JP2018199766A - Modified polysaccharide and use therefor - Google Patents

Abstract

To provide a modified polysaccharide that has excellent adhesiveness even in a non-dry condition (for example, in the presence of moisture), and is suitable as a component of compositions such as bone cement.SOLUTION: The present invention provides a modified polysaccharide in which a phosphate group (a1) and a radical reactive group (a2) are introduced into a polysaccharide.SELECTED DRAWING: None

Description

  The present invention relates to modified polysaccharides and uses thereof.

  Bone cement is used as a filler for missing bone, an adhesive between artificial bone and natural bone, and a filler for balloon kyphoplasty. Patent Document 1 proposes a bone cement containing a polysaccharide having a phosphate group (phosphorylated polysaccharide) and calcium.

  In this bone cement, hydroxyapatite is gradually formed from the phosphoric acid group of phosphorylated polysaccharide and calcium, and since this hydroxyapatite is close in composition to natural bone, it can be chemically combined with natural bone. Known as a cement for use.

International Publication No. 2011/102530

According to the study of the present inventor, the bone cement disclosed in Patent Document 1 has excellent adhesiveness in a dry state, but it becomes clear that the adhesiveness is weak in a non-dry state.
An object of the present invention is to provide a composition such as a bone cement which is excellent in adhesiveness even in a non-dry state (for example, in the presence of moisture), a modified polysaccharide preferably used as a component of the composition, and the composition The object is to provide a gel obtained and a molded body formed from the gel.

The present inventor has intensively studied to solve the above problems. As a result, it has been found that a modified polysaccharide having the following constitution can solve the above-mentioned problems, and the present invention has been completed.
The present invention includes, for example, the following [1] to [11].

[1] A modified polysaccharide in which a phosphate group (a1) and a radical reactive group (a2) are introduced into the polysaccharide.
[2] The modified polysaccharide according to [1], wherein the polysaccharide is pullulan or a derivative thereof.
[3] The modified polysaccharide according to [1] or [2], wherein the radical reactive group (a2) is a group (a21) having a hydroxyaryl group.
[4] The modified polysaccharide according to [3], wherein the group (a21) having a hydroxyaryl group is a group represented by the following formula (1).

[In formula (1), * is a bond with a hydroxyl group residue in the polysaccharide, and R ¹ is an alkanediyl group having 1 to 20 carbon atoms or — (CH ₂ ) _n —O— (CH ₂ ). _{m- represents a} divalent group, and n and m each independently represent an integer of 1 to 10; R ^{2 to} R ⁶ each independently represents a hydrogen atom, a hydroxyl group, an alkoxy group, or a substituent. It is an alkyl group which may have, provided that at least one of R ^{2 to} R ⁶ is a hydroxyl group. ]

[5] A composition comprising the modified polysaccharide according to any one of [1] to [4], an enzyme, and water.
[6] The composition according to [5], further containing a peroxide.
[7] The composition according to [5] or [6] above, further containing at least one metal salt selected from alkali metal salts and Group 2 metal salts of the periodic table.
[8] A gel having a crosslinked structure by oxidative coupling of a hydroxyaryl group contained in the modified polysaccharide according to [3] or [4].
[9] A gel formed from the composition according to any one of [5] to [7].
[10] The gel according to [8] or [9], which is a hydrogel containing 10 to 90% by mass of moisture.
[11] A molded article containing 50% by mass or more of the gel according to any one of [8] to [10].

  According to the present invention, a composition such as a bone cement excellent in adhesiveness even in a non-dried state (for example, in the presence of water), a modified polysaccharide preferably used as a component of the composition, and the composition The resulting gel and a molded body formed from the gel can be provided.

FIG. 1 is a ¹ H NMR chart of the modified pullulan obtained in Synthesis Example 1. FIG. 2 is a ¹ H NMR chart of the modified pullulan obtained in Example 1. FIG. 3 is a ¹ H NMR chart of the modified pullulan obtained in Synthesis Example 2. FIG. 4 is an HPLC analysis chart of the modified pullulan obtained in Example 1 and Synthesis Example 2.

DESCRIPTION OF EMBODIMENTS Hereinafter, embodiments for carrying out the present invention will be described including preferred embodiments.
[Modified polysaccharide]
The modified polysaccharide of the present invention is a compound in which a phosphate group (a1) and a radical reactive group (a2) are introduced into the polysaccharide. Hereinafter, the compound in which the polysaccharide is modified in the above (a1) and / or (a2) is generally referred to as “modified polysaccharide”, and the compound in which the polysaccharide is modified in the above (a1) and (a2) is referred to as “the present invention. It is called “modified polysaccharide”. Such a modified polysaccharide is preferable because it is excellent in biocompatibility or bioabsorbability when used as an adhesive component of a cement for living body hard tissue and an adhesive composition for living body hard tissue described later.

The biological hard tissue means biological tissues such as bones and teeth and tissues containing them.
In the present invention, “modified” means that a phosphate group (a1) or a radical reactive group (a2) is not connected to a functional group such as an alcoholic hydroxyl group of a polysaccharide via a linking group such as an organic group. Means that they are connected. Examples of the organic group in the linking group include a hydrocarbon group and a substituted hydrocarbon group. The substituted hydrocarbon group is a group in which a part of the hydrocarbon group is replaced with an ester bond, an ether bond, an amide bond, a carbonyl group, a hydroxy group, or the like.

  For example, the group (a2) is introduced into the polysaccharide by reacting a functional group such as an alcoholic hydroxyl group possessed by the polysaccharide with a compound having a group capable of reacting with the functional group and a radical reactive group (a2). be able to. In addition, a functional group such as an alcoholic hydroxyl group possessed by the polysaccharide is reacted with a compound having a group capable of reacting with the functional group and a phosphoric acid group (a1), or by reacting phosphoric acid with the functional group. (A1) can be introduced.

<Polysaccharide>
Examples of the polysaccharide before modification, that is, before introduction of the groups (a1) and (a2) include, for example, starch, amylose, amylopectin, glycogen, cellulose, pullulan, dextran, dextrin, hyaluronic acid, alginic acid, xanthan gum, guar gum, fructan, mannan Natural polysaccharides such as carrageenan, chitin, chitosan, pectin and gellan gum; and derivatives of the natural polysaccharide (eg, ether derivatives, ester derivatives).

  As the derivative, for example, the exemplified polysaccharide is a group other than the phosphate group (a1) and the radical reactive group (a2), for example, a methyl ether group, an ethyl ether group, a hydroxyethyl ether group, a hydroxypropyl ether group. , Derivatives having a substituent such as carboxymethyl ether group, acetyl group, stearoxy group, glycerol, propylene glycol and the like. The derivative may have the substituent alone or in combination.

  Specific examples of the derivatives include methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxyethylmethylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, stearoxyhydroxypropylmethylcellulose, acetylated hyaluronic acid, propylene glycol hyaluronate, and propylene alginate. Examples include glycol, methyl guar gum, ethyl guar gum, hydroxyethyl guar gum, hydroxypropyl guar gum, hydroxyethyl methyl guar gum, and hydroxypropyl methyl guar gum.

  The polysaccharide may be a salt, and examples thereof include acid addition salts, metal salts, and ammonium salts. Examples of the acid addition salt include inorganic acid salts such as hydrochloride and sulfate, and organic acid salts such as acetate, maleate, fumarate, tartrate and citrate. Examples of the metal salt include alkali metal salts such as sodium salt and potassium salt, Group 2 metal salts of periodic table such as magnesium salt and calcium salt, aluminum salt and zinc salt. For example, the metal salt of the polysaccharide which has a carboxy group is mentioned.

  Among polysaccharides, a polysaccharide having a glucose unit from the viewpoint of adhesion to a living hard tissue and the strength of the resulting gel, and a group (a21) having a hydroxyaryl group described later can be easily introduced into the polysaccharide. And its derivatives are preferred, and pullulan and its derivatives are more preferred. For example, a modified pullulan and a derivative thereof into which a group (a21) having a hydroxyaryl group, which will be described later, has been introduced, can be gelled by, for example, a peroxidase / peroxide catalyst system.

<Phosphate group (a1)>
The modified polysaccharide of the present invention has a phosphate group (a1).
The phosphate group is represented by, for example, —OP (═O) (OH) ₂ , and depending on the pH, part or all of the phosphate group may take the form of —OP (═O) (O ⁻ ) ₂ , A part or all of them may be a salt, and examples thereof include alkali metal salts such as sodium salt and potassium salt, Group 2 metal salts of periodic table such as magnesium salt and calcium salt, and ammonium salts.

  The modified polysaccharide having a phosphate group (a1) is excellent in adhesion to calcium. For example, the phosphate group (a1) dissolves apatite, which is an inorganic component constituting the living hard tissue, and releases calcium ions. It is considered that the modified polysaccharide adheres to the living hard tissue by the chelate bonding of the phosphate group (a1) of the modified polysaccharide to the released calcium ions and the calcium atoms remaining on the apatite surface. Similarly, the phosphate property (a1) of the modified polysaccharide is chelate-bonded to metal or ceramics which are prosthetic restorations of living hard tissues, and exhibits adhesiveness. Further, hydroxyapatite is gradually formed from the phosphate group (a1) of the modified polysaccharide and calcium, and since this hydroxyapatite is close to the component of natural bone, it can be chemically bonded to natural bone.

Thus, since the modified polysaccharide of this invention has a phosphate group (a1), it can be used as an adhesive component of the cement for living body hard tissues and the adhesive composition for living body hard tissues.
The modified polysaccharide having a phosphate group can be produced, for example, by a known method for phosphorylating a hydroxyl group of a polysaccharide described in paragraph [0019] of International Publication No. 2011/102530. In addition to heating and stirring.

  For example, a polysaccharide and phosphoric acid, or a modified polysaccharide having a radical reactive group (a2) and phosphoric acid are reacted. For example, the solvent is distilled off from the solution containing the polysaccharide or the modified polysaccharide having the group (a2) and phosphoric acid, and then the residue is subjected to heat dehydration condensation. The solvent is distilled off, for example, under reduced pressure, usually at 0 to 150 ° C., preferably 20 to 80 ° C. Heat dehydration condensation is performed by heating the residue after evaporation of the solvent under reduced pressure, and the heating temperature here is usually 50 to 150 ° C., preferably 80 to 130 ° C., and the reaction time is usually 1 -92 hours, preferably 2-48 hours. The decompression condition is, for example, less than 760 mmHg, preferably 1 to 500 mmHg. The product after heat dehydration condensation may be dissolved in water and purified by reprecipitation with a poor solvent such as ethanol, ethyl acetate, or n-hexane.

  In the reaction, phosphoric acid is usually 10 to 1000 parts by weight, preferably 50 to 700 parts by weight, more preferably 100 parts by weight of polysaccharide or 100 parts by weight of modified polysaccharide having a radical reactive group (a2). Can be used in an amount of 100 to 500 parts by mass. In addition, in the said reaction, in order to adjust reaction rate, you may adjust pH with basic compounds, such as sodium hydroxide.

  The reaction is preferably performed in a solvent. Examples of the solvent include water, methanol, ethanol, isopropyl alcohol, N, N′-dimethylformamide, dimethyl sulfoxide, and dimethylacetamide. The reaction may be performed in an inert gas atmosphere such as nitrogen or argon.

<Radical reactive group (a2)>
As described above, the modified polysaccharide having a phosphate group (a1) is excellent in adhesion to a biological hard tissue containing calcium, but has low adhesion persistence in a non-dry state, particularly in water, Considering that the bond between the phosphate group (a1) and calcium is an ionic bond, it is considered that the adhesive force is likely to change with respect to the change in pH. Since the modified polysaccharide of the present invention has a radical reactive group (a2) in addition to the phosphate group (a1), it is an adhesive component with excellent adhesion persistence in water.

  In the present invention, the radical reactive group (a2) means a group that causes a chemical reaction by a radical. Examples of the radical reactive group include a group (a21) having a hydroxyaryl group and a group (a22) having a carbon-carbon unsaturated double bond. By introducing the group (a21) into the polysaccharide, the polysaccharide The group (a21) is preferred from the standpoint of weakening the water solubility of the resin, having water retention, and maintaining adhesion in a non-dry state.

  The hydroxyaryl group is a group capable of radical reaction as described later. As a hydroxyaryl group, C6-C14 hydroxyaryl groups, such as a hydroxyphenyl group and a hydroxy naphthyl group, are mentioned, for example. The number of hydroxyl groups in the hydroxyaryl group is usually 1 to 3, preferably 1. Further, the hydroxyaryl group may have an alkoxy group, an alkyl group which may have a substituent, or the like in addition to the hydroxyl group.

  The group (a21) is preferably a group represented by the following formula (1). For example, the modified polysaccharide of the present invention has a group represented by the formula (1) bonded to a hydroxyl group residue in the polysaccharide.

Each symbol in Formula (1) is demonstrated below.

  * Is a bond with a hydroxyl group residue (—O—) in the polysaccharide. When the polysaccharide has a glucose unit, for example, it is preferable that the hydroxyl group residue in the hydroxymethyl group in the glucose unit is bonded to the group represented by the formula (1).

R ¹ is an alkanediyl group having 1 to 20 carbon atoms or a divalent group represented by — (CH ₂ ) _n —O— (CH ₂ ) _m —, and n and m are each independently 1 to 10 Indicates an integer.
Examples of the alkanediyl group include methane-1,1-diyl group, ethane-1,1-diyl group, ethane-1,2-diyl group, propane-1,1-diyl group, propane-1,2- Diyl group, propane-1,3-diyl group, propane-2,2-diyl group, butane-1,2-diyl group, butane-1,3-diyl group, butane-1,4-diyl group, pentane- 1,4-diyl group, pentane-1,5-diyl group, hexane-1,5-diyl group, hexane-1,6-diyl group, heptane-1,7-diyl group, octane-1,8-diyl Groups. Carbon number of alkanediyl group becomes like this. Preferably it is 1-10, More preferably, it is 2-5.

R ^{2 to} R ⁶ are each independently a hydrogen atom, a hydroxyl group, an alkoxy group, or an alkyl group that may have a substituent, provided that at least one of R ^{2 to} R ⁶ is a hydroxyl group. It is preferable that only R ⁴ is a hydroxyl group among R ^{2 to} R ⁶ because oxidative coupling proceeds preferentially in the gelation described later and a gel superior in mechanical strength is obtained. It is preferable that a hydrogen atom is bonded to a carbon atom in at least one ortho position with respect to the hydroxyl group.

As an alkoxy group, C1-C6 alkoxy groups, such as a methoxy group, an ethoxy group, a propoxy group, a butoxy group, a pentyloxy group, a hexyloxy group, are mentioned, for example.
As an alkyl group, C1-C6 alkyl groups, such as a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, are mentioned, for example. One or more hydrogen atoms in the alkyl group may be substituted. Examples of the substituent include halogen atoms such as fluorine atom, chlorine atom and bromine atom, phenyl group, hydroxyl group, acetoxy group, alkoxy group, carboxy group, A cyano group and a nitro group are mentioned.

Of the R ^{2 to} R ⁶ , the remaining group other than the hydroxyl group is preferably a hydrogen atom or an alkoxy group, and more preferably a hydrogen atom.
As a method for introducing a group (a21) having a hydroxyaryl group into a polysaccharide, for example, a polysaccharide having a functional group such as an amino group, an alcoholic hydroxyl group and a carboxy group, and a functional group capable of reacting with the functional group (for example: And a method of reacting a compound having a carboxy group, an acid halide thereof, an amino group, an alcoholic hydroxyl group, an epoxy group, an oxetanyl group) and a hydroxyaryl group. For example, by reacting a hydroxyl group in a polysaccharide with —COX in a compound represented by the following formula (21) (hereinafter also referred to as “compound (21)”), a hydroxyphenyl group derived from the compound (21) is converted into an ester. It can be introduced into a polysaccharide via a bond.

In formula (21), R < ¹ > -R < ⁶ > is synonymous with the same symbol in Formula (1). X is —OH or a halogen atom. As a halogen atom, a fluorine atom, a chlorine atom, and a bromine atom are mentioned, for example, A chlorine atom is preferable.

  Examples of the compound (21) include 4-hydroxyphenylacetic acid, 3- (4-hydroxyphenyl) propionic acid, 4- (4-hydroxyphenyl) butanoic acid, 2- (2,5-dihydroxyphenyl) acetic acid, and These acid halides are mentioned.

  For example, the polysaccharide and the compound (21) or the modified polysaccharide having a phosphate group (a1) and the compound (21) are reacted. For example, the solvent is distilled off from a solution containing the polysaccharide or the modified polysaccharide having the group (a1) and the compound (21), and then the residue is subjected to heat dehydration condensation. The solvent is distilled off, for example, under reduced pressure, usually at 0 to 150 ° C., preferably 20 to 80 ° C. Heat dehydration condensation is performed by heating the residue after evaporation of the solvent under reduced pressure, and the heating temperature here is usually 50 to 150 ° C., preferably 80 to 130 ° C., and the reaction time is usually 1 -92 hours, preferably 2-48 hours. The decompression condition is, for example, less than 760 mmHg, preferably 1 to 500 mmHg. The product after heat dehydration condensation may be dissolved in water and purified by reprecipitation with a poor solvent such as ethanol, ethyl acetate, or n-hexane.

  In the reaction, the compound (21) is usually 1 to 100 parts by mass, preferably 5 to 80 parts by mass with respect to 100 parts by mass of the polysaccharide or 100 parts by mass of the modified polysaccharide having a phosphate group (a1). Preferably, it can be used in an amount of 10 to 60 parts by mass.

  The reaction is preferably performed in a solvent. Examples of the solvent include water, methanol, ethanol, isopropyl alcohol, N, N′-dimethylformamide, dimethyl sulfoxide, and dimethylacetamide. The reaction may be performed in an inert gas atmosphere such as nitrogen or argon.

  The carbon-carbon unsaturated double bond is preferably a radically polymerizable double bond, for example, as long as it is contained in the modified polysaccharide as a (meth) acryloyl group, a vinyl group or the like, and the (meth) acryloyl group is Preferably, an acryloyl group is more preferable.

  The group (a22) having a carbon-carbon unsaturated double bond is preferably a group ((meth) acryloyl group) represented by the following formula (2). For example, the modified polysaccharide of the present invention has a group represented by the formula (2) bonded to a hydroxyl group residue in the polysaccharide.

In the formula (2), R ^A represents a hydrogen atom or a methyl group.

  * Is a bond with a hydroxyl group residue (—O—) in the polysaccharide. When the polysaccharide has a glucose unit, for example, it is preferable that the hydroxyl group residue in the hydroxymethyl group in the glucose unit is bonded to the group represented by the formula (2).

  As a method for introducing a group (a22) having a carbon-carbon unsaturated double bond into a polysaccharide, for example, a polysaccharide having a functional group such as an amino group, an alcoholic hydroxyl group and a carboxy group can react with the functional group. A compound having a functional group (eg, carboxy group, acid halide thereof, amino group, alcoholic hydroxyl group, epoxy group, oxetanyl group) and a carbon-carbon unsaturated double bond (hereinafter referred to as “double bond-containing compound (31)”) (Also called).

  The double bond-containing compound (31) has a carboxy group or an acid halide thereof (group represented by —COX (X is a halogen atom such as a chlorine atom)) capable of reacting with an alcoholic hydroxyl group possessed by many polysaccharides. Compounds are preferred, for example, unsaturated monocarboxylic acids such as (meth) acrylic acid and crotonic acid; unsaturated dicarboxylic acids such as maleic acid, fumaric acid, citraconic acid, mesaconic acid and itaconic acid; and anhydrides of the unsaturated dicarboxylic acids A mono [(meth) acryloyloxyalkyl] ester of a polyvalent carboxylic acid such as mono [2- (meth) acryloyloxyethyl] succinate, mono [2- (meth) acryloyloxyethyl] phthalate, and the like; Acid halides such as (meth) acrylic acid chloride are mentioned.

  Other examples of the double bond-containing compound (31) include glycidyl (meth) acrylate, 2-methylglycidyl (meth) acrylate, 3,4-epoxybutyl (meth) acrylate, and (meth) acrylic acid 6,7. Epoxy group-containing (meth) acrylates such as epoxyheptyl, 3,4-epoxycyclohexylmethyl (meth) acrylate; o, m or p-vinylphenylglycidyl ether, o, m or p-isopropenylphenylglycidyl ether, Epoxy group-containing vinyl compounds such as o, m or p-vinylbenzylglycidyl ether, o, m or p-isopropenylbenzylglycidyl ether; 3-((meth) acryloyloxymethyl) oxetane, 3-((meth) acryloyloxy Methyl) -2-methyloxetane, 3-((meth) ac Royloxymethyl) -3-ethyloxetane, 3-((meth) acryloyloxymethyl) -2-phenyloxetane and other oxetanyl group-containing (meth) acrylates; 3- (p-vinylphenyloxymethyl) -3-methyloxetane , Oxetanyl group-containing vinyl compounds such as 3- (p-isopropenylphenyloxymethyl) -3-methyloxetane; 2-hydroxyethyl (meth) acrylate, 3-hydroxypropyl (meth) acrylate, (meth) acrylic acid Examples include hydroxyalkyl (meth) acrylates such as 4-hydroxybutyl.

  For example, a polysaccharide and a double bond-containing compound (31) or a modified polysaccharide having a phosphate group (a1) and a double bond-containing compound (31) are reacted in the presence or absence of a basic compound. Let The reaction temperature of the reaction is usually −10 to 50 ° C., preferably −5 to 30 ° C., and the reaction time is usually 1 to 48 hours.

Examples of the basic compound include trimethylamine, triethylamine, tributylamine, pyridine, N, N-dimethyl-4-aminopyridine, and piperidine.
In the reaction, the double bond-containing compound (31) is usually 0.1 to 50 parts by mass, preferably 0 to 100 parts by mass of the polysaccharide or 100 parts by mass of the modified polysaccharide having a phosphate group (a1). .3 to 20 parts by mass, more preferably 0.5 to 10 parts by mass.

  The reaction is preferably performed in a solvent. Examples of the solvent include water, methanol, ethanol, isopropyl alcohol, N, N′-dimethylformamide, dimethyl sulfoxide, and dimethylacetamide. The reaction may be performed in an inert gas atmosphere such as nitrogen or argon.

<Preferred embodiment>
The modified polysaccharide of the present invention is preferably a modified polysaccharide in which a phosphate group (a1) and a group having a hydroxyaryl group (a21) are introduced into the polysaccharide. The above group is introduced into a polysaccharide having a glucose unit or a derivative thereof. A modified polysaccharide is more preferable, and a modified polysaccharide in which the above group is introduced into pullulan or a derivative thereof is particularly preferable.

In the modified polysaccharide of the present invention, a phosphate group (a1) is introduced into the polysaccharide. The degree of substitution (DS _P ) with the group (a1) in the modified polysaccharide is usually 0.1 to 900, preferably 10 to 500, more preferably 100 to 300. Moreover, the content rate of the phosphorus atom contained in the modified polysaccharide of this invention is 0.1-25 mass% normally, Preferably it is 1-20 mass%.

In the modified polysaccharide of the present invention, a radical reactive group (a2) is introduced into the polysaccharide. The degree of substitution (DS _R ) by the group (a2) in the modified polysaccharide, preferably the degree of substitution (DS _Ar ) by the group (a21) having a hydroxyaryl group is usually 0.1 to 40, preferably 0. It is 5-20, More preferably, it is 1-15.

The phosphorus atom content can be determined by elemental analysis by atomic absorption spectrometry, the degree of substitution (DS _P ) can be determined by ³¹ P NMR, and the degree of substitution (DS _R ) can be determined by ¹ H NMR. Can be sought. The degree of substitution of the modified polysaccharide indicates the average number of groups (for example, the group (a1) or (a2)) introduced per 100 units of the constituent monosaccharide of the modified polysaccharide. Since the modified polysaccharide has such a substitution degree, the gelation rate of the modified polysaccharide is improved.

  The degree of substitution of the modified polysaccharide can be controlled, for example, by changing the reaction time, reaction temperature, and concentration of each reaction component described above. For example, the substitution degree can be lowered by shortening the reaction time, lowering the reaction temperature, or lowering the concentration of each reaction component.

  The number average molecular weight by the absolute molecular weight measurement of the modified polysaccharide of the present invention is usually 1,000 to 10,000,000, preferably 3,000 to 1,000,000, more preferably 5,000 to 500,000. is there.

[Composition]
The composition of the present invention contains the above-described modified polysaccharide of the present invention, an enzyme, and water, and preferably further contains a peroxide. One embodiment of the composition comprises a first solution comprising a modified polysaccharide, comprising either a peroxide and an enzyme, but not both, and a first solution of the aforementioned peroxide and enzyme. And a second solution including the one not included.

  The composition of the present invention contains the above-described modified polysaccharide of the present invention. In the composition, the content of the modified polysaccharide of the present invention is usually 8% by mass or more and less than 90% by mass, preferably 30 to 80% by mass. The modified polysaccharides can be used alone or in combination of two or more.

  The composition of the present invention contains an enzyme. The enzyme is preferably an enzyme capable of directly or indirectly catalyzing the crosslinking reaction of the radical reactive group (a2), preferably the group (a21) having a hydroxyaryl group.

  Examples of the enzyme include phenol oxidases such as laccase and tyrosinase, catalase, and peroxidase. Laccase and tyrosinase are excellent in that there is no possibility that the function-expressing substance is denatured during gelation because there is no need to add any other chemicals that do not constitute gel components. Peroxidase can be instantly gelled by using it together with a peroxide such as hydrogen peroxide. Examples of the origins of copper enzymes such as laccase and tyrosinase include urushi, mushrooms (Tuchikaburi, mushrooms), and molds (Polyporus vericolor). The origin of catalase and peroxidase is, for example, bovine liver, horse blood cells, human blood cells, M. lysodeikticus, horseradish (horseradish), soybean, radish, turnip, thyroid, milk, intestine, leukocytes, erythrocytes, yeast, Caldariomyces fumago, Steptococcus faecalis. Among these, tyrosinase is preferably derived from mushrooms and peroxidase is preferably derived from horseradish.

In the present invention, from the viewpoint of efficiently promoting the crosslinking reaction of the radical reactive group (a2) contained in the modified polysaccharide, it is preferable to use a peroxide and at least one or more selected from peroxidase and catalase as an enzyme. More preferably, peroxide is used and at least peroxidase is used as the enzyme.
Enzymes can be used alone or in combination of two or more.

  In the composition of the present invention, the amount of the enzyme is preferably 0.01 U / mL or more, more preferably 1 U / mL or more; 1,000 U / mL or less, more preferably 500 U / mL or less. U represents a unit of enzyme activity, and is the amount of enzyme that can change 1 micromole of substrate per minute at a temperature of 30 ° C. under optimum conditions.

  The composition of the present invention preferably contains a peroxide. An example of the peroxide is hydrogen peroxide. In the composition, the amount of peroxide is usually 0.01 to 1000 mol, preferably 1 mol to 1 mol of the radical reactive group (a2) in the modified polysaccharide of the present invention, preferably 1 mol of phenolic hydroxyl group. Is 0.1 to 500 mol, more preferably 0.5 to 200 mol. The phenolic hydroxyl group is, for example, a phenolic hydroxyl group contained in a hydroxyaryl group in the group (a21).

  The composition of the present invention preferably further contains at least one metal salt selected from alkali metal salts and Group 2 metal salts of the periodic table. However, the aforementioned modified polysaccharide is excluded from the metal salt. By using the metal salt, for example, the mechanical strength of the gel can be increased by chelate bonding between the phosphate group (a1) of the modified polysaccharide and the metal ion.

  Examples of the alkali metal salt include a lithium salt, a sodium salt, and a potassium salt. Examples of Group 2 metals of the periodic table include magnesium salts, calcium salts, strontium salts, and barium salts. The metal salt is preferably a halide or hydroxide of an alkali metal and Group 2 metal of the periodic table, and examples thereof include sodium chloride, magnesium chloride, calcium chloride, barium chloride, magnesium hydroxide, and calcium hydroxide.

Among these, when the composition of the present invention is used as a cement for living body hard tissue and an adhesive composition for living body hard tissue, a calcium salt is preferable.
The metal salts can be used alone or in combination of two or more.
The metal salt can be used in a form dissolved or dispersed in water.

  The content of the metal salt in the composition of the present invention is usually 0.1 to 30 parts by mass, preferably 0.5 to 20 parts by mass, more preferably 1 with respect to 100 parts by mass of the modified polysaccharide of the present invention. -10 parts by mass. It is preferable from a viewpoint of the mechanical strength of the gel obtained that content is more than the said lower limit. It is preferable for the content to be equal to or less than the upper limit since the fluidity when using the composition of the present invention is not insufficient.

The composition of the present invention contains water.
The content rate of the water of the composition of this invention is 10-90 mass% normally. With such an embodiment, the mechanical strength of the resulting gel tends to be excellent.

  The composition of the present invention may further contain a polysaccharide other than the modified polysaccharide. As this polysaccharide, the polysaccharide described in the column of [modified polysaccharide] is mentioned, for example. Polysaccharides can be used alone or in combination of two or more.

  The composition of the present invention may further contain a filler. Examples of the filler include tricalcium phosphate, magnesium phosphate, calcium hydrogen phosphate, hydroxyapatite, barium sulfate, silica, alumina, zirconia, and titania. Among these, barium sulfate can be used as a contrast agent. The filler can be used alone or in combination of two or more.

  In addition, the composition of the present invention may contain one or more selected from phosphoric acid, polyphosphoric acid, a pH adjuster, an ultraviolet absorber, a thickener, a colorant, and a filler. It may contain a function-expressing substance according to the above. Thereby, a function expression substance can be included in the gel obtained. Examples of the function expressing substance include drugs and cells.

  The composition of the present invention can be suitably used as a raw material composition for the following gel (cured product). The gel formed from the composition of the present invention has high strength, and exhibits high adhesion to biological hard tissues such as bones and teeth; metals such as titanium and stainless steel; ceramics and glass. Furthermore, the gel is excellent in adhesiveness even in a non-dry state (for example, in the presence of moisture).

  Therefore, the composition of the present invention is suitable as a cement for living hard tissues such as bone cement and dental cement. The cement has curability and is, for example, a material for repairing or filling bone or tooth defects or voids or low density parts.

  In one example, the composition of the present invention stabilizes a vertebral body collapsed by a spinal compression fracture by bringing the vertebral body into a shape prior to the fracture with a balloon and forms a space for cement injection. It is useful as the bone cement (filling material) in the balloon kyphoplasty (BKP) treatment method for injecting cement for use.

  In addition, the composition of the present invention is used for bone-to-bone fixation, bone-to-medical device fixation such as a fixation screw, artificial joint to surrounding bone fixation, vertebral fracture fixation, and limb fracture fixation. It is also preferable as an adhesive or a dental adhesive for prosthetic restorations such as stuffing and covering in dentistry. Thus, the composition of the present invention is useful as an adhesive composition for biological hard tissue.

  Further, the composition of the present invention can be used as a lubricant in a joint cavity used for the treatment of osteoarthritis or a damage covering material. Osteoarthritis can occur in each joint of the body such as knees, shoulders, hips, hips, ankles, wrists, and fingers, but the composition of the present invention can be applied to any joint.

  In the composition of the present invention, each component is mixed, and the aqueous solution temperature (crosslinking temperature) is usually 4 to 50 ° C., preferably 10 to 45 ° C., more preferably 20 to 40 ° C., and the reaction time is usually 1. It can be cured under conditions of min to 48 hours, preferably 5 minutes to 30 hours.

[gel]
The gel of the present invention is formed from the composition of the present invention described above. In one embodiment, the gel of the present invention has a crosslinked structure by oxidative coupling of a hydroxyaryl group preferably contained in the above-described modified polysaccharide of the present invention, that is, contains a crosslinked product of the modified polysaccharide. Details of the oxidative coupling will be described later.

  In one embodiment, the gel is a hydrogel that normally contains 10 to 90% by weight of moisture. The hydrogel usually contains 90 to 10% by mass of the crosslinked polysaccharide. The hydrogel is excellent in mechanical strength and flexibility.

  The hydrogel of the present invention can be obtained by mixing a modified polysaccharide and an enzyme in water and crosslinking the modified polysaccharide as described later. Moreover, a xerogel can be obtained by removing the water | moisture content by drying the said hydrogel suitably. Alternatively, the gel of the present invention is gelled by light irradiation using a photosensitizer such as eosin Y, methylene blue, or rose bengal together with a modified polysaccharide as described in Macromolecules (2015) 2624-2630, for example. It can also be obtained.

  A gel can be formed by using the composition of the present invention. For example, in one embodiment, the modified polysaccharide, a peroxide such as hydrogen peroxide, and an enzyme are mixed in water to crosslink the modified polysaccharide to form a hydrogel. Here, for example, it is presumed that the hydroxyaryl group in the modified polysaccharide is oxidized, oxidative coupling (crosslinking) between the groups occurs, and gelation proceeds. In more detail about oxidative coupling, for example, when a hydroxyaryl group of a modified polysaccharide is reacted with a peroxide in the presence of an enzyme, a hydrogen atom is extracted from the phenolic hydroxyl group to form a free radical species, which is isomerized. It is considered that a free-radical species in which unpaired electrons exist on the carbon at the ortho position is formed, and then the two free-radical species are combined and then enolized to form a crosslinked structure.

  One embodiment of the method for producing a hydrogel of the present invention includes, for example, mixing a modified polysaccharide, an enzyme, and, if necessary, a peroxide and a metal salt in water to crosslink the modified polysaccharide. Manufacturing. The modified polysaccharide, enzyme, peroxide and metal salt may be used as an aqueous solution.

  Another embodiment of the method for producing a hydrogel of the present invention is, for example, (1) preparing a first solution containing a modified polysaccharide and containing either a peroxide or an enzyme but not both, 2) preparing a second solution containing one of the above-mentioned peroxides and enzymes not included in the first solution; (3) mixing the first solution and the second solution to denature the polysaccharide. Are crosslinked to produce a hydrogel. Specifically, a first solution containing a modified polysaccharide, an enzyme and water and a second solution containing a peroxide and water are mixed to crosslink the modified polysaccharide. Here, for example, a mold having a cavity having a desired shape is prepared, and the first solution and the second solution are mixed and reacted in the cavity to thereby obtain a hydrogel having the shape. Can be formed.

The aqueous solution temperature (crosslinking temperature) at the time of hydrogel formation is usually 4 to 50 ° C., preferably 10 to 45 ° C., more preferably 20 to 40 ° C., and the reaction time is usually 1 minute to 48 hours, preferably Is 5 minutes to 30 hours. Hydrogel formation may be performed under pressure, normal pressure (atmospheric pressure), or reduced pressure, but normal pressure is preferred. By using the composition of the present invention, a hydrogel can be formed under mild conditions of about room temperature.
The gel and hydrogel of the present invention can be suitably used as, for example, artificial cartilage and artificial bone when a part of living tissue such as cartilage and bone is damaged.

[Molded body]
The molded object of this invention contains the gel mentioned above. For example, the gel of the present invention is a molded body formed into a desired shape. In the molded body, the amount of gel is usually 50% by mass or more, preferably 60% by mass or more, and more preferably 70% by mass or more.

  One use of the molded body is a medical instrument. The medical instrument has the molded body. Since the said medical instrument has the said molded object which is excellent in mechanical strength and a softness | flexibility, it can be used suitably for an implant material especially among medical instruments. Examples of the implant material include an implant material used when a part of biological tissue such as cartilage and bone is damaged.

  EXAMPLES Hereinafter, although this invention is demonstrated further more concretely based on an Example, this invention is not limited to these Examples. In the following description of Examples and the like, “part” means “part by mass” unless otherwise specified.

[Synthesis Example 1] Hydroxyphenyl group-modified pullulan (P1)
In a 100 ml eggplant-shaped flask, 8.1 g of pullulan (CAS number: 9057-02-7, manufactured by Tokyo Chemical Industry Co., Ltd.) is added, and 40 g of distilled water is added and dissolved, and 3- (4-hydroxyphenyl) propion is added thereto. 1.38 g of acid was added and stirred to obtain a uniform solution. This liquid was distilled off under reduced pressure while heating at 60 ° C. to obtain a residue. The eggplant-shaped flask containing the residue was heated at 120 ° C. for 24 hours while reducing the pressure at 400 mmHg.

  After completion of the reaction, 80 g of distilled water was added to the reaction residue to dissolve it, and this aqueous solution was reprecipitated with 300 ml of ethanol. The precipitate was subjected to suction filtration, and the resulting precipitate was decompressed while heating at 60 ° C., and the solvent was distilled off to obtain hydroxyphenyl group-modified pullulan (P1).

When the hydroxyphenyl group substitution degree (DS _Ar ) of the hydroxyphenyl group-modified pullulan (P1) was calculated by ¹ H NMR (heavy solvent was D ₂ O), it was 2.3. A chart of ¹ H NMR of the hydroxyphenyl group-modified pullulan (P1) is shown in FIG. ¹ H-NMR spectrum was measured using an apparatus name “JNM-ECX400P” (manufactured by JEOL Ltd.).

[Example 1] Modified polysaccharide (A1)
In a 100 ml eggplant-shaped flask, 1 g of hydroxyphenyl group-modified pullulan (P1) is added, and 10 g of distilled water is added and dissolved. A uniform solution was obtained. This liquid was distilled off under reduced pressure while heating at 60 ° C. to obtain a residue. The eggplant-shaped flask containing the residue was heated at 120 ° C. for 24 hours while reducing the pressure at 400 mmHg.

After completion of the reaction, 30 g of distilled water was added to the reaction residue to dissolve it, and this aqueous solution was reprecipitated with 100 ml of ethanol. After dissolution in distilled water and reprecipitation were performed twice, the precipitate was suction filtered, and the resulting precipitate was depressurized while heating at 60 ° C. to distill off the solvent to obtain a white solid. . According to HPLC analysis, the obtained white solid was a pullulan (modified polysaccharide (A1)) having a phosphate group and a hydroxyphenyl group. A chart of the HPLC analysis is shown in FIG. HPLC was measured using the apparatus name “Acquity” (manufactured by Nihon Waters) under the following conditions.
Flow rate: 0.5 ml / min
Column temperature: 37 ° C
Column type: Product name “Presto FF-C18” (Imtakt)
Developing solution: 50 mM formic acid aqueous solution for 0 to 3 minutes, gradient elution from the aqueous formic acid solution to acetonitrile for 3 to 18 minutes, and acetonitrile for 18 to 20 minutes.

When the hydroxyphenyl group substitution degree (DS _Ar ) of the modified polysaccharide (A1) was calculated by ¹ H NMR (heavy solvent was D ₂ O), it was 1.4. A ¹ H NMR chart of the modified polysaccharide (A1) is shown in FIG.
In addition, ³¹ P NMR (heavy solvent is D ₂ O, device name “JNM-ECX400P” (manufactured by JEOL Ltd.)) was measured. ³¹ P NMR (D ₂ O, 400 MHz) σ (ppm): 1.37 (pentavalent phosphate ester)

[Synthesis Example 2] Phosphoric acid modified pullulan (P2)
In a 100 ml eggplant type flask, 2 g of pullulan (CAS number: 9057-02-7, manufactured by Tokyo Chemical Industry Co., Ltd.) is added, and 20 g of distilled water is added and dissolved therein, and 0.85 M phosphoric acid aqueous solution (pH = 5. 5) Add 57 g and stir to make a uniform solution. This liquid was distilled off under reduced pressure while heating at 60 ° C. to obtain a residue. The eggplant-shaped flask containing the residue was heated at 120 ° C. for 24 hours while reducing the pressure at 400 mmHg.

  After completion of the reaction, 70 g of distilled water was added to the reaction residue to dissolve it, and this aqueous solution was reprecipitated with 100 ml of ethanol. After dissolution in distilled water and reprecipitation were performed twice, the precipitate was suction filtered, and the resulting precipitate was depressurized while heating at 60 ° C. to distill off the solvent to obtain a white solid. . The obtained white solid was a pullulan having a phosphate group (phosphate-modified pullulan (P2)) by HPLC analysis. A chart of the HPLC analysis is shown in FIG.

A ¹ H NMR chart of phosphoric acid-modified pullulan (P2) is shown in FIG.
In addition, ³¹ P NMR (heavy solvent is D ₂ O, device name “JNM-ECX400P” (manufactured by JEOL Ltd.)) was measured. ³¹ P NMR (D ₂ O, 400 MHz) σ (ppm): 1.75 (pentavalent phosphate ester)

[Example 2] Production of composition and gel and evaluation thereof Into an Eppendorf tube, 50 mg of the modified polysaccharide (A1) was added, and 25 mg of an aqueous calcium chloride solution (concentration: 5% by mass) was added. Kneaded for 5 minutes. Subsequently, horseradish peroxidase (manufactured by Wako Pure Chemical Co., Ltd., product code “169-10791”, abbreviated as HRP) in aqueous solution (concentration: 150 U / ml) 5 μl (about 5 mg) and hydrogen peroxide solution (concentration: 4.2 mass%) ) 5 μl (about 5 mg) was added and mixed to produce the composition.

Two borosilicate glass substrates were bonded together through a coating film of a composition having an area of 6.25 cm ² and left at 23 ° C. for 24 hours, and the two borosilicate glass substrates were formed into a gel-like material comprising the composition. Glued with adhesive.

  Next, the two glass substrates bonded with the adhesive were left in distilled water at 23 ° C. for 12 hours, and then one of the borosilicate glasses was peeled off, and the state of the adhesive was visually observed. It remained in the acid glass.

[Comparative Example 1] Production of composition and gel, and evaluation thereof In an Eppendorf tube, 50 mg of the phosphoric acid-modified pullulan (P2) was added, and further 25 mg of an aqueous calcium chloride solution (concentration: 5% by mass) was added. The composition was manufactured by kneading for 5 minutes with microspatel.

Two borosilicate glass substrates were bonded together through a coating film of a composition having an area of 6.25 cm ² and left at 23 ° C. for 24 hours, and the two borosilicate glass substrates were formed into a gel-like material comprising the composition. Glued with adhesive.

  Next, the two glass substrates bonded with the adhesive were left in distilled water at 23 ° C. for 12 hours, and then one of the borosilicate glasses was peeled off, and the state of the adhesive was visually observed. It was confirmed that it did not remain on the acid glass and was peeled off. That is, no adhesive remained on any of the two glass substrates.

Claims (11)

  A modified polysaccharide in which a phosphate group (a1) and a radical reactive group (a2) are introduced into the polysaccharide.   The modified polysaccharide according to claim 1, wherein the polysaccharide is pullulan or a derivative thereof.   The modified polysaccharide according to claim 1 or 2, wherein the radical reactive group (a2) is a group (a21) having a hydroxyaryl group. The modified polysaccharide according to claim 3, wherein the group (a21) having a hydroxyaryl group is a group represented by the following formula (1).
[In formula (1), * is a bond with a hydroxyl group residue in the polysaccharide, and R ¹ is an alkanediyl group having 1 to 20 carbon atoms or — (CH ₂ ) _n —O— (CH ₂ ). _{m- represents a} divalent group, and n and m each independently represent an integer of 1 to 10; R ^{2 to} R ⁶ each independently represents a hydrogen atom, a hydroxyl group, an alkoxy group, or a substituent. It is an alkyl group which may have, provided that at least one of R ^{2 to} R ⁶ is a hydroxyl group. ]   A composition comprising the modified polysaccharide according to any one of claims 1 to 4, an enzyme, and water.   Furthermore, the composition of Claim 5 containing a peroxide.   Furthermore, the composition of Claim 5 or 6 containing the at least 1 sort (s) of metal salt chosen from an alkali metal salt and a periodic table group 2 metal salt.   The gel which has the crosslinked structure by the oxidative coupling of the hydroxyaryl group contained in the modified polysaccharide of Claim 3 or 4.   The gel formed from the composition of any one of Claims 5-7.   The gel according to claim 8 or 9, which is a hydrogel containing 10 to 90% by mass of water.   The molded object containing 50 mass% or more of gels of any one of Claims 8-10.