JP2014050412A - Production method of artificial blood vessel - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a tubular structure object that is excellent in hardness and elasticity while having silk original characteristics such as biocompatibility, and that can be used for blood vessel of a small bore diameter such as peripheral blood vessel.SOLUTION: A coating composition of a tubular structure object including a silk fibroin and a polyurethane resin is prepared. The silk fibroin and the polyurethane resin are performed by dispersion or dissolution to water, an organic solvent or a mixture of water and the organic solvent to make a coating solution. A tubular structure object of a silk fibroin fiber is immersed in the coating solution, then frozen, heat-treated, and dehydrated to thereby produce the coating tubular structure object.

Description

  The present invention relates to a coating composition and a method for producing an artificial blood vessel using 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 and the like are required. 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 with a diameter of 5 mm or less are due to thickening of the intima due to biocompatibility of typical artificial materials such as polyethylene terephthalate (PET) and polytetrafluoroethylene (PTFE) and occlusion due to thrombus formation, It has not yet been put to practical use. Therefore, at present, autologous vein transplantation is performed for bypass to the knee joint periphery, etc., but there are many patients who do not have compatible blood vessels and cannot perform autologous vein transplantation because of heavy burden on the patient. There are many problems. In recent years, with the aging of patients and the increase in diabetes, the demand for regenerative treatment of small blood vessels is increasing. Therefore, development of an artificial blood vessel having an antithrombotic property that can be used particularly for a small-diameter blood vessel such as a peripheral blood vessel has been strongly desired.

  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 by bonding the string with sericin.
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 that there is a demand for an artificial blood vessel having superior functionality while maintaining the original characteristics of silk such as biocompatibility and required characteristics during surgery. It had been.

JP 2004-173772 A JP 2010-137041 A

Tubular structures using silk fibroin fibers obtained by scouring silk and raw silk are low in allergic risk due to sericin and excellent in biocompatibility, but are not sufficient in terms of functionality.
Then, when the present inventors examined, it discovered that it can give elasticity if it coats the tubular structure which consists of silk fibroin with the silk fibroin sponge obtained from the aqueous solution which mixed the polyglycol with the silk aqueous solution, and applied for earlier ( Patent Document 2).
However, considering practical use, it has not yet been satisfactory in terms of elasticity and the like, and there has been a demand for an artificial blood vessel that is further excellent in functionality.

  The present invention has been made in view of the above circumstances, and provides a tubular structure that has excellent properties such as biocompatibility and has excellent strength and elasticity and can be used for small-diameter blood vessels such as peripheral blood vessels. About doing.

  The present inventor has made various studies on tubular structures utilizing the characteristics of silk.If the tubular structure is coated with a mixture of silk fibroin and polyurethane resin, it has sufficient tensile strength and excellent elasticity. The present inventors have found that a tubular structure suitable for an artificial blood vessel having a smaller diameter, which is flexible and satisfies biocompatibility and characteristics required at the time of operation, can be obtained.

That is, the present invention provides a coating composition for a tubular structure containing silk fibroin and a polyurethane resin.
Moreover, this invention provides the manufacturing method of a coated tubular structure which coats a tubular structure with the said composition for coating.

  According to the present invention, a tubular structure that has the original properties of silk, that is, high biocompatibility, has sufficient tensile strength, excellent elasticity, is flexible and easily vascularly anastomosed, and has little blood leakage. Can be provided. Since this tubular structure is excellent in antithrombotic properties, it is particularly suitable for a small-diameter artificial blood vessel having a diameter of 5 mm or less.

It is a figure which shows the coating process of a rabbit silk artificial blood vessel. It is a figure which shows the scanning electron microscope image of a coating rabbit silk artificial blood vessel. (A) Example 1-4 It is a figure which shows the scanning electron microscope image of a coating rabbit silk artificial blood vessel. (B) Comparative Example 1-3 It is a figure which shows the water permeability of a coating rabbit silk artificial blood vessel. It is a figure which shows the compression elastic modulus of a coated rabbit silk artificial blood vessel. It is a figure which shows the anastomosis thread retention strength of a coated rabbit silk artificial blood vessel. It is the figure which compared the change of the various parameters before and 2 weeks after an operation. PRE: preoperative native carotid artery data, POST PROX: data obtained on the proximal side of the post-operative artificial blood vessel, POST DISTAL: data obtained on the distal side of the post-operative artificial blood vessel, PSV: Maximum systolic flow velocity (cm / s), EDV end diastolic flow velocity (cm / s), PI pulsatility index (no unit), RI resistance coefficient (no unit), CSD: vessel diameter (mm)

The coating composition of the present invention is a composition used for coating a tubular structure containing silk fibroin and polyurethane resin.
The silk fibroin used in the present invention is obtained by scouring silkworms, raw silk, and the like obtained from silkworms or genetically modified silkworms, and wild silkworm layers such as Eli silkworms, silkworms, 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, 12 w / v% Marcel soap heated to 100 ° C., 8 w / 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 2 w / After performing the operation of boiling in a v% aqueous sodium carbonate solution for 10 minutes and further washing in distilled water heated to 100 ° C. three times, the protein (sericin) covering fibroin and other fats can be removed by drying.

  The silk fibroin is preferably a silk fibroin solution obtained by dissolving silk fibroin fibers in water and / or an organic solvent. For example, a silk fibroin fiber dissolved in water and / or an organic solvent; a silk fibroin fiber dissolved in a neutral salt aqueous solution and heated, and then the obtained silk fibroin / salt aqueous solution is desalted; What melt | dissolved the latter solution | solution further in the organic solvent, etc. are mentioned.

  Here, examples of the neutral salt aqueous solution include lithium bromide, lithium chloride, calcium chloride, and lithium thiocyanate. Moreover, as a method for desalting treatment, a known method such as a dialysis method or a reverse osmosis method can be employed. After desalting, water may be removed from the aqueous solution as necessary to obtain a dry product. In this case, the aqueous solution is usually spread on a plate, and water is evaporated to produce a silk fibroin film, or spray drying or the like to form a powder. Further, distilled water may be added to prepare an aqueous solution having a silk fibroin concentration of 2 w / v% or less, for example, and freeze-dried to form a sponge (porous). Of these, lyophilization is preferred from the viewpoint of handleability and storage stability.

Examples of the organic solvent include hexafluoroisopropanol (HFIP) and hexafluoroacetone (HFA). Usually, since HFA exists stably as a hydrate, it is preferable to use HFA hydrate. HFIP and HFA may be used alone or in combination.
The silk fibroin concentration dissolved in water or an organic solvent is usually about 3 to 20 w / v%, preferably 3 to 15 w / v%.

  The polyurethane-based resin used in the present invention is a resin obtained by reacting polyisocyanate and polyol as main raw materials according to a conventional method. The polyurethane resin is not particularly limited, and examples thereof include polycarbonate urethane resin, polyester urethane resin, polyether urethane resin, silicone modified urethane resin, fluorine modified urethane resin, and polyamino acid urethane resin.

  The polyisocyanate is not particularly limited as long as it is a compound having two or more isocyanate groups in the molecule. For example, aliphatic diisocyanates such as hexamethylene diisocyanate and 2,2,4-trimethylhexamethylene diisocyanate; isophorone diisocyanate, Examples thereof include alicyclic diisocyanates such as hydrogenated xylylene diisocyanate, dicyclohexylmethane diisocyanate and norbornane diisocyanate; aromatic diisocyanates such as tolylene diisocyanate, diphenylmethane diisocyanate, naphthalene diisocyanate, tolidine diisocyanate and xylylene diisocyanate. These can be used alone or in combination of two or more.

  The polyol is not particularly limited. For example, polyethylene adipate diol, polyethylene propylene adipate diol, polybutylene adipate diol, polyethylene butylene adipate diol, polyhexamethylene adipate diol, polydiethylene adipate diol, polyethylene terephthalate diol, polyethylene isophthalate diol Polyester polyols such as polyhexamethylene isophthalate adipate diol, polyethylene succinate diol, polybutylene succinate diol, polyethylene sebacate diol, polybutylene sebacate diol, poly-ε-caprolactone diol; polyoxytetramethylene glycol, polyoxy Propylene glycol, polyoxyethylene Polycarbonate polyols such as polyhexamethylene carbonate diol; polyether polyols such as propylene glycol Patent acrylic polyols such as those described in 2000-119362 JP include dimer diol. These can be used alone or in combination of two or more.

The polyurethane resin used in the present invention is preferably an aqueous (water-soluble or water-dispersible) polyurethane resin.
The aqueous polyurethane resin can be produced by a conventional method. As a method for imparting water to the polyurethane resin, for example, an anionic group or an ionic group of a cationic group is introduced into the urethane skeleton, or a nonion is used. The method of introduce | transducing a sex group is mentioned. Moreover, you may carry out forced emulsification using emulsifiers, such as surfactant. The ionic group, -COO ^- group, -SO ₃ ^- anionic groups such as, = N ⁺ = group, = P ⁺ = include cationic groups such as, in particular, -COO ^- group.

An aqueous polyurethane resin having an ionic group can be obtained by, for example, the above-mentioned polyisocyanate, polyol, a compound imparting an ionic hydrophilic group, a known chain extender, if necessary, and the like by a one-shot method or a prepolymer method. Can be manufactured.
Here, the compound that imparts the ionic hydrophilic group is not particularly limited, but examples of the compound that imparts the anionic hydrophilic group include 2,2-dimethylolpropionic acid, 2,2-dimethylolbutanoic acid, and the like. Can be mentioned. These are used after neutralization with amines such as trimethylamine, triethylamine, tri-n-propylamine, tributylamine, triethanolamine, potassium hydroxide, sodium hydroxide, ammonia and the like.
Moreover, as what gives a cationic group, alkanolamines are preferable, for example, N-methyldiethanolamine, a triethanolamine, etc. can be mentioned. These are used after neutralization with various organic acids such as hydrochloric acid, nitric acid, formic acid and acetic acid, and inorganic acids, or quaternization with dimethyl sulfate, diethyl sulfate, benzyl chloride, epichlorohydrin and the like.
These can be used alone or in combination of two or more.

  A commercially available product may be used as the aqueous polyurethane resin. For example, water-dispersible polyurethane resins such as Neo Sticker 100C (Nika Kagaku) and Evaphanol (Nikka Kagaku) can be used.

  In the coating composition of the present invention, the content of silk fibroin is preferably 0.1 to 10% by mass, and preferably 0.2 to 5% by mass from the viewpoint of biocompatibility.

  In the coating composition of the present invention, the content of the polyurethane resin varies depending on the type of the resin. For example, in the case of a water-dispersible polyurethane resin, 5 to 50% by mass is preferable, and 15 to 30% by mass. Is preferred.

  The blending ratio (mass ratio) of silk fibroin and polyurethane resin is preferably in the range of 50: 1 to 1: 100, from the viewpoint of improving tensile strength and elasticity, and more preferably 10: 1 to 1: 100, especially 1. : 1-1: 100 is preferable.

The coating composition of the present invention is preferably prepared by dissolving or dispersing silk fibroin and polyurethane resin in water, an organic solvent, or a mixture thereof.
When an aqueous polyurethane resin is used as the polyurethane resin, it is preferably dissolved or dispersed in water. On the other hand, when a non-aqueous polyurethane resin is used, it is preferably dissolved in an organic solvent. Among these, a composition in which silk fibroin and an aqueous polyurethane resin are dissolved or dispersed in water is preferable from the viewpoint of improvement in tensile strength and elasticity.
In this case, either silk fibroin or polyurethane resin may be dissolved or dispersed in water and / or an organic solvent first, and then the other may be dissolved or dispersed. Also good. In addition, for example, a silk fibroin solution obtained by dissolving silk fibroin fibers in water or an organic solvent as described above may be mixed with a polyurethane resin.
In addition, as an organic solvent, the same thing as the above is mentioned.

  The coated tubular structure of the present invention is produced by preparing a coating liquid containing silk fibroin and polyurethane resin, and coating the innermost wall and / or outermost wall of the tubular structure with the coating liquid. In addition to silk fibroin and polyurethane-based resin, the coating liquid can appropriately contain arbitrary components. For example, an antithrombotic agent such as heparin, endothelial cell growth factor and the like can be contained.

  The tubular structure in this invention is not specifically limited, The tubular structure comprised from the various raw material can be used. For example, the thing made from PET, PTFE, silk fibroin, etc. are mentioned. Among these, a tubular structure made of silk fibroin is preferable.

  Examples of the tubular structure made of silk fibroin include a tubular structure in which silk fibroin fibers are wound by one or more methods selected from knitting, braiding, weaving, and entanglement. 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. Moreover, you may use the knitted fabric and textile fabric which were previously produced using the silk fibroin fiber, the cylindrical thing, etc. When using a kite string or raw silk, it is preferable to remove sericin after forming it into a tubular shape.

  For example, the method for braiding 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 thermoplastic resin core rod and the like and assembling silk fibroin fibers. Here, as the thermoplastic resin core rod, one having an outer diameter of 1 to 5 mm, preferably 2 to 5 mm, can be used according to the size of the target tubular structure. The thermoplastic resin is not particularly limited, and examples thereof include polyolefin, polyester, fluororesin, and vinyl chloride resin.

  In addition, if silk fibroin fibers are knitted and formed into a tubular shape, a tubular structure that is highly elastic, flexible, excellent in tensile strength, and difficult to fray at the cut end can be obtained. Examples of knit knitting include round knitting (horizontal knitting), vertical knitting, full fashion, raschel, tricot and the like. The knit machine is not particularly limited. The obtained silk cloth may be wound around the thermoplastic resin core rod or the like to form a tubular shape.

The method of coating the tubular structure with the coating composition of the present invention is not particularly limited, but it is preferable to immerse the tubular structure in the coating solution, freeze, heat-treat, and then dry.
The immersion treatment is not particularly limited, but is performed at normal temperature, normal pressure, or reduced pressure. The immersion time is preferably 5 to 30 minutes, particularly 10 to 20 minutes.

  After immersion, it is frozen at -20 ° C to -196 ° C, preferably at -50 ° C to -100 ° C for 0.5 minutes to 2 hours. After the freezing treatment, further freeze-drying may be performed. In the case of freeze-drying, it is preferable to use a coating solution in which silk fibroin and aqueous polyurethane resin are dissolved or dispersed in water. This makes silk fibroin insoluble and has a fine porous structure, making it more elastic. A tubular structure excellent in flexibility is obtained.

  The heat treatment is preferably performed at a temperature of 100 ° C. or higher, and more preferably 120 ° C. or higher. For the heat treatment, a closed pressure device such as an autoclave may be used. When using an autoclave, it is preferably 120 ° C. or higher and 20 to 40 minutes.

  Examples of the drying treatment include natural drying, heat drying, and vacuum drying. The drying temperature is 15 ° C to 150 ° C, preferably 50 ° C to 80 ° C, and the time is preferably 30 minutes to 1 hour.

In this way, the innermost wall and / or the outermost wall of the tubular structure is coated.
In the present invention, the coating layer formed on the tubular structure may be a single layer, or may be a coated tubular structure having a multilayer coating layer such as two layers, three layers, or four layers.
The thickness of the coating layer is not particularly limited, but the innermost wall portion is preferably 0.005 to 0.5 mm, and the outermost wall portion is preferably 0.1 to 1.0 mm.

  In the coated tubular structure of the present invention, only one of the innermost wall and the outermost wall of the tubular structure may be coated with a film coating composition containing silk fibroin and polyurethane resin, and both are coated. May be. From the viewpoint of preventing blood leakage and improving tensile strength and elasticity, the innermost wall of the tubular structure is preferably coated with the film coating composition. In this case, the other may be coated with an arbitrary coating agent. Examples of such other coating agents include general coating agents such as polyurethane resins and gelatin.

  The coated tubular structure of the present invention thus obtained can be used as a substitute for, for example, an artificial blood vessel, an artificial trachea, a stent graft, and other animal 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.

Production Example 1
<Preparation of dissolved silk fibroin solution>
A silk fibroin fiber from which a protein (sericin) covering fibroin and other fats were removed was obtained by finely cutting the rabbit with a scissors (about 2 mm × 10 mm) or spinning the rabbit.
Next, this silk fibroin fiber was dissolved in 9M lithium bromide aqueous solution so as to be 15 w / v%. This aqueous solution is dialyzed with pure water using a cellulose dialysis membrane (Seamless Cellulose Tubing 36/32 manufactured by VISKASESELES COAP) for 3 days to remove lithium chloride, and further centrifuged to remove undissolved residue and dust. Then, a silkworm silk fibroin lysate was obtained. The silk fibroin concentration in the rabbit silk fibroin lysate was 3-8 w / v%.

Production Example 2
<Creating silkworm silk artificial blood vessels>
1. Fabrication of small-diameter silk graft using an assembling machine For the fabrication of an artificial blood vessel, a braided string production device (16 strokes) (manufactured by Kokbun Limited) was used. The bobbin was wrapped with silk fibroin fiber refined by the above-mentioned standard method. Attach a vinyl chloride or polytetrafluoroethylene core rod with an outer diameter of 1 to 5 mm to the center of the machine, assemble silk fibroin fibers around the core rod, 5 mmφ) was obtained.

Production Example 3
<Preparation of coating composition>
Rabbit silk fibroin melt (SI) obtained in Production Example 1 and water-dispersible polyurethane (PU, Neo Sticker 100c, manufactured by Nikka Chemical Co., Ltd., polyurethane concentration 30 w / v%) in a volume ratio of 1:10, 10 Were mixed at a ratio of 1:10, 1: 4 and 1: 2, respectively, to obtain coating compositions (1), (2), (3) and (4).
Further, a coating composition (5) using only the water-dispersible polyurethane and a coating composition (6) using only a rabbit silk fibroin solution were obtained.

Example 1
<Rabbit silk artificial blood vessel coating>
In the coating composition (5), a silkworm silk artificial blood vessel (inner diameter: 4 mmφ) with a core rod attached was immersed for 10 minutes under normal pressure, and then immersed in liquid nitrogen for 1 minute to freeze. This operation was repeated once more. Next, the frozen silkworm artificial blood vessel was autoclaved at 120 ° C. for 20 minutes, separated from the core rod, and dried in a dryer set at 50 ° C. for 1 hour to thereby remove the outer wall of the silkworm silk artificial blood vessel. Two layers of coated rabbit silk artificial blood vessel were obtained.
Thereafter, 6 mL of the coating composition (1) was flowed on the inner wall of the silkworm artificial blood vessel using a syringe, and then immersed in liquid nitrogen for 1 minute to freeze. This operation was repeated once more. Next, after drying at room temperature for 30 minutes, it was dried in a dryer set at 120 ° C. for 2 hours to obtain a coated rabbit silk artificial blood vessel in which the inner wall of the rabbit silk artificial blood vessel was coated in two layers. The coating conditions are shown in Table 1 (hereinafter the same).

Comparative Example 1
<Rabbit silk artificial blood vessel coating>
In the same manner as in Example 1, a coated rabbit silk artificial blood vessel in which the outermost wall of the rabbit silk artificial blood vessel was coated in two layers with the coating composition (5) was obtained.
Then, the coated rabbit silk artificial blood vessel which coated the inner wall of the rabbit silk artificial blood vessel in two layers was obtained in the same manner as in Example 1 except that the coating composition (1) was replaced with the coating composition (6).

Examples 2-3 and Comparative Examples 2-3
<Rabbit silk artificial blood vessel coating>
As shown in FIG. 1, in a coating composition (5), a silkworm silk artificial blood vessel (inner diameter: 4 mmφ) with a core rod attached is immersed for 10 minutes under normal pressure, and then immersed in liquid nitrogen for 1 minute. Frozen. It preserve | saved for one hour in the freezer of -80 degreeC, and processed for 12 hours by freeze-drying. The 10-minute immersion to lyophilization was repeated twice. Next, the silkworm artificial blood vessel was autoclaved at 120 ° C. for 20 minutes, then immersed in pure water for 15 minutes, separated from the core rod, and dried in a dryer set at 50 ° C. for 1 hour. A coated rabbit silk artificial blood vessel was obtained in which the outer wall of the silk artificial blood vessel was coated in two layers.
Thereafter, 6 mL of the coating composition (1), (2), (5), or (6) was flowed on the inner wall of the silkworm silk artificial blood vessel using a syringe, and immersed in liquid nitrogen for 1 minute to freeze. . It preserve | saved for one hour in the freezer of -80 degreeC, and processed for 12 hours by freeze-drying. Then, an autoclave treatment was performed at 120 ° C. for 20 minutes.
Subsequently, the operation of immersing the silkworm silk artificial blood vessel in pure water at 50 ° C. and allowing it to stand for 12 hours was repeated 6 times while changing the water. Subsequently, it dried at 50 degreeC and the coated rabbit silk artificial blood vessel which coated the innermost wall of the rabbit silk artificial blood vessel with a single layer was obtained.

Example 4
<Rabbit silk artificial blood vessel coating>
In the coating composition (5), a silkworm silk artificial blood vessel (inner diameter 4 mmφ) with a core rod attached was immersed for 10 minutes under normal pressure, and then immersed in liquid nitrogen for 1 minute to freeze. It preserve | saved for one hour in the freezer of -80 degreeC, and processed for 12 hours by freeze-drying. Next, the silkworm artificial blood vessel was autoclaved at 120 ° C. for 20 minutes, then immersed in pure water for 15 minutes, separated from the core rod, and dried in a dryer set at 50 ° C. for 1 hour. A coated rabbit silk artificial blood vessel having a single layer coating on the outermost wall of the silk artificial blood vessel was obtained.
Thereafter, 6 mL of the coating composition (1) was flowed on the inner wall of the rabbit silk artificial blood vessel using a syringe, and the innermost wall of the rabbit silk artificial blood vessel was coated as a single layer in the same manner as in Example 2. Got.

Test Example 1 Morphological Observation Test Morphological observation of each coated rabbit silk artificial blood vessel obtained in Examples 1 to 4 and Comparative Examples 1 to 3 was performed using a scanning electron microscope (VE7800 manufactured by Keyence Corporation).
As shown in FIG. 2, the outer surface, inner surface, and cross section of each coated rabbit silk artificial blood vessel formed a structure covered with a fine porous material along the tubular structure. In particular, by placing a vacuum drying operation in the middle of the coating, it was possible to produce an artificial blood vessel with almost no holes. On the other hand, what coated the inner wall of the artificial blood vessel only with silk fibroin (Comparative Example 1) had a large hole.

Test Example 2 Measurement of Water Permeability In order to quantitatively measure the amount of water leakage from the side walls of each coated rabbit silk artificial blood vessel obtained in Examples 1 to 3 and Comparative Example 1, a test was conducted according to the guidance of ISO 7198. Water was dropped on the side wall of each coated rabbit silk artificial blood vessel so that a water pressure of 120 mmHg was applied, and the amount of water leakage (permeability) per unit area discharged from the fixing jig through the artificial blood vessel was measured. . The test was performed 5 times, and the average value was obtained. The results are shown in FIG.

The amount of water leakage was 90.64 (mL / cm ² / min) in Example 1, 3.12 (mL / cm ² / min) in Example 2, and 0.24 (mL / cm ² / min in Example 3). ) And 7.32 (mL / cm ² / min) in Example 4. By placing the vacuum drying operation in the middle of the coating, the amount of water leakage was greatly reduced. On the other hand, it was 647.2 (mL / cm ² / min) in Comparative Example 1.
The water permeability of Dacron artificial blood vessels that do not require pre-clotting (pre-blood treatment) with commercially available artificial blood vessels is a published value of 1 to 10 mL / cm ² / min.

Test Example 3 Measurement of Compression Elasticity Each coated rabbit silk artificial blood vessel obtained in Examples 2-3 and Comparative Examples 2-3 was cut to a length of 0.5 cm, and a compressive force was applied in the circumferential direction. The compression elastic modulus (N / mm ² ) until the diameter of the artificial blood vessel was reduced by 10% (the change in diameter was plotted on the horizontal axis and the compressive force was plotted on the vertical axis and obtained from the slope) was determined. The test was performed 5 times, and the average value was obtained. The results are shown in FIG.

  From FIG. 4, the elastic modulus of the coated rabbit silk artificial blood vessels obtained in Examples 2 and 3 is more flexible in the circumferential direction than that of the artificial blood vessel coated only with silk fibroin (Comparative Example 2). The improvement was confirmed.

Test Example 4 Measurement of anastomotic thread holding strength Each coated rabbit silk artificial blood vessel obtained in Examples 2-3 and Comparative Examples 2-3 was tested according to the guidance of ISO 7198. The test was performed 5 times, and the average value was obtained. The results are shown in FIG.

  FIG. 5 shows that the coated rabbit silk artificial blood vessels obtained in Examples 2 and 3 have anastomotic thread holding strength equivalent to that obtained by coating the inner wall of the artificial blood vessel only with silk fibroin (Comparative Example 2). confirmed.

Example 5
<Rabbit silk artificial blood vessel coating>
In the coating composition (5), a silkworm silk artificial blood vessel (inner diameter 4 mmφ) with a core rod attached was immersed for 10 minutes under normal pressure, and then immersed in liquid nitrogen for 1 minute to freeze. It preserve | saved for one hour in the freezer of -80 degreeC, and processed for 12 hours by freeze-drying. Instead of the coating compositions (3), (4) and (2) in this order, the steps from 10 minutes immersion to freeze-drying were repeated 4 times. Next, the silkworm artificial blood vessel was autoclaved at 120 ° C. for 20 minutes, then immersed in pure water for 15 minutes, separated from the core rod, and dried in a dryer set at 50 ° C. for 1 hour. A coated rabbit silk artificial blood vessel in which the outer wall of the silk artificial blood vessel was coated in four layers was obtained.

Test Example 5 Canine transplantation test <operative method>
Anesthesia for dogs (beagle dogs, female, 1 year old) is atropine (0.1 ml / kg, atropine injection), midazolam (0.04 ml / kg, midazolam injection 10 mg “sand”), butorphanol (0.04 ml / lg) After pretreatment with Betolfal Note), it was introduced and intubated using Propofol (Betolfal), and maintenance of anesthesia was performed using isoflurane. After shaving the operative field, it was disinfected with alcohol and chlorhexidine and draped. Before approaching the blood vessel, heparin (100 IU / kg, heparin injection) was intravenously administered, and after confirming that the activated coagulation time (ACT) was 200 or more, the operation on the blood vessel was started. During the operation, ACT was confirmed every 20 minutes, and heparin was additionally administered when it was confirmed that the ACT was 200 or less.
After approaching with a cervical midline incision, the right common carotid artery was exfoliated over about 5 cm and supported by silk thread. The both ends of the blood vessel peeled off with bulldog forceps are clamped to block blood circulation, and the carotid artery of about 3 cm is excised with scissors, and the coated rabbit silk artificial blood vessel prepared in Example 5 is transplanted in the form of replacing the resected blood vessel. did. Artificial blood vessels are sutured under an enlarged field of view using a surgical loupe, and blood vessel sutures are 9 mm and 3/8 round needles with 7/0 thickness polyvinylidene fluoride monofilament (PREMIO, 26SZ05AG, Peters SURGICAL, End-to-end anastomosis was performed with 10-12 nodal sutures. After the suture, it was confirmed that there was no blood leak, and for the site where the leak was confirmed, a Z suture was added to control bleeding. After the control of bleeding was confirmed, the subcutaneous tissue and the dermis were closed with 3/0 absorbent thread (glycolide, dioxanone, trimethylene carbonate, biocin 1/2 round needle, needle length 22 mm).

<Postoperative management>
After surgery, anticoagulant therapy was performed in addition to general surgical wound disinfection and systemic antibiotic administration. Subcutaneous administration of low molecular weight heparin (Dalteparin sodium injection solution, hepaclon injection 5000, Yale medicine) 2500 IU / day BID was continuously administered for 21 days with Aspirin (0.5 mg / kg BID).

<Evaluation by echoes of transplanted blood vessels>
In order to evaluate the patency of the transplanted blood vessel and the blood flow in the blood vessel lumen, observation using an ultrasonic diagnostic imaging apparatus (Aloka ProSound SSD-α10) was performed over time. Measurements were performed under sedation under intravenous micro doses of propofol to exclude the effects of animal movement. The electronic linear probe used was UST-5524-7.5 for superficial superficial part, peak systolic velocity (PSV), end-diastolic velocity (EDV), pulsation Index (Pulsatility index: PI), resistance index (RI), systolic acceleration time (Acceleration time: AT), and measurement of the inner diameter of the artificial blood vessel, including before and after the artificial blood vessel, a total of 7 locations (1. common carotid artery) Proximal part, 2. proximal anastomosis part, 3. artificial blood vessel proximal part, 4. artificial blood vessel middle part, 5. artificial blood vessel distal part, 6. distal anastomosis part, 7. common carotid artery distal part) went. At the same time, ultrasonic modeling inspection using color Doppler, e-flow, and sonazoid was also used to evaluate turbulence, stenosis, and plaque formation.

<Result>
There were no problems at the time of implantation, the transplantation operation was completed successfully, and there was no problem with the general condition of the animals after the operation. As a result of evaluation by echo, turbulent flow, stenosis, and plaque formation were not observed in the images observed two weeks after the operation, and good blood flow was maintained. In the evaluation using various echo parameters, there was no change in each parameter value before and 2 weeks after the operation, and not only stenosis and occlusion were observed after the operation, but also similar to the natural blood vessel wall before the operation. These parameter values were also confirmed in an artificial blood vessel 2 weeks after implantation (FIG. 6).
In blood vessels, blood flows intermittently according to pulsation, and at that time, the blood vessel wall repeats stretching to create physiological blood flow. As described above, an artificial blood vessel having a physical property close to a natural blood vessel wall while having high biocompatibility can maintain a blood vessel function by providing a more physiological environment. It was thought that there was.

Claims (7)

  A composition for coating a tubular structure containing silk fibroin and polyurethane resin.   The composition for coating a tubular structure according to claim 1, wherein silk fibroin and polyurethane resin are dispersed or dissolved in water, an organic solvent or a mixture thereof.   The composition for coating a tubular structure according to claim 2, wherein the organic solvent is hexafluoroisopropanol and / or hexafluoroacetone.   The composition for coating a tubular structure according to any one of claims 1 to 3, wherein the polyurethane resin is an aqueous polyurethane resin.   The tubular structure according to any one of claims 1 to 4, wherein the tubular structure is a tubular structure in which silk fibroin fibers are wound by one or more methods selected from knitting, braiding, weaving, and entanglement. A composition for coating a product.   The manufacturing method of a coated tubular structure which coats a tubular structure with the composition for coating of any one of Claims 1-5.   The coating is performed by immersing the tubular structure in a coating solution in which silk fibroin and polyurethane resin are dispersed or dissolved in water, an organic solvent, or a mixture thereof, followed by freezing, heat treatment, and drying. A method for producing a coated tubular structure.