JP4686341B2 - Method for producing collagen fiber using electrospinning and collagen fiber produced by the method - Google Patents

Description

  The present invention relates to a method for producing a collagen fiber, wherein a high voltage is applied to a water / organic solvent mixed solution of collagen and the collagen is fibrillated using electrospinning, and the collagen fiber produced by the production method About.

  In recent years, major surgery has been frequently performed in each medical facility. During major surgery, preventing bleeding of the patient during surgery, ensuring hemostasis, and performing the surgery in a short time are important factors that affect the postoperative course of the patient.

  In general, ligation or electrocoagulation is used as a hemostasis method for arterial bleeding with a clear bleeding point during surgery. On the other hand, for venous bleeding, compression alone is sufficient, and hemostasis is easy.

  However, these hemostatic methods may not be effective for parenchymal organ bleeding or capillary bleeding. In particular, if there is a tendency to bleed in the area of liver failure or cardiovascular surgery, hemostasis is difficult. In such a case, a locally absorbed hemostatic material that accelerates the blood coagulation reaction just by contacting the bleeding surface and quickly forms a thrombus to stop bleeding not only shortens the operation time but also re-bleeds after surgery. It is effective because it prevents and contributes to safe postoperative management.

  On the other hand, local hemostats using collagen, which is a protein that can minimize allergic reactions and foreign body reactions, have their own physiological activity and high hemostatic effect, so they may be applied clinically. It is becoming.

  Collagen local hemostats that have been put into practical use include natural collagen fibers pulverized into flakes, and collagen solutions freeze-dried or naturally dried into sponges (Patent Document 1). is there. The flakes have a drawback that they are not expected to have a hemostatic effect because they are spilled and scattered in the blood, and are more likely to be charged with static electricity, so that they tend to adhere to hands and tweezers during use. In addition, the sponge-like product is not sufficiently adhesive to the wound surface having a complicated shape, so that a hemostatic effect cannot be expected so much.

In order to improve such a drawback, a hemostatic material composed of an aggregate of collagen fibers has been proposed. The collagen fiber aggregate can be obtained by coagulating and regenerating a collagen solution with a high-concentration salt solution to obtain fibers, washing and removing the salts, and then drying (Patent Documents 2 and 3).
JP 2000-510357 A JP-A-5-171510 JP-A-7-977714

  However, since the collagen fiber aggregate disclosed in Patent Document 2 and Patent Document 3 is a cotton lump in which fibers are entangled irregularly, it is difficult to apply it uniformly according to the wound surface shape when used. Met. In addition, in order to make the size and fiber density of the irregularly intertwined cotton lump constant, special means such as performing an opening process such as air blow are necessary. It was difficult to implement.

  Further, it is necessary to sufficiently wash the collagen fibers to remove salts. This is because if these remain, the hemostatic effect is impaired. The fiber diameter is on the order of mm.

  On the other hand, electrospinning (electrostatic spinning method) is known as a method for producing a fiber structure having a small fiber diameter. In electrospinning, a solution or the like containing a fiber-forming substance is introduced into an electric field, thereby causing the liquid to flow toward the electrode and forming the fiber-forming substance in the solution into a fibrous structure. Generally, the fiber-forming material is cured while it is squeezed out of solution. Curing is performed, for example, by cooling, chemical curing, solvent evaporation, or the like. The resulting fibrous structure is then collected on an appropriately placed receptor. However, there is no report that collagen fibers having a fiber diameter on the order of μm were actually produced by electrospinning.

  An object of the present invention is to provide a collagen fiber having a uniform fiber diameter on the order of μm and a method for producing the collagen fiber using electrospinning.

  As a result of examining the interaction between collagen and a solvent, the present inventors have found that a mixed solution of hexafluoroisopropanol and water is an excellent solvent for producing collagen fibers by applying electrospinning. It has been found that collagen fibers having a uniform fiber diameter on the order of μm can be obtained when dissolved in this mixed solution and electrospun.

That is, the present invention
A preparation step of preparing a collagen solution containing collagen, water and hexafluoroisopropanol;
A spinning step of applying a high voltage to the collagen solution and fiberizing the collagen by electrospinning;
A method for producing collagen fibers comprising :
The weight ratio of water to hexafluoroisopropanol in the collagen solution is in the range of 8: 2 to 5: 5;
The present invention relates to a method, wherein the collagen content in the collagen solution is 5% by weight or more and 10% by weight or less.

  According to the method for producing a collagen fiber of the present invention, since a micellar aggregate of collagen molecules is formed in a collagen solution containing collagen, water and hexafluoroisopropanol, when this collagen solution is electrospun, Collagen fibers having a uniform fiber diameter on the order of μm can be produced.

The weight ratio of water and hexafluoroisopropanol in the collagen solution, 8: 2 to 5: Ru der range of 5. The collagen aqueous solution has a light white color. By adding hexafluoroisopropanol to the collagen aqueous solution, the whiteness of the entire solution decreases and the viscosity decreases. If the weight ratio of water and hexafluoroisopropanol in the collagen solution is less than 8: 2, a collagen aqueous solution (light white) partially remains, so the collagen solution is separated into two layers, a white layer and a transparent viscosity-decreasing layer. However, the collagen solution may not be homogenized. On the other hand, if the ratio exceeds 5: 5, the solution viscosity will be too low and the surface tension characteristics of the solution necessary for electrospinning will be lost, and the drying efficiency will also be reduced. This is because there is a fear.

The collagen content in collagen solution, Ru der 10 wt% 5 wt% or more. If the collagen content is less than 5% by weight, the concentration may be too low and the fiber diameter may fall below the μm order. On the other hand, if it exceeds 10% by weight, no matter how much hexafluoroisopropanol is added, This is because the collagen solution hardens in a gel state and collagen cannot be fibrillated.

Wherein in the spinning step, from the following inner diameter of the nozzle 0.1 mm or 2.0 mm, the collagen solution has preferably be discharged. Collagen solution is viscous and may clog if the inner diameter of the nozzle is less than 0.1 mm. On the other hand, if it exceeds 2.0 mm, the collagen solution is difficult to break into fine charged mist and the fiber diameter is μm. This is because there is a possibility that the collagen fibers of the order cannot be manufactured.

The applied voltage in the spinning step, it is not preferable or less of the DC voltage 50 kV or more 1 kV. When the DC voltage is less than 1 kV, the collagen solution is difficult to split into fine charged mists. On the other hand, when it exceeds 50 kV, the splitting force of the charged mists is too strong and the collagen solution is difficult to drop properly. It is.

Furthermore, the present invention is related to the collagen fibers produced by the manufacturing method of the collagen fibers according to any one of claims 1 to 3.

  According to the method for producing a collagen fiber of the present invention, a uniform collagen fiber having a fiber diameter on the order of μm, which has not been obtained conventionally, can be obtained. Moreover, it is not necessary to crosslink or treat after fiberizing collagen, nor to wash away salts.

  Embodiments of the present invention will be described below with reference to the drawings as appropriate. Note that the present invention is not limited to these.

  First, an example of an electrospinning apparatus used in the method for producing a collagen fiber of the present invention is shown in FIG. The electrospinning apparatus 1 includes an insulating plate 3 on the top of a sealed container 2. A metal nozzle 5 connected to a metal holder 4 is fixed to the insulating plate 3. A liquid feed pipe 7 is connected to the metal holder 4 on the opposite side of the metal nozzle 5, and a high voltage power source 6 is connected to the metal holder 4.

  The liquid feeding pipe 7 leads to a container 9 accommodated in another sealed container 8, and the container 9 is filled with a collagen solution 10. Furthermore, the sealed container 8 is connected to the compressor 11 so that the inside can be in a pressurized state.

  When the compressor 11 is turned on, the inside of the sealed container 8 is pressurized, and the collagen solution 10 in the container 9 is fed to the metal nozzle 3 through the liquid feeding pipe 7.

  Inside the hermetic container 2, a metal net 12 is installed on an insulating support 13, and the metal net 12 is grounded 14. The metal net 12 is installed so as to be located directly below the metal nozzle 5.

  Here, when the high voltage power supply 6 is turned on, a high voltage is applied to the metal nozzle 5 through the metal holder 4. At this time, as shown in FIG. 2, charges are induced and accumulated in the collagen solution 10 flowing in the metal nozzle 5 by a high voltage. After being ejected from the metallic nozzle 5, the collagen solutions repel each other to become positively charged.

  This repulsive force opposes the surface tension of the collagen solution, and when the charge criticality is exceeded (when the surface tension is exceeded), the collagen solution becomes a charged mist. Since the surface area of the charged mist is very large with respect to the volume, the solvent water and hexafluoroisopropanol are efficiently evaporated, and the charge density is increased due to the decrease in volume, so that the collagen solution is transferred to the charged fine mist 15. And split up.

  Although a high voltage is applied to the metal nozzle 5, a strong electric field is formed between the metal nozzle 5 and the metal mesh 12 because the metal mesh 12 is grounded. While the charged micro mist 15 repels each other and proceeds toward the metal net 12 by the formed electric field, water and hexafluoroisopropanol which are solvents are volatilized on the way, and fiberized collagen (collagen fiber). Is collected on the metal net 12. At this time, a charge having a sign opposite to the charge applied to the metal nozzle 5 may be applied to the metal net.

  The inner diameter of the metallic nozzle 5 is preferably 0.1 mm or more and 2.0 mm or less, and more preferably 0.1 mm or more and 1.0 mm or less.

  The high voltage applied to the metal holder 4 (and the metal nozzle 5) is preferably a DC voltage of 1 kV to 50 kV, and more preferably a DC voltage of 10 kV to 35 kV.

  The discharge rate of the collagen solution from the metal nozzle 5 is preferably 0.01 mL / min or more and 10 mL / min or less. This discharge speed can be adjusted by controlling the output of the compressor 11 that pressurizes the inside of the sealed container 8.

  Here, the holder, the nozzle, and the net are all made of metal. Moreover, you may electrospin a collagen solution by an open system, without using the airtight container 2. FIG.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited only to these Examples.
Example 1
As Example 1 of this invention, the collagen fiber was manufactured with the following manufacturing methods.
1) Preparation Step First, soluble collagen was dissolved in hydrochloric acid adjusted to pH 2, followed by filtration to obtain a collagen aqueous solution (collagen concentration: 8.12% by weight). Next, hexafluoroisopropanol was added at a rate of 20 mL to 100 mL of this collagen aqueous solution to obtain a collagen solution. The weight ratio of water and hexafluoroisopropanol in the collagen solution is preferably 8: 2 to 5: 5.

Collagen aqueous solution has high viscosity, and collagen fibers cannot be produced by electrospinning as it is. It can be manufactured.
2) Spinning Step Next, the collagen solution produced in the above preparation step was fibrillated using the electrospinning apparatus 1 shown in FIG. 1 to produce a collagen fiber nonwoven fabric. Here, a stainless steel nozzle having an inner diameter of 0.2 mm and a length of 1 cm is used as the metal nozzle 5, and the distance from the lower end of the metal nozzle 5 to the center of the stainless steel net (metal net 12) is 50 cm. . A variable voltage device (high voltage power supply 6: manufactured by Palace Electronics Co., Ltd.) was connected to a stainless steel holder (metal holder 4), and a DC voltage of 20 kV was applied. The ejection speed of the collagen solution was 4.4 mL / min.

As a result, white collagen fibers were collected on the stainless steel net. Even after the collagen fibers were collected, water and hexafluoroisopropanol were completely volatilized although they were collagen solvents. Moreover, this collagen fiber was very delicate, and the formed nonwoven fabric was homogeneous.
(Example 2)
Next, as Example 2, a non-woven fabric of collagen fibers was produced in the same manner as in Example 1 except that hexafluoroisopropanol was added at a ratio of 40 mL to 100 mL of collagen aqueous solution.

As a result, white collagen fibers were collected on the stainless steel net, as in Example 1. Even after the collagen fibers were collected, water and hexafluoroisopropanol were completely volatilized although they were collagen solvents. The collagen fibers were very delicate and homogeneous.
(Comparative Example 1)
Next, as Comparative Example 1, an attempt was made to produce a non-woven fabric of collagen fibers in the same manner as in Example 1 except that hexafluoroisopropanol was added at a ratio of 5 mL to 100 mL of collagen aqueous solution.

However, since the collagen solution has a high viscosity, the collagen solution is clogged in the stainless steel nozzle, and collagen fibers cannot be produced.
(Comparative Example 2)
Next, as Comparative Example 2, an attempt was made to produce a non-woven fabric of collagen fibers in the same manner as in Example 1 except that hexafluoroisopropanol was added at a ratio of 80 mL to 100 mL of collagen aqueous solution.

However, the solvent could not be volatilized before reaching the stainless steel net, and collagen could not be fibrillated.
(Comparative Example 3)
Next, as Comparative Example 3, an attempt was made to produce a nonwoven fabric of collagen fibers in the same manner as in Example 1, except that isopropanol was added at a rate of 20 mL to 100 mL of collagen aqueous solution.

However, when isopropanol was added, the collagen solution solidified, and collagen fibers could not be produced. The same result was obtained when methanol, ethanol or acetone was added instead of isopropanol.
(Comparative Example 4)
Next, as Comparative Example 3, an attempt was made to produce a non-woven fabric of collagen fibers in the same manner as in Example 1, except that o- (trifluoromethyl) benzyl alcohol was added at a rate of 20 mL to 100 mL of collagen aqueous solution. .

However, when o- (trifluoromethyl) benzyl alcohol was added, the collagen solution was solidified as in Comparative Example 3, and collagen fibers could not be produced.
(Comparative Example 5)
Next, as Comparative Example 5, an attempt was made to produce a non-woven fabric of collagen fibers in the same manner as in Example 1, except that trifluoroethanol was added at a rate of 20 mL to 100 mL of collagen aqueous solution.

However, as in Comparative Example 2, the solvent could not be volatilized before reaching the stainless steel net, and collagen could not be fibrillated.
(Comparative Example 6)
Next, as Comparative Example 6, an attempt was made to produce a non-woven fabric of collagen fibers in the same manner as in Example 1, except that trifluoroethanol was added at a rate of 20 mL to 100 mL of collagen aqueous solution.

However, as in Comparative Example 2, the solvent could not be volatilized before reaching the stainless steel net, and collagen could not be fibrillated.
3) Morphological observation of collagen fibers by scanning electron microscope (SEM) The collagen fibers obtained in Example 1 and Example 2 were subjected to platinum / palladium deposition (30 mA, 120 seconds), and a scanning electron microscope (Hitachi). Observation was performed using S-800 type manufactured by Seisakusho. The acceleration voltage was 6 kV and the working distance was 20 mm.

  Electron micrographs of the collagen fibers of Example 1 are shown in FIGS. 3 (a) and 3 (b). The collagen fiber of Example 1 has a uniform fiber diameter as shown in FIG. 3 (a), and measured the fiber diameter at five locations based on an electron micrograph, and the average value was calculated to be about 5 μm. Met. Further, as apparent from FIG. 3B, the surface of the collagen fiber of Example 1 was very smooth.

  Regarding the collagen fibers of Example 2, the fiber diameters at five locations were measured based on the electron micrographs, and the average value was calculated. The result was about 5 μm. The fiber surface was very smooth.

  Although the collagen fiber nonwoven fabric of Example 1 and Example 2 may be used alone, it may be used in combination with other members in accordance with handleability and other requirements. For example, a non-woven fabric, a woven fabric, a film, a stent, or the like that can serve as a support substrate is used as a workpiece, and a non-woven fabric is formed on the non-woven fabric to form a member (for example, an implantable stent). Etc.) can also be produced.

  The collagen fiber and the method for producing the same of the present invention are useful as a biocompatible material in the pharmaceutical and medical fields, a method for producing the material, and the like.

It is the schematic which shows an example of the electrospinning apparatus used in the manufacturing method of the collagen fiber of this invention. It is an enlarged schematic diagram which shows the state of the collagen solution of metal nozzle lower end part vicinity when operating an electrospinning apparatus. 2 is a scanning electron micrograph of collagen fibers according to Example 1. FIG.

Explanation of symbols

1: Electrospinning device 2: Sealed container 3: Insulating plate 4: Metal holder 5: Metal nozzle 6: High-voltage power supply 7: Liquid supply pipe 8: Another sealed container 9: Container 10: Collagen solution 11: Compressor 12: Metal net 13: support 14: ground 15: charged minute mist

Claims (4)

A preparation step of preparing a collagen solution containing collagen, water and hexafluoroisopropanol;
A spinning step of applying a high voltage to the collagen solution and fiberizing the collagen by electrospinning;
A method for producing collagen fibers comprising :
The weight ratio of water to hexafluoroisopropanol in the collagen solution is in the range of 8: 2 to 5: 5;
The method wherein the collagen content in the collagen solution is 5% by weight or more and 10% by weight or less. The method for producing a collagen fiber according to claim 1 , wherein in the spinning step, the collagen solution is discharged from a nozzle having an inner diameter of 0.1 mm or more and 2.0 mm or less. The method for producing a collagen fiber according to claim 1 or 2 , wherein the applied voltage in the spinning step is a DC voltage of 1 kV or more and 50 kV or less. The collagen fiber manufactured by the manufacturing method of the collagen fiber of any one of Claims 1 thru | or 3 .