Slowly biodegradable silk fibroin artificial blood vessel for vein replacement

Abstract

To provide an artificial blood vessel for veins.SOLUTION: An artificial blood vessel for veins comprises: a tubular artificial blood vessel body; and a covering layer that covers and reinforces the outside of the artificial blood vessel body, wherein the artificial blood vessel body includes a fibroin fiber structure.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a venous artificial blood vessel.

[0002] The fibroin used in the present invention is derived from silk and may be abbreviated as "SF (Silk Fibroin)". [Background technology]

[0003] Artificial blood vessels are used, for example, to temporarily secure a blood flow path during surgery or as a substitute for diseased blood vessels. Synthetic fibers such as polyester and polytetrafluoroethylene (PTFE) are used as materials for artificial arterial blood vessels.

[0004] However, artificial blood vessels made of polyester or PTFE have a drawback in that the patency rate is extremely low in the case of so-called small-diameter artificial blood vessels with an inner diameter of less than 6 mm, and thus they have not yet been put to practical use. Here, the patency rate refers to the proportion of artificial blood vessels that remain patent when implanted in a living body. In other words, a low patency rate means that the artificial blood vessel is prone to clogging.

[0005] Because blood flows fast in arteries, blood clots are unlikely to form even in artificial blood vessels made of synthetic fibers. However, when it comes to artificial blood vessels for veins, synthetic fibers have a high rate of blockage after transplantation, so the patient's own venous blood vessels are transplanted instead.

[0006] Non-Patent Documents 1 to 8 disclose that an artificial blood vessel having a thin coating layer made of gelatin, SF, etc. formed on a knitted fabric of silk fibroin (hereinafter sometimes abbreviated as "SF") yarn was implanted into the artery of a rat or dog, and no vascular occlusion occurred, and Non-Patent Document 9 discloses that an artificial blood vessel having a thin coating layer formed on a knitted fabric of SF yarn was implanted into the vein of a rat, and no vascular occlusion occurred. Furthermore, Patent Document 1 discloses an artificial blood vessel manufactured by forming a sponge-like covering part on a base part formed by knitting SF fiber bundles into a tubular shape by double raschel knitting. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent No. 6146718 specification [Non-patent literature]

[0008] [Non-Patent Document 1] JTCVS Open Volume 6, June 2021, Pages 148-156 [Non-Patent Document 2] Molecules 2021, 26, 4649 [Non-Patent Document 3] Organogenesis. 2020;16(1):1-13. doi: 10.1080 / 15476278.2019.1686295. [Non-Patent Document 4] ACS Biomater. Sci. Eng. 2019, 5, 5561-5577 [Non-Patent Document 5] J Biomater Appl. 2018 Apr;32(9):1239-1252. [Non-Patent Document 6] Organogenesis. 2015;11(3):137-51 [Non-Patent Document 7] J Artif Organs. 2017 Sep;20(3):221-229. [Non-Patent Document 8] J. Mater. Chem. B, 2016, 4, 938-946 [Non-Patent Document 9] Scientific Reports volume 10, Article number: 21041 (2020) Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a venous artificial blood vessel. [Means for solving the problem]

[0010] Conventionally, there have been many artificial blood vessels for arteries, but artificial blood vessels for veins have a problem that the patency rate decreases over time, so transplantation of the patient's own venous blood vessels has been mainly performed. In animal experiments, the usefulness of artificial blood vessels in which an SF sponge is formed in the voids of a fiber structure of SF yarn or a thin covering layer made of SF sponge is formed on the outside of the fiber structure has been known for the arteries of small animals such as rats and large animals such as dogs (Non-Patent Documents 1 to 8). Furthermore, an artificial blood vessel in which a thin covering layer is formed on a fiber structure of SF yarn had a high patency rate when transplanted into the veins of small animals such as rats, but surprisingly, when transplanted into the veins of large animals such as dogs, as shown in the comparative examples of the present application, the patency rate was greatly reduced. Even in arteries with high blood pressure, defects such as blood leakage are not a problem when an SF sponge is formed in the voids of the fiber structure and a covering layer is not provided, so it was predicted that an outer covering layer to further improve strength would not be necessary in the case of veins. However, it was found that the patency rate is improved by providing a thick covering layer on the outside of the fiber structure of SF yarn, and the present invention was completed.

[0011] The present invention provides the following venous artificial blood vessel. [1] A venous artificial blood vessel comprising a tubular artificial blood vessel body and a covering layer that covers and reinforces the artificial blood vessel body, the artificial blood vessel body including a fibroin fiber structure. [2] The venous artificial blood vessel described in [1], wherein the artificial blood vessel body contains a filler composed of a biocompatible material in the voids within the fibroin fiber structure. [3] The venous artificial blood vessel according to [2], wherein the filler composed of a biocompatible material contained in the voids in the fibroin fiber structure is a porous body. [4] The elastic modulus of the artificial blood vessel is 0.02 to 0.03 N / mm 2 The venous artificial blood vessel according to [1], [5] The venous artificial blood vessel described in [1], wherein the coating layer contains a biocompatible material. [6] The venous artificial blood vessel according to [1], wherein the coating layer is a porous body made of a biocompatible material. [7] The venous artificial blood vessel according to [5] or [6], wherein the biocompatible material is fibroin. [8] The venous artificial blood vessel according to [1], which is used to replace autologous blood vessels in surgery for replacing varicose veins or stenosed veins, surgery for replacing venous blood vessels accompanying tumor resection, and vascular bypass surgery for blocked lower limb veins. [9] The venous artificial blood vessel according to [1], which is for use in the veins of large animals.

[10] The venous artificial blood vessel according to [9], wherein the large animal is a human.

[11] The venous artificial blood vessel according to [1], wherein the ratio (B / A) of the thickness (A) of the fibroin fiber structure to the thickness (B) of the covering layer is 0.8 to 3.1. Effect of the Invention

[0012] According to the present invention, a venous artificial blood vessel can be provided that can maintain a high patency rate for a long period of time when implanted into a vein. The artificial blood vessel or implant material of the present invention can be used for varicose vein replacement surgery, venous blood vessel replacement accompanying tumor resection surgery, vascular stenosis, vascular bypass of blocked lower limb veins, etc., and can suppress inflammation associated with vein replacement surgery or bypass surgery. Furthermore, when the artificial blood vessel or implant material of the present invention is made of a material that is degraded and absorbed in the body, such as fibroin, the artificial blood vessel or implant material starts to be degraded and absorbed 1 to 2 months after implantation, and at the same time, reconstruction of the venous structure, such as the endothelium, smooth muscle, elastic membrane, and adventitia, begins. [Brief description of the drawings]

[0013] [Figure 1] (a) Cress section, (b) Surface, and (c) Implantation of the Thin SF of the comparative example and the Thick SF of the embodiment are shown. [Diagram 2]Micrographs of (a) Cress section and (b) Surface of the Thin SF of the comparative example and the Thick SF of the embodiment. [Diagram 3] A: Breaking strength and B: Elastic modulus of the Thin SF of the comparative example and the Thick SF of the embodiment are shown. [Figure 4] Photographs of (a) intra-luminal diameter and (b) Doppler ultrasonography are shown 24 hours after implantation of a Thin SF of the comparative example and a Thick SF of the example into a dog's vein. [Diagram 5] 1 shows micrographs of the cross sections of artificial blood vessels after vein implantation, the Thin SF of the comparative example and the Thick SF of the example. [Figure 6] The cross-sectional views of the artificial blood vessel of the embodiment, Thick SF, are shown one month after implantation and 12 months after implantation in a dog vein. [Figure 7] Photographs of HE staining, EVG staining, endothelial marker CD31 immunostaining, and αSMA immunostaining of cross sections of artificial blood vessels after vein transplantation of Thin SF of the Comparative Example and Thick SF of the Example are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The venous artificial blood vessel of the present invention comprises a tubular artificial blood vessel main body and a covering layer which covers and reinforces the artificial blood vessel main body.

[0015] The artificial blood vessel main body includes a fibroin fiber structure. The fiber structure may be a woven fabric, knitted fabric, hollow-weave braid, or nonwoven fabric made of fibroin fiber, or a fibroin fiber bundle or fibroin yarn (preferably twisted yarn) made by combining a plurality of fibroin fibers. Any tubular structure may be used, but is preferably a knitted fabric of fibroin fiber, fibroin fiber bundle, or fibroin yarn, and more preferably a tubular structure made by knitting fibroin fiber, fibroin fiber bundle, or fibroin yarn by double raschel knitting. Examples of double raschel knitting include double barcode knitting, double-eye knitting, and Denbigh knitting, and double-eye knitting is preferable. Note that double raschel knitting refers to knitting using a double raschel machine. The fibroin fiber bundle or fibroin yarn includes fibroin fiber as a component, and the core portion may include fibers other than fibroin, such as polyester fiber. Therefore, the surface of the fibroin yarn is covered with fibroin fibers, but the core portion of the fibroin fiber bundle or fibroin yarn may contain fibers other than fibroin, such as polyamides such as nylon, polyester, and polyurethane.

[0016] In one preferred embodiment of the present invention, the fibroin fiber is first twisted, and a fibroin fiber bundle or fibroin yarn made of a plurality of fibroin fibers is second twisted. That is, a plurality of fibroin fibers that have been first twisted are combined to form a fibroin fiber bundle or fibroin yarn, which is then second twisted. It is preferable that the first twist applied to the fibroin fiber and the second twist applied to the fibroin fiber bundle or fibroin yarn are in the opposite directions. The thickness of the fibroin fiber structure is preferably about 0.3 to 0.7 mm, more preferably about 0.4 to 0.6 mm.

[0017] In one preferred embodiment of the artificial blood vessel body, the fibroin fiber structure contains a filler made of a biocompatible material in the gaps between the fibroin threads. Examples of the biocompatible material include fibroin, collagen, gelatin, albumin, casein, zein, fucoidin, alginate, laminin, agarose, dextran, fibronectin, mucopolysaccharides, mucin, etc., and fibroin is preferred. The filler can contain a substance that inhibits thrombus formation, such as heparin, vascular endothelial growth factor protein sustained release gel, and cell membrane mimicking polymer material.

[0018] The filler is preferably porous. The porosity of the porous filler is preferably 50 to 80%, more preferably 60 to 70%. The filler made of a biocompatible material is intended to prevent blood from leaking out of the artificial blood vessel, but it does not have to be completely filled with the filler as long as blood leakage is suppressed to an acceptable level. For example, the voids near the inner surface of the artificial blood vessel may have some parts that are not filled with the filler.

[0019] The covering layer that covers and reinforces the outside of the artificial blood vessel body may be made of a non-biocompatible material such as polyamides such as nylon, polyester, or polyurethane, or a hybrid material of a non-biocompatible material and the above-mentioned biocompatible material, but is preferably made of the biocompatible material exemplified above, and particularly preferably made of fibroin. When both the filling material and the covering layer are made of a biocompatible material, it is preferable to use the same biocompatible material. In addition, the covering layer is preferably porous. The porosity of the covering layer is preferably 50 to 80%, more preferably 60 to 70%. The outside of the fibroin fiber structure is covered with the covering layer, but it is preferable that the fibroin fiber structure and the filling material made of a biocompatible material are exposed on the inner surface. The exposure of the fibroin fiber structure, especially the fibroin thread that constitutes the fiber structure, promotes the engraftment of vascular endothelial cells on the inner surface, thereby preventing thrombus formation and increasing the patency rate of the artificial blood vessel.

[0020] In a preferred embodiment of the artificial blood vessel of the present invention, the filler and the covering layer allow a certain degree of material transfer between the body fluid outside the artificial blood vessel and the blood inside the artificial blood vessel. This is considered to be capable of suppressing thrombus formation in the artificial blood vessel. In order to allow this material transfer, the filler and the covering layer are preferably porous.

[0021] The venous artificial blood vessel of the present invention is preferably applied to large animals, such as humans, dogs, cats, cows, horses, pigs, sheep, monkeys, chimpanzees, etc., and is preferably applied to humans.

[0022] In a preferred embodiment of the present invention, when the artificial blood vessel is immersed in water, taken out, and the water on the outer and inner surfaces is wiped off and the weight is measured, the weight increases preferably by about 6 to 15 times, more preferably by about 8 to 13 times. The artificial blood vessel of the present invention can be transplanted after being soaked in an aqueous medium such as water or physiological saline, and the flexibility in a state in which a large amount of water is retained in the living body is greatly improved. When transplanted into the vein of a large animal such as a human, the artificial blood vessel can be prevented from stagnation of blood circulation even when the artificial blood vessel is transplanted in a position where it is bent or twisted. Due to this flexibility, even if the artificial blood vessel is twisted, bent, or broken when the body is twisted or bent, it can quickly return to its original state. In order to improve this flexibility, it is preferable that the coating layer is porous.

[0023] The thickness of the coating layer varies depending on the inner diameter of the tubular or cylindrical artificial blood vessel, but is preferably about 0.3 to 1.5 mm, more preferably about 0.5 to 1.0 mm. In the present invention, the thickness of the coating layer means the value measured for the thickness of the coating layer of the artificial blood vessel when it is dried before being impregnated with an aqueous medium. The thickness of the artificial blood vessel, coating layer, fibroin fiber structure, etc. can be measured directly with a vernier caliper or from a micrograph of the cross section of the artificial blood vessel.

[0024] The ratio (B / A) of the thickness (A) of the fibroin fiber structure to the thickness (B) of the covering layer is preferably about 0.8 to 3.1, more preferably about 1.0 to 2.8. In the present invention, the thickness (A) of the fibroin fiber structure and the thickness (B) of the covering layer refer to the respective values ​​measured for the thickness of the fibroin fiber structure and the thickness of the covering layer when dry before impregnating with an aqueous medium.

[0025] The length of the venous artificial blood vessel is preferably about 1 to 10 cm, and more preferably about 3 to 5 cm.

[0026] The inner diameter of the venous artificial blood vessel is preferably about 5 to 35 mm, and more preferably about 10 to 30 mm.

[0027] The elastic modulus of the venous artificial blood vessel is preferably 0.015 to 0.035 N / mm 2 Approximately, more preferably 0.02 to 0.03 N / mm 2 The coating layer does not have a significant effect on the breaking strength of the venous artificial blood vessel, but it does have a significant effect on the elastic modulus. The lower the elastic modulus is below the preferred range, the more likely the venous artificial blood vessel will twist or collapse, and the higher the elastic modulus is above the preferred range, the less flexible the venous artificial blood vessel will be and the more likely it will break when subjected to forces within the body.

[0028] The method for producing the venous artificial blood vessel of the present invention is not particularly limited, but for example, the venous artificial blood vessel of the present invention can be produced by passing a fibroin fiber structure produced by a conventional method through a resin or metal mandrel such as PTFE, placing it in a tubular mold (made of resin or metal) with an inner diameter larger than that of the mandrel, filling it with an aqueous solution of a biocompatible material, freezing it, and freeze-drying it.A pore-forming agent such as polyethylene glycol, glycerin, ethylene glycol, propylene glycol, polyethylene glycol, diglycidyl ether, etc. can be added to the aqueous solution of a biocompatible material, and after freezing, the mixture can be immersed in water or freeze-dried to simultaneously form a porous coating layer and a porous filling material in the voids of the fibroin fiber structure. EXAMPLES

[0029] The present invention will be described in more detail below based on examples and comparative examples, but the present invention is not limited to the following examples. Example 1 According to the following procedures (1) to (9), a venous artificial blood vessel of the present invention having an inner diameter of 8 mm was prepared. (1) A silk fibroin aqueous solution adjusted to 3.5% w / v is mixed with an equal weight of glycerin to the silk fibroin to prepare a silk fibroin-glycerin mixed aqueous solution, which is used as a coating liquid. (2) A two-loop tubular fibroin fiber structure (inner diameter 8 mm) made using a double-raschel knitting machine is passed through a PTFE mandrel with an outer diameter of 8 mm. (3) To perform a thick sponge coating, a PTFE mold (inner diameter 13 mm) is used to prepare a silk artificial blood vessel with an outer diameter of 13 mm and an inner diameter of 8 mm. (4) To this end, a coating solution is poured between the fibroin artificial blood vessel body attached to a PTFE mandrel with an outer diameter of 8 mm and a PTFE mold with an inner diameter of 13 mm, in an amount sufficient to make a length of about 3 cm. (5) The PTFE mold is placed upright in a desiccator and the pressure is reduced at 100 Hp for 30 minutes using a vacuum pump to allow the coating solution prepared in (1) above to fully permeate the artificial blood vessel body. (6) Place it in a -20C freezer and leave it overnight. (7) Remove, place in water, thaw, and remove the venous graft from the mold, at which point the glycerin is removed. (8) Then, the material is placed in liquid nitrogen to freeze, and then placed in a freeze dryer (EYELA freeze dryer FDU-1200, Tokyo Rikaki Co., Ltd.) for freeze-drying to obtain the venous artificial blood vessel (Thick SF) of the present invention. (9) Store in a plastic test tube. Figure 1(a) shows a cross section of Thick SF, Figure 1(b) shows the surface of Thick SF, and Figure 1(c) shows a photograph of Thick SF implanted into a dog's abdominal vein. The cross section and surface of Thick SF are shown in Figure 2. The total thickness of the Thick SF artificial blood vessel was 1.0-1.5 mm, the thickness of the coating layer was 0.6-0.9 mm, and the thickness of the fibroin fiber structure was 0.4-0.6 mm.

[0030] Comparative Example 1 A comparative venous artificial blood vessel (Thin SF) was obtained in the same manner as in Example 1, except that a PTFE mold (inner diameter 10 mm) was used instead of the PTFE mold (inner diameter 13 mm) in Example 1. Figure 1(a) shows a cross section of Thin SF, Figure 1(b) shows the surface of Thin SF, and Figure 1(c) shows a photograph of Thin SF implanted into a dog's abdominal vein. The cross section and surface of Thin SF are shown in Figure 2. The total thickness of the Thin SF artificial blood vessel was less than 0.5 mm, and the thickness of the coating layer was less than 0.1 mm.

[0031] Test Example 1: Measurement of Elastic Modulus The elastic modulus of the venous artificial blood vessels obtained in Example 1 and Comparative Example 1 was measured using the following device. The results are shown in FIG. Elasticity measurement device: EZgraph, manufactured by Shimadzu Corporation

[0032] Test Example 2: Breaking Strength Measurement The rupture strength rates of the venous artificial blood vessels obtained in Example 1 and Comparative Example 1 were measured using the following device. The results are shown in FIG. Breaking strength measuring device: Name: EZgraph, manufactured by Shimadzu Corporation

[0033] Test example 3: Transplantation test The artificial blood vessels obtained in Example 1 and Comparative Example 1 were implanted into the posterior vena cava of beagle dogs. The distribution of the inner diameter of the artificial blood vessel 24 hours after implantation (n=8 for Thick SF, n=3 for Thin SF) and Doppler ultrasonography photographs are shown in FIG. 4, photographs one month after implantation are shown in FIG. 5, and cross-sectional views of the artificial blood vessels one month and one year after implantation of Thick SF are shown in FIG. 6. Furthermore, for the implanted blood vessels one month after vein implantation, HE staining, EVG staining, endothelial marker CD31 immunostaining, and αSMA immunostaining were performed on the cross-section of the artificial blood vessel after vein implantation, and the obtained microscopic photographs are shown in FIG. 7. In the case of the artificial blood vessel (Thick SF) of Example 1, a sufficient inner diameter was maintained both 24 hours and one month later, and a high patency rate was maintained even after 12 months. On the other hand, it was revealed that the inner diameter of the artificial blood vessel (Thin SF) obtained in Comparative Example 1 had already significantly decreased even after 24 hours, and the artificial blood vessel was significantly deformed one month later and could not be used. Furthermore, in the case of the artificial blood vessel of Example 1 (Thick SF), a sufficient endothelial cell layer was formed one month after implantation, making it clear that a practical artificial blood vessel for veins can be obtained even in large animals.

Claims

1. An artificial blood vessel for a vein, comprising a tubular artificial blood vessel body and a covering layer which covers and reinforces the outside of said artificial blood vessel body, said artificial blood vessel body including a fibroin fiber structure.

2. 2. The venous artificial blood vessel according to claim 1, wherein said artificial blood vessel body contains a filling material made of a biocompatible material in voids in the fibroin fiber structure.

3. 3. The venous artificial blood vessel according to claim 2, wherein the filler made of a biocompatible material contained in the voids in the fibroin fiber structure is a porous body.

4. The elastic modulus of the artificial blood vessel is 0.02 to 0.03 N / mm ２ 2. The venous artificial blood vessel according to claim 1,

5. The venous graft of claim 1 , wherein said covering layer comprises a biocompatible material.

6. 2. The venous artificial blood vessel according to claim 1, wherein said covering layer is a porous body made of a biocompatible material.

7. 7. The venous graft of claim 5, wherein the biocompatible material is fibroin.

8. 2. The venous artificial blood vessel according to claim 1, which is used to replace autologous blood vessels in surgery for replacing varicose veins or stenosed veins, surgery for replacing venous blood vessels accompanying tumor resection, and vascular bypass surgery for blocked veins in the lower limbs.

9. 2. The venous artificial blood vessel according to claim 1, which is for use in the veins of large animals.

10. 10. The venous vascular prosthesis of claim 9, wherein the large animal is a human.

11. 2. The venous artificial blood vessel according to claim 1, wherein the ratio (B / A) of the thickness (A) of the fibroin fiber structure to the thickness (B) of the covering layer is 0.8 to 3.1.

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