or porous body obtained by removing moisture from chitin nanofibers and / or chitosan nanofiber dispersions by drying treatment.
Item 7. Item 7. The film, sheet or porous material according to Item 6, wherein the cell adhesion is 2 times or more superior to acid-treated chitin / chitosan.
Item 8. A chitin / chitosan nanofiber dispersion, wherein chitin nanofibers and / or chitosan nanofibers are dispersed in water.
Item 9. Cosmetics containing chitin nanofiber and / or chitosan nanofiber.

  Chitin / chitosan (poorly soluble polysaccharide) is already used in the medical field as a wound dressing (artificial skin). When manufacturing these, chitin / chitosan is dissolved in a solvent and spun or formed into a film. The process to do is necessary. In the case of chitosan, a method of film formation after solubilization with an acid or amide, or fiber formation by electrospinning or the like is employed. However, these prior arts involve complicated processes and use an organic solvent, so there is a concern about the influence on the living body. On the other hand, the present invention is a biomaterial that does not use an organic solvent, and is formed into a slurry as needed after it is made into a slurry by micronizing or nanofibrosis by pressurizing chitin or chitosan in water. In terms of production, there is no environmental load and no danger to the living body.

  The slurry containing nanofibers of chitin / chitosan of the present invention can be formed into a film or a substrate by evaporating water, and should be superior in adhesion and proliferation of somatic cells than conventional methods. Revealed by the present invention. In particular, chitin alone is an excellent biocompatible material, but there is no good solvent, and film formation by the prior art is difficult. The present invention makes it easy.

  In conventional nanospinning by electrospinning, only materials that are soluble in organic solvents could be used, but in the present invention there is no limitation, for example, chitosan with antibacterial properties and chitin with excellent cell adhesion properties at the nano level. It is easy to manufacture biomaterials combined with the above. Moreover, in order to give them appropriate physical properties, they can be easily combined with other biodegradable polymers.

Cell adhesion number 10 days after cell seeding. I: Chitosan acetic acid-dissolved cast film (conventional method). II: Chitosan film treated 5 times with water jet. III: Chitin film treated 5 times with water jet Electron micrograph of SEM observation of the chitin film surface. “Acetic acid”: a sheet prepared by dissolving chitin with acetic acid, “5pass”: a sheet made of chitin treated with a water jet five times. An electron micrograph of SEM observation of the surface of a chitin film adsorbed on cells. “Acetic acid”: a sheet prepared by dissolving chitin with acetic acid, “5pass”: a sheet made of chitin treated with a water jet five times. Observation of chitin / chitosan nanofibers by FE-SEM

  The average diameter (minor axis) of chitin or chitosan nanofibers used in the method of the present invention is about 4 to 100 nm, preferably about 4 to 50 nm, more preferably about 4 to 40 nm, still more preferably about 4 to 25 nm. Most preferably, it is about 20 nm. Such chitin or chitosan nanofibers can be manufactured by high-pressure jetting a chitin or chitosan dispersion fluid.

  According to the present invention, a nanofiber can be formed by high-pressure jet treatment using a crystalline or poorly water-soluble natural polymer such as chitin and chitosan as a water dispersion fluid. Here, the “dispersion fluid” is a dispersion of chitin and chitosan in water. When the concentration is low, the dispersion fluid is a fluid dispersion, but the viscosity is particularly increased as the chitin and chitosan become finer. When it becomes higher and the concentration becomes higher, it becomes a property close to a paste. The concentration of the chitin / chitosan nanofiber dispersion fluid is preferable because the treatment efficiency increases as the concentration increases. In particular, in the case of fine fibers at the nano level, the viscosity becomes too high and high-pressure injection becomes difficult when the paste is formed. . In the present invention, a device capable of high-pressure jetting even when the concentration of chitin / chitosan fiber (preferably chitin / chitosan nanofiber) is high has been developed. %, Preferably about 5 to 20% by weight, more preferably about 10 to 20% by weight, and still more preferably about 11 to 20% by weight. The chitin nanofiber / chitosan nanofiber of the present invention may be applied to a living body (for example, skin, mucous membrane, organ, organ, cell, etc.) in a slurry or paste form. When sprayed, it is not necessary to evaporate water, and it is preferable because it can directly obtain slurry or paste-like nanofibers.

  In the biocompatible material of the present invention, chitin / chitosan nanofibers can be used in combination with other biocompatible materials. Examples of such biocompatible materials include biodegradable polyesters such as polylactic acid, polyglycolic acid, and poly-ε-caprolactone, biological polymers such as collagen, fibronectin, and vitronectin, and synthetic polymers such as polyethylene and polymethyl methacrylate. Inorganic compounds such as hydroxyapatite, titanium dioxide, and barium sulfate.

  The raw material for chitin / chitosan used in the present invention may be in any form such as fibrous or granular. Chitin can be directly used as a raw material for biomass such as shells of crustaceans such as shrimps and crabs. As chitin / chitosan, it is preferable to use purified chitin / chitosan which has been subjected to deproteinization / decalcification treatment by a generally known method as a raw material. Chitin / chitosan may be a commercially available raw material. When biomass is subjected to high-pressure injection treatment with the apparatus of the present invention, chitin / chitosan is untangled between fibers while maintaining the fiber length, but becomes thin by changing treatment conditions such as injection pressure and number of treatments. It is also possible to reduce the molecular weight. In the present specification, the “nanofiber” means a fiber having a nano width. The diameter (width) of the nanofiber can be measured by an electron micrograph. Such a fiber is not nano-sized in length, but has a diameter (width) of nano-size, and is therefore referred to as nanofiber in this specification.

  The material of the present invention is used in the form of paste, gel, slurry, film, sheet, porous body, dispersion, suspension or emulsion (W / O, O / W, W / O / W, etc.). be able to. The material of the present invention is a material containing water, and the water content is 5 to 99.5 parts by weight, preferably 8 to 98.5 parts by weight when the total amount of water and nanofibers is 100 parts by weight. For example, in the case of a semi-solid or low fluidity material such as a paste, gel or slurry, the water content is 10 to 95 parts by weight. In the case of a dispersion or suspension, the water content is 95-98.5 parts by weight. Furthermore, in the case of films, sheets, porous bodies, the water content is 8 to 70 parts by weight. In addition, a porous body can be manufactured by freeze-drying, and a film, a sheet, etc. can be manufactured in accordance with a conventional method such as casting.

  The average diameter of the nanofibers obtained by the treatment by the method of the present invention is about 4 to 100 nm, preferably about 4 to 40 nm, and most preferably about 4 to 25 nm. Since the nanofiber of the present invention has a large fiber length / fiber width (aspect ratio), it can be easily formed into a film or sheet in which nanofibers are entangled like a nonwoven fabric while maintaining strength. It can be suitably used as a compatible material. The nonwoven fabric obtained by forming the nanofiber of the present invention into a film / sheet is hereinafter referred to as “nanofiber film”. The nanofiber film of the present invention is characterized by a very narrow fiber width and high transparency.

  The dispersion fluid of chitin and chitosan can be jetted from a nozzle at a high pressure using an ultrafine device using a water jet developed by Sugino Machine Co., Ltd. The pressure of the high pressure injection is about 30 to 245 MPa. The injection speed is about 242 to 700 m / s.

  The dispersed fluid of chitin and chitosan collided with the collision hard body by high-pressure injection is recovered, and is again injected with high pressure from the nozzle toward the collision hard body, and this operation is performed a required number of times, for example, about 1 to 50 times. Preferably about 1 to 40 times, more preferably about 1 to 30 times, still more preferably about 1 to 20 times, and particularly preferably about 1 to 10 times. When chitin and chitosan collide with the collision-use hard body, the fibers are entangled, the fiber diameter is reduced, and the nano size is reduced. In addition, as a hard body for collision, shapes, such as ball shape and flat plate shape, are mentioned. Examples of the diameter of the nozzle that jets the dispersion fluid at a high pressure include about 0.1 to 0.8 mm.

  Chitin or chitosan nanofibers can be expected to have an effect of increasing the strength by increasing the fiber diameter (fiber width) so that the fibers are entangled with each other at high density. In addition, by increasing the space between the fibers, it is possible to reinforce the function of the scaffold to which the cells adhere and the movement of substances necessary for cell growth.

  Since the nanofiber of the present invention has excellent cell adhesion, healing can be promoted by applying a sheet-like or film-like biomaterial to the suture site and suturing. Similarly, various stem cells, fibroblasts, epithelial cells, endothelial cells, muscle cells, islet cells, liver cells and the like are cultured on the sheet / film of the present invention, and the sheet / film having cells is prosthetic and embedded In addition, it may be applied to a living body or a living body surface for the purpose of tissue regeneration.

  The material of the present invention can be composed of only water and nanofibers, and does not use any catalyst or harmful chemicals. Moreover, the nanofibers obtained by the present invention have an extremely large specific surface area compared to conventional micro-order ultrafine fibers, and are advantageous in terms of cell adhesion efficiency.

  The nanofibers obtained by the present invention have a fiber diameter of 100 nm or less, more preferably 80 nm or less, still more preferably 60 nm or less, particularly 40 nm or less. Since the nanofiber of the present invention has a very thin fiber diameter, it has an appearance close to a transparent solution when dispersed in water, and it is visually recognized that the nanofiber is dispersed in water. In this case, a transparent dispersion can be obtained. The chitin nanofibers treated with the water jet of the present invention can be uniformly dispersed even after centrifugation (15,000 rpm). By increasing the number of water jet treatments, a transparent gel state can be obtained. When the same treatment is performed with a water jet, a similar paste, gel or slurry can be obtained with chitosan. When the thus obtained bionanofiber paste / gel / slurry is cast in a petri dish and dried, a translucent or transparent film is obtained.

  In addition, the bionanofiber of the present invention is useful as a biomaterial to be blended in UV care cosmetics because it has a property of passing visible light but cutting ultraviolet rays. Since bionanofiber also has a moisturizing action, it can be blended into cosmetics and applied to the skin as a film or sheet. In particular, chitin / chitosan is known to give moisture and elasticity to damaged hair and skin, and the chitin / chitosan nanofiber of the present invention is suitable as a cosmetic.

  Drugs that contain biomaterials include ointments, lotions, patches, plasters, aerosols, solutions, extracts, eye ointments, eye drops, transdermal preparations, wound dressings, suspensions Examples of cosmetics include foundations, lipsticks, lotions, emulsions, creams, and the like, which can be suitably used for applications such as skin care and moisturizing.

  Chitin / chitosan nanofibers can be cast to produce sheets or films. By applying this, it is possible to create an artificial skin and an anti-organ adhesion sheet at the time of surgery. The chitin contact lens is obtained by dissolving a chitin in an amide solvent to obtain a transparent and viscous dope, and then removing the solvent with a special coagulating liquid and drying to obtain a transparent lens. When nanofibers are used, a special solvent is not required, and a lens-like transparent molded body can be produced only by drying.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, it cannot be overemphasized that this invention is not limited to these Examples.

Example 1
(1) Adhesion / proliferation of normal human dermal fibroblasts to chitin and chitosan film Using normal human dermal fibroblasts (NHDF) (Kurabo Co., Ltd.) constituting the dermis layer, water jet Films obtained by casting the chitin nanofiber dispersion treated 5 times with the above and the chitosan nanofiber dispersion treated 5 times with the water jet, respectively (in this example, they may be referred to as chitin film and chitosan film, respectively) The cell adhesion number was evaluated and the adhesion form was evaluated by SEM.

(1-1) After culturing NHDF in a T-25 flask, it was detached with trypsin and collected in a medium (Medium 106S) (manufactured by Kurabo Corp.) to prepare a cell suspension. Thereafter, the cell density was adjusted to 2.0 × 10 ⁴ cells / cm ² and seeded on chitin and chitosan films. The culture conditions were 37 ° C. and 5% CO ₂ , and the medium was changed every two days. After culturing for 10 days, the number of cell adhesion to the surface of each film was quantified by MTT assay (TA5355 manufactured by Cosmo Bio Co., Ltd.) (FIG. 1).

There is no significant difference between the number of cell adhesion to the chitosan acetate-dissolved cast film and the number of cell adhesion to the chitosan film. From the viewpoint of cell adhesion number, cell adhesion number to polylactic acid, which is considered to be excellent in cell adhesion performed under the same conditions, 5.8 × 10 ⁴ cell / cm ² , or adhesion number to polyglycolic acid 6.3 × 10 ⁴ cell / cm ² , which has a clearer number of adhesions and high biocompatibility. Furthermore, the chitin cast film has a cell adhesion number more than twice that of the chitosan film, and the cast film of the present invention is high. It can be said that it has biocompatibility.

  (1-2) After washing the cell-adhered chitosan film with PBS (−) and fixing with 2.5% glutaraldehyde for 2 hours, the ethanol rising series (20%, 50%, 75%, 90%, 95%, The film dehydrated with ethanol was replaced with t-butyl alcohol solution and freeze-dried.The dried sample was observed using SEMEDX Type N made by Hitachi. (Figure 2).

  The chitosan acetic acid solution cast film surface is smooth and adheres in a state where fibroblasts are aggregated, but the surface is not completely covered. On the other hand, the chitosan film has fine irregularities, and the artificial legs extend to cover them, and the cell is seen to extend. This is due to the difference in the surface structure of the base material used as the substrate, but it does not inhibit the cell's adhesion extensibility, and the chitosan cast film is superior from the viewpoint of adhesive strength with the substrate. I can guess.

2) Field emission scanning electron microscope (FE-SEM) observation The water jet-treated chitin and chitosan dispersion was replaced with t-butanol and freeze-dried. A mixture of platinum and palladium was deposited on the dried sample with a film thickness of about 3 nanometers. An electron microscope was observed under the condition of an acceleration voltage of 2.0 kV using a JEOL JSM-6700. Fig. 3 shows FE-SEM photographs of chitin and chitosan nanofiberized by water jet treatment.

Example 2
Apply an appropriate amount of an aqueous dispersion containing 1% by weight of chitin nanofiber or chitosan nanofiber treated with water jet 5 times thinly on the back of 13 panelists, and evaluate the moisturizing effect 10 minutes after the coating solution is completely dried did. The results are shown in Table 1 below.

  As mentioned above, chitin nanofibers or dispersions containing chitosan nanofibers have a strong and lasting moisturizing effect, and especially chitosan has a tendency to dry after drying, so the skin care, moisturizing and other cosmetic fields, or dry skin, etc. It became clear that it is excellent as a medicine for the treatment.

Claims (9)

Biomaterials containing chitin nanofibers and / or chitosan nanofibers. The biocompatible material according to claim 1, which is in the form of a paste, gel, slurry, film, sheet, porous body, dispersion, suspension or emulsion. The biomaterial according to claim 1, comprising chitin nanofibers and chitosan nanofibers. The biomaterial according to any one of claims 1 to 3, which is used as a cell culture substrate as a scaffold for medical materials such as artificial skin and wound dressing or various organ regeneration. The biomaterial according to any one of claims 1 to 4, wherein the nanofiber has an average fiber width (minor axis) of 4 to 100 nm, preferably 4 to 40 nm. A film, sheet or porous body obtained by removing moisture from chitin nanofibers and / or chitosan nanofiber dispersions by drying treatment. 7. The film, sheet or porous body according to claim 6, wherein cell adhesion is 2 times or more superior to acid-treated chitin / chitosan. A chitin / chitosan nanofiber dispersion, wherein chitin nanofibers and / or chitosan nanofibers are dispersed in water. Cosmetics containing chitin nanofiber and / or chitosan nanofiber.

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