JPWO2013172021A1 - Artificial blood vessel and manufacturing method thereof - Google Patents

Abstract

An artificial blood vessel capable of being maintained for a long period of time and a method for producing the same are provided. The present invention provides a silk fibroin fiber bundle by combining a plurality of silk fibroin fibers having a surface with silk sericin attached thereto, the silk fibroin fiber bundle is knitted into a tubular shape by double raschel knitting, and silk sericin is removed. Base portion 1 obtained and a sponge-like covering portion 2 whose surface is covered with silk fibroin, the thickness of the covering portion 2 of the front side portion of the base portion 1 is 0.8 mm or more, Is a non-cracking artificial blood vessel 10 having a ratio of 60% or more. [Selection] Figure 1

Description

  The present invention relates to an artificial blood vessel and a method for manufacturing the same, and more particularly to an artificial blood vessel that can be maintained for a long period of time and a method for manufacturing the same.

The artificial blood vessel is used, for example, for a purpose of temporarily securing a blood flow path during surgery or a use as a substitute blood vessel for a diseased blood vessel.
Polyester, polytetrafluoroethylene (PTFE) and the like have been developed as materials for such artificial blood vessels.

  Among these, as a material that relatively satisfies the functional requirements (for example, strength, elasticity, etc.) of an artificial blood vessel, a knitted or woven fabric using polyester yarn, which is knitted into a tubular shape, is commercially available.

  However, an artificial blood vessel using polyester or PTFE has a drawback that the patency rate is remarkably lowered in the case of a so-called small-diameter artificial blood vessel having an inner diameter of less than 6 mm, and has not been put into practical use. Here, the patency rate means the rate at which the artificial blood vessel is patent when transplanted in a living body. That is, a low patency rate means that the artificial blood vessel is easily clogged.

By the way, in recent years, an artificial blood vessel using silk used as a surgical suture has been developed as an alternative material for polyester.
For example, a small-diameter artificial blood vessel in which a multilayered tubular structure formed by braiding silk fibroin yarn in multiple layers is coated with silk fibroin (see, for example, Patent Document 1), silk fibroin fiber is knitted, assembled, Sericin-removed tubular structure obtained by smoothing the outer wall surface of the tubular structure wound by one or more methods selected from woven and entangled (see Patent Document 2), silk fibroin dissolution Silk nanofibers formed by electrospinning using a liquid are collected around a rotating support rod covered with a resin tube, and then the support rod is pulled out to produce a tubular structure (Patent Document) 3) is known.

JP 2008-73408 A JP 2009-279214 A JP 2010-137041 A

  However, the artificial blood vessel using silk described in Patent Documents 1 to 3 is superior to the artificial blood vessel made of polyester in that endothelial cells are fixed and thrombus is hardly formed, but the patency rate Is not enough.

  The present invention has been made in view of the above circumstances, and an object thereof is to provide an artificial blood vessel that can be maintained for a long period of time and a method for manufacturing the same.

  The inventors of the present invention have intensively studied to solve the above-mentioned problems. As a result, the silk fibroin fiber is formed into a tubular shape by double raschel knitting, and the surface is thickly covered with silk fibroin. Surprisingly, the inventors have found that the above-described problems can be solved by having a water content of 5%, and have completed the present invention.

  In the present invention, (1) a silk fibroin fiber bundle is formed by combining a plurality of silk fibroin fibers having silk sericin attached to the surface, the silk fibroin fiber bundle is knitted into a tubular shape by double raschel knitting, and the silk sericin is removed. A base part obtained and a sponge-like covering part whose surface is covered with silk fibroin, the thickness of the covering part of the front side part of the base part is 0.8 mm or more, and the moisture content is 60% or more It exists in non-cracking artificial blood vessels.

This invention exists in the artificial blood vessel of the said (1) description whose water permeability measured according to (2) ISO7198 is 70-240 ml / cm < ² > / min.

This invention exists in the artificial blood vessel as described in said (1) or (2) whose elasticity modulus is 0.05-0.09 N / mm < ² >.

  The present invention relates to (4) the artificial fission according to any one of (1) to (3), wherein the silk fibroin fiber is subjected to a lower twist, and the silk fibroin fiber bundle is subjected to an upper twist. It exists in blood vessels.

  The present invention resides in the artificial blood vessel according to (4) above, wherein (5) the number of twists of the upper twist is smaller than the number of twists of the lower twist.

  The present invention is (6) the method for producing an artificial blood vessel according to any one of claims 1 to 5, wherein raw silk composed of silk thread is scoured, and a portion of silk sericin remains on the surface of silk fibroin A scouring step to produce a silk fibroin fiber, a yarn-making step to combine a plurality of silk fibroin fibers into a silk fibroin fiber bundle, a knitting step to knitting the silk fibroin fiber bundle into a tubular body by double raschel knitting, The removal process of silk sericin remaining in the tubular body to remove the base and the sponge-like covering portion covered with silk fibroin on the surface of the base portion, the thickness of the covering portion of the front portion of the base portion is 0.8 mm or more And forming a temporary artificial blood vessel, and an immersion step of immersing the temporary artificial blood vessel in pure water. After the coating step, the immersion is performed in a state in which the wet state of the temporary artificial blood vessel is maintained. Consists in the production method of the artificial blood vessel is performed.

  The present invention resides in (7) the method for producing an artificial blood vessel according to the above (6), wherein the immersion step is performed by immersing the temporary artificial blood vessel in pure water for 1 hour or more.

  This invention exists in the manufacturing method of the artificial blood vessel as described in said (6) or (7) by which (8) immersion processes are performed until just before transplantation of an artificial blood vessel.

  This invention exists in the manufacturing method of the artificial blood vessel as described in any one of said (6)-(8) by which a coating process is performed by immersing a base in a silk fibroin solution and freeze-drying. .

  The method for producing an artificial blood vessel according to any one of the above (6) to (8), wherein (10) the coating step is performed by performing silk fibroin sponge coating on the base using a hole source material. Exist.

The artificial blood vessel of the present invention can be maintained for a long time by setting the moisture content to 60% or more.
Although it is not clear why it can be maintained for a long period of time, by keeping the moisture content within the above range, it prevents the sponge-like coating part coated at the time of living transplantation from peeling off from the base surface at the time of transplanting. In addition, it is presumed that oxygen, moisture, and biological material can smoothly pass through the wall of the sponge-like covering portion of the artificial blood vessel after transplantation.

  In the above artificial blood vessel, the inner surface of the double raschel knitted base is also covered with the covering portion, so that the endothelial cells are easily fixed and thrombus is hardly formed.

Further, since the entire base portion is covered with a sponge-like covering portion, the artificial blood vessel is not easily refracted even when bent into a U shape.
At this time, when the elastic modulus is 0.05 to 0.09 N / mm ² , it is more difficult to refract even if it is bent into a U shape.

  Furthermore, durability of the artificial blood vessel is improved by making the covering portion thick and non-cracking. Here, the non-cracking property means that, for example, the generation of strain and cracks that occur when the moisture content of the artificial blood vessel is temporarily reduced (for example, less than 60%) is suppressed.

When the water permeability measured according to ISO 7198 is 70 to 240 ml / cm ² / min, the artificial blood vessel of the present invention has an advantage that it can be transplanted smoothly and the organization is improved after transplantation.

In the artificial blood vessel of the present invention, when the silk fibroin fiber is subjected to a lower twist and the silk fibroin fiber bundle is subjected to an upper twist, an appropriate focusing effect between the silk fibroin fibers can be obtained, and at the time of knitting It can also suppress that a cross section collapses.
Further, since contact friction is reduced, problems such as yarn breakage and fluffing can be prevented.
At this time, when the number of twists of the upper twist is less than the number of twists of the lower twist, a silk fibroin fiber bundle in a stable state without rotational torque is obtained.

  According to the method for producing an artificial blood vessel of the present invention, the above-described artificial blood vessel can be obtained, so that it can be maintained for a long time.

  Further, in the above artificial blood vessel manufacturing method, the coating portion is thickened, and after the coating step, the immersion step is performed in a state where the temporary artificial blood vessel is kept wet. It is possible to suppress the occurrence of strain and cracks that occur when the amount is reduced (for example, less than 60%). In addition, if an immersion process is performed until just before transplantation of an artificial blood vessel, an artificial blood vessel can be transplanted in a state where generation of strain and cracks is suppressed as much as possible.

  In the method for producing an artificial blood vessel of the present invention, when the immersion of the temporary artificial blood vessel in the pure water in the immersion step is 1 hour or more, the pure water can sufficiently penetrate into the temporary artificial blood vessel, In addition, the resulting artificial blood vessel is difficult to dry.

Fig.1 (a) is a perspective view which shows typically the artificial blood vessel concerning this embodiment, FIG.1 (b) is an enlarged view which shows the part P of Fig.1 (a). Fig.2 (a) is a schematic diagram which shows the apparatus which measures a (compression) elastic modulus, FIG.2 (b) is a photograph of Fig.2 (a). FIG. 3A is a schematic diagram showing an apparatus for measuring the anastomosis retention strength, and FIG. 3B is a photograph of FIG. FIG. 4 is a schematic diagram showing an apparatus for measuring the porosity. Fig.5 (a) is a schematic diagram which shows the apparatus which measures circumferential-axis intensity | strength, FIG.5 (b) is a photograph of Fig.5 (a). FIG. 6 is a flowchart showing a method for manufacturing an artificial blood vessel according to the present embodiment. FIG. 7 is a partial explanatory view showing the positional relationship between the ridges, needles, and knitting yarns of the double raschel machine. FIG. 8 is an enlarged structural diagram showing a double denby structure in double raschel knitting. FIG. 9 is an enlarged structural view showing an inverted half structure in double raschel knitting. FIG. 10 is a solid ¹³ C DD / MAS spectrum obtained in Evaluation 3 of the example. FIG. 11 is a two-dimensional spectrum of ² H longitudinal relaxation time (T ₁ ) and transverse relaxation time (T ₂ ) obtained in Evaluation 4 of the example.

  Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings as necessary. In the drawings, the same elements are denoted by the same reference numerals, and redundant description is omitted. Further, the positional relationship such as up, down, left and right is based on the positional relationship shown in the drawings unless otherwise specified. Further, the dimensional ratios in the drawings are not limited to the illustrated ratios.

FIG. 1A is a perspective view schematically showing an artificial blood vessel according to this embodiment.
As shown in FIG. 1 (a), an artificial blood vessel 10 according to the present embodiment is made of silk fibroin and includes a tubular base 1 and a sponge-like covering 2 covering the surface of the base 1.

  The artificial blood vessel 10 is configured such that blood circulates in the base 1 by, for example, stitching both ends to the blood vessel.

  Since the artificial blood vessel 10 is made of silk fibroin excellent in biocompatibility, when transplanted into the body, the endothelial cells are easily fixed and thrombus is hardly formed.

  The base 1 is formed by combining a plurality of silk fibroin fibers into a silk fibroin fiber bundle, and the silk fibroin fiber bundle is knitted into a tubular shape by double raschel knitting.

Since the silk fibroin fiber before being applied to the double raschel machine has a structure in which silk sericin is adhered to the surface, the silk fibroin is protected by the silk sericin. Thereby, for example, during knitting, damage to silk fibroin is suppressed even if there is contact with a guide, a needle or the like, and as a result, a high-quality artificial blood vessel with excellent strength can be obtained.
Moreover, since silk sericin is excellent in moisture retention, it plays the role of a lubricant. For this reason, problems such as yarn breakage and fluffing during knitting can also be prevented.

  The silk fibroin fiber bundle is a bundle formed by combining a plurality of silk fibroin fibers, and is knitted into a tube by double raschel knitting using a double raschel machine. By using double raschel knitting, it is possible to obtain fraying difficulty, elasticity, and flexibility that cannot be obtained with a conventional artificial blood vessel made of silk.

  Then, after knitting into a tubular shape by double raschel knitting, the base 1 is formed by removing silk sericin. Details of the manufacturing method will be described later.

  In the artificial blood vessel 10, the silk fibroin fiber is subjected to a lower twist, and the silk fibroin fiber bundle obtained by combining a plurality of silk fibroin fibers is subjected to an upper twist. That is, a plurality of silk fibroin fibers subjected to a lower twist are combined to form a silk fibroin fiber bundle, and an upper twist is applied thereto. Note that the lower twist applied to the silk fibroin fiber and the upper twist applied to the silk fibroin fiber bundle are in opposite directions.

When the silk fibroin fiber is under twisted, an appropriate bundling effect between the silk fibroin fibers can be obtained, so that there is an advantage that a silk fibroin fiber bundle can be easily obtained.
Moreover, since the cross section can be prevented from being crushed during knitting and the contact friction between silk fibroin fibers is reduced, problems such as yarn breakage and fluffing can be prevented.
When the upper twist is applied to the silk fibroin fiber bundle, the strength is improved and the contact friction between the silk fibroin fiber bundles is also reduced as described above, so that problems such as yarn breakage and fluffing can be prevented.

  At this time, it is preferable that the number of twists of the upper twist applied to the silk fibroin fiber bundle is smaller than the number of twists of the lower twist applied to the silk fibroin fiber. In this case, a silk fibroin fiber bundle in a stable state without rotational torque is obtained.

The covering portion 2 is obtained by covering the back and front surfaces of the base portion 1 with silk fibroin. In addition, when sericin and other fat are contained in silk fibroin, these are removed and used.
Although the removal method is not particularly limited, for example, boiling washing may be performed using Marcel soap.

  The covering portion 2 is formed by applying silk fibroin to the base portion 1. At this time, since the base 1 is knitted by double raschel knitting, the silk fibroin aqueous solution passes through the stitches and easily reaches the surface on the back side of the base 1. Thereby, since the surface of the back side of the base 1 becomes smooth, the endothelial cells are more easily fixed and thrombus is less easily formed. Details of the manufacturing method will be described later.

Since the artificial blood vessel 10 includes the sponge-like covering portion 2, the flexibility is improved. For example, even when the artificial blood vessel 10 is bent in a U shape, the bending is maintained and it is difficult to refract.
Thereby, even if it is a case where the artificial blood vessel 10 is transplanted in the position which bends, it can suppress that the circulation | circulation of the blood is refracted and stagnate.

In the artificial blood vessel 10, the thickness of the covering portion 2 is such that the thickness H1 of the covering portion of the front side portion of the base portion is 0.8 mm or more (see FIG. 1B). When the thickness H1 of the covering portion on the front side portion of the base is less than 0.8 mm, there is a drawback that the number of holes is reduced and the blocking rate is improved.
Moreover, the artificial blood vessel 10 is non-cracking. That is, generation | occurrence | production of a distortion and a crack is suppressed. In order to make the artificial blood vessel 10 non-crackable, it is preferable not to dry it. Specifically, it is preferable that the water content is not less than 60%.
Once the moisture content is lowered, even if the moisture content is later set to 60% or more, strain and cracks are generated.
Thus, durability of the artificial blood vessel 10 is improved by making the covering portion thick and non-cracking.

The artificial blood vessel 10 has a moisture content of 60% or more, preferably 80% or more, and more preferably 90% or more. In particular, a saturated state is most preferable.
Here, the moisture content is calculated by dividing a value obtained by subtracting the weight of a sufficiently dried artificial blood vessel from the weight of the water-containing artificial blood vessel by the weight of the water-containing artificial blood vessel.
If the water content is less than 60%, there is a disadvantage that it cannot be maintained for a long time.

The artificial blood vessel 10 preferably has a water permeability measured according to ISO 7198 of 70 to 240 ml / cm ² / min.
When the water permeability is less than 70 ml / cm ² / min%, there is a drawback that organization is difficult to proceed after transplantation compared to the case where the water permeability is within the above range, and the water permeability is 240 ml / cm ² / min. When it exceeds%, there is a drawback that blood may leak after transplantation, compared with the case where the water permeability is in the above range.

The artificial blood vessel 10 preferably has a (compression) elastic modulus of 0.05 to 0.09 N / mm ² .
Here, the (compression) elastic modulus is measured using a pair of compression bases A1 and A2 as shown in FIGS. 2 (a) and 2 (b). That is, a sample P (tubular artificial blood vessel) having a length of 1 cm in the longitudinal direction is placed on the lower compression table A2 so that the longitudinal direction is horizontal, and the upper compression table A1 is placed in the operating cell 5N and the descent speed 2 mm. The value is calculated from the value when the compression table A1 is compressed up to 25% of the inner diameter of the sample P.
When the elastic modulus is less than 0.05%, the elastic modulus is easily refracted when bent in a U shape as compared with the elastic modulus being in the above range, and the elastic modulus is 0.09%. If it exceeds, the elastic modulus is in the above range, which makes it difficult to sew.

The artificial blood vessel 10 preferably has an anastomosis holding strength of 4.9 N or more, and more preferably 4.9 to 6.5 N.
Here, as shown in FIGS. 3A and 3B, the anastomosis holding strength refers to a sample P (tubular artificial blood vessel) having a longitudinal length of 2 cm so that the longitudinal direction is vertical. The anastomosis thread Q (surgical thread) is anastomosed at a distance of 2 mm from the end of the sample P to form an anastomosis part P1, and the anastomosis part P1 of the anastomosis thread is 2 cm away from the anastomosis part P1. It is calculated from the strength at the time of rupture when pulled in the direction at a working cell 100N and a speed of 3 mm / min.
If the anastomosis holding strength is less than 4.9 N, the anastomosis holding strength may be broken during the anastomosis as compared with the case where the anastomosis holding strength is within the above range.

The artificial blood vessel 10 preferably has a porosity of 66 to 77%.
Here, the porosity means the proportion of the pore portion (void portion) in the total volume. For example, as shown in FIG. 4, hexane is placed in a graduated cylinder, and its amount (V1) is measured. Sample P is placed therein, and after placing under reduced pressure of 500 hPa for 10 minutes, the total amount (V2). Measure. Then, the sample P is removed, and the amount of remaining hexane (V3) is measured. And the porosity can be calculated by the following formula.
ε (porosity) (%) = (V1−V2) / (V2−V3) × 100

  When the porosity is less than 66%, there is a possibility that the endothelial cells may be insufficiently fixed as compared with the case where the porosity is within the above range. When the porosity exceeds 78%, the porosity There is a possibility that the strength becomes insufficient as compared with the case where is in the above range.

The artificial blood vessel 10 preferably has a circumferential strength of 30N or more, more preferably 30 to 48N.
Here, as shown in FIGS. 5 (a) and 5 (b), the circumferential axis strength refers to two bars B1 and B2 (for example, hexagons) inside a sample P having a longitudinal length of 5 mm. Wrench or the like) is inserted, and the rods B1 and B2 are calculated from the strength at the time of breakage when they are pulled in the opposite direction to each other with the working cell 100N or 1 kN at a measurement speed of 2 mm / min.
When the circumferential strength is less than 30 N, the circumferential strength is more easily broken compared to the case where the circumferential strength is within the above range.

  The artificial blood vessel 10 is preferably stored in a bag filled with pure water in order to maintain the moisture content. The transplantation of the artificial blood vessel 10 is performed in a state where the moisture content is maintained.

  The artificial blood vessel 10 according to this embodiment can be a large-diameter artificial blood vessel or a small-diameter artificial blood vessel having an inner diameter of less than 6 mm. It can be used not only as an artificial blood vessel but also as a substitute for an artificial trachea, a stent graft, and other biological tubular structures.

Next, a method for manufacturing the artificial blood vessel 10 will be described.
FIG. 6 is a flowchart showing a method for manufacturing an artificial blood vessel according to the present embodiment.
As shown in FIG. 6, the artificial blood vessel 10 is produced by scouring raw silk composed of silk thread to obtain silk fibroin fiber in which silk sericin partially remains on the surface of silk fibroin, and silk fibroin. A yarn forming step S2 in which a plurality of fibers are combined to form a silk fibroin fiber bundle, a silk fibroin fiber bundle is knitted into a tubular body by double raschel knitting, and a knitting step S3 is formed to remove the silk sericin remaining in the tubular body. , Removal step S4 to be the base 1, and a coating step S5 to form a sponge-like covering portion 2 covered with silk fibroin on the surface of the base to make a temporary artificial blood vessel, and immersion to immerse the temporary artificial blood vessel in pure water Step S6.

  According to the method for manufacturing an artificial blood vessel according to the present embodiment, since the artificial blood vessel 10 is obtained, it can be maintained for a long period of time.

Hereinafter, each step will be described in more detail. In addition, the description which overlaps with description of the artificial blood vessel mentioned above is abbreviate | omitted.
(Scouring process)
The scouring step S1 is a step of scouring raw silk composed of silk thread to form silk fibroin fibers in which part of the silk sericin remains on the surface of the silk fibroin.

The silk thread is a thread that the silk thread discharges from the spout, and has a core-sheath structure in which a pair of silk fibroin is covered with silk sericin.
A silk fibroin fiber in which part of the surface silk sericin remains is obtained from the silk thread. In addition, it is preferable that the weight of the silk sericin to remain | survive is 20 to 40% of a total weight from a viewpoint of protecting silk fibroin.

  The scouring method is not particularly limited, but it is preferable to use an enzyme. Such enzymes include proteases.

  After scouring, it is preferable to perform a washing step and a drying step. Thereby, dirt and useless sericin can be removed, and generation of mold and the like due to drying can be prevented.

(Yarn making process)
The yarn making step S2 is a step in which a plurality of silk fibroin fibers are combined to form a silk fibroin fiber bundle.

It is preferable to apply a lower twist to the silk fibroin fiber and to apply an upper twist to the silk fibroin fiber bundle.
Specifically, the silk fibroin fiber is subjected to a lower twist of 1100 t / m by left twist.
Then, at least two silk fibroin fibers subjected to a lower twist are combined, and an upper twist of 900 t / m is applied by a right twist. As a result, a so-called twisted silk fibroin fiber bundle is obtained. In addition, as a range of the above-mentioned twist processing, from the viewpoint of the twist effect mentioned later, 900 t / m to 1200 t / m for the lower twist and 700 t / m to 1000 t / m for the upper twist are preferably employed.

Here, it is preferable to reverse the twist direction of the lower twist and the upper twist and to make the number of the upper twists smaller than the number of the lower twists. In this case, a silk fibroin fiber bundle in a stable state without rotational torque is obtained. It should be noted that how much the number of upper twists should be made smaller than the number of lower twists is determined to be the most stable state with no rotational torque.
Further, by twisting, it is possible to prevent the cross section from being crushed during knitting, and the contact friction between silk fibroin fibers is reduced, so that problems such as yarn breakage and fluffing can be prevented. Thereby, even a so-called small-diameter artificial blood vessel having an inner diameter of 6 mm or less can be manufactured.

(Knitting process)
The knitting step S3 is a step in which the silk fibroin fiber bundle is knitted into a tubular shape by double raschel knitting to form a tubular body.
Here, “double raschel knitting” means a knitting method in which elastic fibers are knitted in a columnar shape by a double raschel machine. By performing double raschel knitting, there is an advantage that fraying difficulty, elasticity, and flexibility are improved as compared with conventional woven fabrics.

Here, an example of double raschel knitting using a silk fibroin fiber bundle will be described.
FIG. 7 is a partial explanatory view showing the positional relationship between the ridges, needles and knitting yarns of the double raschel machine.
As shown in FIG. 7, a silk fibroin fiber bundle is used as a knitting yarn Y (Y1 to Y6), and a tubular body is knitted by a 6-sheet double Russell machine R (28 gauge). The gauge is the number of needles N existing between 1 inch (2.54 cm).

FIG. 8 is an enlarged structural diagram showing a double denby structure in double raschel knitting.
As shown in FIG. 8, as the knitting structure, a double denby structure B (knitting 1-0 / 1-2 with oscillating L1 and 1-2 / 1-0 with oscillating L3) is adopted. The double denby structure B is obtained by simultaneously knitting two denby structures D.

Returning to FIG. 7, the knitting yarns Y1 and Y3 are controlled by the ribs L1 and L3, and the front knitted fabric K1 of the double denby structure B is knitted by the front needle FN.
Then, the knitting yarns Y4 and Y6 are controlled by the loops L4 and L6, and the back knitted fabric K2 of the double denby structure B is knitted by the back needle BN.
A tubular tubular body W is formed by connecting the knitted fabrics K1 and K2 of the two double denby structures B knitted in this way by knitting yarns Y2 and Y5 controlled by the loops L2 and L5.
In the case of this double denby structure B, since the knitting yarn is swung from needle to needle during knitting, the above-described twisting effect and covering effect can be further exhibited.

(Removal process)
The removal step S4 is a step of removing the silk sericin remaining in the tubular body to form the base 1. That is, in the removal step S4, the sericin adhered in the manufacturing process of the tubular body is completely removed.

  The removal method is not particularly limited, and a known method can be used. For example, a mixed aqueous solution of Marcel soap and sodium carbonate is heated to 100 ° C., a tubular body is added, boiled and washed with stirring for several hours, then boiled and washed together in the order of sodium carbonate aqueous solution and distilled water, and dried. By doing so, the base 1 from which sericin has been removed is obtained. These boiling washings may be repeated a plurality of times.

(Coating process)
In the coating step S5, a sponge-like covering portion 2 covered with silk fibroin on the surface of the base portion is formed so that the thickness H1 of the covering portion 2 on the front side portion of the base portion is 0.8 mm or more to form a temporary artificial blood vessel. It is a process. Note that the thickness H1 of the covering portion 2 is the same as described above, and a description thereof will be omitted.

For example, the coating step S5 is performed by immersing the base 1 in a silk fibroin solution, attaching silk fibroin to the back and front surfaces of the base 1, and freeze-drying.
Here, the silk fibroin solution is obtained by removing silk sericin, then dissolving the silk fibroin fiber, sponge, and film in a neutral salt such as lithium bromide or calcium chloride, and then removing the neutral salt by dialysis. .

  Or coating process S5 is performed by coating the base 1 with the liquid mixture of a hole source and silk fibroin. At this time, the liquid mixture passes through the stitches of the base 1 and reaches the surface on the back side of the base 1. Thereafter, if the pore source material is removed by freezing and then sufficiently immersed in water, the coating portion becomes a silk sponge. Examples of the hole source material include polyglycols.

It should be noted that the silk fibroin solution in both steps may further contain an antithrombotic agent such as heparin.
Moreover, the base 1 that has undergone the coating step S5 may be subjected to autoclave sterilization at 120 ° C. for 20 minutes.

  By performing the coating step S <b> 5, a sponge-like (porous) covering portion 2 is formed on the surface of the base portion 1.

(Immersion process)
The immersing step S6 is a step of immersing the temporary artificial blood vessel in pure water in a state where the wet state of the temporary artificial blood vessel is maintained after the coating step S5.
After the coating step S5, in order to maintain the wet state of the temporary artificial blood vessel, the strain generated when the moisture content of the temporary artificial blood vessel is temporarily reduced (for example, less than 60%) by performing the immersion step S6 promptly. Generation of cracks can be suppressed.

The dipping step S6 is performed, for example, by dipping in a bag filled with pure water.
Although the period immersed in a pure water is not specifically limited, It is preferable that it is 1 hour or more, and it is more preferable that it is 1 hour-1 week. If the immersion period is less than 1 hour, the moisture content may not be sufficient as compared to the case where the immersion period is within the above range. If the immersion period exceeds 1 week, There is a risk that the silk sponge on the surface of the artificial blood vessel may be removed or hardened as compared with the case where the period of time is within the above range.

It is preferable that the dipping step S6 is performed until just before the transplantation of the artificial blood vessel 10. In this case, the artificial blood vessel 10 can be transplanted in a state where generation of strain and cracks is suppressed as much as possible.
By these steps, the artificial blood vessel 10 having the water content described above is obtained.

  As mentioned above, although preferred embodiment of this invention was described, this invention is not limited to embodiment mentioned above.

  In the artificial blood vessel 10 according to the present embodiment, the silk fibroin fiber is subjected to a lower twist, and the silk fibroin fiber bundle obtained by combining a plurality of silk fibroin fibers is subjected to an upper twist, but is not necessarily essential. It is not a configuration.

The artificial blood vessel 10 according to the present embodiment can be subjected to bellows processing or the like in order to improve flexibility.
Moreover, in order to maintain a moist state, you may further coat with glycerin.

In the method for manufacturing the artificial blood vessel 10 according to the present embodiment, a double denby structure in double raschel knitting is shown in FIG. 8, but the present invention is not limited to this. Good.
For example, the reverse half structure H shown in FIG. 9 (knitting 1-2 / 1-0 with oscillating L4 and 1-0 / 2-3 with oscillating L6) is formed by Denby organization D and chord organization C. In the tissue, the sinker loop S1 of the cord tissue C is longer than the double denby tissue B by one stitch compared to the sinker loop S2 of the Denby tissue D, and thus a dense knitted fabric is obtained and used as the tubular body W. In some cases, it is effective in preventing blood leakage.
In the case of this reverse half structure H, since the way of knitting yarn to the needle during knitting becomes larger than that of the Denby structure, a twisting effect and a covering effect are further exhibited.

  EXAMPLES Hereinafter, although this invention is demonstrated more concretely based on an Example and a comparative example, this invention is not limited to a following example.

(Examples 1 and 2 and Comparative Examples 1 and 2)
First, raw silk composed of silk thread and protease ("Esperase" (trade name); manufactured by Novozymes Japan Co., Ltd.) are added to water and scoured under mild conditions, and silk sericin is formed on the surface of silk fibroin. Part of the remaining silk fibroin fiber was obtained (scouring process). At this time, the weight of the remaining silk sericin was 10 to 15% of the total weight.

  After washing and drying, the silk fibroin fiber is subjected to a lower twist of 1100 t / m by left twist, and 30 silk fibroin fibers are combined to form a silk fibroin fiber bundle, and further, an upper twist of 900 t / m is twisted by right twist. The silk fibroin fiber bundle was obtained by applying (spinning process).

  Next, the silk fibroin fiber bundle was knitted into a tubular shape by double raschel knitting according to the double denby structure shown in FIG. 8 to obtain a tubular body (knitting step).

  Next, the tubular body is put into a mixed aqueous solution of 12 w / v% Marcel soap and 8 w / v% sodium carbonate heated to 95 ° C., boiled for 120 minutes, and then boiled for 10 minutes with 2 w / v% sodium carbonate aqueous solution. Then, the base 1 from which silk sericin remaining in the tubular body was removed was obtained by performing the operation of washing in distilled water heated to 95 ° C. three times and the operation of washing with warm water three times (removal step). ).

Next, silk fibroin solution is dissolved in 9 mol of neutral salt aqueous solution (lithium bromide, lithium chloride, calcium chloride or lithium thiocyanate), and then neutral salt is removed by dialysis. Got.
And the base 1 was immersed in the silk fibroin melt | dissolution of 4 w / v% density | concentration under normal pressure for 30 minutes, silk fibroin was made to adhere to the surface of the back side and front side of the base 1, and it was left to stand at -20 degreeC for 1 hour. After putting it in a freeze dryer overnight, it was put in a pouch together with water, sterilized by an autoclave, and stored in distilled water to obtain a temporary artificial blood vessel in which the covering portion 2 was formed (coating step).

  Next, in order to maintain a wet state, the temporary artificial blood vessel was immediately immersed in pure water. By adjusting the immersion time, a sample having a diameter of 3.5 mm was obtained (immersion step). Table 1 shows the physical property data of the obtained sample.

(Example 3)
Instead of the coating process described above, the base 1 is immersed for 10 minutes under normal pressure in a coating solution in which polyethylene glycol diglycidyl ether is mixed at a weight ratio of 1: 1 with a 4 w / v% silk fibroin aqueous solution. Silk fibroin was attached to the surface of the back side and the front side of the base 1 and left at −20 ° C. overnight. After thawing at room temperature, a sample having a diameter of 3.5 mm was obtained in the same manner as in Example 1 except that the coating part 2 was formed by immersing in water for 3 days to remove polyethylene glycol diglycidyl ether. Table 1 shows the physical property data of the obtained sample.

(Comparative Example 3)
A sample having a diameter of 3.5 mm was obtained in the same manner as in Example 1 except that the dipping process was not performed. Table 1 shows the physical property data of the obtained sample.

(Table 1)

(Evaluation 1)
Surgery for transplanting the samples obtained in Examples 1 to 3 and Comparative Examples 1 and 2 into the carotid artery of the dog was performed, and the patency rate after 2 weeks was evaluated by echo. In the evaluation, “◯” indicates the existing one, “Δ” indicates that the existing one is likely to be blocked, and “×” indicates that the one is closed. The obtained results are shown in Table 2.

(Evaluation 2)
An operation for transplanting the samples obtained in Examples 1 to 3 and Comparative Examples 1 and 2 into the carotid artery of a dog was performed, and the patency period was measured. The obtained results are shown in Table 2.

(Table 2)

Example 4
In the same manner as in Example 1, except that raw silk having the same composition in which carbons of ¹³ amino acids such as Ala, Tyr, Gly, and Ser were marked with hydrogen and deuterium was used instead of raw silk, A 5 mm sample was obtained. In addition, marking was performed by mixing [3- ¹³ C] Tyr and [3- ¹³ C] Ser in an artificial feed and orally administering it to a rabbit at the age of 5 years.

(Comparative Example 4)
A sample with a diameter of 3.5 mm was obtained in the same manner as in Example 4 except that the dipping process was not performed.

(Evaluation 3)
Changes in the local structure and motility of silk were investigated for the sample of Example 4 (hydrated state) and the sample of Comparative Example 4 (dried state). Specifically, solid ¹³ C DD / MAS spectra were measured for both samples using a Bruker Biospin Avance DSX400 NMR apparatus. The obtained result is shown in FIG. In FIG. 10, “wet” is the result of measurement using the sample obtained in Example 4, and “dry” is the result of measurement using the sample obtained in Comparative Example 4.

As shown in FIG. 10, in the sample obtained in Example 4, in addition to the broad component in the sample obtained in Comparative Example 4, Ala-Cβ, Tyr-Cβ, Gly-Cα, Ser-Cβ are sharp. Peak was observed.
This means that the sample obtained in Example 4 has a site where water is easily accessible in the structure.
In addition, serine residues are mainly present in the crystalline region, and tyrosine residues are present in the semicrystalline region, which means that the site is present not only in the semicrystalline region but also in the crystalline region.
Therefore, it was found from the above that the artificial blood vessel of the present invention can easily have a moisture content of 60% or more, and can retain moisture effectively through an immersion process.

(Evaluation 4)
The mobility of water in silk was investigated for the sample of Example 4 (containing water). Specifically, heavy water was immersed in the sample of Example 4, and a two-dimensional spectrum of ² H longitudinal relaxation time (T ₁ ) and transverse relaxation time (T ₂ ) was measured using a Varian Unity 200 NMR apparatus. The obtained results are shown in FIG. In FIG. 11, each peak indicates the amount of heavy water (log scale), which means that it increases as it goes to the top.

As shown in FIG. 11, in the sample obtained in Example 4, it was confirmed that four types of water molecules were present in the system. That is, the heavy water in the state of the strongest interaction with silk and the restricted movement was T ₁ = 0.14 s, T ₂ = 0.01 s, and the correlation time τc = 2.2 × 10 ⁻⁸ s.
This means that the sample obtained in Example 4 interacts with a lot of water.
Therefore, from this, it was found that the artificial blood vessel of the present invention interacts with water with certainty, so that it is important to set the water content to 60% or more and to undergo an immersion step.

  From the above, it was confirmed that the artificial blood vessel according to this embodiment can be maintained for a long period of time.

The artificial blood vessel of the present invention is used for a purpose of temporarily securing a blood flow path during surgery or a use as a substitute blood vessel for a diseased blood vessel.
Moreover, the artificial blood vessel of the present invention can be a large-diameter artificial blood vessel or a small-diameter artificial blood vessel having an inner diameter of less than 6 mm.
According to the artificial blood vessel of the present invention, it becomes possible to make it open for a long time.

DESCRIPTION OF SYMBOLS 1 ... Base part 2 ... Covering part 10 ... Artificial blood vessel A1 ... Upper compression stand A2 ... Lower compression stand B ... Double Denby tissue B1, B2 ... Bar BN ... -Back needle C ... Cord structure D ... Denby structure FN ... Front needle H ... Reverse half structure H1 ... Thickness K1 ... Front knitted fabric K2 ... Back knitted fabric L1-L6 ... Osa N ... Needle P ... Sample P1 ... Anastomosis part Q ... Anastomosis thread R ... Double Russell machine S1 ... Sinker loop of cord tissue S2 ... Sinker of Denby tissue Loop W ... Tubular body Y (Y1-Y6) ... Knitting yarn

Claims (10)

A plurality of silk fibroin fibers with silk sericin attached to the surface are combined into a silk fibroin fiber bundle, the silk fibroin fiber bundle is knitted into a tubular shape by double raschel knitting, and the base obtained by removing the silk sericin,
A sponge-like covering portion in which the surface of the base portion is covered with silk fibroin;
With
The thickness of the covering portion of the front side portion of the base portion is 0.8 mm or more,
A non-cracking artificial blood vessel having a water content of 60% or more. The artificial blood vessel according to claim 1, wherein the water permeability measured according to ISO 7198 is 70 to 240 ml / cm ² / min. Artificial blood vessel according to claim 1 or 2 elastic modulus is 0.05~0.09N / mm ^2. The silk fibroin fiber is under twisted,
The artificial blood vessel according to any one of claims 1 to 3, wherein an upper twist is applied to the silk fibroin fiber bundle.   The artificial blood vessel according to claim 4, wherein the number of twists of the upper twist is less than the number of twists of the lower twist. A method for producing an artificial blood vessel according to any one of claims 1 to 5,
Scouring raw silk composed of silk thread to make silk fibroin fiber with some silk sericin remaining on the surface of silk fibroin,
A yarn-making process in which a plurality of silk fibroin fibers are combined to form a silk fibroin fiber bundle;
A knitting step in which the silk fibroin fiber bundle is knitted into a tubular body by double raschel knitting, and a tubular body;
Removing the silk sericin remaining in the tubular body, and using as a base,
Forming a sponge-like covering portion covered with silk fibroin on the surface of the base portion so that the thickness of the covering portion of the front side portion of the base portion is 0.8 mm or more, and forming a temporary artificial blood vessel;
An immersion step of immersing the temporary artificial blood vessel in pure water;
With
A method of manufacturing an artificial blood vessel, wherein the immersion step is performed after the coating step while maintaining the wet state of the temporary artificial blood vessel.   The method for producing an artificial blood vessel according to claim 6, wherein the immersing step is performed by immersing the temporary artificial blood vessel in the pure water for 1 hour or more.   The method for manufacturing an artificial blood vessel according to claim 6 or 7, wherein the immersion step is performed until immediately before transplantation of the artificial blood vessel.   The method for producing an artificial blood vessel according to any one of claims 6 to 8, wherein the coating step is performed by immersing the base in a silk fibroin solution and freeze-drying.   The method of manufacturing an artificial blood vessel according to any one of claims 6 to 8, wherein the coating step is performed by coating the base with a silk fibroin sponge using a hole source material.

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