JPH01303151A - Artificial skin and wound covering material and production thereof - Google Patents

Abstract

PURPOSE:To obtain the medical material which can be applied to a wound covering material, artificial skin, artificial blood vessel, hemostatic, etc., by forming the material of a composite material consisting of a composite material of N-acylchitosan and collagen as well as various derivatives. CONSTITUTION:A water holding property can be imparted to the medical material by adding chondroitin, chondroitin-4-sulfuric acid, dermatane sulfuric acid, chondroitin-6-sulfuric acid or hyaluronic acid to the composite material composed of the N-acylchitosan and collagen. These mucopolysaccharides are contained in the skin, etc., play an important role in holding of moisture and have an effect of preventing drying of a wound surface when the medical material is applied to the treatment of a wound. The restoration of the wounded part can be accelerated by adding fibronectin or fibrin thereto. The reaction with blood can be controlled by adding heparin or protamine thereto. The strength and water absorptivity of the medical material are improved by crosslinking the medical material consisting of the composite material of the N-acylchitosan and collagen.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a biocompatible soaked medical material made of a composite of N-acyl chitosan and collagen and a method for producing the same, and more particularly, to a wound dressing material, The present invention relates to medical materials that can be applied to artificial skin, artificial blood vessels, hemostatic materials, etc., and methods for producing the same.

[Technical background and explanation of conventional technology]

Chitosan is a combination of N-acetyl-D-glucosamine and D-glucosamine obtained by deacetylating chitin with concentrated alkali.
A complex polysaccharide consisting of glucosamine, which can be divided into N-acetyl-D-glucosamine and D-glucosamine depending on the degree of deacetylation.
-Various glucosamine ratios 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 implants between different species, but since the antigenicity of collagen is relatively low, collagen is widely used as a cosmetic or medical material. Furthermore, trobocollagen (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. (Japanese Unexamined Patent Publication No. 56-133344) However, chitosan is an 11-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 larger foreign body reaction will occur than with collagen. , many giant cells are observed around chitosan using an optical microscope. In this way, chitosan alone
It is difficult to apply chitosan as a medical material, and the method of adding collagen to improve biocompatibility is still insufficient, so chitosan has not been put to clinical use.

The present inventors have continued research on chitosan and collagen for many years, and have chemically modified chitosan to produce chitosan derivatives, namely N-acyl chitosan.
It was found that these chitosan derivatives can be satisfactorily used as medical materials, and furthermore, it was found that the combination of these chitosan derivatives and collagen can be used clinically.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 objective is to provide composite materials that are of the highest quality.

The present invention is a medical material made of a composite material of N-acyl chitosan and collagen.

The collagen in the composite material of N-acyl chitosan and collagen of the present invention may be either a fullerogen body or a collagen derivative obtained by chemically modifying collagen.

The medical material made of the composite material of N-acyl chitosan and collagen of the present invention can be obtained by freeze-drying a solution, dispersion or gel of the composite material of N-acyl chitosan and collagen and processing it into a sponge shape, or It is manufactured by molding a composite material of chitosan and collagen and processing it into a film or membrane.

The N-acyl chitosan in the N-acyl chitosan and collagen composite material of the present invention 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. It can be a chitosan derivative, and the N-acylchitosan can also be N-acetylchitosan or N-succinylchitosan.

The collagen in the N-acyl chitosan and collagen composite of the present invention can be succinylated collagen, acetylated collagen or methylated collagen.

The N-acyl chitosan and collagen composite material of the present invention includes chondroitin, chondroitin-4-sulfate, dermatan sulfate, chondroitin-6-sulfate, hyaluronic acid,
Products containing one or more of fibronectin, fibrin, epidermal growth factor and vertebroblast growth factor have excellent effects on wound healing and are suitable for use as artificial skin and wound covering materials. suitable for N-
A composite material of acyl chitosan and collagen impregnated with heparin has excellent antithrombotic properties and is suitable for application to blood vessel walls or as an artificial blood vessel.
Furthermore, a composite material of N-acyl chitosan and collagen 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 of N-acyl chitosan and collagen processed into a sponge or applied to a substrate is crosslinked by treatment with a bifunctional crosslinking agent or by irradiation with radiation. You can also. This can also be further sterilized before use.

[Detailed description of the invention]

The N-acyl chitosan in the N-acyl chitosan and collagen composite material of the present invention is obtained by acylating chitosan, but the chitosan used for preparing N-acyl chitosan is obtained by deacetylating chitin with an alkali. Any acid-soluble material can be used, but it is preferable to use one with a degree of deacetylation of at least 45%. preferable.

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

The collagen in the composite material of N-acyl chitosan and collagen of the present invention can be any collagen or collagen derivative obtained by chemically processing collagen, but biocompatibility must be ensured. It is also possible to use improved atelocollagen or collagen that has been chemically modified to have improved biocompatibility. 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. Modifications can also be made at the same time.

Chemical modification of collagen is mainly carried out on the amino groups or carboxyl groups of collagen.
It is preferable to carry out a chemical modification of 0096 (preferably 30 to 100%).

The composite material of N-acyl chitosan and collagen of the present invention contains chondroitin, chondroitin-4-1
Water retention properties can be imparted to medical materials by adding acids, dermatan sulfate, chondroitin-6-sulfate or hyaluronic acid. These mucopolysaccharides are contained in the skin and play an important role in retaining moisture. For example, when the medical material of the present invention is applied to wound treatment, it can be used to prevent the wound surface from drying out. effective.

Furthermore, by adding fibronectin or fibrin to the composite material of N-acyl chitosan and collagen of the present invention, repair of a wound can be promoted.

Furthermore, by adding heparin or protamine to the N-acyl chitosan and collagen composite material of the present invention, the reaction with blood can be reduced.

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 of N-acyl chitosan and collagen described above. When the composite material is applied to the surface of a substrate and dried, a film-like or membranous medical material can be produced, and when the composite material is freeze-dried, a sponge-like medical material can be collapsed.

The N-acylchitosan and collagen composite medical material S of the present invention is treated with a bifunctional cross-linking agent or irradiated with radiation to improve the strength and water absorption of the medical material. can be improved.

Any difunctional crosslinking agent for crosslinking medical materials can be used as long as it has two or more functional groups, but hexamethylene diisocyanate or glutaraldehyde is preferably used. is preferable. A bifunctional cross-linking agent can also be added to a solution or dispersion of a composite material of N-acyl chitosan and collagen in advance and subjected to a cross-reaction after forming into a film, membrane, or sponge. - After molding the acyl chitosan and collagen composite into a film, membrane or sponge, the molded product is immersed in a layer solution of a crosslinking agent or irradiated with radiation, thereby causing a cross-linking reaction. You can also make them do it. 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 N-acyl chitosan and collagen composite medical material of the present invention is used in the form of a film, membranous, or sponge-like molded product, 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 dressings, 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, there are two methods: immersing the synthetic polymer medical material in a solution of a composite of N-acyl chitosan and collagen and then drying it; A viscous solution or dispersion may be applied to a synthetic polymer medical material and then dried, or the composite may be chemically treated by reacting with a carboxylic acid anhydride or other reactant prior to drying. There is a way to dry it after washing. Such a composite material of N-acyl chitosan and collagen of the present invention combined with other medical materials has its surface coated with the composite material of N-acyl chitosan and collagen of the present invention, and is dried. The surface of the medical material obtained by lyophilization is film-like or 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 placed in 5% hydrochloric acid 21 and stirred at room temperature for 5 hours, then the solution was filtered and the remaining solids were washed with water. Add this solid to a 5% aqueous sodium hydroxide solution for 27 hours! After heating at 90° C. for 2.5 hours with stirring, 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 21, heated at 90°C for 2.5 hours with stirring, the solution was filtered, the precipitated solid was thoroughly washed with water, and the mixture was heated at 95°C. After drying, 41 g of chitosan with a degree of deacetylation of 99% was obtained.

(Preparation of atelocollagen) Finely grind fresh calf dermis, and add 100g of this fine powder.
was washed repeatedly with 0.111 aqueous sodium acetate solution and then with water. 0.5M to the fine powder IOg obtained here
Acetic acid aqueous solution 4I! After stirring at 5°C for 3 days, the precipitated insoluble collagen was filtered out. This insoluble collagen IOg was placed in 0.1M acetic acid + 00mjl, and pepsin (Sigma product, 1: ao, ooo) was added.
o, otg were added and stirred at 20°C for 3 days, 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 to adjust its pH to 7.5, thereby producing a fiber-rich precipitate. The resulting solution was centrifuged at 7,000 G and 8,000 rpm, and the resulting precipitate was washed three times with distilled water to obtain 7 g of atelocollagen.

(Preparation of N-acetyl chitosan-acetylated collagen sponge) 18 g of chitosan with a degree of deacetylation of 99% (0
, 12 moles) was mixed with 2 g of atelocollagen obtained above, and 20 g of this mixture was added to a 2% acetic acid aqueous solution lI! This was diluted with methanol 2A. 24 g of acetic anhydride (corresponding to 2 molar equivalents per hexosaminyl residue of chitosan) was mixed with this solution, and the mixture was left at room temperature overnight and immersed in running water to remove acetic acid and methanol. 350 to 380 g of a hydrated gel of a complex of collagen was obtained. Place this gel in a metal container with dry ice-methanol solution 5I! immerse it in water and freeze it quickly, then freeze it at 0. Lyophilization was performed in a vacuum of 2+s+Hg to obtain 20 g of N-acetylchitosan-acetylated collagen sponge.

(Preparation of wound dressing material) An N-acetyl chitosan-acetylated collagen sponge was immersed in 10 t of a 2% methanol solution of hexamethylene diisocyanate, and a bridge was introduced.

After 2 hours, the N-acetylchitosan-acetylated collagen sponge was taken out, washed thoroughly with methanol and water, and then freeze-dried in a vacuum of 0.2 JllHg. The sponge thus obtained was sliced to a thickness of 11111 mm to obtain a wound covering material.

When this wound covering material was applied to human wounds, a smooth healing process was observed.

Example 2 (Preparation of N-succinyl chitosan) After dissolving 2 g of the chitosan of Example 1 in 40 mm of a 5% acetic acid aqueous solution, this was diluted with 160 mm of methanol. Separately, 1.4 g of succinic anhydride (corresponding to 0.98 mol per 1 mol of the amino group of chitosan) was dissolved in 50-mL of acetone, and the entire amount of the resulting acetone solution of succinic anhydride was dissolved. The above chitosan solution was added to the apparatus overnight.

After filtering the precipitate, it was dried to obtain 2 g of N-succinyl chitosan powder. 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 concentrated hydrochloric acid (1.6 mm) and water (100 mm), and while stirring, a 5N aqueous sodium hydroxide solution was gradually added thereto to adjust the pH to 13. .

Apart from this, succinic anhydride o, o79 (corresponding to 1 mol per 1 mol of ε-amino group in the side chain of collagen)
was dissolved in acetone IO-, and the entire amount thereof was gradually added to the above collagen solution, followed by stirring overnight while constantly adjusting the pH to 13 with a 5N aqueous sodium hydroxide solution. The precipitate was filtered, thoroughly washed with water, and then dried to obtain 2 g of succinylated collagen. The modification rate of ε-amino groups in this succinylated collagen was 30%.

(Preparation of artificial blood vessel) 0.9 g of N-succinyl chitosan and 0.1 g of succinylated collagen obtained above were dissolved in water +00-, and the surface was plasma-treated to prepare a Dacron artificial blood vessel (inner diameter 6jIj11 length 5Ci). was soaked, taken out, and air-dried. After repeating this operation 20 times, the Dacron artificial blood vessel was treated with glutaraldehyde adjusted to pH 13.
% aqueous solution for 1 hour to introduce crosslinking. After thoroughly washing this Dacron artificial blood vessel with water and drying it for three times, the surface became N-
An artificial blood vessel consisting of a film of a mixture of succinyl chitosan and succinylated collagen was obtained.

This artificial blood vessel was implanted into a living body and showed good antithrombotic properties.

Example 3 (Preparation of N-succinyl-N-octanoyl chitosan and succinylated collagen mixture) 9 g of N-succinyl chitosan of Example 2 and succinylated collagen 1,9
was dissolved in 500 g of water and diluted with 21 g of methanol. Add octanoic anhydride (caprylic anhydride) to this solution.
2219 (corresponding to 2 molar equivalents per hexosaminyl residue of chitosan) was added, and this was left at room temperature overnight and N-
A solution of succinyl-N-octanoyl chitosan and succinylated collagen mixture was obtained.

(Preparation of medical material film) This solution of N-succinyl-N-octanoyl chitosan and succinylated collagen mixture was applied on a glass plate and air-dried to form a mixture of N-succinyl-N-octanoyl chitosan and succinylated collagen. film (thickness Q
, l*m) and was irradiated with ultraviolet rays to introduce crosslinking.

When this film was applied to a false membrane as an adhesion prevention film, no adhesion was observed and good results were obtained.

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

(Preparation of sponge) 18 g of chitosan from Example 1 was mixed with 2 g of the methylated collagen obtained above and 1 g of commercially available protamine sulfate (product of Wako Pure Chemical Industries, Ltd.), and the resulting mixture was mixed with 1 t of a 2% acetic acid aqueous solution. dissolved in This is methanol 47! This was mixed with acetic anhydride 249 (corresponding to 2 molar equivalents per hexosaminyl residue of chitosan), and the mixture was incubated overnight at room temperature to form a mixture of N-acetyl chitosan, methylated collagen, and protamine. Got 9 on gel 5. This gel was lyophilized in a vacuum of 0.2 Hg and N-
A sponge of acetyl chitosan-methylated collagen-protamine was obtained.

After slicing this sponge into 5 mm thick slices, the sponge was irradiated with 5 megarad gamma rays to introduce crosslinking into the sponge and sterilized.

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

Example 5 A solution of the mixture of N-succinyl-N-octanoylchitosan and succinylated collagen obtained in Example 3 was dried under vacuum (0.1 torr) L, I, N-succinyl-N-octanoylchitosan and succinylated collagen. A powder 109 of the mixture was obtained. Dissolve lOy of this powder in 50 ml of water, and add 0.0 ml of chondroitin-4-sulfate. IQ, dermatan sulfate 0.1 g, hyaluronic acid 0.05 g, fibronectin o, ooot g and fibrin 0.001
1 was added and the solution was stirred with a homogenizer. This solution was vacuum freeze-dried at 0.2 torr, and the resulting sponge-like dry product was immersed in 2% hexamethylene diisocyanate (methanol solution), then taken out and thoroughly washed with methanol. This was vacuum-dried again at 0.2 torr and then sliced to a thickness of 1111+. This sponge (20s + m x 20 dragon x I 111
1 (thickness)] and cultured at 37°C for 2 weeks to obtain artificial skin incorporating the cells. When the artificial skin obtained here was transplanted to an area on the back of a rat where the skin was completely missing, and a small piece of epidermis was transplanted onto the artificial skin, the epidermis regenerated and complete healing was observed.

Example 6 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% of water were added and fractionated well, and the pH was adjusted to 3 with IN hydrochloric acid. did. After adding 1 g of protamine to this solution and dissolving it, this solution was applied to a Tetron mesh, which was dried at 2560. Application of the solution and drying were repeated 10 times to obtain a wound cover a in which a composite material of N-acetyl chitosan, acetylated collagen, methylated collagen, and protamine was applied in the form of a film on Tetron mesh. This wound covering material was irradiated with 1.5 Megarad gamma rays to sterilize it, and when it was applied to the wound surface of the skin with medical adhesive tape, it suppressed bleeding and protected the wound area. However, the wound showed good healing.

Example 7 (Preparation of artificial blood vessel) N-succinyl chitosan obtained in Example 2, 0.9 J7°, 0.1 g of succinylated collagen obtained in Example 2, and 20 μg of heparin were dissolved in water +00-, and this was carried out. I lived with Example 2 and prepared an artificial blood vessel. This artificial blood vessel had excellent antithrombotic properties.

〔Effect of the invention〕

The medical material made of the chitosan derivative and collagen 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 flW wound covering material or artificial skin in combination, it promotes the healing of the wound surface, and if it has thrombotic or thrombotic properties, it can be used as a hemostatic material or an artificial blood vessel, Shows excellent effects.

Applicant: Katakura Chikkarin Co., Ltd. Takaken Co., Ltd.

Claims (25)

[Claims] (1) Composite material of N-acyl chitosan and collagen,
and chondroitin, chondroitin-4-sulfate,
An artificial skin made of a composite material selected from the group consisting of dermatan sulfate, chondroitin-6-sulfate, hyaluronic acid, fibronectin, fibrin, epidermal growth factor, and fibroblast growth factor; and Wound dressing material. (2) A patent claim 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 artificial skin and wound covering material according to item 1. (3) The artificial skin and wound covering material according to claim 1, wherein the N-acyl chitosan is N-acetyl chitosan obtained by acylating chitosan. (4) The artificial skin and wound covering material according to claim 1, wherein the N-acyl chitosan is N-succinyl chitosan. (5) The artificial skin and wound covering material according to any one of claims 1 to 4, wherein the collagen is succinylated collagen. (6) The artificial skin and wound covering material according to any one of claims 1 to 4, wherein the collagen is acetylated collagen. (7) The artificial skin and wound covering material according to any one of claims 1 to 4, wherein the collagen is methylated collagen. (8) The artificial skin and wound covering material according to any one of claims 1 to 7, wherein the composite material is processed into a sponge shape. (9) The artificial skin and wound covering material according to any one of claims 1 to 7, wherein the composite material is processed into a film. (10) Consisting of a composite of N-acyl chitosan and collagen, as well as chondroitin, chondroitin-4-sulfate, dermatan sulfate, chondroitin-6-sulfate, hyaluronic acid, fibronectin, fibrin, epidermal growth factor and fibroblast growth factor 1. A method for producing artificial skin and wound covering materials, which comprises freeze-drying a solution of a composite material selected from the group and processing it into a sponge-like material. (11) The method for producing artificial skin and wound covering materials according to claim 10, wherein the N-acyl chitosan is N-acetyl chitosan obtained by acetylating chitosan. (12) The method for producing artificial skin and wound covering materials according to claim 10, wherein the N-acyl chitosan is N-succinyl chitosan. (13) Claims 10 to 12, characterized in that the collagen is succinylated collagen.
A method for producing the artificial skin and wound covering material according to any of the above. (14) The method for producing artificial skin and wound covering materials according to any one of claims 10 to 12, wherein the collagen is acetylated collagen. (15) The method for producing artificial skin and wound covering materials according to any one of claims 10 to 12, wherein the collagen is methylated collagen. (16) The artificial skin and wound covering material according to any one of claims 10 to 15, characterized in that the composite material processed into a sponge shape is treated with a bifunctional crosslinking agent. manufacturing method. (17) The artificial skin and wound cover 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. How the material is manufactured. (18) Composed of a composite of N-acyl chitosan and collagen, as well as chondroitin, chondroitin-4-sulfate, dermatan sulfate, chondroitin-6-sulfate, hyaluronic acid, fibronectin, fibrin, epidermal growth factor, and fibroblast growth factor. A method for producing artificial skin and wound covering materials, which comprises applying a solution of a composite material to a substrate and then drying the solution. (19) The method for producing artificial skin and wound covering materials according to claim 18, wherein the N-acyl chitosan is N-acetyl chitosan obtained by acetylating chitosan. (20) The method for producing artificial skin and wound covering materials according to claim 18, wherein the N-acyl chitosan is N-succinyl chitosan. (21) Claims 18 to 20, characterized in that the collagen is succinylated collagen.
A method for producing the artificial skin and wound covering material according to any of the above. (22) The method for producing artificial skin and wound covering materials according to any one of claims 18 to 20, wherein the collagen is acetylated collagen. (23) The method for producing artificial skin and wound covering materials according to any one of claims 18 to 20, wherein the collagen is methylated collagen. (24) The artificial skin and wound covering 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. Manufacturing method. (25) The artificial skin and wound covering material according to any one of claims 18 to 23, wherein the composite material applied to the base is cross-linked with radiation and then sterilized. manufacturing method.