JPH0567301B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a medical material with excellent biocompatibility consisting of a composite material of N-acyl chitosan and collagen, and a composite material consisting of protamine, and a method for producing the same. Specifically, the present invention relates to medical materials that can be applied to wound covering materials, hemostatic materials, etc., and methods for producing the same.

[Technical background and explanation of conventional technology]

Chitosan is a complex polysaccharide consisting of N-acetyl-D-glucosamine and D-glucosamine obtained by deacetylating chitin with concentrated alkali. - Different ratios of glucosamine and D-glucosamine are known.

Collagen is a major component of the connective tissue of living organisms, and is the most suitable material as a cell matrix, so it is a material with extremely high biocompatibility. Since collagen is a protein, antigenicity is a problem in xenogeneic implants, but collagen's antigenicity is relatively low, so collagen is widely used as a cosmetic or medical material. Furthermore, tropocollagen (collagen molecules) is treated with proteolytic enzymes other than collagenase to remove the non-helical portion (telopeptide) at the end of the molecule, which has extremely low antigenicity and is called atelocollagen. Atelocollagen has outstanding characteristics such as having the best biocompatibility among currently used medical materials due to its low antigenicity. Since it will eventually be absorbed by the living body and is more expensive than chitosan, its range of use is limited.

Chitosan-collagen composite materials are known to take advantage of the characteristics of chitosan and collagen and compensate for their respective drawbacks. (Unexamined Japanese Patent Publication No. 56-133344
However, chitosan is a polysaccharide that does not exist in living organisms, and is obviously a foreign substance to living organisms, so if it is directly applied to living organisms, a foreign body reaction will occur that is larger than that of collagen, and an optical microscope shows that chitosan is a foreign substance. Many giant cells are observed in the surrounding area. As described above, it is difficult to apply chitosan alone as a medical material, and the method of adding collagen to improve biocompatibility is still insufficient, so chitosan has not been put into clinical use. .

The present inventors have continued research on chitosan and collagen for many years, and have found that chitosan derivatives, namely N-acyl chitosan, obtained by chemically modifying chitosan can be used satisfactorily as medical materials. Furthermore, the inventors discovered that the combination of these chitosan derivatives and collagen can be put to practical use clinically, and based on these findings, the present invention was achieved.

[Object of the invention and summary of the invention]

The purpose of the present invention is to provide biocompatible and clinically practical chitosan derivatives, and more specifically, to provide chitosan derivatives and collagen that are biocompatible and clinically practical. Our goal is to provide composite materials that are of great quality.

The present invention is a medical material made of a composite material of N-acylchitosan and collagen and a composite material of protamine.

Collagen in the present invention may be either a simple collagen or a collagen derivative obtained by chemically modifying collagen.

The medical material of the present invention can be obtained by freeze-drying a solution, dispersion, or gel of the composite material to form a sponge, or by molding these composite materials to form a film or membrane. It is manufactured by

The N-acyl chitosan in the present invention can be a chitosan derivative obtained by modifying the amino group of chitosan with a saturated or unsaturated fatty acid having 1 to 32 carbon atoms and/or a dicarboxylic acid having 2 to 8 carbon atoms. N-acyl chitosan can also be N-acetyl chitosan or N-succinyl chitosan.

Collagen in the present invention can be succinylated collagen, acetylated collagen or methylated collagen.

The composite material of N-acyl chitosan and collagen of the present invention containing protamine has an excellent blood coagulation effect and is suitable for application as a hemostatic material.

Furthermore, in the medical material of the present invention, a composite material processed into a sponge shape or applied to a substrate can be crosslinked by treatment with a bifunctional crosslinking agent or by irradiation with radiation. This can also be further sterilized before use.

[Specific description of the invention]

N-acyl chitosan in the present invention is obtained by acylating chitosan, and N-acyl chitosan is obtained by acylating chitosan.
The chitosan used in the preparation of acyl chitosan is obtained by deacetylating chitin with a concentrated alkali, and any chitosan can be used as long as it is soluble in acid. but the degree of deacetylation is preferably at least 45%.

N-acyl chitosan is prepared by reacting chitosan with a carboxylic acid anhydride to perform N-acylation of chitosan, but N-acylation of chitosan is performed after adding collagen to chitosan. It can also be prepared by

Collagen in the present invention may be any collagen or collagen derivative obtained by chemically processing collagen, but atelocollagen or collagen with improved biocompatibility may be chemically modified. It is also possible to use collagen whose biocompatibility has been improved. For example, succinylated collagen, which is made by treating collagen with succinic anhydride, has excellent antithrombotic properties, and methylated collagen, which is made by treating collagen with anhydrous methanol, has unique properties such as increasing the reaction with platelets. Therefore, medical materials suitable for purposes can be obtained according to their properties.

Chemical modification of collagen is carried out by reaction of collagen with a reactant, but can also be carried out after adding collagen to N-acyl chitosan. When the reactive agent for chemically modifying collagen is a carboxylic anhydride, a composite material obtained by adding collagen to chitosan is reacted with the carboxylic anhydride to N-acylate the chitosan and chemically modify the collagen. It is also possible to make modifications at the same time.

Chemical modification of collagen is mainly carried out on the amino groups or carboxyl groups of collagen, and the amino groups or carboxyl groups of collagen are modified by 5 to 100% (preferably 30 to 100%).
%) is preferably applied.

The present invention can control the reaction with blood by adding protamine to the composite material of N-acyl chitosan and collagen.

In order to improve biocompatibility, it is necessary to create medical materials that are similar to living organisms, and the chemical modifications and addition of biologically useful components described above are suitable for this purpose.

The medical material of the present invention is produced by drying the solution, dispersion or gel of the composite material described above, and can be produced by applying the solution or dispersion of the composite material to the surface of a substrate such as a glass plate. When the composite material is lyophilized, a film-like or membranous medical material can be produced.
Sponge-like medical materials can be made.

The medical material of the composite of the present invention can be crosslinked by treating it with a bifunctional crosslinking agent or by irradiating it with radiation, thereby improving the strength and water absorption of the medical material.

Bifunctional crosslinking agents in crosslinking medical materials are
Any material having two or more functional groups can be used, but hexamethylene diisocyanate or glutaraldehyde is preferably used. A bifunctional crosslinking agent can be added to a solution or dispersion of a composite material in advance, and a crosslinking reaction can be carried out after forming the composite material into a film, membrane or sponge. After being molded into a shape, the molded product can be immersed in a solution of a crosslinking agent or irradiated with radiation, thereby causing a crosslinking reaction. As the radiation, any particle beam such as ultraviolet rays, gamma rays, or alpha rays can be used, but it is preferable to use ultraviolet rays or gamma rays.

The medical material of the present invention is used in the form of a film, membranous, or sponge-like molded article, but it can also be used in combination with other medical materials. Examples of use in the form of film-like, membranous or sponge-like molded articles include use as wound covering materials, artificial skin, hemostatic materials or anti-adhesion membranes, and examples of use in combination with other medical materials include: It is used as a coating on the inner and outer surfaces of artificial blood vessels and catheters made of synthetic polymeric materials. To use such a synthetic polymer medical material as a coating, the synthetic polymer medical material is immersed in a composite solution and then dried. There is a method in which the composite material is applied to the material and then dried, or a method in which the composite material is reacted with a carboxylic acid anhydride or other reactant prior to drying, and then a method is performed in which the composite material is chemically treated and then dried. When the composite material of the present invention is combined with other medical materials, the surface thereof is coated with the composite material of the present invention, and the surface of the medical material obtained by drying is film-like or The medical material is membrane-like, and the surface of the medical material obtained by freeze-drying is spongy, and its biocompatibility is improved.

EXAMPLES Below, the present invention will be explained in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 (Preparation of chitosan) 200 g of crushed red snow crab shell was added to 5% hydrochloric acid 2 and stirred at room temperature for 5 hours, then the solution was filtered and the remaining solids were washed with water. This solid was placed in a 5% aqueous sodium hydroxide solution 2 and heated to 90° C. for 2.5 hours with stirring, then the solution was filtered and the remaining solid was washed with water.

The chitin obtained here was placed in a 50% aqueous sodium hydroxide solution 2 and heated to 90°C for 2.5 hours while stirring, then the solution was filtered, the precipitated solid was thoroughly washed with water, and then dried at 95°C. , 41 g of chitosan with a degree of deacetylation of 99% was obtained.

(Preparation of atelocollagen) Fresh calf dermis is finely ground, and this fine powder is
100 g was washed repeatedly with 0.1M aqueous sodium acetate solution and then with water. 10g of fine powder obtained here
Add 0.5M acetic acid aqueous solution 4 to
After stirring for several days, the precipitated insoluble collagen was filtered off. 10g of this insoluble collagen was mixed with 0.1M acetic acid.
Add pepsin (Sigma product, 1:
60000) was added and stirred for 3 days at 20°C, thereby obtaining an atelocollagen (pepsin-solubilized collagen) solution. This atelocollagen solution was filtered through a glass filter, and an aqueous sodium hydroxide solution was added to the resulting filtrate.
The pH was adjusted to 7.5, which produced a fibrous precipitate. 7,
The mixture was centrifuged at 000G and 8,000 rpm, and the resulting precipitate was washed three times with distilled water to obtain 7 g of atelocollagen.

Example 2 (Preparation of N-succinyl chitosan) 2 g of chitosan from Example 1 was added to 40 ml of 5% aqueous acetic acid solution.
This was diluted with 160 ml of methanol. Separately, 1.4 g of succinic anhydride (corresponding to 0.98 mol per 1 mol of chitosan amino group) was dissolved in 50 ml of acetone, and the entire amount of the obtained acetone solution of succinic anhydride was added to the chitosan solution. was added and left overnight. After filtering the precipitate, dry it to obtain 2g of N-succinylchitosan powder.
I got it. The amino group modification rate of this N-succinyl chitosan was 35%.

(Preparation of succinylated collagen) 2 g of collagen was dissolved in a mixture of 1.6 ml of concentrated hydrochloric acid and 100 ml of water, and a 5N aqueous sodium hydroxide solution was gradually added thereto while stirring to adjust the pH to 13.

Separately, 0.07 g of succinic anhydride (equivalent to 1 mol per 1 mol of ε-amino group in the side chain of collagen) was dissolved in 10 ml of acetone, and the entire amount was gradually added to the collagen solution. Thereafter, the mixture was stirred overnight while constantly adjusting the pH to 13 with a 5N aqueous sodium hydroxide solution. After filtering the precipitate and thoroughly washing with water, dry it to obtain 2g of succinylated collagen.
I got it. The modification rate of ε-amino groups in this succinylated collagen was 30%.

Example 3 (Preparation of mixture of N-succinyl-N-octanoyl chitosan and succinylated collagen) 9 g of N-succinyl chitosan of Example 2 and 1 g of succinylated collagen were dissolved in 500 ml of water,
This was diluted with 2 parts of methanol. 22 g of octanoic anhydride (caprylic anhydride) (corresponding to 2 molar equivalents per hexosaminyl residue of chitosan) was added to this solution, and this was left at room temperature overnight, and N-succinyl-N-octanoyl chitosan and succinyl A solution of collagen mixture was obtained.

Example 4 (Preparation of methylated collagen) 2 g of atelocollagen from Example 1 was immersed in an acidic hydrochloric acid solution 1 of anhydrous methanol for 2 weeks to methylate the carboxyl groups in the side chains of collagen, thereby obtaining 2 g of methylated collagen.

(Preparation of sponge) 18 g of the chitosan of Example 1 was mixed with 2 g of the methylated collagen obtained above and 1 g of commercially available protamine sulfate (manufactured by Wako Pure Chemical Industries, Ltd.), and the resulting mixture was mixed with 1 g of a 2% acetic acid aqueous solution. dissolved in. This was diluted with 4 methanol, 24 g of acetic anhydride (corresponding to 2 molar equivalents per hexosaminyl residue in chitosan) was mixed with it, and the mixture was left at room temperature overnight.
5 kg of a mixed gel of N-acetyl chitosan, methylated collagen and protamine was obtained. this gel
A sponge of N-acetylchitosan-methylated collagen-protamine was obtained by freeze-drying in a vacuum of 0.2 mmHg.

After slicing this sponge into 5mm thick slices,
Cross-linking was introduced into the sponge by irradiation with megarad gamma rays, and sterilization was performed.

When the sponge obtained here was used as a hemostatic material in surgical operations, it showed excellent effectiveness as a hemostatic material.

Example 5 To 5 g of the gel of N-acetyl chitosan and acetylated collagen obtained in Example 1, 1 g of methylated collagen obtained in Example 4 and 100 ml of water were added and well dispersed, and the pH was adjusted to 3 with 1N hydrochloric acid. After adding and dissolving 1 g of protamine in this solution, this solution was applied to a mesh of Tetron, and this was dried at 25°C. Repeat application and drying of the solution 10 times.
A wound covering material was obtained by applying a composite material of N-acetyl chitosan, acetylated collagen, methylated collagen, and protamine to Tetoron mesh in the form of a film. This wound dressing is cross-linked and sterilized by irradiating it with 1.5 Megarad gamma rays.
When attached to the wound surface of the skin with medical adhesive tape, it suppresses bleeding and protects the wound area.
The wound showed good healing.

〔Effect of the invention〕

The medical material made of the chitosan derivative, collagen composite, and protamine composite material of the present invention has excellent biocompatibility, so even when used in living organisms, antigenicity will not be a problem and it can be used safely. When used as a wound covering material in combination with various derivatives, it promotes healing of the wound surface, and those with thrombotic or antithrombotic properties can be used as a hemostatic material and show excellent effects. .

Claims (1)

[Scope of Claims] 1. A hemostatic material made of a composite material of N-acyl chitosan and collagen, and a composite material of protamine. 2. Claims characterized in that the N-acyl chitosan is obtained by modifying the amino group of chitosan with a saturated or unsaturated fatty acid having 1 to 32 carbon atoms and/or a dicarboxylic acid having 2 to 8 carbon atoms. The hemostatic material according to item 1. 3. The hemostatic material according to claim 1, wherein the N-acyl chitosan is N-acetyl chitosan obtained by acylating chitosan. 4. Claim 1, wherein the N-acyl chitosan is N-succinyl chitosan.
Hemostatic material as described in section. 5. The hemostatic material according to any one of claims 1 to 4, wherein the collagen is succinylated collagen. 6. The hemostatic material according to any one of claims 1 to 4, wherein the collagen is acetylated collagen. 7. The hemostatic material according to any one of claims 1 to 4, wherein the collagen is methylated collagen. 8. The hemostatic material according to any one of claims 1 to 7, wherein the composite material is processed into a sponge shape. 9. The hemostatic material according to any one of claims 1 to 7, wherein the composite material is processed into a film shape. 10 A method for producing a hemostatic material, which comprises freeze-drying a solution of a composite material of N-acyl chitosan and collagen, and processing it into a sponge. 11. The method for producing a hemostatic material according to claim 10, wherein the N-acyl chitosan is N-acetyl chitosan obtained by acetylating chitosan. 12. The method for producing a hemostatic material according to claim 10, wherein the N-acyl chitosan is N-succinyl chitosan. 13. The method for producing a hemostatic material according to any one of claims 10 to 12, wherein the collagen is succinylated collagen. 14. The method for producing a hemostatic material according to any one of claims 10 to 12, wherein the collagen is acetylated collagen. 15. The method for producing a hemostatic material according to any one of claims 10 to 12, wherein the collagen is methylated collagen. 16. The method for producing a hemostatic material according to any one of claims 10 to 15, characterized in that a composite material processed into a sponge shape is treated with a bifunctional crosslinking agent. 17. The method for producing a hemostatic material according to any one of claims 10 to 15, characterized in that the composite material processed into a sponge shape is cross-linked with radiation and then sterilized. 18 A method for producing a hemostatic material, which comprises applying a solution of a composite material of N-acyl chitosan and collagen, and protamine to a substrate, and then drying the solution. 19. The method for producing a hemostatic material according to claim 18, wherein the N-acyl chitosan is N-acetyl chitosan obtained by acetylating chitosan. 20. The method for producing a hemostatic material according to claim 18, wherein the N-acyl chitosan is N-succinyl chitosan. 21. The method for producing a hemostatic material according to any one of claims 18 to 20, wherein the collagen is succinylated collagen. 22. The method for producing a hemostatic material according to any one of claims 18 to 20, wherein the collagen is acetylated collagen. 23. The method for producing a hemostatic material according to any one of claims 18 to 20, wherein the collagen is methylated collagen. 24. The method for producing a hemostatic material according to any one of claims 18 to 23, characterized in that the composite material applied to the substrate is treated with a bifunctional crosslinking agent. 25. The method for producing a hemostatic material according to any one of claims 18 to 23, characterized in that the composite material applied to the substrate is cross-linked with radiation and then sterilized.