JPWO2006022430A1 - Fiber structure containing phospholipid - Google Patents

Abstract

A fiber structure containing 0.01 to 100 parts by weight of a phospholipid with respect to 100 parts by weight of an aliphatic polyester and having an average fiber diameter of 0.05 to 50 μm. More preferably, the fiber structure has pores on the fiber surface and serves as a base material suitable for cell culture.

Description

  The present invention relates to a fibrous structure containing a phospholipid. More preferably, the present invention relates to a fiber structure which has pores on the fiber surface and serves as a base material suitable for cell culture.

In the field of regenerative medicine, a porous body may be used as a substrate when culturing cells. As a porous body, a foam or a fibrous structure obtained by freeze-drying is known. These porous materials are required to have affinity with cells, biodegradability and safety.
Polylactic acid is available at a relatively low cost among these materials known to be biodegradable and safe. In particular, polylactic acid, which is mainly composed of L-lactic acid component, has recently been produced in a large amount.
For example, the use of a porous body of polylactic acid, which is known to be biodegradable and safe, as a cell culture substrate has been studied (for example, "Regenerative Medicine" by Noriya Ohno and Masuo Aizawa. See TS, January 31, 2002, page 262.).
However, in these methods, the area to which cells can adhere is insufficient, and a porous body having a larger surface area is desired, and a fiber structure having a small fiber diameter has been studied as one of them.
An electrostatic spinning method is known as a method for producing a fiber structure having a small fiber diameter (see, for example, JP-A-63-145465 and JP-A-2002-249966). The electrospinning method includes a step of introducing a liquid, for example, a solution containing a fiber-forming substance, into an electric field, and thereby pulling the liquid toward an electrode to form a fibrous substance. Usually, the fiber-forming substance cures while being drawn from the solution. Curing is done, for example, by cooling (eg, when the spinning liquid is a solid at room temperature), chemical curing (eg, treatment with curing vapors), solvent evaporation, or the like. Further, the obtained fibrous substance is collected on an appropriately arranged receptor and, if necessary, can be peeled therefrom. Further, since the electrospinning method can directly obtain a non-woven fibrous substance, it is not necessary to form a fiber structure once the fibers are once spun, and the operation is simple.
It is also known to use the fiber structure obtained by the electrospinning method as a substrate for culturing cells. For example, regeneration of blood vessels has been studied by forming a fibrous structure made of polylactic acid by an electrostatic spinning method and culturing smooth muscle cells on the fibrous structure (for example, Joel D. Stitzel, Kristin J. Powlowski, Gary E. Wnek, David G. Simpson, Gary L. Bowlin, Journal of Biomaterials Applications 2001, 16, 22-33).
However, the reports so far have not yet obtained a cell adhesion effect sufficient to satisfy the demand.

An object of the present invention is to provide a substrate suitable for cell culture in the field of regenerative medicine.
That is, the present invention relates to a fiber structure characterized by containing 0.01 to 100 parts by weight of phospholipid with respect to 100 parts by weight of aliphatic polyester and having an average fiber diameter of 0.05 to 50 μm. Is. The present invention more preferably relates to a fiber structure having an average porosity of 3% to 90% on the fiber surface of the fiber structure.
EFFECTS OF THE INVENTION The fiber structure containing the phospholipid of the present invention can provide a fiber structure with improved cell adhesion, and is particularly effective in the field of regenerative medicine.

It is an example of an apparatus used in the electrospinning method of discharging a spinning solution into an electrostatic field in the production method of the present invention. It is an example of an apparatus used in the electrospinning method of introducing fine droplets of a spinning solution into an electrostatic field in the production method of the present invention. Scanning electron micrograph of the fiber structure obtained in Example 1. Scanning electron micrograph of the fiber structure obtained in Example 2. Scanning electron micrograph of the fiber structure obtained in Example 3. Scanning electron micrograph of the fiber structure obtained in Example 4. Scanning electron micrograph of the fiber structure obtained in Example 5. Scanning electron micrograph of the fiber structure obtained in Comparative Example 1. It is a result of measurement of Alamarblue of Example 6 and Comparative Example 2.

Explanation of symbols

1. Nozzle 2. Spinning solution 3. Spinning liquid holding tank 4. Electrode 5. Fibrous substance collecting electrode 6. High voltage generator 7. Nozzle 8. Spinning solution 9. Spinning liquid holding tank 10. Electrode 11. Fibrous substance collecting electrode 12. Preferred Embodiments of High Voltage Generator Invention Hereinafter, the present invention will be described in detail. It should be noted that these examples and the like and the explanations only exemplify the present invention and do not limit the scope of the present invention. It goes without saying that other embodiments can be included in the scope of the present invention as long as they match the spirit of the present invention.
The present invention is a fiber structure containing 0.01 to 100 parts by weight of a phospholipid with respect to 100 parts by weight of an aliphatic polyester. The content of the phospholipid is preferably 0.1 to 20 parts by weight, more preferably 0.1 to 10 parts by weight, based on 100 parts by weight of the aliphatic polyester. If the phospholipid content is 100 parts by weight or more, the stability of the fiber may be insufficient.
The average fiber diameter of the fiber structure used in the present invention is preferably 0.05 to 50 μm. When the average fiber diameter is smaller than 0.05 μm, it is not preferable because the decomposition at the time of cell culture or when transplanted in vivo after cell culture is too fast. Further, if the average fiber diameter is larger than 50 μm, a sufficient surface area for cell culture cannot be obtained, which is not preferable. The average fiber diameter is more preferably 0.1 to 20 μm.
The average porosity of the fiber structure used in the present invention is preferably 3 to 90%, more preferably 11 to 90%, and further preferably 30 to 90%.
The average porosity here means the ratio of the pore area to the area of the entire fiber surface, and a scanning electron micrograph of 20,000 times of the fiber structure is obtained using image processing software (next New Qube) It is the one obtained by performing binarization processing.
When the average porosity is 3 to 11%, a sufficient cell adhesion effect may not be obtained, and therefore by providing the surface with unevenness, a sufficient cell adhesion effect can be preferably provided. Even when the average porosity is 11% or more, it is preferable that the surface has irregularities to obtain a more excellent cell adhesion effect.
When the average porosity is more than 30%, a better cell adhesion effect can be obtained.
If the average porosity is greater than 90%, the strength of the fiber may not be maintained.
The average pore diameter (diameter) of the concave portion on the surface of the fiber structure is preferably 0.001 to 10 μm. The thickness is more preferably 0.005 to 5 μm, and more preferably 0.01 to 1 μm. The average pore diameter of the depressions can be obtained by taking a scanning electron microscope photograph and calculating the average value of the diameters of the depressions measured at n=20 from the photograph.
Examples of the aliphatic polyester used in the present invention include polylactic acid, polyglycolic acid, polycaprolactone, polybutylene succinate, polyethylene succinate, and copolymers thereof. Among these, as the aliphatic polyester, polylactic acid, polyglycolic acid, lactic acid-glycolic acid copolymer, polycaprolactone and lactic acid-caprolactone copolymer are preferable, and polylactic acid and polycaprolactone are particularly preferable.
The weight average molecular weight of the aliphatic polyester is preferably 20,000 to 1,000,000. More preferably, the weight average molecular weight is 50,000 to 500,000.
The phospholipid used in the present invention can be used regardless of its origin, whether it is extracted from animal tissue or artificially synthesized. As the phospholipid, it is desirable to use one selected from the group consisting of phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylglycerol and derivatives thereof. Preferred is phosphatidyl ethanolamine or phosphatidyl choline. More preferred is L-α-phosphatidylethanolamine dioleoyl, or L-α-phosphatidylcholine dioleoyl.
The fibrous structure used in the present invention refers to a three-dimensional structure formed by laminating one kind or plural kinds of fibers, weaving, knitting or other methods. It also includes the collected yarns. Preferred specific examples of the fiber structure include non-woven fabric, woven fabric, knitted fabric, tube and mesh. A more preferable form is a non-woven fabric.
It also includes a complex of fibers containing phospholipids and fibers not containing phospholipids.
The average apparent density of the fibrous structure is preferably 10 to 350 kg/m ³ . Here, the average apparent density means the density calculated from the area, average thickness, and mass of the fiber structure.
If the average apparent density is more than 350 kg/m ³ , the solution containing nutrients or the like does not sufficiently penetrate into the inside of the fiber structure during cell culture, and thus cells may be cultured only on the surface of the fiber structure. If the average apparent density is less than 10 kg/m ³ , the mechanical strength required during cell culture may not be maintained. A preferable average apparent density is 10 to 250 kg/m ³ .
Examples of the method for producing the fiber structure of the present invention include an electrostatic spinning method, a spun bond method, a melt blow method, a flash spinning method and the like. Among them, the electrostatic spinning method is preferable from the viewpoint of operability and simplicity. The method of manufacturing by the electrostatic spinning method will be described in detail below.
In the electrospinning method used in the present invention, a solution of an aliphatic polyester dissolved in a volatile solvent is discharged into an electrostatic field formed between electrodes, and the solution is spun to form a fibrous fiber. A fibrous structure can be obtained by accumulating substances on the collection substrate. The fibrous substance indicates not only a state in which the solvent of the solution has been completely distilled off to form a fibrous structure, but also a state in which the solvent of the solution is still contained.
First, an apparatus used in the electrostatic spinning method will be described. The electrode used in the present invention may be any metal, inorganic material, or organic material as long as it exhibits conductivity. Further, it may have a thin film of a conductive metal, an inorganic material, or an organic material on the insulator. The electrostatic field in the present invention is formed between a pair of or a plurality of electrodes, and a high voltage may be applied to any of the electrodes. This includes, for example, two high voltage electrodes with different voltage values (for example, 15 kV and 10 kV) and a total of three electrodes connected to the ground, or a case where more than three electrodes are used. Shall be included.
Next, a preferable method for producing the fiber structure of the present invention by the electrostatic spinning method will be described in detail. First there is the step of producing a solution consisting of an aliphatic polyester, a phospholipid and a volatile solvent. The concentration of the aliphatic polyester in the solution in the production method of the present invention is preferably 1 to 30% by weight. If the concentration of the aliphatic polyester is less than 1% by weight, the concentration is too low and it is difficult to form a fiber structure, which is not preferable. On the other hand, if it is more than 30% by weight, the viscosity of the solution increases, so that it is necessary to apply a higher voltage between the electrodes, which is not preferable. The more preferable concentration of the aliphatic polyester is 2 to 25% by weight.
The volatile solvent of the present invention is not particularly limited as long as it can dissolve the aliphatic polyester. As the volatile solvent, for example, water-insoluble methylene chloride, chloroform, a halogen element-containing hydrocarbon such as carbon tetrachloride, and acetone that can dissolve water at an arbitrary ratio, methanol, ethanol, propanol, isopropanol, Toluene, tetrahydrofuran, 1,1,1,3,3,3-hexafluoroisopropanol, water, 1,4-dioxane, cyclohexane, cyclohexanone, N,N-dimethylformamide, acetonitrile and the like can be mentioned. Of these, methylene chloride, chloroform, acetone, and tetrahydrofuran, which have particularly high volatility, are particularly preferable for promoting the porosity of the fiber surface.
These solvents may be used alone or a plurality of solvents may be combined. Further, other solvents may be used in combination as long as the object of the present invention is not impaired.
Next, the step of spinning the solution by electrostatic spinning will be described. Any method can be used to discharge the solution into an electrostatic field. For example, the following description will be given with reference to FIG. 1 as an example. By supplying the solution 2 to the nozzle, the solution is placed at an appropriate position in the electrostatic field, and the solution is spun by the electric field from the nozzle to be fiberized. For this purpose, an appropriate device can be used, for example, an appropriate means at the tip of the cylindrical solution holding tank 3 of the syringe, for example, an injection needle-shaped solution ejection nozzle in which a voltage is applied by the high voltage generator 6. Install 1 and lead the solution to its tip. The tip of the ejection nozzle 1 is arranged at an appropriate distance from the grounded fibrous substance collection electrode 5, and when the solution 2 exits the tip of the ejection nozzle 1, the tip of the ejection nozzle 1 and the fibrous substance collection electrode 5 are separated from each other. To form a fibrous material.
Those skilled in the art will also be able to introduce microdroplets of the solution into the electrostatic field by methods that are obvious. An example will be described below with reference to FIG. The only requirement in that case is to place the droplet in an electrostatic field and keep it away from the fibrous material collecting electrode 11 at a distance where fibrosis can occur. For example, the electrode 10 that directly opposes the fibrous substance collecting electrode 11 may be directly inserted into the solution 8 in the solution holding tank 9 having the nozzle 7.
When the solution is supplied from the nozzle into the electrostatic field, the production rate of the fibrous substance can be increased by using several nozzles. The distance between the electrodes depends on the charge amount, the nozzle size, the spinning solution flow rate, the spinning solution concentration, etc., but a distance of 5 to 20 cm was suitable when it was about 10 kV. The applied electrostatic potential is generally 3 to 100 kV, preferably 5 to 50 kV, and more preferably 5 to 30 kV. The desired potential may be created by any suitable method.
The above description is for the case where the electrode also serves as the collecting substrate, but by providing an object that can serve as the collecting substrate between the electrodes, a collecting substrate is provided separately from the electrodes, and the fiber structure is collected there. Can be done. In this case, for example, a belt-shaped substance is installed between the electrodes and is used as a collection substrate to enable continuous production.
In the present invention, maintaining the relative humidity between the nozzle and the collection substrate at 20% or more is preferable because the fiber having the above-mentioned surface structure can be easily obtained. A more preferable relative humidity is 25 to 95%.
Finally, the step of obtaining a fiber laminate that is accumulated on the collection substrate will be described. In the present invention, the solvent evaporates depending on the conditions to form a fibrous substance while the solution is drawn toward the collection substrate. At normal room temperature, the solvent completely evaporates before being collected on the collecting substrate, but if the solvent evaporation is insufficient, the solvent may be drawn under reduced pressure. The spinning temperature depends on the evaporation behavior of the solvent and the viscosity of the spinning solution, but is usually 0 to 50°C.
The fiber structure of the present invention may be composed of the above fiber structure alone, or may be combined with other members. The cell culture substrate of the present invention may be combined with proteins such as cell growth factors and cell growth factors, extracellular matrix such as collagen, etc., as long as the characteristics thereof are not impaired.

Hereinafter, embodiments of the present invention will be described with reference to examples, but these do not limit the invention of the present invention.
The average porosity was obtained by performing a binarization process on a scanning electron micrograph of the obtained fiber structure at a magnification of 20,000 using image processing software (next New Qube). The pore diameter of the fiber surface was calculated by averaging the diameter of the pores measured at n=20 from a scanning electron micrograph of the obtained fiber structure at a magnification of 20,000.
Example 1
100 parts by weight of polylactic acid (Shimadzu Corporation: trade name "Lacty 9031", weight average molecular weight 168,000) and 0.5 parts by weight of phosphatidylethanolamine dioleoyl (Wako Pure Chemical Industries) are added to methylene chloride (Wako Pure Chemical Industries, special grade) 899. It was dissolved in 0.5 parts by weight at room temperature (29° C.) to prepare a solution. The solution was discharged to the fibrous substance collecting electrode 5 for 15 minutes using the device shown in FIG. The inner diameter of the ejection nozzle 1 was 0.8 mm, the voltage was 12 kV, the distance from the ejection nozzle 1 to the fibrous material collecting electrode 5 was 20 cm, and the relative humidity was 39%. When the obtained fiber structure was measured by a scanning electron microscope (Hitachi S-2400), the average fiber diameter was 10 μm, the average porosity was 36.7%, the average pore size was 0.52 μm, and the apparent density was 209 kg. /M ³ . FIG. 3 shows a scanning electron micrograph.
Example 2
Polylactic acid (Shimadzu: trade name "Lacty 9031", weight average molecular weight 168,000) 100 parts by weight and phosphatidylethanolamine dioleoyl (Wako Pure Chemical Industries) 10 parts by weight methylene chloride (Wako Pure Chemical Industries, special grade) 890 parts by weight Was dissolved at room temperature (26° C.) to prepare a solution. The solution was discharged to the fibrous substance collecting electrode 5 for 15 minutes using the device shown in FIG. The inner diameter of the ejection nozzle 1 was 0.8 mm, the voltage was 12 kV, the distance from the ejection nozzle 1 to the fibrous material collecting electrode 5 was 20 cm, and the relative humidity was 39%. When the obtained fiber structure was measured by a scanning electron microscope (Hitachi S-2400), the average fiber diameter was 10 μm, the average porosity was 66.1%, the average pore size was 0.98 μm, and the apparent density was 212 kg. /M ³ . FIG. 4 shows a scanning electron micrograph.
Example 3
Polylactic acid (Shimadzu: trade name "Lacty 9031", weight average molecular weight 168,000) 100 parts by weight and phosphatidylcholine dioleoyl (Wako Pure Chemical Industries) 0.5 parts by weight are methylene chloride (Wako Pure Chemical Industries, special grade) 899.5. A solution was prepared by dissolving in parts by weight at room temperature (29° C.). The solution was discharged to the fibrous substance collecting electrode 5 for 15 minutes using the device shown in FIG. The inner diameter of the ejection nozzle 1 was 0.8 mm, the voltage was 12 kV, the distance from the ejection nozzle 1 to the fibrous material collecting electrode 5 was 20 cm, and the relative humidity was 39%. When the obtained fiber structure was measured with a scanning electron microscope (Hitachi S-2400), the average fiber diameter was 8 μm, the average porosity was 25.8%, the average pore size was 0.11 μm, and the apparent density was 208 kg. /M ³ . FIG. 5 shows a scanning electron micrograph.
Example 4
100 parts by weight of polylactic acid (Shimadzu: trade name "Lacty 9031", weight average molecular weight 168,000) and 0.5 parts by weight of phosphatidylethanolamine dioleoyl (Wako Pure Chemical Industries) were added to methylene chloride (Wako Pure Chemical Industries, special grade) 449. 0.75 parts by weight and N,N-dimethylformamide 449.75 parts by weight were dissolved at room temperature (29° C.) to prepare a solution. The solution was discharged to the fibrous substance collecting electrode 5 for 15 minutes using the device shown in FIG. The inner diameter of the ejection nozzle 1 was 0.8 mm, the voltage was 12 kV, the distance from the ejection nozzle 1 to the fibrous substance collecting electrode 5 was 10 cm, and the relative humidity was 39%. When the obtained fiber structure was measured with a scanning electron microscope (Hitachi S-2400), the average fiber diameter was 0.7 μm, the average porosity was 3%, the average pore diameter was 0.032 μm, and the apparent density was 210 kg. /M ³ . FIG. 6 shows a scanning electron micrograph.
Example 5
100 parts by weight of polylactic acid (Shimadzu Corporation: trade name "Lacty 9031", weight average molecular weight 168,000) and 0.1 part by weight of phosphatidylethanolamine dioleoyl (Wako Pure Chemical Industries) are added to methylene chloride (Wako Pure Chemical Industries, special grade) 899. It was dissolved in room temperature (29° C.) in 0.9 parts by weight to prepare a solution. The solution was discharged onto the fibrous substance collecting electrode 5 for 13 minutes by using the device shown in FIG. The inner diameter of the ejection nozzle 1 was 0.8 mm, the voltage was 12 kV, the distance from the ejection nozzle 1 to the fibrous substance collection electrode 5 was 20 cm, and the relative humidity was 33%. When the obtained fiber structure was measured with a scanning electron microscope (Hitachi S-2400), the average fiber diameter was 8 μm, the average porosity was 35.4%, the average pore size was 0.63 μm, and the apparent density was 214 kg. /M ³ . FIG. 7 shows a scanning electron micrograph.
Comparative Example 1
10 parts by weight of polylactic acid (Shimadzu Corporation: trade name "Lacty 9031", weight average molecular weight 168,000) was dissolved in 90 parts by weight of methylene chloride (Wako Pure Chemical Industries, special grade) at room temperature (22°C) to prepare a solution. Created. The solution was discharged onto the fibrous substance collecting electrode 5 for 13 minutes by using the device shown in FIG. The inner diameter of the ejection nozzle 1 was 0.8 mm, the voltage was 12 kV, the distance from the ejection nozzle 1 to the fibrous substance collection electrode 5 was 20 cm, and the relative humidity was 36%. When the obtained fiber structure was measured with a scanning electron microscope (Hitachi S-2400), the average fiber diameter was 3 μm, the average porosity was 10.5%, and the apparent density was 215 kg/m ³ . FIG. 8 shows a scanning electron micrograph.
Example 6: Evaluation of cell culture The fibrous structure obtained in Example 1 was cut into a circle having a diameter of 12 mm, immersed in a 70% aqueous ethanol solution (Wako Pure Chemical Industries) for sterilization, air-dried, and then a cell incubator. PAE (porcine aorta-derived vascular endothelial cells) was seeded at 0.25×10 5 cells/ml/well, placed in a 6-well plate, and cultured in an incubator under the conditions of 5% CO 2 and 37° C. Cell proliferation capacity was measured on Alamarblue on days 1 and 4. In the measurement, the fluorescence of 590 nm that appeared using the excitation light of wavelength 530 nm was detected.
The results are shown in Fig. 9.
Comparative example 2
The same operation as in Example 6 was performed except that the fiber structure of Comparative Example 1 was used. The results are shown together in FIG.
As a result of performing a significant difference test using two specimens, in one day after culturing, Example 6 showed a statistically significant difference in adhesiveness and proliferation between the two specimens of Comparative Example 2 and Comparative Example 2 at a risk rate of 0.001%. The result was good. After 4 days of culturing, the results of Example 6 showed that the two specimens of Comparative Example 2 and Adhesiveness and Proliferativeness were good with a statistically significant difference at a risk rate of 0.001%.

Claims (8)

  A fiber structure containing 0.01 to 100 parts by weight of a phospholipid with respect to 100 parts by weight of an aliphatic polyester and having an average fiber diameter of 0.05 to 50 μm.   The fibrous structure according to claim 1, which contains 0.01 to 20 parts by weight of a phospholipid with respect to 100 parts by weight of an aliphatic polyester.   The fiber structure according to claim 1, wherein the average porosity of the fiber surface of the fiber structure is 3% to 90%.   The aliphatic polyester is at least one polymer selected from the group consisting of polylactic acid, polyglycolic acid, polycaprolactone, polybutylene succinate, polyethylene succinate, and copolymers thereof. Fiber structure.   The fibrous structure according to claim 1, wherein the phospholipid is selected from the group consisting of phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, cardiolipin, phosphatidylinositol, sphingomyelin, phosphatidic acid, plasmalogen and derivatives thereof. .   The fibrous structure according to claim 1, wherein the phospholipid is phosphatidylethanolamine or phosphatidylcholine.   The fibrous structure according to claim 6, wherein the phospholipid is L-α-phosphatidylethanolamine dioleoyl or L-α-phosphatidylcholine dioleoyl.   A method comprising: producing a solution of an aliphatic polyester and a phospholipid dissolved in a volatile solvent and spinning the solution by an electrostatic spinning method to obtain a fibrous structure accumulated on a collection substrate. The method for producing a fiber structure according to.

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