JP5392744B2 - Artificial blood vessel and manufacturing method thereof - Google Patents

Description

  The present invention relates to an artificial blood vessel using silk fibroin fiber and a method for producing the same.

In recent years, with the increase of vascular diseases such as arteriosclerosis, the importance of artificial blood vessels has definitely increased. In artificial blood vessels, (1) safety (acute toxicity, intradermal reaction test, hemolysis test, pyrogen test, skin sensitization test, cytotoxicity, etc.), (2) functionality (stretchability, stitching) Easiness, flexibility, difficulty in fraying the cut end, difficulty in bleeding from the artificial blood vessel wall), and (3) durability. Various types of artificial blood vessels are required depending on the site to be implanted in the body.
Artificial blood vessels with large diameters have already been put into practical use and can withstand clinical use. However, small-diameter artificial blood vessels having a diameter of 5 mm or less have not yet been put into practical use due to thickening of the intima due to biocompatibility of typical artificial materials such as polyethylene terephthalate and PTFE and occlusion due to thrombus formation. Therefore, at present, autologous vein transplantation is performed for bypass to the knee joint periphery, etc., but the burden on the patient is large, and there are many patients who do not have compatible blood vessels and cannot perform autologous vein transplantation. There are many problems. In recent years, with the aging of patients and the increase in diabetes, regenerative treatment of small blood vessels has increased. Therefore, development of an artificial blood vessel having antithrombotic properties that can be used for small-diameter blood vessels such as peripheral blood vessels has been strongly desired for a long time.

On the other hand, silk thread has high biocompatibility, is thin, strong, has moderate elasticity and flexibility, has good sliding properties, and is easy to tie and hard to fray. It is a natural fiber used as Various regenerated silk materials that utilize the high biocompatibility of silk have been developed so far, and are expected to be used in a wide range of fields such as medicine, biochemistry, food, and cosmetics. In particular, it is attracting attention as a material for regenerative medicine.
As an attempt to produce an artificial blood vessel using silk, a kite thread structure is known which is wound by combining braiding operations according to the principle of braid fabrication, and the kite yarns and mixed fibers are glued together with sericin held on the kite surface. (Patent Document 1). This string structure has a tensile strength that can withstand practical use because the string is glued together with seriline.
However, since sericin is highly likely to cause an allergic reaction, it is desirable to remove sericin from the viewpoint of reducing its risk. In addition, the silk thread structure is not flexible enough, and the cut end is easily frayed, so there is a demand for an artificial blood vessel that is superior in functionality while maintaining the original characteristics of silk such as biocompatibility and required characteristics during surgery. It had been.
JP 2004-173772 A

  The present invention has been made in view of the above circumstances, and has a tubular structure that can be used for small-diameter blood vessels such as peripheral blood vessels, which have inherent properties such as biocompatibility and have reduced allergic risk due to sericin. About providing.

  The present inventor has made various studies on tubular structures utilizing the characteristics of silk. As a result, the outer wall of the tubular structure in which silk fibroin fibers are wound by one or more methods selected from knitting, braiding, weaving and entanglement. It has been found that by applying a smoothing process to smooth the surface, it is possible to obtain a tubular structure which is flexible and has very little fraying at the cut end and extremely little blood leakage. Further, the present inventors have found that a tubular structure suitable for an artificial blood vessel having a smaller diameter that satisfies the required characteristics at the time of surgery can be obtained by further raising the smoothed surface.

That is, the present invention is obtained by performing a smoothing treatment on the outer wall surface of a tubular structure in which silk fibroin fibers are wound by one or more methods selected from knitting, braiding, weaving and entanglement. A tubular structure from which sericin has been removed is provided.
In the present invention, the silk fibroin fiber is formed into a tubular shape by one or more methods selected from knitting, braiding, weaving, and entanglement, and then the outer wall surface of the obtained tubular structure is subjected to smoothing treatment and raising treatment. The present invention provides a method for producing a tubular structure from which sericin has been removed.

  According to the present invention, it is possible to provide a tubular structure that has sufficient tensile strength, is flexible and easily vascularly anastomosed while reducing the risk of allergy due to sericin, and has very little fraying at the cut end and extremely little blood leakage. it can. This tubular structure is suitable for a small-diameter artificial blood vessel having a diameter of 5 mm or less because it has the original properties of silk, that is, high biocompatibility and excellent antithrombotic properties.

Hereinafter, the present invention will be described in detail.
The tubular structure from which sericin has been removed in the present invention is formed by winding silk fibroin fibers by one or more methods selected from knitting, braiding, weaving and entanglement. Here, the silk fibroin fiber is a scoured raw silk, raw silk, etc., obtained from rabbit silkworms and wild silkworm layers such as Eli silkworm, silkworm, and tengu. Sericin is removed by the scouring treatment.

  The scouring method is not particularly limited, and a known method can be used. For example, 12w / v% Marcel soap heated to 100 ° C, 8w / v% sodium carbonate mixed aqueous solution, and the above-mentioned cocoon layer, silk thread, raw silk, etc. are put in, boiled for 120 minutes with stirring, then 2w / After performing the operation of boiling in a v% aqueous sodium carbonate solution for 10 minutes and washing in distilled water heated to 100 ° C. three times, the protein (sericin) covering fibroin and other fats can be removed by drying.

  In order to construct the silk fibroin fiber into a tubular shape, a known knitting method, braiding method, weaving method, and entanglement method can be used. In the present invention, braiding is preferable from the viewpoint of improvement in tensile strength and blood leakage. The method for braiding the silk fibroin fiber is not particularly limited, and for example, a known braiding technique such as eight punching, twelve punching, sixteen punching can be used. Specifically, it is carried out by winding a silk thread or refined thread unraveled from boiled rice cake around a core rod made of a thermoplastic resin, etc., and assembling silk fibroin fibers. A thermoplastic resin core rod having an outer diameter of 1 to 5 mm, preferably 1.5 to 5 mm can be used depending on the size of the target tubular structure. Moreover, you may use the knitted fabric and textile fabric previously produced using the silk fibroin fiber, the cylindrical thing, etc.

  When the silk fibroin fiber is formed into a tubular shape, the silk fibroin fiber may be used alone or may be used by mixing with other fibrous materials. Examples of such fibers include synthetic fibers such as nylon, polyester, polylactic acid, and polytetrafluoroethylene, inorganic fibers, and metal fibers.

  The tubular structure may have a single layer structure, but preferably has a multilayer structure of 2 to 4 layers, more preferably 3 to 4 layers, from the viewpoint of tensile strength and flexibility.

The smoothing treatment in the present invention is not limited as long as the silk fibroin fiber on the outer wall surface of the tubular structure can be defibrated and flattened. Pressure and friction are applied to the outer wall surface of the tubular structure, and the braided yarn portion is rolled and flattened. It is done to become. Thereby, the improvement of blood leakage and the fraying of a cut surface can be aimed at. Moreover, the removal effect of sericin can be improved.
The method of applying pressure is not particularly limited, and examples thereof include a rolling process in which a metal bar-like material is brought into contact with and rotated on the outside of the tubular structure, and the braid portion on the surface thereof is rolled and smoothed. The pressure applied to the outer wall surface of the tubular structure is about 1 to 10 kg / cm ² , but preferably varies depending on the size, depth, and height of the unevenness of the braided yarn portion.

  Subsequently, by raising the hair, a tubular structure that is more flexible and less frayed at the cut end and less leaked blood can be obtained. Moreover, the removal effect of sericin can be improved. Here, the raising treatment is not limited as long as the silk fibroin fiber on the outer wall surface of the tubular structure is disentangled and the fibers can be entangled with each other, and the surface is fluffed by pulling the fiber to the outer wall surface of the tubular structure. Put it in a state. The degree of raising is not particularly limited as long as the inner surface of the tubular structure is held. The treatment can be, for example, polishing with an abrasive or brushing with a brush-like material. Of these, from the viewpoint of workability, it is preferable to brush using various brush-like materials such as metal, plastic, and pig hair.

It is preferable to immerse the sericin-removed tubular structure thus obtained in a silk fibroin solution so that silk fibroin fibers or mixed fibers are glued together with silk fibroin. Thereby, the state of the tubular structure formed by the smoothing process or the raising process can be held and fixed.
The silk fibroin solution can be prepared by a known method. For example, silk fibroin is obtained by scouring a silkworm layer, silk thread, raw silk, etc., dissolved in a neutral salt aqueous solution, heated, and then obtained silk It can be prepared by desalting a fibroin / salt aqueous solution. Examples of the neutral salt aqueous solution include lithium bromide, lithium chloride, calcium chloride, and lithium thiocyanate. As a desalting treatment method, a known method such as a dialysis method or a reverse osmosis method can be employed.

Although the immersion in the silk fibroin solution is performed by a normal method, penetration into the inside may be promoted by rolling or decompression. The silk fibroin concentration in the solution is preferably about 0.1 to 5 w / v%, but if the concentration is low, the immersion and drying may be repeated.
Furthermore, the silk fibroin solution may be heated, or the tubular structure may be immersed in alcohols such as ethanol and isopropyl alcohol, whereby the silk fibroin gels and a sterilizing effect can be added to the tubular structure.

  After dipping, a tubular structure is obtained by heating and drying to separate from the thermoplastic resin core rod. The drying temperature is not particularly limited as long as the core rod made of thermoplastic resin is softened, but is generally 90 to 120 ° C, preferably 100 to 110 ° C.

  The tubular structure of the present invention can be used as a substitute for, for example, artificial blood vessels, artificial trachea, stent grafts, and other biological tubular structures. In particular, because of the excellent biocompatibility and antithrombogenicity of silk, it is suitable for small-diameter artificial blood vessels having a diameter of 5 mm or less, preferably 1 to 5 mm or less.

  Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.

Reference example <Preparation of rabbit silk fibroin solution>
Rabbits were finely cut with a scissors (about 2 mm × 10 mm) and refined by a conventional method to obtain silk fibroin from which protein (sericin) covering fibroin and other fats were removed. Scanning electron microscope observation was performed to confirm the residual sericin deposits. For the measurement, a real surface view microscope VE-7800 (manufactured by Keyence) was used, the sample was fixed with carbon tape, and measurement was performed without vapor deposition. The acceleration voltage was 1.3 kV and the working distance was 7.3 mm. The obtained silk fibroin is shown in FIG.
Next, this silk fibroin was dissolved in 9M lithium chloride aqueous solution so as to be 15 w / v%. This aqueous solution is dialyzed with pure water using cellulose dialysis membrane (VISKASESELES COAP Co., Ltd. Seamless Cellulose Tubing 36/32) for 3 days to remove lithium chloride, and further centrifuged to remove undissolved residue and dust. Then, a silkworm silk fibroin solution was obtained.

Example 1 <Creation of Rabbit Silk Artificial Blood Vessel>
1. Fabrication of small-diameter silk graft using an assembling machine For the fabrication of an artificial blood vessel, a braided string producing device (16 strokes) (manufactured by Kokbun Limited) was used. The bobbin was wrapped with silk fibroin fiber scoured by a conventional method. A 1.5mm outer diameter core rod (vinyl chloride core rod) was attached to the center of the machine, and silk fibroin fibers were assembled around the core rod.

2. 1. Smoothing treatment to silkworm silk artificial blood vessel Place the artificial blood vessel core rod prepared in step 1 on a hard flat surface, apply a load of 5 kg / cm ^{2 on} the outer wall surface with a metal rod (stainless steel roll), and roll it for 10 minutes in the longitudinal direction of the artificial blood vessel. The part was flattened. This greatly reduced the gap between the silk threads. The results of scanning electron microscope observation of the artificial blood vessel before and after the rolling treatment are shown in FIG.

3. 1. Raising treatment of artificial blood vessel The artificial blood vessel obtained in (1) was fixed to an electric drill manufactured by Ryobi, and a metal brush was brought into contact while rotating at 300 rpm. At this time, raising the rotation direction improved the efficiency of raising. As a result, the outer wall surface of the artificial blood vessel was raised. A scanning electron microscope image after the brushing treatment is shown in FIG.
As is clear from FIG. 3, the fibers were dispersed and entangled in each direction.

4). Immersion in silk fibroin solution The artificial blood vessel is immersed in the silk fibroin solution prepared in <Preparation of silk silk fibroin solution> and subjected to 5 times reduced pressure infiltration at -60 mmHg pressure, then 110 ° C. It dried in the dryer set to.
After drying, the artificial blood vessel is immersed in a 50% ethanol aqueous solution to fix the silk fibroin, and then dried again in a dryer set at 110 ° C. and separated from the core rod, so that the silkworm artificial blood vessel (caliber 1.5 mm).

Example 2 <Production of 3 mmφ artificial blood vessel using torsion race>
1. Production of 1.3 mmφ silk graft A 3 mmφ artificial blood vessel was produced using a rabbit silk torsion lace (manufactured by Futoshi lace). A 3 mm thermoplastic resin core rod (vinyl chloride core rod) was covered with a 5 mm wide torsion race.

2.3 Smoothing treatment to 3 mmφ artificial blood vessel Was placed on a hard flat surface, a 5 kg / cm ² load was applied to the outer wall surface with a metal rod (stainless steel roll), and the braided yarn portion was flattened by rolling in the longitudinal direction of the artificial blood vessel for 10 minutes. . This greatly reduced the gap between the silk threads.

3. Brushing treatment on 3 mmφ artificial blood vessel The artificial blood vessel produced in (1) was fixed to an electric drill manufactured by Ryobi and contacted with a metal brush while rotating at 300 rpm.

4). Immersion in silk fibroin solution 3 mmφ artificial blood vessel is immersed in the silk fibroin solution prepared in the above <Preparation of silkworm silk fibroin solution>, and after 5 times under reduced pressure infiltration at -60 mmHg pressure, 110 It dried in the dryer set to ° C.
After drying, the artificial blood vessel is immersed in a 50% ethanol aqueous solution to fix the silk fibroin, and then dried again in a dryer set at 110 ° C., and separated from the core rod, so that the silkworm artificial blood vessel (diameter 3 mm) )
The rabbit silkworm artificial blood vessel (diameter 3 mm) obtained in this way was flexible and could not be cut at the cut end even if it was cut in any direction (FIG. 4).

It is a figure which shows the scanning electron microscope image of the silk fibroin fiber after scouring. It is a figure which shows the scanning electron microscope image of the silkworm silk artificial blood vessel before and behind a rolling process. It is a figure which shows the scanning electron microscope image of the rabbit silk artificial blood vessel after a brushing process. It is a figure which shows the cutting end of a rabbit silk artificial blood vessel. It is the figure which illustrated the manufacturing method of the tubular structure of this invention.

Claims (4)

A sericin-removed tubular product obtained by rolling the outer wall surface of a tubular structure in which silk fibroin fiber is wound by one or more methods selected from knitting, braiding, weaving, and entanglement Structure. Further napping treatment is performed comprising Claim 1 sericin-extracted tubular structure according. 3. The tubular structure from which sericin has been removed according to claim 1, wherein the tubular structure is a small-diameter artificial blood vessel. A sericin characterized in that silk fibroin fiber is formed into a tubular shape by one or more methods selected from knitting, braiding, weaving and entanglement, and then the outer wall surface of the obtained tubular structure is subjected to rolling treatment and raising treatment. A method for producing the removed tubular structure.