JPH0931100A - Denaturated collagen and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new denaturated collagen having gel characteristics, etc., changed stepwise without changing the properties of collagen and useful as cell culture base, medical material, cosmetic material, etc., by treating a collagen with a denaturation agent and undenaturating the product. SOLUTION: Collagen is extracted from the cord of pig with a pepsin by conventional method and purified by salting-out. The obtained collagen is added to a 10mM phosphate buffer solution, denaturated with one or more denaturants such as urea, N-acylurea, O-acylisourea, alkylated urea, guanidine, etc., and undenaturated by dialysis, etc. The objective denaturated collagen having gel characteristics, etc., changed stepwise without changing the basic properties of collagen and useful as cell culture base, medical material, cosmetic material, etc., is produced by treating the above product by collagen extraction method and/or enzymatic decomposition method.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to denatured collagen and a method for producing the same. More specifically, the cell culture substrate,
The present invention relates to denatured collagen used as a medical material, a cosmetic material, etc., a method for producing the same, and its use.

[0002]

BACKGROUND OF THE INVENTION Collagen is 1/3 of all proteins in the animal body.
It is a protein that occupies the above and is abundant in animal skin, tendons, bones, and the like. Collagen was considered to be a protein responsible for physical functions such as morphological maintenance of the animal body, but recently, it has been revealed that collagen exerts various biological actions on the animal body. Since collagen is a biomaterial, it has excellent biocompatibility, and various applications utilizing this property have been proposed. For example, collagen functions as a matrix between cells and affects the development and differentiation of cells. Even cells that cannot grow in a glass or plastic culture dish, collagen-coated culture dish or collagen It has been reported to be able to grow in gel matrices. From such knowledge, collagen is used as a cell culture substrate, a cell culture carrier (microcarrier), or the like. It is also used as a moisturizer / moisturizer in cosmetics because of its excellent biocompatibility.As medical materials, it is used for wound cover materials, artificial skin, artificial esophagus, artificial organs such as artificial trachea, and cell diagnostics. Substrate and the like.

[0003]

As described above, collagen is widely used for various purposes, and collagen gel has a wide range of applications. When a collagen gel is used, it is necessary to change gel properties such as gel strength according to its application. As a method of adjusting the strength of the collagen gel, a method of changing the collagen concentration can be considered, but in order to obtain high gel strength, it is necessary to remarkably increase the collagen concentration, which is not practical. Further, chemical modification of collagen, heat treatment, cross-linking and the like are not preferable because the basic properties of collagen are changed.
From this point of view, the applicant has proposed a method of mixing collagen having different degrees of association as a method for modifying a collagen gel (see Japanese Patent Laid-Open No. 149200/1992).
According to this method, collagen containing a large amount of aggregates has a high gel strength, and collagen containing few aggregates has a low gel strength. Therefore, the gel strength can be adjusted by mixing collagens having different aggregate contents. It was revealed that this method can achieve the intended purpose sufficiently, but the fiber diameter becomes thin in proportion to the gel strength, and when culturing cells, it may not be suitable for growth depending on the type of cells. There was such a problem.

In order to solve the above problems, the present inventors can adjust the gel strength without affecting the characteristics in the solution state, the fiberization rate in the gel state, and the fiber diameter. Have been repeatedly examined. As a result, it has been conventionally considered that collagen is not denatured by a low concentration denaturant (such as 2M urea), but the present inventors can achieve the intended purpose by treating collagen with a denaturant. It was also found that collagen treated in this way has different properties from untreated collagen in addition to the gelling property. The present invention has been made based on such findings, and an object of the present invention is to provide a denatured collagen in which properties such as gelling property are stepwise modified and a method for producing the same.

[0005]

Means for Solving the Problems The present invention, which has been made to solve the above-mentioned problems, is a modified collagen obtained by treating collagen with a denaturing agent and then treating with a dedenaturing agent; treating collagen with a denaturing agent. After that, a method for producing denatured collagen, which comprises treating with a dedenaturing agent to obtain denatured collagen; treating a collagen-containing raw material with a denaturing agent, then treating with a dedenaturing agent, and then using a collagen extraction method and / or an enzymatic decomposition method. Denatured collagen obtained by treatment; Denatured collagen obtained by treating a collagen-containing raw material with a collagen extraction method and / or an enzymatic decomposition method in the presence of a denaturing agent; the denaturing agent is urea, N-acylurea, O -The modified collagen according to the above or any of the above, which uses one or more selected from acylisourea, alkylated urea, guanidine and salts thereof. A cell culture substrate, a medical material, or a cosmetic material containing the modified collagen according to any one of the above and or.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, treatment of collagen or a collagen-containing raw material with a denaturing agent means bringing the collagen or collagen-containing raw material into contact with the denaturing agent in a suitable solvent. The collagen is not particularly limited, and various collagens are targeted, but type I collagen is preferably used. As a collagen-containing raw material,
Any material can be used as long as it is a raw material containing collagen, and mammals (for example, cow, pig, rabbit, sheep,
Examples thereof include skin, bone, cartilage, tendon, and organs (such as mice), but bone, cartilage, skin, tendon, placenta, and the like are preferably used because of their high collagen content.

As the denaturing agent, any agent can be used as long as it is an agent capable of denaturing proteins, and examples thereof include urea, urea derivatives (eg N-acylurea, O-acylisourea, alkylated urea, etc.) and guanidine. And conventional modifiers such as salts thereof can be used. In some cases, a concentrated solution of an inorganic salt such as calcium salt, potassium salt, thiocyanate, or perchlorate may be used. The dedenaturing agent treatment performed after denaturation means an operation of removing the denaturing agent, and if the collagen state after denaturing is in a solution state, dialysis, gel filtration,
It can be carried out by a method such as affinity chromatography, and when the collagen after denaturation is in a solid state, it can be carried out by a method such as solubilization, dialysis, filtration or washing.

The treatment by the collagen extraction method means a treatment for extracting collagen from a collagen-containing raw material, which can be carried out by a conventional method such as acid solubilization, alkali solubilization, neutral salt solubilization and enzyme solubilization. it can. The above-mentioned acid solubilization is performed by using, for example, an inorganic acid such as hydrochloric acid and / or an organic acid such as acetic acid or citric acid, and the pH is about 2 to 4, preferably pH.
It can be performed by extracting collagen with an aqueous solution of about 3. Alkali solubilization is performed by using a basic substance such as sodium hydroxide or potassium hydroxide,
H12 ~ 14, preferably pH 12.5-13.5
It can be carried out by extracting collagen with an aqueous solution of a certain degree (Japanese Patent Publication No. 46-15033).
-106807 gazette etc.). Neutral salt solubilization includes, for example, inorganic salts such as sodium chloride (0.15-0.5M
It can be carried out by extracting collagen with a Tris-hydrochloric acid buffer solution (about pH 7 to 8, preferably about pH 7.5) containing the same. Enzyme solubilization, for example, using proteolytic enzymes such as pepsin and pronase,
It can be performed by solubilizing and extracting collagen. In addition, the enzymatic decomposition method refers to treating collagen with a proteolytic enzyme, a fat hydrolase, a sugar hydrolase, or the like, which is a means for purifying collagen.

Hereinafter, the denatured collagen of the present invention and the method for producing the same will be described in more detail. One of the denatured collagens of the present invention is a denatured collagen obtained by treating collagen with a denaturant and then dedenaturing the collagen, and the collagen is obtained by treating collagen with a denaturant in a suitable solvent. ,
It can be obtained by treating with a denaturing agent. As the above collagen, it is preferable to use purified collagen, but the collagen-containing raw material is subjected to the above-mentioned extraction treatment and / or
Alternatively, crude collagen obtained by enzymatic treatment can also be used. The solvent to be used is not particularly limited as long as it is a solvent capable of denaturing collagen, but for example, a phosphate buffer solution having a pH of about 7 to 8, preferably a pH of about 7.5 is used. When denaturing with a low denaturant concentration, it is preferable to carry out denaturing under acidic conditions (for example, about pH 3).

The amount of the denaturing agent used can be appropriately changed depending on the desired properties of the denatured collagen. That is, in the present invention, the properties of collagen change stepwise depending on the denaturing conditions such as the amount of denaturant used, so that the amount of denaturant used can be determined according to the required properties. Further, the amount of the denaturant used can be appropriately changed depending on the kind of the denaturant, the state of collagen, the denaturation temperature and time, and the like. For example, when urea is used as a modifier, it is generally about 0.05 to 10 M, preferably 1 to 8 M.
It is used in the range of about M. The denaturing time is appropriately set depending on the type of denaturing agent, denaturing temperature, etc., but is generally at room temperature for about 0.5 days to 1 month, preferably for 1 week to 3 weeks, more preferably for about 2 weeks, with stirring. While denaturing. The collagen denatured in this way is subjected to the above-mentioned dedenaturing agent treatment. The denaturing agent treatment is preferably a dialysis method. For example, the denaturing agent treatment is performed by dialysis against a hydrochloric acid acidic solution (pH about 3.0). The denatured collagen of the present invention can be obtained by the above method.

Another modified collagen of the present invention is a modified collagen obtained by treating (A) a collagen-containing raw material with a denaturing agent, followed by a dedenaturing agent, and then a collagen extraction method and / or an enzymatic decomposition method. Collagen; and (B) a denatured collagen obtained by treating a collagen-containing raw material with a collagen extraction method and / or an enzymatic decomposition method in the presence of a denaturing agent. These modified collagens are obtained by treating (a) a collagen-containing raw material with a denaturing agent in a suitable solvent, followed by a dedenaturing agent, and then a collagen extraction method and / or an enzymatic decomposition method; ) In the presence of a denaturant,
It can be obtained by treating a collagen-containing raw material with a collagen extraction method and / or an enzymatic decomposition method. According to these methods, the denatured collagen of the present invention can be prepared from a collagen-containing raw material, which is simple and cost can be reduced.

In the method (a), first, the collagen-containing raw material is denatured with a denaturing agent in a suitable solvent. The amount of the denaturant used can be appropriately changed depending on the desired properties of the denatured collagen as described above.
The solvent is not particularly limited as long as it does not interfere with the denaturation of collagen in the raw material, but a phosphate buffer or the like is generally used. The denaturing time is appropriately set depending on the type of denaturing agent, denaturing temperature, etc., but is generally at room temperature for about 0.5 days to 1 month, preferably for 1 week to 3 weeks, more preferably for about 2 weeks, with stirring. While denaturing. The collagen denatured in this way is subjected to the above-mentioned dedenaturing agent treatment. A washing method is preferable as the de-denaturing agent treatment, and for example, the de-denaturing agent treatment is performed by a running water washing method. The collagen-containing raw material that has been subjected to the dedenaturing agent treatment is subjected to the above-mentioned collagen extraction method and / or enzymatic decomposition method,
Extraction and purification of collagen is performed. For example, in the case of the acid extraction method, the modified collagen-containing raw material is
The denatured collagen of the present invention can be obtained by adding a citric acid or acetic acid solution of about 3.5 to dissolve it by stirring, separating the insoluble portion, and dialyzing or salting out the soluble portion.

In the method (b), the collagen-containing raw material is treated by the above-mentioned collagen extraction method and / or enzymatic decomposition method in the presence of a denaturing agent, and the denaturation of collagen and the extraction / purification of collagen are performed. Can be done at the same time.
The amount of the denaturant used can be appropriately changed depending on the desired properties of the denatured collagen as described above. As an example of this method, the case of performing a collagen extraction method using a proteolytic enzyme will be explained. A collagen-containing raw material is added to an acidic solution (for example, acetic acid acidity, hydrochloric acid acidity, etc.), and a proteolytic enzyme such as pepsin is further added. In addition, it can be performed by stirring at room temperature for about 1 day to 1 week. After completion of the reaction, the soluble portion is removed and salted out to obtain the denatured collagen of the present invention.

The denatured collagen of the present invention thus obtained may be used as a solution, or may be powdered by means such as freeze-drying and used. The denatured collagen of the present invention, as shown in the test examples below, does not change the basic properties of collagen (for example, specific optical rotation, molecular weight, fiber diameter, fibrosis rate, etc.) and the concentration of the denaturing agent. It has the feature that characteristics such as gel strength are changed stepwise by adjusting.

In addition, it has other characteristics different from those of conventional collagen. For example, most of the antigenicity of collagen originates from telopeptides, but in addition to that, a very small amount of collagen has a higher-order structure (triple helix) and a peptide that has undergone protease degradation after denaturation (gelatin). Is said to exist. In the peptide of the present invention, it is considered that the higher order structure of collagen is slightly changed by the treatment with the denaturing agent, which makes it difficult to recognize. In addition, since it has not been completely denatured, it is considered that it is resistant to protease degradation and the peptide having antigenicity is less likely to be produced, resulting in a decrease in antigenicity. Therefore, the denatured collagen of the present invention has low antigenicity and high biocompatibility.

Further, the collagen of the present invention is used to form fibers (gel).
It is expected that when the composition is constructed, the fiber diameter is not affected, but the fiber spacing is affected, and the difference in the fiber spacing is considered to appear as the difference in gel strength. Depending on the type of cell, the most suitable environment (scaffold) of the cell can be artificially adjusted by adjusting the difference in fiber spacing. This property can regulate the reaction to cells in the tissue when it is implanted not only as a substrate for cell culture but also as a medical material into a living body. For example, regulation of in vivo degradation rate, fibroblasts and epithelial cells. It is possible to control the invasion speed of the in the matrix.

The denatured collagen of the present invention has the above-mentioned features and can be used in various applications in which conventional collagen is used. Particularly preferably, it is used as a cell culture substrate, a medical material, and a cosmetic material. The cell culture substrate is used as a cell culture substrate, a cell culture microcarrier, or the like. When used as a cell culture substrate, a gel matrix is prepared using the denatured collagen of the present invention, and cells are cultured using the gel matrix. Examples of the cell culture method include a collagen gel culturing method, a floating collagen gel culturing method, and a collagen gel embedding culturing method. The cell culture microcarrier is an insoluble carrier used for culturing adherent cells, and is prepared by molding the denatured collagen of the present invention into porous beads.

Examples of the medical material include a wound cover material, artificial skin, an artificial organ in which cells are embedded in collagen gel to have a function, a substrate for cytodiagnosis, and the like. Collagen has excellent moldability, so that materials of various shapes can be obtained, and examples thereof include gel, spun fiber, film, membrane, tube, hollow fiber, sponge, and the like. Depending on the shape, a wound cover material, It is used as sutures, artificial tendons, hemodialysis membranes, artificial eardrums, artificial blood vessels, tubular organs, hemostatic agents, etc. By using the denatured collagen of the present invention, the physical properties (for example, tensile strength, elastic force, etc.) of materials having these shapes can be adjusted without changing the basic properties of collagen. Further, it is clear that the sponge prepared by drying the gel obtained using the modified collagen of the present invention has improved surface smoothness and flexibility as compared with the product prepared using unmodified collagen. became.

As a cosmetic material, it is mixed with cream, ointment, lotion, etc. as a material that retains moisture of the skin and imparts elasticity and smoothness to the skin by utilizing the moisturizing / wetting property and film forming property of collagen. To be done. The use of the denatured collagen of the present invention is not limited to the above-mentioned uses, and it can be used in all industrial fields utilizing collagen.

[0020]

The present invention will be described in more detail based on the following examples and test examples, but the present invention is not limited to these examples. Example 1 Preparation of denatured collagen As collagen, enzyme-soluble type I collagen was used which was extracted from pig tendon with pepsin according to a conventional method and purified by salting out. The above collagen was added to a 10 mM phosphate buffer solution (pH 7.4) containing 2, 4, 6, 7, and 8 M urea and stirred for 2 weeks. 1M urea treatment, 1 collagen
It was added to a hydrochloric acid solution (pH 3.0) containing M urea and stirred. After the urea treatment, it was dialyzed against a hydrochloric acid solution (pH 3.0) to completely remove the urea to obtain the denatured collagen of the present invention. In the following test examples, each denatured collagen prepared by changing the concentration of the denaturant by the above method was used.

Test Example 1 Measurement of gel strength Denatured collagen solution diluted to 1.5 mg / ml,
200 mM phosphate buffer (pH 7.4) containing 1.4 M sodium chloride was mixed at a ratio of 9: 1. This is 3
Dispense 7.5 ml each into a 5 mm dish and mix at 37 ° C for 2
Allow to gel for hours. Using a tensipressor (manufactured by Taketomo Electric Co., Ltd.), the strength of 1 byte was measured. The result is shown in FIG. The horizontal axis represents the denaturant concentration (urea concentration) when preparing denatured collagen (the same applies hereinafter). As shown in FIG. 1, it was revealed that the gel strength gradually decreased depending on the concentration of the modifier.

Test Example 2 Gel Melting Temperature Measurement Denatured collagen solution diluted to 1.0 mg / ml,
200 mM phosphate buffer (pH 7.4) containing 1.4 M sodium chloride was mixed at a ratio of 9: 1. 3 ml of this was put in a cell and gelled at 37 ° C. for 90 minutes.
After gelation, the temperature at 37 ° C. to 52 ° C. was raised at 1 ° C./10 minutes, the absorbance at 400 nm was measured, and the melting temperature was measured. The result is shown in FIG. As shown in FIG.
It was revealed that the gel melting temperature decreased depending on the denaturant concentration except when the urea concentration was 8M.

Test Example 3 Measurement of optical rotation Using SEPA-300 (manufactured by Horiba Ltd.), 20 ° C.
20m capacity in a constant temperature cell holder with circulating constant temperature water
The optical rotation of each denatured collagen was measured using 1 and a cell length of 10 cm. For comparison, the optical rotation of the denatured collagen before dialysis (denatured collagen in the presence of urea) was also measured. The result is shown in FIG. As shown in FIG. 3, in the denatured collagen before dialysis, the specific rotation was shifted to the + side depending on the concentration of the denaturant. On the other hand, the denatured collagen after dialysis showed a substantially constant specific optical rotation regardless of the concentration of the denaturant, which was similar to that of undenatured collagen. From this, the denatured collagen of the present invention is
It was revealed that the optical rotation did not change.

Test Example 4 Measurement of Fibrosis Ratio Denatured collagen solution diluted to 1.0 mg / ml,
A 200 mM phosphate buffer solution (pH 7.4) containing 1.4 M sodium chloride was mixed at a ratio of 9: 1, and the mixture was mixed at 37 ° C. for 90 minutes.
Allow to gel for minutes. Centrifuge (3500 rpm, 30 minutes) after gelation
Then, the fibrosis rate was determined by quantifying the protein in the supernatant. FIG. 4 shows the results. As shown in FIG. 4, except for the case where the urea concentration was 8 M, the fiberization rate was substantially constant regardless of the modifier concentration, and it was found that the fiberization rate did not change.

Test Example 5 Intrinsic Viscosity Measurement A Viscotimer (Viscotimer S / 4, manufactured by Lauda) was used.
The intrinsic viscosity at 20.2 ° C was measured for each modified collagen. The result is shown in FIG. As shown in FIG. 5, except for the case where the urea concentration was 8 M, the intrinsic viscosity was a substantially constant value regardless of the modifier concentration, and it was found that the intrinsic viscosity (that is, the molecular weight) did not change.

Test Example 6 Measurement of Transition Temperature Using a Viscotimer S / 4 (manufactured by Lauda),
The viscosity was measured from 30 ° C. to 45 ° C. at a temperature rising rate of 1 ° C./30 minutes, and the transition temperature of each denatured collagen was measured. FIG. 6 shows the result. As shown in FIG. 6, it was found that the transition temperature was a substantially constant value regardless of the denaturant concentration, and the transition temperature did not change.

Test Example 7 Fiber Diameter Measurement From an electron micrograph of collagen gel, image processing apparatus L
The fiber diameter of each denatured collagen was measured using A-525 (manufactured by Pierce). FIG. 7 shows the result. As shown in FIG. 7, it was found that the fiber diameter had a substantially constant value regardless of the concentration of the modifier, and the fiber diameter did not change.

Test Example 8 Cell Culture Test A solution of denatured collagen obtained by treating with 4 M urea (3 mg
/ Ml) 10 times the concentration of DMEM and pH adjusting buffer (2.2% NaHCO ₃ , 4.77% HEPE).
S / 0.05 N NaOH) was mixed with each one under ice-cooling, and 50 μl of each was dispensed into a 96-well microplate, followed by gelation at 37 ° C. for 1 hour. Rat skin-derived fibroblasts were seeded at 1 × 10 ³ cells / well at 37 ° C.
The cells were cultured in a CO ₂ incubator, and the proliferation of cells after the first day was measured. The cells were grown by measuring the absorbance at 570 nm and 600 nm with a microplate reader using AlamarBlue (sold by Kanto Kagaku). FIG. 8 shows the result. Note that in FIG. 8, the cells grow as the absorbance increases. As shown in FIG. 8, good growth of rat skin-derived fibroblasts was observed on the gel using the denatured collagen of the present invention.

Example 2 In Example 1, 3M guanidine hydrochloride was used instead of 4M urea, and denaturation / dialysis was performed in the same manner as in Example 1 to obtain denatured collagen.

[0030]

INDUSTRIAL APPLICABILITY As described above, the denatured collagen of the present invention can gradually change specific properties such as gelling property without changing the basic properties of collagen. Therefore, by using the denatured collagen of the present invention, a gel or the like having a desired gel strength can be simply and quickly prepared.

[Brief description of drawings]

FIG. 1 is a graph showing the influence of denaturant concentration on gel strength.

FIG. 2 is a graph showing the effect of denaturant concentration on gel melting temperature.

FIG. 3 is a diagram showing the effect of denaturant concentration on optical rotation.

FIG. 4 is a graph showing the influence of a modifier concentration on the fiberization rate.

FIG. 5 is a diagram showing the influence of a modifier concentration on the intrinsic viscosity.

FIG. 6 is a graph showing the effect of denaturant concentration on transition temperature.

FIG. 7 is a diagram showing the influence of a modifier concentration on the fiber diameter.

FIG. 8 shows the results of culturing rat skin-derived fibroblasts on a gel prepared using the denatured collagen of the present invention.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Toshikazu Makihara 3-3 Midorigahara, Tsukuba-shi, Ibaraki Central Research Laboratory, Nippon Ham Co., Ltd.

Claims (6)

[Claims] 1. After treating collagen with a denaturing agent,
Denatured collagen obtained by treatment with a dedenaturing agent. 2. After treating collagen with a denaturing agent,
A method for producing denatured collagen, which comprises treating with a dedenaturing agent to obtain denatured collagen. 3. Denatured collagen obtained by treating a collagen-containing raw material with a denaturing agent, then treating with a dedenaturing agent, and then treating with a collagen extraction method and / or an enzymatic decomposition method. 4. A denatured collagen obtained by treating a collagen-containing raw material with a collagen extraction method and / or an enzymatic decomposition method in the presence of a denaturing agent. 5. The one or more kinds selected from urea, N-acylurea, O-acylisourea, alkylated urea, guanidine and salts thereof are used as a modifier. The denatured collagen according to 1. 6. A cell culture substrate, a medical material or a cosmetic material containing the modified collagen according to any one of claims 1, 3, 4 and 5.