JP6402984B2 - Transparent fibroin nanofiber nonwoven fabric, cell culture substrate, cell sheet, and method for producing transparent fibroin nanofiber nonwoven fabric - Google Patents

Description

  The present invention relates to a transparent fibroin nanofiber nonwoven fabric, a cell culture substrate, a cell sheet, and a method for producing a transparent fibroin nanofiber nonwoven fabric.

The silk thread discharged by a silkworm is composed of a pair of fibers made of a protein called fibroin and a protein called sericin, which is a glue component that bundles them together.
Silk fibroin is processed into various shapes such as cast film, hydrogel, sponge, and fiber nonwoven fabric. The fibroin fiber can be made into an aqueous fibroin solution by dissolving in a lithium bromide solution and then dialyzing and desalting.
For example, a fibroin cast film obtained by drying a silk fibroin aqueous solution on a flat plate has extremely high transparency. Moreover, the technique of producing a nanofiber nonwoven fabric from the fibroin aqueous solution is reported (nonpatent literature 1).

  Silk fibroin is widely studied as a tissue regeneration material because of its excellent biocompatibility. The regenerated fibroin material, like silk used as a surgical suture, has biocompatibility without causing an immune response in the human body, and has excellent mechanical properties such as high tensile strength, It is known that it is a material having biodegradability that is decomposed and absorbed in the body.

Hyoung-Joon Jin, Sergey V. Fridrikh, Gregory C. Rutledge, and David L. Kaplan. Electrospinning Bombyx mori Silk with Poly (ethylene oxide), Biomacromolecules, 3, 1233-1239, 2002

Fibroin cast film has extremely high transparency, but has drawbacks such as poor cell adhesion and substance permeability, which may be a problem depending on application conditions. For example, if a fibroin cast film is to be used as a material for regenerating cornea, a poor permeability to the substance is not preferable in a cornea without a vascular system.
A fibroin nanofiber nonwoven fabric produced from an aqueous fibroin solution has good material permeability, but there is a problem that the transparency is remarkably low depending on application conditions. For example, when a fibroin nanofiber nonwoven fabric is used as a material for regenerating cornea, improvement of low transparency becomes an important issue for eyes that are photoreceptor organs.

  The present invention has been made in view of the above circumstances, and has a transparent fibroin nanofiber nonwoven fabric having excellent substance permeability, cell adhesiveness and transparency, a cell culture substrate, a cell sheet, and a transparent An object is to provide a method for producing a fibroin nanofiber nonwoven fabric.

  The present invention provides a transparent fibroin nanofiber nonwoven fabric having the following characteristics, a cell culture substrate, a cell sheet, and a method for producing the transparent fibroin nanofiber nonwoven fabric.

[1] A transparent fibroin nanofiber nonwoven fabric having an apparent transparency of 60% or more obtained by the following test.
(Test method)
In the photograph obtained by immersing the transparent fibroin nanofiber nonwoven fabric in water, (I) the brightness of the background color observed without passing through the transparent fibroin nanofiber nonwoven fabric, and (II) the transparent fibroin nanofiber Transparency is calculated | required by following formula (1) from the ratio of the brightness of the said background color observed through a nonwoven fabric.
Transparency (%) = (I) / (II) × 100 (1)
[2] The average density of the whole transparent fibroin nanofiber nonwoven fabric is 0.05 g / cm ^{3 to} 0.4 g / cm ³ , and the average diameter of the fibroin nanofibers constituting the transparent fibroin nanofiber nonwoven fabric is 1 nm to 1500 nm. The transparent fibroin nanofiber nonwoven fabric according to [1] above.
[3] The transparent fibroin nanofiber nonwoven fabric according to [1] or [2], which has glucose permeability.
[4] The transparent fibroin nanofiber nonwoven fabric according to any one of [1] to [3], which has albumin permeability.
[5] The transparent fibroin nanofiber nonwoven fabric according to [4], wherein the albumin permeability is 0.001 mg / mm ² · min or more at 1 atmosphere.
[6] A cell culture substrate comprising the transparent fibroin nanofiber nonwoven fabric according to any one of [1] to [5].
[7] A cell sheet comprising cells adhered to the transparent fibroin nanofiber nonwoven fabric according to any one of [1] to [5].
[8] The cell sheet according to [7], wherein the cells are cells of corneal tissue.
[9] A transparentization treatment step in which an insolubilized fibroin nanofiber nonwoven fabric in which the fibroin nanofibers are insolubilized is brought into contact with a solution containing a Group 2 element, and drying to dry the insolubilized fibroin nanofiber nonwoven fabric that has undergone the above-mentioned transparency treatment step And a process for producing a transparent fibroin nanofiber nonwoven fabric.
[10] The method for producing a transparent fibroin nanofiber nonwoven fabric according to [9], wherein the Group 2 element is calcium or magnesium.
[11] Prior to the clearing treatment step, the fibroin nanofiber nonwoven fabric is contacted with 95% by volume or more of an alcohol aqueous solution, and then contacted with 60% by volume or more and less than 95% by volume of an alcohol aqueous solution, thereby insolubilized fibroin nanofibers. The method for producing a transparent fibroin nanofiber nonwoven fabric according to the above [9] or [10], which comprises an insolubilization treatment step for obtaining a fiber nonwoven fabric.
[12] A process of spinning fibroin nanofibers using an electrospinning method, and winding the fibroin nanofibers using a rotating collector, and aligning the fibroin nanofibers in the rotating direction of the rotating collector. The method for producing a transparent fibroin nanofiber nonwoven fabric according to [11], comprising a step of obtaining a nonwoven fabric.

  According to the present invention, there is provided a transparent fibroin nanofiber nonwoven fabric having excellent substance permeability, cell adhesion and transparency, a cell culture substrate having the same, a cell sheet, and a method for producing a transparent fibroin nanofiber nonwoven fabric. Can be provided.

It is a schematic diagram explaining the flow of each process of an insolubilization process, a transparentization process, a drying process, and a washing | cleaning in an Example. It is a macroscopic photograph and scanning electron microscope (SEM) image of an oriented fibroin nanofiber nonwoven fabric and a random fibroin nanofiber nonwoven fabric. In Examples, the appearance and position of the fibroin non-woven fabric after being insolubilized by immersion in 100% by volume ethanol for 24 hours and then by insolubilization by immersion in 95% by volume ethanol for 24 hours, after being subjected to a transparentization treatment by an aqueous magnesium chloride solution It is a phase-contrast microscope and a scanning electron microscope (SEM) image. In the examples, the appearance and position of the fibroin nonwoven fabric after immersion in 100 volume% ethanol for 24 hours followed by insolubilization treatment in 95 volume% ethanol for 24 hours, followed by clearing treatment with an aqueous calcium chloride solution. It is a phase-contrast microscope and a scanning electron microscope (SEM) image. In the examples, the appearance and phase difference of the fibroin nonwoven fabric after being immersed in 100% by volume ethanol for 144 hours and then immersed in 70% by volume ethanol for 144 hours and then subjected to a clearing treatment with an aqueous calcium chloride solution. It is a microscope and a scanning electron microscope (SEM) image. In Examples, after being immersed in 100% by volume ethanol for 144 hours and then immersed in 70% by volume ethanol for 144 hours, the fibroin nonwoven fabric was subjected to a transparentizing treatment with an aqueous calcium chloride solution. It is a figure which shows the analysis result of transparency. In Examples, human corneal epithelium engrafted on a fibroin non-woven fabric after being insolubilized by being immersed in 100% by volume ethanol for 24 hours and then by being soaked in 95% by volume ethanol for 24 hours. It is a photograph which shows the mode of a cell. It is a schematic diagram explaining the procedure of the permeability test in an Example. It is the photograph which copied the progress of the permeability test in an Example. It is a graph which shows the result of the permeability test in an example. It is a graph which shows the result of the permeability test in an example.

≪Transparent fibroin nanofiber nonwoven fabric≫
The transparent fibroin nanofiber nonwoven fabric of this embodiment has an apparent transparency of 60% or more obtained by the following test.
(Test method)
In the photograph obtained by immersing the transparent fibroin nanofiber nonwoven fabric in water, (I) the brightness of the background color observed without passing through the transparent fibroin nanofiber nonwoven fabric, and (II) the transparent fibroin nanofiber Transparency is calculated | required by following formula (1) from the ratio of the brightness of the said background color observed through a nonwoven fabric.
Transparency (%) = (I) / (II) × 100 (1)

The fibroin nanofiber nonwoven fabric is a sheet-like molded body formed by collecting fibroin nanofibers while having a fiber shape. Here, the “sheet shape” refers to a shape in which the thickness of the molded body is smaller than the spread in the two-dimensional direction orthogonal to the thickness direction, and the thickness is extremely thin, Includes up to thick plate-like ones. The transparent fibroin nanofiber nonwoven fabric of this embodiment has pores as gaps between fibroin nanofibers. On the other hand, the fibroin cast film does not have pores as a gap between fibroin nanofibers, and the transparent fibroin nanofiber nonwoven fabric of this embodiment is also different from the fibroin cast film in this respect.
The porosity of the transparent fibroin nanofiber nonwoven fabric is not particularly limited, but the average porosity of the entire transparent fibroin nanofiber nonwoven fabric is preferably 30% or more, and more preferably 40% or more. 55% or more is more preferable, and 75% or more is particularly preferable.

  Fibroin nanofibers constituting the nonwoven fabric are mainly composed of silkworm fibroin. As the fibroin, a fibrous material obtained by removing sericin from the kite string can be preferably used. Fibroin can be obtained, for example, by boiling a cocoon cut into fine pieces with a basic aqueous solution and then washing and removing the remaining basic aqueous solution and sericin. The basic aqueous solution used here has a low concentration so as not to dissolve fibroin excessively. In addition, other methods can be adopted as long as sericin can be removed from the cocoon.

  In the test method for the transparency of the transparent fibroin nanofiber nonwoven fabric, the background color is preferably black. The lightness can be measured by commercially available image analysis software, for example, it can be measured by histogram analysis of image analysis software ImageJ.

  The apparent transparency of the fibroin nanofiber nonwoven fabric calculated by the above formula is preferably 60% or more, more preferably 85% or more, still more preferably 90% or more, and particularly preferably 94% or more.

  Fibroin nanofibers are fibrous structures formed by the assembly of fibroin. The average diameter of the fibroin nanofibers constituting the fibroin nanofiber nonwoven fabric is preferably 1 to 1500 nm, more preferably 10 to 1000 nm, and further preferably 50 to 500 nm.

  The thickness of the transparent fibroin nanofiber nonwoven fabric is preferably 0.1 to 1000 μm, more preferably 0.2 to 200 μm, and further preferably 0.5 to 30 μm. Transparent fibroin nanofibers having a thickness in the above range are also preferred in terms of excellent handling. Moreover, when a transparent fibroin nanofiber nonwoven fabric is used for a cell culture substrate or a cell sheet as described later, it can be a preferable material from the viewpoint of biocompatibility in addition to excellent handling.

Transparent fibroin nanofiber nonwoven fabric of the present embodiment, the density of the average of the nonwoven fabric in the entire nonwoven fabric ^is preferably from ^{0.05g / cm 3 ~0.4g / cm 3} , 0.1g / cm 3 ~0.3g / more preferably cm is ^3, even more preferably from ^{0.1g / cm 3 ~0.25g / cm 3} . In the case where the fibroin nanofiber itself constituting the nonwoven fabric is porous, the density value can be adopted as a value obtained as an apparent density.

In the transparent fibroin nanofiber nonwoven fabric of this embodiment, the degree of aggregation of the fibroin nanofibers is such that the nonwoven fabric has pores. The pores are preferably formed between the fibroin nanofibers due to a slight irregularity of the overlap (orientation) between the fibroin nanofibers and the fibroin nanofibers. The fibroin nanofibers constituting the transparent fibroin nanofiber nonwoven fabric have a fiber shape, but the fibroin nanofibers may be fused together at the contact portion between the fibroin nanofibers generated by the assembly of the fibroin nanofibers.
Transparent fibroin nanofiber nonwoven fabric of the present embodiment is preferably the density of the average in the entire nonwoven fabric is ^{0.05g / cm 3 ~0.4g / cm 3} , 0.1g / cm 3 ~0.3g / cm 3 more ^preferably, it is preferable that further preferably from ^{0.1g / cm 3 ~0.25g / cm 3} , a and an average diameter 1~1500nm fibroin nanofibers constituting the transparent fibroin nanofiber nonwoven fabric 10 to 1000 nm is more preferable, and 50 to 500 nm is even more preferable.
When the average density value of the transparent fibroin nanofiber nonwoven fabric and the average diameter value of the fibroin nanofibers constituting the transparent fibroin nanofiber nonwoven fabric are within the above ranges, the transparent fibroin is used from the viewpoint of using the fibroin nanofiber nonwoven fabric as a biomaterial. From the viewpoint of substance permeability, the pore size of the nanofiber nonwoven fabric can be set to a preferable value. The permeated substance is preferably a biological substance constituting the living body, a substance existing in the living body represented by various nutritional components, or the like.

Since the transparent fibroin nanofiber nonwoven fabric of this embodiment has pores as described above, it has excellent material permeability. The degree of substance permeability is not particularly limited, but from the viewpoint of using a transparent fibroin nanofiber nonwoven fabric as a biological material, biological substances constituting the living body and substances existing in the living body represented by various nutrients are used. It is preferable that the material has a permeability that allows it to pass through. This is because, when a transparent fibroin nanofiber nonwoven fabric is used as a biomaterial, it is preferably a material that transmits signal molecules such as various nutrients and growth factors required by surrounding cells.
The transparent fibroin nanofiber nonwoven fabric preferably transmits molecules having a molecular weight of 150 or more, more preferably transmits molecules having a molecular weight of 60000 or more, and further preferably transmits molecules having a molecular weight of 150,000 or more. More specifically, it preferably has glucose permeability, more preferably has glucose permeability and albumin permeability, and further preferably has glucose permeability, albumin permeability, and globulin IgG permeability. The molecular weight of glucose is about 180.16, the molecular weight of albumin is about 66000, and the molecular weight of globulin IgG is about 150,000. Since albumin is a relatively high molecular weight molecule as an in vivo substance, if it is a non-woven fibroin nanofiber nonwoven fabric that has albumin permeability, it can easily pass through signal molecules such as various nutrients and growth factors required by cells. Conceivable and highly preferred as a biomaterial.

  When it is assumed that a transparent fibroin nanofiber nonwoven fabric is used as a biomaterial, the permeability of the substance is preferably measured under a 1 atm condition.

The material permeability of the transparent fibroin nanofiber nonwoven fabric is determined from the amount of the substance contained in the solution that has passed through the nonwoven fabric by placing a solution containing the substance to be transmitted through the surface of the transparent fibroin nanofiber nonwoven fabric to be measured. To do.
Substance permeability (%) = (Amount of substance contained in solution before passing through nonwoven fabric / Amount of substance contained in solution after passing through nonwoven fabric) × 100
The glucose permeability and albumin permeability of the transparent fibroin nanofiber nonwoven fabric are not particularly limited, but the glucose permeability when applied to the above formula is preferably 90% or more, and preferably 95% or more. 98% or more is preferable. Similarly, the albumin permeability is preferably 90% or more, preferably 95% or more, and preferably 98% or more. The means for measuring and calculating the amount of permeation of the substance is not particularly limited as long as the amount of the substance before and after permeation can be compared, and may be an indirect amount measurement by absorbance or the like.

The rate of glucose permeability and albumin permeability is not particularly limited, but the glucose permeability is preferably 0.009 mg / mm ² · min or more under 1 atm, and 0.01 mg / mm ² · min. It is more preferable that it is 0.05 mg / mm ² · min or more. The albumin permeability is preferably at 0.001 mg / mm ² · min or more at 1 atmosphere, and more preferably 0.075 mg / mm ² · min.

Fibroin nanofibers can be obtained by electrospinning. The electrospinning method (electrospinning) is a method of spinning a fiber by applying a high voltage to a polymer. In general, by applying a voltage between a syringe filled with a polymer solution-filled syringe and a spun yarn, fibers are released from the needle of the syringe toward the collector and spun.
Fibroin nanofibers can be spun by an electrospinning method using a fibroin aqueous solution in which fibroin is dissolved in water. An aqueous fibroin solution can be obtained, for example, by dissolving fibroin in a basic aqueous solution having a pH higher than that used for the removal of sericin and then dialysis and desalting the resulting solution.

  In the electrospinning method, the fibroin nanofibers collected to the collector are unfolded and laminated on the collector in a state of natural irregularity. Therefore, a fibroin nanofiber non-woven fabric (random fibroin nanofiber nonwoven fabric) can be obtained simply by collecting fibroin nanofibers in a collector.

  If fibroin nanofibers can be spun, fibroin nanofibers may be spun by a method other than the above electrospinning method, for example, spinning of fibroin nanofibers by spray drying, wet spinning, etc. .

The transparent fibroin nanofiber nonwoven fabric may be a nonwoven fabric in which fibroin nanofibers are oriented with orientation (orientated fibroin nanofiber nonwoven fabric). When producing nonwoven fabrics using electrospinning, oriented fibroin nanofiber nonwoven fabrics can be obtained by collecting fibroin nanofibers using a rotating collector as the collector. By using a rotating collector, oriented fibroin nanofibers oriented in the rotating direction of the rotating collector can be obtained. Even when a rotating collector is used, fibroin nanofibers can be deployed on the collector with some irregularities, but at this time, the position of the syringe relative to the rotating collector during spinning should be adjusted accordingly. Depending on, the state of the overlap between the fibroin nanofiber and the fibroin nanofiber may be adjusted. As the rotating collector, it is possible to use a known rotating drum type collector. The rotating degree of the rotating collector is about 1500 to 3000 rpm as a guide when a drum having a diameter of 10 cm is used.
By increasing the degree of orientation of the fibroin nanofibers constituting the transparent fibroin nanofiber nonwoven fabric, the transparency of the fibroin nanofiber nonwoven fabric can be made higher.

  The thickness of the transparent fibroin nanofiber nonwoven fabric can be arbitrarily adjusted by spinning and laminating the fibroin nanofibers by adjusting factors such as the diameter, volume and number of fibroin nanofibers constituting the transparent fibroin nanofiber and the concentration of the fibroin aqueous solution. It is possible to obtain a transparent fibroin nanofiber nonwoven fabric having a thickness of.

  The transparent fibroin nanofiber nonwoven fabric may be composed only of fibroin nanofibers, and if the apparent transparency is achieved to be 60% or more, any material other than fibroin nanofibers may be used. May be included. An arbitrary substance may be contained in the fibroin nanofiber, and may be contained in the transparent fibroin nanofiber nonwoven fabric independently of the fibroin nanofiber. Examples of optional substances that can be contained in the transparent fibroin nanofiber nonwoven fabric include biodegradable polyesters such as collagen, gelatin, polylactic acid and derivatives thereof, sericin, polyvinyl alcohol (PVA), polyethylene glycol (PEG) polysaccharides, and the like. The material which can form a fiber like fibroin is preferable.

≪Cell culture substrate and cell sheet≫
The cell culture substrate of the present embodiment is provided with the transparent fibroin nanofiber nonwoven fabric, and may be composed of the transparent fibroin nanofiber nonwoven fabric. Fibroin has excellent biocompatibility, and the transparent fibroin nanofiber nonwoven fabric has excellent material permeability, cell adhesion and transparency, and is therefore suitable as a cell culture substrate. is there.
The cell culture substrate can serve as a scaffold for the cells after being transplanted. Moreover, it can be used as a transplant material in the form of a cell sheet in which cells adhere to a transparent fibroin nanofiber nonwoven fabric.
Due to the excellent transparency of the transparent fibroin nanofiber nonwoven fabric, the cell culture substrate does not interfere with the visibility of the transplant target site. For example, since the transparent fibroin nanofiber nonwoven fabric has excellent transparency, the state can be easily observed even in a tissue covered with a substrate for cell culture and covered with the substrate. It is also excellent in that it is possible to grasp the state of the.

  The cell type that adheres to or is cultured on the transparent fibroin nanofiber nonwoven fabric is not particularly limited, but the cells that make up the fibrous tissue are those of the fibrous scaffold rather than the scaffold on a flat plate such as a cell culture dish. The findings that more active proliferation and expression of functions (for example, Phu D, Lindsay S, Wray S, Warren RV, Haskell RC, Orwin EJ (2011), Effect of substrate composition and alignment on corneal cell phenotype, Tissue Eng, 17, 799-807. Lee CH, Shin HJ, Cho, IH, Kang YM, Kim I, Park KD, Shin, JW (2005) .Nanofiber alignment and direction of mechanical strain affect the ECM production of human ACL fibroblast. Biomaterials, 26, 1261-1270.), And it is preferably a cell constituting a fibrous tissue. Examples of fibrous tissue cells include ligament, cartilage, bone, adipose tissue, corneal tissue, skin tissue cells, or progenitor cells or stem cells that can differentiate into these tissues. Examples of corneal tissue cells include corneal endothelial cells, corneal parenchymal cells, corneal epithelial cells, corneal epithelial limbal stem cells, and precursor cells or stem cells that can differentiate into them.

When a cell culture substrate is used as a transplant material, the transplant destination is not particularly limited, but is preferably transplanted to the eye, more preferably to the cornea. Examples of the cornea include corneal endothelium, corneal epithelium or corneal stroma, and corneal epithelium or corneal stroma is more preferable. Cell culture substrate transplantation to the cornea is performed to supplement the cornea, to provide a scaffold for regenerating the cells of the cornea, and transplanted cells cultured on the cell culture substrate Including things to do.
Since the eye is a photoreceptor organ, a cell culture substrate having excellent transparency is extremely useful as a material for transplantation into the eye. Furthermore, since the corneal parenchyma is an avascular tissue, a material excellent in substance permeability is very useful. In addition, it is known that the regeneration speed is slow compared to other tissues because of its low regenerative ability because it is an avascular tissue. Therefore, as a material for regenerating corneal stroma, a material that exists in a living body for a certain long period is preferable. Since fibroin is not easily decomposed in vivo, it can be said that it is a material having very useful characteristics in addition to biocompatibility from the viewpoint of corneal substantial regeneration.
The overwhelming donor shortage with respect to the number of corneal transplantation desired patients has become a global problem not only in Japan, but the development of an artificial cornea is awaited. Until now, many attempts have been made to develop an artificial cornea using various materials. With the development of technology, technologies that are actually applied clinically have been created for corneal epithelial cell layers and corneal endothelial cell layers. Several excellent technologies have also been developed for the corneal stroma, but no clinically satisfactory one has yet been developed.
The cell culture substrate of the present embodiment is a material for regenerating cornea due to the above-mentioned properties such as excellent transparency, cell adhesion, substance permeability, and degradation rate. Can also be suitably used.

≪Method for producing transparent fibroin nanofiber nonwoven fabric≫
In the present embodiment, the fibroin nanofiber nonwoven fabric is a sheet-like molded body in which fibroin nanofibers are aggregated while having a fiber shape. Here, the “sheet shape” refers to a shape in which the thickness of the molded body is smaller than the spread in the two-dimensional direction orthogonal to the thickness direction, and the thickness is extremely thin, Includes up to thick plate-like ones. The fibroin nanofibers constituting the transparent fibroin nanofiber nonwoven fabric have a fiber shape, but the fibroin nanofibers may be fused together at the contact portion between the fibroin nanofibers generated by the assembly of the fibroin nanofibers. The transparent fibroin nanofiber nonwoven fabric has pores as gaps between fibroin nanofibers. Hereinafter, when the matters common to the fibroin nanofiber nonwoven fabric are described regardless of the orientation of the fibroin nanofibers and the transparency of the fibroin nanofiber nonwoven fabric, they may be simply described as a fibroin nanofiber nonwoven fabric or a nonwoven fabric.

  In the method for producing a transparent fibroin nanofiber nonwoven fabric according to this embodiment, an oriented fibroin nanofiber nonwoven fabric obtained by orienting fibroin nanofibers with orientation is brought into contact with an aqueous alcohol solution of 96% by volume or more and then 60% by volume. An insolubilization treatment step of obtaining an insolubilized fibroin nanofiber nonwoven fabric by contacting with an aqueous alcohol solution of less than 96 volume% and an insolubilized fibroin nanofiber nonwoven fabric obtained by the insolubilization treatment step are brought into contact with a solution containing a Group 2 element A clearing treatment step, and a drying treatment step of drying the insolubilized fibroin nanofiber nonwoven fabric that has undergone the clearing treatment step.

As shown in the present embodiment, the insolubilized fibroin nanofiber nonwoven fabric subjected to the transparentization treatment step is preferably an oriented fibroin nanofiber nonwoven fabric in which fibroin nanofibers are oriented with orientation.
When producing nonwoven fabrics using electrospinning, oriented fibroin nanofiber nonwoven fabrics can be obtained by collecting fibroin nanofibers using a rotating collector as the collector. By using a rotating collector, oriented fibroin nanofibers oriented in the rotating direction of the rotating collector can be obtained.
By increasing the degree of orientation of the fibroin nanofibers constituting the fibroin nanofiber nonwoven fabric, a transparent fibroin nanofiber nonwoven fabric with high transparency can be obtained more easily. The use of the oriented fibroin nanofiber nonwoven fabric is very compatible with the transparency treatment process. This is presumably because fibrin nanofibers are made transparent by using a highly transparent material that has been previously oriented to fibroin nanofibers, and the fibroin nanofibers are made transparent enough to dissolve the fiber surface. It is because it can raise remarkably. If it is a weak clearing treatment, it is easy to control the clearing treatment so as not to cause the fiber shape of the fibroin nanofibers to collapse.

As shown in this embodiment, the insolubilized fibroin nanofiber nonwoven fabric subjected to the clarification treatment step is such that the oriented fibroin nanofiber nonwoven fabric is contacted with 96% by volume or more of an alcohol aqueous solution and then 60% by volume or more 96. It is preferable that it is the insolubilized fibroin nanofiber nonwoven fabric manufactured through the insolubilization process made to contact the alcohol aqueous solution of less than volume%.
When the fibroin nanofiber nonwoven fabric is brought into contact with the alcohol aqueous solution, the water inside the fibroin nanofiber nonwoven fabric oozes out due to the osmotic pressure generated by the difference in concentration between the water contained in the fibroin nanofiber nonwoven fabric and the aqueous alcohol solution. It becomes easy to contact each other. It is considered that fibroin is crystallized by intermolecular force or hydrogen bond at the part where fibroin is in contact with each other, and as a result, fibroin is insolubilized.

As the insolubilization treatment, generally, an alcohol aqueous solution of about 70% by volume to 80% by volume is prepared using an alcohol such as methanol or ethanol, and a process of immersing in the aqueous alcohol solution for about 15 minutes to 60 minutes is performed. Fibroin is said to form II-type crystals having a β-sheet structure, and the degree of crystallinity rises to a certain equilibrium value, regardless of the concentration of the alcohol aqueous solution used in the insolubilization treatment.
However, when the inventor examined in detail, the fiber shape of the fibroin nanofibers was first brought into contact with a 95% by volume or higher alcohol aqueous solution having a concentration much higher than that generally used by bringing the nonwoven fabric into contact with the fibroin nanofiber nonwoven fabric. It became clear that the operability was improved by being easily maintained, and a transparent fibroin nanofiber nonwoven fabric could be easily obtained even after the subsequent transparentization treatment. The concentration of the 95% by volume or higher alcohol aqueous solution is preferably 96% by volume or higher, more preferably 98% by volume or higher, and still more preferably 99% by volume or higher.

  Thereafter, the crystallinity of the fibroin nanofibers is increased by contacting with an alcohol aqueous solution of 60% by volume or more and less than 95% by volume. The concentration of the alcohol aqueous solution of 60% by volume or more and less than 95% by volume is preferably 60% by volume or more and less than 90% by volume, more preferably 60% by volume or more and less than 80% by volume, and 65% by volume or more and 75% by volume. More preferably, it is less than volume%. By bringing the fibroin nanofiber nonwoven fabric into contact with the alcohol aqueous solution having the above concentration, the fiber shape of the fibroin nanofiber can be easily maintained even after the subsequent transparentization treatment, and the transparent fibroin nanofiber nonwoven fabric can be easily obtained.

  As said alcohol, methanol and ethanol can be used conveniently. Also, transparent fibroin nanofiber nonwoven fabric contains a small amount of alcohol such as propanol, butanol, and PEG (polyethylene glycol) as long as it does not have an adverse effect when cell culture is used as a cell culture substrate or biomaterial. It may be.

  As a method of bringing alcohol into contact with the fibroin nanofiber nonwoven fabric, various methods can be selected as long as insolubilization treatment is possible. For example, small pieces of fibroin nanofiber nonwoven fabric may be immersed in an aqueous alcohol solution, or a sufficient amount of an alcohol aqueous solution may be sprayed or applied to the small pieces.

  The time for insolubilizing the nonwoven fabric with each of the above alcohols is preferably from 10 seconds to 720 hours, more preferably from 16 hours to 480 hours, and particularly preferably from 72 hours to 240 hours.

  The temperature of the insolubilization treatment of the molded product with alcohol is preferably from −97.8 ° C. (freezing point of 100% methanol) to 78.4 ° C. (boiling point of 100% ethanol), and is preferably −38 ° C. (freezing point of 50% ethanol by volume solution). ) To 65 ° C. (boiling point of 100% methanol) is more preferable.

  The manufacturing method of the transparent fibroin nanofiber nonwoven fabric of this embodiment has the transparentization process process which makes the solution containing a group 2 element contact the insolubilized fibroin nanofiber nonwoven fabric obtained in the said insolubilization process process.

  The solution containing a Group 2 element may contain a plurality of types of Group 2 elements in the solution. The solution containing a Group 2 element is preferably a solution containing Group 2 element ions. Group 2 element ions are ions of elements belonging to Group 2 of the periodic table, and examples include beryllium ions, magnesium ions, calcium ions, strontium ions, barium ions, and radium ions. From the viewpoint of biocompatibility, Magnesium ions or calcium ions are preferable, and calcium ions are more preferable.

A solution containing a Group 2 element ion can be obtained, for example, by adding a Group 2 element to a polar solvent. Examples of the polar solvent include water, ethanol, formic acid, acetic acid, and a composition obtained by mixing them. Adding a Group 2 element includes adding a compound such as an inorganic acid salt such as a halide, carbonate, sulfate, nitrate, or phosphate of a Group 2 element; For example, compounds such as BeSO ₄ , BeCl ₂ , Mg (NO ₃ ) ₂ , MgCO ₃ , CaCl ₂ , SrCl ₂ , BaSO ₄ , and RaCl ₂ can be added.

  The method for bringing the insolubilized fibroin nanofiber nonwoven fabric into contact with the solution containing the Group 2 element is not particularly limited, and the insolubilized fibroin nanofiber nonwoven fabric is impregnated with the solution containing the Group 2 element, sprayed, applied, etc. The method is mentioned.

  By bringing the insolubilized fibroin nanofiber nonwoven fabric into contact with a solution containing a Group 2 element, the surface of the fibroin nanofiber is probably dissolved slightly, and the higher order structure of the fibroin nanofiber constituting the insolubilized fibroin nanofiber nonwoven fabric. And the arrangement can be changed to improve the transparency of the insolubilized fibroin nanofibers.

When the Group 2 element is calcium, the calcium concentration in the solution is preferably 10 mM to 500 mM, more preferably 60 mM to 350 mM, further preferably 70 mM to 250 mM, and particularly preferably 80 mM to 200 mM. The above M represents mol / m ³ .
At this time, the time for bringing the insolubilized fibroin nanofiber nonwoven fabric into contact with the solution containing calcium is preferably 0.05 hours to 12 hours, more preferably 0.1 hours to 3 hours, and 0.2 hours to 1 hour. Further preferred.

The concentration of the solution containing calcium and the time for which the insolubilized fibroin nanofiber nonwoven fabric is brought into contact with the solution can be arbitrarily combined.
When using a solution containing a relatively high concentration of calcium, the contact time between the solution and the insolubilized fibroin nanofiber nonwoven fabric is shortened, and when using a solution containing a relatively low concentration of calcium, The contact time between the solution and the insolubilized fibroin nanofiber nonwoven fabric may be set long.

  Qualitatively, the transparency of the fibroin nanofiber nonwoven fabric tends to improve as the calcium concentration of the solution containing calcium is increased, or as the time for contacting the solution with the insolubilized fibroin nanofiber nonwoven fabric is increased. It is believed that there is. However, since treatment with a solution having a too high concentration or treatment for a long time may decrease transparency, in that case, the calcium concentration of the solution is lowered or the solution and the insolubilized fibroin nanofiber nonwoven fabric are brought into contact with each other. What is necessary is just to adjust in the direction which shortens time.

  In consideration of these, when manufacturing a transparent fibroin nanofiber nonwoven fabric, it is good to perform a preliminary experiment beforehand and to set the combination of the density | concentration and time with which desired transparency is obtained.

In the manufacturing method of the transparent fibroin nanofiber nonwoven fabric of this embodiment, a drying process is performed to the insolubilized fibroin nanofiber nonwoven fabric which passed through the transparentization process. By the drying treatment, the solvent of the solution containing the Group 2 element is removed from the nonwoven fabric. The drying process probably maintains or fixes the higher order structure and arrangement of the fibroin nanofibers that have changed due to the clearing process. As the nonwoven fabric is dried, the transparency of the nonwoven fabric is gradually improved. It is not necessary to completely remove the solvent of the nonwoven fabric as the degree of drying of the nonwoven fabric, and as described in the above << Transparent fibroin nanofiber nonwoven fabric >>, the transparency of the transparent fibroin nanofiber nonwoven fabric is dried to an extent of 60% or more. Can be mentioned. The drying process is preferably performed immediately after the clarification process.
As a drying method, a conventionally known method can be adopted as long as the transparent fibroin nanofiber nonwoven fabric is not deteriorated. For example, air drying, heating drying, drying under reduced pressure, etc. left in the air can be mentioned. Of these drying methods, air drying is preferred. Also, drying may be promoted by appropriately blowing air.

The transparent fibroin nanofiber nonwoven fabric that has undergone the transparentization treatment step may be appropriately subjected to a washing step in order to remove the Group 2 element brought into contact with the transparentization treatment step from the nonwoven fabric.
The washing step is, for example, a method of immersing the transparent fibroin nanofiber nonwoven fabric that has undergone the clearing treatment step in water, a transparent fibroin nanofiber nonwoven fabric that has undergone the clearing treatment step into a liquid containing a chelating agent that chelates the Group 2 element. Examples of the method include a method of impregnating with water after the immersion.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated more concretely, this invention is not limited to these.

1. Fabrication experiment of transparent oriented fibroin nonwoven fabric [Production of fibroin aqueous solution]
The koji cut into strips was boiled in 0.02M sodium carbonate for 30 minutes (95-98 ° C.) and then washed with hot water to remove sericin. The obtained fibroin fiber was dissolved in 9 M lithium bromide at room temperature to prepare a solution having a concentration of about 60 g / L. After dialysis against water using a dialysis tube (MWCO: 5000-8000) for 3 days, impurities were removed by centrifugation (20000 rpm × 30 min).

[Production of oriented fibroin nonwoven fabric]
A fibroin aqueous solution was prepared using ultrapure water so as to have a concentration of 8.0 mg / ml. Further, polyethylene oxide (PEO, Mw = 9 × 10 ⁵ g / mol, Sigma-Aldrich) was dissolved in ultrapure water to prepare an aqueous solution having a concentration of 50 mg / ml. A mixture of an aqueous fibroin solution and an aqueous PEO solution at a volume ratio of 4 to 1 was used as a spinning solution (reference document Biomacromolecules 3, 6, 1233-1239.). The prepared spinning solution was filled in a syringe equipped with a 25G stainless needle, and spinning was performed using an automatic spinning device (NANON, manufactured by MECC). The applied voltage was +10 kV, the discharge speed was 0.3 ml / h, and the spinning distance was 18 cm. The relative humidity of the spinning atmosphere was maintained at 15% or less by perfusing dry nitrogen into the spinning apparatus. An aluminum foil was wound around a rotating drum type collector (3,000 rpm) having a diameter of 10 cm, and spinning was performed. After spinning, the fiber was removed from the aluminum foil, and the fiber was collected on a minu sheet (Φ1.3 cm, Iida Trading Chemical). The density of the produced oriented fibroin nonwoven was 0.155 g / cm ³ , the porosity was 85.5%, the average diameter of the fibroin nanofibers was 200 to 300 nm, and the thickness was about 10 μm. FIG. 2 shows photographs comparing macroscopic photographs and SEM images of oriented fibroin nonwoven fabric and random fibroin nanofiber nonwoven fabric.

[Insolubilization treatment]
The insolubilization of the collected fiber with sufficient affinity for water was examined under the following two conditions.
<Insolubilization condition 1> An oriented fibroin nonwoven fabric is immersed in 100 volume% ethanol for 24 hours, and then immersed in 95 volume% ethanol for 24 hours.
<Insolubilization condition 2> An oriented fibroin nonwoven fabric is immersed in 100 volume% ethanol for 144 hours, and then immersed in 70 volume% ethanol for 144 hours.
After the insolubilization treatment, the fiber was sufficiently affinitized with water to carry out a transparency treatment.

[Transparency treatment / Dry treatment]
An 80 mM, 100 mM, 160 mM, 200 mM or 500 mM calcium chloride aqueous solution and a 200 mM magnesium chloride aqueous solution were prepared using ultrapure water. For samples that have been insolubilized under the conditions of <Insolubilization Condition 1>, after 30 minutes of immersion in an 80 mM or 200 mM calcium chloride aqueous solution, the sample is taken out, wiped off excess water, and then dried in a wet box for 24 hours. Then, it was taken out from the wet box and dried for 48 hours or more in an indoor atmosphere. For samples that have been insolubilized under the conditions of <Insolubilization Condition 2>, the sample is immersed in an 80 mM, 100 mM, 160 mM, 200 mM, or 500 mM calcium chloride aqueous solution for 30 minutes, dried in a wet box for 24 hours, and at room temperature. For more than 48 hours. The sample after the drying treatment is immersed in ultrapure water as it is under the above <insolubilization condition 1>, and the sample subjected to the above <insolubilization condition 2> has a molar ratio with the immersed calcium chloride aqueous solution of 5: 3. After soaking in an aqueous solution of sodium dihydrogen phosphate as described above, it was examined separately by soaking in ultrapure water after soaking in 10% EDTA-2Na for 24 hours. FIG. 1 shows the sequence of a series of processes of insolubilization, clearing, drying, and washing.

[Quantitative analysis of transparency]
After the clearing treatment, a photograph was taken with the sample immersed in water. Transparency was calculated by the following equation by comparing the average gray value (brightness) of the black part inside and outside the material using the histogram analysis of the image analysis software Image J.
Transparency = lightness of the black part existing outside the material / lightness of the black part existing inside the material × 100

  The “lightness of the black part existing outside the material” corresponds to “(I) the lightness of the background color observed without using the transparent fibroin nanofiber nonwoven fabric” in the apparent transparency test method described above, The “brightness of the black part existing in the material” corresponds to “(II) brightness of the background color observed through the transparent fibroin nanofiber nonwoven fabric” in the apparent transparency test method described above.

Results After insolubilization using 100 volume% ethanol and 95 volume% ethanol, the sample that was dipped in 200 mM magnesium chloride aqueous solution and dried was more transparent than the sample that was dipped in ultrapure water prepared as a control and then air dried. It was improving. In FIG. 3, the observation image of each sample in an optical microscope and a scanning electron microscope is shown. Observation with an optical microscope confirmed that a fibrous structure remained in both samples. Observation with a scanning electron microscope reveals that the fiber structure of the sample treated with magnesium chloride, which has been visually improved in transparency, has changed compared to the control. In the sample treated with magnesium chloride, an extreme increase in fiber diameter and a decrease in fiber gap were observed. This phenomenon is thought to be due to the fibroin nanofibers partially dissolved when immersed in magnesium chloride and the adjacent nanofibers fused together during drying to take such a fiber structure. As a result, it is considered that the light gap is suppressed by reducing the fiber gap, and the macroscopic transparency is improved.

  Samples that were insolubilized using 100 volume% ethanol and 95 volume% ethanol and then dipped in 200 mM or 80 mM calcium chloride aqueous solution and dried gave a transparent structure regardless of the concentration of the calcium solution. However, a fibrous structure could not be confirmed in the obtained sample, and a structure like an uneven flat plate was confirmed. The appearance after processing, a phase contrast microscope, and a scanning electron microscope (SEM) image are shown in FIG. Such a structure is considered that the fiber of the oriented fibroin non-woven fabric was dissolved by calcium, the fiber structure collapsed, and dried to form a cast film-like flat plate structure.

  A sample that is insolubilized using 100 volume% ethanol and 70 volume% ethanol and then immersed and dried in an 80 mM, 100 mM, 160 mM, or 200 mM calcium chloride aqueous solution can provide an extremely transparent structure. It was. FIG. 5 shows the appearance after processing, a phase contrast microscope, and an SEM image. An analysis by image processing is shown in FIG. Apparent transparency by image analysis is 94.2% for the sample treated with 80 mM, 93.4% for the sample treated with 100 mM, 98.7% for 160 mM, and 97.4% for the sample treated with 200 mM aqueous calcium chloride. It was as high as the macroscopic findings. In these samples, it was confirmed by observation with an SEM image that fibers and pores were present in the samples with improved transparency. The fiber structure observed in the sample with improved transparency was seen as if some of the fibers were fused together. On the other hand, in the sample treated with 500 mM calcium chloride aqueous solution, the transparency was not visually different from the control dried without being immersed in calcium chloride. The apparent transparency by image analysis was also low, reflecting the gross findings of 66.6% for the sample treated with 500 mM and 47.8% for the control. In these samples, fiber fusion was lighter than in samples with improved transparency.

  Under this insolubilization condition (<insolubilization condition 2>), 95% by volume is obtained by taking sufficient time for crystallization formation of fibroin caused by insolubilization and by treatment with ethanol at a lower concentration of 70% by volume than before. It is considered that the crystallinity is higher than the treatment with ethanol. As a result, it is considered that the fiber shape was maintained even after the dissolution treatment with calcium chloride, and that the fiber structure gap that could cause light scattering was filled with fibroin eluted from the fiber, so that the transparency was achieved.

  Comparing these results together, in the insolubilization process, fibroin was more effective when 100 volume% ethanol and 70 volume% ethanol were used than when 100 volume% ethanol and 95 volume% ethanol were used. The fiberization state could be controlled more easily, and a high-quality transparent fibroin nanofiber nonwoven fabric could be manufactured more easily. When 100 volume% ethanol and 95 volume% ethanol were used, the fibroin nanofiber fiber structure collapsed as described above, but by repeatedly investigating the ethanol immersion time, the fiber structure of the fibroin nanofiber was changed. There is a possibility that it can be maintained.

  In addition, in the clarification treatment step, the fibroin nanofiber can be more easily controlled when the calcium chloride aqueous solution is used than when the magnesium chloride aqueous solution is used, and the fibroin fiberization state can be controlled more easily. A nonwoven fabric could be produced.

  The concentration of the aqueous solution of calcium chloride can be more easily controlled by using the 80 mM to 200 mM aqueous solution of calcium chloride than when using the 500 mM aqueous solution of calcium chloride. A fiber nonwoven fabric could be produced. Even when a 500 mM calcium chloride aqueous solution is used, there is a possibility that the transparency of the fibroin nanofibers can be improved by repeatedly examining the immersion time and the like.

2. Cytotoxicity test experiment of fibroin film after clearing treatment After insolubilization treatment with 100 volume% ethanol and 95 volume% ethanol, the sample immersed in 80 mM calcium chloride, dried and then immersed in ultra pure water for 24 hours is ultra pure water. The solution was sterilized by autoclaving at 105 ° C. for 30 minutes while immersed in the solution. Sterilized samples were seeded with human corneal epithelial cells (HCE-T) and cultured for 1 week after seeding. After completion of the culture, the sample was fixed with 4% paraformaldehyde, a paraffin-embedded section was prepared, hematoxylin-eosin (HE) staining was performed, and the state of the cells was observed.

Results FIG. 7 shows a photograph of HE staining. HCE-T was able to be engrafted on the material for one week even on the material that had undergone the fibroin dissolution and drying process by calcium contact. Thereby, it became clear that the denaturation which shows the toxicity to a cell was not produced by the clearing process by calcium contact.

3. Material permeability test experiment of transparent oriented fibroin nonwoven fabric [Preparation of material permeability test sample]
After preparing an oriented fibroin nonwoven fabric by the same procedure as the method described in 1 above (preparing transparent oriented fibroin nonwoven fabric), it was immersed in 100% by volume and 70% by volume ethanol for 144 hours and hydrated (1 ) A non-woven fabric obtained by transparentizing the non-woven fabric of (1) with 80 mM calcium chloride was used as (2) a transparent oriented fibroin non-woven fabric. In addition, as an object for the permeability test, fibroin aqueous solution was allowed to flow on a flat plate and dried (3) three cast films were prepared as test materials. Each produced material was sandwiched between the above-mentioned minu sheets.

[Nutrient permeability test]
As a nutrient component permeability test, a 2.5 mg / ml glucose solution (molecular weight 180.16) and a 2.0 mg / ml bovine serum albumin solution (molecular weight about 66000) were prepared, and the permeability test as shown in FIG. Went. A glass cylindrical container was placed in a hydrophobic film shape, and a material sandwiched between supports was placed thereon. 300 μl of each solution is dropped on each material of the above (1) to (3) of 50.24 mm ² , and the time during which the dropped solution permeates each material and the glucose and albumin concentrations of the permeated droplets are measured. The permeability of each material was compared. A photograph showing the progress of the permeability test is shown in FIG. After dropping the aqueous solution, the non-woven fabric ((1) non-woven fabric) and the transparent fiber ((2) transparent oriented fibroin non-woven fabric) permeated within 1 minute, but the cast film ((3) cast film) still remained as water droplets. Remained.
In addition, the glucose test CII and protein assay kit of Wako Pure Chemical Industries were used for the concentration measurement of glucose and albumin, and the microplate reader (iMark BioRad) was used for the concentration measurement of the solute.

Results The results of the transmission test are shown in FIGS. As a result of the drop test of glucose and albumin on each material, almost no change in permeation rate due to the difference in solute was observed. (1) In the non-woven fabric, the permeation rate of glucose was 29.74 ± 3.22 seconds and albumin was 23.23. 09 ± 4.00 seconds. (2) The transparent oriented fibroin nonwoven had a glucose transmission rate of 49.91 ± 8.62 seconds and albumin of 49.19 ± 5.14 seconds. On the other hand, in the case of (3) cast film, no permeation of the solution occurred even after 30 minutes had passed after dropping. In (1) and (2), the concentration of the solute contained in the solution after passing through the droplet was not different from the concentration of the solution before dropping (transmittance of 95% or more).
(1) albumin permeability of the nonwoven fabric is under 1 atm is 0.08 mg / mm ² · min, (2) albumin permeability of the transparent orientation fibroin nonwoven under 1 atm, 0.04 mg / mm ²・ It was minutes. (1) The glucose permeability of the nonwoven fabric is 0.10 mg / mm ² · min at 1 atmosphere, and (2) the glucose permeability of the transparent oriented fibroin nonwoven fabric is 0.06 mg / mm ^{2 at} 1 atmosphere.・ It was minutes.
The cast film could not measure the concentration because no dripping solution was obtained. Clearly oriented fibroin nonwovens certainly have a lower permeation rate than nonwovens, but are found to be much faster than cast films. In addition, the ability to rapidly permeate proteins with a molecular weight equivalent to albumin can cause problems with the nutrients and biochemical factors in the corneal tissue even after implantation in vivo. Is low. From the above results, it was shown that the transparent oriented fibroin non-woven fabric is a non-woven material with little loss of material permeability and transparency.

  The configurations and combinations thereof in the embodiments described above are examples, and the addition, omission, replacement, and other modifications of the configurations can be made without departing from the spirit of the present invention. Further, the present invention is not limited by each embodiment, and is limited only by the scope of the claims.

  A transparent material having a fibrous structure using fibroin with high biocompatibility can be used not only for the corneal parenchyma but also for various fibrous tissues, so it can be used effectively in industrial fields such as regenerative medicine. .

Claims (12)

  A clearing treatment process in which an insolubilized fibroin nanofiber nonwoven fabric in which fibroin nanofibers are insolubilized is brought into contact with a solution containing a Group 2 element, and a drying treatment process in which the insolubilized fibroin nanofiber nonwoven fabric that has undergone the above-mentioned transparency treatment process is dried. The manufacturing method of the transparent fibroin nanofiber nonwoven fabric characterized by having. The method for producing a transparent fibroin nanofiber nonwoven fabric according to claim 1 , wherein the Group 2 element is calcium or magnesium. Prior to the transparency treatment step,
The fibroin nanofiber nonwoven fabric, into contact with an aqueous alcohol solution of less than 60 vol% to 95 vol% after contacting the alcohol solution of the above 95% by volume, according to claim 1 having an insolubilization treatment step of obtaining the insolubilization fibroin nanofiber nonwoven fabric Or the manufacturing method of the transparent fibroin nanofiber nonwoven fabric of 2 . Spinning the fibroin nanofibers using electrospinning,
Winding the fibroin nanofibers using a rotary collector, transparent fibroin nanofiber according to claim 3 having, a step of obtaining the fibroin nanofiber nonwoven fabric of the fibroin nanofibers is oriented in the rotation direction of the rotary collector Nonwoven fabric manufacturing method. Manufactured by the method for producing a transparent fibroin nanofiber nonwoven fabric according to claim 1,
A transparent fibroin nanofiber nonwoven fabric having an apparent transparency of 60% or more obtained by the following test.
(Test method)
In the photograph obtained by immersing the transparent fibroin nanofiber nonwoven fabric in water, (I) the brightness of the background color observed without passing through the transparent fibroin nanofiber nonwoven fabric, and (II) the transparent fibroin nanofiber Transparency is calculated | required by following formula (1) from the ratio of the brightness of the said background color observed through a nonwoven fabric.
Transparency (%) = (I) / (II) × 100 (1) The average density of the transparent fibroin nanofiber nonwoven fabric as a whole is 0.05 g / cm ^{3 to} 0.4 g / cm ³ , and the average diameter of the fibroin nanofibers constituting the transparent fibroin nanofiber nonwoven fabric is 1 nm to 1500 nm. Item 6. The transparent fibroin nanofiber nonwoven fabric according to Item 5 . The transparent fibroin nanofiber nonwoven fabric according to claim 5 or 6 , which has glucose permeability. The transparent fibroin nanofiber nonwoven fabric according to any one of claims 5 to 7 , which has albumin permeability. The transparent fibroin nanofiber nonwoven fabric according to claim 8 , wherein the albumin permeability is 0.001 mg / mm ² · min or more under 1 atmosphere. A cell culture substrate comprising the transparent fibroin nanofiber nonwoven fabric according to any one of claims 5 to 9 . A cell sheet comprising cells adhered to the transparent fibroin nanofiber nonwoven fabric according to any one of claims 5 to 9 . The cell sheet according to claim 11 , wherein the cells are cells of corneal tissue.