Silk fibroin tubular body

Abstract

To provide a silk fibroin tubular body used for reproducing a biological tissue.SOLUTION: The silk fibroin tubular body exhibits a porous structure at room temperature, and the patency rate when a 1 N load is applied to the silk fibroin tubular body in a water-saturated state is 10% to 50%.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a silk fibroin tubular body used for regenerating a living tissue such as nerve tissue.

Background Art

[0002] When a nerve is damaged by external factors (e.g., trauma, surgery) or internal factors (e.g., malignant tumor), it causes significant obstacles in daily life due to motor sensory disorders, pain, numbness, etc. Also, in the case of extensive nerve defects, although autologous nerve transplantation, which involves transplanting the patient's own nerve to the defect area, is the standard treatment, this method has drawbacks such as functional deficiency in the donor area because the patient's own nerve is taken from another part, and there is a limit to the length of nerve that can be harvested. Furthermore, since the surgery time for harvesting the patient's own nerve is long, there is a problem of a large burden on both the doctor and the patient.

[0003] Therefore, there is a need for a nerve regeneration device for regenerating nerve tissue. For example, in Patent Document 1, a support (A) formed of a biodegradable material or a bioabsorbable material is disclosed as a device for regenerating a human tissue or organ, such as nerve fibers or microvessels, that has been severed due to a lesion or injury. The device for regenerating a living tissue or organ is characterized by comprising a sponge-like fine matrix (B) formed of a biodegradable material or a bioabsorbable material and a linear living tissue or organ induction path (C).

[0004] In this regeneration device, the sponge-like fine matrix and the linear induction path formed in the lumen can be used as scaffolds for cell regeneration, and the regeneration of living tissues or organs is carried out.

[0005] In Non-Patent Document 1, after degumming treatment with a 0.1 wt% solution of Na2CO3 or NaHCO3, a silk fibroin solution obtained by dissolving the degummed product in 9.3 M LiBr at 60°C over 2 hours was injected into a tube-shaped mold and then subjected to a freezing process. The resulting silk fibroin tube was shown to have stretchability in the longitudinal direction and biodegradability.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Non-Patent Documents

[0007]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0008] However, in Patent Document 1, although a sponge-like fine matrix and a linear guiding path can be used as scaffolds, due to their presence, the void space decreases, and it is impossible to deny the possibility that the entry of regenerated tissue is inhibited, and further improvement is required.

[0009] In addition, although Non-Patent Document 1 focuses on the stretchability of the silk fibroin tube in the longitudinal direction, such a tube has low compressive resistance in the direction orthogonal to the longitudinal direction as it has stretchability, so it is easily crushed by external pressure and cannot ensure the void space after being implanted in the body.

[0010] Accordingly, one object of the present invention is to provide a silk fibroin tubular body having a hollow structure that promotes the entry of regenerated tissue and having excellent ability to induce the regeneration of living tissues such as nerve tissue.

Means for Solving the Problems

[0011] As a result of intensive studies to solve the above-described problems of the prior art, the present inventors have found that a tubular body composed of silk fibroin that is not easily crushed even when a load is applied has an excellent ability to regenerate living tissues such as nerve tissue while maintaining a hollow structure after implantation into the body, and have completed the present invention.

[0012] That is, the present invention can be configured in the following aspects. 〔Aspect 1〕 A silk fibroin tubular body having a porous structure at room temperature and having a survival rate of 10.00 to 50.00% (preferably 13.00 to 45.00%, more preferably 15.00 to 40.00%) when a 1N load is applied in a state saturated with water. 〔Aspect 2〕 In the silk fibroin tubular body of Aspect 1, the compression stress when compressed by 25% in a state 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), a silk fibroin tubular body. 〔Aspect 3〕 In the silk fibroin tubular body of Aspect 1 or 2, the compression stress when compressed by 50% in a state saturated with water is 0.040 to 0.500 N (preferably 0.050 to 0.300 N, more preferably 0.060 to 0.200 N), a silk fibroin tubular body. 〔Aspect 4〕 In the silk fibroin tubular body of Aspects 1 to 3, after performing a 50% compression test, the recovery rate after leaving it for 10 minutes is 95.50% or more (preferably 96.00% or more, more preferably 97.00% or more), a silk fibroin tubular body. 〔Aspect 5〕 In the silk fibroin tubular body of any one of Aspects 1 to 4, the fibroin elution rate when stored in water for 24 hours is 2.00 to 6.50% (preferably 2.50 to 6.30%, more preferably 3.00 to 6.00%), the silk fibroin tubular body. [Aspect 6] In the silk fibroin tubular body of any one of Aspects 1 to 5, the silk fibroin tubular body having protein permeability using ovalbumin as a standard substance. [Aspect 7] In the silk fibroin tubular body of any one of Aspects 1 to 6, the silk fibroin tubular body used as a nerve regeneration inducer.

Advantages of the Invention

[0013] In the silk fibroin tubular body of the present invention, by transplanting the silk fibroin tubular body in a state of bridging both cut ends of a severed or damaged nerve, not only can nerve regeneration be induced in the hollow part of the tubular body, but after a predetermined period has elapsed, Since the silk fibroin tubular body is decomposed and absorbed, the harmfulness to the living body can be reduced.

Brief Description of the Drawings

[0014] This 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 only and should not be used to define the scope of this invention. The scope of this invention is determined by the appended claims.

[0015]

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Mode for Carrying Out the Invention

[0016] [Silk Fibroin Tubular Body] The silk fibroin tubular body is a tubular body composed of silk fibroin. Here, the tubular body means an elongated shaft whose inner cavity is surrounded by an outer wall. The tubular body may be an elongated hollow cylinder as shown in FIG. 1, but the cross-sectional shapes of the inner cavity and the outer wall may be other shapes than circular, for example, elliptical, substantially polygonal, etc.

[0017] The silk fibroin tubular body has a porous structure. In this specification, the porous structure is a structure formed by the presence of a large number of fine amorphous pores, and by having the porous structure, liquid can permeate from the outside of the tubular body into the inner cavity part through the pores.

[0018] Silk fibroin mainly contains glycine, alanine, serine, and tyrosine, and is composed of a crystalline part where the molecules are regularly arranged and an amorphous part with a random arrangement. It is usually known as a type of fibrous protein. Note that silk fibroin may be chemically modified as long as it does not inhibit the effects of the present invention. In this specification, when simply described as "silk fibroin", its definition also includes chemically modified silk fibroin. However, the silk fibroin present on the film surface may be silk fibroin that has not been modified by polyethylene glycol (PEG).

[0019] As the silk fibroin raw material, silk raw materials (cocoons, raw silk, etc.) produced by insects (Lepidoptera insects such as silkworms, wild silkworms, and tussah silkworms, and Hymenoptera insects such as bees and wasps) or spiders can be used, and there is no particular limitation as long as it is a silk raw material containing fibroin and sericin. By refining the silk raw material, silk fibroin from which sericin has been removed can be obtained. Also, silk fibroin can be obtained from the silk gland.

[0020] Refining can be carried out by known methods. For example, methods such as swelling and removing sericin using an alkaline refining agent such as sodium carbonate, sodium silicate, or sodium phosphate, decomposing and removing sericin using a sericin-degrading enzyme, and rotting and removing sericin can be mentioned. From the perspective of simplicity in setting conditions, refining using an alkaline refining agent is preferred.

[0021] Although it varies depending on the type of silk raw material and refining agent, etc., in refining mainly aimed at removing sericin, from the perspective of suppressing the decomposition of silk fibroin, for example, when using an alkaline refining agent, the refining time may be, for example, 5 to 60 minutes, preferably 10 to 50 minutes, and more preferably 15 to 45 minutes. Also, the temperature during refining may be, for example, 90 to 100 °C, preferably 93 to 99 °C, and more preferably 95 to 98 °C.

[0022] Next, for the refined silk fibroin, a water-soluble solvent diluted with water (such as methanol, ethanol, propanol, butanol, DMSO (dimethyl sulfoxide), acetone, etc.) is used as a solvent, and stirring is performed as necessary to prepare an aqueous silk fibroin solution with 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 according to the type of the solvent. For example, the concentration of DMSO can be, for example, 0.5 to 10% by volume, preferably 1 to 5% by volume, more preferably 1 to 3% by volume.

[0023] The concentration of the aqueous silk fibroin solution can be, for example, 5 to 15% by weight, preferably 5.5 to 13% by weight, more preferably 6 to 10% by weight. Here, the concentration of the aqueous silk fibroin solution means the proportion of the weight of silk fibroin in the total weight of the aqueous solution containing silk fibroin as a solute, a water-soluble solvent, and water as a solvent.

[0024] The prepared aqueous silk fibroin solution is injected into a mold designed according to the shape of the target tubular body. For example, FIGS. 3 and 4 illustrate an example of a mold for forming a cylindrical tubular body.

[0025] FIG. 3 is a schematic perspective view for showing the components for assembling an example of the mold. FIG. 3 shows a cylindrical mandrel 2 made of Teflon, an aluminum cylinder 4 disposed around the mandrel, a base 6 having grooves 6a and 6b for fixing the mandrel 2 and the cylinder 4 respectively, and a lid 8 having grooves 8a and 8b (not shown) for fixing the mandrel 2 and the cylinder 4 respectively.

[0026] Figure 4 shows a schematic cross-sectional view of the assembled mold 9. As shown in Figure 4, by fixing the mandrel 2 and the cylinder 4 to the grooves 6a and 6b of the base 6 and standing them upright, a cylindrical void 7 is formed, and an aqueous solution is injected into this void 7. After injecting the aqueous solution, a lid 8 is put on, and the mandrel 2 and the cylinder 4 are fixed to the grooves 8a and 8b of the lid 8. Note that the mandrel 2 and the cylinder 4 are fixed by the base 6 and the lid 8 in a state where there is no liquid leakage. In Figure 4, the aqueous solution is injected into the entire void 7 of the mold and exists as a precursor 5 of the silk fibroin tubular body before forming a porous structure.

[0027] Thereafter, the mold into which the silk fibroin aqueous solution has been injected is directly transferred to a freezer, and freezing treatment is performed, for example, at -40°C to -10°C, preferably at -30°C to -15°C. The time for the freezing treatment 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 it may be, for example, 10 hours. After the freezing treatment, it can be taken out as appropriate according to the timing of the thawing treatment.

[0028] After the freezing treatment, a thawing treatment is performed, and a silk fibroin tubular body having a porous structure can be obtained. The thawing treatment is not particularly limited as long as the frozen silk fibroin tubular body can be thawed. For example, at room temperature (for example, 15 to 30°C), the thawing treatment is completed by standing for, for example, 60 to 180 minutes.

[0029] After the thawing treatment, the silk fibroin tubular body is taken out from the mold and washed at room temperature (15 to 30°C). The washing operation is performed by immersing the silk fibroin tubular body in a desired liquid at a sufficient bath ratio. The bath ratio may be, for example, a volume ratio of (silk fibroin tubular body: liquid) of 1:10 to 1:50, preferably 1:15 to 1:45, more preferably 1:20 to 1:40. The immersion time may be, for example, 5 to 20 hours, preferably 8 to 18 hours, more preferably 10 to 16 hours. Such a washing operation may be performed multiple times, for example, 3 to 8 times, preferably 4 to 7 times.

[0030] After the washing treatment, the silk fibroin tubular body may be stored until use while immersed in a desired liquid. From the viewpoint of simplicity, the storage temperature may be, for example, 1 to 7 °C, preferably 2 to 6 °C, and more preferably 3 to 5 °C. For example, examples of the liquid include water, a buffer solution (for example, phosphate buffered saline), and Ringer's solution.

[0031] Since the silk fibroin tubular body of one embodiment has a porous structure, it may contain the above-described liquid after being taken out of the stored liquid.

[0032] The obtained silk fibroin tubular body can be used as, for example, a nerve regeneration induction device as described below.

[0033] Note that the silk fibroin tubular body may be distributed in a state where the frozen silk fibroin tubular body is placed in a mold, and thawed and washed at the time of use for use, or the silk fibroin tubular body in a thawed state may be distributed in a state where it is placed in a mold, and thawed and washed at the time of use for use. Further, as described above, it may be distributed in a state of being immersed in a desired liquid after thawing and washing treatments.

[0034] The silk fibroin tubular body of one embodiment only needs to have an appropriate size according to the target object and the treatment site, and its 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. Also, the outer diameter may be 0.5 to 20 mm, preferably 1 to 18 mm, and more preferably 2 to 16 mm. Here, the inner diameter and outer diameter of the silk fibroin tubular body are values measured by the method described in the examples below. Note that when the cross-section orthogonal to the longitudinal direction of the tubular body is non-circular, a circumscribed circle may be drawn for each cross-sectional shape of the inner wall and outer wall of the tubular body, and the diameter of the circumscribed circle may be used as the inner diameter and outer diameter, respectively.

[0035] In addition, the wall thickness of the tubular body can be appropriately set according to the inner diameter. When the size of the inner diameter is D (mm), it may be 0.05×D to 0.50×D mm, preferably 0.10×D to 0.45×D mm, and more preferably 0.15×D to 0.40×D mm. The wall thickness means the thickness measured in the radial direction from the inner diameter to the outer diameter by randomly selecting 3 to 10 locations (the more complex the shape, the more measurement locations are preferred) using an electronic caliper or the like. In the case of a simple shape such as a cylinder, the value calculated simply by (outer diameter - inner diameter)÷2 may be used as the wall thickness, and the value calculated as a ratio to the inner diameter may also be used.

[0036] The length of the silk fibroin tubular body in one aspect can be appropriately determined according to the length required at the target treatment site and is not particularly limited. For example, it may be 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.

[0037] In one aspect, the silk fibroin tubular body is formed of a large number of pores having a high proportion of non-circular shapes and uneven pore diameters of various sizes, surrounded by pore walls. As an index of non-circularity, for example, the proportion of pores that are substantially circular among the pore shapes including the shape supplemented by the extrapolated line connecting the notches may be 0 to 30%. Here, "substantially circular" means a circular shape drawn by a line where the distance (r) from the center is in the range of 0.85×r to 1.15×r. For example, in a perfect circle, r means the radius of the circle, and it becomes a circle drawn by a line in the range of 1×r. For example, in FIG. 2, an enlarged photograph (400 times) observed in a cross section perpendicular to the longitudinal direction of the tubular body is shown. It can be seen from FIG. 2 that the proportion of non-circular pores is high.

[0038] In one aspect, the porosity of the silk fibroin tubular body may be, for example, 85.00 to 98.00%, preferably 88.00 to 97.00%, and more preferably 90.00 to 96.00%. Here, the porosity is a value measured by the method described in the examples below.

[0039] In one aspect, the silk fibroin tubular body may have a survival rate of, for example, 10.00 to 50.00%, preferably 13.00 to 45.00%, more preferably 15.00 to 40.00% when a 1N load is applied in a water-saturated state. Here, the survival rate is an index indicating that the inner cavity portion is not excessively crushed even when compressed, and specifically, the survival rate when a 1N load is applied is a value measured by the method described in the examples below.

[0040] In one aspect, the silk fibroin tubular body may have a stress against 50% compression in a water-saturated state of, for example, 0.040 to 0.500 N, preferably 0.050 to 0.300 N, more preferably 0.060 to 0.200 N. Here, the stress against 50% compression is a value measured by the method described in the examples below.

[0041] In one aspect, the silk fibroin tubular body may have a stress against 25% compression in a water-saturated state of, for example, 0.020 to 0.300 N, preferably 0.030 to 0.200 N, more preferably 0.050 to 0.100 N. Here, the stress against 25% compression is a value measured by the method described in the examples below.

[0042] In one aspect, after performing the 50% compression test, the silk fibroin tubular body may have a recovery rate of, for example, 95.50% or more, preferably 96.00% or more, more preferably 97.00% or more after being left for 10 minutes. Here, the survival rate when a 1N load is applied is a value measured by the method described in the examples below.

[0043] The silk fibroin tubular body of one embodiment may have a water content ratio (water content rate) of, for example, 85.00 to 98.00%, preferably 88.00 to 97.00%, more preferably 90.00 to 96.00%. Here, the water content rate is a value measured by the method described in the examples below. Also, the silk fibroin tubular body is preferably measured in a normal state in which the liquid contained inside is replaced with water as much as possible by immersing it in a sufficient amount (for example, a volume 20 times or more the volume of the sample) of pure water before measuring the water content rate.

[0044] Since the silk fibroin tubular body of one embodiment has a porous structure, it has excellent protein permeability. For example, using ovalbumin as a standard substance, the permeability of the protein through the silk fibroin tubular body can be grasped by the method described in the examples below.

[0045] Since the silk fibroin tubular body of one embodiment has biodegradability, as an index of short-term degradability, the fibroin elution rate when stored in water for 24 hours may be, for example, 2.00 to 6.50%, preferably 2.50 to 6.30%, more preferably 3.00 to 6.00%. Here, the fibroin elution rate is a value measured by the method described in the examples below.

[0046] The silk fibroin tubular body of one embodiment can cover the nerve bundle existing at one end of the severed or damaged nerve bundle stump with one end of the tubular body and fix it by suturing or the like, cover the nerve bundle existing at the other end of the stump with the other end of the tubular body and fix it by suturing or the like, and form a state in which both ends of the severed end are bridged by the silk fibroin tubular body.

[0047] For example, FIG. 5 is a schematic perspective view for explaining a state in which a silk fibroin tubular body of one aspect is fixed to both cut ends of a nerve bundle. In FIG. 5, a nerve bundle a having a 15-mm defect site is shown, which is divided into a nerve bundle fragment a1 and a nerve bundle fragment a2 via the defect site. Here, the nerve bundle fragment a1 is located on the proximal side of the nerve bundle a, and the nerve bundle fragment a2 is located on the distal side of the nerve bundle. The distal end of the nerve bundle fragment a1 and the proximal end of the nerve bundle fragment a2 are each covered by about 2 mm at 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.

[0048] Since the silk fibroin tubular body of one aspect has a predetermined compressive stress against compression, it is possible to secure a lumen portion having a void portion that promotes the entry of regenerated tissue. Furthermore, because it has a porous structure, it is possible to permeate various nutrients and growth factors into the lumen portion of the silk fibroin tubular body, and in the lumen portion of the silk fibroin tubular body, it is possible to promote the induction of vascular endothelial cells and Schwann cells. Subsequently, it is possible to promote the extension of axons and myelin sheaths from the cut end of the nerve bundle, and as a result, it is possible to exhibit excellent nerve regeneration induction ability.

[0049] The silk fibroin tubular body of the present invention can be used to induce regeneration against fractures and defects of nerves (such as various peripheral nerves or spinal nerves) in humans or non-human animals. Here, non-human animals include all vertebrates, for example, mammals and non-mammals such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, but mammals such as non-human primates, sheep, dogs, cats, cows, and horses are preferred.

Examples

[0050] Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited by these. In the following examples and comparative examples, various physical properties were measured by the following methods.

[0051] In addition, in order to remove the influence of the liquid in the tubular body, the silk fibroin tubular body used was washed 6 times by immersing the silk fibroin tubular body at a bath ratio of silk fibroin tubular body: water of 1:20 for 10 to 14 hours at room temperature (15 to 30 °C).

[0052] Also, until immediately before measuring the physical properties, the silk fibroin tubular body was stored at 2 to 6 °C while immersed in water. After taking it out of the water, the silk fibroin tubular body was drained, and if necessary, the outer surface of the silk fibroin tubular body was gently wiped with Kimwipe to remove the water droplets adhering to the surface and used for various measurements.

[0053] [Inner diameter, outer diameter, length] The inner diameter, outer diameter, and length (mm) of the sample were measured using an electronic caliper (Mitutoyo, MDC-25MX). The test was performed 3 times and the average value was adopted.

[0054] [Porosity] The sample was immersed in water with a volume 20 times or more that of the sample and left in a normal state. Then, the sample was taken out of the water and the water on the surface was removed by gently wiping. Subsequently, the weight of the sample was quickly measured to quantify the sample in the water-containing state (X g). Next, the sample was freeze-dried to quantify the sample in the non-water-containing state (Y g). Assuming the density of water and silk fibroin in the water-containing state is 1 g / cm 3 , and the density of silk fibroin in the non-water-containing state is 1.2 g / cm 3 , the porosity was calculated using the following formula: Porosity (%) = {(X - Y / 1.2) / X} × 100 The test was performed 3 times and the average value was adopted.

[0055] [Survival rate when a 1 N load is applied] As shown in Fig. 6, the sample was placed between two smooth and parallel pressure plates of a compression testing machine. The two pressure plates were such that the lower one was a fixed plate and the upper one was a moving plate. The moving plate was moved at a rate of 3 mm / min, and when a 1 N load was applied to the sample, the outer diameter (Y mm) of the sample in the compression direction was measured, and the survival rate was calculated as the ratio to the outer diameter (X mm) of the sample before the load was applied. The test was conducted 3 times and the average value was adopted.

[0056] [Stress for 25% Compression and 50% Compression] As shown in Fig. 7, the sample was placed between two smooth and parallel pressure plates of a compression testing machine. The two pressure plates were such that the lower one was a fixed plate and the upper one was a moving plate. The moving plate was moved at a rate of 3 mm / min, and when the sample was compressed by 25% with respect to the outer diameter (X mm) of the sample before the load was applied, in other words, when the outer diameter of the sample in the compression direction was 0.75X mm, the compression stress shown by the sample was measured as the 25% compression stress. Also, when the sample was compressed by 50%, in other words, when the outer diameter of the sample in the compression direction was 0.50X mm, the compression stress shown by the sample was measured as the 50% compression stress. The test was conducted 3 times and the average value was adopted.

[0057] [Recovery Rate after 50% Compression] In the same manner as when measuring the above-mentioned 50% compression stress, after the sample was compressed by 50% with respect to the outer diameter (X mm) of the sample before the load was applied, the load was quickly removed, and the outer diameter (Y mm) of the sample in the compression direction after 10 minutes was measured, and the recovery rate (%) was calculated as the ratio to the outer diameter (X mm) of the sample before the load was applied. The test was conducted 3 times and the average value was adopted.

[0058] [Water Content] The sample was immersed in water with a volume more than 20 times that of the sample to bring it to a normal state. Then, the sample was taken out of the water and the surface water was removed by gently blotting. Thereafter, the weight of the sample was quickly measured to quantify the sample in the water-containing state (X g). Next, the sample was freeze-dried to quantify the sample in the non-water-containing state (Y g), and the water content was calculated using the following formula: Water content (%) = {(X - Y) / X} × 100 It was calculated as such. The test was conducted in triplicate, and the average value was adopted.

[0059] [Cross-sectional shape] The sample was freeze-dried, and in the direction orthogonal to the longitudinal direction of the tubular sample, the surface of the sample on one side (hereinafter referred to as the sample cross-section) was observed at 400 times magnification with a scanning electron microscope (SEM).

[0060] [Protein permeability] Figure 8A shows a Franz cell for conducting a protein permeability test. The Franz cell is equipped with a donor chamber on the upper side and a receptor chamber on the lower side, and a sample is fixed between them. After the sample was cut open at one location in the longitudinal direction to form a sheet shape, the sample was fixed between the donor chamber and the receptor chamber. FITC (Fluorescein Isothiocyanate)-labeled ovalbumin was dropped from the donor chamber towards the sample, and the presence or absence of ovalbumin that had moved to the receptor chamber containing water as the receiving solution was confirmed by the presence or absence of fluorescence emission by UV irradiation from the bottom of the receptor chamber.

[0061] [Fibroin elution rate] The sample in the freeze-dried state was quantified (X g), 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, 100 μL of the extract was collected from the immersion solution, the fibroin in the extract was quantified (Y g), and the elution rate (Y / X) was calculated. For the quantification of fibroin, a BCA Protein Assay Kit was used. The test was conducted in triplicate, and the average value was adopted.

[0062] [Long-term degradability] Three samples were placed one by one in each well of a 24-well plate (each well diameter: 15.7 mm), 1 mL of water was added to each well, and the degradability of the samples was visually evaluated in the following 5 grades over time (0, 3, and 6 hours, 1, 2, 7, 14, and 28 days). The evaluation criteria indicate that the degree of degradation progresses as the grade goes from 1 to 5. The average value of the three samples was used for the test. 1: The sample is white and opaque and retains its shape. 2: The sample has changed color from white but retains its shape. 3: At least a part of the sample is transparent, but it retains its shape. 4: At least a part of the sample has collapsed. 5: The sample has completely collapsed.

[0063] [Nerve Regeneration Test] A sciatic nerve regeneration test was conducted on Sprague-Dawley (SD) rats (8 weeks old, male) using the samples. A part of the left sciatic nerve of the rat was resected to create a 15-mm defect site, and a 19-mm silk fibroin tubular body was introduced into this defect site with both cut ends of the defect site covered 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 suturing at multiple locations. A total of 10 rats were transplanted and evaluated.

[0064] One month and three months after implantation, the implanted part was excised. As shown in Figure 11, a sagittal section was made at the 1 / 2 position from the proximal side of the excised part, and an axial section was made at approximately the central part of the excised part to confirm nerve fiber regeneration by immunohistochemical staining. The myelin sheath was confirmed by MBP, the axon was confirmed by NFH, and the vascular endothelium was confirmed by CD31. In addition, cell nucleus proliferation was confirmed by Hoechst.

[0065] (Example 1) The cocoons of silkworms were shredded, and the cocoons were scoured in boiling 0.02 M aqueous sodium carbonate solution for 30 minutes to obtain scoured silk fibroin.

[0066] Refined 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 cut-off of about 12,000 - 14,000) to prepare an aqueous silk fibroin solution. After concentrating the obtained aqueous fibroin solution, a DMSO aqueous solution diluted to a final concentration of 1% by volume was gradually mixed with the aqueous silk fibroin solution diluted to a final concentration of 6% by weight to prepare a 6% by weight silk fibroin / DMSO solution.

[0067] The obtained aqueous solution was injected into the cylindrical mold shown in Figure 4, covered and sealed, then the mold was placed in a programmable precision low-temperature constant temperature water bath freezer (EYELA), cooled from room temperature to -20°C in 6 hours, and then left standing at -20°C for 6 hours or more. Then the mold was placed at room temperature and the contents in the mold were thawed to obtain a silk fibroin tubular body having a porous structure.

[0068] This silk fibroin tubular body was immersed in water at a bath ratio of (1:20) and left standing for 10 - 14 hours to perform a washing operation. This washing operation was repeated 6 times. Thereafter, the silk fibroin tubular body was stored in water at 4°C until use.

[0069] Note that the size of the silk fibroin tubular body used in the nerve regeneration test was made with an inner diameter of 3 mm, an outer diameter of 5 mm, and a length of 19 mm, and the size of the silk fibroin tubular body used in other tests was made with an inner diameter of 3 mm, an outer diameter of 5 mm, and a length of 10 mm.

[0070] (Example 2) A silk fibroin tubular body was prepared in the same manner as in Example 1 except that an aqueous solution with a silk fibroin concentration of 8% by weight was used.

[0071] (Comparative Example 1) A silk fibroin tubular body was prepared in the same manner as in Example 1, except that an aqueous solution having a silk fibroin concentration of 4% by weight was used.

[0072] (Comparative Example 2) In the nerve regeneration test, a silicone tube (inner diameter 3 mm, outer diameter 5 mm, length 19 mm) was used, and in a part of the physical property test, a silicone tube (inner diameter 3 mm, outer diameter 5 mm, length 10 mm) was used.

[0073] The various physical properties measured in the examples and comparative examples are shown in Table 1.

Table 1

[0074] As shown in Table 1, in Examples 1 and 2, even when a 1 N load was applied, the survival rate was high compared to Comparative Example 1, and furthermore, the stress against 25% compression also showed extremely high values. On the other hand, in Comparative Example 2 which is a silicone tube, although the survival rate when a 1 N load was applied was higher than that in Examples 1 and 2, since it has no voids and thus has no protein permeability, as will be described later, almost no tissue regeneration was confirmed in the central part of the tubular body even in the nerve regeneration test. Regarding the long-term degradability score, in Comparative Example 1, discoloration of the tubular body started 6 hours after immersion, part of it collapsed in 7 days, and the whole collapsed in 14 days. On the other hand, in Examples 1 and 2, even after 28 days of immersion, the collapse of the tubular body as a whole could be suppressed.

[0075] The results of the nerve regeneration test of Example 1 are shown in FIGS. 12A to 12E. FIG. 12A is a photograph showing the state in which the silk fibroin tubular body was taken out from a rat 1 month after transplantation. The silk fibroin tubular body could be taken out without adhesion to the outside while maintaining the lumen as a whole.

[0076] Figure 12B is a photograph (magnification: 4×) showing the state of immunostaining of vascular endothelial cells and axons in an axial section prepared by removing the silk fibroin tubular body of Example 1 from a rat 1 month after transplantation. In the axial section, although the lumen portion of the silk fibroin tubular body was slightly deformed, it was maintained without being crushed. It was confirmed that the lumen portion was filled with vascular endothelial cells and that an axon was present at the center of the lumen portion. Also, as shown in Figure 12C, in the enlarged photograph (magnification: 20×) showing the state of immunostaining of axons in the axial section, it was confirmed that the axons were regenerated well in the axial section.

[0077] As shown in Figure 12D, in the sagittal fragment, it was confirmed that the vascular endothelial cells extended along the inner wall of the silk fibroin tubular body and that the axons extended following them. Also, as shown in Figure 12E, in the enlarged photograph (magnification: 20×) showing the state of immunostaining of axons and myelin sheaths in the sagittal section, it was confirmed that the axons and myelin sheaths were regenerated well in the sagittal section.

[0078] Also, the results of the nerve regeneration test of Example 1 are shown in Figures 13A to 13D. Figure 13A is a photograph showing the state of removing the silk fibroin tubular body from a rat 3 months after transplantation. The silk fibroin tubular body maintained its lumen as a whole and could be removed without adhesion to the outside.

[0079] Figure 13B is a photograph (magnification: 4×) showing the state of immunostaining of vascular endothelial cells and axons in an axial section prepared by removing the silk fibroin tubular body of Example 1 from a rat 3 months after transplantation. In the axial section, although the lumen portion of the silk fibroin tubular body was slightly deformed, it was maintained without being crushed. It was confirmed that proliferated vascular endothelial cells were observed in the lumen portion and that the axons were regenerated well in the central portion.

[0080] Next, as shown in Fig. 13C, in the axial section, in a photograph (4x magnification) showing the state of immunostaining of axons and myelin sheaths, it was confirmed that the myelin sheath had regenerated well around the axons present in the center.

[0081] In the magnified photograph (20x magnification) shown in Fig. 13D, in the axial section, it was confirmed that the axons had regenerated well and the presence of proliferated vascular endothelial cells around them.

[0082] Also, the results of the nerve regeneration test of Comparative Example 1 are shown in Figs. 14A to 14C. Fig. 14A is a photograph showing the state immediately after transplantation of a silk fibroin tubular body into a rat, and Fig. 14B is a photograph showing the state one month after transplantation. When compared with the state of the silk fibroin tubular body one month after transplantation in Example 1 shown in Fig. 12A, the lumen part of the silk fibroin tubular body one month after transplantation shown in Fig. 14B was significantly collapsed. Also, as shown in Fig. 14C, only slight regeneration of axons was observed. Also, no regeneration of the myelin sheath was observed.

[0083] Also, the results of the nerve regeneration test of Comparative Example 2 are shown in Figs. 15A and 15B. Fig. 15A is a photograph showing the state immediately after transplantation of a silicone tubular body into a rat, and Fig. 15B is a photograph showing the state three months after transplantation. As shown in Fig. 15B, when the silicone tubular body was cut open and the lumen part was confirmed three months after transplantation, almost no tissue regeneration was confirmed in the central part of the tubular body.

[0084] The results of the nerve regeneration test of Example 2 are shown in Figs. 16A to 16E. Fig. 16A is a photograph showing the state immediately after transplantation of a silk fibroin tubular body into a rat, Fig. 16B is the state one month after transplantation, and Fig. 16C is a photograph showing the state three months after transplantation. Also, Fig. 16D is a photograph (4x magnification) showing the state of immunostaining of axons and myelin sheaths in the axial section, and Fig. 16E is the magnified photograph (20x magnification).

[0085] Even in the state one month after transplantation, the silk fibroin tubular body of Example 2 existed without the lumen part being crushed as in Comparative Example 1. Furthermore, three months after transplantation, the silk fibroin tubular body could be taken out without adhesion to the outside while maintaining the lumen as a whole. Good axon and myelin regeneration were observed by immunostaining.

[0086] Although the above test was conducted on the regenerative ability of peripheral nerve tissue, since various nutrients and growth factors can permeate into the lumen part of the silk fibroin tubular body to promote the induction of vascular endothelial cells, it is predicted that excellent effects will also be exhibited when regenerating other tissues and organs such as the central nervous system, ligaments, and tendons.

[0087] As described above, although the preferred embodiments of the present invention have been described, various additions, changes, or deletions are possible without departing from the spirit of the present invention, and such things are also included within the scope of the present invention.

Explanation of Reference Numerals

[0088] 2... mandrel 4... cylinder 5... precursor of silk fibroin tubular body 6... base 6a, 6b... grooves of the base 7... void part 8... lid 8a, 8b... grooves of the 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 that has a porous structure at room temperature and has a survival rate of 10.00 to 50.00% when a 1 N load is applied in a water-saturated state.

2. The silk fibroin tubular body according to Claim 1, wherein the compression stress when compressed by 25% in a water-saturated state is 0.020 to 0.300 N.

3. The silk fibroin tubular body according to Claim 1 or 2, wherein the compression stress when compressed by 50% in a water-saturated state is 0.040 to 0.500 N.

4. The silk fibroin tubular body according to Claims 1 to 3, wherein the recovery rate after leaving it for 10 minutes after performing a 50% compression test is 95.50% or more.

5. The silk fibroin tubular body according to any one of Claims 1 to 4, wherein the fibroin elution rate when stored in water for 24 hours is 2.00 to 7.00%.

6. The silk fibroin tubular body according to any one of Claims 1 to 5, which has protein permeability using ovalbumin as a standard substance.

7. The 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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