JP2015213675A - Soluble collagen fiber porous body - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a soluble collagen compact constituted of collagen fibers, and having a porous spongy shape capable of three-dimensional cell cultivation.SOLUTION: A soluble collagen fiber porous body is obtained by freeze-drying fibrotic collagen deposited by an alkali metal bicarbonate. A soluble collagen compact produced by the method has a porous spongy shape capable of three-dimensional cell cultivation, while keeping a fibrous structure by the collagen.

Description

  The present invention relates to a soluble collagen fiber porous body, and is particularly suitable for cosmetics and medical materials (for example, wound dressing materials, adhesion prevention materials, cosmetic materials, etc.).

  Collagen is an important protein that accounts for 30% of proteins in the body and has functions such as skeletal support and cell adhesion. For example, bone, cartilage, ligaments, tendons, corneal stroma, skin, liver, muscle and other tissues Is made of collagen fibers. Collagen molded bodies prepared from this collagen are widely used as cosmetics and medical materials.

  In Patent Document 1, bubbles are removed from a collagen dispersion, solution or mixture thereof having a collagen concentration of 50 mg / ml or more, and then freeze-dried and then subjected to insolubilization treatment by physical crosslinking or chemical crosslinking. A technology relating to a cell culture carrier having a stress of 10 to 30 kPa when loaded and having a pore structure on the surface and inside is disclosed.

  Patent Document 2 discloses a technique relating to a collagen structure that is composed of collagen fibers having an average diameter of 1 to 5 μm.

Japanese Patent No. 4915693 International Publication No. 2013/105665 Pamphlet

  Since collagen exists in a living body in a fibrous form, it is considered that the collagen constituting the collagen molded body is preferably not in an amorphous form but in a fibrous form from the viewpoint of biocompatibility.

  On the other hand, from the viewpoint of solubility, a porous material having a large pore diameter is advantageous.

  However, in the conventional technique, a porous body composed of collagen fibers and having a large pore diameter, particularly a porous collagen fiber body having a porous sponge shape having a pore diameter capable of three-dimensional cell culture ( Hereinafter, it was difficult to produce a “fiber macroporous body”. By the way, there are various theories about the size of the pore diameter that enables three-dimensional cell culture, and it is said to be at least 50 μm or 70 μm. On the other hand, 100 μm and 150 μm are also proposed, but this is considered to realize sufficiently smooth cell migration.

  In order to fibrillate collagen, for example, as described in JP-A-2010-273847, a neutral buffer solution is used, and the ionic strength of the solubilized collagen solution is appropriately increased so that the pH is close to neutral. It is well known that this is important, and in this publication, salt aqueous solutions having buffering capacity such as phosphate, acetate, carbonate, citrate and Tris are exemplified as neutral buffers. Phosphate buffer is mentioned as preferred.

The present applicant performed the following methods (a) to (c) as an attempt to obtain a fibrous macroporous material.
(a) A method in which phosphate buffered saline (PBS) is added to a solubilized collagen solution to fibrillate collagen, and then freeze-dried.
(b) After PBS was added to the solubilized collagen solution to fibrillate the collagen, the fibrillated collagen was mixed with ethanol series (mixed solution of ethanol and water whose ethanol concentration was increased stepwise. Ethanol concentration: 50%, 70%, 90%, 100%), followed by dehydration and desalting, then replacing the solvent with t-butanol, freezing, and freeze-drying.
(c) A method of freeze-drying a solubilized collagen solution and then fibrillating the collagen with PBS.

  However, no fibrous macroporous material was obtained by any of the methods (a) to (c).

  That is, in the method (a), the fibrillated collagen becomes non-fibrotic (defibrinated) by a high concentration salt concentrated during freeze-drying, and the pore diameter is small. Since the cell culture carrier described in Patent Document 1 is also produced by the same method as the method (a), it is difficult to say that the collagen is fibrous.

  In the method (b), a porous body composed of collagen fibers was obtained, but it did not have a pore size large enough to enable three-dimensional cell culture. This is probably because the frozen crystals of t-butanol were not as large as the frozen crystals of water. By the way, when the solubilized collagen solution in an aqueous solvent is freeze-dried as it is, a porous body having a pore size that allows three-dimensional cell culture can be obtained, but non-fibrous (amorphous) dissolved collagen is It is known that it is non-fibrous (amorphous) because it is freeze-dried as it is.

  Further, in the method (c), a stable shape could not be obtained.

  Regarding the collagen structure described in Patent Document 2, judging from FIGS. 3 and 4 of the electron microscope image according to Example 1, the pore size was small enough to be less than 50 μm.

  An object of the present invention is to provide a collagen molded body that is composed of collagen fibers and has a porous sponge-like shape capable of three-dimensional cell culture and has solubility.

  As a result of intensive studies to solve the above problems, the present inventors surprisingly lyophilized fibrotic collagen precipitated by alkali metal bicarbonate, while maintaining the fibrous structure of the collagen, The present inventors have found that a soluble collagen molded body having a porous sponge-like (sponge-like) shape capable of three-dimensional cell culture can be obtained, and the present invention has been completed.

That is, the present invention is as follows.
[1] A soluble collagen fiber porous body composed of collagen fibers and having a porous spongy shape capable of three-dimensional cell culture.
[2] In an electron microscopic image of the soluble collagen fiber porous body, when the maximum number of holes is n in a fixed section where the number of holes observed on the outermost surface is at least 30, the average pore diameter = {Σ (maximum width of hole i + minimum width of hole i) / 2} / n (where i = 1 to n), the average pore diameter is in the range of 50 to 300 μm above [1 ] The collagen porous body of description.
[3] The above [1] or [2], comprising a first step of mixing a solubilized collagen solution and an alkali metal bicarbonate to precipitate fibrotic collagen, and a second step of freeze-drying. The manufacturing method of the soluble collagen fiber porous body of description.
[4] When the amount of alkali metal bicarbonate applied is 300,000, the collagen in the alkali metal bicarbonate / solubilized collagen solution (molar ratio) = 3 × 10 ^{2 to} 3 × 10 ^4. The method for producing a soluble collagen fiber porous body according to the above [3], which is in the range of ⁴ .

  The soluble collagen fiber porous body of the present invention is composed of a collagen fiber and has a porous sponge-like shape capable of three-dimensional cell culture, and thus has excellent biocompatibility and also has solubility. In addition, cells can enter inside. Therefore, it is a material suitable for cosmetics (for example, preparations for use at the time of use) and medical materials (for example, wound dressings, adhesion prevention materials, cosmetic surgery materials, etc.).

It is a photograph when the soluble collagen fiber porous body in Example 1 of this invention is pinched with metal tweezers. It is an electron microscope image (magnification: 100 times) of the soluble collagen fiber porous material in Example 1 of the present invention. The inside of the outermost layer hole is filled so that only the outermost layer hole of FIG. 2 can be identified. It is a photograph when the non-fibrotic collagen porous body in Comparative Example 1 of the present invention is pinched with metal tweezers. It is a photograph when the collagen fiber porous body in the comparative example 2 of this invention is pinched with metal tweezers. It is an electron microscopic image (magnification: 100 times) of the collagen fiber porous body in the comparative example 2 of this invention.

Hereinafter, the soluble collagen fiber porous body of the present invention (hereinafter referred to as “the porous body of the present invention”) which is a fiber macroporous body will be described in detail.
The porous body of the present invention is characterized by being composed of collagen fibers and having a porous spongy shape capable of three-dimensional cell culture. In addition, in order that the porous body of this invention may exhibit a soluble characteristic, it is desirable that the crosslinking process is not performed. Examples of the crosslinking treatment include physical crosslinking treatment by γ-ray irradiation, electron beam irradiation, plasma irradiation, UV irradiation, thermal dehydration, etc., chemical crosslinking treatment with a water-soluble chemical crosslinking agent or a chemical crosslinking agent having vaporization ability. It is done.

  The porous body of the present invention has solubility, and has, for example, the following characteristics as solubility in an aqueous solvent. That is, although it tends to become gradually soluble in an aqueous solvent near the isoelectric point of collagen, the solubility tends to improve as the distance from the isoelectric point increases. As a specific example, when collagen fibers are derived from acid-solubilized collagen or enzyme-solubilized collagen, the dissolution rate tends to increase as the acidity of the aqueous solvent increases. One preferred form of use utilizing the above-mentioned dissolution characteristics is, for example, when the porous body of the present invention is used as a cosmetic preparation for use, slowing the dissolution rate of collagen and allowing the collagen to slowly act on the skin. When it is desired to use, a weakly acidic lotion for dissolving the porous body of the present invention is used, and when it is desired to act quickly, a highly acidic lotion is used. Here, the aqueous solvent is not particularly limited as long as it is a solvent containing water, and examples thereof include water, an aqueous solution, and a buffer solution.

  Further, the porous body of the present invention is recognized by those skilled in the art as having a large pore diameter in the present technical field. Although it is difficult to accurately represent the size of the pore diameter, for example, whether or not three-dimensional cell culture is possible can be an index. In three-dimensional cell culture, it is required to have a porous sponge-like structure having continuous pores with a diameter suitable for smooth cell migration so that cells can enter the inside of the substrate. The porous body of the present invention has a porous sponge-like structure capable of three-dimensional cell culture. The following experiment clearly showed this. That is, when the porous body of the present invention was subjected to γ-ray crosslinking and subjected to cell culture, when the focal position of the microscope was moved from the upper surface to the lower surface of the cultured porous body, the cells were uniformly distributed at any focal position. Was confirmed.

  A preferred embodiment relating to the pore diameter is an electron microscopic image of the porous body of the present invention, wherein the maximum number of holes is n in a fixed section where the number of holes observed on the outermost surface is at least 30. Average pore diameter = {Σ (maximum width of hole i + minimum width of hole i) / 2} / n (where i = 1 to n), the average pore diameter is in the range of 50 to 300 μm. Is. A more preferable range of the average pore diameter is 70 to 250 μm. In addition, as said hole, a complete hole is object and the incomplete hole by which the hole was divided | segmented by the division boundary line is not included.

  The porous body of the present invention is excellent in mechanical strength in a dried state before being immersed in a solution, and has a high resistance to external forces such as compression and extension. Further, even when handled with metal tweezers, the appearance is hardly damaged. This is thought to be due to collagen being fibrillated.

  Examples of uses of the porous body of the present invention include cosmetics and medical materials, but are not limited thereto. Examples of the cosmetics include, but are not limited to, cosmetics prepared at the time of use, and examples of the medical materials include wound dressing materials, adhesion prevention materials, and cosmetic surgery materials.

Next, the manufacturing method of the porous body of this invention is demonstrated.
The method for producing a porous body of the present invention includes a first step of mixing a solubilized collagen solution and an alkali metal bicarbonate to precipitate fibrotic collagen, and then a second step of freeze-drying. To do. In addition, since the mixed liquid in which the collagen fibers are precipitated in the first step exhibits a gel shape, the mixed liquid is hereinafter referred to as “fibrotic collagen gel”.

  A solubilized collagen solution is an aqueous solution in which collagen is dissolved. The collagen is preferably water-soluble collagen having a triple helical structure. The solubilized collagen solution may partially contain peptides, amino acids, gelatin, etc., but these are preferably excluded as much as possible.

  Water-soluble collagen having a triple helix structure can be obtained by known methods from collagen-containing tissues of biological materials such as mammals, seafood, birds and reptiles. For example, [1] dilute acid Examples include acid-solubilized collagen obtained by the extraction method, [2] enzyme-solubilized collagen obtained by a method of solubilizing with an enzyme, and [3] alkali-solubilized collagen obtained by a method of solubilizing with an alkali. . Acid-solubilized collagen and enzyme-solubilized collagen are soluble under acidic conditions, and alkali-solubilized collagen is soluble under alkaline conditions, but both collagens have an ionic strength and pH within an appropriate range. It is known to fibrosis.

  As the type of collagen, collagen derived from fish that does not have a virus common to humans is particularly suitable, and those having a relatively high denaturation temperature are preferable from the viewpoint of applicability to various uses. It is derived collagen. Of the genus Oreochromis, tilapia, which is cultivated mainly for food from China to Southeast Asia and is readily available, is particularly preferred. Furthermore, atelocollagen from which telopeptide which is an antigenic determinant of collagen is removed is preferable. There is no particular limitation on the type of collagen, but type I, which is abundant in living organisms, is preferred.

  Here, the method for obtaining the enzyme-solubilized collagen of [2] will be described. The acquisition method is not particularly limited, and may be a conventional method. For example, the method described in JP 2006-257014 A or JP 2010-193808 A can be exemplified. One aspect of the acquisition method will be briefly explained with an example of scales. By treating scales decalcified with acid with a protease such as pepsin, the collagen is atelolated and purified as necessary to produce enzyme. Solubilized collagen can be obtained. For the purification treatment, for example, salting out or a method using activated carbon having a pH of 7 or less described in JP2013-116875A can be applied.

  As the alkali metal bicarbonate, sodium bicarbonate and potassium bicarbonate are preferable. The present invention does not exclude the mixing of alkali metal carbonates as long as the effects of the present invention are not impaired.

The amount of alkali metal bicarbonate applied is not particularly limited as long as fibrotic collagen is precipitated and the porous body of the present invention is finally obtained. If the amount of alkali metal bicarbonate is too small, collagen fibrillation will be insufficient, while if too large, collagen may become non-fibrotic (defibrillated) during lyophilization in the second step. Therefore, it is desirable to appropriately set the amount of alkali metal bicarbonate so that the porous body of the present invention can be obtained. As an example of a preferred embodiment relating to the amount of alkali metal bicarbonate applied, collagen in the alkali metal bicarbonate / solubilized collagen solution (molar ratio) = 3 × 10 ² when the molecular weight of collagen is 300,000. The amount is in the range of ˜3 × 10 ⁴ .

  The method for mixing the solubilized collagen solution and the alkali metal bicarbonate in the first step is not particularly limited. For example, from the viewpoint of workability, an alkali metal bicarbonate aqueous solution is used as the alkali metal bicarbonate. It is preferable.

  Next, the second step of freeze-drying the fibrillated collagen gel obtained in the first step is performed. A known method may be employed as the freeze-drying method. The lyophilization conditions may be appropriately set so that a porous material can be obtained by a conventional method. For example, the freezing temperature is preferably in the range of -20 to -60 ° C, and the lyophilization time is preferably 1 to 60 hours. .

  In order to obtain the porous body of the present invention, it is important to prevent fibrous collagen from becoming non-fibrotic (defibrotic) during lyophilization. For example, when PBS is used for fibrosis of collagen, collagen is non-fibrotic (defibrotic) by PBS concentrated at the time of freeze-drying. Therefore, if the amount of PBS in collagen fibrillation is reduced so as not to be non-fibrotic (defibrotic) during lyophilization, fibrosis becomes insufficient.

  On the other hand, alkali metal bicarbonate is presumed not to inhibit the crystal growth of frozen crystals (ice) of water, and alkali metal bicarbonate can fibrosis collagen even at a low concentration compared to PBS, It is possible to obtain the porous body of the present invention which is composed of collagen fibers and has a porous spongy shape capable of three-dimensional cell culture.

  EXAMPLES The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto. In Examples, “%” means “% by mass” unless otherwise specified. In addition, an analytical scanning electron microscope JSM-6010LA manufactured by JEOL Ltd. was used as the electron microscope, and a solid and straight stainless steel tweezers were used as the metal tweezers.

[Example 1]
As a solubilized collagen solution, a colorless and transparent solution prepared by dissolving “Cell Campus FD-08G” sponge manufactured by Taki Kagaku Co., Ltd. in tilapia scales in an HCl solution of pH 3 and adjusting the collagen concentration to 1.1% ( Hereinafter, “collagen solution A”) was used.

Add 1 part by volume of sodium bicarbonate aqueous solution to 9 parts by volume of collagen solution A so that the collagen (molar ratio) in sodium bicarbonate / collagen solution A is 1.5 × 10 ³ to precipitate fibrotic collagen. It was. Next, after 2 ml of fibrillated collagen gel containing the fibrillated collagen is dispensed into a 12-well plate, freeze-dried at −35 ° C. for 3 hours to obtain a soluble collagen fiber porous body composed of collagen fibers. Obtained.

  The obtained porous body was not deformed even when handled with metal tweezers (FIG. 1). Damage due to metal tweezers was not observed in appearance.

  Further, FIG. 3 shows the inside of the outermost layer hole filled so that only the outermost layer hole of FIG. 2 can be identified. With reference to FIG. 3, the maximum width and the minimum width were measured for all of the complete holes (holes not divided by the partition boundary line), and the average hole diameter was calculated from the above formula. The average hole diameter was 115.0 μm. .

[Comparative Example 1]
9 parts by volume of collagen solution A and 1 part by volume of PBS prepared to a concentration 10 times higher were mixed to precipitate fibrotic collagen. Next, 2 ml each of the fibrillated collagen gel containing the fibrillated collagen was dispensed into a 12-well plate and then freeze-dried at -35 ° C. for 3 hours to form a non-fibrotic collagen porous material composed of non-fibrotic collagen. Got the body.

  As shown in FIG. 4, the obtained porous body was found to be deformed such as curved.

[Comparative Example 2]
9 parts by volume of collagen solution A and 1 part by volume of PBS prepared to a concentration 10 times higher were mixed to precipitate fibrotic collagen. Next, the solvent of the fibrillated collagen gel containing the fibrillated collagen was successively desalted and dehydrated with an ethanol series (ethanol concentration: 50%, 70%, 90%, 100%) by mixing ethanol and water. Thereafter, the solvent was replaced with t-butanol and freeze-dried at −35 ° C. for 3 hours to obtain a collagen fiber porous body composed of collagen fibers.

  The obtained porous body was not deformed even when handled with metal tweezers (FIG. 5). However, as shown in FIG. 6, the presence of pores was clearly observed in an electron microscope image (magnification: 100 times). It was a porous body composed of small pores that were not recognized.

Claims (4)

A soluble collagen fiber porous body composed of a collagen fiber and having a porous spongy shape capable of three-dimensional cell culture. In the electron microscope image of the soluble collagen fiber porous body, in a fixed section where the number of holes observed on the outermost surface is at least 30, when the maximum number of the holes is n,
Average hole diameter = {Σ (maximum width of hole i + minimum width of hole i) / 2} / n (where i = 1 to n)
2. The collagen porous body according to claim 1, wherein the average pore diameter calculated by the mathematical formula is in the range of 50 to 300 μm. A first step of mixing solubilized collagen solution and alkali metal bicarbonate to precipitate fibrotic collagen;
The method for producing a soluble collagen fiber porous body according to claim 1 or 2, comprising a second step of freeze-drying. When the amount of alkali metal bicarbonate applied is 300,000, the collagen in the alkali metal bicarbonate / solubilized collagen solution (molar ratio) = 3 × 10 ^{2 to} 3 × 10 ⁴ The method for producing a soluble collagen fiber porous body according to claim 3.

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