JPS6134798B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is a method for producing a collagen porous body mainly for medical purposes, which comprises converting a collagen substance into a lump-like solubilized collagen using an enzyme, purifying and dissolving the lump, freezing it, and vacuum-drying it.

It has been known for a long time that collagen has high water absorption and blood coagulation properties, and these properties are utilized as absorbent materials, hemostatic materials, and the like. The blood coagulation effect of collagen is caused by the interaction of collagen and platelets. In addition, collagen acts as a foothold for the growth of living tissue, and when made porous, it has a high water absorption capacity of more than 100 times its weight, making it suitable for hemostatic materials and wound treatment materials. It is the material. Collagen is found in animal dermis, cartilage,
A protein that makes up bones, tendons, etc., and its molecular size is made up of a basic unit called tropocollagen, which is approximately 2800 Å long and 15 Å thick. These tropocollagen combine to form fibrils. The terminal portion of the molecule that crosslinks tropocollagen is called telopeptide, and its amino acid composition is very different from that of the entire molecule. Moreover, this telopeptide moiety is the main factor in the antigenicity of collagen.

Collagen itself is originally a protein with low antigenicity, but when applied to a living body as a medical material, antigenicity becomes a problem as an effect on the living body.

The present inventors solubilized the collagen molecule by selectively digesting the telopeptide portion without cleaving the peptide portion, and the purified and solubilized collagen was sufficiently purified and purified. It has been found that the material meets the purpose of the invention.

The collagen porous material obtained in the present invention may be impregnated with or coated with substances important for wound and healing, such as buffering agents, antiseptics, antibiotics, pharmaceutically effective substances, nutrients, growth factors, etc.

Conventionally, when solubilizing collagen, for example using enzymes, a proteolytic enzyme that has solubilizing power in an acidic region is generally applied to the raw collagen, and as the reaction progresses, the collagen is gradually dissolved into an acidic aqueous solution as a mother liquor. After completion of the reaction, purified collagen was produced by dissolving it in a solution, precipitating it by means such as neutralization and dialysis, recovering the precipitated collagen by means such as filtration, and then repeating washing, dissolution and recovery. Similarly, the alkali solubilization method also required recovery and purification from an aqueous solution.

However, the solubilized collagen precipitated by neutralization and dialysis is extremely fine and feather-like, as it is sometimes cut into single molecules, and the solution is viscous, so it is difficult to absorb solutes. The amount of solvent is usually about 20 to 200 times the amount of solvent, and when neutralization and dialysis are repeated, the ratio becomes even larger. Therefore, it is extremely difficult to purify it easily and with little loss when carried out on an industrial scale.

As a result of various studies regarding the production of medical collagen, the present inventors have found that by allowing enzymes to act in a weakly alkaline region and solubilizing the collagen while retaining its small block shape, the subsequent cleaning and purification process can be greatly improved. The present invention has been completed to facilitate the production of highly purified solubilized collagen at a high yield.

That is, in the method of the present invention, a collagen raw material from which impurities have been removed, purified, and shredded is pretreated in a mixed aqueous solution of caustic soda and sodium sulfate, and this is heated to a pH of 7 to 10.
A proteolytic enzyme that has the ability to solubilize collagen is reacted for 10 to 30 hours in a weakly alkaline region of Then, a porous collagen material is produced by vacuum drying.

During the process, pretreatment with a mixed aqueous solution of caustic soda and sodium sulfate is performed to first swell the collagen raw material and relax the tissue due to the action of the caustic soda, making it easier to react with enzymes, and the sodium sulfate salting out the collagen. This is to prevent denaturation due to caustic soda. Next, the enzymatic reaction is carried out until the pH of the solution is 7.
-10 is not suitable for this purpose because if the pH of the solution is 7 or less, the dissolution of collagen will gradually proceed as the reaction progresses.
This is because when the number exceeds 10, alkaline swelling of collagen rapidly progresses, making it impossible to obtain stable lump-like solubilized collagen. In addition, if the reaction time is less than 10 hours, the solubilization reaction will not proceed sufficiently, and if it exceeds 30 hours, the reaction with substances other than telopeptide will be significant, resulting in a low yield of solubilized collagen in both cases. Since it contains impurities, it becomes necessary to repeat purification.

The lump-like solubilized collagen thus obtained has almost the same molecular arrangement as the raw collagen, with only unnecessary intermolecular crosslinks being digested. Then, in an aqueous acid solution, for example, a 2.4% by weight acetic acid aqueous solution,
Solubilized collagen is dissolved at 0.2-2 weight/dissolution.
The acid to be used may be an organic acid such as acetic acid or butyric acid, or an inorganic acid such as hydrochloric acid, and the concentration is adjusted so that the pH of the solubilized collagen solution is 2.5 to 3.5. If the concentration of solubilized collagen exceeds 2% by weight, it becomes difficult to handle due to its high viscosity.
If it is less than 0.2% by weight, the amount of water to be evaporated is too large and is not economical.

The enzyme used in the present invention is, for example, a proteolytic enzyme (manufactured by Kaken Chemical Co., Ltd., product name: Pronase) that has an optimal pH of around 8.0 to 9.0 for casein produced by Streptomyces griseus, a streptomycin-producing bacterium. ) can be given. Of course, it is obvious that any protease having collagen solubilizing power in the slightly alkaline pH range of 7 to 10 can be used as the protease of the present invention, and is not limited to the above-mentioned enzymes.

The method of the present invention will be explained below with reference to Examples.

Example 1 100 g of purified collagen raw material (steerhide cowhide from North America) shredded into 3-8 mm squares was immersed in mixed aqueous solution 2 containing 4% by weight caustic soda and 15% by weight sodium sulfate, while being gently stirred. It was left for 3 days. Thereafter, this lumped collagen was washed and neutralized sequentially with a 5% by weight saline solution and a 0.12% by weight acetic acid aqueous solution, and then washed under running ion-exchanged water for 24 hours. Next, the washed bulk collagen was immersed in PH9 solution 2 containing 0.2% by weight of pronase (Streptomyces griseus protease, manufactured by Kaken Chemical Co., Ltd.) for 18 hours, and reacted with gentle stirring.
After the reaction, the lumps were washed sequentially with water and a 0.12 wt% acetic acid aqueous solution, washed and purified under running ion-exchanged water for 24 hours, and then the purified solubilized collagen lumps were mixed with 2.4 wt% so that the concentration was 0.5 wt%. It was completely dissolved in an acetic acid aqueous solution, put into a mold, frozen in a methanol-dry ice refrigerant at -70°C, and then vacuum dried to obtain 91 g of porous collagen. The absorption capacity of this product for ion-exchanged water is 180g/g product.
When implanted into the peritoneal cavity of an ICR nut (male, 6-7 weeks old), it was completely absorbed into the body and disappeared on the 4th day after surgery.

Claims (1)

[Claims] 1. After pre-treating the collagen raw material which has been purified and shredded to remove impurities in a mixed aqueous solution of caustic soda and sodium sulfate, it is treated with a proteolytic enzyme that has the ability to solubilize collagen in a slightly alkaline region of pH 7 to 10 for 10 to 30 hours, Then, after thorough washing with water and a dilute acid aqueous solution, a block of solubilized collagen is obtained, which is then used as solubilized collagen at a concentration of 0.2 to 2% by weight.
A method for producing a porous collagen material, which comprises dissolving it in an acid solution so as to give the following properties, freezing it, and vacuum drying it.