Silk fibroin tubular substance

Abstract

Provided is a silk fibroin tubular substance used for reproducing a biological tissue. The silk fibroin tubular substance exhibits a porous makeup at room temperature, the patency rate of the silk fibroin tubular substance when a 1N load is applied thereon in a water-saturated state being 10%-50%.

Description

Silk fibroin tubular body Related Applications

[0001] This application claims priority from Japanese Patent Application No. 2023-205384, filed on December 5, 2023, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to a silk fibroin tubular body used for regenerating biological tissue such as nerve tissue.

[0003] When nerves are damaged by external factors (e.g., trauma, surgery) or internal factors (e.g., malignant tumors), motor and sensory impairments, pain, numbness, and other symptoms can significantly impair daily life. In cases of widespread nerve defects, autologous nerve transplantation, in which a self-nurse is transplanted into the defect, is the standard treatment. However, this procedure involves harvesting the autologous nerve from another part of the body, which can result in the loss of function in the donor area and limits the length of the nerve that can be harvested. Furthermore, the long surgical time required to harvest the autologous nerve places a significant burden on both the physician and the patient.

[0004] Therefore, there is a demand for a nerve regeneration instrument for regenerating nerve tissue. For example, Patent Document 1 discloses a biological tissue or organ regeneration instrument for regenerating human tissue or organs, such as nerve fibers or microvessels, that have been severed due to a lesion or injury, which is characterized by comprising a support (A) made of a biodegradable or bioabsorbable material, a sponge-like fine matrix (B) made of a biodegradable or bioabsorbable material, and a linear biological tissue or organ guide path (C).

[0005] In this regenerative device, the sponge-like micro-matrix and linear guided pathways formed in the lumen can be used as a scaffold for cell regeneration, allowing for the regeneration of biological tissues or organs.

[0006] In addition, in Non-Patent Document 1, Na ２ CO ３ or NaHCO ３After scouring the silk fibroin in a 0.1 wt% solution, the scouring material was dissolved in 9.3 M LiBr at 60°C for 2 hours to form a silk fibroin solution, which was then poured into a mold for forming a tube and frozen. The resulting silk fibroin tube was shown to be stretchable in the longitudinal direction and biodegradable.

[0007] Japanese Patent Application Laid-Open No. 2002-320630

[0008] X. Li et al. Polymer Degradation and Stability 164 (2019) 61-68

[0009] However, although Patent Document 1 allows the use of a sponge-like fine matrix and linear guidance pathways as scaffolds, the presence of these reduces the amount of void space, which may hinder the penetration of regenerated tissue, and further improvements are required.

[0010] Furthermore, although Non-Patent Document 1 focuses on the longitudinal elasticity of silk fibroin tubes, the elasticity of such tubes reduces their resistance to compression in a direction perpendicular to the longitudinal direction, making them susceptible to being crushed by external pressure and making it impossible to secure voids after implantation in the body.

[0011] Therefore, one object of the present invention is to provide a silk fibroin tubular body that has a hollow structure that promotes the infiltration of regenerated tissue and has excellent regeneration-inducing ability for biological tissues such as nervous tissue.

[0012] As a result of extensive research conducted by the inventors to solve the problems of the prior art described above, they discovered that a tubular body made of silk fibroin, which is resistant to crushing even when a load is applied, can maintain its hollow structure after implantation in the body and have excellent regenerative properties for biological tissues such as nervous tissue, leading to the completion of the present invention.

[0013] That is, the present invention can be configured in the following aspects. [Aspect 1] A silk fibroin tubular body that has a porous structure at room temperature and that has a patency rate of 10.00 to 50.00% (preferably 13.00 to 45.00%, more preferably 15.00 to 40.00%) when a load of 1 N is applied while the body is saturated with water. [Aspect 2] The silk fibroin tubular body of Aspect 1, wherein the compressive stress when compressed by 25% while the body is saturated with water is 0.020 to 0.300 N (preferably 0.030 to 0.200 N, more preferably 0.050 to 0.100 N). [Aspect 3] The silk fibroin tubular body of Aspect 1 or 2, wherein the compressive stress when compressed 50% in a water-saturated state is 0.040 to 0.500 N (preferably 0.050 to 0.300 N, more preferably 0.060 to 0.200 N). [Aspect 4] The silk fibroin tubular body of Aspects 1 to 3, wherein the silk fibroin tubular body has a recovery rate of 95.50% or more (preferably 96.00% or more, more preferably 97.00% or more) after being subjected to a 50% compression test and left for 10 minutes. [Aspect 5] The silk fibroin tubular body of any one of Aspects 1 to 4, wherein the fibroin elution rate when stored for 24 hours in 1 mL of a protease XIV / phosphate buffered saline (PBS) solution (2 U / mL) is 2.00 to 6.50% (preferably 2.50 to 6.30%, more preferably 3.00 to 6.00%). [Aspect 6] The silk fibroin tubular body of any one of Aspects 1 to 5, wherein the silk fibroin tubular body has protein permeability using ovalbumin as a standard substance. [Aspect 7] The silk fibroin tubular body of any one of Aspects 1 to 6, wherein the silk fibroin tubular body is used as a nerve regeneration guide material.

[0014] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms "at least one," unless the content clearly dictates otherwise. As used herein, the terms "and / or," "at least one," and "one or more" include any and all combinations of the associated listed items.

[0015] It should be noted that any combination of at least two elements disclosed in the claims and / or the specification and / or the drawings is included in the present invention, and in particular any combination of two or more of the claims set forth in the claims is included in the present invention.

[0016] The silk fibroin tubular body of the present invention is transplanted in a state in which it bridges both ends of a severed or missing nerve, thereby not only inducing nerve regeneration in the hollow part of the tubular body, but also reducing its harmfulness to the living body because the silk fibroin tubular body is decomposed and absorbed after a predetermined period of time has passed.

[0017] The present invention will be more clearly understood from the following description of preferred embodiments with reference to the accompanying drawings. However, the embodiments and drawings are for illustration and explanation purposes only and should not be used to define the scope of the present invention, which is defined by the appended claims.

[0018] 1 is a photograph showing a silk fibroin tubular body of one embodiment. FIG. 2 is an enlarged photograph of the silk fibroin tubular body of FIG. 1. FIG. 3 is a schematic perspective view showing components for assembling a mold used to form a silk fibroin tubular body of one embodiment. FIG. 4 is a schematic cross-sectional view illustrating the assembled state of the mold of FIG. 3. FIG. 5 is a schematic cross-sectional view illustrating a state in which a silk fibroin tubular body of one embodiment is fixed to a transected nerve. FIG. 6 is a schematic cross-sectional view illustrating a method for measuring the patency rate when a 1 N load is applied. FIG. 7 is a schematic cross-sectional view illustrating a method for measuring stresses at 25% and 50% compression. FIG. 8 is a photograph showing a Franz cell used to confirm protein permeability. FIG. 9 is a photograph showing a part of a 12-well plate used in the examples to confirm long-term degradability, showing the state immediately after the start of the test (0 h), 3 hours (3 h), 6 hours (6 h), and 24 hours (24 h). 12B is a photograph showing a portion of a 12-well plate used in the examples to confirm long-term degradability, showing the state after 2 days (2d), 7 days (7d), 14 days (14d), and 28 days (28d). 12C is a photograph showing the state immediately after transplanting the silk fibroin tubular body of Example 1 into a sciatic nerve defect site of a Sprague-Dawley (SD) rat in a nerve regeneration test. 12D is a schematic cross-sectional view illustrating how to cut out sagittal and axial sections. 12E is a photograph showing the silk fibroin tubular body of Example 1 removed from the rat one month after transplantation. 12F is a photograph (magnification 4x) showing the state of immunostained vascular endothelial cells and axons in an axial section prepared by removing the silk fibroin tubular body of Example 1 from the rat one month after transplantation. 12G is an enlarged photograph (magnification 20x) showing the state of immunostained axons in the axial section of FIG. 12B. 12A and 12B are photographs (magnification 4x) showing immunostained axons and myelin sheaths in a sagittal section prepared by removing the silk fibroin tubular body of Example 1 from a rat one month after transplantation. 12C and 12D are enlarged photographs (magnification 20x) showing immunostained axons and myelin sheaths in the sagittal section of FIG.13B is a photograph showing the silk fibroin tubular body of Example 1 removed from a rat three months after implantation. 13C is a photograph (magnification 4x) showing the state of immunostained vascular endothelial cells and axons in an axial section prepared by removing the silk fibroin tubular body of Example 1 from a rat three months after implantation. 13D is a photograph (magnification 4x) showing the state of immunostained axons and myelin sheaths in the axial section of FIG. 13B. 13E is an enlarged photograph (magnification 20x) showing the state of immunostained axons in the axial section of FIG. 13B. 13F is a photograph showing the state immediately after implantation of the silk fibroin tubular body of Comparative Example 1 into a rat. 13G is a photograph showing the state of the silk fibroin tubular body of Comparative Example 1 one month after implantation. 13H is a photograph (magnification 4x) showing the state of immunostained myelin sheaths and axons in an axial section prepared by removing the silk fibroin tubular body of Comparative Example 1 from a rat one month after implantation. 13I is a photograph showing the state immediately after implantation of the silicone tubular body of Comparative Example 2 into a rat. 16B is a photograph showing the state of the silicone tubular body of Comparative Example 2 three months after implantation. 16C is a photograph showing the state of the silk fibroin tubular body of Example 2 immediately after implantation into a rat. 16D is a photograph showing the state of the silk fibroin tubular body of Example 2 one month after implantation. 16E is a photograph showing the state of the silk fibroin tubular body of Example 2 three months after implantation. 16F is a photograph (magnification 4x) showing the state of immunostained axons and myelin sheaths in an axial section prepared by removing the silk fibroin tubular body of Example 2 from a rat three months after implantation. 16G is an enlarged photograph (magnification 20x) showing the state of immunostained axons in the axial section of FIG. 16C.

[0019] [Silk fibroin tubular body] The silk fibroin tubular body is a tubular body made of silk fibroin. Here, the tubular body means an elongated shaft with an inner lumen surrounded by an outer wall. The tubular body may be an elongated hollow cylinder as shown in Figure 1, but the cross-sectional shape of the inner lumen and the outer wall may be other shapes than circular, such as an ellipse or a substantially polygonal shape.

[0020] The silk fibroin tubular body has a porous structure. In this specification, the term "porous structure" refers to a structure formed by the presence of numerous minute, irregularly shaped pores, and the porous structure allows liquid to permeate from the outside of the tubular body to the lumen through the pores.

[0021] Silk fibroin mainly contains glycine, alanine, serine, and tyrosine, and is composed of a crystalline portion in which the molecules are regularly arranged and an amorphous portion in which the molecules are randomly arranged. Silk fibroin may be chemically modified to the extent that the effects of the present invention are not impaired. In this specification, when the term "silk fibroin" is used, the definition also includes chemically modified silk fibroin. However, the silk fibroin present on the surface of the film may be silk fibroin that has not been modified with polyethylene glycol (PEG).

[0022] As the raw material for silk fibroin, silk raw materials (such as cocoons and raw silk) produced by insects (Lepidoptera insects such as domestic silkworms, wild silkworms, and wild silkworms, and silkworms that produce silk threads, such as Hymenoptera insects such as hornets and honeybees) or spiders can be used, and are not particularly limited as long as they contain fibroin and sericin. Silk fibroin from which sericin has been removed can be obtained by scouring the silk raw material. Silk fibroin can also be obtained from silk glands.

[0023] Scouring can be carried out by known methods, including various methods such as a method of removing sericin by swelling it with an alkaline scouring agent such as sodium carbonate, sodium silicate, or sodium phosphate, a method of removing sericin by decomposing it with a sericin-degrading enzyme, and a method of removing sericin by decaying it. From the viewpoint of ease of setting conditions, scouring using an alkaline scouring agent is preferred.

[0024] Although it varies depending on the type of silk raw material and scouring agent, in scouring whose main purpose is to remove sericin, from the viewpoint of suppressing the decomposition of silk fibroin, when an alkaline scouring agent is used, the scouring time may be, for example, 5 to 60 minutes, preferably 10 to 50 minutes, and more preferably 15 to 45 minutes. The scouring temperature may be, for example, 90 to 100°C, preferably 93 to 99°C, and more preferably 95 to 98°C.

[0025] Next, the refined silk fibroin is diluted with water and used as a solvent (e.g., methanol, ethanol, propanol, butanol, DMSO (dimethyl sulfoxide), acetone, etc.), and stirred as necessary to prepare a silk fibroin aqueous solution of a predetermined concentration. DMSO is preferred as the water-soluble solvent. The concentration of the water-soluble solvent in the solvent can be appropriately set depending on the type of solvent. For example, the concentration of DMSO may be, for example, 0.5 to 10% by volume, preferably 1 to 5% by volume, and more preferably 1 to 3% by volume.

[0026] The concentration of the silk fibroin aqueous solution may be, for example, 5 to 15 wt %, preferably 5.5 to 13 wt %, more preferably 6 to 10 wt %. Here, the concentration of the silk fibroin aqueous solution refers to the weight ratio of silk fibroin to the total weight of the aqueous solution containing silk fibroin as a solute and a water-soluble solvent and water as a solvent.

[0027] The prepared silk fibroin aqueous solution is poured into a mold designed according to the desired shape of the tubular body. For example, Figures 3 and 4 show an example of a mold for forming a cylindrical tubular body.

[0028] Fig. 3 is a schematic perspective view showing parts for assembling an example of a mold, showing a cylindrical core rod 2 made of Teflon (registered trademark), an aluminum cylinder 4 arranged around the core rod, a base 6 having grooves 6a and 6b for fixing the core rod 2 and the cylinder 4, respectively, and a lid 8 having grooves 8a and 8b (not shown) for fixing the core rod 2 and the cylinder 4, respectively.

[0029] Figure 4 shows a schematic cross-sectional view of an assembled mold 9. As shown in Figure 4, the core rod 2 and cylinder 4 are fixed upright in the grooves 6a and 6b of the base 6, forming a cylindrical cavity 7, into which an aqueous solution is poured. After the aqueous solution is poured, the lid 8 is placed over the mold, and the core rod 2 and cylinder 4 are fixed in the grooves 8a and 8b of the lid 8. The core rod 2 and cylinder 4 are fixed by the base 6 and the lid 8 in a liquid-tight manner. In Figure 4, the aqueous solution is poured into the entire cavity 7 of the mold, and exists as a silk fibroin tubular precursor 5 before the porous structure is formed.

[0030] Thereafter, the mold into which the silk fibroin aqueous solution has been poured is transferred as is to a freezer and subjected to a freezing treatment, for example, at a temperature of −40° C. to −10° C., preferably −30° C. to −15° C. The freezing treatment time may be, for example, 4 hours or more, preferably 5 hours or more. The upper limit of the freezing treatment is not particularly limited and may be, for example, 10 hours. After the freezing treatment, the mold can be removed as appropriate depending on the timing of the thawing treatment.

[0031] After the freezing treatment, a thawing treatment is carried out to obtain a silk fibroin tubular body having a porous structure. The thawing treatment is not particularly limited as long as it can thaw the frozen silk fibroin tubular body, but the thawing treatment can be completed, for example, by leaving the tubular body at room temperature (e.g., 15 to 30°C) for, for example, 60 to 180 minutes.

[0032] After thawing, the silk fibroin tubular body is removed from the mold and washed at room temperature (15 to 30°C). The washing operation is carried out by immersing the silk fibroin tubular body in a desired liquid at a sufficient bath ratio. The bath ratio, expressed as a volume ratio of silk fibroin tubular body to liquid, may be, for example, 1:10 to 1:50, preferably 1:15 to 1:45, and more preferably 1:20 to 1:40. The immersion time may be, for example, 5 to 20 hours, preferably 8 to 18 hours, and more preferably 10 to 16 hours. Such washing operation may be carried out multiple times, for example, 3 to 8 times, and preferably 4 to 7 times.

[0033] After the washing treatment, the silk fibroin tubular body may be stored immersed in a desired liquid until use. From the viewpoint of convenience, the storage temperature may be, for example, 1 to 7°C, preferably 2 to 6°C, and more preferably 3 to 5°C. Examples of the liquid include water, a buffer solution (e.g., phosphate buffered saline), and Ringer's solution.

[0034] In one embodiment, the silk fibroin tubular body has a porous structure, and therefore may contain the liquid after being removed from the liquid in which it is stored.

[0035] The obtained silk fibroin tubular body can be used, for example, as a nerve regeneration-inducing device as described below.

[0036] The silk fibroin tubular body may be distributed in a frozen state in a mold and then thawed and washed before use, or may be distributed in a thawed state in a mold and then thawed and washed before use. Furthermore, as described above, the silk fibroin tubular body may be distributed in a state immersed in a desired liquid after thawing and washing.

[0037] The silk fibroin tubular body of one embodiment may have an appropriate size depending on the target subject and treatment site, and the size is not particularly limited. For example, the inner diameter may be 0.1 to 10 mm, preferably 0.5 to 7 mm, and more preferably 1 to 5 mm. The outer diameter may be 0.5 to 20 mm, preferably 1 to 18 mm, and more preferably 2 to 16 mm. The inner and outer diameters of the silk fibroin tubular body are values ​​measured by the method described in the Examples section below. When the cross section perpendicular to the longitudinal direction of the tubular body is noncircular, the inner and outer diameters may be determined by drawing circumscribing circles around the cross-sectional shapes of the inner and outer walls of the tubular body, and the diameters of the circumscribing circles may be used as the inner and outer diameters, respectively.

[0038] The wall thickness of the tubular body can be set appropriately depending on the inner diameter, and where D (mm) is the size of the inner diameter, it may be 0.05 x D to 0.50 x D mm, preferably 0.10 x D to 0.45 x D mm, and more preferably 0.15 x D to 0.40 x D mm. The wall thickness refers to the thickness measured in the radial direction from the inner diameter to the outer diameter using an electronic caliper or the like at 3 to 10 randomly selected locations (the more complex the shape, the more preferably the number of measurement locations). However, in the case of a simple shape such as a cylinder, the wall thickness may be calculated simply by dividing (outer diameter - inner diameter) by 2, and the value calculated as the ratio to the inner diameter may be used.

[0039] The length of the silk fibroin tubular body in one embodiment can be appropriately determined depending on the length required for the target treatment site, and is not particularly limited, but may be, for example, 5 to 70 mm, preferably 7 to 65 mm, and more preferably 10 to 60 mm. Here, the length of the silk fibroin tubular body is a value measured by the method described in the Examples below.

[0040] In one embodiment, the silk fibroin tubular body is formed with numerous pores surrounded by pore walls, each of which has a high proportion of non-circular pores and a heterogeneous pore size with a mixture of large and small pores. As an indicator of non-circularity, for example, the proportion of pores that are approximately circular, including pore shapes formed by extrapolating lines connecting notches, may be 0 to 30%. Here, "approximately circular" refers to a circular shape described by a line whose distance from the center (r) is in the range of 0.85 × r to 1.15 × r. For example, a perfect circle, where r represents the radius of the circle, is described by a line whose distance is in the range of 1 × r. For example, Figure 2 shows a magnified photograph (400x magnification) of a cross section perpendicular to the longitudinal direction of the tubular body. It can be seen from Figure 2 that the pores have a high proportion of non-circular pores.

[0041] In one embodiment, the silk fibroin tubular body may have a porosity of, for example, 85.00 to 98.00%, preferably 88.00 to 97.00%, and more preferably 90.00 to 96.00%, as measured by the method described in the Examples section below.

[0042] The silk fibroin tubular body of one embodiment may have a patency rate of, for example, 10.00 to 50.00%, preferably 13.00 to 45.00%, and more preferably 15.00 to 40.00% when saturated with water and subjected to a load of 1 N. Here, the patency rate is an index showing that the lumen portion is not excessively crushed even when compressed, and specifically, the patency rate when a load of 1 N is applied is a value measured by the method described in the Examples below.

[0043] In one embodiment, the silk fibroin tubular body may have a stress at 50% compression when saturated with water of, for example, 0.040 to 0.500 N, preferably 0.050 to 0.300 N, and more preferably 0.060 to 0.200 N. Here, the stress at 50% compression is a value measured by the method described in the Examples below.

[0044] In one embodiment, the silk fibroin tubular body may have a stress at 25% compression when saturated with water of, for example, 0.020 to 0.300 N, preferably 0.030 to 0.200 N, and more preferably 0.050 to 0.100 N. Here, the stress at 25% compression is a value measured by the method described in the Examples below.

[0045] In one embodiment, the silk fibroin tubular body may have a recovery rate of, for example, 95.50% or more, preferably 96.00% or more, and more preferably 97.00% or more after being left for 10 minutes after the 50% compression test. Here, the patency rate when a load of 1 N is applied is a value measured by the method described in the Examples below.

[0046] The silk fibroin tubular body of one embodiment may have a water content (water content) of, for example, 85.00 to 98.00%, preferably 88.00 to 97.00%, and more preferably 90.00 to 96.00%. Here, the water content is a value measured by the method described in the Examples section below. Furthermore, it is preferable to measure the water content of the silk fibroin tubular body under normal conditions by immersing it in a sufficient amount of pure water (e.g., a volume at least 20 times the volume of the sample) before measuring the water content, so that as much of the liquid contained therein as possible has been replaced with water.

[0047] The silk fibroin tubular structure of one embodiment has a porous structure and therefore has excellent protein permeability. For example, the protein permeability through the silk fibroin tubular structure can be determined by the method described in the Examples below, using ovalbumin as a standard substance.

[0048] The silk fibroin tubular body of one embodiment is biodegradable, and therefore, as an indicator of short-term degradability, the silk fibroin tubular body of one embodiment may exhibit a fibroin elution rate of, for example, 2.00 to 6.50%, preferably 2.50 to 6.30%, and more preferably 3.00 to 6.00% when stored in 1 mL of a protease XIV / phosphate buffered saline (PBS) solution (2 U / mL) for 24 hours. Here, the fibroin elution rate is a value measured by the method described in the Examples section below.

[0049] In one embodiment, the silk fibroin tubular body can be formed by covering the nerve bundle present at one end of the stump of a torn or missing nerve bundle with one end of the tubular body and fixing it by suturing or the like, and covering the nerve bundle present at the other end of the stump with the other end of the tubular body and fixing it by suturing or the like, thereby forming a state in which both stumps are bridged by the silk fibroin tubular body.

[0050] For example, Fig. 5 is a schematic perspective view illustrating one embodiment of a silk fibroin tubular body fixed to both cut portions of a nerve bundle. Fig. 5 depicts nerve bundle a, which has a 15 mm defect in the nerve, and which is divided into nerve bundle fragments a1 and a2 across the defect. Here, nerve bundle fragment a1 is located on the proximal side of nerve bundle a, and nerve bundle fragment a2 is located on the distal side of nerve bundle a. The distal end of nerve bundle fragment a1 and the proximal end of nerve bundle fragment a2 can each be covered by approximately 2 mm of both ends of the silk fibroin tubular body 10, and the silk fibroin tubular body 10 and the nerve bundle fragments a1 and a2 can be fixed by suturing or the like.

[0051] One embodiment of the silk fibroin tubular body has a predetermined compressive stress against compression, thereby ensuring a lumen portion with voids that promote the infiltration of regenerated tissue. Furthermore, the porous structure allows various nutrients and growth factors to permeate the lumen portion of the silk fibroin tubular body, thereby promoting the induction of vascular endothelial cells and Schwann cells in the lumen portion of the silk fibroin tubular body. Subsequently, the extension of axons and myelin sheaths from the stumps of nerve bundles can be promoted, resulting in excellent nerve regeneration induction ability.

[0052] The silk fibroin tubular body of the present invention can be used to induce regeneration of ruptured or defective nerves (such as various peripheral nerves or spinal nerves) in humans or non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, with mammals such as non-human primates, sheep, dogs, cats, cows, and horses being preferred.

[0053] The present invention will be described in more detail below with reference to examples, but the present invention is not limited by these examples. In the following examples and comparative examples, various physical properties were measured by the following methods.

[0054] In order to remove the influence of the liquid in the silk fibroin tubular body, the silk fibroin tubular body was washed by immersing the silk fibroin tubular body in a bath of 1:20 silk fibroin tubular body:water at room temperature (15 to 30°C) for 10 to 14 hours, and repeating this washing process six times.

[0055] The silk fibroin tubular body was stored immersed in water at 2 to 6° C. until immediately before the physical properties were measured. After being removed from the water, the silk fibroin tubular body was drained, and if necessary, the outer surface of the silk fibroin tubular body was lightly wiped with Kimwipe to remove water droplets adhering to the surface, and then subjected to various measurements.

[0056] [Inner diameter, outer diameter, length] The inner diameter, outer diameter, and length (mm) of the sample were measured using an electronic caliper (manufactured by Mitutoyo, MDC-25MX). The test was repeated three times, and the average value was used.

[0057] [Porosity] The sample was immersed in water with a volume 20 times or more the volume of the sample, and after returning to a normal state, the sample was taken out of the water and gently wiped to remove the surface water. The weight of the sample was then immediately measured, and the amount of the hydrated sample was quantified (X g). The sample was then freeze-dried, and the amount of the non-hydrated sample was quantified (Y g). The densities of the water and hydrated silk fibroin were determined to be 1 g / cm3, respectively. ３ , the density of non-hydrated silk fibroin is 1.2 g / cm ３ The porosity was calculated using the following formula: Porosity (%)={(X−Y / 1.2) / X}×100 The test was repeated three times, and the average value was used.

[0058] [Patency Rate When a 1 N Load is Applied] As shown in Figure 6, a sample was mounted between two smooth and parallel pressure plates of a compression tester. The two pressure plates were a fixed plate on the lower side and a movable plate on the upper side. The movable plate was moved at a rate of 3 mm / min, and a 1 N load was applied to the sample. The outer diameter (Y mm) of the sample on the compression side was measured, and the patency rate was calculated as a ratio to the outer diameter (X mm) of the sample before the load was applied. The test was performed three times, and the average value was used.

[0059] [Stress at 25% Compression and 50% Compression] As shown in Figure 7, a sample was mounted between two smooth, parallel pressure plates of a compression tester. The two pressure plates were a fixed plate (lower) and a movable plate (upper). The movable plate was moved at 3 mm / min. The compressive stress of the sample was measured when it was compressed by 25% of the outer diameter (X mm) of the sample before the load was applied, i.e., when the outer diameter of the sample on the compression side was 0.75X mm. The compressive stress of the sample was measured when it was compressed by 50% of the outer diameter, i.e., when the outer diameter of the sample on the compression side was 0.50X mm. The test was performed three times, and the average value was used.

[0060] [Restoration Rate after 50% Compression] In the same manner as in the measurement of the 50% compressive stress described above, the sample was compressed by 50% of the outer diameter (X mm) of the sample before the load was applied, and then the load was quickly removed. After 10 minutes, the outer diameter (Y mm) of the sample on the compressed side was measured, and the restoration rate (%) was calculated as a ratio to the outer diameter (X mm) of the sample before the load was applied. The test was repeated three times, and the average value was used.

[0061] [Moisture Content] A sample was immersed in water with a volume 20 times or more the volume of the sample, and after returning to a normal state, the sample was removed from the water and gently wiped to remove the surface water. The weight of the sample was then quickly measured, and the amount of the hydrated sample (X g) was determined. The sample was then freeze-dried, and the amount of the non-hydrated sample (Y g) was determined, and the moisture content was calculated using the following formula: Moisture Content (%) = {(X - Y) / X} × 100. The test was performed three times, and the average value was used.

[0062] [Cross-sectional shape] The sample was freeze-dried, and the surface of one side of the sample in a direction perpendicular to the longitudinal direction of the tubular sample (hereinafter referred to as the sample cross section) was observed at 400x magnification using a scanning electron microscope (SEM).

[0063] [Protein Permeability] Figure 8A shows a Franz cell used to test protein permeability. The Franz cell has a donor chamber on the upper side and a receptor chamber on the lower side, with the sample immobilized between them. The sample was cut open in one location longitudinally to form a sheet, and then immobilized between the donor and receptor chambers. FITC (fluorescein isothiocyanate)-labeled ovalbumin was dropped from the donor chamber onto the sample, and the presence or absence of ovalbumin that had migrated to the receptor chamber, which contained water as the receptor liquid, was confirmed by the presence or absence of fluorescence emitted by UV irradiation from the bottom of the receptor chamber.

[0064] [Fibroin elution rate] The sample was quantified (Xg) in a freeze-dried state, and then immersed in 1 mL of protease XIV / phosphate buffered saline (PBS) solution (2 U / mL) at 37 ° C. After immersion, on the first day (24 hours of storage), 100 μL of extract was collected from the immersion solution, and the fibroin in the extract was quantified (Yg), and the elution rate (Y / X) was calculated. A BCA Protein Assay Kit was used to quantify the fibroin. The test was performed three times, and the average value was used.

[0065] [Long-term degradability] Three samples of each type were placed in each well of a 24-well plate (well diameter: 15.7 mm), 1 mL of water was added to each well, and the degradability of the samples was visually evaluated over time (0, 3, and 6 hours, and 1, 2, 7, 14, and 28 days) using the following five-point scale. The evaluation scale indicates an increasing degree of decomposition, with scores ranging from 1 to 5. The average score for the three samples was used. 1: The sample was white and opaque, and retained its shape. 2: The sample had changed color from white, but retained its shape. 3: At least some parts of the sample had become transparent, but retained their shape. 4: At least some of the sample had disintegrated. 5: The sample had completely disintegrated.

[0066] [Nerve regeneration test] A sciatic nerve regeneration test was performed on Sprague-Dawley (SD) rats (8 weeks old, male) using the sample. The left sciatic nerve of each rat was partially resected to create a 15 mm defect. A 19 mm silk fibroin tubular body was inserted into the defect, covering both cut ends of the defect by 2 mm each. As shown in Figure 10, the silk fibroin tubular body and both cut ends of the sciatic nerve were fixed by multiple sutures. The transplants were evaluated in 10 rats.

[0067] The implanted area was excised one and three months after implantation. Sagittal sections were prepared at the proximal half of the excised area, and axial sections were prepared at approximately the center of the excised area, as shown in Figure 11. Nerve fiber regeneration was confirmed by immunohistochemical staining. Myelin sheaths were confirmed by MBP, axons by NFH, and vascular endothelium by CD31. Cell nuclei proliferation was also confirmed by Hoechst staining.

[0068] Example 1 Domestic silkworm cocoons were cut into small pieces and degummed in a boiling 0.02 M aqueous sodium carbonate solution for 30 minutes to obtain degummed silk fibroin.

[0069] Purified silk fibroin (3 g) was placed in a beaker and dissolved in 50 ml of 9 M lithium bromide aqueous solution at room temperature for 12 hours or more. The dissolved silk fibroin lithium bromide solution was dialyzed against pure water using a cellulose dialysis membrane (Visking tube, molecular weight cutoff approximately 12,000-14,000) to prepare a silk fibroin aqueous solution. The resulting fibroin aqueous solution was concentrated, and then gradually mixed with a DMSO aqueous solution diluted to a final concentration of 1 vol% to prepare a 6 wt% silk fibroin / DMSO solution.

[0070] The obtained aqueous solution was poured into a cylindrical mold shown in Figure 4, and the mold was sealed with a lid. The mold was then placed in a programmable precision low-temperature constant-temperature water bath freezer (EYELA) and cooled from room temperature to -20°C over 6 hours, and then allowed to stand at -20°C for 6 hours or more. The mold was then placed at room temperature, and the contents within the mold were thawed to obtain a silk fibroin tubular body with a porous structure.

[0071] The silk fibroin tubular body was washed by immersing it in water at a bath ratio of 1:20 and leaving it for 10 to 14 hours. This washing procedure was repeated six times. Thereafter, the silk fibroin tubular body was stored in water at 4°C until use.

[0072] The silk fibroin tubular bodies used in the nerve regeneration test had an inner diameter of 3 mm, an outer diameter of 5 mm, and a length of 19 mm, while the silk fibroin tubular bodies used in other tests had an inner diameter of 3 mm, an outer diameter of 5 mm, and a length of 10 mm.

[0073] Example 2 A silk fibroin tubular body was produced in the same manner as in Example 1, except that an aqueous solution with a silk fibroin concentration of 8 wt % was used.

[0074] Comparative Example 1 A silk fibroin tubular body was produced in the same manner as in Example 1, except that an aqueous solution with a silk fibroin concentration of 4 wt % was used.

[0075] Comparative Example 2 A silicone tube (inner diameter 3 mm, outer diameter 5 mm, length 19 mm) was used in the nerve regeneration test, and a silicone tube (inner diameter 3 mm, outer diameter 5 mm, length 10 mm) was used in some of the physical property tests.

[0076] Table 1 shows various physical properties measured in the examples and comparative examples.

[0077] As shown in Table 1, Examples 1 and 2 showed higher patency rates than Comparative Example 1 even when a 1 N load was applied, and also showed extremely high stress values ​​against 25% compression. On the other hand, Comparative Example 2, which is a silicone tube, showed a higher patency rate when a 1 N load was applied than Examples 1 and 2, but because it had no voids and was therefore impermeable to proteins, as described below, almost no tissue regeneration was observed in the central portion of the tubular body in nerve regeneration tests. Regarding the long-term degradability score, in Comparative Example 1, the tubular body began to discolor 6 hours after immersion, partially disintegrated by 7 days, and completely disintegrated by 14 days, while in both Examples 1 and 2, the tubular body was able to suppress complete disintegration even 28 days after immersion.

[0078] The results of the nerve regeneration test in Example 1 are shown in Figures 12A to 12E. Figure 12A is a photograph showing the silk fibroin tubular body removed from the rat one month after transplantation. The silk fibroin tubular body was removed with its lumen maintained throughout and without adhesion to the outside.

[0079] Figure 12B is a photograph (magnification 4x) showing immunostained vascular endothelial cells and axons in an axial section prepared by removing the silk fibroin tubular body of Example 1 from a rat one month after implantation. In the axial section, the lumen of the silk fibroin tubular body was slightly deformed but maintained without collapse, and it was confirmed that the lumen was filled with vascular endothelial cells and that an axon was present in the center of the lumen. Furthermore, as shown in Figure 12C, a magnified photograph (magnification 20x) showing the immunostained axons in the axial section confirmed that axons had regenerated well in the axial section.

[0080] As shown in Figure 12D, in the sagittal section, it was confirmed that vascular endothelial cells extended along the inner wall of the silk fibroin tubular body, and axons extended in a manner that followed them. Furthermore, as shown in Figure 12E, a magnified photograph (20x magnification) showing the immunostained axons and myelin sheaths in the sagittal section confirmed that the axons and myelin sheaths had regenerated well in the sagittal section.

[0081] The results of the nerve regeneration test in Example 1 are shown in Figures 13A to 13D. Figure 13A is a photograph showing the silk fibroin tubular body removed from the rat three months after transplantation. The silk fibroin tubular body was removed with its lumen maintained throughout and without adhesion to the outside.

[0082] 13B is a photograph (magnification 4x) showing immunostained vascular endothelial cells and axons in an axial section prepared by removing the silk fibroin tubular body of Example 1 from the rat three months after implantation. In the axial section, the lumen of the silk fibroin tubular body was slightly deformed but maintained without collapse, and vascular endothelial cells were observed to have proliferated in the lumen, confirming that axons had regenerated well in the central part.

[0083] Next, as shown in Figure 13C, a photograph (4x magnification) showing the state of immunostained axons and myelin sheaths in an axial section confirmed that myelin sheaths had regenerated well around the axon located in the center.

[0084] In the enlarged photograph (magnification 20x) shown in Figure 13D, good axonal regeneration was confirmed in the axial section, and the presence of vascular endothelial cells proliferating around the axons was also confirmed.

[0085] The results of the nerve regeneration test of Comparative Example 1 are shown in Figures 14A to 14C. Figure 14A is a photograph showing the state immediately after implantation of the silk fibroin tubular body into a rat, and Figure 14B is a photograph showing the state one month after implantation. Compared to the state of the silk fibroin tubular body of Example 1 one month after implantation shown in Figure 12A, the lumen portion of the silk fibroin tubular body one month after implantation shown in Figure 14B was significantly collapsed. Furthermore, as shown in Figure 14C, only slight axon regeneration was observed. Furthermore, no myelin regeneration was observed.

[0086] The results of the nerve regeneration test for Comparative Example 2 are shown in Figures 15A and 15B. Figure 15A is a photograph showing the condition immediately after implantation of the silicone tubular body into a rat, and Figure 15B is a photograph showing the condition three months after implantation. As shown in Figure 15B, three months after implantation, the silicone tubular body was cut open to examine the lumen, and almost no tissue regeneration was observed in the central part of the tubular body.

[0087] The results of the nerve regeneration test of Example 2 are shown in Figures 16A to 16E. Figure 16A is a photograph showing the condition immediately after transplantation of the silk fibroin tubular body into a rat, Figure 16B is a photograph showing the condition one month after transplantation, and Figure 16C is a photograph showing the condition three months after transplantation. Figure 16D is a photograph (4x magnification) showing the immunostained axons and myelin sheaths in an axial section, and Figure 16E is an enlarged photograph (20x magnification).

[0088] Even one month after transplantation, the silk fibroin tubular structure of Example 2 maintained its lumen without collapse, as in Comparative Example 1. Furthermore, three months after transplantation, the silk fibroin tubular structure could be removed without adhesion to the outside, with the lumen maintained throughout. Immunostaining confirmed good regeneration of axons and myelin sheaths.

[0089] The above-mentioned tests were conducted on the regenerative ability of peripheral nervous tissue, but because various nutrients and growth factors can be permeated into the lumen of the silk fibroin tubular body to promote the induction of vascular endothelial cells, it is predicted that the silk fibroin tubular body will also be highly effective in regenerating other tissues and organs, such as the central nervous system, ligaments, and tendons.

[0090] As described above, the preferred embodiment of the present invention has been described, but various additions, modifications, or deletions can be made without departing from the spirit of the present invention, and such additions, modifications, or deletions are also included within the scope of the present invention.

[0091] 2: Core rod 4: Cylinder 5: Precursor of silk fibroin tubular body 6: Base 6a, 6b: Groove in base 7: Void portion 8: Lid 8a, 8b: Groove in lid 9: Mold 10: Silk fibroin tubular body a: Nerve bundle having a defect site a1: Proximal fragment of nerve bundle a a2: Distal fragment of nerve bundle a

Claims

1. A silk fibroin tubular body which has a porous structure at room temperature and has a patency rate of 10.00 to 50.00% when a load of 1 N is applied when saturated with water.

2. The silk fibroin tubular body according to claim 1, which has a compressive stress of 0.020 to 0.300 N when compressed 25% in a water-saturated state.

3. The silk fibroin tubular body according to claim 1 or 2, which has a compressive stress of 0.040 to 0.500 N when compressed 50% in a water-saturated state.

4. A silk fibroin tubular body according to any one of claims 1 to 3, which has a recovery rate of 95.50% or more after being left for 10 minutes after a 50% compression test.

5. The silk fibroin tubular body according to any one of claims 1 to 4, wherein the fibroin dissolution rate when stored for 24 hours in 1 mL of a protease XIV / phosphate buffered saline (PBS) solution (2 U / mL) is 2.00 to 7.00%.

6. A silk fibroin tubular body according to any one of claims 1 to 5, having protein permeability using ovalbumin as a standard substance.

7. A silk fibroin tubular body according to any one of claims 1 to 6, which is used as a nerve regeneration inducer.

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