JP2000230002A - Bone restoration material - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a bone restoratin material excellent in biocompatibility, moldability, and in-vivo decompsability by incorporating, into the same, a gel which is formed from hyaluronic acid alone and is hardly soluble in a neutral aqueous solution. SOLUTION: The hardly water-souble hyaluronic acid gel used here exhibits a solubility of 50% or lower in a neutral aqueous solution at 37 deg.C for 12 hr. The gel can be decomposed and solubilized by treating it under the reaction conditions of accelerated acid hydrolysis of hyaluronic acid. A pH of an aqueous solution of 1.5 and a temperature of 60 deg.C are suitable as the reaction conditions. The resultant acidic aqueous solution of hyaluronic acid is subjected to freezing and thawing, each at least one time. Preferably, the freezing is done at -5 deg.C or lower; and the thawing, at 5 deg.C or higher. Then, the gel is cleaned to remove an acid, etc. Water, a physiological salt solution, etc., can be used for the cleaning. The cleaned gel in the state of being immersed in a solvent, in the state of being wet by a solvent or in the dry state is used as a bone restoration material.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a surgical treatment for bone loss, damage, tooth extraction, etc. in humans or animals containing a gel formed of hyaluronic acid alone which is hardly soluble in a neutral aqueous solution, orthopedic surgery and the like. A bone repair material used for

[0002]

2. Description of the Related Art Among biomaterials, as a block-like filler, for example, a porous sponge disclosed in JP-A-62-39506 is a filler in which chitin is crosslinked with a drug. JP-A-3-23864 is a block-shaped composite material composed of collagen sponge and polylactic acid. Japanese Patent Publication No. 7-505643 is a biocompatible and bioabsorbable excellent bone substitute composed of a hyaluronic acid ester.

[0003]

However, the prior art has the following problems. That is, since the porous sponge disclosed in JP-A-62-39506 is non-absorbable in a living body and is not completely replaced by bone itself, there is a risk of infection and a risk of detachment of the material itself. On the other hand, the composite material of JP-A-3-23864 has a problem that atelocollagen has some antigenicity.

[0004] These problems of non-absorbability and biocompatibility can be solved by using hyaluronic acid. However, the bone repair material of hyaluronic acid by the conventional chemical modification method contains a cross-linking substance or metal in the molecule of hyaluronic acid, so its physiological action and biocompatibility are essentially equivalent to hyaluronic acid. It was difficult to completely avoid security problems.

An object of the present invention is to provide a bone repair material having excellent biocompatibility, moldability and biodegradability.

The present inventors have found a poorly water-soluble hyaluronic acid gel consisting essentially of only hyaluronic acid without using a cross-linking agent (PCT / JP98 / 03536). The inventors found usefulness as a material, and completed the present invention.

[0007]

That is, the present invention provides:
(1) a bone repair material containing a gel formed of hyaluronic acid alone which is hardly soluble in a neutral aqueous solution; (2) the following (a):
(B) a bone repair material comprising a gel formed of hyaluronic acid alone which satisfies the requirement of (b), and (a) a dissolution rate in a neutral aqueous solution at 37 ° C for 12 hours of 50% or less. (B) Hyaluronic acid solubilized by treating the hyaluronic acid gel under conditions of accelerated acid hydrolysis of hyaluronic acid has a branched structure, and the solubilized hyaluronic acid has a degree of branching of 0 (3) a gel formed solely of hyaluronic acid, which partially contains a molecular weight fraction of .5 or more,
The bone repair material according to claim 2, wherein the bone repair material is one selected from the group consisting of a film, a sheet, a slurry, a crush, a sponge, a lump, and a paste.
50% dissolution rate in 12 hours in neutral 37 ° C aqueous solution
Hyaluronic acid solubilized by treating the hyaluronic acid gel under conditions of accelerated acid hydrolysis of hyaluronic acid has a branched structure, and the degree of branching in the solubilized hyaluronic acid is 0. A hyaluronic acid gel partially containing a molecular weight fraction of 5 or more, and a bone repair material containing at least one selected from the group consisting of ungelled hyaluronic acid, bioactive substances, bone granules, and antibiotics.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The bone repair material referred to in the present invention is a human or animal bone defect,
It promotes the growth and repair of bone-deficient tissue during injury and tooth extraction.

The hyaluronic acid gel referred to in the present invention is a polymer having a three-dimensional network structure and a swollen body thereof. The three-dimensional network is formed by a cross-linked structure of hyaluronic acid.

The hyaluronic acid used in the present invention can be used regardless of its origin, whether it is extracted from animal tissues or manufactured by fermentation. The strain used in the fermentation method is a microorganism having hyaluronic acid-producing ability, such as Streptococcus sp.
No. 3-123392, Streptococcus equi FM-100 (Microtechnical Laboratories No. 9027), and Streptococcus equi FM-300 described in Japanese Patent Application Laid-Open No. Hei 2-234689 (Microtechnical Labs No. 2319) )), A mutant which stably produces hyaluronic acid in high yield is desirable. Those cultured and purified using the above mutant strains are used.

The molecular weight of the hyaluronic acid used in the present invention is preferably in the range of about 1 × 10 ⁵ to about 1 × 10 ⁷ daltons. In addition, as long as it has a molecular weight within the above range, a higher molecular weight one and a low molecular weight one obtained by hydrolysis or the like can also be preferably used.
It should be noted that the hyaluronic acid according to the present invention is used in a concept that also includes alkali metal salts thereof, for example, sodium, potassium and lithium salts.

The term "hyaluronic acid alone" as used in the present invention means that no chemical cross-linking agent or chemical modifier other than hyaluronic acid is used, and that it is not complexed with a cationic polymer. .

The hyaluronic acid gel referred to in the present invention can be decomposed and solubilized by treating the hyaluronic acid gel under the conditions of the accelerated acid hydrolysis reaction of hyaluronic acid. When the solubilized hyaluronic acid retains a crosslinked structure,
Hyaluronic acid having a branch point can be distinguished from linear hyaluronic acid in terms of polymer solution theory.

The conditions for the accelerated acid hydrolysis reaction of hyaluronic acid referred to in the present invention include a pH of an aqueous solution of 1.5 and a temperature of 60 ° C.
Is appropriate. The main chain cleavage reaction due to hydrolysis of the glycosidic bond of hyaluronic acid, compared with neutral aqueous solution,
Significantly promoted in acidic or alkaline aqueous solutions. Further, the acid hydrolysis reaction is promoted at a higher reaction temperature.

In the present invention, the GPC-MALLS method is used,
The molecular weight and the degree of branching of the molecular weight fraction separated by GPC were continuously measured online. In the present invention, the degree of branching is measured by using the elution volume method of calculating the degree of branching by comparing the molecular weight of the solubilized hyaluronic acid of the fraction having the same elution volume with the molecular weight of the linear hyaluronic acid as a control. Was. The degree of branching is the number of branch points present per one polymer chain of solubilized hyaluronic acid, and is plotted against the molecular weight of solubilized hyaluronic acid.

The solubilized hyaluronic acid was diluted with a GPC solvent to adjust the concentration, filtered through a 0.2 μm membrane filter, and used for measurement. In the case where the hyaluronic acid gel referred to in the present invention has a cross-linked structure that is stably present even under the accelerated acid hydrolysis conditions of hyaluronic acid, a branched structure is confirmed in the solubilized hyaluronic acid in terms of a polymer solution.

The following are specific examples of bone repair materials using a poorly water-soluble hyaluronic acid gel. These examples are given only by way of example.
The production method is not particularly limited as long as it is a bone repair material comprising a poorly water-soluble hyaluronic acid gel formed of hyaluronic acid alone having a dissolution rate of 50% or less in No. 2.

An example of the manufacturing process of the bone repair material of the present invention will be described step by step. The aqueous solution of hyaluronic acid is obtained by mixing hyaluronic acid powder and water and stirring. The aqueous solution of hyaluronic acid is preferably adjusted to be acidic.

As the acid used to adjust the pH of the aqueous solution of hyaluronic acid, any acid can be used as long as it can adjust the pH to 3.5 or less. In order to reduce the amount of acid used, it is desirable to use a strong acid, for example, hydrochloric acid, nitric acid, sulfuric acid and the like.

The pH of the aqueous solution of hyaluronic acid is adjusted so that the carboxyl group of hyaluronic acid is protonated at a sufficient ratio.
Adjust to H. The pH to be adjusted is appropriately determined according to various kinds of properties such as the type of the counter ion of the hyaluronic acid salt, the molecular weight of the hyaluronic acid, the concentration of the aqueous solution, the conditions for freezing and thawing, and the strength of the gel to be formed. The pH is adjusted to 0.5 or less, preferably to 2.5 or less.

For freezing and thawing, an operation of placing an acidic aqueous solution of hyaluronic acid in an arbitrary container, freezing it at a predetermined temperature, and thawing at a predetermined temperature after freezing is performed one or more times. As the temperature for freezing and thawing, a freezing temperature of -5 ° C or less and a thawing temperature of 5 ° C or more are preferably selected, and the time is not particularly limited. It is preferable that the operation is repeated at least once.

Next, this poorly water-soluble hyaluronic acid gel is
It is subjected to washing to remove components such as acids. The liquid used for washing is not particularly limited, but for example, water, physiological saline, phosphate buffer and the like are preferably used.

The washing method is not particularly limited, but usually, a batch method, a filtration method, a method of filling a column or the like and passing the solution, and the like are used. These cleaning conditions are:
Any condition can be used as long as the components to be removed can be reduced to a target concentration or less, including the number of times, and can be appropriately selected depending on the form and use of the hyaluronic acid gel.

The washed poorly water-soluble hyaluronic acid gel is converted into a state in which it is immersed in a solvent, a state in which the solvent is moistened, a state in which it has been dried through air drying, reduced pressure drying, or freeze drying. Served as

In the preparation of the poorly water-soluble hyaluronic acid gel, for example, at the time of preparation, a restoration material for the poorly water-soluble hyaluronic acid gel in a desired form can be prepared by selecting a container and a method for freezing the hyaluronic acid acidic solution. It is possible. For example, lumps and sponges are obtained depending on the shape of the container. As the processing after the preparation, a fine crushed state by mechanical pulverization is exemplified.

Further, before freezing and thawing the acidic solution of hyaluronic acid, a bone repair material of a poorly water-soluble hyaluronic acid gel containing them is obtained by mixing bone granules, physiologically active substances, antibiotics and the like. It is also possible. Also,
After freezing, thawing and washing the hyaluronic acid acidic solution, by mixing ungelled hyaluronic acid, bone granules, bioactive substances, antibiotics, etc., the poorly water-soluble hyaluronic acid gel containing them is obtained. It is also possible to obtain bone repair materials.

Bone granules, like hyaluronic acid, function as cell scaffolds and have the function of enhancing bone repair. Further, the physiologically active substance has a function of activating osteoblasts and accelerating bone repair. Antibiotics also have the function of suppressing the growth of pathogenic bacteria that can infect the site of bone damage.

There are no particular restrictions on the non-gelled hyaluronic acid, bone granules, physiologically active substances, antibiotics, etc. used in the bone, but the application site of the bone repair material is bone, joints, roots, etc. From the following are listed.

The origin of hyaluronic acid which is not gelled is not restricted at all, it also includes various alkali metal salts thereof, and there is no particular restriction on its molecular weight, and its molecular weight is about 1 × 10 ^2. It can be used up to the range of １1 × 10 ⁷ Dalton.

Examples of the physiologically active substance include bone cell metabolism promoters such as TGFβ and BMP, and calcitonin, elcitonin, salmon calcitonin and the like.

Examples of the bone granules include natural or artificial bone granules, such as hydroxyapatite, tricalcium phosphate and calcium carbonate granules, and are not particularly limited. Generally, what is already used usually can be used. For example, the bone granules have a diameter of 50 μm to 5 mm, and they may be either porous or non-porous.

The type of antibiotic is not particularly limited, but it is preferable to use an antibiotic effective against osteomyelitis and osteomyelitis. In particular, cepham antibiotics such as cephalexin, penicillin antibiotics such as ampicillin, aminoglucoside antibiotics such as gentamicin, macrolide antibiotics such as erythromycin, tetracycline antibiotics such as minocyclone, and peptide antibiotics such as bacitracin. Substances and the like.

The bone repair material of the poorly water-soluble hyaluronic acid gel is
The present invention can be applied to any animal in which bone damage occurs, and can suitably repair bone in mammals, particularly humans. The bone repair material of poorly water-soluble hyaluronic acid gel is used for surgery in the form of paste, crushed, sponge, lump, slurry, film or the like. It is preferable to directly fill the bone damage site with the above method, or to insert it into the bone damage site as a sponge or mass.

[0034]

The present invention will be described in more detail with reference to the following examples. Note that the present invention is not limited to this.

Example 1 Sodium hyaluronate having a molecular weight of 2 × 10 ⁶ daltons was dissolved in distilled water to prepare a 1% by weight aqueous solution of hyaluronic acid. The pH of this aqueous solution was adjusted to pH 1.5 with 1N hydrochloric acid to obtain an acidic aqueous solution of hyaluronic acid. 25 ml of this aqueous hyaluronic acid solution was placed in a plastic petri dish, and freezing at -20 ° C for 22 hours and thawing at 25 ° C for 2 hours were repeated twice. Then put this in saline for 50m
After being immersed in 100 ml of a phosphate buffered physiological saline of pH 7 adjusted by adding a phosphate buffer component at M concentration at 5 ° C. for 24 hours, it was sufficiently washed with distilled water. This was freeze-dried to obtain a sheet-like poorly water-soluble hyaluronic acid gel bone repair material.

Example 2 The same operation as in Example 1 was performed using sodium hyaluronate having a molecular weight of 6 × 10 ⁵ daltons to obtain a sheet-like bone-repair material of a poorly water-soluble hyaluronic acid gel.

Example 3 25 ml of an acidic aqueous solution of hyaluronic acid having a molecular weight of 2 × 10 ⁶ daltons and a pH of 1.5 was placed in a plastic petri dish, frozen at −20 ° C. for 22 hours, and thawed at 25 ° C. 2 The time was repeated twice. Next, this was immersed in a phosphate buffered saline solution and washed in the same manner as in Example 1, and then dried in an oven at 40 ° C. for 3 hours to obtain a film-shaped poorly water-soluble hyaluronic acid gel bone repair material.

Example 4 50 mg of the sheet-form poorly soluble hyaluronic acid of Example 1 was aseptically put into 10 ml of physiological saline, and pulverized with a microhomogenizer (POLYTORON, manufactured by KINEMATICA AG) to form a slurry and crushed. A bone-repair material made of a poorly water-soluble hyaluronic acid gel was obtained.

Example 5 15 ml of an acidic aqueous solution of hyaluronic acid having a molecular weight of 2 × 10 ⁶ dalton and having a pH of 1.5 and placed in a 30 ml glass bottle was frozen at −20 ° C. for 22 hours and thawed at 25 ° C. for 2 hours. Was repeated twice. This was immersed in phosphate buffered saline, washed, and freeze-dried in the same manner as in Example 1 to obtain a sponge-like poorly water-soluble hyaluronic acid gel bone repair material.

Example 6 15 ml of an acidic aqueous solution of hyaluronic acid having a molecular weight of 2 × 10 ⁶ daltons and having a pH of 1.5 and placed in a 30 ml glass bottle was frozen at −20 ° C. for 22 hours and thawed at 25 ° C. for 2 hours. Was repeated twice. This was immersed in a phosphate buffered saline solution, washed and freeze-dried while being compacted by centrifugal dehydration in the same manner as in Example 1 to obtain a massive, poorly water-soluble hyaluronic acid gel bone repair material.

Example 7 The sponge-like hyaluronic acid gel of Example 5 was crushed with a homogenizer until the particle size became 1 mm or less, and collected by centrifugation. This was mixed with a 2% by weight aqueous solution of hyaluronic acid having a molecular weight of 2 × 10 ⁶ daltons to obtain a paste-like poorly water-soluble hyaluronic acid gel bone repair material using hyaluronic acid in combination.

Comparative Example 1 The pH of the aqueous solution of hyaluronic acid adjusted in Example 1 was adjusted to pH 7.0 with 1N sodium hydroxide, and 15 ml thereof was placed in a 30 ml glass bottle, frozen at -20 ° C., and freeze-dried. A sheet-shaped hyaluronic acid bone repair material was obtained.

Comparative Example 2 The pH of the aqueous solution of hyaluronic acid adjusted in Example 1 was adjusted to pH 7.0 with 1N sodium hydroxide, and 25 ml thereof was placed in a plastic petri dish and air-dried at 60 ° C. A bone repair material was obtained.

Comparative Example 3 The pH of the aqueous solution of hyaluronic acid adjusted in Example 1 was adjusted to pH 7.0 with 1N sodium hydroxide, 25 ml of the solution was placed in a beaker, frozen at -20 ° C., freeze-dried and sponge-like. Hyaluronic acid bone repair material was obtained.

Comparative Example 4 0.6 g of sodium hyaluronate having an average molecular weight of 600,000 was dissolved in a solution of 1.1 g of disodium hydrogen phosphate dissolved in 30 g of water and adjusted to pH 10 with 2% sodium hydroxide. 0.05 g of cyanuric chloride was dissolved in 1.5 ml of dioxane, added to the above hyaluronic acid solution, and reacted at room temperature for 3 hours. Thereafter, the mixture was placed in a dialysis membrane, dialyzed against water for 1 day, and 15 ml thereof was placed in a 30 ml glass bottle and freeze-dried to obtain a sponge-like cyanuric chloride-crosslinked hyaluronic acid gel bone repair material.

Example 8 Solubility Test of Bone Repair Material of Poorly Water-Soluble Hyaluronic Acid Gel A phosphate buffer component was added to physiological saline at a concentration of 50 mM.
A pH 7 phosphate buffered saline was prepared. The resulting poorly water-soluble hyaluronic acid gel was washed with distilled water and dehydrated on filter paper. A poorly water-soluble hyaluronic acid gel containing 150 mg of hyaluronic acid by dry weight was immersed in a phosphate-buffered saline at a ratio of 50 ml of a phosphate buffered saline. The percentage of hyaluronic acid eluted in phosphate buffered saline at 37 ° C. was determined from the concentration of hyaluronic acid in phosphate buffered saline.

The concentration of hyaluronic acid in the phosphate buffered saline was determined from the peak area of the differential detector using GPC.

As described above, Examples 1 to 7 and Comparative Example 1
3 were tested for solubility. Table 1 shows the results.

[0049]

[Table 1]

From Table 1, it was confirmed that the hyaluronic acid gels obtained in Examples 1 to 7 were poorly soluble in neutral aqueous solutions.

Example 9 Measurement of Branching Degree of Bone Repair Material of Poorly Water-Soluble Hyaluronic Acid Gel The poorly water-soluble hyaluronic acid gel obtained in Examples 1 to 7 was
Immerse in 15 ml of hydrochloric acid aqueous solution of pH 1.5,
Time hydrolysis was performed. The gel was solubilized by hydrolysis and diluted twice with GPC solvent to a concentration of 0.0
After adjusting to 5% by weight and filtering through a 0.2 μm membrane filter, 0.1 ml was injected and GPC-MALLS was added.
Was measured. The degree of branching of the poorly water-soluble hyaluronic acid gels obtained in Examples 1 to 7 was all 0.5 or more.

Example 10 Bone repairing effect test of bone repair material of poorly water-soluble hyaluronic acid gel using rabbit skull defect model Bone repair material of sponge-like poorly water-soluble hyaluronic acid gel (1 cm in diameter, high Further, the paste-like poorly water-soluble hyaluronic acid gel bone repair material of Example 7 was subjected to the following test. As a control, an untreated group to which only physiological saline was administered, and a sample obtained by cutting the cyanuric chloride-crosslinked hyaluronic acid of Comparative Example 4 to a diameter of 1 cm and a height of 3 mm were subjected to the following tests.

20 Japanese white rabbits (about 2.5 kg)
Among them, 5 were sponge-like poorly water-soluble hyaluronic acid gels,
Five of them were used for paste-like poorly water-soluble hyaluronic acid gel, five for comparison were administered with physiological saline, and the other five were used for implantation of cyanuric chloride cross-linked hyaluronic acid. Implantation was performed by cutting the hair of the rabbit head, creating a bone defect (5 mm in diameter) using a microdrill on the skull under ether anesthesia, filling the bone defect with various hyaluronic acids, and suturing. .

Nine weeks after the implantation, the hardly soluble hyaluronic acid-implanted rabbit and the freeze-dried hyaluronic acid-implanted rabbit were killed, the head was incised, and the state of the implanted portion was observed. (5 mm in diameter) was regenerated and the skull was repaired. Table 2 shows the results of calculating the hyaluronic acid residual ratio based on the amount of hyaluronic acid in the embedded sheet, together with the observation results of the state of the embedded portion.

[0055]

[Table 2]

According to Table 2, all rabbits grew normally.
As for the condition of the tissue, the poorly water-soluble hyaluronic acid gel and freeze-dried hyaluronic acid were implanted, and no abnormalities were found in the local tissue condition. Was done.

[0057]

According to the present invention, it is possible to provide a bone repair material containing a poorly water-soluble hyaluronic acid formed of hyaluronic acid alone. Since the bone repair material of the present invention does not use a crosslinking agent or the like, it maintains the structure of the original hyaluronic acid existing in the living body, is excellent in non-toxicity and biocompatibility, and further remarkably improves bone repair. It is to improve.

Claims (4)

[Claims] 1. A bone repair material containing a gel formed of hyaluronic acid alone, which is hardly soluble in a neutral aqueous solution. 2. A bone repair material containing a gel formed of hyaluronic acid alone, which satisfies the following requirements (a) and (b): (A) The dissolution rate in a neutral 37 ° C. aqueous solution at 12 hours is 50% or less. (B) Solubilized by treating the hyaluronic acid gel under conditions of accelerated acid hydrolysis of hyaluronic acid. Hyaluronic acid has a branched structure, and the solubilized hyaluronic acid partially contains a molecular weight fraction having a degree of branching of 0.5 or more. 3. A gel formed with hyaluronic acid alone,
The bone repair material according to claim 2, wherein the bone repair material is one selected from the group consisting of a film, a sheet, a slurry, a crush, a sponge, a lump, and a paste. 4. Hyaluronic acid having a solubility of 50% or less in 12 hours in a neutral aqueous solution at 37 ° C. and solubilized by treating a hyaluronic acid gel under conditions of accelerated acid hydrolysis of hyaluronic acid. The acid has a branched structure, and in the solubilized hyaluronic acid, a hyaluronic acid gel partially containing a molecular weight fraction having a degree of branching of 0.5 or more, hyaluronic acid not gelled, a physiologically active substance, A bone repair material comprising at least one selected from the group consisting of bone granules and antibiotics.