WO2020067014A1 - Composition for bone regeneration, kit of composition for bone regeneration, member for bone regeneration and bone regeneration method - Google Patents

Composition for bone regeneration, kit of composition for bone regeneration, member for bone regeneration and bone regeneration method Download PDF

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WO2020067014A1
WO2020067014A1 PCT/JP2019/037277 JP2019037277W WO2020067014A1 WO 2020067014 A1 WO2020067014 A1 WO 2020067014A1 JP 2019037277 W JP2019037277 W JP 2019037277W WO 2020067014 A1 WO2020067014 A1 WO 2020067014A1
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bone regeneration
composition
bone
component
solution
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PCT/JP2019/037277
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French (fr)
Japanese (ja)
Inventor
林　英治
愛 舟▲崎▼
純 諫山
健太郎 内田
亘 齋藤
真太郎 庄司
晶士 高相
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Jsr株式会社
学校法人北里研究所
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Priority to JP2020549215A priority Critical patent/JP7313370B2/en
Publication of WO2020067014A1 publication Critical patent/WO2020067014A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/728—Hyaluronic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a bone regeneration composition, a bone regeneration composition kit, a bone regeneration member, and a bone regeneration method.
  • Regeneration of bone after fracture involves an inflammatory phase in which the fracture site is inflamed due to hematoma occupation of the fracture gap, callus formation due to the conversion of the hematoma to granulocytes, and new bone formation due to osteogenic cells present in the periosteum It is known that the process progresses during a bone repair period, in which the bone is deformed due to repeated bone resorption and formation of new bone, and a remodeling period in which the correction of bone deformation progresses.
  • Patent Documents 1 and 2 Conventionally, a method of using a bone regeneration member containing a growth factor such as a bone morphogenetic protein (BMP) for bone regeneration treatment for a fracture has been known (see Patent Documents 1 and 2).
  • a growth factor such as a bone morphogenetic protein (BMP)
  • the present invention provides a bone regeneration composition, a bone regeneration composition kit, and a bone regeneration member useful for intractable fractures including periosteal damage and the like, and bone regeneration using the composition and the like.
  • the aim is to provide a method.
  • the present inventors have studied to solve the above problems. As a result, the inventors have found that the following problems can be solved by the following bone regeneration composition and the like, and have completed the present invention.
  • the present invention relates to, for example, the following [1] to [10].
  • a composition for bone regeneration comprising at least one component (C) selected from c1) and a substance (c2) that produces peroxide, an osteogenic protein (D), and water (E).
  • the hydroxyaryl group is a group derived from tyramine.
  • [3] The bone regeneration composition according to the above [1] or [2], wherein the degree of substitution of the component (A) with the hydroxyaryl group is 0.1 to 50.
  • [4] The composition for bone regeneration according to any of [1] to [3], wherein the oxidative coupling enzyme (B) is horseradish peroxidase.
  • [5] The composition for bone regeneration according to any of [1] to [4], wherein the component (C) is the peroxide (c1).
  • [6] The composition for bone regeneration according to any of [1] to [5], which is used for intractable fractures.
  • a bone regeneration composition kit having a first solution, a second solution, and a third solution, wherein the first solution is a modified hyaluronic acid in which a hydroxyaryl group is introduced into hyaluronic acid. It contains at least one component (A) selected from an acid and a salt thereof and water (E), and contains the oxidative coupling enzyme (B) for the hydroxyaryl group, and the peroxide (c1) and the peroxide.
  • the second solution contains at least one component (C) selected from the substance (c2) to be formed, but not both, and the second solution contains the oxidative coupling enzyme (B) and the component (C).
  • a composition kit for bone regeneration comprising the component (C) that is not contained in the first solution, wherein the third solution contains an osteogenic protein (D).
  • a bone regeneration method for regenerating bone using a regeneration member is regenerating bone using a regeneration member.
  • a bone regeneration method can be provided.
  • FIG. 1 is a scheme of a bone regeneration process using the bone regeneration composition of the present invention.
  • FIG. 2 is a microCT image measured in Examples and the like.
  • composition for bone regeneration of the present invention comprises: At least one component (A) selected from a modified hyaluronic acid in which a hydroxyaryl group is introduced into hyaluronic acid and a salt thereof; Said hydroxyaryl group oxidative coupling enzyme (B); At least one component (C) selected from a peroxide (c1) and a substance (c2) that generates peroxide; Osteogenic protein (D), Water (E).
  • the component (A) is at least one selected from a modified hyaluronic acid in which a hydroxyaryl group has been introduced into hyaluronic acid, and a salt of the modified hyaluronic acid. Since the bone morphogenetic protein (D) is easily circulated by body fluids, it is generally difficult to maintain its effect at the administration site of the bone morphogenetic protein (D) for a long period of time. In the present embodiment, by using the component (A) together with the bone morphogenetic protein (D), the bone morphogenetic protein (D) can be stably present at the administration site of the composition of the present embodiment, and the bone morphogenetic protein (D) Is expected to be maintained for a long time. Therefore, the composition of the present embodiment has a high bone regeneration effect. Specifically, in the present embodiment, it is considered that by using the component (A) and the bone morphogenetic protein (D), new bone formation can be promoted and bone regeneration speed can be improved.
  • the composition of the present embodiment is useful for treating intractable fractures, particularly for treating periosteal injury-type fractures, and by using the composition of the present embodiment, it is possible to increase the union rate of a fractured bone. .
  • the reason for this is that the affinity between the gel formed from the component (A) and the bone morphogenetic protein (D), which is the active ingredient, is moderately excellent, so that the sustained release rate of the active ingredient from the gel decreases in the fracture. This is probably because the speed is suitable for bone regeneration.
  • the intractable fracture is a fracture of a tissue important in bone formation, such as a damage of a periosteum, or a fracture having impaired blood flow accompanying the damage, and may be accompanied by an infection. Intractable fractures include, for example, open fractures.
  • Hyaluronic acid is a polysaccharide having a disaccharide unit of N-acetylglucosamine and glucuronic acid as a repeating structural unit.
  • Examples of the salt of the modified hyaluronic acid include physiologically or pharmaceutically acceptable salts, and include alkali metal salts such as sodium salt and potassium salt, and ammonium salts.
  • Component (A) has a hydroxyaryl group.
  • the number of carbon atoms of the hydroxyaryl group is usually 6 to 50, preferably 6 to 30.
  • the number of hydroxyl groups bonded to the benzene nucleus is usually 1 or more, preferably 1 to 3, and more preferably 1 or 2.
  • the hydroxyaryl group is not limited to a monohydroxyaryl group.
  • the hydroxyaryl group examples include a hydroxyphenyl group, a hydroxynaphthyl group, and a hydroxyanthracenyl group.
  • the hydroxyaryl group can have a substituent such as an alkyl group.
  • a hydrogen atom is bonded to a carbon atom at least one ortho position to the phenolic hydroxyl group. Since it is considered that the bone morphogenetic protein (D) can be stably present at the administration site of the composition of the present embodiment and can maintain its effective concentration, the hydroxyaryl group is a group derived from tyramine. preferable.
  • Modified hyaluronic acid is preferably a compound in which a hydroxyaryl group is bonded to hyaluronic acid by an ester bond, a urethane bond or an ether bond, directly or via an organic group.
  • the ester bond, urethane bond and ether bond are preferably bonded to a carbon atom to which a hydroxyl group contained in hyaluronic acid has been bonded.
  • the organic group is, for example, an alkanediyl group having 1 to 20 carbon atoms, or a substitution in which at least one selected from an ether bond and a divalent amino group (such as —NH—) is added or inserted to the alkanediyl group.
  • Examples include an alkanediyl group and an organic group having a peptide bond (specifically, a group derived from an oligopeptide such as a dipeptide or an ester thereof).
  • Examples of the carbonylating reagent include nitrophenyl chloroformate, phenyl chloroformate, trichloromethyl chloroformate, and bis (trichloromethyl) carbonate.
  • the amount of the carbonylation reagent is usually 1 to 100 parts by mass based on 100 parts by mass of hyaluronic acid.
  • Examples of the amine compound having a hydroxyaryl group include tyramine.
  • the amount of the amine compound having a hydroxyaryl group is usually 1 to 100 parts by mass relative to 100 parts by mass of the carbonate derivative of hyaluronic acid.
  • the conversion reaction is preferably performed in the presence of a basic compound.
  • the basic compound include trimethylamine, triethylamine, tributylamine, pyridine, and piperidine.
  • the conversion reaction and the hydroxyaryl group introduction reaction are preferably performed in a solvent.
  • the solvent include amide solvents such as dimethylformamide and dimethylacetamide; sulfoxide solvents such as dimethylsulfoxide; halogenated hydrocarbon solvents such as dichloromethane, 1,2-dichloroethane and chloroform; and aromatic hydrocarbons such as benzene, toluene and xylene. Hydrogen solvents; ether solvents such as diethyl ether, tetrahydrofuran, dioxane and the like.
  • an alkali metal salt such as lithium chloride can be used.
  • hyaluronic acid and a functional group capable of reacting with an alcoholic hydroxyl group in hyaluronic acid eg, a carboxy group, an acid halide thereof, an epoxy group, an oxetanyl group
  • a compound having a hydroxyaryl group for example, hyaluronic acid and a functional group capable of reacting with an alcoholic hydroxyl group in hyaluronic acid (eg, a carboxy group, an acid halide thereof, an epoxy group, an oxetanyl group) ) And a compound having a hydroxyaryl group.
  • Examples of the compound having a carboxy group capable of reacting with an alcoholic hydroxyl group in hyaluronic acid or an acid halide thereof and a hydroxyaryl group include, for example, 4-hydroxyphenylacetic acid, 4-hydroxyphenylpropionic acid, 4-hydroxyphenylbutane Acids, 2- (2,5-dihydroxyphenyl) acetic acid, and acid halides thereof.
  • the degree of substitution (DS) of the component (A) with the hydroxyaryl group is usually 0.1 to 50, preferably 1 to 40, more preferably 2 to 30.
  • the degree of substitution can be determined by 1 H NMR.
  • the substitution degree of the component (A) indicates the average number of hydroxyaryl groups introduced per 100 units of the constituent monosaccharide of the component (A).
  • the component (A) having such a degree of substitution improves the gelation rate.
  • the number average molecular weight of the component (A) as measured by absolute molecular weight measurement using a multi-angle light scattering detector is usually 1,000 to 10,000,000, preferably 3,000 to 1,000,000, more preferably 5,000 to 500,000.
  • the molecular weight distribution of the component (A) is usually 1 to 20, preferably 1 to 10, and more preferably 1 to 5.
  • the component (A) one type or two or more types can be used.
  • the content ratio of the component (A) in the composition of the present embodiment is usually 0.05 to 49% by mass, preferably 0.5 to 30% by mass, and more preferably 3 to 15% by mass. Such an embodiment is preferable from the viewpoint of the mechanical strength and handleability of the gel.
  • the composition of the present embodiment contains an oxidative coupling enzyme (B) for a hydroxyaryl group (hereinafter also referred to as “enzyme (B)”).
  • the enzyme (B) is an enzyme capable of directly or indirectly catalyzing an oxidative coupling reaction of a hydroxyaryl group.
  • Examples of the enzyme (B) include phenol oxidase such as laccase and tyrosinase, catalase, and peroxidase.
  • the composition of the present embodiment can be gelled by using peroxidase in combination with a peroxide (c1) such as hydrogen peroxide.
  • a peroxide (c1) such as hydrogen peroxide.
  • the origin of copper enzymes such as laccase and tyrosinase includes, for example, sorghum, mushrooms (Acacia fulica, mushroom), and mold (Polyporus vericolor).
  • Catalase the origin of peroxidase, for example, bovine liver, horse blood cells, human blood cells, M.
  • tyrosinase is preferably derived from mushroom
  • peroxidase is preferably derived from horseradish, that is, horseradish peroxidase.
  • the component (C) is used, and at least one selected from peroxidase and catalase is used as the enzyme (B). More preferably, component (C) is used, and horseradish peroxidase is used as enzyme (B).
  • One or more enzymes (B) can be used.
  • the amount of the enzyme (B) is preferably at least 0.01 U / mL, more preferably at least 0.1 U / mL; preferably at most 1,000 U / mL, and more preferably at most 500 U / mL. preferable.
  • U represents a unit of enzyme activity, and is an amount of enzyme capable of changing 1 ⁇ mol of substrate per minute at a temperature of 30 ° C. under optimum conditions.
  • the composition of the present embodiment contains at least one component (C) selected from a peroxide (c1) and a substance (c2) that generates a peroxide.
  • the peroxide (c1) include hydrogen peroxide.
  • the amount of the peroxide (c1) is usually based on 1 mol of the hydroxyaryl group in the component (A). The amount is 0.01 to 1000 mol, preferably 0.1 to 500 mol, and more preferably 0.5 to 200 mol.
  • the content of the peroxide (c1) in the composition of the present embodiment is preferably from 10 to 1,000 mM, more preferably from 100 to 800 mM, from the viewpoint of reactivity.
  • the peroxide-generating substance (c2) is usually used together with the oxidase (c3).
  • the oxidase (c3) is an enzyme that catalyzes a reaction that consumes the substance (c2) to generate a peroxide such as hydrogen peroxide.
  • Examples of the oxidase (c3) include glucose oxidase, choline oxidase, amino acid oxidase, alcohol oxidase, pyruvate oxidase, and cholesterol oxidase. These enzymes can be appropriately selected depending on the application. For example, glucose oxidase is preferable because the gelation rate is greatly improved.
  • the substance (c2) is consumed by, for example, the oxidase (c3) to generate a peroxide such as hydrogen peroxide, and is selected according to the oxidase (c3).
  • hydrogen peroxide can be generated by the enzyme treatment of the substance (c2) by the oxidase (c3) in the presence of oxygen (eg, oxygen in the air).
  • oxygen eg, oxygen in the air
  • the oxidase (c3) is glucose oxidase, choline oxidase, amino acid oxidase, alcohol oxidase, pyruvate oxidase or cholesterol oxidase
  • the substance (c2) is glucose, choline, amino acid, alcohol, pyruvate or Cholesterol.
  • the content of the oxidase (c3) in the composition of the present embodiment is preferably 0.01 U / mL or more, and more preferably 1 U / mL or more from the viewpoint of reactivity. Is more preferable; 1,000 U / mL or less is preferable, and 500 U / mL or less is more preferable. Further, the content of the substance (c2) is appropriately selected such that the peroxide is appropriately generated.
  • One or more components (C) can be used.
  • Bone morphogenetic protein (D) is a protein that promotes bone formation and repair of fractures. Specifically, it is a factor that enhances the activity of osteoblasts, and a component of mesenchymal stem cells. It is a factor that induces differentiation into osteoblasts.
  • Preferred examples of the bone morphogenetic protein (D) include, for example, BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, and BMP-9.
  • the bone morphogenetic protein (D) may be derived from a human or a non-human animal.
  • the bone morphogenetic protein (D) has an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of naturally occurring BMP, and has the same amino acid sequence as naturally occurring BMP. And a protein having the activity of Thus, the bone morphogenetic protein (D) may be recombinant.
  • BMP-2 is preferable.
  • BMP-2 is a type of factor that controls and / or promotes cell growth.
  • BMP-2 is excellent in bone formation ability and promotes differentiation of mesenchymal stem cells present in periosteum and bone marrow.
  • bone morphogenetic protein (D) for example, bone morphogenetic proteins prepared by various methods can be used as long as the bone morphogenetic protein (D) is purified to a degree that can be used as a medicine.
  • One or more bone morphogenetic proteins (D) can be used.
  • the content of the bone morphogenetic protein (D) is usually 0.01 to 10 parts by mass, preferably 0.05 to 5 parts by mass with respect to 100 parts by mass of the component (A). is there.
  • the bone morphogenetic protein (D) is favorably retained in the hydrogel formed from the composition of the present embodiment.
  • the composition of the present embodiment contains water (E).
  • the content ratio of water (E) in the composition of the present embodiment is usually 50 to 99.9% by mass, preferably 65 to 99% by mass, and more preferably 70 to 97% by mass.
  • the composition can be gelled under conditions of low invasiveness to a living body, and the resulting gel tends to have excellent mechanical strength.
  • the composition of the present embodiment can further contain a polysaccharide other than the component (A).
  • the polysaccharide include, for example, unmodified hyaluronic acid, pullulan, xanthan gum, cellulose, guar gum, fructan, mannan, carrageenan, chitin, chitosan, pectin, starch, amylose, amylopectin, glycogen, dextran into which a hydroxyaryl group has not been introduced. Dextrin, gellan gum and alginic acid.
  • the composition of the present embodiment may further contain one or more selected from phosphoric acid, polyphosphoric acid, a pH adjuster, an ultraviolet absorber, a thickener, a coloring agent and a filler, It may contain a substance expressing function such as a drug or a cell. Further, the composition of the present embodiment can further contain sodium chloride. For example, the composition of the present embodiment can contain a buffer such as a phosphate buffered saline.
  • the composition kit for bone regeneration of the present invention includes a first solution, a second solution, and a third solution described below. And a solution.
  • the first solution contains at least one component (A) selected from a modified hyaluronic acid in which a hydroxyaryl group has been introduced into hyaluronic acid and a salt thereof, and water (E). It contains the ring enzyme (B) and at least one component (C) selected from the peroxide (c1) and the peroxide-generating substance (c2), but not both.
  • the second solution contains one of the oxidative coupling enzyme (B) and the component (C) that is not contained in the first solution.
  • the third solution contains the bone morphogenetic protein (D).
  • Each of the first to third solutions is preferably an aqueous solution. Details of the components (A) to (E) in the first to third solutions are as described above. Further, the amount ratio of the first to third solutions and the content of each component contained in each of the first to third solutions are determined in the composition obtained by mixing the first to third solutions. The amount ratio and content of each of the components (A) to (E) are appropriately set so as to fall within the ranges described in the [Composition for bone regeneration] column.
  • the composition of the present embodiment can be prepared.
  • the order of mixing the respective solutions is not particularly limited, but a mode in which the second solution is mixed with the mixture obtained by mixing the first solution and the third solution, or the second solution and the third solution are mixed. And a solution obtained by mixing the first solution with the first solution.
  • the bone regeneration member of the present invention is formed using the bone regeneration composition or the bone regeneration composition kit of the present embodiment, and specifically, the bone regeneration composition or the bone regeneration composition A gel formed from a composition composition kit.
  • the gel has a crosslinked structure by oxidative coupling of the hydroxyaryl group of component (A), that is, contains a crosslinked product derived from component (A).
  • component (A) contains a crosslinked product derived from component (A).
  • the gel is a hydrogel, specifically a hydrogel containing usually 50 to 99.9% by mass, preferably 70 to 97% by mass of water.
  • the hydrogel generally contains 50 to 0.1% by mass, preferably 30 to 3% by mass of a crosslinked product derived from the component (A).
  • the hydrogel has excellent mechanical strength and flexibility.
  • a xerogel can be obtained by removing water by appropriately drying the hydrogel.
  • the gel is formed by irradiating light with a photosensitizer such as eosin Y, methylene blue, or rose bengal together with the component (A) as described in Macromolecules (2015) 2624-2630, for example. You can also get it.
  • An example of a method for manufacturing a bone regeneration member is as follows.
  • the components (A) to (E) in the composition of the present embodiment or the first to third solutions in the kit of the present embodiment are mixed, and the aqueous solution temperature (crosslinking temperature) is usually 4 to 50 ° C.
  • Gelation can be carried out under the conditions of preferably 10 to 45 ° C., more preferably 20 to 40 ° C., and a reaction time of usually 1 minute to 48 hours, preferably 5 minutes to 30 hours.
  • Each of the components (A) to (D) can be used as an aqueous solution of each component.
  • the formation of the hydrogel can be performed under any of pressurized, normal pressure (atmospheric pressure), and reduced pressure, but is preferably performed under normal pressure.
  • a mold having a cavity having a desired shape is prepared, and the components (A) to (E) of the composition of the present embodiment or the first to third solutions in the kit of the present embodiment are added to the mold.
  • a hydrogel having the above shape can be formed.
  • a film-like hydrogel can be obtained by casting a composition of the present embodiment or a mixture of the first to third solutions in the kit of the present embodiment on a predetermined substrate.
  • the film-shaped hydrogel can be punched into, for example, a columnar shape, a polygonal column shape, or the like.
  • the shape of the bone regeneration member is not particularly limited, and examples thereof include a film shape, a cylindrical shape, a polygonal column shape, a nonwoven fabric shape, a fiber shape, a tube shape, a particle shape, and a mesh shape.
  • the bone morphogenetic protein (D) is carried. Therefore, in the present embodiment, the bone regeneration component is applied to a site to which the bone regeneration member is applied (eg, a bone defect site or a fracture site). It is thought that protein (D) can be maintained for a long period of time.
  • the hydrogel functions as an extracellular matrix in the affected area, and the extracellular matrix is considered to be a scaffold for bone-regenerating cells. Therefore, the bone regeneration member of the present embodiment has a high bone regeneration effect.
  • the composition for bone regeneration, the composition kit for bone regeneration, and the member for bone regeneration of the present embodiment are used for bone regeneration.
  • a bone regeneration composition is administered to an affected area such as a bone defect site or a fracture site of a subject requiring bone regeneration (for example, a human or a mammal other than a human), or a bone regeneration composition kit.
  • a bone regeneration member is formed in an affected part, or by applying the bone regeneration member to the affected part, bone regeneration is induced, and bone regeneration is induced. Can improve the fusion rate.
  • FIG. 1 shows an example of a scheme for estimating bone regeneration when the composition for bone regeneration of the present embodiment is used.
  • the composition for bone regeneration administered to the affected area 30 forms a hydrogel 40, and the bone morphogenetic protein 50 is favorably retained in the hydrogel 40.
  • the bone morphogenetic protein 50 promotes differentiation or proliferation of a mesenchymal stem cell, which is a precursor cell of the bone cell, and the osteoblast 20. As a result, a large new bone (callus) 70 is formed.
  • FIG. 1 bone cells are omitted.
  • the bone to be regenerated in the present embodiment is formed by depositing hydroxyapatite on a collagen fiber formed in a network, and most of the organic matter of the bone is collagen.
  • the usage, dosage, and shape of the composition for bone regeneration, the composition kit for bone regeneration, or the member for bone regeneration of the present embodiment can be appropriately determined according to the purpose of use.
  • the composition for bone regeneration or the mixture of the first to third solutions of the composition kit for bone regeneration is directly applied to an affected part such as a bone defect site or a fracture site in a living body by, for example, injection, application, or the like. Can be administered.
  • the composition or mixture may be mixed with an appropriate excipient to form an ointment, gel, or cream, and then applied to the affected area.
  • the target disease of the bone regeneration composition, the bone regeneration composition kit and the bone regeneration member of the present embodiment includes, as described above, intractable fracture.
  • the obtained solution was cooled to 0 ° C, 9.2 ml of pyridine and 23.8 g of nitrophenyl chloroformate were added, and the mixture was stirred at 0 ° C for 1 hour. Then, 2000 ml of cooled ethanol was added to the flask, the precipitate was separated by filtration, and the precipitate was washed with cooled ethanol and dried.
  • a hydroxyphenyl group-modified hyaluronic acid (A1) was obtained.
  • the degree of substitution (DS) with the hydroxyphenyl group was 3.2.
  • Preparation Example 2 Preparation of First Solution (c1 ) 0.2 g of the hydroxyphenyl group-modified dextran (AR1) obtained in Synthesis Example 2 was mixed with phosphate buffered saline (product name "PBS (-)", The polymer solution was prepared by dissolving in 0.8 g of Kojun Pharmaceutical Co., Ltd. 1.0 g of the above polymer solution and 1.0 g of an aqueous solution of horseradish peroxidase (manufactured by Wako Pure Chemical, product code "169-10791”) (concentration: 0.8 U / ml) were uniformly mixed to form a first solution ( c1) was prepared.
  • PBS (-) phosphate buffered saline
  • composition for bone regeneration was prepared in the same manner as in Example 1 except that the first solution (c1) was used instead of the first solution (1).
  • the composition for bone regeneration was administered to the fracture site of the mouse in the same manner as in Example 1, and after 6 weeks, the mouse was sacrificed and the bone fusion rate was measured by microCT.
  • the bone union rate was 10% (1/10).
  • the microCT image after 6 weeks is shown in FIG.
  • Example 1 was the same as Example 1 except that the third solution (1) was replaced with a phosphate buffered saline (product name “PBS ( ⁇ )”, manufactured by Wako Pure Chemical Industries, Ltd.).
  • a composition for bone regeneration was prepared.
  • the composition for bone regeneration was administered to the fracture site of the mouse in the same manner as in Example 1, and after 6 weeks, the mouse was sacrificed and the bone fusion rate was measured by microCT.
  • the bone union rate was 10% (1/10).
  • the microCT image after 6 weeks is shown in FIG.
  • control A C57BL / 6J mouse (male, 9 weeks old) in which the femur was fractured after cauterizing the periosteum of the femur with an electric scalpel was used as control.
  • the bone union rate was 10% (1/10).
  • the microCT image after 6 weeks is shown in FIG.
  • Example 1 has a higher bone union rate than Comparative Examples 1 and 2 and Experimental Example 1, and the composition for bone regeneration of this embodiment is useful for intractable fractures including periosteal damage. It was revealed.
  • Bone 15 bone marrow 20: mesenchymal stem cells, osteoblasts (periosteum) 30: Affected area (bone defect site, fracture site) 40: Hydrogel 50: Bone morphogenetic protein 60: Proliferated osteoblast 70: New bone 80: Neoperiosteum

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Abstract

A composition for bone regeneration that comprises: at least one component (A) selected from a modified hyaluronic acid having a hydroxyaryl group introduced into hyaluronic acid and a salt thereof; an oxidative coupling enzyme (B) for the hydroxyaryl group; at least one component (C) selected from a peroxide (c1) and a substance (c2) forming a peroxide; a bone-forming protein (D); and water (E).

Description

骨再生用組成物、骨再生用組成物キット、骨再生用部材および骨再生方法Bone regeneration composition, bone regeneration composition kit, bone regeneration member and bone regeneration method
　本発明は、骨再生用組成物、骨再生用組成物キット、骨再生用部材および骨再生方法に関する。 The present invention relates to a bone regeneration composition, a bone regeneration composition kit, a bone regeneration member, and a bone regeneration method.
　骨折後の骨の再生は、骨折間隙の血腫占拠による骨折部位の炎症が起こる炎症期、血腫が肉芽細胞に変わることによる仮骨の形成、骨膜に存在する骨形成細胞による新生骨の形成が進行する骨修復期、および新生骨の骨吸収・形成の繰り返しによる骨変形の矯正が進行する再造形期、により進行することが知られている。 Regeneration of bone after fracture involves an inflammatory phase in which the fracture site is inflamed due to hematoma occupation of the fracture gap, callus formation due to the conversion of the hematoma to granulocytes, and new bone formation due to osteogenic cells present in the periosteum It is known that the process progresses during a bone repair period, in which the bone is deformed due to repeated bone resorption and formation of new bone, and a remodeling period in which the correction of bone deformation progresses.
　交通事故のような過剰な外圧が骨に加わると、不全骨折や開放骨折などの骨膜の損傷を伴う場合が多く、新生骨の形成に支障をきたすことがある。一般的に、新生骨が形成されれば、骨折部位は生活に支障のない程度の強度を有することから、骨膜の損傷を伴う交通事故において骨修復期が長いと、患者の日常生活に大きな影響を与えることが知られている。 過 剰 Excessive external pressure, such as that in a traffic accident, is often associated with periosteal damage, such as incomplete or open fractures, which can hinder new bone formation. In general, if new bone is formed, the fracture site has a strength that does not hinder daily life.If the bone repair period is long in a traffic accident involving periosteum damage, it will have a significant effect on the patient's daily life. It is known to give.
　従来、骨折に対する骨再生治療のために、骨形成タンパク質（Bone Morphogenetic Protein、ＢＭＰ）等の成長因子を含む骨再生用部材を用いる方法が知られている（特許文献１～２参照）。 Conventionally, a method of using a bone regeneration member containing a growth factor such as a bone morphogenetic protein (BMP) for bone regeneration treatment for a fracture has been known (see Patent Documents 1 and 2).
特開２０１２－０８２１８０号公報JP 2012/082180 A 特表２０１５－５２３１７６号公報JP-T-2015-523176
　本発明者らの検討によれば、骨膜の損傷などの、骨形成において重要な組織の損傷や、それに伴う血流障害のある難治性骨折においては、従来の骨再生用部材を用いる方法では治療効果が充分ではないことがわかった。 According to the study of the present inventors, in the case of damage to tissues important in bone formation, such as damage to periosteum, and intractable fractures accompanied by impaired blood flow, treatment using a conventional method of using a bone regeneration member is not effective. It turned out that the effect was not enough.
　本発明は、骨膜の損傷などを含む難治性骨折等に有用な骨再生用組成物、骨再生用組成物キット、および骨再生用部材を提供すること、ならびに前記組成物等を用いた骨再生方法を提供することを目的とする。 The present invention provides a bone regeneration composition, a bone regeneration composition kit, and a bone regeneration member useful for intractable fractures including periosteal damage and the like, and bone regeneration using the composition and the like. The aim is to provide a method.
　本発明者らは上記課題を解決すべく検討した。その結果、以下の骨再生用組成物などにより上記課題を解決できることを見出し、本発明を完成するに至った。
　本発明は、例えば以下の［１］～［１０］に関する。 The present inventors have studied to solve the above problems. As a result, the inventors have found that the following problems can be solved by the following bone regeneration composition and the like, and have completed the present invention.
The present invention relates to, for example, the following [1] to [10].
　［１］ヒアルロン酸にヒドロキシアリール基が導入された変性ヒアルロン酸およびその塩から選ばれる少なくとも１種の成分（Ａ）と、前記ヒドロキシアリール基の酸化カップリング酵素（Ｂ）と、過酸化物（ｃ１）および過酸化物を生成する物質（ｃ２）から選ばれる少なくとも１種の成分（Ｃ）と、骨形成タンパク質（Ｄ）と、水（Ｅ）とを含有する骨再生用組成物。
　［２］前記ヒドロキシアリール基が、チラミン由来の基である前記［１］に記載の骨再生用組成物。
　［３］前記成分（Ａ）における前記ヒドロキシアリール基による置換度が、０．１～５０である前記［１］または［２］に記載の骨再生用組成物。
　［４］前記酸化カップリング酵素（Ｂ）が、西洋わさびぺルオキシダーゼである前記［１］～［３］のいずれかに記載の骨再生用組成物。
　［５］前記成分（Ｃ）が、前記過酸化物（ｃ１）である前記［１］～［４］のいずれかに記載の骨再生用組成物。
　［６］難治性骨折用である前記［１］～［５］のいずれかに記載の骨再生用組成物。
　［７］第１の溶液と、第２の溶液と、第３の溶液とを有する骨再生用組成物キットであり、前記第１の溶液が、ヒアルロン酸にヒドロキシアリール基が導入された変性ヒアルロン酸およびその塩から選ばれる少なくとも１種の成分（Ａ）と水（Ｅ）とを含有し、前記ヒドロキシアリール基の酸化カップリング酵素（Ｂ）、ならびに過酸化物（ｃ１）および過酸化物を生成する物質（ｃ２）から選ばれる少なくとも１種の成分（Ｃ）、のいずれかを含有するが両方は含有せず、前記第２の溶液が、前記酸化カップリング酵素（Ｂ）および前記成分（Ｃ）のうち第１の溶液中で含有されない方を含有し、前記第３の溶液が、骨形成タンパク質（Ｄ）を含有する、骨再生用組成物キット。
　［８］難治性骨折用である前記［７］に記載の骨再生用組成物キット。
　［９］前記［１］～［６］のいずれかに記載の骨再生用組成物、または前記［７］もしくは［８］に記載の骨再生用組成物キットを用いて形成した骨再生用部材。
　［１０］前記［１］～［６］のいずれかに記載の骨再生用組成物、前記［７］もしくは［８］に記載の骨再生用組成物キット、または前記［９］に記載の骨再生用部材を用いて、骨を再生させる骨再生方法。 [1] At least one component (A) selected from a modified hyaluronic acid in which a hydroxyaryl group is introduced into hyaluronic acid and a salt thereof, an oxidative coupling enzyme (B) of the hydroxyaryl group, and a peroxide ( A composition for bone regeneration comprising at least one component (C) selected from c1) and a substance (c2) that produces peroxide, an osteogenic protein (D), and water (E).
[2] The composition for bone regeneration according to [1], wherein the hydroxyaryl group is a group derived from tyramine.
[3] The bone regeneration composition according to the above [1] or [2], wherein the degree of substitution of the component (A) with the hydroxyaryl group is 0.1 to 50.
[4] The composition for bone regeneration according to any of [1] to [3], wherein the oxidative coupling enzyme (B) is horseradish peroxidase.
[5] The composition for bone regeneration according to any of [1] to [4], wherein the component (C) is the peroxide (c1).
[6] The composition for bone regeneration according to any of [1] to [5], which is used for intractable fractures.
[7] A bone regeneration composition kit having a first solution, a second solution, and a third solution, wherein the first solution is a modified hyaluronic acid in which a hydroxyaryl group is introduced into hyaluronic acid. It contains at least one component (A) selected from an acid and a salt thereof and water (E), and contains the oxidative coupling enzyme (B) for the hydroxyaryl group, and the peroxide (c1) and the peroxide. The second solution contains at least one component (C) selected from the substance (c2) to be formed, but not both, and the second solution contains the oxidative coupling enzyme (B) and the component (C). A composition kit for bone regeneration, comprising the component (C) that is not contained in the first solution, wherein the third solution contains an osteogenic protein (D).
[8] The composition kit for bone regeneration according to the above [7], which is used for intractable fractures.
[9] A bone regeneration member formed using the bone regeneration composition according to any one of [1] to [6], or the bone regeneration composition kit according to [7] or [8]. .
[10] The composition for bone regeneration according to any of [1] to [6], the composition kit for bone regeneration according to [7] or [8], or the bone according to [9]. A bone regeneration method for regenerating bone using a regeneration member.
　本発明によれば、骨膜の損傷などを含む難治性骨折等に有用な骨再生用組成物、骨再生用組成物キット、および骨再生用部材を提供すること、ならびに前記組成物等を用いた骨再生方法を提供することができる。 According to the present invention, it is possible to provide a composition for bone regeneration, a composition kit for bone regeneration, and a member for bone regeneration useful for intractable fracture including periosteal damage and the like, and using the composition and the like. A bone regeneration method can be provided.
図１は、本発明の骨再生用組成物を使用した骨再生過程のスキームである。FIG. 1 is a scheme of a bone regeneration process using the bone regeneration composition of the present invention. 図２は、実施例等で測定したｍｉｃｒｏＣＴ画像である。FIG. 2 is a microCT image measured in Examples and the like.
　以下、本発明を実施するための形態について説明する。
　本明細書で例示する各物質、例えば、組成物中に含まれるまたは各工程で用いられる物質は、特に言及しない限り、それぞれ１種用いることができ、または２種以上を併用して用いることができる。 Hereinafter, embodiments for carrying out the present invention will be described.
Each substance exemplified in the present specification, for example, a substance contained in the composition or used in each step can be used alone or in combination of two or more, unless otherwise specified. it can.
　［骨再生用組成物］
　一実施形態において、本発明の骨再生用組成物（以下、単に「本実施形態の組成物」ともいう）は、
　ヒアルロン酸にヒドロキシアリール基が導入された変性ヒアルロン酸およびその塩から選ばれる少なくとも１種の成分（Ａ）と、
　前記ヒドロキシアリール基の酸化カップリング酵素（Ｂ）と、
　過酸化物（ｃ１）および過酸化物を生成する物質（ｃ２）から選ばれる少なくとも１種の成分（Ｃ）と、
　骨形成タンパク質（Ｄ）と、
　水（Ｅ）と
を含有する。 [Bone regeneration composition]
In one embodiment, the composition for bone regeneration of the present invention (hereinafter, also simply referred to as “composition of the present embodiment”) comprises:
At least one component (A) selected from a modified hyaluronic acid in which a hydroxyaryl group is introduced into hyaluronic acid and a salt thereof;
Said hydroxyaryl group oxidative coupling enzyme (B);
At least one component (C) selected from a peroxide (c1) and a substance (c2) that generates peroxide;
Osteogenic protein (D),
Water (E).
　＜成分（Ａ）＞
　成分（Ａ）は、ヒアルロン酸にヒドロキシアリール基が導入された変性ヒアルロン酸、および当該変性ヒアルロン酸の塩から選ばれる少なくとも１種である。
　骨形成タンパク質（Ｄ）は体液によって循環しやすいため、骨形成タンパク質（Ｄ）の投与部位にその効果を長期間維持することは一般的に困難である。本実施形態では、骨形成タンパク質（Ｄ）とともに成分（Ａ）を用いることにより、本実施形態の組成物の投与部位において骨形成タンパク質（Ｄ）が安定的に存在でき、骨形成タンパク質（Ｄ）の有効濃度が長期間維持されると考えられる。このため、本実施形態の組成物は骨の再生効果が高い。具体的には本実施形態では、成分（Ａ）および骨形成タンパク質（Ｄ）を用いることにより、新生骨形成を促進し、骨の再生速度を向上させることができると考えられる。 <Component (A)>
The component (A) is at least one selected from a modified hyaluronic acid in which a hydroxyaryl group has been introduced into hyaluronic acid, and a salt of the modified hyaluronic acid.
Since the bone morphogenetic protein (D) is easily circulated by body fluids, it is generally difficult to maintain its effect at the administration site of the bone morphogenetic protein (D) for a long period of time. In the present embodiment, by using the component (A) together with the bone morphogenetic protein (D), the bone morphogenetic protein (D) can be stably present at the administration site of the composition of the present embodiment, and the bone morphogenetic protein (D) Is expected to be maintained for a long time. Therefore, the composition of the present embodiment has a high bone regeneration effect. Specifically, in the present embodiment, it is considered that by using the component (A) and the bone morphogenetic protein (D), new bone formation can be promoted and bone regeneration speed can be improved.
　本実施形態の組成物は、難治性骨折の治療用、特に骨膜損傷型骨折の治療用に有用であり、本実施形態の組成物を用いることにより、骨折した骨の癒合率を高めることができる。この理由は、成分（Ａ）より形成されるゲルと有効成分である骨形成タンパク質（Ｄ）との親和性が適度に優れているため、前記ゲルからの有効成分の徐放速度が前記骨折における骨再生に適した速度であるためであると考えられる。なお、難治性骨折とは、骨膜の損傷などの、骨形成において重要な組織の損傷や、当該損傷に伴う血流障害のある骨折であり、感染症を伴う場合もある。難治性骨折としては、例えば、開放骨折が挙げられる。 The composition of the present embodiment is useful for treating intractable fractures, particularly for treating periosteal injury-type fractures, and by using the composition of the present embodiment, it is possible to increase the union rate of a fractured bone. . The reason for this is that the affinity between the gel formed from the component (A) and the bone morphogenetic protein (D), which is the active ingredient, is moderately excellent, so that the sustained release rate of the active ingredient from the gel decreases in the fracture. This is probably because the speed is suitable for bone regeneration. The intractable fracture is a fracture of a tissue important in bone formation, such as a damage of a periosteum, or a fracture having impaired blood flow accompanying the damage, and may be accompanied by an infection. Intractable fractures include, for example, open fractures.
　ヒアルロン酸は、Ｎ－アセチルグルコサミンとグルクロン酸との二糖単位を反復構造単位として有する多糖である。変性ヒアルロン酸の塩としては、例えば、生理学的または薬学的に許容される塩が挙げられ、ナトリウム塩、カリウム塩等のアルカリ金属塩、アンモニウム塩が挙げられる。 Hyaluronic acid is a polysaccharide having a disaccharide unit of N-acetylglucosamine and glucuronic acid as a repeating structural unit. Examples of the salt of the modified hyaluronic acid include physiologically or pharmaceutically acceptable salts, and include alkali metal salts such as sodium salt and potassium salt, and ammonium salts.
　成分（Ａ）は、ヒドロキシアリール基を有する。ヒドロキシアリール基の炭素数は、通常、６～５０、好ましくは６～３０である。前記ヒドロキシアリール基において、ベンゼン核に結合する水酸基数は通常、１以上、好ましくは１～３、より好ましくは１～２である。このように、ヒドロキシアリール基は、モノヒドロキシアリール基に限定されるわけではない。 Component (A) has a hydroxyaryl group. The number of carbon atoms of the hydroxyaryl group is usually 6 to 50, preferably 6 to 30. In the hydroxyaryl group, the number of hydroxyl groups bonded to the benzene nucleus is usually 1 or more, preferably 1 to 3, and more preferably 1 or 2. Thus, the hydroxyaryl group is not limited to a monohydroxyaryl group.
　ヒドロキシアリール基としては、例えば、ヒドロキシフェニル基、ヒドロキシナフチル基、ヒドロキシアントラセニル基が挙げられる。ヒドロキシアリール基は、アルキル基等の置換基を有することができる。ヒドロキシアリール基において、フェノール性水酸基に対して少なくとも１つのオルト位置の炭素原子には水素原子が結合していることが好ましい。本実施形態の組成物の投与部位において、骨形成タンパク質（Ｄ）が、安定的に存在でき、且つその有効濃度を保持できると考えられることから、ヒドロキシアリール基はチラミン由来の基であることが好ましい。 Examples of the hydroxyaryl group include a hydroxyphenyl group, a hydroxynaphthyl group, and a hydroxyanthracenyl group. The hydroxyaryl group can have a substituent such as an alkyl group. In the hydroxyaryl group, it is preferable that a hydrogen atom is bonded to a carbon atom at least one ortho position to the phenolic hydroxyl group. Since it is considered that the bone morphogenetic protein (D) can be stably present at the administration site of the composition of the present embodiment and can maintain its effective concentration, the hydroxyaryl group is a group derived from tyramine. preferable.
　変性ヒアルロン酸は、好ましくは、ヒドロキシアリール基が、直接または有機基を介して、エステル結合、ウレタン結合またはエーテル結合によりヒアルロン酸に結合した化合物である。エステル結合、ウレタン結合およびエーテル結合は、ヒアルロン酸に含まれる水酸基が結合していた炭素原子と結合していることが好ましい。 Modified hyaluronic acid is preferably a compound in which a hydroxyaryl group is bonded to hyaluronic acid by an ester bond, a urethane bond or an ether bond, directly or via an organic group. The ester bond, urethane bond and ether bond are preferably bonded to a carbon atom to which a hydroxyl group contained in hyaluronic acid has been bonded.
　前記有機基としては、例えば、炭素数１～２０のアルカンジイル基、前記アルカンジイル基にエーテル結合および２価のアミノ基（－ＮＨ－等）から選ばれる少なくとも１種が付加又は挿入された置換アルカンジイル基、ペプチド結合を有する有機基（具体的には、ジペプチド等のオリゴペプチドまたはそのエステル体に由来する基）が挙げられる。 The organic group is, for example, an alkanediyl group having 1 to 20 carbon atoms, or a substitution in which at least one selected from an ether bond and a divalent amino group (such as —NH—) is added or inserted to the alkanediyl group. Examples include an alkanediyl group and an organic group having a peptide bond (specifically, a group derived from an oligopeptide such as a dipeptide or an ester thereof).
　以下、成分（Ａ）の製造例の一実施態様について説明する。
　例えば、ヒアルロン酸が有するアルコール性水酸基を、カルボニル化試薬を用いて、炭酸エステル誘導体基に変換した後（以下「変換反応」ともいう）、得られたヒアルロン酸の炭酸エステル誘導体と、ヒドロキシアリール基を有するアミン化合物とを反応させる（以下「ヒドロキシアリール基導入反応」ともいう）。 Hereinafter, one embodiment of the production example of the component (A) will be described.
For example, after converting the alcoholic hydroxyl group of hyaluronic acid into a carbonate derivative group using a carbonylating reagent (hereinafter also referred to as “conversion reaction”), the resulting carbonate derivative of hyaluronic acid and a hydroxyaryl group (Hereinafter, also referred to as “hydroxyaryl group introduction reaction”).
　前記カルボニル化試薬としては、例えば、クロロギ酸ニトロフェニル、クロロギ酸フェニル、クロロギ酸トリクロロメチル、炭酸ビス（トリクロロメチル）が挙げられる。カルボニル化試薬の量は、通常、ヒアルロン酸１００質量部に対して１～１００質量部である。中間体である炭酸エステル誘導体が安定である場合は、一旦単離した後に次の反応を行うことができる。 Examples of the carbonylating reagent include nitrophenyl chloroformate, phenyl chloroformate, trichloromethyl chloroformate, and bis (trichloromethyl) carbonate. The amount of the carbonylation reagent is usually 1 to 100 parts by mass based on 100 parts by mass of hyaluronic acid. When the carbonate derivative as an intermediate is stable, the following reaction can be carried out after isolation.
　前記ヒドロキシアリール基を有するアミン化合物としては、例えば、チラミンが挙げられる。前記ヒドロキシアリール基を有するアミン化合物の量は、通常、ヒアルロン酸の炭酸エステル誘導体１００質量部に対して１～１００質量部である。 ア ミ ン Examples of the amine compound having a hydroxyaryl group include tyramine. The amount of the amine compound having a hydroxyaryl group is usually 1 to 100 parts by mass relative to 100 parts by mass of the carbonate derivative of hyaluronic acid.
　前記変換反応は、塩基性化合物の存在下で行うことが好ましい。塩基性化合物としては、例えば、トリメチルアミン、トリエチルアミン、トリブチルアミン、ピリジン、ピペリジンが挙げられる。 The conversion reaction is preferably performed in the presence of a basic compound. Examples of the basic compound include trimethylamine, triethylamine, tributylamine, pyridine, and piperidine.
　前記変換反応およびヒドロキシアリール基導入反応は、溶媒中で行うことが好ましい。溶媒としては、例えば、ジメチルホルムアミド、ジメチルアセトアミド等のアミド溶媒；ジメチルスルホキシド等のスルホキシド溶媒；ジクロロメタン、１，２－ジクロロエタン、クロロホルム等のハロゲン化炭化水素溶媒；ベンゼン、トルエン、キシレン等の芳香族炭化水素溶媒；ジエチルエーテル、テトラヒドロフラン、ジオキサン等のエーテル溶媒が挙げられる。ヒアルロン酸の溶媒中への溶解性を向上させるため、塩化リチウム等のアルカリ金属塩を用いることができる。 The conversion reaction and the hydroxyaryl group introduction reaction are preferably performed in a solvent. Examples of the solvent include amide solvents such as dimethylformamide and dimethylacetamide; sulfoxide solvents such as dimethylsulfoxide; halogenated hydrocarbon solvents such as dichloromethane, 1,2-dichloroethane and chloroform; and aromatic hydrocarbons such as benzene, toluene and xylene. Hydrogen solvents; ether solvents such as diethyl ether, tetrahydrofuran, dioxane and the like. In order to improve the solubility of hyaluronic acid in a solvent, an alkali metal salt such as lithium chloride can be used.
　成分（Ａ）の製造例の他の実施態様としては、例えば、ヒアルロン酸と、ヒアルロン酸中のアルコール性水酸基と反応可能な官能基（例：カルボキシ基、その酸ハロゲン化物、エポキシ基、オキセタニル基）およびヒドロキシアリール基を有する化合物とを、反応させる方法が挙げられる。 As another embodiment of the production example of the component (A), for example, hyaluronic acid and a functional group capable of reacting with an alcoholic hydroxyl group in hyaluronic acid (eg, a carboxy group, an acid halide thereof, an epoxy group, an oxetanyl group) ) And a compound having a hydroxyaryl group.
　ヒアルロン酸中のアルコール性水酸基と反応可能なカルボキシ基またはその酸ハロゲン化物と、ヒドロキシアリール基とを有する化合物としては、例えば、４－ヒドロキシフェニル酢酸、４－ヒドロキシフェニルプロピオン酸、４－ヒドロキシフェニルブタン酸、２－（２，５－ジヒドロキシフェニル）酢酸、これらの酸ハロゲン化物が挙げられる。 Examples of the compound having a carboxy group capable of reacting with an alcoholic hydroxyl group in hyaluronic acid or an acid halide thereof and a hydroxyaryl group include, for example, 4-hydroxyphenylacetic acid, 4-hydroxyphenylpropionic acid, 4-hydroxyphenylbutane Acids, 2- (2,5-dihydroxyphenyl) acetic acid, and acid halides thereof.
　成分（Ａ）におけるヒドロキシアリール基による置換度（ＤＳ）は、通常、０．１～５０であり、好ましくは１～４０、より好ましくは２～３０である。置換度は、1Ｈ ＮＭＲにより求めることができる。成分（Ａ）の置換度とは、成分（Ａ）の構成単糖１００単位あたりに導入されたヒドロキシアリール基の平均数を示す。成分（Ａ）はこのような置換度を有することにより、そのゲル化速度が向上する。 The degree of substitution (DS) of the component (A) with the hydroxyaryl group is usually 0.1 to 50, preferably 1 to 40, more preferably 2 to 30. The degree of substitution can be determined by 1 H NMR. The substitution degree of the component (A) indicates the average number of hydroxyaryl groups introduced per 100 units of the constituent monosaccharide of the component (A). The component (A) having such a degree of substitution improves the gelation rate.
　成分（Ａ）の多角度光散乱検出器を用いた絶対分子量測定による数平均分子量は、通常、１，０００～１０，０００，０００、好ましくは３，０００～１，０００，０００、より好ましくは５，０００～５００，０００である。また、成分（Ａ）の分子量分布（重量平均分子量／数平均分子量；いずれも前記絶対分子量測定による）は、通常、１～２０、好ましくは１～１０、より好ましくは１～５である。 The number average molecular weight of the component (A) as measured by absolute molecular weight measurement using a multi-angle light scattering detector is usually 1,000 to 10,000,000, preferably 3,000 to 1,000,000, more preferably 5,000 to 500,000. The molecular weight distribution of the component (A) (weight average molecular weight / number average molecular weight; both measured by the absolute molecular weight) is usually 1 to 20, preferably 1 to 10, and more preferably 1 to 5.
　成分（Ａ）は１種または２種以上用いることができる。
　本実施形態の組成物中の成分（Ａ）の含有割合は、通常、０．０５～４９質量％、好ましくは０．５～３０質量％、より好ましくは３～１５質量％である。このような態様であると、ゲルの機械的強度および取扱い性の観点から好ましい。 As the component (A), one type or two or more types can be used.
The content ratio of the component (A) in the composition of the present embodiment is usually 0.05 to 49% by mass, preferably 0.5 to 30% by mass, and more preferably 3 to 15% by mass. Such an embodiment is preferable from the viewpoint of the mechanical strength and handleability of the gel.
　＜ヒドロキシアリール基の酸化カップリング酵素（Ｂ）＞
　本実施形態の組成物は、ヒドロキシアリール基の酸化カップリング酵素（Ｂ）（以下「酵素（Ｂ）」ともいう）を含有する。酵素（Ｂ）は、ヒドロキシアリール基の酸化カップリング反応を直接的または間接的に触媒しうる酵素である。 <Hydroxyaryl group oxidative coupling enzyme (B)>
The composition of the present embodiment contains an oxidative coupling enzyme (B) for a hydroxyaryl group (hereinafter also referred to as “enzyme (B)”). The enzyme (B) is an enzyme capable of directly or indirectly catalyzing an oxidative coupling reaction of a hydroxyaryl group.
　酵素（Ｂ）としては、例えば、ラッカーゼ、チロシナーゼ等のフェノールオキシダーゼ、カタラーゼ、ペルオキシダーゼが挙げられる。ペルオキシダーゼは、過酸化水素等の過酸化物（ｃ１）と併用することで本実施形態の組成物をゲル化させることができる。ラッカーゼ、チロシナーゼ等の銅酵素類の起源は、例えば、ウルシ、キノコ（ツチカブリ、マッシュルーム）、カビ（Polyporus vericolor）が挙げられる。カタラーゼ、ペルオキシダーゼの起源は、例えば、ウシ肝臓、ウマ血球、ヒト血球、M. lysodeikticus、西洋わさび（ホースラディッシュ）、大豆、ダイコン、カブ、甲状腺、牛乳、腸、白血球、赤血球、酵母、Caldariomyces fumago、Steptococcus faecalisが挙げられる。これらの中でも、チロシナーゼとしてはマッシュルーム由来、ペルオキシダーゼとしては西洋わさび由来のもの、すなわち西洋わさびペルオキシダーゼが好ましい。 Examples of the enzyme (B) include phenol oxidase such as laccase and tyrosinase, catalase, and peroxidase. The composition of the present embodiment can be gelled by using peroxidase in combination with a peroxide (c1) such as hydrogen peroxide. The origin of copper enzymes such as laccase and tyrosinase includes, for example, sorghum, mushrooms (Acacia fulica, mushroom), and mold (Polyporus vericolor). Catalase, the origin of peroxidase, for example, bovine liver, horse blood cells, human blood cells, M. lysodeikticus, horseradish (horseradish), soybean, radish, turnip, thyroid, milk, intestine, white blood cells, red blood cells, yeast, Caldariomyces fumago, Steptococcus faecalis. Among them, tyrosinase is preferably derived from mushroom, and peroxidase is preferably derived from horseradish, that is, horseradish peroxidase.
　本実施形態では、成分（Ａ）が有するヒドロキシアリール基の架橋反応を効率的に進める観点から、成分（Ｃ）を用い、酵素（Ｂ）としてペルオキシダーゼおよびカタラーゼから選ばれる少なくとも１種を用いることが好ましく、成分（Ｃ）を用い、酵素（Ｂ）として西洋わさびペルオキシダーゼを用いることがより好ましい。 In the present embodiment, from the viewpoint of efficiently promoting the cross-linking reaction of the hydroxyaryl group of the component (A), the component (C) is used, and at least one selected from peroxidase and catalase is used as the enzyme (B). More preferably, component (C) is used, and horseradish peroxidase is used as enzyme (B).
　酵素（Ｂ）は１種または２種以上用いることができる。
　本実施形態の組成物において、酵素（Ｂ）の量は、０．０１Ｕ／ｍＬ以上が好ましく、０．１Ｕ／ｍＬ以上がより好ましく；１，０００Ｕ／ｍＬ以下が好ましく、５００Ｕ／ｍＬ以下がより好ましい。なお、Ｕとは酵素活性の単位を示し、至適条件下で、温度３０℃で毎分１マイクロモルの基質を変化させることができる酵素量である。 One or more enzymes (B) can be used.
In the composition of the present embodiment, the amount of the enzyme (B) is preferably at least 0.01 U / mL, more preferably at least 0.1 U / mL; preferably at most 1,000 U / mL, and more preferably at most 500 U / mL. preferable. U represents a unit of enzyme activity, and is an amount of enzyme capable of changing 1 μmol of substrate per minute at a temperature of 30 ° C. under optimum conditions.
　＜成分（Ｃ）＞
　本実施形態の組成物は、過酸化物（ｃ１）および過酸化物を生成する物質（ｃ２）から選ばれる少なくとも１種の成分（Ｃ）を含有する。
　過酸化物（ｃ１）としては、例えば、過酸化水素が挙げられる。
　成分（Ｃ）として過酸化物（ｃ１）を用いる場合、本実施形態の組成物において、過酸化物（ｃ１）の量は、成分（Ａ）中のヒドロキシアリール基１モルに対して、通常、０．０１～１０００モル、好ましくは０．１～５００モル、より好ましくは０．５～２００モルである。また、一実施態様において、本実施形態の組成物中の過酸化物（ｃ１）の含有量は、反応性の点から、好ましくは１０～１，０００ｍＭ、より好ましくは１００～８００ｍＭである。 <Component (C)>
The composition of the present embodiment contains at least one component (C) selected from a peroxide (c1) and a substance (c2) that generates a peroxide.
Examples of the peroxide (c1) include hydrogen peroxide.
When the peroxide (c1) is used as the component (C), in the composition of the present embodiment, the amount of the peroxide (c1) is usually based on 1 mol of the hydroxyaryl group in the component (A). The amount is 0.01 to 1000 mol, preferably 0.1 to 500 mol, and more preferably 0.5 to 200 mol. In one embodiment, the content of the peroxide (c1) in the composition of the present embodiment is preferably from 10 to 1,000 mM, more preferably from 100 to 800 mM, from the viewpoint of reactivity.
　過酸化物を生成する物質（ｃ２）は、通常、酸化酵素（ｃ３）とともに用いられる。酸化酵素（ｃ３）は、物質（ｃ２）を消費して過酸化水素等の過酸化物を生成する反応を触媒する酵素である。酸化酵素（ｃ３）としては、例えば、グルコースオキシダーゼ、コリンオキシダーゼ、アミノ酸オキシダーゼ、アルコールオキシダーゼ、ピルビン酸オキシダーゼ、コレステロールオキシダーゼが挙げられる。これらの酵素は、用途に応じて適宜選択できる。例えば、ゲル化速度が大幅に向上することから、グルコースオキシダーゼが好ましい。 物質 The peroxide-generating substance (c2) is usually used together with the oxidase (c3). The oxidase (c3) is an enzyme that catalyzes a reaction that consumes the substance (c2) to generate a peroxide such as hydrogen peroxide. Examples of the oxidase (c3) include glucose oxidase, choline oxidase, amino acid oxidase, alcohol oxidase, pyruvate oxidase, and cholesterol oxidase. These enzymes can be appropriately selected depending on the application. For example, glucose oxidase is preferable because the gelation rate is greatly improved.
　物質（ｃ２）は、例えば、酸化酵素（ｃ３）により消費されて過酸化水素等の過酸化物を生成し、酸化酵素（ｃ３）に対応して選択される。例えば、酸化酵素（ｃ３）が物質（ｃ２）を、酸素の存在下（例：空気中の酸素）に酵素処理することによって、過酸化水素を生成させることができる。例えば、酸化酵素（ｃ３）が、グルコースオキシダーゼ、コリンオキシダーゼ、アミノ酸オキシダーゼ、アルコールオキシダーゼ、ピルビン酸オキシダーゼまたはコレステロールオキシダーゼである場合、物質（ｃ２）は、それぞれ、グルコース、コリン、アミノ酸、アルコール、ピルビン酸またはコレステロールである。 The substance (c2) is consumed by, for example, the oxidase (c3) to generate a peroxide such as hydrogen peroxide, and is selected according to the oxidase (c3). For example, hydrogen peroxide can be generated by the enzyme treatment of the substance (c2) by the oxidase (c3) in the presence of oxygen (eg, oxygen in the air). For example, when the oxidase (c3) is glucose oxidase, choline oxidase, amino acid oxidase, alcohol oxidase, pyruvate oxidase or cholesterol oxidase, the substance (c2) is glucose, choline, amino acid, alcohol, pyruvate or Cholesterol.
　成分（Ｃ）として物質（ｃ２）を用いる場合、本実施形態の組成物中の酸化酵素（ｃ３）の含有量は、反応性の点から、０．０１Ｕ／ｍＬ以上が好ましく、１Ｕ／ｍＬ以上がより好ましく；１，０００Ｕ／ｍＬ以下が好ましく、５００Ｕ／ｍＬ以下がより好ましい。また、物質（ｃ２）の含有量は、過酸化物を適度に生成するような量が適宜選択される。
　成分（Ｃ）は１種または２種以上用いることができる。 When the substance (c2) is used as the component (C), the content of the oxidase (c3) in the composition of the present embodiment is preferably 0.01 U / mL or more, and more preferably 1 U / mL or more from the viewpoint of reactivity. Is more preferable; 1,000 U / mL or less is preferable, and 500 U / mL or less is more preferable. Further, the content of the substance (c2) is appropriately selected such that the peroxide is appropriately generated.
One or more components (C) can be used.
　＜骨形成タンパク質（Ｄ）＞
　本実施形態の組成物は、骨形成タンパク質（Ｄ）を含有する。
　骨形成タンパク質（ＢＭＰ：Bone Morphogenetic Protein）（Ｄ）は、骨の形成および骨折の修復を促進するタンパク質であり、具体的には、骨芽細胞の活性を増進する因子、および間葉系幹細胞の骨芽細胞への分化を誘導する因子である。骨形成タンパク質（Ｄ）の好適例としては、例えば、ＢＭＰ－２、ＢＭＰ－４、ＢＭＰ－５、ＢＭＰ－６、ＢＭＰ－７、ＢＭＰ－８、ＢＭＰ－９が挙げられる。骨形成タンパク質（Ｄ）は、ヒト由来であっても、非ヒト動物由来であってもよい。 <Bone morphogenetic protein (D)>
The composition of the present embodiment contains an osteogenic protein (D).
Bone morphogenetic protein (BMP) (D) is a protein that promotes bone formation and repair of fractures. Specifically, it is a factor that enhances the activity of osteoblasts, and a component of mesenchymal stem cells. It is a factor that induces differentiation into osteoblasts. Preferred examples of the bone morphogenetic protein (D) include, for example, BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, and BMP-9. The bone morphogenetic protein (D) may be derived from a human or a non-human animal.
　また、骨形成タンパク質（Ｄ）としては、天然に存在するＢＭＰのアミノ酸配列において１個または数個のアミノ酸が欠失、置換または付加されたアミノ酸配列を有し、天然に存在するＢＭＰと同程度の活性を有するタンパク質も挙げられる。このように、骨形成タンパク質（Ｄ）は、遺伝子組換え型であってもよい。 Further, the bone morphogenetic protein (D) has an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of naturally occurring BMP, and has the same amino acid sequence as naturally occurring BMP. And a protein having the activity of Thus, the bone morphogenetic protein (D) may be recombinant.
　骨形成タンパク質（Ｄ）の中でも、ＢＭＰ－２が好ましい。ＢＭＰ－２は、細胞の増殖を制御および／または促進する因子の一種である。ＢＭＰ－２は、骨形成能に優れており、骨膜や骨髄に存在する間葉系幹細胞の分化を促進する。 Ｂ Among the bone morphogenetic proteins (D), BMP-2 is preferable. BMP-2 is a type of factor that controls and / or promotes cell growth. BMP-2 is excellent in bone formation ability and promotes differentiation of mesenchymal stem cells present in periosteum and bone marrow.
　骨形成タンパク質（Ｄ）は、例えば医薬として使用できる程度に精製されていれば、種々の方法で調製された骨形成タンパク質を用いることができる。
　骨形成タンパク質（Ｄ）は１種または２種以上用いることができる。 As the bone morphogenetic protein (D), for example, bone morphogenetic proteins prepared by various methods can be used as long as the bone morphogenetic protein (D) is purified to a degree that can be used as a medicine.
One or more bone morphogenetic proteins (D) can be used.
　本実施形態の組成物において、骨形成タンパク質（Ｄ）の含有量は、成分（Ａ）１００質量部に対して、通常、０．０１～１０質量部、好ましくは０．０５～５質量部である。このような態様であると、本実施形態の組成物から形成されるハイドロゲル中に骨形成タンパク質（Ｄ）が良好に保持される。 In the composition of the present embodiment, the content of the bone morphogenetic protein (D) is usually 0.01 to 10 parts by mass, preferably 0.05 to 5 parts by mass with respect to 100 parts by mass of the component (A). is there. With such an aspect, the bone morphogenetic protein (D) is favorably retained in the hydrogel formed from the composition of the present embodiment.
　＜水（Ｅ）＞
　本実施形態の組成物は、水（Ｅ）を含有する。
　本実施形態の組成物中の水（Ｅ）の含有割合は、通常、５０～９９．９質量％、好ましくは６５～９９質量％、より好ましくは７０～９７質量％である。このような態様であると、生体に対して低浸襲性な条件でゲル化が可能な組成物となり、さらに得られるゲルの機械的強度が優れる傾向にある。 <Water (E)>
The composition of the present embodiment contains water (E).
The content ratio of water (E) in the composition of the present embodiment is usually 50 to 99.9% by mass, preferably 65 to 99% by mass, and more preferably 70 to 97% by mass. In such an embodiment, the composition can be gelled under conditions of low invasiveness to a living body, and the resulting gel tends to have excellent mechanical strength.
　＜他の成分＞
　本実施形態の組成物は、成分（Ａ）以外の多糖をさらに含有することができる。前記多糖としては、例えば、ヒドロキシアリール基が導入されていない未変性のヒアルロン酸、プルラン、キサンタンガム、セルロース、グアーガム、フルクタン、マンナン、カラギーナン、キチン、キトサン、ペクチン、デンプン、アミロース、アミロペクチン、グリコーゲン、デキストラン、デキストリン、ゲランガム、アルギン酸が挙げられる。 <Other components>
The composition of the present embodiment can further contain a polysaccharide other than the component (A). Examples of the polysaccharide include, for example, unmodified hyaluronic acid, pullulan, xanthan gum, cellulose, guar gum, fructan, mannan, carrageenan, chitin, chitosan, pectin, starch, amylose, amylopectin, glycogen, dextran into which a hydroxyaryl group has not been introduced. Dextrin, gellan gum and alginic acid.
　本実施形態の組成物は、その他、リン酸、ポリリン酸、ｐＨ調整剤、紫外線吸収剤、増粘剤、着色剤およびフィラーから選ばれる１種または２種以上を含有することができ、また、薬剤、細胞等の機能発現物質を含有することができる。
　また、本実施形態の組成物は、さらに塩化ナトリウムを含有することができる。例えば本実施形態の組成物は、リン酸緩衝生理食塩水等の緩衝液を含有することができる。 The composition of the present embodiment may further contain one or more selected from phosphoric acid, polyphosphoric acid, a pH adjuster, an ultraviolet absorber, a thickener, a coloring agent and a filler, It may contain a substance expressing function such as a drug or a cell.
Further, the composition of the present embodiment can further contain sodium chloride. For example, the composition of the present embodiment can contain a buffer such as a phosphate buffered saline.
　［骨再生用組成物キット］
　一実施形態において、本発明の骨再生用組成物キット（以下、単に「本実施形態のキット」ともいう）は、以下に説明する、第１の溶液と、第２の溶液と、第３の溶液とを有する。
　第１の溶液は、ヒアルロン酸にヒドロキシアリール基が導入された変性ヒアルロン酸およびその塩から選ばれる少なくとも１種の成分（Ａ）と水（Ｅ）とを含有し、前記ヒドロキシアリール基の酸化カップリング酵素（Ｂ）、ならびに過酸化物（ｃ１）および過酸化物を生成する物質（ｃ２）から選ばれる少なくとも１種の成分（Ｃ）、のいずれかを含有するが両方は含有しない。 [Composition kit for bone regeneration]
In one embodiment, the composition kit for bone regeneration of the present invention (hereinafter, also simply referred to as “kit of the present embodiment”) includes a first solution, a second solution, and a third solution described below. And a solution.
The first solution contains at least one component (A) selected from a modified hyaluronic acid in which a hydroxyaryl group has been introduced into hyaluronic acid and a salt thereof, and water (E). It contains the ring enzyme (B) and at least one component (C) selected from the peroxide (c1) and the peroxide-generating substance (c2), but not both.
　第２の溶液は、前記酸化カップリング酵素（Ｂ）および前記成分（Ｃ）のうち第１の溶液中で含有されない方を含有する。
　第３の溶液は、骨形成タンパク質（Ｄ）を含有する。 The second solution contains one of the oxidative coupling enzyme (B) and the component (C) that is not contained in the first solution.
The third solution contains the bone morphogenetic protein (D).
　第１～第３の溶液は、いずれも水溶液であることが好ましい。
　第１～第３の溶液中の各成分（Ａ）～（Ｅ）の詳細は上述したとおりである。また、第１～第３の溶液の量比、および第１～第３の各溶液中に含まれる各成分の含有量は、第１～第３の溶液を混合して得られる組成物中の各成分（Ａ）～（Ｅ）の量比や含有量が［骨再生用組成物］欄に記載した範囲となるよう適宜設定される。 Each of the first to third solutions is preferably an aqueous solution.
Details of the components (A) to (E) in the first to third solutions are as described above. Further, the amount ratio of the first to third solutions and the content of each component contained in each of the first to third solutions are determined in the composition obtained by mixing the first to third solutions. The amount ratio and content of each of the components (A) to (E) are appropriately set so as to fall within the ranges described in the [Composition for bone regeneration] column.
　第１～第３の溶液を混合することにより、本実施形態の組成物を調製することができる。この際、各溶液の混合順序は特に限定されないが、第１の溶液と第３の溶液とを混合して得られた混合物に第２の溶液を混合する態様、あるいは第２の溶液と第３の溶液とを混合して得られた混合物に第１の溶液を混合する態様が挙げられる。 組成 By mixing the first to third solutions, the composition of the present embodiment can be prepared. At this time, the order of mixing the respective solutions is not particularly limited, but a mode in which the second solution is mixed with the mixture obtained by mixing the first solution and the third solution, or the second solution and the third solution are mixed. And a solution obtained by mixing the first solution with the first solution.
　［骨再生用部材］
　一実施形態において、本発明の骨再生用部材は、本実施形態の骨再生用組成物または骨再生用組成物キットを用いて形成され、具体的には、前記骨再生用組成物または骨再生用組成物キットから形成されるゲルからなる。 [Bone regeneration members]
In one embodiment, the bone regeneration member of the present invention is formed using the bone regeneration composition or the bone regeneration composition kit of the present embodiment, and specifically, the bone regeneration composition or the bone regeneration composition A gel formed from a composition composition kit.
　前記ゲルは、成分（Ａ）が有するヒドロキシアリール基の酸化カップリングによる架橋構造を有し、すなわち成分（Ａ）由来の架橋体を含有する。例えば、成分（Ａ）中のヒドロキシアリール基が酸化され、前記基同士の酸化カップリング（架橋）が起こり、ゲル化が進行すると推測される。 The gel has a crosslinked structure by oxidative coupling of the hydroxyaryl group of component (A), that is, contains a crosslinked product derived from component (A). For example, it is presumed that the hydroxyaryl group in the component (A) is oxidized, oxidative coupling (crosslinking) between the groups occurs, and gelation proceeds.
　一実施態様において、前記ゲルは、ハイドロゲルであり、具体的には水を通常、５０～９９．９質量％、好ましくは７０～９７質量％含有するハイドロゲルである。前記ハイドロゲルは、成分（Ａ）由来の架橋体を通常、５０～０．１質量％、好ましくは３０～３質量％含有する。前記ハイドロゲルは、機械的強度および柔軟性に優れる。 に お い て In one embodiment, the gel is a hydrogel, specifically a hydrogel containing usually 50 to 99.9% by mass, preferably 70 to 97% by mass of water. The hydrogel generally contains 50 to 0.1% by mass, preferably 30 to 3% by mass of a crosslinked product derived from the component (A). The hydrogel has excellent mechanical strength and flexibility.
　前記ハイドロゲルを適宜乾燥するなどして水分を除去することにより、キセロゲルを得ることができる。あるいは、前記ゲルは、例えば、Ｍａｃｒｏｍｏｌｅｃｕｌｅｓ（２０１５）２６２４－２６３０に記載のように、成分（Ａ）とともにエオシンＹ、メチレンブルー、ローズベンガル等の光増感剤を用い、光照射を行うことでゲル化させて得ることもできる。 (4) A xerogel can be obtained by removing water by appropriately drying the hydrogel. Alternatively, the gel is formed by irradiating light with a photosensitizer such as eosin Y, methylene blue, or rose bengal together with the component (A) as described in Macromolecules (2015) 2624-2630, for example. You can also get it.
　骨再生用部材の製造方法の一例は、以下のとおりである。
　本実施形態の組成物における各成分（Ａ）～（Ｅ）、または本実施形態のキットにおける第１～第３の溶液を混合し、水溶液温度（架橋温度）が、通常、４～５０℃、好ましくは１０～４５℃、より好ましくは２０～４０℃、反応時間が、通常、１分～４８時間、好ましくは５分～３０時間の条件で、ゲル化させることができる。前記各成分（Ａ）～（Ｄ）は、各成分の水溶液として用いることができる。ハイドロゲル形成は、加圧下、常圧（大気圧）下および減圧下のいずれかで行うことができるが、常圧下が好ましい。 An example of a method for manufacturing a bone regeneration member is as follows.
The components (A) to (E) in the composition of the present embodiment or the first to third solutions in the kit of the present embodiment are mixed, and the aqueous solution temperature (crosslinking temperature) is usually 4 to 50 ° C. Gelation can be carried out under the conditions of preferably 10 to 45 ° C., more preferably 20 to 40 ° C., and a reaction time of usually 1 minute to 48 hours, preferably 5 minutes to 30 hours. Each of the components (A) to (D) can be used as an aqueous solution of each component. The formation of the hydrogel can be performed under any of pressurized, normal pressure (atmospheric pressure), and reduced pressure, but is preferably performed under normal pressure.
　なお、所望の形状のキャビティーを有する金型を用意し、本実施形態の組成物の各成分（Ａ）～（Ｅ）、または本実施形態のキットにおける第１～第３の溶液を前記キャビティー中で混合して反応させることにより、前記形状を有するハイドロゲルを形成することができる。また、所定の基材上に本実施形態の組成物、または本実施形態のキットにおける第１～第３の溶液の混合液をキャストすることにより、フィルム状のハイドロゲルを得ることができ、前記フィルム状のハイドロゲルを例えば円柱形状、多角柱状等に打ち抜くこともできる。 A mold having a cavity having a desired shape is prepared, and the components (A) to (E) of the composition of the present embodiment or the first to third solutions in the kit of the present embodiment are added to the mold. By mixing and reacting in a tea, a hydrogel having the above shape can be formed. Further, a film-like hydrogel can be obtained by casting a composition of the present embodiment or a mixture of the first to third solutions in the kit of the present embodiment on a predetermined substrate. The film-shaped hydrogel can be punched into, for example, a columnar shape, a polygonal column shape, or the like.
　骨再生用部材の形状は特に限定されないが、例えば、フィルム状、円柱形状、多角柱状、不織布状、ファイバー状、チューブ状、粒子状、メッシュ状が挙げられる。
　本実施形態の骨再生用部材中には骨形成タンパク質（Ｄ）が担持しており、したがって本実施形態では前記骨再生用部材の適用部位（例：骨の欠損部位、骨折部位）に骨形成タンパク質（Ｄ）を長期間維持できると考えられる。また、前記ハイドロゲルは患部において細胞外マトリックスとして機能し、この細胞外マトリックスは骨再生する細胞の足場となり、骨再生する細胞が定着して、良好に増殖および分化できると考えられる。したがって、本実施形態の骨再生用部材は、骨再生効果が高い。 The shape of the bone regeneration member is not particularly limited, and examples thereof include a film shape, a cylindrical shape, a polygonal column shape, a nonwoven fabric shape, a fiber shape, a tube shape, a particle shape, and a mesh shape.
In the bone regeneration member of the present embodiment, the bone morphogenetic protein (D) is carried. Therefore, in the present embodiment, the bone regeneration component is applied to a site to which the bone regeneration member is applied (eg, a bone defect site or a fracture site). It is thought that protein (D) can be maintained for a long period of time. In addition, the hydrogel functions as an extracellular matrix in the affected area, and the extracellular matrix is considered to be a scaffold for bone-regenerating cells. Therefore, the bone regeneration member of the present embodiment has a high bone regeneration effect.
　［骨再生方法］
　本実施形態の骨再生用組成物、骨再生用組成物キットおよび骨再生用部材は、骨再生用途に用いられる。例えば、骨再生を必要とする対象（例えば、ヒト、ヒトを除く哺乳動物）の骨の欠損部位、骨折部位等の患部に、骨再生用組成物を投与する、もしくは骨再生用組成物キットの第１～第３の溶液を混合してなる混合物を投与することにより、骨再生用部材を患部に形成する、または前記患部に骨再生用部材を適用することにより、骨再生を誘導し、骨の癒合率を向上させることができる。 [Bone regeneration method]
The composition for bone regeneration, the composition kit for bone regeneration, and the member for bone regeneration of the present embodiment are used for bone regeneration. For example, a bone regeneration composition is administered to an affected area such as a bone defect site or a fracture site of a subject requiring bone regeneration (for example, a human or a mammal other than a human), or a bone regeneration composition kit. By administering a mixture obtained by mixing the first to third solutions, a bone regeneration member is formed in an affected part, or by applying the bone regeneration member to the affected part, bone regeneration is induced, and bone regeneration is induced. Can improve the fusion rate.
　図１に、一例として本実施形態の骨再生用組成物を用いた場合における骨再生の推定スキームを記載する。患部３０に投与された骨再生用組成物はハイドロゲル４０を形成し、ハイドロゲル４０中には骨形成タンパク質５０が良好に保持される。骨形成タンパク質５０は、骨細胞の前駆細胞である間葉系幹細胞、骨芽細胞２０の分化または増殖を促進する。その結果、大きな新生骨（仮骨）７０が形成される。なお、図１中では骨細胞は省略している。 FIG. 1 shows an example of a scheme for estimating bone regeneration when the composition for bone regeneration of the present embodiment is used. The composition for bone regeneration administered to the affected area 30 forms a hydrogel 40, and the bone morphogenetic protein 50 is favorably retained in the hydrogel 40. The bone morphogenetic protein 50 promotes differentiation or proliferation of a mesenchymal stem cell, which is a precursor cell of the bone cell, and the osteoblast 20. As a result, a large new bone (callus) 70 is formed. In FIG. 1, bone cells are omitted.
　本実施形態の再生対象である骨は、網目状に形成されたコラーゲン線維にヒドロキシアパタイトが沈着したものであり、骨の有機質の大部分はコラーゲンである。
　本実施形態の骨再生用組成物、骨再生用組成物キットまたは骨再生用部材は、その使用目的に合わせて、用法、用量および形状を適宜決定することができる。例えば、骨再生用組成物、または骨再生用組成物キットの第１～第３の溶液の混合物は、生体内の骨の欠損部位、骨折部位等の患部に、直接、例えば注射、塗布等により投与することができる。また、前記組成物または混合物を適当な賦形剤と混合し、軟膏状、ゲル状またはクリーム状にしてから、前記患部に塗布することもできる。 The bone to be regenerated in the present embodiment is formed by depositing hydroxyapatite on a collagen fiber formed in a network, and most of the organic matter of the bone is collagen.
The usage, dosage, and shape of the composition for bone regeneration, the composition kit for bone regeneration, or the member for bone regeneration of the present embodiment can be appropriately determined according to the purpose of use. For example, the composition for bone regeneration or the mixture of the first to third solutions of the composition kit for bone regeneration is directly applied to an affected part such as a bone defect site or a fracture site in a living body by, for example, injection, application, or the like. Can be administered. Alternatively, the composition or mixture may be mixed with an appropriate excipient to form an ointment, gel, or cream, and then applied to the affected area.
　本実施形態の骨再生用組成物、骨再生用組成物キットおよび骨再生用部材の対象疾患としては、前述したように、難治性骨折が挙げられる。 疾患 The target disease of the bone regeneration composition, the bone regeneration composition kit and the bone regeneration member of the present embodiment includes, as described above, intractable fracture.
　以下、本実施形態を実施例に基づいてさらに具体的に説明するが、本実施形態はこれら実施例に限定されない。
　＜変性多糖の製造＞
　［合成例１］ヒドロキシフェニル基変性ヒアルロン酸（Ａ１）の合成
　窒素雰囲気下、フラスコに、ヒアルロン酸（品名「HA-LF5-A」、キューピー株式会社製）４０ｇ、ジメチルホルムアミド１６００ｍｌ、および塩化リチウム３０．９ｇを加え、９０℃で９０分間攪拌した。得られた溶液を０℃に冷却し、ピリジン９．２ｍｌとクロロギ酸ニトロフェニル２３．８ｇとを加え、０℃で１時間撹拌した。次いで、前記フラスコに、冷やしたエタノール２０００ｍｌを加え、析出物を濾別し、濾物を冷やしたエタノールで洗浄し、乾燥した。 Hereinafter, the present embodiment will be described more specifically based on examples, but the present embodiment is not limited to these examples.
<Production of modified polysaccharide>
[Synthesis Example 1] Synthesis of hydroxyphenyl group-modified hyaluronic acid (A1) Under a nitrogen atmosphere, 40 g of hyaluronic acid (product name "HA-LF5-A", manufactured by Kewpie Co., Ltd.), 1600 ml of dimethylformamide, and lithium chloride 30 were placed in a flask. Then, the mixture was stirred at 90 ° C. for 90 minutes. The obtained solution was cooled to 0 ° C, 9.2 ml of pyridine and 23.8 g of nitrophenyl chloroformate were added, and the mixture was stirred at 0 ° C for 1 hour. Then, 2000 ml of cooled ethanol was added to the flask, the precipitate was separated by filtration, and the precipitate was washed with cooled ethanol and dried.
　窒素雰囲気下、フラスコに、乾燥後の前記濾物をジメチルホルムアミド７４０ｍｌで溶解した溶液を入れ、次いでチラミン９．１ｇを入れ、室温で３時間撹拌した。次いで、前記フラスコに、冷やしたエタノール８００ｍｌを加え、析出物を濾別し、濾物を純水に溶解させ透析した。 (4) Under a nitrogen atmosphere, a solution prepared by dissolving the dried residue in 740 ml of dimethylformamide was added to a flask, and 9.1 g of tyramine was added to the flask. The mixture was stirred at room temperature for 3 hours. Next, 800 ml of cooled ethanol was added to the flask, the precipitate was separated by filtration, and the filtrate was dissolved in pure water and dialyzed.
　このようにして、ヒドロキシフェニル基変性ヒアルロン酸（Ａ１）を得た。ヒドロキシフェニル基変性ヒアルロン酸（Ａ１）を1Ｈ ＮＭＲにて分析したところ、ヒドロキシフェニル基による置換度（ＤＳ）は３．２であった。 Thus, a hydroxyphenyl group-modified hyaluronic acid (A1) was obtained. When the hydroxyphenyl group-modified hyaluronic acid (A1) was analyzed by 1 H NMR, the degree of substitution (DS) with the hydroxyphenyl group was 3.2.
　［合成例２］ヒドロキシフェニル基変性デキストラン（ＡＲ１）の合成
　合成例１において、ヒアルロン酸の代わりにデキストラン（品名「Dextran 40 powder」、名糖工業株式会社製）４０ｇを用いたこと以外は同様の手法にて、ヒドロキシフェニル基変性デキストラン（ＡＲ１）を得た。ヒドロキシフェニル基変性デキストラン（ＡＲ１）を1Ｈ ＮＭＲにて分析したところ、ヒドロキシフェニル基によるＤＳは１２であった。 [Synthesis Example 2] Synthesis of hydroxyphenyl group-modified dextran (AR1) The same procedure as in Synthesis Example 1 except that 40 g of dextran (trade name “Dextran 40 powder”, manufactured by Meito Industry Co., Ltd.) was used instead of hyaluronic acid. By the method, hydroxyphenyl group-modified dextran (AR1) was obtained. When the hydroxyphenyl group-modified dextran (AR1) was analyzed by 1 H NMR, the DS by the hydroxyphenyl group was 12.
　＜骨再生用組成物キットの第１～第３の溶液の製造＞
　［製造例１］第１の溶液（１）の製造
　合成例１で得られたヒドロキシフェニル基変性ヒアルロン酸（Ａ１）０．２ｇを、リン酸緩衝生理食塩水（品名「ＰＢＳ（－）」、和光純薬工業株式会社（製））０．８ｇに溶解しポリマー溶液を準備した。前記ポリマー溶液１．０ｇと、西洋わさびペルオキシダーゼ（和光純薬製、商品コード「１６９－１０７９１」）水溶液（濃度：３０Ｕ／ｍｌ）１．０ｇとを均一に混合し、第１の溶液（１）を製造した。 <Production of first to third solutions of composition kit for bone regeneration>
[Production Example 1] Production of First Solution (1 ) 0.2 g of the hydroxyphenyl group-modified hyaluronic acid (A1) obtained in Synthesis Example 1 was added to a phosphate buffered saline (trade name “PBS (−)”, The polymer was dissolved in 0.8 g of Wako Pure Chemical Industries, Ltd. (manufactured by Wako Pure Chemical Industries, Ltd.) to prepare a polymer solution. 1.0 g of the polymer solution and 1.0 g of an aqueous solution of horseradish peroxidase (manufactured by Wako Pure Chemical, product code “169-10791”) (concentration: 30 U / ml) were uniformly mixed to form a first solution (1). Was manufactured.
　［製造例２］第１の溶液（ｃ１）の製造
　合成例２で得られたヒドロキシフェニル基変性デキストラン（ＡＲ１）０．２ｇを、リン酸緩衝生理食塩水（品名「ＰＢＳ（－）」、和光純薬工業株式会社（製））０．８ｇに溶解しポリマー溶液を準備した。前記ポリマー溶液１．０ｇと、西洋わさびペルオキシダーゼ（和光純薬製、商品コード「１６９－１０７９１」）水溶液（濃度：０．８Ｕ／ｍｌ）１．０ｇとを均一に混合し、第１の溶液（ｃ１）を製造した。 [Preparation Example 2] Preparation of First Solution (c1 ) 0.2 g of the hydroxyphenyl group-modified dextran (AR1) obtained in Synthesis Example 2 was mixed with phosphate buffered saline (product name "PBS (-)", The polymer solution was prepared by dissolving in 0.8 g of Kojun Pharmaceutical Co., Ltd. 1.0 g of the above polymer solution and 1.0 g of an aqueous solution of horseradish peroxidase (manufactured by Wako Pure Chemical, product code "169-10791") (concentration: 0.8 U / ml) were uniformly mixed to form a first solution ( c1) was prepared.
　［調製例１］第２の溶液（１）の準備
　過酸化水素水（濃度：８８２ｍＭ）を、第２の溶液（１）とした。 [Preparation Example 1] Preparation of Second Solution (1) Hydrogen peroxide solution (concentration: 882 mM) was used as a second solution (1).
　［製造例３］第３の溶液（１）の製造
　ＢＭＰ－２（ＰＥＰＲＯＴＥＣＨ社製）を前記ＰＢＳ（－）で濃度が２μｇ／μｌとなるように調整し、第３の溶液（１）を準備した。 [Production Example 3] Production of third solution (1) BMP-2 (manufactured by PEPROTECH) was adjusted to a concentration of 2 µg / µl with the PBS (-) to prepare a third solution (1). did.
　＜患部への骨再生用組成物の適用および評価＞
　［実施例１］
　第３の溶液（１）１４．４μｌと、第１の溶液（１）１６５．６μｌとを均一に混合し、混合液を得た。前記混合液１５μｌに第２の溶液（１）１５μｌを混合し、骨再生用組成物を準備した。
　Ｃ５７ＢＬ／６Ｊマウス（オス、生後９週齢）の大腿骨を、骨膜を電気メスで焼灼した後に骨折させた。前記マウスの骨折部位に前記骨再生用組成物２５μｌを投与した（図１参照）。６週間後、Ｃ５７ＢＬ／６Ｊマウスを屠殺し、骨癒合率をｍｉｃｒｏＣＴにて測定した。骨癒合率は８０％（８／１０）であった。６週間後のｍｉｃｒｏＣＴの画像を図２に示す。 <Application and evaluation of composition for bone regeneration to affected area>
[Example 1]
14.4 μl of the third solution (1) and 165.6 μl of the first solution (1) were uniformly mixed to obtain a mixed solution. 15 μl of the second solution (1) was mixed with 15 μl of the mixture to prepare a composition for bone regeneration.
The femur of a C57BL / 6J mouse (male, 9 weeks old) was fractured after cauterizing the periosteum with an electric scalpel. 25 μl of the composition for bone regeneration was administered to the fracture site of the mouse (see FIG. 1). Six weeks later, C57BL / 6J mice were sacrificed, and the rate of bone fusion was measured by microCT. The bone union rate was 80% (8/10). The microCT image after 6 weeks is shown in FIG.
　［比較例１］
　実施例１において、第１の溶液（１）の代わりに、第１の溶液（ｃ１）を用いたこと以外は実施例１と同様にして、骨再生用組成物を準備した。次いで、実施例１と同様に、マウスの骨折部位に前記骨再生用組成物を投与し、６週間後マウスを屠殺し、骨癒合率をｍｉｃｒｏＣＴにて測定した。骨癒合率は１０％（１／１０）であった。６週間後のｍｉｃｒｏＣＴの画像を図２に示す。 [Comparative Example 1]
A composition for bone regeneration was prepared in the same manner as in Example 1 except that the first solution (c1) was used instead of the first solution (1). Next, the composition for bone regeneration was administered to the fracture site of the mouse in the same manner as in Example 1, and after 6 weeks, the mouse was sacrificed and the bone fusion rate was measured by microCT. The bone union rate was 10% (1/10). The microCT image after 6 weeks is shown in FIG.
　［比較例２］
　実施例１において、第３の溶液（１）の代わりに、リン酸緩衝生理食塩水（品名「ＰＢＳ（－）」、和光純薬工業株式会社（製））を用いたこと以外は実施例１と同様にして、骨再生用組成物を準備した。次いで、実施例１と同様に、マウスの骨折部位に前記骨再生用組成物を投与し、６週間後マウスを屠殺し、骨癒合率をｍｉｃｒｏＣＴにて測定した。骨癒合率は１０％（１／１０）であった。６週間後のｍｉｃｒｏＣＴの画像を図２に示す。 [Comparative Example 2]
Example 1 was the same as Example 1 except that the third solution (1) was replaced with a phosphate buffered saline (product name “PBS (−)”, manufactured by Wako Pure Chemical Industries, Ltd.). In the same manner as in the above, a composition for bone regeneration was prepared. Next, the composition for bone regeneration was administered to the fracture site of the mouse in the same manner as in Example 1, and after 6 weeks, the mouse was sacrificed and the bone fusion rate was measured by microCT. The bone union rate was 10% (1/10). The microCT image after 6 weeks is shown in FIG.
　［実験例１］ｃｏｎｔｒｏｌ
　大腿骨の骨膜を電気メスで焼灼した後に大腿骨を骨折させたＣ５７ＢＬ／６Ｊマウス（オス、生後９週齢）をｃｏｎｔｒｏｌとした。骨癒合率は１０％（１／１０）であった。６週間後のｍｉｃｒｏＣＴの画像を図２に示す。 [Experimental example 1] control
A C57BL / 6J mouse (male, 9 weeks old) in which the femur was fractured after cauterizing the periosteum of the femur with an electric scalpel was used as control. The bone union rate was 10% (1/10). The microCT image after 6 weeks is shown in FIG.
　＜考察＞
　図２の上側が前記大腿骨の横視図、下側が前記大腿骨の骨断面図であり、濃灰色部が新生骨である。実施例１は、比較例１～２および実験例１と比較して骨癒合率が高く、本実施形態の骨再生用組成物は、骨膜の損傷などを含む難治性骨折に有用であることが明らかになった。 <Discussion>
The upper side of FIG. 2 is a lateral view of the femur, the lower side is a cross-sectional view of the femur, and the dark gray portion is a new bone. Example 1 has a higher bone union rate than Comparative Examples 1 and 2 and Experimental Example 1, and the composition for bone regeneration of this embodiment is useful for intractable fractures including periosteal damage. It was revealed.
１０：骨
１５：骨髄
２０：間葉系幹細胞、骨芽細胞（骨膜）
３０：患部（骨の欠損部位、骨折部位）
４０：ハイドロゲル
５０：骨形成タンパク質
６０：増殖した骨芽細胞
７０：新生骨
８０：新生骨膜 10: bone 15: bone marrow 20: mesenchymal stem cells, osteoblasts (periosteum)
30: Affected area (bone defect site, fracture site)
40: Hydrogel 50: Bone morphogenetic protein 60: Proliferated osteoblast 70: New bone 80: Neoperiosteum

Claims (10)

  1. 　ヒアルロン酸にヒドロキシアリール基が導入された変性ヒアルロン酸およびその塩から選ばれる少なくとも１種の成分（Ａ）と、
    　前記ヒドロキシアリール基の酸化カップリング酵素（Ｂ）と、
    　過酸化物（ｃ１）および過酸化物を生成する物質（ｃ２）から選ばれる少なくとも１種の成分（Ｃ）と、
    　骨形成タンパク質（Ｄ）と、
    　水（Ｅ）と
    を含有する骨再生用組成物。     At least one component (A) selected from a modified hyaluronic acid in which a hydroxyaryl group is introduced into hyaluronic acid and a salt thereof;
    Said hydroxyaryl group oxidative coupling enzyme (B);
    At least one component (C) selected from a peroxide (c1) and a substance (c2) that generates peroxide;
    Osteogenic protein (D),
    A composition for bone regeneration containing water (E).
  2. 　前記ヒドロキシアリール基が、チラミン由来の基である請求項１に記載の骨再生用組成物。 The composition for bone regeneration according to claim 1, wherein the hydroxyaryl group is a group derived from tyramine.
  3. 　前記成分（Ａ）における前記ヒドロキシアリール基による置換度が、０．１～５０である請求項１または２に記載の骨再生用組成物。 (4) The composition for bone regeneration according to (1) or (2), wherein the degree of substitution of the component (A) with the hydroxyaryl group is 0.1 to 50.
  4. 　前記酸化カップリング酵素（Ｂ）が、西洋わさびペルオキシダーゼである請求項１～３のいずれか１項に記載の骨再生用組成物。 The composition for bone regeneration according to any one of claims 1 to 3, wherein the oxidative coupling enzyme (B) is horseradish peroxidase.
  5. 　前記成分（Ｃ）が、前記過酸化物（ｃ１）である請求項１～４のいずれか１項に記載の骨再生用組成物。 骨 The bone regeneration composition according to any one of claims 1 to 4, wherein the component (C) is the peroxide (c1).
  6. 　難治性骨折用である請求項１～５のいずれか１項に記載の骨再生用組成物。 The composition for bone regeneration according to any one of claims 1 to 5, which is used for intractable fractures.
  7. 　第１の溶液と、第２の溶液と、第３の溶液とを有する骨再生用組成物キットであり、
    　前記第１の溶液が、ヒアルロン酸にヒドロキシアリール基が導入された変性ヒアルロン酸およびその塩から選ばれる少なくとも１種の成分（Ａ）と水（Ｅ）とを含有し、前記ヒドロキシアリール基の酸化カップリング酵素（Ｂ）、ならびに過酸化物（ｃ１）および過酸化物を生成する物質（ｃ２）から選ばれる少なくとも１種の成分（Ｃ）、のいずれかを含有するが両方は含有せず、
    　前記第２の溶液が、前記酸化カップリング酵素（Ｂ）および前記成分（Ｃ）のうち第１の溶液中で含有されない方を含有し、
    　前記第３の溶液が、骨形成タンパク質（Ｄ）を含有する、
    骨再生用組成物キット。     A bone regeneration composition kit comprising a first solution, a second solution, and a third solution,
    The first solution contains at least one component (A) selected from a modified hyaluronic acid in which a hydroxyaryl group has been introduced into hyaluronic acid and a salt thereof, and water (E), and oxidizes the hydroxyaryl group. It contains a coupling enzyme (B) and at least one component (C) selected from a peroxide (c1) and a peroxide-generating substance (c2), but not both,
    The second solution contains one of the oxidative coupling enzyme (B) and the component (C) that is not contained in the first solution;
    The third solution contains an osteogenic protein (D);
    A composition kit for bone regeneration.
  8. 　難治性骨折用である請求項７に記載の骨再生用組成物キット。 The composition kit for bone regeneration according to claim 7, which is used for intractable fractures.
  9. 　請求項１～６のいずれか１項に記載の骨再生用組成物、または請求項７もしくは８に記載の骨再生用組成物キットを用いて形成した骨再生用部材。 部 材 A bone regeneration member formed using the bone regeneration composition according to any one of claims 1 to 6, or the bone regeneration composition kit according to claim 7 or 8.
  10. 　請求項１～６のいずれか１項に記載の骨再生用組成物、請求項７もしくは８に記載の骨再生用組成物キット、または請求項９に記載の骨再生用部材を用いて、骨を再生させる骨再生方法。 Bone regeneration using the composition for bone regeneration according to any one of claims 1 to 6, the composition kit for bone regeneration according to claim 7 or 8, or the member for bone regeneration according to claim 9 Regenerate bone regeneration method.
PCT/JP2019/037277 2018-09-28 2019-09-24 Composition for bone regeneration, kit of composition for bone regeneration, member for bone regeneration and bone regeneration method WO2020067014A1 (en)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006517598A (en) * 2003-01-10 2006-07-27 ザ クリーヴランド クリニック ファウンデーション Hydroxyphenyl cross-linked polymer network and uses thereof
JP2015508118A (en) * 2012-02-28 2015-03-16 コンティプロ ビオテック スポレチノスト エス ルチェニム オメゼニム Hydrogel-formable derivatives based on hyaluronic acid, their preparation, hydrogels based on said derivatives, their preparation and use
JP2017529208A (en) * 2014-08-28 2017-10-05 バイオヴェンタス・エルエルシー Improved osteoinductive substrate and method for making the same
JP2018123241A (en) * 2017-02-01 2018-08-09 Jsr株式会社 Modified polysaccharide and use therefor
JP2018162440A (en) * 2017-03-27 2018-10-18 Jsr株式会社 Composition and use therefor
JP2018188561A (en) * 2017-05-09 2018-11-29 Jsr株式会社 Denatured polysaccharide and use therefor
JP2018199766A (en) * 2017-05-26 2018-12-20 Jsr株式会社 Modified polysaccharide and use therefor

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006517598A (en) * 2003-01-10 2006-07-27 ザ クリーヴランド クリニック ファウンデーション Hydroxyphenyl cross-linked polymer network and uses thereof
JP2015508118A (en) * 2012-02-28 2015-03-16 コンティプロ ビオテック スポレチノスト エス ルチェニム オメゼニム Hydrogel-formable derivatives based on hyaluronic acid, their preparation, hydrogels based on said derivatives, their preparation and use
JP2017529208A (en) * 2014-08-28 2017-10-05 バイオヴェンタス・エルエルシー Improved osteoinductive substrate and method for making the same
JP2018123241A (en) * 2017-02-01 2018-08-09 Jsr株式会社 Modified polysaccharide and use therefor
JP2018162440A (en) * 2017-03-27 2018-10-18 Jsr株式会社 Composition and use therefor
JP2018188561A (en) * 2017-05-09 2018-11-29 Jsr株式会社 Denatured polysaccharide and use therefor
JP2018199766A (en) * 2017-05-26 2018-12-20 Jsr株式会社 Modified polysaccharide and use therefor

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHANG, Y. ET AL.: "Injectable hydrogels from enzyme-catalyzed crosslinking as BMSCs-laden scaffold for bone repair and regeneration", MATER. SCI. ENG. C MATER. BIOL. APPL., vol. 9 6, March 2019 (2019-03-01), pages 841 - 849, XP085569824 *

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