JP7018631B2 - Bone regeneration composition, bone regeneration composition kit, bone regeneration member and bone regeneration method - Google Patents

Description

The present invention relates to a bone regeneration composition, a bone regeneration composition kit, a bone regeneration member, and a bone regeneration method.

When a part of the bone is damaged, hydrogel is used as an extracellular matrix at the damaged part of the bone to proliferate and differentiate the cells. On the other hand, since growth factors such as basic fibroblast growth factor are known to contribute to bone regeneration, research on hydrogels containing growth factors is underway (see Patent Documents 1 and 2). ).

International Publication No. 2012/157339 International Publication No. 2011/07850

In bone regeneration therapy, it is difficult to maintain the transplantation effect at the administration site of the growth factor for a long period of time because the growth factor is easily circulated by the body fluid. For example, it is easy to enclose a growth factor in a hydrogel, but the encapsulation alone is not sufficient for the transplantation effect, and the combination of the hydrogel and the growth factor is very important.

The present invention provides, for the above-mentioned problems, a bone regeneration composition, a bone regeneration composition kit, and a bone regeneration member, which are useful for bone regeneration, and a method for providing a bone regeneration method. The purpose.

The present inventors have studied to solve the above problems. As a result, they have found that the above problems can be solved by the following composition for bone regeneration and the like, and have completed the present invention.
The present invention relates to, for example, the following [1] to [8].

[1] Select from modified dextran (A) in which a hydroxyaryl group is introduced into dextran, oxidation coupling enzyme (B) of the hydroxyaryl group, peroxide (c1), and a substance (c2) that produces a peroxide. Contains at least one component (C), at least one growth factor (D) selected from basic fibroblast growth factor (D1) and bone-forming protein (D2), and water (E). Composition for bone regeneration.

[2] The composition for bone regeneration according to the above [1], wherein the hydroxyaryl group is a group derived from tyramine.
[3] The composition for bone regeneration according to the above [1] or [2], wherein the degree of substitution with the hydroxyaryl group in the modified dextran (A) is 0.1 to 50.

[4] The composition for bone regeneration according to any one of the above [1] to [3], wherein the oxidative coupling enzyme (B) is horseradish peroxidase.
[5] The composition for bone regeneration according to any one of the above [1] to [4], wherein the component (C) is the peroxide (c1).

[6] A composition kit for bone regeneration comprising a first solution, a second solution, and a third solution. The first solution is a modified dextran in which a hydroxyaryl group is introduced into dextran (6). At least one component selected from the oxidation coupling enzyme (B) of the hydroxyaryl group, which contains A) and water (E), and a peroxide (c1) and a substance (c2) that produces a peroxide. (C), which contains either but not both, and the second solution is not contained in the first solution of the oxidation coupling enzyme (B) and the component (C). A composition for bone regeneration, wherein the third solution contains at least one growth factor (D) selected from basic fibroblast growth factor (D1) and bone-forming protein (D2). kit.

[7] A bone regeneration member formed by using the bone regeneration composition according to any one of the above [1] to [5] or the bone regeneration composition kit according to the above [6].
[8] The bone regeneration composition according to any one of [1] to [5], the bone regeneration composition kit according to [6], or the bone regeneration composition according to [7]. A bone regeneration method that regenerates bone using a member.

According to the present invention, it is possible to provide a bone regeneration composition, a bone regeneration composition kit, and a bone regeneration member useful for bone regeneration, and to provide a bone regeneration method.

FIG. 1 is a scheme of a bone regeneration process using the composition for bone regeneration of the present invention. FIG. 2 is a graph showing the measurement results of the new bone mass at the fracture site in Example 1, Comparative Examples 1 and 2, and the control group.

Hereinafter, embodiments for carrying out the present invention will be described.
Unless otherwise specified, each substance exemplified in the present specification, for example, a substance contained in a composition or used in each step, may be used alone or in combination of two or more. be able to.

[Bone regeneration composition]
The composition for bone regeneration of the present invention (hereinafter, also simply referred to as “composition of the present invention”) is
Modified dextran (A), in which a hydroxyaryl group is introduced into dextran,
Oxidation coupling enzyme (B) of the hydroxyaryl group,
At least one component (C) selected from the peroxide (c1) and the substance (c2) that produces the peroxide,
At least one growth factor (D) selected from basic fibroblast growth factor (D1) and bone-forming protein (D2), and water (E).
Contains.

<Denatured dextran (A)>
The modified dextran (A) is a compound in which a hydroxyaryl group is introduced into dextran. Dextran is a polysaccharide whose main chain is α-1,6 bond, with glucose as a constituent monosaccharide.

Denatured dextran (A) can function as a receptor for growth factor (D). Therefore, it is considered that the growth factor (D) can be stably present at the administration site of the composition of the present invention and the effective concentration of the growth factor (D) can be maintained for a long period of time, so that the bone regeneration effect is high. That is, in the present invention, by using a specific growth factor (D) together with denatured dextran (A), callus formation can be promoted and the bone regeneration rate can be improved.

The modified dextran (A) has a hydroxyaryl group. The hydroxyaryl group usually has 6 to 50 carbon atoms, preferably 6 to 30 carbon atoms. 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 to 2. As described above, the hydroxyaryl group is not limited to the monohydroxyaryl group.

Examples of the hydroxyaryl group include a hydroxyphenyl group, a hydroxynaphthyl group, and a hydroxyanthrasenyl group. The hydroxyaryl group can have a substituent such as an alkyl group. In a hydroxyaryl group, it is preferable that a hydrogen atom is bonded to a carbon atom at at least one ortho position with respect to a hydroxyl group. The hydroxyaryl group is preferably a tyramine-derived group because it is considered that the growth factor can be stably present and the effective concentration thereof can be maintained at the administration site of the composition of the present invention.

The modified dextran (A) is preferably a compound in which a hydroxyaryl group is bonded to dextran by an ester bond, a urethane bond or an ether bond, either directly or via an organic group. The ester bond, urethane bond and ether bond are preferably bonded to the carbon atom to which the hydroxyl group contained in dextran was bonded.

As the organic group, for example, an alcandiyl group having 1 to 20 carbon atoms, a substitution in which at least one selected from an ether bond and a divalent amino group (-NH-, etc.) is added or inserted into the alcandiyl group. Examples thereof include an arcandyl group and an organic group having a peptide bond (specifically, a group derived from an oligopeptide such as a dipeptide or an ester thereof).

Hereinafter, an embodiment of a production example of the modified dextran (A) will be described.
For example, after converting the alcoholic hydroxyl group of dextran into a carbonic acid ester derivative group using a carbonylation reagent (hereinafter, also referred to as "conversion reaction"), the obtained dextran's carbonic acid ester derivative and a hydroxyaryl group are provided. Reacts with an amine compound (hereinafter also referred to as "hydroxyaryl group introduction reaction").

Examples of the carbonylation reagent include nitrophenyl chloroformate, phenyl chloroformate, trichloromethyl chloroformate, and bis carbonate (trichloromethyl). The amount of carbonylation reagent is usually 1 to 100 parts by mass with respect to 100 parts by mass of dextran. If the carbonic acid ester derivative as an intermediate is stable, the following reaction can be carried out after isolation once.

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 with respect to 100 parts by mass of the carbonic acid ester derivative of dextran.

The conversion reaction is preferably carried out in the presence of a basic compound. Examples of the basic compound include trimethylamine, triethylamine, tributylamine, pyridine and piperidine.

The conversion reaction and the hydroxyaryl group introduction reaction are preferably carried out in a solvent. Examples of the solvent include amide solvents such as dimethylformamide and dimethylacetamide; sulfoxide solvents such as dimethyl sulfoxide; halogenated hydrocarbons such as dichloromethane, 1,2-dichloroethane and chloroform; aromatics such as benzene, toluene and xylene. Hydrocarbons; examples include ethers such as diethyl ether, tetrahydrofuran and dioxane. In order to improve the solubility of dextran in the solvent, an alkali metal salt such as lithium chloride can be used.

Other embodiments of the production of the modified dextran (A) include, for example, dextran and a functional group capable of reacting with an alcoholic hydroxyl group (eg, a carboxy group, an acid halide thereof, an epoxy group, an oxetanyl group) and a hydroxyaryl. Examples thereof include a method of reacting with a compound having a group.

Examples of the compound having a carboxy group capable of reacting with an alcoholic hydroxyl group in dextran or an acid halide thereof and a hydroxyaryl group include 4-hydroxyphenylacetic acid, 4-hydroxyphenylpropionic acid and 4-hydroxyphenylbutanoic acid. , 2- (2,5-dihydroxyphenyl) acetic acid, acid halides thereof.

The degree of substitution (DS) with the hydroxyaryl group in the modified dextran (A) is usually 0.1 to 50, preferably 1 to 40, and more preferably 2 to 30. The degree of substitution can be determined by ^{1 1} H NMR. The degree of substitution of the modified dextran (A) indicates the average number of hydroxyaryl groups introduced per 100 units of the constituent monosaccharides of the modified dextran (A). By having such a degree of substitution, the modified dextran (A) has an improved gelation rate.

The number average molecular weight measured by absolute molecular weight measurement using the multi-angle light scattering detector of the modified dextran (A) is usually 1,000 to 10,000,000, preferably 3,000 to 1,000,000, more preferably. Is between 5,000 and 500,000. The molecular weight distribution of the modified dextran (A) (weight average molecular weight / number average molecular weight; both by the absolute molecular weight measurement) is usually 1 to 20, preferably 1 to 10, and more preferably 1 to 5.

The modified dextran (A) can be used alone or in combination of two or more.
The content of the modified dextran (A) in the composition of the present invention 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 mechanical strength and handleability of the gel.

<Oxidation coupling enzyme of hydroxyaryl group (B)>
The composition of the present invention contains an oxidative coupling enzyme (B) of a hydroxyaryl group (hereinafter, also referred to as "enzyme (B)"). Enzyme (B) is an enzyme that can directly or indirectly catalyze the oxidative coupling reaction of hydroxyaryl groups.

Examples of the enzyme (B) include phenol oxidases such as laccase and tyrosinase, catalase, and peroxidase. Peroxidase can gel the composition of the present invention when used in combination with a peroxide (c1) such as hydrogen peroxide. The origin of copper enzymes such as laccase and tyrosinase includes, for example, sumac, mushrooms (Tsuchikaburi, mushrooms), and mold (Polyporus vericolor). The origins of catalase and peroxidase are, for example, bovine liver, horse blood cells, human blood cells, M. lysodeikticus, horseradish, soybeans, radishes, cubs, thyroid, milk, intestines, white blood cells, red blood cells, yeast, Caldariomyces fumago, Steptococcus faecalis can be mentioned. Among these, mushroom-derived tyrosinase and horseradish-derived peroxidase, that is, horseradish peroxidase are preferable.

In the present invention, from the viewpoint of efficiently promoting the cross-linking reaction of the hydroxyaryl group of the modified dextran (A), the component (C) is used, and at least one selected from peroxidase and catalase is used as the enzyme (B). Is preferable, and it is more preferable to use the component (C) and at least the horseradish peroxidase as the enzyme (B).

One type or two or more types of enzyme (B) can be used.
In the composition of the present invention, the amount of the enzyme (B) is preferably 0.01 U / mL or more, more preferably 0.1 U / mL or more; preferably 1,000 U / mL or less, more preferably 500 U / mL or less. .. U is a unit of enzyme activity, and is an amount of enzyme capable of changing a substrate of 1 micromolar per minute at a temperature of 30 ° C. under optimum conditions.

<Component (C)>
The composition of the present invention contains at least one component (C) selected from the peroxide (c1) and the substance (c2) that produces the 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 invention, the amount of the peroxide (c1) is usually 1 mol with respect to 1 mol of the hydroxyaryl group in the modified dextran (A). It is 0.01 to 1000 mol, preferably 0.1 to 500 mol, and more preferably 0.5 to 200 mol. Further, in one embodiment, the content of the peroxide (c1) in the composition of the present invention is preferably 10 to 1,000 mM, more preferably 100 to 800 mM from the viewpoint of reactivity.

The substance (c2) that produces a peroxide is usually used together with an oxidase (c3). The oxidase (c3) is an enzyme that catalyzes a reaction that consumes a substance (c2) to produce 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. Although these enzymes can be appropriately selected depending on the intended use, glucose oxidase is preferable because the gelation rate is significantly improved.

The substance (c2) is, for example, consumed by the oxidase (c3) to produce a peroxide such as hydrogen peroxide, and is selected corresponding to the oxidase (c3). For example, the oxidase (c3) can generate hydrogen peroxide by enzymatically treating the substance (c2) in the presence of oxygen (eg, oxygen in the air). For example, if 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 pyruvate, respectively. It is cholesterol.

When the substance (c2) is used as the component (C), the content of the oxidase (c3) in the composition of the present invention is preferably 0.01 U / mL or more, preferably 1 U / mL or more, from the viewpoint of reactivity. More preferably; 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 in an amount that appropriately produces a peroxide.
The component (C) may be used alone or in combination of two or more.

<Growth factor (D)>
The composition of the present invention contains at least one growth factor (D) selected from basic fibroblast growth factor (D1) and bone-forming protein (D2).

Basic fibroblast growth factor (D1) (hereinafter, also referred to as "bFGF") is a kind of factor that controls and promotes cell growth. bFGF has excellent bone formation ability and not only promotes the proliferation of fibroblasts, but also promotes the proliferation of mesenchymal cells present in the periosteum and bone marrow.

As the bFGF, for example, a natural bFGF extracted from an organ such as the brain or the pituitary gland of a mammal such as a human or a cow, or a recombinant bFGF produced by genetic engineering technology can be used. In addition, bFGF also includes variants of basic fibroblast growth factor in which a part of the amino acid sequence is deleted or replaced with another amino acid, or another amino acid sequence is inserted.

Bone-forming proteins (D2) include proteins belonging to the TGF-β superfamily that promote bone formation and fracture repair. Specifically, it is a factor that promotes the activity of osteoblasts and a factor that induces the differentiation of mesenchymal stem cells into osteoblasts. Preferable examples of the bone-forming protein (D2) include BMP (Bone Morphogenic Protein), which is a protein belonging to the TGF-β superfamily, for example, BMP2, BMP4, BMP5, BMP6, BMP7, BMP8, and BMP9.

One kind or two or more kinds of growth factors (D) can be used.
In the composition of the present invention, the content of the growth factor (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 modified dextran (A). .. In such an embodiment, the growth factor (D) is well retained in the hydrogel formed from the composition of the present invention.

<Water (E)>
The composition of the present invention preferably contains water (E).
The content of water (E) in the composition of the present invention 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 that are less invasive to the living body, and the mechanical strength of the obtained gel tends to be excellent.

<Other ingredients>
The composition of the present invention can further contain a polysaccharide other than the modified dextran (A). Examples of the polysaccharide include unmodified dextran in which hyaluronic acid, pullulan, xanthan gum, cellulose, guar gum, fructan, mannan, carrageenan, chitin, chitosan, pectin, starch, amylose, amylopectin, glycogen, and hydroxyaryl group are not introduced. , Dextrin, gellan gum, alginic acid.

The composition of the present invention may also contain one or more selected from phosphoric acid, polyphosphoric acid, pH adjusters, ultraviolet absorbers, thickeners, colorants and fillers, and may also contain agents. , A substance expressing a function such as cells can be contained.
In addition, the composition of the present invention can further contain sodium chloride. For example, the composition of the present invention can contain a buffer solution such as phosphate buffered saline.

[Composition kit for bone regeneration]
The composition kit for bone regeneration of the present invention (hereinafter, also simply referred to as “kit of the present invention”) has a first solution, a second solution, and a third solution, which will be described below.

The first solution contains a modified dextran (A) and water (E) in which a hydroxyaryl group has been introduced into the dextran, the oxidation coupling enzyme (B) of the hydroxyaryl group, and the peroxide (c1) and. It contains at least one component (C) selected from the peroxide-producing substance (c2), but does not contain both.

The second solution contains the oxidation-coupling enzyme (B) and the component (C) whichever is not contained in the first solution.
The third solution contains at least one growth factor (D) selected from basic fibroblast growth factor (D1) and bone-forming protein (D2).

It is preferable that the first to third solutions are all aqueous solutions.
The details of each component (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 component (A) to (E) are appropriately set so as to be within the range described in the [Bone regeneration composition] column. The composition of the present invention can be prepared by mixing the first to third solutions. At this time, the mixing order of each solution is not particularly limited, but the second solution is mixed with the mixture obtained after mixing the first solution and the third solution, or the second solution and the third solution are mixed. An embodiment in which the first solution is mixed with the mixture obtained after mixing with the solution can be mentioned.

[Bone regeneration member]
The bone regeneration member of the present invention is formed by using the bone regeneration composition or the bone regeneration composition kit of the present invention, and specifically, is formed from the bone regeneration composition or the bone regeneration composition kit. Consists of gel to be made.

The gel has a crosslinked structure by oxidation coupling of the hydroxyaryl group possessed by the modified dextran (A), that is, contains a crosslinked product derived from the modified dextran (A). For example, it is presumed that the hydroxyaryl group in the modified dextran (A) is oxidized, oxidation coupling (crosslinking) between the groups occurs, and gelation proceeds.

In one embodiment, the gel is a hydrogel, specifically a hydrogel containing 50-99.9% by weight, preferably 70-97% by weight of water. The hydrogel usually contains 50 to 0.1% by mass, preferably 30 to 3% by mass, of a crosslinked product derived from modified dextran (A). The hydrogel is excellent in mechanical strength and flexibility.

A xerogel can be obtained by removing water by appropriately drying the hydrogel. Alternatively, the gel may be gelled by irradiating with light using a photosensitizer such as eosin Y, methylene blue, or rose bengal together with modified dextran (A), for example, as described in Macromolecules (2015) 2624-2630. It can also be obtained by converting it to.

An example of a method for manufacturing a bone regeneration member is as follows.
Each component (A) to (E) in the composition of the present invention or the first to third solutions in the kit of the present invention is mixed, and the aqueous solution temperature (crosslinking temperature) is usually 4 to 50 ° C., preferably 10. Gelation can be carried out under the conditions of ~ 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. Hydrogel formation can be performed under normal pressure, normal pressure (atmospheric pressure), or reduced pressure, but normal pressure is preferable.

A mold having a cavity having a desired shape is prepared, and each component (A) to (E) of the composition of the present invention or the first to third solutions in the kit of the present invention is placed in the cavity. By mixing and reacting, a hydrogel having the above-mentioned shape can be formed, and the composition of the present invention or the mixed solution of the first to third solutions in the kit of the present invention is placed on a predetermined substrate. By casting, a film-shaped hydrogel can be obtained, and the film-shaped hydrogel can be punched into, for example, a columnar shape or a polygonal columnar shape.

The shape of the bone regeneration member is not particularly limited, and examples thereof include a film shape, a columnar shape, a polygonal columnar shape, a non-woven fabric shape, a fiber shape, a tube shape, a particle shape, and a mesh shape.
The growth factor (D) is carried in the bone regeneration member of the present invention. Therefore, the growth factor (D) is applied to the application site (eg, bone defect site, fracture site) of the bone regeneration member for a long period of time. It is possible to maintain. In addition, the hydrogel functions as an extracellular matrix in the affected area, and this extracellular matrix serves as a scaffold for bone regenerating cells, and the bone regenerating cells can be established and can proliferate and differentiate well. Therefore, the bone regeneration member of the present invention has a high bone regeneration effect.

[Bone regeneration method]
The bone regeneration composition, the bone regeneration composition kit, and the bone regeneration member of the present invention are used for bone regeneration applications. 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, human or a mammal excluding 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 the affected area, or by applying the bone regeneration member to the affected area, bone regeneration is induced to induce bone regeneration. The playback speed can be improved.

FIG. 1 shows, as an example, an estimation scheme of bone regeneration when the composition for bone regeneration of the present invention is used. The bone regeneration composition administered to the affected area 30 forms a hydrogel 40, and the growth factor 50 is well retained in the hydrogel 40. Growth factor 50 promotes the proliferation of periosteal cells 20, which are progenitor cells of bone cells. As a result, a large new bone (callus) 70 is formed. In addition, bone cells are omitted in FIG.

The bone to be regenerated in the present invention is formed by depositing hydroxyapatite on collagen fibers formed in a network shape, and most of the organic matter of the bone is collagen.
The usage, dose, and shape of the bone regeneration composition, bone regeneration composition kit, or bone regeneration member of the present invention can be appropriately determined according to the purpose of use thereof. 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 administered to the affected part such as a bone defect site or a fracture site in the living body by injection, application or the like. can do. It is also possible to mix the composition or mixture with a suitable excipient to make an ointment, gel or cream, and then apply to the affected area.

Target diseases of the bone regeneration composition, bone regeneration composition kit, and bone regeneration member of the present invention include, for example, bone defect when a part of bone is damaged due to trauma such as a fracture, oral surgery. Diseases include osteoporosis.

Hereinafter, the present invention will be described in more detail based on Examples, but the present invention is not limited to these Examples.
<Manufacturing of denatured polysaccharides>
[Synthesis Example 1] Synthesis of hydroxyphenyl group-modified dextran (A1)
Under a nitrogen atmosphere, 40 g of dextran (product name "dextran 40", manufactured by Meito Sangyo Co., Ltd.), 1600 ml of dimethylformamide, and 30.9 g of lithium chloride were added to the flask, and 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 nitrophenylchloroformate were added, and the mixture was stirred at 0 ° C. for 1 hour. Then, 2000 ml of chilled ethanol was added to the flask, the precipitate was filtered off, and the filtrate was washed with chilled ethanol and dried.

Under a nitrogen atmosphere, a solution prepared by dissolving the dried filter medium in 740 ml of dimethylformamide was placed in a flask, then 9.1 g of tyramine was added, and the mixture was stirred at room temperature for 3 hours. Next, 800 ml of chilled ethanol was added to the flask, the precipitate was filtered off, and the filtrate was dissolved in pure water for dialysis.

In this way, a hydroxyphenyl group-modified dextran (A1) was obtained. When the obtained hydroxyphenyl group-modified dextran (A1) was analyzed by ¹ H NMR, the degree of substitution (DS) by the hydroxyphenyl group was 12.

[Comparative Synthesis Example 1] Synthesis of hydroxyphenyl group-modified hyaluronic acid (AR1) In the synthesis synthesis example 1, the hydroxyphenyl group-modified hyaluronic acid (AR1) was prepared by the same method except that hyaluronic acid was used instead of dextran. Obtained. When the obtained hydroxyphenyl group-modified hyaluronic acid (AR1) was analyzed by ¹ H NMR, its DS was 3.2.

<Manufacturing of the first to third solutions of the composition kit for bone regeneration>
[Production Example 1] Production of First Solution (1 ) 0.2 g of hydroxyphenyl group-modified dextran (A1) obtained in Synthesis Example 1 was added to a phosphate buffered saline solution (product name "PBS (-)", sum. A polymer solution was prepared by dissolving in 0.8 g of Kojunyaku Kogyo Co., Ltd. (manufactured by). 1.0 g of the polymer solution and 1.0 g of a horseradish peroxidase (manufactured by Wako Pure Chemical Industries, Ltd., product code "169-10791", abbreviated as "HRP") aqueous solution (concentration: 0.8 U / ml) are uniformly mixed. , The first solution (1) was produced.

[Comparative Production Example 1] Production of First Solution (2 ) 0.2 g of hydroxyphenyl group-modified hyaluronic acid (AR1) obtained in Comparative Synthesis Example 1 is dissolved in 0.8 g of the PBS (-) to provide a polymer solution. Prepared. 1.0 g of the polymer solution and 1.0 g of the HRP aqueous solution (concentration: 30 U / ml) were uniformly mixed to prepare the first solution (2).

[Preparation Example 1] Preparation of the second solution (1) The hydrogen peroxide solution (concentration: 882 mM) was used as the second solution (1).
[Preparation Example 2] Production of the third solution (1)
Basic fibroblast growth factor (product name "Fiblast Spray", manufactured by Kaken Pharmaceutical Co., Ltd.) was adjusted to a concentration of 1 μg / μl with the above-mentioned PBS (-), and a third solution (1) was prepared. ..

<Application and evaluation of bone regeneration materials to affected areas>
[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. A composition for bone regeneration was prepared by mixing 15 μl of the second solution (1) with 15 μl of the mixed solution.

25 μl of the bone regeneration composition was administered to the fractured site of a C57BL / 6J mouse (male, 9 weeks old) with a fractured femur (see FIG. 1), and 4 weeks later, the C57BL / 6J mouse was slaughtered. .. The amount of new bone at the fracture site was measured by microCT. The new bone mass was about 13.1 mm ³ .

[Comparative Example 1]
In Example 1, a composition for bone regeneration was prepared in the same manner as in Example 1 except that the first solution (2) was used instead of the first solution (1).

25 μl of the bone regeneration composition was administered to the fractured site of a C57BL / 6J mouse (male, 9 weeks old) in which the femur was fractured, and the same test as in Example 1 was performed. The amount of new bone at the fracture site was measured by microCT. The new bone mass was about 6.1 mm ³ .

[Comparative Example 2]
In Example 1, the test was carried out in the same manner as in Example 1 except that the PBS (−) was used instead of the third solution (1). The amount of new bone at the fracture site was measured by microCT. The new bone mass was about 5.0 mm ³ .

[Experimental Example 1] control
As a control group, the amount of new bone at the fracture site of C57BL / 6J mice (male, 9 weeks old) having a fractured femur was measured by microCT after 4 weeks. The new bone mass was about 5.6 mm ³ .
As described above, the results of new bone mass are summarized in FIG.

<Discussion>
It was revealed that Example 1 significantly produced new bone as compared with Comparative Examples 1 and 2 and Experimental Example 1 (control), and was superior to bone regeneration.

10: Bone 15: Bone marrow 20: Periosteal cells (periosteum)
30: Affected area (bone defect site, fracture site)
40: Hydrogel 50: Growth factor 60: Proliferated periosteal cells 70: New bone 80: New periosteum

Claims (11)

Modified dextran (A), in which a hydroxyaryl group is introduced into dextran,
Oxidation coupling enzyme (B) of the hydroxyaryl group,
At least one component (C) selected from the peroxide (c1) and the substance (c2) that produces the peroxide,
Basic fibroblast growth factor (D1 ), as well as water (E)
A composition for bone regeneration containing. The composition for bone regeneration according to claim 1, wherein the basic fibroblast growth factor (D1) is a recombinant bFGF produced by a genetic engineering technique. The composition for bone regeneration according to claim 1 or 2, wherein the content of the basic fibroblast growth factor (D1) is 0.01 to 10 parts by mass with respect to 100 parts by mass of the denatured dextran (A). thing. The composition for bone regeneration according to any one of claims 1 to 3, which does not substantially contain a growth factor other than the basic fibroblast growth factor (D1). The composition for bone regeneration according to any one of claims 1 to 4 , wherein the hydroxyaryl group is a group derived from tyramine. The composition for bone regeneration according to any one of claims 1 to 5, wherein the degree of substitution with the hydroxyaryl group in the modified dextran (A) is 0.1 to 50. The composition for bone regeneration according to any one of claims 1 to 6 , wherein the oxidation-coupling enzyme (B) is horseradish peroxidase. The composition for bone regeneration according to any one of claims 1 to 7 , wherein the component (C) is the peroxide (c1). A composition kit for bone regeneration comprising a first solution, a second solution, and a third solution.
The first solution contains a modified dextran (A) and water (E) in which a hydroxyaryl group has been introduced into the dextran, the oxidation coupling enzyme (B) of the hydroxyaryl group, and the peroxide (c1). And at least one component (C) selected from the peroxide-producing substance (c2), but not both.
The second solution contains the oxidation-coupling enzyme (B) and the component (C), whichever is not contained in the first solution.
The third solution contains basic fibroblast growth factor (D1 ) .
Bone regeneration composition kit. A bone regeneration member formed by using the bone regeneration composition according to any one of claims 1 to 8 or the bone regeneration composition kit according to claim 9 . The subject (human ) using the bone regeneration composition according to any one of claims 1 to 8 , the bone regeneration composition kit according to claim 9 , or the bone regeneration member according to claim 10 . Bone regeneration method to regenerate bone (excluding) .

Images