JP2017057248A - Composition, medical composition, and process for producing composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To solve a problem that a material in which a silk fibroin is utilized has stiffness and lacks flexibility.SOLUTION: The composition comprises fibroin and a polymeric material different from fibroin, and in which the polymeric material has a decomposition period in the rat abdominal cavity of 4 days or more and 360 days or less and has a Young's modulus of 0.4 MPa or more and 4,000 MPa or less. The polymeric material may comprise at least one of polyethylene carbonate and polydioxanone. The fibroin and the polymeric material may form a phase separation structure having a first phase made of fibroin and a second phase made of polymeric material in the composition.SELECTED DRAWING: Figure 34

Description

  The present invention relates to a composition, a medical composition, and a method for producing the composition.

Silk fibroin contained in silk is a material excellent in tensile strength, biocompatibility, water retention, etc., and research on materials using silk fibroin has been actively conducted (see Patent Publications 1 to 5).
[Prior art documents]
[Patent Literature]
[Patent Literature 1] International Publication No. 2012/090553 [Patent Literature 2] International Publication No. 2013/172202 [Patent Literature 3] Japanese Patent Laid-Open No. 6-345958 [Patent Literature 4] Japanese Patent Laid-Open No. 2012-219100 [Patent Literature] Reference 5] Japanese Patent Publication No. 2006-519664

  Silk fibroin-based materials are stiff and lack flexibility.

  In a first aspect of the invention, a composition is provided. The above composition comprises fibroin. The above composition comprises a polymeric material that is different from fibroin. In the above composition, the polymer material has a degradation period in the rat abdominal cavity of 4 days to 360 days and a Young's modulus of 0.4 MPa to 4,000 MPa.

  In the above composition, the polymer material may include at least one of polyethylene carbonate and polydioxanone. In the above composition, the fibroin and the polymer material may form a phase separation structure having a first phase made of fibroin and a second phase made of the polymer material in the composition. In the above composition, the phase separation structure may be confirmed in an observation range of 100 μm × 100 μm to 2,000 μm × 2,000 μm when the composition is observed with a scanning electron microscope.

  Said composition may contain 1 to 99 mass% polymeric material with respect to the total of fibroin and polymeric material. Said composition may contain 50 to 90 mass% polymeric material with respect to the sum total of a fibroin and a polymeric material. The Young's modulus of the above composition may be 0.5 MPa or more and 300 MPa or less.

  In the 2nd aspect of this invention, the medical composition containing said composition is provided. In the above-described medical composition, the composition may include 40% by mass or more and 80% by mass or less of the polymer material with respect to the total of fibroin and the polymer material. In the medical composition described above, the composition may include 60% by mass or more and 99% by mass or less of the polymer material with respect to the total of fibroin and the polymer material. In the above medical composition, the composition may contain 1% by mass or more and 60% by mass or less of the polymer material with respect to the total of fibroin and the polymer material.

  In a third aspect of the invention, a method for producing a composition comprising fibroin and a polymeric material is provided. The above method includes a step of removing at least a part of the solvent from the mixed solution in which the fibroin and the polymer material are dissolved in the solvent to form a composite material of the fibroin and the polymer material. The above method comprises the step of insolubilizing the composite material using a poor solvent for fibroin. In the above method, the polymer material is a material different from fibroin, the degradation period in the rat abdominal cavity is 4 days or more and 360 days or less, and the Young's modulus is 0.4 MPa or more and 4,000 MPa or less. is there.

  In a fourth aspect of the invention, a method for producing a composition comprising fibroin and a polymeric material is provided. The above method has a step of forming a fibrous or non-woven material by electrospinning using a mixed solution in which fibroin and a polymer material are dissolved in a solvent. Said method has the step of insolubilizing a fibrous or nonwoven material using the poor solvent of fibroin. In the above method, the polymer material is a material different from fibroin, the degradation period in the rat abdominal cavity is 4 days or more and 360 days or less, and the Young's modulus is 0.4 MPa or more and 4,000 MPa or less. is there.

  It should be noted that the above summary of the invention does not enumerate all the necessary features of the present invention. In addition, a sub-combination of these feature groups can also be an invention.

The external appearance of Example 1 is shown. The external appearance of Example 2 is shown. The external appearance of Example 3 is shown. The external appearance of Example 4 is shown. The external appearance of Example 5 is shown. The external appearance of Example 6 is shown. The external appearance of Example 7 is shown. The external appearance of Example 8 is shown. The external appearance of the comparative example 1 is shown. The external appearance of the comparative example 2 is shown. The SEM image of Example 2 is shown. The enlarged image of FIG. 11 is shown. The SEM image of Example 4 is shown. The SEM image of Example 5 is shown. The enlarged image of FIG. 14 is shown. The SEM image of Example 6 is shown. The enlarged image of FIG. 16 is shown. The SEM image of Example 7 is shown. The enlarged image of FIG. 18 is shown. The SEM image of Example 8 is shown. The enlarged image of FIG. 20 is shown. The SEM-EDS analysis of Example 2 is shown. The SEM-EDS analysis of Example 4 is shown. The SEM-EDS analysis of Example 6 is shown. An example of the tensile test result of an Example and a comparative example is shown. An example of the tensile test result of an Example and a comparative example is shown. An example of the tensile test result of an Example and a comparative example is shown. The SEM image of Example 9 is shown. The SEM image of Example 10 is shown. The SEM image of Example 11 is shown. The SEM image of the comparative example 3 is shown. The SEM image of the comparative example 4 is shown. The average fiber diameter of Examples 9-11 and Comparative Examples 3-4 is shown. An example of the tensile test result of an Example and a comparative example is shown. An example of the tensile test result of an Example and a comparative example is shown. An example of the tensile test result of an Example and a comparative example is shown. An example of the transplantation experiment result of the repair patch of Example 12 is shown. An example of the transplantation experiment result of the repair patch of Example 12 is shown. An example of the transplantation experiment result of the repair patch of Example 12 is shown.

  Hereinafter, the present invention will be described through embodiments of the invention, but the following embodiments do not limit the invention according to the claims. In addition, not all the combinations of features described in the embodiments are essential for the solving means of the invention. In the drawings, the same or similar parts are denoted by the same reference numerals, and redundant description may be omitted.

[Composition of composition]
In this embodiment, the composition includes fibroin and a polymeric material. Here, “including fibroin” means not only including fibroin but also including a component derived from fibroin. Similarly, “including a polymer material” means not only including a polymer material but also including a component derived from the polymer material.

(Fibroin)
In this embodiment, fibroin provides excellent tensile strength to the composition. In one embodiment, the fibroin may be silk fibroin derived from natural silk made by silkworms or spiders. The fibroin is preferably silk fibroin derived from silk produced by silkworm (sometimes referred to as silkworm silk). In other embodiments, the fibroin may be derived from a genetically engineered silk protein. Examples of genetically engineered silk proteins include silk proteins produced by bacteria, yeasts, animal and plant cells, transgenic plants, transgenic animals, etc. whose genes have been modified to produce silk proteins. Can do.

  In silkworm silk, fibroin is coated with sericin. Fibroin derived from natural silkworm silk can be obtained by removing sericin from silkworm silk. In one embodiment, the composition may comprise 10-35% by weight of sericin as an impurity relative to the weight of fibroin. In another embodiment, the content of sericin in the composition is preferably less than 20% (mass ratio), more preferably less than 10% (mass ratio) with respect to the mass of fibroin. More preferably, it is less than% (mass ratio).

(Polymer material)
In the present embodiment, the polymer material is a material different from fibroin. The polymer material may be a polymer composed of one type of monomer or a polymer composed of two or more types of monomers. The polymer material may be a material containing two or more kinds of polymers. The polymer material preferably includes a polymer that has been used as a material for medical devices, and more preferably a polymer that has been used as a material for medical devices.

  In this embodiment, the polymer material has a Young's modulus smaller than that of fibroin. The polymer material is a material having a Young's modulus (sometimes referred to as a tensile elastic modulus) of 0.2 MPa or more and 6,000 MPa or less before being combined with fibroin. The Young's modulus of the polymer material is preferably 0.4 MPa or more and 4,000 MPa or less, more preferably 0.5 MPa or more and 400 MPa or less, and further preferably 0.5 MPa or more and 10 MPa or less. Thereby, a composition using fibroin, which is excellent in breaking strength (sometimes referred to as tensile strength) and breaking elongation (sometimes called tensile elongation) is provided. can do. The breaking strength of the composition is measured according to, for example, ASTM-D-882.

  By using a polymer material having a Young's modulus of 6,000 MPa or less in a state before being combined with fibroin, compared with a case where a polymer material having a Young's modulus exceeding 6,000 MPa is used, the polymer material and The difference between the Young's modulus of the composition combined with fibroin and the Young's modulus of living tissue (for example, heart tissue) is reduced. Thereby, the foreign material reaction at the time of arrange | positioning a composition in a biological body can be suppressed. On the other hand, by using a polymer material having a Young's modulus of 0.2 MPa or more in a state before being combined with fibroin, the mechanical strength of the composition in which the polymer material and fibroin are combined can be ensured. In particular, by using a polymer material having a Young's modulus of 0.5 MPa or more in a state before being combined with fibroin, a mechanical strength as required for a medical composition can be obtained.

  In the present embodiment, the Young's modulus of the polymer material is calculated according to ISO 527-1 and JIS K 7161. Specifically, first, a 40 mm × 5 mm test piece of a polymer material to be measured is prepared. Moreover, the thickness of a test piece is measured. The thickness of the test piece may be the thickness at a single location of the test piece, or may be the average value of the thickness at multiple locations. Next, a tensile load of 10 mm / min per minute is applied to the test piece in the atmosphere at 20 ° C., and the test piece is pulled in the long side direction, and sometimes referred to as tensile stress (normal stress). ) And strain (sometimes referred to as elongation).

The tensile stress [MPa] is calculated by dividing the tensile load [N] by the cross-sectional area [mm ² ] of the test piece before starting the test. Said cross-sectional area is the area of the surface which cut | disconnected the sample piece by the surface substantially perpendicular | vertical to the tension direction. The strain [%] is calculated by the following formula 1.
[Formula 1]
Strain [%] = 100 × (L−Lo) / Lo
In Equation 1, Lo is the length of the sample before the start of the test, and L is the length of the sample at the time of the test.

The Young's modulus is calculated as the ratio of tensile stress to strain within the tensile proportional limit (sometimes referred to as the elastic region). In the present embodiment, the Young's modulus is calculated from the slope of the tangent line of the SS curve (sometimes referred to as a stress-strain diagram). The slope of the tangent is calculated from, for example, strain data when the stress is 0.2 N / mm ² and strain data when the stress is 0.6 N / mm ² .

  In the present embodiment, the polymer material is superior in biodegradability compared to fibroin. Thereby, the biodegradability of a composition can be adjusted with the composition ratio of a polymeric material and fibroin. The biodegradability of the material may be assessed by the period of degradation within the rat abdominal cavity. The polymer material may be a material having a degradation period in the rat abdominal cavity of 4 days or more and 360 days or less before being complexed with fibroin. The degradation period of the polymeric material in the rat abdominal cavity is preferably 4 days or more and 200 days or less, more preferably 4 days or more and 120 days or less, and further preferably 4 days or more and 90 days or less. .

  By using a polymer material having a degradation period of 4 days or more, for example, when a composition in which a polymer material and fibroin are combined is used for the repair or regeneration of a living tissue, the living tissue is sufficient. The function of the composition can be maintained until it is regenerated. On the other hand, by using the polymer material having a degradation period of 360 days or less, inhibition of tissue regeneration due to the polymer material remaining in the living body can be suppressed. Moreover, the calcification in a composition can be suppressed.

  In the present embodiment, the degradation period in the rat abdominal cavity is measured by the following procedure. First, a test piece of a polymer material to be measured is prepared. The shape of the test piece is a pellet having a diameter of 20 mm and a thickness of 0.6 mm. Next, the test piece is implanted in the rat abdominal cavity. Thereafter, the specimen is removed at a fixed interval and sacrificed, and the specimen is carefully washed. After the washed test piece is sufficiently dried, the mass of the test piece is measured. Repeat the above operation until the implanted specimen has completely disappeared. By data fitting, a period until the implanted specimen is completely disappeared is estimated, and the estimated value is calculated as a decomposition period.

  In the present embodiment, the molecular weight of the polymer material may be 100,000 or more and 1,000,000 or less. The molecular weight of the polymer material is preferably 150,000 or more and 500,000 or less, more preferably 150,000 or more and 300,000 or less, and further preferably 150,000 or more and 250,000 or less. .

  The polymer material may be a synthetic polymer having biodegradability. The polymer material may include at least one of polyethylene carbonate and polydioxanone. The polymeric material may be polyethylene carbonate or polydioxanone. Polyethylene carbonate and polydioxanone may have a substituted or unsubstituted arbitrary functional group introduced therein. The polymer material may be polyethylene carbonate having an arbitrary functional group introduced into the polymer main chain.

  The Young's modulus of polyethylene carbonate is 11.74 MPa. The degradation period of polyethylene carbonate in the rat abdominal cavity is 16 days. The Young's modulus of polydioxanone is 3,332 MPa. The degradation period of polydioxanone in the rat peritoneal cavity is 240 days. Thereby, it is a composition using fibroin and can provide a composition excellent in breaking strength and breaking elongation.

(Composition ratio of polymer material)
In the present embodiment, the composition preferably contains 1% by mass or more and 99% by mass or less of the polymer material with respect to the total of fibroin and the polymer material. Thereby, compared with a fibroin single-piece | unit, breaking strength and breaking elongation can be improved. In the present embodiment, fibroin is a material excellent in biocompatibility, and the polymer material is superior in biodegradability compared to fibroin. Therefore, the composition of this embodiment is particularly suitable for a medical composition. Show the characteristics. Examples of the medical composition include materials used for sutures, materials used for repair or regeneration of living tissue, materials used for artificial organs, and the like.

  In one embodiment, the composition includes 50% by mass or more and 90% by mass or less of the polymer material with respect to the total of fibroin and the polymer material. The composition may include more than 50% by weight and less than 90% by weight of the polymer material based on the total of fibroin and the polymer material. The composition may contain 55% by mass or more and 85% by mass or less of the polymer material with respect to the total of fibroin and the polymer material, and preferably contains 60% by mass or more and 80% by mass or less of the polymer material. More preferably, the polymer material is contained in an amount of 65% by mass or more and 75% by mass or less, and more preferably 67% by mass or more and 73% by mass or less. Thereby, the breaking strength and breaking elongation of a composition improve. In particular, when the polymer material is polyethylene carbonate, a composition having significantly improved breaking strength and breaking elongation can be obtained as compared to fibroin alone and polycarbonate alone.

  In another embodiment, the composition includes 1% by mass or more and 60% by mass or less of the polymer material based on the total of fibroin and the polymer material. The composition may include 1% by mass or more and 50% by mass or less of the polymer material, or 10% by mass or more and 40% by mass or less of the polymer material with respect to the total of fibroin and the polymer material. Thereby, the material excellent in intensity | strength can be provided. The strength of the composition can be evaluated by, for example, the maximum tensile stress. The composition of the present embodiment exhibits properties particularly suitable for a medical composition used for a medical device related to hard tissue. Examples of the hard tissue include elastic cartilage such as femur, rib, nerve, spine and outer ear, hyaline cartilage and the like.

  The specific value of the maximum tensile stress is adjusted according to the hard tissue to which the composition is applied. For example, if the hard tissue to which the composition is applied is the femur, the maximum tensile stress is about 162 MPa. When the hard tissue is a rib, the maximum tensile stress is about 54 MPa. When the hard tissue is a nerve, the maximum tensile stress is about 13 MPa. When the hard tissue is the spine, the maximum tensile stress is about 3.5 MPa. When the hard tissue is an elastic cartilage such as the outer ear, the maximum tensile stress is about 3.1 MPa. When the hard tissue is hyaline cartilage, the maximum tensile stress is about 2.9 MPa. The maximum tensile stress of the composition can be controlled by adjusting the composition ratio of fibroin and polymer material in the composition, the material of the polymer material, the molecular weight of the polymer material, and the like.

  In another embodiment, the composition comprises 40% by weight or more and 80% by weight or less of the polymer material with respect to the total of fibroin and the polymer material. The composition may include 45% by mass or more and 75% by mass or less of the polymer material, or 50% by mass or more and 70% by mass or less of the polymer material with respect to the total of fibroin and the polymer material. Thereby, the material excellent in breaking strength and breaking elongation can be provided. The composition of the present embodiment exhibits properties particularly suitable for a medical composition used for a medical device related to a circulatory system tissue. Examples of medical devices related to circulatory system tissues include heart repair patches, artificial heart valves, vascular repair patches, artificial blood vessels, tubes, and catheters.

  For example, a conventional medical composition containing silk fibroin and a polyurethane resin is excellent in strength but poor in flexibility. Therefore, it has been difficult to use it as a medical device material that requires flexibility, such as a heart repair patch, an artificial heart valve, a blood vessel repair patch, and an artificial blood vessel. Further, when the material and the organ are anastomosed or sutured, there is a problem that the amount of blood leakage from the patch is large and the amount of bleeding from the passage hole of the suture thread is large.

However, in one embodiment, the composition of this embodiment exhibits a Young's modulus of 1 MPa or less in 37 ° C. water. In another embodiment, the composition of this embodiment exhibits a storage modulus of 1 MPa or less in 37 ° C. water. Thereby, compared with the case where the patch of a fibroin single-piece | unit or the patch which consists of a composite material of a silk fibroin and a polyurethane resin is utilized, the amount of bleeding from the passage hole of a suture can be suppressed. Furthermore, the amount of blood leakage from the patch can be suppressed to 1 ml / cm ⁻² · min. In addition, although the composition according to the present embodiment has biodegradability, the degradation rate in the living body is smaller than the repair rate of the living tissue. Therefore, after the composition of this embodiment is transplanted in the living body, it gradually replaces the living tissue over a period of about one year.

  In another embodiment, the composition includes 60% by mass or more and 99% by mass or less of the polymer material with respect to the total of fibroin and the polymer material. The composition may include 65% by mass or more and 90% by mass or less of the polymer material, or 70% by mass or more and 80% by mass or less of the polymer material with respect to the total of fibroin and the polymer material. The composition may include 70% by mass or more and 99% by mass or less of the polymer material with respect to the total of fibroin and the polymer material. Thereby, the material excellent in elasticity can be provided. The composition of the present embodiment exhibits properties particularly suitable for a medical composition used for a medical device related to a membrane tissue. Examples of medical devices related to the membrane tissue include artificial pericardium, angiogenic pericardium sheet, artificial dura mater, artificial skin, digestive organ substitute material having a lumen structure such as large intestine and small intestine.

(Other ingredients)
In this embodiment, the composition may contain inevitable impurities resulting from raw materials or manufacturing processes. The composition may contain various additives as long as the properties of the composition are not impaired. An additive may be used independently and may be used in combination of 2 or more type. When an additive is added, the lower limit value of the additive amount is preferably 0.01% by mass and more preferably 0.05% by mass with respect to the total of fibroin and the polymer material. Preferably, it is 0.1 mass%. On the other hand, the upper limit of the additive amount is preferably 10% by mass, preferably 5% by mass, and more preferably 1% by mass with respect to the total of fibroin and the polymer material. .

  Additives include heat stabilizers, light stabilizers, colorants, inorganic fillers, flame retardants, compatibilizers, surfactants, lubricants, mold release agents, UV absorbers, UV inhibitors, antioxidants, Examples thereof include an anti-sticking agent and an antistatic agent. The additive is preferably a compound that has been used in medical devices.

  Examples of the heat stabilizer include phosphorus compounds, phenol derivatives, compounds containing sulfur, tin compounds, and the like. Phosphorus compounds include phosphoric acid, phosphorous acid aliphatic, aromatic or alkyl group-substituted aromatic esters and hypophosphorous acid derivatives, phenylphosphonic acid, phenylphosphinic acid, diphenylphosphonic acid, polyphosphonate, dialkylpentaerythritol diphosphite Moreover, dialkyl bisphenol A diphosphite etc. can be illustrated. Examples of the phenol-based derivatives include hindered phenol compounds such as Irganox 1010 (trade name: manufactured by Ciba Geigy) and Irganox 1520 (trade name: manufactured by Ciba Geigy).

  Examples of sulfur-containing compounds include thioether, dithioate, mercaptobenzimidazole, thiocarbanilide, and thiodipropionic acid ester compounds. Examples of thioether compounds include dilauryl thiopropionate (DLTP) and distearyl thiopropionate (DSTP). Examples of the tin compound include tin malate and dibutyltin monoxide.

  Examples of the light stabilizer include benzotriazole compounds and benzophenone compounds. Specifically, “TINUVIN 622LD”, “TINUVIN 765” (manufactured by Ciba Specialty Chemicals), “SANOL LS-2626” and “SANOL LS-765” (manufactured by Sankyo) are used. Good.

  Examples of the colorant include dyes, inorganic pigments, and organic pigments. Examples of the dye include direct dyes, acid dyes, basic dyes, and metal complex dyes. Examples of inorganic pigments include carbon black, titanium oxide, zinc oxide, iron oxide, and mica. Examples of organic pigments include coupling azo, condensed azo, anthraquinone, thioindigo, dioxazone, and phthalocyanine compounds.

  Examples of the inorganic filler include short glass fibers, carbon fibers, alumina, talc, graphite, melamine, and clay. Examples of the flame retardant include phosphorus and halogen-containing organic compounds, bromine or chlorine-containing organic compounds, ammonium polyphosphate, aluminum hydroxide, and antimony oxide.

[Physical properties of the composition]
In the present embodiment, the composition has a Young's modulus of 0.4 MPa to 4,000 MPa. The Young's modulus of the composition is preferably from 0.5 MPa to 1,000 MPa, more preferably from 0.5 MPa to 400 MPa, and even more preferably from 0.5 MPa to 10 MPa.

  In the present embodiment, the composition has a breaking strength of 0.1 MPa or more and 100 MPa or less. The breaking strength of the composition is preferably 0.3 MPa or more and 20 MPa or less, more preferably 0.3 MPa or more and 10 MPa or less, and further preferably 0.3 MPa or more and 5 MPa or less. The breaking strength of the composition is measured according to ASTM-D-882.

  In this embodiment, the composition has a breaking elongation of 10% or more and 2,000% or less. The elongation at break of the composition is preferably 75% or more and 2,000% or less, more preferably 154% or more and 1,500% or less, and further preferably 154% or more and 1,400% or less. preferable. The breaking strength of the composition is measured according to ASTM-D-882.

  In the present embodiment, the lower limit of the decomposition period of the composition in the rat abdominal cavity is, for example, 30 days. The lower limit may be 50 days, 60 days, or 90 days. The upper limit of the degradation period of the composition in the rat abdominal cavity is, for example, 480 days. The upper limit value may be 360 days, 240 days, or 120 days.

[Shape and structure of composition]
Although the shape of a composition is not specifically limited, Shapes, such as a thread form, a fiber form, a cloth form, a nonwoven fabric form, a film | membrane form, a gel form, sponge form, a particle form, can be illustrated. In the present embodiment, the fibroin and the polymer material may be referred to as a phase separation structure (sea island structure) having a first phase made of fibroin and a second phase made of the polymer material in the composition. ) May be formed. In one embodiment, the first phase composed of fibroin is an isolated phase (sometimes referred to as an island structure) and the second phase composed of a polymer material is sometimes referred to as a continuous phase (sometimes referred to as a sea structure). .) In another embodiment, the first phase composed of fibroin is a continuous phase and the second phase composed of a polymer material is an isolated phase. A plurality of isolated phases may be dispersed in the continuous phase. The shape of each isolated phase may be spherical or approximately spherical.

  In one embodiment, for example, when the composition is formed into a film, a sea-island structure composed of an aggregate of a polymer material and fibroin is, for example, a scanning electron microscope (sometimes referred to as SEM). Is observed within an observation range of 1 μm × 1 μm to 2,000 μm × 2,000 μm. The sea-island structure composed of a polymer material and fibroin agglomerates is preferably confirmed in an observation range of 5 μm × 5 μm to 1,000 μm × 1,000 μm, and confirmed in an observation range of 50 μm × 50 μm to 500 μm × 500 μm More preferably, it can be confirmed in an observation range of 100 μm × 100 μm to 300 μm × 300 μm.

  In another embodiment, when the composition is formed into a nonwoven fabric, the composite of the polymer material and fibroin may have compatibility before and after the combination. The presence or absence of compatibility is a loss factor that is a ratio (G ″ / G ′) of loss elastic modulus (sometimes referred to as G ″) to storage elastic modulus (sometimes referred to as G ′). You may evaluate using (it may be called tan-delta). Storage elastic modulus and loss elastic modulus can be measured using a dynamic viscoelastic device.

  The presence or absence of compatibility may be evaluated by calculating the temperature at which the loss coefficient is maximized (sometimes referred to as glass transition temperature Tg) from the above measurement results. The presence or absence of compatibility is, for example, the temperature at which the loss factor of the polymer material (before complexing) is maximized and the temperature at which the loss factor of the composition (in which the polymer material is complexed with fibroin) is maximized. The difference is evaluated. If the difference between the temperature at which the loss factor of the polymer material is maximized and the temperature at which the loss factor of the composition is maximized is 1 ° C. or more and 30 ° C. or less, it may be determined that the polymer material is compatible. The temperature difference is preferably 1 ° C. or higher and 20 ° C. or lower, and more preferably 1.5 ° C. or higher and 10 ° C. or lower.

[Method for producing composition]
In this embodiment, the manufacturing method of a composition is not specifically limited. In one embodiment, the composition comprises removing at least a portion of the solvent from the mixed solution of the fibroin and the polymeric material in the solvent to form a composite material of fibroin and the polymeric material; Using a poor solvent to insolubilize the composite material. Examples of the poor solvent for fibroin include lower alcohols such as methanol.

  The shape of the composite material is determined by a molding method in the step of forming a composite material of fibroin and a polymer material. In one embodiment, a film-like composite material is obtained by evaporating the solvent in a state where a mixed solution in which fibroin and the polymer material are dissolved in the solvent is applied onto the substrate. In another embodiment, a sponge-like composite material is obtained by evaporating the solvent while the mixed solution is placed in a container. In still another embodiment, a fibrous or non-woven composite material can be obtained by applying a known technique such as an electrospinning method using a mixed solution in which fibroin and a polymer material are dissolved in a solvent. .

Hereinafter, the present invention will be described more specifically with reference to examples and comparative examples. It should be noted that the present invention is not limited to the following examples without departing from the gist thereof.
[Example 1]
A film-like composition was prepared by the following procedure. First, after adding 0.1200 g of silk fibroin (may be described as SF) to 2.2638 g of hexafluoroisopropanol (which may be expressed as HFIP), conditions of 22 ° C. and stirring speed of 100 rpm Under stirring for 2 hours, an SF solution was obtained. As silk fibroin, silk sericin was removed by spinning and scouring cocoons harvested at the Field Science Center, Tokyo University of Agriculture and Technology. Next, 0.4795 g of polyethylene carbonate (may be referred to as PEC) (QPAC25 poly (ethylene carbonate), manufactured by EMPOWER MATERIALS Co., Ltd.) was added to 19.404 g of HFIP. The mixture was stirred for 2 hours under the above conditions to obtain a PEC solution.

Next, the SF solution and the PEC solution were mixed and stirred for 24 hours under conditions of 22 ° C. and a stirring speed of 100 rpm to obtain a mixed solution of SF and PEC. Next, a mixed solution of 13.4 cm ³ was injected into a PFA petri dish having a diameter of 5.0 cm. The PFA petri dish containing the mixed solution was allowed to stand in a reduced-pressure atmosphere at room temperature (20 to 22 ° C.) for 13.5 hours, and the mixed solution was dried under reduced pressure. Furthermore, the PFA petri dish containing the mixed solution was allowed to stand in a vacuum atmosphere at room temperature (20 to 22 ° C.) for 24 hours, and the mixed solution was vacuum dried. Thereby, a film-like composite material of SF and PEC was obtained.

  Next, the film-like composite material was allowed to stand for 24 hours in an environment of 37 ° C. and a relative humidity of 100%, and SF was insolubilized. Thereby, the composition which is SF: PEC = 8: 2 (mass ratio) was obtained. The thickness of the composition was 297 μm at the thinnest place and 373 μm at the thickest place. A photograph of the composition is shown in FIG.

[Examples 2 to 8]
Except having changed the addition amount of PEC and changing the ratio of SF: PEC in a composition, it carried out similarly to Example 1, and obtained the composition of Examples 2-8. SF: PEC in each Example is 7: 3 (Example 2), 6: 4 (Example 3), 5: 5 (Example 4), 4: 6 (Example 5), and 3: 7 (implemented). Example 6), 2: 8 (Example 7), and 1: 9 (Example 8). The average thickness of each composition was about 150 μm to 250 μm. The photograph of the composition of Examples 2-8 is shown to FIGS.

[Comparative Examples 1 and 2]
A composition of Comparative Example 1 was obtained in the same manner as in Example 1 except that PEC was not added. A composition of Comparative Example 2 was obtained in the same manner as in Example 1 except that SF was not added. Photographs of the compositions of Comparative Examples 1 and 2 are shown in FIGS. 9 and 10.

[Evaluation method]
Examples and comparative examples were evaluated by (1) SEM observation, (2) SEM-EDS (Energy Dispersive X-ray Spectroscopy) analysis, and (3) tensile test.

(1) SEM observation The compositions of Examples 2, 4, 5, 6, 7, and 8 were observed using JSM-6510 manufactured by JEOL. In each of the examples, the sample piece of the composition was not sliced and inserted into the sample chamber in a non-deposited state. The acceleration voltage was set to 15 kV. The SEM image of Example 2 is shown in FIG. An enlarged image of FIG. 11 is shown in FIG. The SEM image of Example 4 is shown in FIG. The SEM image of Example 5 is shown in FIG. The enlarged image of FIG. 14 is shown in FIG. The SEM image of Example 6 is shown in FIG. An enlarged image of FIG. 16 is shown in FIG. The SEM image of Example 7 is shown in FIG. The enlarged image of FIG. 18 is shown in FIG. The SEM image of Example 8 is shown in FIG. An enlarged image of FIG. 20 is shown in FIG. From FIG. 11 to FIG. 21, it can be seen that a phase separation structure in which isolated phases are dispersed is formed in the continuous phase.

(2) SEM-EDS analysis The SEM-EDS analysis was implemented about the composition of Example 2, 4, and 6 in order to investigate the composition of each phase of a phase-separation structure. The SEM-EDS analysis result of the composition of Example 2 is shown in FIG. The SEM-EDS analysis result of the composition of Example 4 is shown in FIG. The SEM-EDS analysis result of the composition of Example 6 is shown in FIG. 22 to 24, the upper left drawing shows the SEM image, the upper right drawing shows the carbon (C) distribution in the region indicated by the SEM image, and the lower left drawing shows the SEM image. The distribution of nitrogen (N) in the indicated region is shown, and the lower right drawing shows the distribution of oxygen (O) in the region shown in the SEM image.

  Since silk fibroin contains nitrogen and polyethylene carbonate does not contain nitrogen, the main component of the isolated phase and the continuous phase can be determined by comparing the distribution of nitrogen and the SEM image. When the nitrogen distribution and the SEM image are compared, the distribution of the isolated phase and the nitrogen distribution agree well. Therefore, it can be seen that the main component of the isolated phase is silk fibroin and the main component of the continuous phase is polyethylene carbonate.

(3) Tensile test Tensile tests were performed on the compositions of Examples 2, 4 and 6 and the compositions of Comparative Examples 1 and 2, respectively. The tensile test was performed according to the following procedure. In addition, the composition of 5 mm x 40 mm was cut out from the center part of each composition, and was used as a sample for a tensile test. The sample for the tensile test was performed using the central portion of the film. For the tensile test, EZ-Test / CE manufactured by Shimadzu Corporation was used. The displacement speed was set to 10 mm / min. Moreover, the measurement was implemented at room temperature (20.4 degreeC).

  FIG. 25 shows a stress-strain curve obtained by the tensile test. FIG. 26 shows an enlarged part of FIG. In FIG. 25 and FIG. 26, a curve 2512 shows the stress-strain curve of Example 2. A curve 2514 shows the stress-strain curve of Example 4. Curve 2516 shows the stress-strain curve of Example 6. A curve 2522 shows the stress-strain curve of Comparative Example 1. A curve 2524 shows the stress-strain curve of Comparative Example 2.

  FIG. 27 shows changes in the breaking stress and the breaking strain obtained by the tensile test depending on the PEC ratio. In FIG. 27, a broken line 2710 indicates the relationship between the ratio [% by mass] of PEC in the composition and the breaking stress [MPa] of the composition. A polygonal line 2720 indicates the relationship between the proportion [% by mass] of PEC in the composition and the breaking strain [−] of the composition.

  From FIG. 25 to 27, it can be seen that the flexibility of the composition is improved by adding PEC to SF. Moreover, when the ratio of PEC in a composition exceeds 30%, it turns out that the softness | flexibility of a composition improves rapidly. In particular, when the proportion of PEC in the composition is 50% or more, it can be seen that a composition having superior flexibility was obtained as compared with the case of using SF alone.

[Example 9]
A non-woven composition was prepared by the following procedure. First, in 6.0 ml of hexafluoroisopropanol (sometimes referred to as HFIP), 252 mg silk fibroin sponge and 108 mg polyethylene carbonate (sometimes referred to as PEC) (QPAC25 pellet foam, EMPOWER MATERIALS Co., Ltd.) And a mixture solution of SF and PEC was obtained by stirring at room temperature (22 to 27 ° C.) at a stirring rate of about 100 rpm for 2 to 3 hours. The silk fibroin sponge used was one obtained by spinning and scouring cocoons harvested at the Field Science Center, Tokyo University of Agriculture and Technology to remove sericin.

  Next, a non-woven composite material was produced using the above mixed solution by using Esprayer ES-2000S2A manufactured by Fusion. Electrospinning was performed under the conditions of a temperature of 29 to 32 ° C., a relative humidity of 28 to 34%, a solution discharge speed of 15 μL / min, an applied voltage of 22 kV, a syringe-target distance of 10 cm, and a spinning time of 2 hours.

  Next, the non-woven composite material was allowed to stand for 24 hours in an environment of 37 ° C. and relative humidity of 100%, and SF insolubilization treatment was performed. Thereafter, the non-woven composite material was immersed in purified water for 72 hours to remove the residual solvent. Thereby, the composition which is SF: PEC = 7: 3 (mass ratio) was obtained. The prepared nonwoven composition was put in a sterilization bag and sterilized at 121 ° C. for 20 minutes to obtain a heart repair patch. An SEM image of the nonwoven fabric composition is shown in FIG. The average fiber diameter [μm] is shown in FIG. The average fiber diameter was calculated from the SEM image using image analysis software ImageJ.

[Examples 10 and 11]
The compositions of Examples 10 and 11 were obtained in the same manner as in Example 9, except that the ratio of SF: PEC in the composition was changed by changing the amount of PEC added. SF: PEC in each Example was 5: 5 (Example 10) and 3: 7 (Example 11). SEM images of the compositions of Examples 10 and 11 are shown in FIGS. The average fiber diameter [μm] is shown in FIG.

[Comparative Examples 3 and 4]
A composition of Comparative Example 3 was obtained in the same manner as in Example 9 except that PEC was not added. A composition of Comparative Example 4 was obtained in the same manner as in Example 9 except that SF was not added. SEM images of the compositions of Comparative Examples 3 and 4 are shown in FIGS. 31 and 32. The average fiber diameter [μm] is shown in FIG.

[Evaluation method]
Examples 9 to 11 and Comparative Example 3 were evaluated by a tensile test. FIG. 34 shows the stress-strain curve obtained by the tensile test. The tensile test regarding the stress-strain curve was carried out based on the same procedure as in Examples 2, 4 and 6, and Comparative Examples 1 and 2, except that it was measured in water at 37 ° C. In FIG. 34, a curve 3412 shows the stress-strain curve of Example 9. Curve 3414 shows the stress-strain curve of Example 10. Curve 3416 shows the stress-strain curve of Example 11. A curve 3422 shows the stress-strain curve of Comparative Example 3.

  FIG. 35 shows the tensile strength [MPa] obtained by the tensile test. The tensile test regarding tensile strength was carried out using EZ-Test / CE manufactured by Shimadzu Corporation. The displacement speed was set to 10 mm / min. The measurement was carried out in 37 ° C. water. Samples were prepared based on the same procedures as in Examples 2, 4 and 6, and Comparative Examples 1 and 2. The tensile test was carried out under the above conditions, and the maximum value of the nominal stress reached up to the break, that is, the value obtained by dividing the maximum load by the cross-sectional area before loading was taken as the tensile strength.

FIG. 36 shows the elongation at break [%] obtained by the tensile test. The tensile test regarding elongation at break was carried out using a model made by Shimadzu Corporation: EZ-Test / CE. The displacement speed was set to 10 mm / min. The measurement was carried out in 37 ° C. water. Samples were prepared based on the same procedures as in Examples 2, 4 and 6, and Comparative Examples 1 and 2. A tensile test was carried out under the above conditions, and the strain reached up to the break was defined as the elongation at break. The elongation at break was calculated by the following formula 2.
[Formula 2]
Elongation at break [%] = 100 × (L-Lo) / Lo
In Equation 2, Lo is the length of the sample before the start of the test, and L is the length of the sample at the time of the test.

  34 to 36 show that the flexibility of the composition is improved by adding PEC to SF. Moreover, when the ratio of PEC in a composition exceeds 50%, it turns out that the softness | flexibility of a composition improves rapidly.

[Example 12]
The composition obtained in Example 9 was implanted in the posterior vena cava of a beagle dog as a heart repair patch. A photograph of the heart repair patch immediately after implantation is shown in FIG. Specifically, a non-woven patch (5 cm × 5 cm) of the composition produced in Example 9 was cut into an elliptical shape having a major axis of 2.5 cm and a minor axis of 1.0 cm to obtain a heart repair patch 3710. . A midline incision was made in the posterior vena cava of the beagle dog over a length of about 1 cm, and the prepared cardiac repair patch 3710 was implanted. After a period of time, the beagle dog was sacrificed and the implanted portion of the heart repair patch 3710 was collected. The collected implants were evaluated with the naked eye and histologically.

[Comparative Example 5]
A commercially available ePTFE heart repair patch (manufactured by W. El Gore & Associates (USA), Gore-Tex EPTF Patch 35273100) was implanted in the posterior vena cava of Beagle dogs. The above-mentioned heart repair patch can be obtained from Japan Gore Co., Ltd. in Japan. The implantation and evaluation of the cardiac repair patch was performed in the same manner as in Example 12.

[Evaluation method]
Example 12 and Comparative Example 5 were evaluated based on (1) the amount of blood leakage from the patch and the amount of bleeding from the suture hole, and (2) biocompatibility.

(1) Amount of blood leakage from patch and amount of bleeding from passage hole of suture The amount of blood leakage from the patch itself during surgery and the amount of bleeding from the passage hole of suture were visually confirmed. Compared with the repair patch of Comparative Example 5, the repair patch of Example 12 had less blood leakage from the patch itself and less bleeding from the suture passage hole.

(2) Biocompatibility Hematoxylin and eosin staining of a cross-section of a cardiac repair patch implant taken along the longitudinal axis of the posterior vena cava extracted from a beagle dog sacrificed 98 days after implantation ( An HE staining image is shown in FIG. A hematoxylin and eosin stained (HE stained) image of a cross-section of a heart repair patch implant taken along the longitudinal axis of the posterior vena cava extracted from a beagle dog sacrificed on day 203 after implantation 39.

  38 and 39, it can be seen that the patch 3810 of Example 12 is gradually decomposed over time and replaced with living tissue. According to FIG. 38, patch 3810 is coated with thick collagen fibers 3820. According to FIG. 39, the patch 3910 was split and several fragmented patches 3910 were observed. It can be seen that the fragmented patch 3910 is rounded to erode. Around the patch, macrophage cell layers containing giant cells were observed in several places, and active degradation and absorption of the patch at the interface were observed. From the above, the biodegradability of the patch could be confirmed.

[Compatibility evaluation by dynamic viscoelasticity measurement]
The dynamic viscoelasticity of the compositions of Example 4 and Example 9 was measured to evaluate the compatibility of the compositions of each Example. The dynamic viscoelasticity of the composition of each Example was measured using DVA-225 of IT Measurement Control Co., Ltd. A test piece of 5 mm × 2.5 mm was cut out from the center of the sample of each example. The test piece was fixed in the sample chamber of DVA-225, and the dynamic viscoelasticity was measured in the tensile mode. The measurement temperature range was set to −100 to 200 ° C., and the rate of temperature increase was set to 2 ° C./min. As a reference example for evaluating the compatibility, the same measurement was performed on a test piece cut out from the data of Comparative Example 4.

  For each of the reference example (PEC), Example 4 (SF: PEC = 5: 5), and Example 9 (SF: PEC = 7: 3), the loss factor is maximal from the change of the loss factor accompanying the temperature change. The temperature to be determined. The difference between the temperature of the reference example and the temperature of Example 4 was 0.7 ° C. Therefore, it can be seen that the composition of Example 4 is not in a compatible state and a phase separation structure is formed. On the other hand, the difference between the temperature of the reference example and the temperature of Example 9 was 3.7 ° C. Therefore, it can be seen that the composition of Example 9 is in a compatible state.

  As described above, the composition of the present embodiment includes fibroin and a polymer material having elasticity and biodegradability. Thereby, the fibroin containing composition excellent in the softness | flexibility can be provided. Moreover, the composition excellent in breaking strength and breaking elongation can be provided.

  As mentioned above, although this invention was demonstrated using embodiment, the technical scope of this invention is not limited to the range as described in the said embodiment. It will be apparent to those skilled in the art that various modifications or improvements can be added to the above-described embodiment. In addition, the matters described in the specific embodiment can be applied to other embodiments within a technically consistent range. It is apparent from the scope of the claims that the embodiments added with such changes or improvements can be included in the technical scope of the present invention.

  The execution order of each process such as operations, procedures, steps, and stages in the materials shown in the claims, the description, and the drawings and the manufacturing method thereof is particularly “Before”, “Before”, etc. It should be noted that it is not specified and can be realized in any order unless the product of the previous process is used in the subsequent process. Even though the operations in the claims, the description, and the drawings are described using “first,” “next,” etc. for the sake of convenience, this means that it is essential to carry out the operations in this order. is not.

  2512 curve, 2514 curve, 2516 curve, 2522 curve, 2524 curve, 2710 line, 2720 line, 3412 curve, 3414 curve, 3416 curve, 3422 curve, 3710 heart repair patch, 3810 patch, 3820 collagen fiber, 3910 patch

Claims (12)

With fibroin,
A polymer material different from the fibroin;
Including
The polymer material is
The degradation period in the rat abdominal cavity is 4 days or more and 360 days or less,
Young's modulus is 0.4 MPa or more and 4,000 MPa or less,
Composition. The polymer material includes at least one of polyethylene carbonate and polydioxanone.
The composition of claim 1. The fibroin and the polymer material form a phase separation structure having a first phase composed of the fibroin and a second phase composed of the polymer material in the composition.
The composition according to claim 1 or claim 2. The phase separation structure is confirmed in an observation range of 1 μm × 1 μm to 2,000 μm × 2,000 μm when the composition is observed with a scanning electron microscope.
The composition according to claim 3. 1% by mass or more and 99% by mass or less of the polymer material with respect to the total of the fibroin and the polymer material,
The composition according to any one of claims 1 to 4. 50% by mass or more and 90% by mass or less of the polymer material with respect to the total of the fibroin and the polymer material,
The composition according to any one of claims 1 to 5. The Young's modulus of the composition is 0.5 MPa or more and 300 MPa or less,
The composition according to any one of claims 1 to 6. Comprising the composition according to any one of claims 1 to 5;
The composition includes 40% by mass or more and 80% by mass or less of the polymer material with respect to the total of the fibroin and the polymer material.
Medical composition. Comprising the composition according to any one of claims 1 to 5;
The composition includes 60% by mass or more and 99% by mass or less of the polymer material with respect to the total of the fibroin and the polymer material.
Medical composition. Comprising the composition according to any one of claims 1 to 5;
The composition includes 1% by mass or more and 60% by mass or less of the polymer material with respect to the total of the fibroin and the polymer material.
Medical composition. Removing at least a portion of the solvent from a mixed solution in which fibroin and the polymer material are dissolved in a solvent to form a composite material of the fibroin and the polymer material;
Utilizing a poor solvent for fibroin to insolubilize the composite material;
Having
A method for producing a composition comprising the fibroin and the polymer material,
The polymer material is
A material different from the fibroin,
The degradation period in the rat abdominal cavity is 4 days or more and 360 days or less,
Young's modulus is 0.4 MPa or more and 4,000 MPa or less,
Method. Forming a fibrous or non-woven material by electrospinning using a mixed solution of fibroin and polymer material dissolved in a solvent;
Utilizing a poor solvent for fibroin to insolubilize the fibrous or non-woven material;
Having
A method for producing a composition comprising the fibroin and the polymer material,
The polymer material is
A material different from the fibroin,
The degradation period in the rat abdominal cavity is 4 days or more and 360 days or less,
Young's modulus is 0.4 MPa or more and 4,000 MPa or less,
Method.