JP2013194084A - Organic/inorganic composite hydrogel - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an organic/inorganic composite hydrogel which ensures easy cell adhesion and growth on the surface thereof, and to provide a method for producing the organic/inorganic composite hydrogel.SOLUTION: An organic/inorganic composite hydrogel includes water (C) in a three-dimensional network structure formed by a polymer of a water-soluble radically polymerizable organic monomer (A) and a water-swellable clay mineral (B), wherein the organic/inorganic composite hydrogel is characterized by having a layer consisting mainly of the water-swellable clay mineral (B) on a part of the surface of the hydrogel or on the entire surface thereof. The greatest feature of the organic/inorganic composite hydrogel and a method for producing the organic/inorganic composite hydrogel is the fact that the water-swellable clay mineral (B) serving also as a constituent of the hydrogel is adsorbed to the outermost surface of the hydrogel, and by adsorption of cell adhesion factors and protein to the adsorbed water-swellable clay mineral layer, the organic/inorganic composite hydrogel bears the adhesion and growth of a cell.

Description

  The present invention relates to an organic-inorganic composite hydrogel whose surface is coated with a water-swellable clay mineral, and specifically relates to an organic-inorganic composite hydrogel capable of easily adhering and proliferating cells on the surface, and a method for producing the same.

  Polymer hydrogels are organic polymer three-dimensional cross-linked products that contain water and swell, and as soft materials with swelling and rubber-like elasticity, such as medical / medicine, food, civil engineering, bioengineering, sports-related, etc. Widely used in the field (for example, see Non-Patent Document 1). To date, the present inventors have found that a polymer hydrogel having a three-dimensional network formed by combining a water-soluble organic polymer and a layered clay mineral has characteristics such as excellent water absorption and extremely high extensibility. (For example, refer to Patent Documents 1 and 2).

  Furthermore, the present inventors, among the above water-soluble organic polymers, poly N-isopropylacrylamide having a lower critical solution temperature (LCST) or lower, poly-2-methoxyethyl acrylate which is a hydrophobic polymer, and layered clay minerals. It has been reported that a polymer hydrogel having a three-dimensional network formed by combining and has excellent cell culture characteristics (see, for example, Patent Documents 3 and 4). However, the technology to adhere and proliferate cells on the surface of a polymer hydrogel with a three-dimensional network formed by combining a hydrophilic polymer such as dimethylacrylamide, acryloylmorpholine, and polyethylene glycol with a layered clay mineral has been disclosed. Was not.

  On the other hand, for the purpose of promoting cell adhesion to the culture scaffold, a technique (1) for coating the surface of the scaffold with a cell adhesion factor such as collagen or fibronectin is widely performed on the surface of the plastic container.

  However, when the method (1) is applied to a polymer hydrogel having a three-dimensional network formed by combining a water-soluble organic polymer and a layered clay mineral, collagen and fibronectin can be absorbed into the gel. However, since it cannot be immobilized on the surface of the hydrogel, it was difficult to cause the cells to adhere and proliferate.

  In addition, a method (2) has been developed in which collagen is chemically immobilized using a crosslinker such as carbodiimide or succinimide on the surface of an acrylamide gel, and is applied to cell culture on the gel surface. (For example, refer nonpatent literature 2). However, the method (2) is not effective when the water-soluble polymer does not have OH groups, and is a three-dimensional structure formed by combining a water-soluble organic polymer having no OH groups and a layered clay mineral. It cannot be applied to a polymer hydrogel having a network.

  On the other hand, the present inventors have found that a culture bed containing a water-swellable clay mineral exhibits good culturing properties for various cells (for example, see Patent Document 5). However, this patent does not disclose an organic-inorganic composite hydrogel or a method for producing an organic-inorganic composite hydrogel in which protein is adsorbed on the surface of the hydrogel or cells adhere and proliferate.

  As described above, among the water-soluble polymers, a polymer hydrogel having a three-dimensional network formed by combining a hydrophilic polymer such as dimethylacrylamide, acryloylmorpholine, and polyethylene glycol with a layered clay mineral is particularly transparent. Although it is excellent in physical properties such as property and strength, it has not been realized that cells adhere to and proliferate on the surface.

JP 2002-53629 A US Pat. No. 6,767,710 JP 2005-110604 A JP 2008-237088 A Japanese Patent Application No. 2011-196012

Adv. Mater., 14, 1120-1124 (2002) Proc. Natl. Acad. Sci. USA, vol. 94, pp13661-13665, (1997)

  The problem to be solved by the present invention is to solve the above-mentioned problems of the prior art and to provide an organic-inorganic composite hydrogel that allows cells to adhere and grow easily on the surface, and a method for producing the same.

  As a result of diligent research to solve the above problems, the inventors of the present invention have found that the gel surface is a mixture of clay mineral or clay mineral and protein, particularly in a gel composed of a polymer of a water-soluble organic monomer and a layered exfoliated clay mineral and medium. By covering with the body, a gel material is obtained in which the flexibility is controlled over a wide range and the culturing property to various cells is good while maintaining the high stretchability, transparency and compressibility of the gel. As a result, the present invention has been completed.

  That is, the present invention relates to an organic-inorganic composite hydrogel comprising water (C) in a three-dimensional network structure formed by a polymer of a water-soluble radically polymerizable organic monomer (A) and a water-swellable clay mineral (B). An organic-inorganic composite hydrogel comprising a layer mainly composed of a water-swellable clay mineral (B) on a part or the whole of the surface of the hydrogel.

The present invention also provides a method for producing the above organic-inorganic composite hydrogel,
(1) An organic-inorganic composite hydrogel containing water (C) in a three-dimensional network structure formed by a water-soluble radical polymerizable organic monomer (A) polymer and a water-swellable clay mineral (B) is produced. Process,
(2) Dispersing the water-swellable clay mineral (B) in water to produce a water-swellable clay mineral (B) dispersion;
(3) The water-swellable clay mineral (B) is applied to a part or the entire surface of the hydrogel by immersing the hydrogel in the dispersion or by applying the dispersion to the surface of the hydrogel. Forming a layer having a main component;
The manufacturing method of the organic inorganic composite hydrogel characterized by including this.

  The greatest feature of the organic-inorganic composite hydrogel and the method for producing the organic-inorganic composite hydrogel of the present invention is that the water-swellable clay mineral (B), which is also a constituent of the hydrogel, is adsorbed on the outermost surface of the hydrogel and adsorbed. It is responsible for cell adhesion and proliferation by adsorbing cell adhesion factors and proteins to the mineral layer.

  The present invention has high stretchability and high compressibility, flexibility is controlled over a wide range, and a large amount of cell adhesion factors and protein components in the culture solution can be adsorbed on the surface, followed by good cell adhesion / Provided is an organic-inorganic composite gel capable of extending / proliferating (highly cultivated) and adhering biological tissues. The obtained organic-inorganic composite gel can have excellent mechanical properties, humidity control, flame retardancy, and the like in addition to high cell culture properties. Moreover, the obtained organic-inorganic composite gel can be controlled so that part of the surface has cell adhesiveness and the other part has cell non-adhesiveness.

  The organic-inorganic composite hydrogel and the method for producing the organic-inorganic composite hydrogel can be organic-inorganic composite hydrogels of various shapes including a columnar shape, a rod shape, a film shape, and a thread shape. The organic-inorganic composite hydrogel of the present invention can be used as a physicochemical material as a scaffold for cell culture, and further in the medical / pharmaceutical field, particularly as a regenerative medical material such as cornea, lens, cartilage, tendon, bone. Further, it is also effectively used as a material for artificial organs such as artificial valves, artificial blood vessels, and artificial cartilages excellent in biocompatibility and flexibility, and a therapeutic material such as catheters. Furthermore, it is also used in various fields such as agriculture, industry, electronic materials, civil engineering, and packaging materials as various industrial materials.

  The present invention forms a hydrogel composed of a polymer of a water-soluble organic monomer, a swellable clay mineral, and a medium, and the water-swellable clay mineral adheres to the surface by coating the surface with the water-swellable clay mineral. It is found that an organic-inorganic composite gel having a surface with protein, cell, and tissue adhesiveness can be obtained. On the other hand, the gel in which the water-swellable clay mineral was not applied remained a problem in cell culture and tissue adhesion.

  The organic-inorganic composite hydrogel referred to in the present invention is a composite of a water-soluble organic monomer polymer (hereinafter simply referred to as polymer (A)) and a water-swellable clay mineral (B) that can be uniformly dispersed in water. Water (C) is contained in the three-dimensional network formed in this way, and is a hydrogel having a thin layer of water-swellable clay mineral (B) on the surface.

  The water-soluble organic monomer polymer (A) used in the present invention is capable of forming a three-dimensional network by interaction with the water-swellable clay mineral (B), preferably an amide group, amino group, ester. Containing one or more of a group, a hydroxyl group and a cationic group in the side chain or main chain and exhibiting hydrophilicity or amphiphilicity, particularly preferably swelled in water or a mixed solvent of water and an organic solvent. It has the property of dissolving. Preferable examples of such a polymer (A) include a polymer of a water-soluble acrylamide derivative and a copolymer containing at least a part thereof. Examples of the water-soluble acrylamide derivative include water-soluble N-alkyl acrylamide or N, N-dialkyl acrylamide having an alkyl group having 1 or more carbon atoms.

  The water-soluble organic monomer used in the polymer (A) is one selected from acrylamide, methacrylamide, alkyl methacrylamide having an alkyl group having 1 or more carbon atoms, and alkyl acrylate in addition to the acrylamide derivative. Or a plurality is used. Here, specific examples of N-alkylacrylamide, N, N-dialkylacrylamide, alkylmethacrylamide, and alkylacrylate include N-methylacrylamide, N-ethylacrylamide, N-cyclopropylacrylamide, N-isopropylacrylamide, and N-methyl. Methacrylamide, N-cyclopropylmethacrylamide, N-isopropylmethacrylamide, N, N-dimethylacrylamide, N, N-dimethylaminopropylacrylamide, N-methyl-N-ethylacrylamide, N-methyl-N-isopropylacrylamide, N-methyl-Nn-propylacrylamide, N, N-diethylacrylamide, N-acryloylpyrrolidine, N-acryloylpiperidine, N-acryloylmethylphospho Piperadin, N- acryloyl methylpiperazinyl Laden, such as 2-methoxyethyl acrylate are exemplified. In addition, it is possible to use these monomers together with other organic monomers as long as the organic-inorganic composite gel referred to in the present invention is formed.

  Further, a high refractive index polymer hydrogel having a three-dimensional network formed by combining a polymer obtained by copolymerizing chemical formulas (2) and (3) and a layered clay mineral, the main component being the following chemical formula (1) Is mentioned.

  The water-swellable clay mineral (B) in the present invention is preferably one that swells in water or an aqueous solution, and more preferably can be exfoliated and dispersed finely and uniformly in a solution containing a water-soluble organic monomer. Further, it is particularly preferable that such a fine and uniformly dispersed state is maintained even in water. In the organic-inorganic composite gel of the present invention, the swellable clay mineral (B) is preferably dispersed at a nanometer level (thickness) of 10 layers or less, more preferably 3 layers or less, particularly preferably 1 layer or 2 layers. It is what. The dispersion of the swellable clay mineral was confirmed by observing an ultrathin section of the dried foam gel with a transmission electron microscope, as well as by small-angle X-ray diffraction measurement using a similar sample. (2θ) is preferably 3 ° to 8 °, more preferably 2 ° to 8 °, and particularly preferably 1 ° to 8 °, which is confirmed by the fact that no clear diffraction peak based on clay mineral lamination is observed. . The water-swellable clay mineral (B) in the present invention is preferably one that can form a three-dimensional network with the water-soluble organic monomer polymer (A), more preferably an organic crosslinking agent such as methylenebisacrylamide. It is possible to form a three-dimensional network composed of (A) and (B) without using. More preferably, the formed three-dimensional network structure is maintained even in water. As the water-swellable clay mineral (B), for example, a swellable inorganic clay mineral that swells in water such as water-swellable smectite or water-swellable mica and can be finely dispersed in a layered state is used. Specific examples thereof include water-swellable hectorite containing sodium as an interlayer ion, water-swellable montmorillonite, water-swellable saponite, and water-swellable synthetic mica. In addition, if the layer can be peeled in a solvent together with the water-soluble organic monomer, a clay mineral partially organicized with a surfactant or the like can be used.

  On the other hand, in addition to the formation of a three-dimensional network composed of a polymer (A) of a water-soluble organic monomer and a water-swellable clay mineral (B), the simultaneous inclusion of a small amount of chemical cross-linking can reduce the hysteresis in repeated compression and stretching tests. Effectively used to decrease, increase initial elastic modulus, etc. The degree of chemical crosslinking used is such that the concentration of the crosslinking agent is 0.3 mol% or less of the monomer, more preferably 0.2 mol% or less, and particularly preferably 0.01 to 0.1 mol%.

  In the organic-inorganic composite gel in the present invention, the ratio of the (inorganic) clay mineral constituting the gel can be set in a wide range, and in particular, an organic-inorganic composite gel having a low to high wide clay mineral ratio is obtained. is there. The amount ratio of the polymer (A) of the water-soluble organic monomer and the swellable clay mineral (B) contained in the organic-inorganic composite gel of the present invention is preferably within the range in which both form a three-dimensional network in water, (B) The mass ratio of / (A) is 0.01 to 3, preferably 0.1 to 3, particularly preferably 0.3 to 2.5. When the mass ratio of (B) / (A) is less than 0.01, it is difficult to form an effective three-dimensional network of (A) and (B), while when it exceeds 3, the uniform (B) In many cases, delamination dispersion becomes difficult.

  In the present invention, for the formation of a three-dimensional network of the polymer (A) of the water-soluble organic monomer and the water-swellable clay mineral (B), the interaction between the surface of (B) and the terminal of (A), or (B) The interaction between the surface of and the functional group of the main chain or side chain of (A) is used alone or in combination. Various types of interaction can be selected depending on the types and combinations of (A) and (B). For example, ionic interaction, coordination bond, hydrogen bond, covalent bond, hydrophobic interaction, etc. alone Or a plurality are used in combination.

  As the medium (C) in the present invention, water or other functional molecules (for example, organic compounds or organic or inorganic salts having an effect of reinforcing a three-dimensional network or having medicinal effects) are included. Water is used.

  As the water-swellable clay mineral (B) for surface coating in the present invention, those that effectively adhere to the surface of the organic-inorganic composite gel and give surface cell adhesion and tissue adhesion are used. Particularly preferably, an inorganic clay mineral that can be uniformly dispersed in water at a molecular level (single layer) or at a level close thereto is used. Specific examples include water-swellable hectorite containing sodium as an interlayer ion, water-swellable montmorillonite, water-swellable saponite, and water-swellable synthetic mica. You may mix and use these clay minerals.

  The form of coating the surface of the water-swellable clay mineral (B) is not particularly limited, but is preferably 10 layers or less, more preferably 3 layers or less, and particularly preferably 1 layer or 2 layers of nanometer level (of (Thickness) is preferably dispersed in a medium such as water. The amount of the water-swellable clay mineral (B) in the medium is 0.025 or less, preferably 0.015 or less, more preferably 0.01 or less, by mass ratio with the medium. When the mass ratio of the water-swellable clay mineral (B) to the aqueous medium is as described above, a good dispersion can be obtained, and the coating onto the surface is easy, and a smooth and uniform thin coating film can be obtained.

  In the water-swellable clay mineral (B) dispersion that covers the surface, water-soluble organic compounds, such as preservatives, antibacterial agents, coloring agents, fragrances, enzymes, proteins, to the extent that no aggregation or precipitation occurs due to interaction , Synthetic peptides, saccharides, amino acids, vitamins, cells, DNAs, salts, water-soluble organic solvents, surfactants, polymer compounds, leveling agents and the like. Preferably, alkali-treated collagen and alkali-treated gelatin can be included. The amount of these water-soluble organic compounds is desirably such that the mass ratio to the water-swellable clay mineral is 5 or less, preferably 2 or less, more preferably 1 or less.

  As the coating thickness on the gel surface, a thickness that improves cell adhesion on the gel surface, more preferably exhibits cell proliferation, and is in a range that does not desorb from the gel surface is used. Specifically, a range of preferably 3 μm or less, more preferably 2 μm or less, and even more preferably 1 μm or less is selected and used depending on the type of the water-swellable clay mineral (B). If it is 3 μm or more, the organic-inorganic composite hydrogel may crack on the surface, and the layer containing the water-swellable clay mineral may fall off. With the above-mentioned thickness, the water-swellable clay mineral on the surface of the hydrogel does not swell and crack or fall off, and is effective for cell adhesion and proliferation. The water-swellable clay mineral (B) used in the present invention is not limited as long as it can adhere to the organic-inorganic composite gel and give its surface cell adhesion. ˜1000 nm, more preferably 0.5 to 300 nm, particularly preferably 1 to 50 μm.

  In the present invention, the surface coating with the water-swellable clay mineral (B) may be performed on the entire surface of the organic-inorganic composite gel, and is preferably performed partially according to the purpose. For example, by coating only one surface of a film-like organic-inorganic composite gel with a water-swellable clay mineral (B), it is possible to obtain a gel whose lower surface has cell non-adhesiveness and whose upper surface has cell-adhesive properties. . Moreover, the organic-inorganic composite gel whose surface cell adhesion is controlled by dividing the surface of the organic-inorganic composite gel film into grid-like or strip-like sections and coating a part thereof with the water-swellable clay mineral (B) Is obtained. This makes it possible to co-culture suspension cells, such as macrophages and lymphocytes, and adherent cells, such as vascular endothelial cells and mesenchymal stem cells, co-culture of adherent cells, and peel cells in sheet form. become.

  Since the surface coated with the water-swellable clay mineral (B) is easy to adsorb proteins, it is possible to adsorb cell adhesion factors such as type I collagen, type IV collagen, fibronectin, vitronectin and laminin. In addition to these living cell-derived cell adhesion factors, poly-L-lysine, polyamine, polyethyleneimine, or a synthetic polymer containing an RGD (arginine-glycine-aspartic acid) sequence may be used. Moreover, what mixed the biological origin cell adhesion factor and the synthetic polymer may be used. The concentration (mass%) with respect to these aqueous media is not particularly limited as long as it is effective for the cells to adhere and proliferate, but it is 5% or less, preferably 1%, more preferably 0.1% or less. Cells adhere and grow well.

  The cells cultured in the present invention are not particularly limited as long as they are adherent cells, but preferably include cells derived from humans or animals other than humans. Examples of adhesive cells include fibroblasts, skeletal muscle cells, myoblasts, myotube cells, corneal endothelial cells, corneal epithelial cells, retinal pigment epithelial cells, lens epithelial cells, vascular endothelial cells, lymphatic endothelial cells Smooth muscle cells, cardiomyocytes, epidermal keratinocytes, respiratory epithelial cells, mammary epithelial cells, mucosal epithelial cells, mesenchymal stem cells, stromal cells, ES cells, iPS cells, osteoblasts, bone cells, chondrocytes, fat Examples include cells, nerve cells, kidney cells, bladder cells, prostate cells, hair root cells, dental pulp stem cells, pancreatic beta cells, and liver parenchymal cells. In addition, in the present invention, a cell means an individual cell, and includes a cell that is collected from a living body and constitutes a tissue.

  These cells are preferably autologous cells in view of their use in tissue engineering and regenerative medicine for humans, etc., but are cells derived from different animals as long as they have acceptable immunocompatibility. Alternatively, it may be an allogeneic cell in the same type of cell.

   The method of seeding the cells on the organic-inorganic composite hydrogel is not particularly limited, but preferably includes a method of directly seeding the primary adherent cells removed from the tissue. More preferred is a method of seeding adherent cells that have become a single cell population by trypsin treatment or the like. More preferably, a method of seeding adherent cells cultured in a petri dish or the like while maintaining intercellular adhesion can be mentioned.

The following method is used for the method for producing the organic-inorganic composite gel of the present invention.
(1) An organic-inorganic composite hydrogel containing water (C) in a three-dimensional network structure formed by a water-soluble radical polymerizable organic monomer (A) polymer and a water-swellable clay mineral (B) is produced. Process,
(2) Dispersing the water-swellable clay mineral (B) in water or water containing collagen or gelatin to produce a water-swellable clay mineral (B) dispersion;
(3) The water-swellable clay mineral (B) is applied to a part or the entire surface of the hydrogel by immersing the hydrogel in the dispersion or by applying the dispersion to the surface of the hydrogel. Forming a layer having a main component;
If necessary, (4) the water-swellable clay mineral (B) by immersing the hydrogel in an aqueous solution of collagen or fibronectin, or by applying an aqueous solution of collagen or fibronectin to a part of or the entire surface. Forming a layer containing collagen or fibronectin on the layer mainly composed of
It is.

  After preparing a solution containing water, a water-swellable clay mineral and a water-soluble organic monomer, the water-soluble organic monomer is polymerized in the presence of them to form a three-dimensional network composed of the water-soluble organic monomer polymer and the layered exfoliated clay mineral. Let it form. A polymerization initiator necessary for containing a water-soluble organic monomer or a catalyst required depending on polymerization conditions is added to the solution in advance.

  The surface of the structure formed with the three-dimensional network is covered with a water-swellable clay mineral or a mixture of water-swellable clay mineral and gelatin or collagen. The surface coating of the structure with the water-swellable clay mineral may be performed immediately after the structure is synthesized or after a medium such as water is swollen. If necessary, the structure is further coated with collagen or fibronectin.

  The method for coating the surface with a water-swellable clay mineral or a mixture of water-swellable clay mineral and gelatin, collagen or the like on the surface may be a known and conventional method. For example, a method in which the dispersion is cast into an organic-inorganic composite hydrogel, a method in which the structure is immersed in the dispersion, a coater method using a bar coater or a spin coater, or a spray method such as spraying, a dispersion on a patterned rubber plate And a method of transferring to a support after applying the coating, a pattern coating method in which a portion not coated on the structure is shielded in advance and the shielding portion is removed after coating, and a dispersion coating method using an ink jet printer method. Preferably, a method of immersing the structure in the dispersion liquid is used.

  Specific examples of the polymerization initiator include water-soluble peroxides such as potassium peroxodisulfate and ammonium peroxodisulfate, water-soluble azo compounds such as VA-044 and V-50 manufactured by Wako Pure Chemical Industries, Ltd. V-501 or the like is preferably used. In addition, a water-soluble radical initiator having a polyethylene oxide chain is also used. As the catalyst, tertiary amine compounds such as N, N, N ′, N′-tetramethylethylenediamine and β-dimethylaminopropionitrile are preferably used. The polymerization temperature is set in the range of 0 ° C. to 100 ° C. according to the type of water-soluble organic polymer, polymerization catalyst and initiator used. The polymerization time also varies depending on the polymerization conditions such as the catalyst, initiator, polymerization temperature, polymerization solution amount (thickness) and cannot be generally defined, but it is generally carried out in the range of several tens of seconds to several tens of hours.

  In addition, even if the same preparation is performed on a conventional organic cross-linked gel that is cross-linked by an organic cross-linking agent without containing a water-swellable clay mineral, it is difficult to obtain a gel excellent in stretchability and strength. Have. For example, in the case of an organic cross-linked gel that does not contain a water-swellable clay mineral and is prepared by adding 1 mol% of a water-soluble organic monomer instead of methylenebisacrylamide, the mechanical properties are weak and brittle regardless of density. Indicates.

  The organic-inorganic composite gel in the present invention exhibits surface cell adhesion by surface coating with a water-swellable clay mineral (B). In addition, characteristics other than surface adhesiveness, for example, mechanical properties such as stretchability and compressibility, are the types of the polymer (A) and the water-swellable clay mineral (B), the ratio of both, and the type of medium. The ratio varies depending on the ratio (for example, the ratio of the low-volatile medium and water) and the amount thereof, and can be set according to the purpose.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, the scope of the present invention is not limited only to these Examples.

Example 1
In this example, the surface of the organic / inorganic composite hydrogel is modified.
[Preparation of organic-inorganic composite hydrogel (before coating)]
As a clay mineral, a water-swellable synthetic hectorite having a composition of [Mg _5.34 Li _0.66 Si ₈ O ₂₀ (OH) ₄ ] Na ⁺ _0.66 (trademark Laponite XLG (manufactured by Rockwood Additives Ltd.)) ) Was used after vacuum drying at 100 ° C. for 2 hours. As the organic monomer, N, N-dimethylacrylamide (DMAA: Wako Pure Chemical Industries, Ltd.) was used, and the polymerization inhibitor was removed using a silica gel column (manufactured by Merck) in a volume of 80 ml with respect to 100 ml of the organic monomer. Used from.

  As the polymerization initiator, potassium peroxodisulfate (PPS: manufactured by Kanto Chemical Co., Inc.) was diluted with pure water at a ratio of PPS / water = 0.384 / 20 (g / g) and used as an aqueous solution. As the water, pure water obtained by distilling ion-exchanged water was used. All water was used after bubbling high-purity nitrogen in advance for 3 hours or more to remove oxygen.

  In a constant temperature room at 20 ° C, put 100 g of pure water and a Teflon (registered trademark) stirrer into a flat bottom glass container with nitrogen inside, and be careful not to let air bubbles into 1.6 g of Laponite XLG while stirring. While adding a small amount, a colorless and transparent solution was prepared. To this was added 9.9 g of DMAA and stirred until a colorless transparent solution was obtained.

  To this was added 0.1 g of an aqueous PPS solution, and the mixture was stirred for 5 minutes to obtain a colorless transparent solution. This solution was left in a nitrogen atmosphere and transferred to a glass container (9 cm × 15 cm) from which oxygen in the container had been removed so as not to come into contact with oxygen, and then sealed and sealed at a constant temperature of 20 ° C. The polymerization was carried out by standing in a water bath for 20 hours.

Twenty hours after the start of polymerization, a substantially colorless and transparent sheet-like hydrogel was obtained in a glass container. The sheet-like hydrogel was immersed in physiological saline at 50 ° C. for 15 minutes, then punched out with a punch having a diameter of 10 mm, and swollen overnight with pure water. The hydrogel was used for surface coating.
[Preparation of dispersion containing water-swellable clay mineral (B)]
As a water-swellable clay mineral (B), 0.08 g of Laponite XLG (manufactured by Rockwood Additives Ltd.) and 20 g of water were uniformly mixed to prepare a dispersion (L).
[Application of aqueous solution containing water-swellable clay mineral (B)]
The hydrogel was placed in a 6-well plate for cell culture (Falcon 353046) containing 2 mL of the dispersion (L), and allowed to stand at 37 ° C. for 3 hours. After washing with pure water three times, an organic-inorganic composite hydrogel (after coating) coated with an aqueous solution containing a water-swellable clay mineral (B) was used for the culture test.
[Culture test]
As cells to be cultured, normal human dermal fibroblasts (DS Pharma Biomedical Co., Ltd.) were used. Cultivation was performed in a 37 ° C. incubator containing 5% carbon dioxide using Dulbecco's modified minimum essential eagle medium (Wako Pure Chemical Industries) containing 10% fetal bovine serum (Bio West). . The number of cells to be seeded was 20,000 cells per gel. The seeding amount of the medium was 4 ml. 1 day, 4 days, and 7 days after seeding, the surface of the organic-inorganic composite hydrogel was observed with an optical microscope, and it was clear that the cells adhered and grew sufficiently on the organic-inorganic composite hydrogel. I was able to confirm.
(Example 2)
Experiments were conducted in the same manner as in Example 1 except that 0.01 g of alkali-treated collagen (Nitta Gelatin Co., Ltd.) was added to the preparation of the dispersion containing the water-swellable clay mineral (B).
(Example 3)
A dispersion containing a water-swellable clay mineral (B) was applied to an organic-inorganic composite hydrogel (before coating) produced in the same manner as in Example 1, followed by 0.1 g of acid-treated collagen (Nitta Gelatin Co., Ltd.). 100 μL of a solution in which 20 g of water was dissolved in water (pH 3) was applied. A culture test was conducted in the same manner as in Example 1.
Example 4
A dispersion containing a water-swellable clay mineral (B) and alkali-treated collagen was applied to the organic-inorganic composite hydrogel (before coating) produced in the same manner as in Example 1, followed by acid-treated collagen (manufactured by Nitta Gelatin Co., Ltd.). 100 μL of a solution prepared by dissolving 0.1 g in 20 g of water (pH 3) was applied. A culture test was conducted in the same manner as in Example 1.
(Example 5)
A dispersion containing a water-swellable clay mineral (B) and alkali-treated collagen was applied to the organic-inorganic composite hydrogel (before coating) produced in the same manner as in Example 1, followed by fibronectin (Becton Dickinson Co., Ltd.). 100 μL of a solution dissolved in water and adjusted to 50 μg / mL was applied. A culture test was conducted in the same manner as in Example 1.
(Comparative Example 1)
An organic-inorganic composite hydrogel (before coating) was prepared in the same manner as in Example 1, and a culture test was conducted in the same manner as in Example 1 without applying an aqueous solution containing the water-swellable clay mineral (B).
(Comparative Example 2)
An organic-inorganic composite hydrogel (before coating) was prepared in the same manner as in Example 1, and a dispersion (containing a water-swellable clay mineral) in which 0.1 g of alkali-treated collagen (Nitta Gelatin Co., Ltd.) was uniformly mixed with 20 g of water A culture test was carried out in the same manner as in Example 1 using an organic-inorganic composite hydrogel coated with 100 μL of No).
(Comparative Example 3)
An organic-inorganic composite hydrogel (before coating) was prepared in the same manner as in Example 1, and a dispersion (containing a water-swellable clay mineral) in which 0.1 g of acid-treated collagen (Nitta Gelatin Co., Ltd.) was uniformly mixed with 20 g of water A culture test was carried out in the same manner as in Example 1 using an organic-inorganic composite hydrogel coated with 100 μL of No).

In order to count the number of cells after 1 day, 4 days, and 1 week after culturing in Examples 1 to 5 and Comparative Examples 1 to 3, first, 2 ml of phosphate buffered saline (PBS, manufactured by Wako Pure Chemical Industries, Ltd.) The culture surface was washed. Subsequently, 0.2 mL of cell lysate A (manufactured by Chemometech) was added, and then 0.2 mL of neutralizing liquid B (manufactured by Chemometech) was added. This solution was collected with a cell counting cassette (Nucleo Cassette, manufactured by Chemometech), and the number of cells was measured with a cell number measuring device (Nucleo Counter ^™ , manufactured by Chemometech).

  Table 1 shows the results of cell number measurement in the culture test.

  As shown in Table 1, cells did not adhere and proliferate in the organic-inorganic composite hydrogel (before coating) (Comparative Example 1). Furthermore, the cells did not adhere and proliferate only by applying the alkali treatment (Comparative Example 2) to the organic-inorganic composite hydrogel (before coating). In addition, when acid treatment (Comparative Example 3) collagen was applied to the organic-inorganic composite hydrogel (before coating), cell adhesion was confirmed the day after cell seeding (day 1), but adherent cells could be confirmed on day 4. There wasn't. However, a clay solution (Example 1), a mixed solution of alkali-treated collagen and clay (Example 2) was applied to the surface of the organic-inorganic composite hydrogel (before coating), and acid-treated collagen was further applied to Example 2 (Example 3). In some cases, it was possible to achieve the effect of cell adhesion and proliferation.

That is, it was clarified in Table 1 that cells can be proliferated well by applying a solution containing a water-swellable clay mineral on the surface of the organic-inorganic composite hydrogel (before coating).

  If the organic-inorganic composite hydrogel and the method for producing an organic-inorganic composite hydrogel of the present invention are performed, a large amount of cell adhesion factors and protein components can be adsorbed on the surface of the organic-inorganic composite hydrogel, and then good adhesion, extension, and proliferation of cells and living tissues (High cultureability) is possible.

  The organic-inorganic composite hydrogel of the present invention can be used as a regenerative medical material such as cornea, lens, cartilage, tendon and bone, particularly in the medical / pharmaceutical field. Further, it is also effectively used as a material for artificial organs such as artificial valves, artificial blood vessels, and artificial cartilages excellent in biocompatibility and flexibility, and a therapeutic material such as catheters. Furthermore, it is also used in various fields such as agriculture, industry, electronic materials, civil engineering, and packaging materials as various industrial materials.

Claims (8)

  An organic-inorganic composite hydrogel comprising water (C) in a three-dimensional network structure formed by a polymer of a water-soluble radically polymerizable organic monomer (A) and a water-swellable clay mineral (B), An organic-inorganic composite hydrogel comprising a layer mainly composed of a water-swellable clay mineral (B) on a part or the whole of the surface.   The organic-inorganic composite hydrogel according to claim 1 or 2, wherein the layer mainly composed of the water-swellable clay mineral (B) contains at least one selected from gelatin and collagen.   The organic-inorganic composite hydrogel according to claim 3, wherein the gelatin and collagen are treated with an alkali.   The organic-inorganic composite hydrogel according to any one of claims 1 to 4, further comprising a layer containing collagen or fibronectin on the layer mainly composed of the water-swellable clay mineral (B). A method for producing the organic-inorganic composite hydrogel according to claim 1,
(1) An organic-inorganic composite hydrogel containing water (C) in a three-dimensional network structure formed by a water-soluble radical polymerizable organic monomer (A) polymer and a water-swellable clay mineral (B) is produced. Process,
(2) Dispersing the water-swellable clay mineral (B) in water to produce a water-swellable clay mineral (B) dispersion;
(3) The water-swellable clay mineral (B) is applied to a part or the entire surface of the hydrogel by immersing the hydrogel in the dispersion or by applying the dispersion to the surface of the hydrogel. Forming a layer having a main component;
The manufacturing method of the organic inorganic composite hydrogel characterized by including.   In order to form a layer mainly composed of the water-swellable clay mineral (B), the water-swellable clay mineral (B) is dispersed in an aqueous solution containing at least one selected from gelatin and collagen. The manufacturing method of the organic inorganic composite hydrogel of Claim 6 which has the process of manufacturing the dispersion liquid of a water-swellable clay mineral (B).   The method for producing an organic-inorganic composite hydrogel according to claim 7, wherein the gelatin and collagen are alkali-treated. After performing a step of forming a layer mainly composed of the water-swellable clay mineral (B) on a part or the entire surface of the hydrogel,
By immersing the hydrogel in an aqueous solution of collagen or fibronectin, or by applying an aqueous solution of collagen or fibronectin to a part of or the entire surface,
Forming a layer containing collagen or fibronectin on the water-swellable clay mineral (B) as a main component;
The manufacturing method of the organic inorganic composite hydrogel in any one of Claims 6-8 performed.