JP2664930B2 - Artificial skin material and manufacturing method thereof - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an artificial skin material and a method for producing the same.

[Problems to be Solved by the Prior Art and the Invention] When polyvinyl alcohol is gelled without chemical treatment with formaldehyde, glutaraldehyde, hexamethylenediamine or the like, a polymer network structure is formed due to entanglement between polymer chains. , And a characteristic characteristic of the gel is produced by a synergistic effect with the solvent that has entered the interstices of the network.
If the degree of entanglement between the polymer chains is small, not only the tensile strength and the elasticity of the formed gel are low, but also the gel property is easily lost due to external factors such as temperature, which is not practical.

Many methods have already been proposed as means for increasing the degree of entanglement between polymer chains of polyvinyl alcohol. However, all have difficulty in properties and operation of the product.

The typical gelling methods of conventional polyvinyl alcohol are summarized as follows: 1) a method of irradiating an aqueous solution of polyvinyl alcohol with cobalt 60 (Japanese Patent Application Laid-Open No. 50-55647);
3) a method of cooling the aqueous solution of polyvinyl alcohol to a temperature of 18 ° C. or lower, and drying or dehydrating the aqueous solution of polyvinyl alcohol in a vacuum without thawing it (JP-A-57-130543 and JP-A-58-36630). 3)
The gel obtained by the method described above is relatively durable and sufficiently practical depending on the application, but it becomes a soft gel in an aqueous solution of 50 to 60 ° C or higher, easily dissolves at 100 ° C, Since it is still weak, it leaves concerns about its sterility when used as a biomedical material and its strength when implanted in vivo for a long time. Furthermore, considering that hyaluronic acid is essential for the biosynthesis of gels, it is necessary to elute hyaluronic acid little by little for a suitable period of time, which requires heat or physically. If weak, hyaluronic acid elutes easily and is unsuitable for application to living organisms.

Therefore, the present inventors, when applied to the living body, as a result of diligently examining whether it is biocompatible and effective if you devised a gel of polyvinyl alcohol only, it has biocompatibility, and The inventors succeeded in developing a safe and novel artificial skin material having anti-deterioration properties, and completed the present invention.

[Means for Solving the Problems] The present invention relates to an artificial skin material obtained by treating polyvinyl alcohol and hyaluronic acid with alkali.

Polyvinyl alcohol (hereinafter referred to as “PVA”) used in the present invention
), The viscosity average degree of polymerization is preferably 500 or more, more preferably 1000 or more. If the viscosity average degree of polymerization of PVA is less than 500, it becomes a gel which is physically and thermally weak, which is not practical.

As hyaluronic acid (hereinafter referred to as "HA"), those having an average molecular weight of preferably 100,000 or more, more preferably about 1,000,000 are used. As the HA, a salt thereof, usually, a sodium salt is preferably used, but a potassium salt or the like may be used. If the average molecular weight of HA is less than 100,000, PVA
Since the entanglement of the molecule with the compound is weak, the elution from the gel is accelerated, which is not practical.

The artificial cartilage material of the present invention includes, in addition to PVA and HA, chondroitin sulfate (hereinafter referred to as “CS”), heparin (hereinafter “He”).
p)), dermatan sulfate (hereinafter referred to as "DS"),
Glucosaminoglycans other than HA (hereinafter, referred to as “GAG”) such as keratan sulfate and heparan sulfate: may include hydroxyapatite (hereinafter, referred to as “HAP”), collagen (type I or atelocollagen), and the like.

The artificial skin material of the present invention can be produced by alkali-treating the above-mentioned PVA, HA and other optional components.

For example, it can be produced by adding an aqueous solution of HA to an aqueous solution of PVA and, if necessary, the other optional components described above and stirring the mixture, followed by alkali treatment.

The concentration of the PVA aqueous solution is not particularly limited, but the concentration at the time of molding is preferably 1% or more. The concentration is 1%
If it is less than 3, the bubbles disappear during molding, and it is difficult to form a sponge.

The amount of HA is preferably 0.1 to 10% based on PVA. If this ratio is less than 0.1%, the HA content in the gel is small, so that the biocompatibility becomes poor.
The physical strength of the gel decreases.

The rotation speed during stirring is preferably 800 rpm or more, more preferably 1,000 to 10,000 rpm. Stirring at 800 rpm or more not only obtains uniformity, but also stabilizes generated bubbles, so it exists in the gel even after freeze-drying and hot-air drying, and the gel itself becomes a sponge-like, artificial skin material It will be very excellent.

Examples of the alkali include alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, and alkaline earth metal hydroxides such as calcium hydroxide. These alkalis are preferably used in such an amount that the pH becomes 9 or more.

After the alkali treatment as described above, bubbles are sufficiently formed. After molding, it is frozen, preferably at -20 ° C or less. Dry without thawing and wash with water (hereinafter referred to as “lyophilization method”), or after freezing for at least 15 hours, thaw and remove the alkali by washing with water,
Dehydrate with hot air at ℃ (the dehydration rate is 5% or more of the gel weight) (hereinafter referred to as “hot air drying method”). Unpolymerized PVA, HA
And, the artificial skin material of the present invention can be obtained by removing the alkali by washing with water.

The artificial skin material of the present invention obtained as described above,
Normally, tensile strength 5 kg / cm ² or more, dynamic elastic modulus 3.0 to 5.5
× 10 ⁵ N / m ² , water content 50 to 95% by weight, elongation 300 to 500%. The added GAGs such as HA, CS, and Hep are eluted very slowly and continuously in small amounts when the gel is immersed in water, but low molecular weight substances such as inorganic salts, alkalis, and unpolymerized PVA Is easily eluted. Further, the artificial skin material of the present invention does not change in moisture content and tensile strength even when immersed in water at room temperature for 3 months. Furthermore, no change is observed even when immersed in water at 50 ° C. for several tens of hours.

On the other hand, the conventional method, that is, the gel obtained without alkali treatment, does not have the above-mentioned physical properties, becomes a very soft gel only by immersing it in water at 50 ° C. for several hours, 100 ° C. 30
Dissolves in minutes.

It is necessary from the viewpoint of biocompatibility that HA is eluted even in trace amounts when the gel is in contact with the living body, or that HA is always present on the surface of the gel in contact with the living body. It is very important when considering that the elution rate of HA from a PVA gel containing HA prepared by any conventional method in water at 37 to 50 ° C. is very important because the gel is soft. It is very fast and is not sufficient for practical use as a biomedical material that is kept in contact with a living body for a long time.

In the present invention, alkali treatment, preferably making the pH 9 or more, is a tough gel, that is, an important factor for increasing the degree of polymerization of GAG such as PVA and HA, and PVA-H
The viscosity gradually increases as the alkali is added to the A mixed aqueous solution or the PVA-CS mixed aqueous solution, and becomes gel when the alkali is further added. This suggests that PVA and GAG molecules are entangled with each other, and that the entanglement becomes firm with alkali. In this way, the alkali-treated gel becomes a water-insoluble gel even when frozen and immersed in water once for 5 hours, compared to the non-alkali-treated gel, but the pH is 7.0 or less with the alkali-free gel and hydrochloric acid. What was made is easily dissolved in water by the same operation. After immersion in water to remove alkali, the gel is dried at a temperature of 20 to 50 ° C., and immersed in water again to form a more durable gel. On the other hand, a product that has been frozen for 5 hours and dried with warm air without being subjected to alkali treatment will also be dissolved when immersed in water. Furthermore, the product subjected to alkali treatment and freeze-dried had a tensile strength several tens of times higher than the product freeze-dried without alkali treatment. A neutral inorganic salt instead of an alkali increased the viscosity of the PVA-HA mixed aqueous solution, but did not form a gel like the product of the present invention.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples.
These examples do not limit the scope of the invention in any way.

Example 1 Freeze-drying method 20 ml of water was added to 1.5 g of PVA having a degree of polymerization (DP) of 2000, and dissolved in an autoclave at 120 ° C. 1% in this aqueous solution
10 ml of an HA (molecular weight: 1,000,000) aqueous solution was added, and 0.05 ml of 0.1N-NaOH was added while stirring at 1000 rpm. This aqueous solution was put in a predetermined mold and frozen. It was dried without melting as it was, and unpolymerized PVA, HA and alkali were washed out with purified water at 20 to 50 ° C. The gel thus obtained (lot 01
Table 1 compares the physical properties of the gel (Control 1) prepared without the alkali treatment with 62-1).

Further, an elution experiment of HA at each temperature was performed as follows.

The gel was cut into a size of 1.0 cm × 1.0 cm × 1 mm, and 30 pieces were prepared. 200 ml of water was added thereto, and the mixture was gently stirred at each temperature. Collect the filtrate and DEAE-cellulose 100m
After adsorbing HA through a column of l and washing with 500 ml of purified water, elution was carried out with a 1 M saline solution, and HA was eluted with a carbazole sulfate method.
Was quantified. The results are shown in FIG. In FIG. 1, (○)
The mark indicates the change in the elution amount of the product of the present invention, and the mark (●) indicates the change in the elution amount of Control 1. The elution conditions 1 to 6 were 20 ° C, 24 hours; 50 ° C, 1.5 hours; 50 ° C, 3.5 hours; 5 hours; 100
° C, 0.5 hours; 120 ° C, 0.25 hours.

From FIG. 1, the correlation between the stable life of the gel and the elution rate of HA from the gel is clear, and the effect of the alkali treatment is clear.

Example 2 A sample was prepared according to Example 1. Table 2 shows the preparation conditions. The analytical values are shown in Table 3.

 In Table 3, biocompatibility was examined as follows.

1.0cm subcutaneously on the chest or abdomen of an adult cat (2.5-3.5kg)
× 1.0 cm × 1 mm plate-shaped specimens are implanted in 12 animals per specimen. Three, two, four, eight and twelve weeks later, three animals were sacrificed, and the implants and surrounding tissues were observed for histopathological findings. In the case where the proliferation of the capillaries and the proliferation of the fibroblasts were observed in 2 weeks, x was shown, and in the case where the macrophages appeared, they were completely disappeared after 8 weeks, and the case of ○ was evaluated. Although the presence of macrophages was recognized after 8 weeks, those completely disappeared after 12 weeks.
And

 The effect was determined as follows.

Cut back skin (1.0 × 1.0cm) of C3H / He mouse,
The specimen was placed on the same size and sutured at eight places, and the top was fixed with band-aid. Two weeks later, the tape was removed, and the effect was indicated by the number of days required until the cut-out portion became 0.3 × 0.3 cm or less (Rejection). The control group used saline, and was 16, 18, 18, 18, and 18.

Example 3 Warm air drying method 20 ml of water was added to 1.5 g of PVA having a polymerization degree (DP) of 2000, and
Autoclaved at 0 ° C to dissolve. 1% in this aqueous solution
Of HA (molecular weight: 1,000,000) was added, and 0.05 ml of 10N-NaOH was added while stirring at 1000 rpm. This aqueous solution was placed in a predetermined mold and frozen at -20 ° C for 20 hours. It was melted and immersed in purified water to remove unpolymerized PVA, HA and alkali.
The gel was dried with warm air at 50 ° C. and swollen again with purified water.
Table 4 compares the physical properties of the thus obtained gel (lot 1062-1) and the gel prepared without alkali treatment (Control 3).

Further, an elution experiment of HA at each temperature was performed in the same manner as in Example 1. The results are shown in FIG. In FIG.
(○) indicates the change of the elution amount of the product of the present invention, and (●) indicates the change of the elution amount of Control 3. Elution conditions 1 to 6 were at 20 ° C., respectively.
24 hours; 50 ° C, 1.5 hours; 50 ° C, 3.5 hours; 50 ° C, 5 hours; 10
0 ° C, 0.5 hours; 120 ° C, 0.25 hours.

Example 4 A specimen was prepared according to Example 3. Table 5 shows the preparation conditions. The analytical values are shown in Table 6.

The determination of biocompatibility and effect was performed in the same manner as in Example 2.

Test Example The products of the present invention (lots 0162-1 and 1062-1) and Controls 1 and 3 as controls were each 1.0 cm × 1.0 cm × 1 mm.
, And 30 pieces each were put into 100 ml of purified water and slowly stirred at 37 ° C. After filtration for 24 hours, the filtrate was quantified for HA according to Example 1.

The HA content of the starting gel was as follows: Lot 162-1: 59.2 mg / dry g, 11.7 mg / wet g Lot 1062-1: 48.1 mg / dry g, 5.58 mg / wet g Control 1: 15.4 mg / dry g, 1.74 mg / wet g Control 3: 6.6 mg / dry g, 0.49 mg / wet g, and taking this value as 100, the% HA elution in the filtrate was determined, and the results are shown in FIGS.

In FIG. 3, a mark (○) indicates a product of the present invention (lot 0162−
In (1), the mark (●) indicates the change in the amount of elution of Control 1, and in FIG. 4, the mark (）) indicates the product of the present invention (lot 1062-
In (1), the mark (●) indicates the change in the amount of elution of Control 3.

3 and 4, the HA content at the start was lower in the control group than in the control group.
It is clear that the efficiency used for gelling due to entanglement with A is poor. Moreover, while the product of the present invention is eluted slowly over a long period of time, the control group is less than 30 days at best.
HA has been eluted. From this, it was judged that the control group was unsuitable as artificial skin that was in contact with the living body for a long period of time.

[Effects of the Invention] According to the present invention, safe and novel artificial skin having biocompatibility and having anti-deterioration properties can be provided.

[Brief description of the drawings]

1 and 2 are diagrams showing the results of an HA elution experiment at each temperature. 3 and 4 are diagrams showing the daily change of HA elution at 37 ° C.

Claims (4)

(57) [Claims] 1. An artificial skin material obtained by treating polyvinyl alcohol and hyaluronic acid with alkali. 2. The artificial skin material according to claim 1, wherein the alkali is an alkali metal hydroxide or an alkaline earth metal hydroxide. 3. A method for producing an artificial skin material, comprising adding an aqueous solution of hyaluronic acid to an aqueous solution of polyvinyl alcohol, stirring the mixture, and subjecting the mixture to an alkali treatment. 4. The method according to claim 3, wherein the alkali is an alkali metal hydroxide or an alkaline earth metal hydroxide.