JP5807329B2 - Aggregate of collagen fibers and method for producing the same - Google Patents

Description

  The present invention relates to an aggregate of collagen fibers and a method for producing the same.

  Until now, films and fibers made of biodegradable polymers have been expected to be applied to cell scaffold materials and medical materials.

  As medical materials, collagen as animal protein, hyaluronic acid as polysaccharide, chitin, chitosan, carboxymethylcellulose, dextrin, polyglycolic acid as aliphatic polyester, glycolic acid / L-lactic acid copolymer, poly-L -Lactic acid, L-lactic acid / ε-caprolactone copolymer, poly-p-dioxanone, and other synthetic polymers such as polyvinylpyrrolidone and polyethylene glycol have been used. Collagen in particular has a feature of excellent cell adhesion.

  As a method for producing a collagen fiber assembly, a technique is known in which collagen is dissolved in a fluorine-based organic solvent such as trifluoroethanol (TFE) or hexafluoroisopropanol (HFIP), and collagen fibers are produced by charge spinning. (See Patent Documents 1 and 2 and Non-Patent Document 1).

JP-T-2006-501949 JP 2007-138364 A

Jamil A. Mattews, Gary E. Wnek, David G. Simpson, and Gary L. Bowlin. , Biomacomolecules, 2002, 3, 232

  As described above, conventionally, fibers made from collagen have been known. However, this technique uses collagen as a raw material, but uses a solution of a fluorine-based organic solvent such as HFIP, so that the obtained fiber does not maintain the structure of collagen and is made of gelatin. Become. Therefore, it has the problem that it is inferior to cell adhesiveness. In addition, when an organic solvent containing halogen is used, there is a problem in that it adversely affects the human body and the environment.

  The present invention was invented in view of the current state of the prior art, and an object thereof is an aggregate of collagen fibers having a collagen structure substantially maintaining a collagen structure, and having a fiber diameter of 10 nm or more and 1 μm or less, and its It is to provide a manufacturing method.

  As a result of intensive studies to achieve the above object, the present inventors have found that when a collagen solution prepared using an organic acid aqueous solution that does not contain a halogen in the molecular structure is subjected to charge spinning, the fiber diameter is 10 nm to 1 μm. The present inventors have found that a collagen fiber assembly composed of collagen fibers substantially maintaining the collagen structure is obtained, and the present invention has been achieved.

  The method for producing an aggregate of collagen fibers according to the present invention comprises a step of preparing a collagen solution by dissolving collagen in an organic acid aqueous solution that does not contain halogen in the molecular structure, and spinning by using the collagen solution by a charged spinning method. Including the step of:

  The aggregate of collagen fibers according to the present invention is characterized in that the fiber diameter is 10 nm or more and 1 μm or less.

FIG. 1 is an electron micrograph of the collagen fiber assembly obtained in Example 1. FIG. 2 is an electron micrograph of the collagen fiber assembly obtained in Example 3. FIG. 3 is an electron micrograph of the collagen fiber assembly obtained in Example 4. FIG. 4 is a circular dichroism dispersometer measurement result of the collagen fiber aggregate obtained in Example 4.

  The present invention is described in detail below. First, a method for producing a collagen fiber assembly according to the present invention will be described.

1. Collagen Solution Preparation Step In the method of the present invention, collagen is dissolved in an organic acid aqueous solution that does not contain halogen in the molecular structure to prepare a collagen solution.

  By carrying out charge spinning using an organic acid aqueous solution containing no halogen in the molecular structure, a fiber assembly composed of collagen fibers substantially maintaining the collagen structure can be obtained. As described above in the background art, examples have been introduced in which a collagen solution is prepared using a fluorine-based organic solvent such as TFE or HFIP, and charged spinning is performed using this solution. Since these fluorine-based organic solvents such as TFE and HFIP have fluorine atoms in their molecular structure, they do not correspond to organic acid aqueous solutions that do not contain halogen in the molecular structure. When these fluorinated organic solvents are used, they may remain in the collagen fiber assembly, and depending on the intended use, they may affect the human body. In addition, they can be dissolved in a fluorinated organic solvent and charged. When spinning, almost all the collagen is transformed into gelatin, so that a fiber assembly composed of collagen fibers substantially maintaining the collagen structure cannot be obtained.

  The kind of water contained in the organic acid aqueous solution not containing halogen in the molecular structure used in the present invention is not particularly limited. For example, tap water, well water, distilled water, purified water, pure water, ultrapure water, etc. can be used, and since they are low in impurities and suitable for the charged spinning method, distilled water, purified water, pure water, ultrapure water, etc. Water is preferred, and distilled water and purified water are more preferred.

  The solvent for dissolving collagen may contain a solvent having good volatility such as ethanol, 1-propanol, 2-propanol and the like that is harmless to the human body as long as it does not inhibit the dissolution of collagen. As the solvent, a mixed solvent of an aqueous organic acid solution and an alcohol is particularly preferable because it can easily stabilize the higher-order structure of collagen and can evaporate easily during charge spinning to obtain finer collagen fibers. When adding alcohol or the like, the content of alcohol or the like is preferably 1 wt% or more and 30 wt% or less with respect to the organic acid aqueous solution. If the said content rate is 1 wt% or more, a solvent will evaporate easily during spinning and it will become easy to obtain a better quality collagen fiber aggregate. On the other hand, if the content is too high, collagen may be difficult to dissolve, so 30 wt% or less is preferable. As the said ratio, 5 wt% or more is more preferable, 7 wt% or more is further more preferable, 25 wt% or less is more preferable, and 20 wt% or less is further more preferable.

As the organic acid contained in the aqueous organic acid solution used in the present invention, those which do not contain a halogen in the molecular structure are preferred, like the solvent. The organic acid is not particularly limited, and formic acid, propionic acid, butyric acid, isobutyric acid, lactic acid, acetic acid, and the like can be used. Two or more of these may be mixed. Inorganic acids such as hydrochloric acid and sulfuric acid are not preferable because they are harmful to the human body. Further, in the case of hydrochloric acid, the chlorine ion Cl ⁻ is heavy, so that it does not fly during spinning and may remain in the resulting fiber.

  The concentration of these organic acids in the aqueous organic acid solution is preferably 5 to 99 wt%, more preferably 40 to 95 wt%, and still more preferably 50 to 90 wt%.

  Of these, acetic acid is preferable as the organic acid not containing halogen in the molecular structure, and an acetic acid aqueous solution or an acetic acid buffer is preferably used as the organic acid aqueous solution. The concentration of acetic acid in the acetic acid aqueous solution is preferably 5 to 99 wt%. When the concentration of acetic acid is less than 5 wt%, the viscosity becomes high and spinning may be difficult. If it exceeds 99 wt%, collagen may not be dissolved and spinning may be difficult. The concentration is more preferably 40 to 95 wt%, still more preferably 50 to 90 wt%.

  The collagen used in the present invention is, for example, type I, type II, type III, type IV, type V, type VI, type VII, type VIII, type IX, type X, type XI, type XII, type XIII, XIV. Type, XV type, XVI type, XVII type, XVIII type and XIX type collagen, but are not limited thereto. Preferred collagens are type I, type II and type III. Many of these types are distributed in the living body and can be easily obtained.

  The structure of the collagen used in the present invention is not particularly limited, but it is preferably so-called atelocollagen in which the telopeptide which is the antigenic expression site of collagen is removed to reduce immunotoxicity.

  Although the density | concentration of the collagen in a collagen solution is not specifically limited, 3-20 wt% is preferable. If the concentration is less than 3 wt%, spinnability may be impaired. When the concentration exceeds 20 wt%, the viscosity becomes high, and it may be difficult to discharge from the nozzle.

  The collagen solution can be prepared by using a normal dissolution method, but the solution temperature is preferably 0 ° C. or higher and 40 ° C. or lower. Below 0 ° C., the solution may freeze, and above 40 ° C., the collagen may be thermally denatured and gelatinized. The temperature is more preferably 3 ° C. or more and 35 ° C. or less, and further preferably 5 ° C. or more and 30 ° C. or less.

2. Charge Spinning Step In the method of the present invention, a collagen fiber aggregate is then obtained by spinning with a collagen solution using a charge spinning method.

  The charged spinning method is a method in which a fine fibrous material is obtained by discharging a charged polymer solution from a nozzle tip while being charged, and using the charge repulsive force of the solution. As a method for applying the voltage, the nozzle side may be positive and the collecting part may be grounded or negative, and conversely, the nozzle may be grounded or negative and the collecting part may be positive. Although the potential difference between the nozzle and the collection part is not particularly limited, it is generally preferable to set the potential difference between 3 kV and 100 kV. When the voltage is less than 3 kV, charge repulsion between polymers may be difficult to occur. If it exceeds 100 kV, discharge may occur, which may be unfavorable for safety. The potential difference is more preferably 5 kV to 50 kV, and still more preferably 5 kV to 40 kV.

  The inner diameter of the nozzle is not particularly limited, but is preferably 0.05 mm or more and 3 mm or less. If it is less than 0.05 mm, the amount of discharged solution may be small and productivity may be lacking. If it exceeds 3 mm, spinnability may be lacking. The material of the nozzle is not particularly limited, and a metallic or non-metallic material can be used.

  Although the discharge speed of a nozzle is not specifically limited, 0.005 mm / min or more and 0.5 mm / min or less are preferable. If it is less than 0.005 mm / min, the production efficiency may not be good. If it exceeds 0.5 mm / min, the amount of the solution increases and the resulting sample may be easily formed into a film.

  The temperature of the collagen solution during spinning is preferably 0 ° C or higher and 40 ° C or lower. If it is less than 0 ° C., the solution may freeze, and if it exceeds 40 ° C., collagen may be heat-denatured and gelatinized. The temperature is more preferably 3 ° C. or more and 35 ° C. or less, and further preferably 5 ° C. or more and 30 ° C. or less.

  Although the distance between a nozzle and a collection part is not specifically limited, 1 cm or more and 40 cm or less are preferable. If it is less than 1 cm, solvent evaporation hardly occurs during spinning, and there is a possibility of film formation. If it exceeds 40 cm, the sample may be deposited not only on the collecting part but also on a conductive part other than the collecting part, and the production efficiency may be reduced.

  The spinning atmosphere is not particularly limited, and may be performed in air or a gas having a higher discharge start voltage than air, such as carbon dioxide.

  In the charged spinning method, while the polymer solution reaches the collection part, the solvent evaporates depending on the conditions, and a fiber assembly is obtained. The spinning atmosphere temperature is not particularly limited. Normally, the solvent evaporates when carried out at room temperature, but if the solvent evaporation is insufficient, the atmosphere temperature may be raised. However, the spinning atmosphere temperature is preferably 40 ° C. or lower so that the collagen fibers are not heat-denatured and gelatinized.

  The fiber aggregate obtained by the present invention may be collected alone, but may be combined with other members depending on the handleability and application. For example, cloth (nonwoven fabric, woven fabric, knitted fabric) serving as a support substrate as a collection substrate, a film, a drum, a net, a flat plate, a belt-like conductive material made of metal or carbon, a non-polymer made of an organic polymer, or the like. Conductive materials can be used. By forming the fiber assembly thereon, a member in which the support base material and the fiber assembly are combined can be produced.

3. Insolubilization process The collagen fiber aggregate of the present invention is insoluble in water, but may be further insolubilized for the purpose of improving the resistance to an acidic solution. Insolubilization methods include methods using chemical reagents such as glutaraldehyde, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), ultraviolet radiation, gamma irradiation, and thermal dehydration. Although there is no particular limitation, a method using thermal dehydration is preferable. When a chemical reagent is used, cytotoxicity due to the remaining reagent is considered, and ultraviolet radiation and γ-ray radiation may destroy the structure of the collagen. A thermal dehydration method under reduced pressure (1 kPa or less) can be used in order to produce a collagen fiber aggregate that does not collapse the collagen structure and is acid resistant.

  The temperature of the insolubilization treatment by thermal dehydration is preferably 100 to 160 ° C. If it is less than 100 degreeC, a dehydration reaction may not fully be performed but an insolubilization process may become inadequate. Further, when the temperature exceeds 160 ° C., the structure may change due to thermal degradation of collagen molecules, and there may be a problem that the color changes to yellow. The insolubilization time is preferably 12 hours or longer. If the time is less than 12 hours, problems such as insufficient insolubilization and non-uniform dehydration may occur.

  The aggregate of collagen fibers of the present invention is a fiber aggregate composed of collagen fibers having a fiber diameter of 10 nm to 1 μm and substantially maintaining a collagen structure. The fiber diameter can be obtained by observing the surface of the fiber assembly with a scanning electron microscope, randomly selecting 10 to 20 fibers from the photograph, measuring the fiber diameter, and calculating the average value.

  The fiber diameter of the aggregate of collagen fibers according to the present invention is 10 nm or more and 1 μm or less. If it is less than 10 nm, there is a problem that the fibers are too thin and cell recognition is poor. When it exceeds 1 μm, the effect as a cell scaffold material is hardly exhibited.

  The maintenance of the collagen structure means that a triple helical structure, which is a structure peculiar to collagen, is maintained. A material in which the triple helix structure is solved is called gelatin. In the collagen fiber aggregate of the present invention, the collagen structure is maintained, and the ratio of gelatin can be kept lower than before. A fiber aggregate having a gelatin structure is not preferable because of poor cell adhesion. In addition, the maintenance rate of the collagen structure, that is, the maintenance rate of the triple helical structure is preferably 30% or more, more preferably 40% or more, and still more preferably 50% or more. Of course, the upper limit is preferably 100%.

  Confirmation that the collagen fiber maintains the triple helical structure can be confirmed by circular dichroism dispersometer measurement. CD spectrum obtained by circular dichroism dispersion meter measurement shows a negative cotton effect at any wavelength in the 185 to 205 nm wavelength range, and a positive cotton effect at any wavelength in the 210 to 230 nm wavelength range. By showing, it can confirm that at least one part or all of polypeptide maintains the triple helix structure. The circular dichroism refers to a phenomenon in which a difference in absorbance occurs between the left circularly polarized light and the right circularly polarized light when the optically active substance absorbs the circularly polarized light. Further, the circular dichroism cotton effect refers to a phenomenon in which a difference in absorbance and transmission speed occurs simultaneously with respect to left and right circularly polarized light at a specific wavelength.

The basis weight of the collagen fiber aggregate of the present invention is determined according to the intended use and is not particularly limited, but is preferably 0.01 g / m ² or more and 50 g / m ² or less. If it is less than 0.01 g / m ² , the strength may be low, and if it exceeds 50 g / m ² , the cost may be high. The basis weight can be adjusted by the discharge amount of the collagen solution in the charge spinning process, the spinning time, and the like.

  The thickness of the collagen fiber aggregate of the present invention can be determined according to the intended use and is not particularly limited, but is preferably 0.1 μm or more and 500 μm or less. If the thickness is less than 0.1 μm, the strength may be low, and if it exceeds 500 μm, the cost may be high. The thickness is more preferably 0.5 μm or more and 400 μm or less, and still more preferably 1 μm or more and 300 μm or less.

  ADVANTAGE OF THE INVENTION According to this invention, the fiber diameter is 10 nm or more and 1 micrometer or less, The aggregate | assembly of the collagen fiber which consists of a collagen fiber with which the collagen structure was substantially maintained can be provided. The aggregate of collagen fibers according to the present invention maintains a collagen structure and has excellent cell adhesion and biocompatibility because the fiber diameter is a size with excellent cell recognition. Moreover, according to this invention, the method of manufacturing the aggregate | assembly of the said collagen fiber can be provided, without using the organic solvent containing the halogen harmful | toxic to a human body.

  EXAMPLES The present invention will be specifically described below with reference to examples and comparative examples, but the present invention is not limited to the examples.

  Below, the measuring method used in the Example is demonstrated.

1. Measurement of Fiber Diameter Platinum sputtering was applied to the obtained fiber assembly using ion sputtering (E-1030, manufactured by Hitachi, Ltd.). The surface of the fiber assembly is observed using a field emission scanning electron microscope (FE-SEM, S-800, S-4500, manufactured by Hitachi) at an acceleration voltage of 10 kV and a magnification of 10,000 times. And took a photo. 10 to 20 fibers were randomly selected from the photograph, and the fiber diameter was measured. The average value of the measured fiber diameter was calculated and used as the fiber diameter.

2. Measurement of circular dichroism dispersometer Measurement was performed using a circular dichroism dispersometer (J-820, manufactured by JASCO Corporation). Cell optical path length is 0.2 mm, measurement temperature is 4 ° C., measurement wavelength range is 260 to 185 nm, scanning speed is 50 nm / min, bandwidth is 1 nm, response is 2 sec, sensitivity is standard, data capture interval is 0.1 nm The number of integrations was 1, the fluorescence side sensitivity was 0 V, the solution concentration was 1 mg / mL, and the solvent was a hydrochloric acid aqueous solution (pH 3). In Table 2, ○ and × are evaluated as follows.
○: CD spectrum measured by a circular dichroism dispersometer, showing a negative cotton effect at any wavelength in the wavelength range of 185 to 205 nm, and showing a positive cotton effect at any wavelength in the wavelength range of 210 to 230 nm. And maintains a triple helix structure.
X: The CD spectrum does not show the above effects, and the triple helical structure is not maintained.

3. Maintenance rate of triple helix structure The circular dichroism dispersometer measurement of collagen before charge spinning and the fiber aggregate obtained in each example was performed according to the above 2. From the obtained CD spectrum, the triple helix structure, The maintenance rate of collagen structure was evaluated.

The retention rate of the triple helical structure, that is, the collagen structure, is based on the average residue ellipticity (deg · cm ² · dmol ^-1 ) at a wavelength of 222 nm of a solution obtained by dissolving collagen before charge spinning in an aqueous pH 3 hydrochloric acid solution. Thus, the following formula was used.
Triple helical structure maintenance rate (%) = {average residue ellipticity of aqueous solution of hydrochloric acid of fiber aggregate obtained in each example (λ222) / collagen (before charge spinning) average residue ellipticity of aqueous solution of hydrochloric acid ( λ222)} × 100

The average residue ellipticity: [θ] (deg · cm ² · dmol ⁻¹ ) was determined using the following equation.
[Θ] = θ / (10 · Cr · l)
[Wherein, Cr = nCp, θ is the ellipticity (mdeg), Cr is the average residue molar concentration (mol / L), l is the cell length (cm), and n is the polymer It is the number of constituent residues, and Cp is the molar concentration of the polymer (mol / L)]

4). TEM Observation A sample was fixed overnight in a 1% aqueous solution of OsO ₄ , washed with water, dehydrated with ethanol, and embedded in a resin to obtain an embedded sample. An ultrathin section was prepared from the embedded sample and stained with a 1% aqueous phosphotungstic acid solution at room temperature for 30 minutes. Observation was performed with a JEM2010 transmission electron microscope manufactured by JEOL at an acceleration voltage of 200 kV and direct magnifications of 5,000 and 40,000.

Example 1
Add collagen powder (mixed type I and type III, porcine skin NMP collagen PS, manufactured by Nippon Ham Co., Ltd.) to a mixed solution of acetic acid and water (acetic acid / water = 9/1 (wt ratio)) The mixture was stirred to obtain a 10 wt% collagen solution. Charge spinning was performed using this collagen solution.

  Spinning was performed under the following conditions. The apparatus used was “Nanofiber Electrospinning Unit” manufactured by Kato Tech. As the polymer solution discharge port, an ejection nozzle having an inner diameter of 0.8 mm was used, and the solution supply speed was 0.05 cm / min. The potential difference between the nozzle part and the ground part was set to 13 kV, and the distance (AG length) between the nozzle part and the ground part was 10 cm. Moreover, it implemented on temperature 27 degreeC and relative humidity 42RH% conditions. The produced fiber assembly was observed with a scanning electron microscope and measured by a circular dichroism dispersometer. An electron micrograph of the obtained collagen fiber aggregate is shown in FIG. Moreover, when observed with TEM, no stripe pattern was observed. It is considered that no striped pattern is observed because collagen is dissolved before spinning.

Example 2
After spinning under the same spinning conditions as in Example 1, insolubilization treatment was carried out at 140 ° C. under reduced pressure for 24 hours.

Example 3
Add collagen powder (mixed type I and type III, porcine skin NMP collagen PS, manufactured by Nippon Ham Co., Ltd.) to an acetic acid water mixed solution (acetic acid / water = 1/1 (wt ratio)) The mixture was stirred to obtain a 10 wt% collagen solution. Charge spinning was performed using this collagen solution.

  Spinning was performed under the following conditions. The apparatus used was “Nanofiber Electrospinning Unit” manufactured by Kato Tech. As the polymer solution discharge port, an ejection nozzle having an inner diameter of 0.8 mm was used, and the solution supply speed was set to 0.05 cm / min. The potential difference between the nozzle part and the ground part was set to 33 kV, and the distance (AG length) between the nozzle part and the ground part was 10 cm. Moreover, it implemented on temperature 27 degreeC and relative humidity 44RH% conditions. The produced fiber assembly was observed with a scanning electron microscope and measured by a circular dichroism dispersometer. An electron micrograph of the resulting collagen fiber aggregate is shown in FIG. Moreover, when observed with TEM, no stripe pattern was observed. It is considered that no striped pattern is observed because collagen is dissolved before spinning.

Example 4
Add collagen (mixed type I and type III, porcine skin NMP collagen PS, Nippon Ham Co.) powder to acetic acid water ethanol mixed solution (acetic acid / water / ethanol = 9/1 / 1.3 (wt ratio)) The mixture was stirred while cooling to 5 ° C. to obtain a 10 wt% collagen solution. Charge spinning was performed using this collagen solution.

  Spinning was performed under the following conditions. The apparatus used was “Nanofiber Electrospinning Unit” manufactured by Kato Tech. As the polymer solution discharge port, an ejection nozzle having an inner diameter of 0.8 mm was used, and the solution supply speed was 0.05 cm / min. The potential difference between the nozzle part and the ground part was set to 11 kV, and the distance (AG length) between the nozzle part and the ground part was 10 cm. Moreover, it implemented on temperature 26 degreeC and relative humidity 38RH% conditions. The produced fiber assembly was observed with a scanning electron microscope and measured by a circular dichroism dispersometer. FIG. 3 shows an electron micrograph of the obtained collagen fiber assembly, and FIG. 4 shows the results of circular dichroism dispersometer measurement. Moreover, when observed with TEM, no stripe pattern was observed. It is considered that no striped pattern is observed because collagen is dissolved before spinning.

Comparative Example 1
Collagen powder (mixed type I and type III, porcine skin NMP collagen PS, manufactured by Nippon Ham) was added to HFIP and stirred while cooling to obtain a 5 wt% collagen solution. The solution was spun under the same spinning conditions as in Example 1.

Comparative Example 2
Collagen powder (mixture of type I and type III, pig skin NMP collagen PS, manufactured by Nippon Ham) was added to hydrochloric acid (pH 3) and stirred while cooling to obtain a collagen solution. The solution was subjected to charge spinning under the same spinning conditions as in Example 1, but spinning was not possible.
Below, the measurement result of each fiber is shown.

  Examples 1 to 4 use an acetic acid aqueous solution, and the resulting collagen fibers have a fiber diameter of 10 nm to 1 μm and maintain a triple helical structure. It was excellent in properties. In addition, there is no data of a triple helix structure maintenance factor in Example 2 because it was not able to be measured since it was insolubilized. However, since the fiber of Example 2 is obtained by insolubilizing the fiber of Example 1, it is considered that the triple helical structure is maintained as in Example 1.

  On the other hand, since HFIP was used as a solvent in Comparative Example 1, the fiber did not maintain a triple helical structure and had a gelatin structure, and therefore the cell adhesion was poor.

  In Comparative Example 2, hydrochloric acid was used, and collagen fibers could not be obtained.

  ADVANTAGE OF THE INVENTION According to this invention, the fiber diameter is 10 nm or more and 1 micrometer or less, The aggregate | assembly of the collagen fiber which consists of a collagen fiber with which the collagen structure was substantially maintained can be provided. The aggregate of collagen fibers of the present invention maintains the collagen structure, and its fiber diameter is a size with excellent cell recognizability, so it has excellent cell adhesion and biocompatibility, and is used as a cell scaffold material, medical material, etc. Can be used.

Claims (3)

In a mixed solvent of acetic acid aqueous solution and an alcohol, preparing a collagen solution to dissolve the collagen,
A CD spectrum obtained by a circular dichroism dispersometer, comprising a step of spinning by a charged spinning method using the collagen solution, and exhibits a negative cotton effect at any wavelength in a wavelength range of 185 to 205 nm. The manufacturing method of the aggregate | assembly of the collagen fiber which shows the positive cotton effect in any wavelength within the wavelength range 210-230 nm.   The method for producing an aggregate of collagen fibers according to claim 1, wherein the collagen solution is obtained by dissolving collagen in a solvent containing 5-99 wt% acetic acid.   The method for producing an aggregate of collagen fibers according to claim 1 or 2, wherein the mixed solvent has an alcohol content of 1 wt% to 30 wt% with respect to the acetic acid aqueous solution.

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