WO2012090553A1 - Method for manufacturing artificial blood vessel - Google Patents

Method for manufacturing artificial blood vessel Download PDF

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Publication number
WO2012090553A1
WO2012090553A1 PCT/JP2011/069691 JP2011069691W WO2012090553A1 WO 2012090553 A1 WO2012090553 A1 WO 2012090553A1 JP 2011069691 W JP2011069691 W JP 2011069691W WO 2012090553 A1 WO2012090553 A1 WO 2012090553A1
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Prior art keywords
tubular structure
blood vessel
artificial blood
silk
coating
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PCT/JP2011/069691
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French (fr)
Japanese (ja)
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朝倉　哲郎
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国立大学法人東京農工大学
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Publication of WO2012090553A1 publication Critical patent/WO2012090553A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/507—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials for artificial blood vessels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/28—Materials for coating prostheses
    • A61L27/34—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/04—Hollow or tubular parts of organs, e.g. bladders, tracheae, bronchi or bile ducts
    • A61F2/06—Blood vessels
    • A61F2/062—Apparatus for the production of blood vessels made from natural tissue or with layers of living cells

Definitions

  • the present invention relates to a coating composition and a method for producing an artificial blood vessel using the same.
  • the large-diameter artificial blood vessels have already been put into practical use and can withstand clinical use.
  • small-diameter artificial blood vessels with a diameter of 5 mm or less are caused by 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.
  • PET polyethylene terephthalate
  • PTFE polytetrafluoroethylene
  • 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.
  • kite thread structure 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.
  • sericin is highly likely to cause an allergic reaction, it is desirable to remove sericin from the viewpoint of reducing its risk.
  • 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.
  • a tubular structure using silk fibroin fibers obtained by scouring warp and raw silk has a low allergy risk due to sericin and is excellent in biocompatibility, but is 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.
  • 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 composition for coating of the tubular structure containing a fibroin and a polyurethane-type resin. Moreover, this invention provides the coated tubular structure obtained by the said manufacturing method.
  • 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.
  • 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.
  • FIG. 1 is a diagram showing a coating process of a silkworm silk artificial blood vessel.
  • Fig.2 (a) is a figure which shows the scanning electron microscope image of a coating rabbit silk artificial blood vessel.
  • FIG.2 (b) is a figure which shows the scanning electron microscope image of a coating rabbit silk artificial blood vessel.
  • FIG. 3 is a diagram showing the water permeability of the coated rabbit silk artificial blood vessel.
  • FIG. 4 is a view showing the compression elastic modulus of the coated rabbit silk artificial blood vessel.
  • FIG. 5 is a view showing anastomotic thread holding strength of a coated rabbit silk artificial blood vessel.
  • FIG. 6 is a diagram comparing changes in various parameters before and 2 weeks after the operation.
  • PRE pre-operative 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.
  • the silk fibroin is preferably a silk fibroin solution obtained by dissolving silk fibroin fibers in water and / or an organic solvent.
  • 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
  • examples of the neutral salt aqueous solution include lithium bromide, lithium chloride, calcium chloride, and lithium thiocyanate.
  • a known method such as a dialysis method or a reverse osmosis method can be employed.
  • water may be removed from the aqueous solution as necessary to obtain a dry product.
  • 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.
  • 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).
  • lyophilization is preferred from the viewpoint of handleability and storage stability.
  • the organic solvent examples include hexafluoroisopropanol (HFIP) and hexafluoroacetone (HFA).
  • HFA hexafluoroisopropanol
  • HFA hydrate hexafluoroacetone
  • the concentration of silk fibroin 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.
  • 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.
  • Polyester polyols such as polyhexamethylene isophthalate adipate diol, polyethylene succinate diol, polybutylene succinate diol, polyethylene sebacate diol, polybutylene sebacate diol, poly- ⁇ -caprolactone diol; polyoxytetramethylene glycol, polyoxy Propylene glycol, polyoxyethylene
  • Polycarbonate polyols such as polyhexamethylene carbonate diol
  • polyether polyols such emissions propylene glycol
  • Patent acrylic polyols such as those described in 2000-119362 J
  • 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.
  • 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.
  • transducing a sex group is mentioned.
  • 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.
  • the compound imparting the ionic hydrophilic group is not particularly limited, but examples of the compound imparting the anionic hydrophilic group include 2,2-dimethylolpropionic acid, 2,2-dimethylolbutanoic acid and the like. Can be mentioned. These are used after neutralizing with amines such as trimethylamine, triethylamine, tri-n-propylamine, tributylamine, triethanolamine, potassium hydroxide, sodium hydroxide, ammonia and the like. Further, alkanolamines are preferred as those imparting a cationic group, and examples thereof include N-methyldiethanolamine and triethanolamine.
  • a commercially available product may be used as the aqueous polyurethane resin.
  • water-dispersible polyurethane resins such as Neo Sticker 100C (Nika Kagaku) and Evaphanol (Nikka Kagaku) can be used.
  • 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.
  • the content of the polyurethane resin varies depending on the type of the resin.
  • the content of the polyurethane resin varies depending on the type of the 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 to 1: 100 are preferred.
  • 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.
  • an aqueous polyurethane resin is used as the polyurethane resin, it is preferably dissolved or dispersed in water.
  • a non-aqueous polyurethane resin is used, it is preferably dissolved in an organic solvent.
  • 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.
  • 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.
  • 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.
  • 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.
  • the coating liquid can appropriately contain arbitrary components.
  • an antithrombotic agent such as heparin, endothelial cell growth factor and the like can be contained.
  • the tubular structure in the present invention is not particularly limited, and tubular structures composed of various materials can be used.
  • the thing made from PET, PTFE, silk fibroin, etc. are mentioned.
  • a tubular structure made of silk fibroin is preferable.
  • tubular structure made of silk fibroin examples include a tubular structure in which silk fibroin fibers are wound by one or more methods selected from knitting, braiding, weaving and entanglement.
  • a known knitting method, braiding method, weaving method, and entanglement method can be used.
  • the method for braiding silk fibroin fiber is not particularly limited, and for example, known braiding techniques such as eight punching, twelve punching, and 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.
  • a thermoplastic resin core rod 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.
  • silk fibroin fibers are knit knitted to form a tubular structure, it can be made into a tubular structure that is highly elastic, flexible, excellent in tensile strength, and difficult to fray at the cut end.
  • 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 for 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 room temperature, normal pressure, or reduced pressure.
  • the immersion time is preferably 5 to 30 minutes, particularly 10 to 20 minutes.
  • the heat treatment is preferably performed at a temperature of 100 ° C. or higher, more preferably 120 ° C. or higher.
  • a closed pressure device such as an autoclave may be used.
  • an autoclave it is preferably at 120 ° C. or higher for 20 to 40 minutes.
  • drying treatment examples 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.
  • 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.
  • the innermost wall and the outermost wall of the tubular structure may be coated with a coating composition containing silk fibroin and polyurethane resin, and both are coated. It may be.
  • the innermost wall of the tubular structure is preferably coated with the coating composition.
  • 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.
  • coated tubular structure of the present invention can be used, for example, as a substitute for an artificial blood vessel, an artificial trachea, a stent graft, and other animal biological tubular structures.
  • an artificial blood vessel an artificial trachea, a stent graft, and other animal biological tubular structures.
  • it is suitable for small-diameter artificial blood vessels having a diameter of 5 mm or less, preferably 1 to 5 mm or less.
  • 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. A vinyl chloride or polytetrafluoroethylene core rod with an outer diameter of 1 to 5 mm is attached to the center of the machine, and silk fibroin fibers are assembled around the core rod, and a silkworm artificial blood vessel with an inner diameter of 1.5 to 5 mm ⁇ )
  • 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>
  • a silkworm silk artificial blood vessel (inner diameter: 4 mm ⁇ ) 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.
  • 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.
  • 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>
  • 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.
  • 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 to 3 and Comparative Examples 2 to 3 ⁇ Rabbit silk artificial blood vessel coating>
  • a silkworm silk artificial blood vessel (inner diameter: 4 mm ⁇ ) with a core rod attached is immersed for 10 minutes under normal pressure, and then immersed in liquid nitrogen for 1 minute. Frozen. It was stored in a freezer at ⁇ 80 ° C. for 1 hour, and treated by freeze vacuum drying for 12 hours. The 10-minute immersion to lyophilization was repeated twice.
  • 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 was stored in a freezer at ⁇ 80 ° C. for 1 hour, and treated by freeze vacuum drying for 12 hours. 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>
  • a silkworm silk artificial blood vessel (inner diameter 4 mm ⁇ ) 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 was stored in a freezer at ⁇ 80 ° C. for 1 hour, and treated by freeze vacuum drying for 12 hours.
  • 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.
  • 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
  • tests were conducted in accordance with ISO 7198 guidance. 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.
  • the amount of water leakage was greatly reduced.
  • 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 Elastic Modulus
  • Each coated rabbit silk artificial blood vessel obtained in Examples 2 to 3 and Comparative Examples 2 to 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 retention 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>
  • a silkworm silk artificial blood vessel (inner diameter 4 mm ⁇ ) 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 was stored in a freezer at ⁇ 80 ° C. for 1 hour, and treated by freeze vacuum drying for 12 hours.
  • the steps from 10 minutes immersion to freeze-drying were repeated 4 times.
  • 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.
  • Betolfal Propofol
  • heparin 100 IU / kg, heparin injection
  • ACT activated coagulation time
  • 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.

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  • Oral & Maxillofacial Surgery (AREA)
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Abstract

The present invention relates to a tubular structure coating composition containing silk fibroin and polyurethane resin.

Description

人工血管の製造方法Artificial blood vessel manufacturing method
　本発明は、コーティング用組成物及びこれを用いた人工血管の製造方法に関する。 The present invention relates to a coating composition and a method for producing an artificial blood vessel using the same.
　近年、動脈硬化症等血管疾患の増加に伴い、人工血管の重要性は確実に高まっている。人工血管においては、（１）安全性（急性毒性、皮内反応試験、溶血性試験、発熱性物質試験、皮膚感作性試験、細胞毒性等）、（２）機能性（伸縮性、縫合し易さ、柔軟性、切断端のほつれ難さ、人工血管壁からの出血し難さ）、（３）耐久性等が要求される。また、人工血管は、体内に移植する部位によって様々な種類が必要とされる。 In recent years, with the increase in 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.
　人工血管のうち大口径のものは既に実用化され、臨床使用に耐えられるものとなっている。しかしながら、口径５ｍｍ以下の小口径人工血管は、ポリエチレンテレフタレート（ＰＥＴ）やポリテトラフロロエチレン（ＰＴＦＥ）等の代表的な人工素材の生体不適合性による血管内膜の肥厚や血栓形成による閉塞が原因で、未だ実用化されるに至っていない。そのため、現行では、膝関節末梢等へのバイパス術は自家静脈移植が行われているが、患者への負担が大きいこと、適合する血管を持たず自家静脈移植を行うことができない患者が多数いる等問題は多い。近年、患者の高齢化や糖尿病の増加に伴い、細小血管の再生治療の需要は増加している。従って、特に末梢血管等小口径の血管に利用できる抗血栓性のある人工血管の開発が、以前から強く望まれていた。 The large-diameter artificial blood vessels 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 caused by 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 antithrombotic properties that can be used particularly for a small-diameter blood vessel such as a peripheral blood vessel has been strongly desired.
　一方、絹糸は、高い生体親和性を有しており、細くて強く適度な弾性と柔軟性を持ち、糸の滑りがよく、結びやすくほつれ難い特性を持っていることから、手術用の縫合糸として用いられる天然繊維である。これまでに絹の高い生体適合性を利用した様々な再生絹材料が開発され、医療、生化学、食品、化粧料等幅広い分野での利用が期待されている。特に、再生医療のための材料として注目されている。 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.
　絹を用いた人工血管作製の試みとしては、組紐作製原理により編み込む動作を組み合わせて巻かれ、且つ繭糸相互や混繊維相互が繭糸表面に保有されているセリシンにより膠着されてなる繭糸構造物が知られている（特許文献１）。この繭糸構造物は繭糸相互がセリシンで膠着されることで、実用に耐え得る引っ張り強度になっている。
　しかし、セリシンはアレルギー反応を引き起こす可能性が高いため、そのリスクを低減する観点からセリシンを除去することが望ましい。また、前記繭糸構造物は柔軟性が十分ではなく、切断端もほつれ易いため、生体適合性と術時の要求特性等絹本来の特性を保持しつつもより機能性に優れた人工血管が要望されていた。 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.
特開２００４－１７３７７２号公報JP 2004-173772 A 特開２０１０－１３７０４１号公報JP 2010-137041 A
　繭糸や生糸等を精練して得られる絹フィブロイン繊維を用いた管状構造物は、セリシンによるアレルギーリスクが低く、生体適合性に優れるものの、機能性の点で十分ではない。
　そこで、本発明者が検討したところ、絹水溶液にポリグリコールを混合した水溶液から得られる絹フィブロインスポンジで絹フィブロインからなる管状構造物をコーティングすれば弾力性を付与できることを見出し、先に出願した（特許文献２）。
　しかしながら、実用を考慮すると、未だ弾力性等の点で満足できるものではなく、より一層機能性に優れた人工血管が要望されていた。 A tubular structure using silk fibroin fibers obtained by scouring warp and raw silk has a low allergy risk due to sericin and is excellent in biocompatibility, but is 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.
　本発明は、上記実情に鑑みなされたものであり、生体適合性等絹本来の特性を有しつつも、強度、弾力性に優れ、末梢血管等小口径の血管へ利用できる管状構造物を提供することに関する。 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.
　本発明者は、絹の特性を活かした管状構造物について種々検討したところ、管状構造物を絹フィブロインとポリウレタン系樹脂の混合物でコーティングすれば、十分な引っ張り強度、優れた弾力性を有し、柔軟で、生体適合性と術時の要求特性を満足するより小口径の人工血管に適した管状構造物が得られることを見出し、本発明を完成した。 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.
　すなわち、本発明は、絹フィブロインとポリウレタン系樹脂を含有する管状構造物のコーティング用組成物を提供するものである。
　また、本発明は、フィブロインとポリウレタン系樹脂を含有する管状構造物のコーティング用組成物により管状構造物をコーティングする、コーティング管状構造物の製造方法を提供するものである。
　また、本発明は、上記製造方法により得られたコーティング管状構造物を提供するものである。 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 composition for coating of the tubular structure containing a fibroin and a polyurethane-type resin.
Moreover, this invention provides the coated tubular structure obtained by the said manufacturing method.
　本発明によれば、絹本来の特性、すなわち高い生体適合性を有しつつも、十分な引っ張り強度、優れた弾力性を有し、柔軟で血管吻合し易く、血液の漏出が少ない管状構造物を提供することができる。この管状構造物は、抗血栓性に優れるため、特に直径５ｍｍ以下の小口径人工血管に好適である。 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.
図１は、家蚕絹人工血管のコーティング工程を示す図である。FIG. 1 is a diagram showing a coating process of a silkworm silk artificial blood vessel. 図２（ａ）は、コーティング家蚕絹人工血管の走査型電子顕微鏡像を示す図である。（ａ）実施例１－４Fig.2 (a) is a figure which shows the scanning electron microscope image of a coating rabbit silk artificial blood vessel. (A) Example 1-4 図２（ｂ）は、コーティング家蚕絹人工血管の走査型電子顕微鏡像を示す図である。（ｂ）比較例１－３FIG.2 (b) is a figure which shows the scanning electron microscope image of a coating rabbit silk artificial blood vessel. (B) Comparative Example 1-3 図３は、コーティング家蚕絹人工血管の透水率を示す図である。FIG. 3 is a diagram showing the water permeability of the coated rabbit silk artificial blood vessel. 図４は、コーティング家蚕絹人工血管の圧縮弾性率を示す図である。FIG. 4 is a view showing the compression elastic modulus of the coated rabbit silk artificial blood vessel. 図５は、コーティング家蚕絹人工血管の吻合糸保持強度を示す図である。FIG. 5 is a view showing anastomotic thread holding strength of a coated rabbit silk artificial blood vessel. 図６は、術前、術後２週間の各種パラメータの変化を比較した図である。ＰＲＥ：術前の生来の頸動脈のデータ、ＰＯＳＴ　ＰＲＯＸ：術後の人工血管の近位側で得られたデータ、ＰＯＳＴ　ＤＩＳＴＡＬ：術後の人工血管の遠位側で得られたデータ、ＰＳＶ：収縮期最大流速（ｃｍ／ｓ）、ＥＤＶ拡張末期流速（ｃｍ／ｓ）、ＰＩ拍動指数（単位なし）、ＲＩ抵抗係数（単位なし）、ＣＳＤ：血管径（ｍｍ）FIG. 6 is a diagram comparing changes in various parameters before and 2 weeks after the operation. PRE: pre-operative 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)
　本発明のコーティング用組成物は、絹フィブロインとポリウレタン系樹脂を含む、管状構造物のコーティングに用いられる組成物である。 The coating composition of the present invention is a composition used for coating a tubular structure containing silk fibroin and polyurethane resin.
　本発明に用いられる絹フィブロインは、家蚕又は遺伝子組み換え蚕、及びエリ蚕、柞蚕、天蚕等の野蚕の繭層から得た繭糸や生糸等を精練したものである。当該精練処理によりセリシンが除去される。
　精練方法は、特に制限されず、公知の方法を使用できる。例えば１００℃に加熱した１２ｗ／ｖ％マルセル石鹸、８ｗ／ｖ％炭酸ナトリウム混合水溶液、及び上述した繭層や繭糸、生糸等を入れ、操糸後、撹拌しながら１２０分煮沸し、その後２ｗ／ｖ％炭酸ナトリウム水溶液で１０分煮沸、更に１００℃に加熱した蒸留水中で洗浄する操作を３回行った後、乾燥することでフィブロインを覆う蛋白質（セリシン）や、その他脂肪分等を除去できる。 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.
　絹フィブロインは、絹フィブロイン繊維を水及び／又は有機溶媒に溶解した絹フィブロイン溶解物を用いるのが好ましい。例えば、絹フィブロイン繊維を水及び／又は有機溶媒に溶解したもの；絹フィブロイン繊維を中性塩水溶液に溶解、加熱した後、得られた絹フィブロイン／塩水溶液を脱塩処理したもの；脱塩処理後の溶解液を、更に有機溶媒に溶解したもの、等が挙げられる。 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.
　ここで、中性塩水溶液としては、例えば臭化リチウム、塩化リチウム、塩化カルシウム、チオシアン酸リチウム等が挙げられる。また、脱塩処理の方法としては、公知の方法、例えば透析法、逆浸透法等を採用することができる。脱塩後、必要に応じて水溶液から水を除去し乾燥物としてもよい。この場合、通常は水溶液をプレートに展開し、水を蒸発させて絹フィブロインのフィルムを作製したり、スプレー乾燥等を行ったりして粉末状とする。また、蒸留水を加えて、例えば絹フィブロイン濃度２ｗ／ｖ％以下の水溶液を調製し、凍結乾燥を行ってスポンジ状（多孔質状）としてもよい。これらのうち、取扱性・保存性の点から、凍結乾燥するのが好ましい。 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.
　有機溶媒としては、例えばヘキサフロロイソプロパノール（ＨＦＩＰ）、ヘキサフロロアセトン（ＨＦＡ）が挙げられる。通常、ＨＦＡは水和物として安定に存在するため、ＨＦＡ水和物を用いるのが好ましい。ＨＦＩＰとＨＦＡはそれぞれ単独で用いてもよいし、混合して用いてもよい。
　水、有機溶媒に溶解する絹フィブロイン濃度は、通常３～２０ｗ／ｖ％程度であり、好ましくは３～１５ｗ／ｖ％である。 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 concentration of silk fibroin dissolved in water or an organic solvent is usually about 3 to 20 w / v%, preferably 3 to 15 w / v%.
　本発明に用いられるポリウレタン系樹脂は、ポリイソシアネートとポリオールを主原料に、常法に従って反応させることにより得られる樹脂である。ポリウレタン系樹脂としては、特に限定されず、ポリカーボネート系ウレタン樹脂、ポリエステル系ウレタン樹脂、ポリエーテル系ウレタン樹脂、シリコーン変性ウレタン樹脂、フッ素変性ウレタン樹脂、ポリアミノ酸系ウレタン樹脂等が挙げられる。 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.
　ポリイソシアネートとしては、分子内に２個以上のイソシアネート基を有する化合物であれば特に限定されず、例えば、ヘキサメチレンジイソシアネート、２，２，４－トリメチルヘキサメチレンジイソシアネート等の脂肪族ジイソシアネート；イソホロンジイソシアネート、水添キシリレンジイソシアネート、ジシクロヘキシルメタンジイソシアネート、ノルボルナンジイソシアネート等の脂環式ジイソシアネート；トリレンジイソシアネート、ジフェニルメタンジイソシアネート、ナフタレンジイソシアネート、トリジンジイソシアネート、キシリレンジイソシアネート等の芳香族ジイソシアネート等が挙げられる。
　これらは単独又は２種以上を組み合わせて用いることができる。 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.
　また、ポリオールとしては特に限定されず、例えば、ポリエチレンアジペートジオール、ポリエチレンプロピレンアジペートジオール、ポリブチレンアジペートジオール、ポリエチレンブチレンアジペートジオール、ポリヘキサメチレンアジペートジオール、ポリジエチレンアジペートジオール、ポリエチレンテレフタレートジオール、ポリエチレンイソフタレートジオール、ポリヘキサメチレンイソフタレートアジペートジオール、ポリエチレンサクシネートジオール、ポリブチレンサクシネートジオール、ポリエチレンセバケートジオール、ポリブチレンセバケートジオール、ポリ－ε－カプロラクトンジオール等のポリエステルポリオール；ポリオキシテトラメチレングリコール、ポリオキシプロピレングリコール、ポリオキシエチレンプロピレングリコール等のポリエーテルポリオール；ポリヘキサメチレンカーボネートジオール等のポリカーボネートポリオール；特開２０００－１１９３６２号公報に記載されているようなアクリルポリオール、ダイマージオールが挙げられる。これらは単独又は２種以上を組み合わせて用いることができる。 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 emissions 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.
　本発明に用いられるポリウレタン系樹脂は、水性（水溶性又は水分散性）ポリウレタン系樹脂であることが好ましい。
　水性ポリウレタン系樹脂は、常法により製造することができるが、ポリウレタン系樹脂に水性を付与する方法として、例えば、ウレタン骨格中にアニオン性基又はカチオン性基のイオン性基を導入するか、ノニオン性基を導入する方法が挙げられる。また、界面活性剤等の乳化剤を使用して強制乳化させてもよい。イオン性基としては、－ＣＯＯ-基、－ＳＯ3 -基等のアニオン基、＝Ｎ+＝基、＝Ｐ+＝基等のカチオン基が挙げられ、特に－ＣＯＯ-基が好ましい。 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.
　イオン性基を有する水性ポリウレタン系樹脂は、例えば、上記ポリイソシアネート、ポリオール、イオン性親水基を付与する化合物、必要により公知の鎖伸張剤等を適宜用いて、ワンショット法やプレポリマー法等により製造することができる。 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.
　ここで、イオン性親水基を付与する化合物としては、特に限定されないが、アニオン性親水基を付与するものとして、例えば、２，２－ジメチロールプロピオン酸、２，２－ジメチロールブタン酸等を挙げることができる。これらはトリメチルアミン、トリエチルアミン、トリ－ｎ－プロピルアミン、トリブチルアミン、トリエタノールアミン等のアミン類、水酸化カリウム、水酸化ナトリウム、アンモニア等で中和して用いられる。
　また、カチオン性基を付与するものとして、アルカノールアミン類が好ましく、例えばＮ－メチルジエタノールアミン、トリエタノールアミン等を挙げることができる。これらは塩酸、硝酸、蟻酸、酢酸等の各種有機酸や無機酸等で中和して、あるいはジメチル硫酸、ジエチル硫酸、塩化ベンジル、エピクロルヒドリン等で４級化して用いられる。
　これらは単独又は２種以上を組み合わせて用いることができる。 Here, the compound imparting the ionic hydrophilic group is not particularly limited, but examples of the compound imparting the anionic hydrophilic group include 2,2-dimethylolpropionic acid, 2,2-dimethylolbutanoic acid and the like. Can be mentioned. These are used after neutralizing with amines such as trimethylamine, triethylamine, tri-n-propylamine, tributylamine, triethanolamine, potassium hydroxide, sodium hydroxide, ammonia and the like.
Further, alkanolamines are preferred as those imparting a cationic group, and examples thereof include N-methyldiethanolamine and triethanolamine. 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.
　水性ポリウレタン系樹脂は、市販品を用いてもよい。例えば、ネオステッカー１００Ｃ（日華化学）、エバファノール（日華化学）等の水分散性ポリウレタン系樹脂を用いることができる。 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.
　本発明のコーティング用組成物中、絹フィブロインの含有量は、生体適合性の点から、０．１～１０質量％が好ましく、０．２～５質量％が好ましい。 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.
　また、本発明のコーティング用組成物中、ポリウレタン系樹脂の含有量は、樹脂の種類によって相違するが、例えば水分散性ポリウレタン系樹脂の場合、５～５０質量％が好ましく、１５～３０質量％が好ましい。 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.
　絹フィブロインとポリウレタン系樹脂の配合比（質量比）は、５０：１～１：１００の範囲とすることが引っ張り強度、弾力性向上の点から好ましく、更に１０：１～１：１００、特に１：１～１：１００が好ましい。 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 to 1: 100 are preferred.
　本発明のコーティング用組成物は、絹フィブロインとポリウレタン系樹脂を水、有機溶媒又はこれらの混合物に溶解又は分散したものであるのが好ましい。
　ポリウレタン系樹脂として水性ポリウレタン系樹脂を用いる場合は、水に溶解又は分散したものであるのが好ましく、他方、非水性ポリウレタン系樹脂を用いる場合は、有機溶媒に溶解したものであるのが好ましい。なかでも、引っ張り強度、弾力性向上の点から、絹フィブロインと水性ポリウレタン系樹脂を水に溶解乃至分散させた組成物が好ましい。
　この場合、絹フィブロインとポリウレタン系樹脂はどちらか一方を先に水及び／又は有機溶媒に溶解又は分散した後に、他方を溶解又は分散してもよく、両者を混合してから溶解又は分散させてもよい。また、例えば、上記のように絹フィブロイン繊維を水、有機溶媒に溶解させた絹フィブロイン溶解物と、ポリウレタン系樹脂を混合してもよい。
　なお、有機溶媒としては、上記と同様のものが挙げられる。 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.
　本発明のコーティング管状構造物は、絹フィブロインとポリウレタン系樹脂を含有するコーティング液を調製し、該コーティング液で管状構造物の最内壁及び／又は最外壁をコーティングすることにより製造される。当該コーティング液には、絹フィブロインとポリウレタン系樹脂の他、適宜任意成分を含有させることができる。例えば、ヘパリン等の抗血栓剤、内皮細胞成長因子等を含有させることができる。 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.
　本発明における管状構造物は、特に限定されず、種々の素材から構成された管状構造物を用いることができる。例えば、ＰＥＴやＰＴＦＥ製、絹フィブロイン製のもの等が挙げられる。なかでも、絹フィブロインからなる管状構造物が好ましい。 The tubular structure in the present invention is not particularly limited, and tubular structures composed of various materials 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.
　絹フィブロインからなる管状構造物としては、例えば絹フィブロイン繊維が編、組、織及び絡から選ばれる１又は２以上の方法により巻かれてなる管状構造物が挙げられる。絹フィブロイン繊維を管状に構成するには、公知の編法、組法、織法、絡法を用いることができる。また、絹フィブロイン繊維を用いて予め作製した編物や織物、その筒状物等を用いてもよい。繭糸、生糸を用いる場合は、管状に形成した後、セリシンを除去するのが好ましい。 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.
　例えば、絹フィブロイン繊維を編組する方法としては、特に制限されず、例えば八つ打ち、十二打ち、十六打ち等公知の組紐技術を用いることができる。具体的には、煮繭から解いた繭糸又は精錬糸を熱可塑性樹脂製芯棒等に巻き付け、絹フィブロイン繊維を組むことにより行われる。ここで、熱可塑性樹脂製芯棒は、目的とする管状構造物の大きさに応じて、外径１～５ｍｍ、好ましくは２～５ｍｍのものを用いることができる。熱可塑性樹脂としては、特に制限されず、例えばポリオレフィン、ポリエステル、フッ素樹脂、塩化ビニル樹脂等が挙げられる。 For example, the method for braiding silk fibroin fiber is not particularly limited, and for example, known braiding techniques such as eight punching, twelve punching, and 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, a thermoplastic resin core rod 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.
　また、絹フィブロイン繊維をニット編みして管状に形成すれば、弾力性に富み、且つ柔軟で、引っ張り強度に優れ、切断端のほつれ難い管状構造物とできる。ニット編みには、例えば、丸編（横編）、縦編、フルファッション、ラッシェル、トリコット等がある。ニット編み機は、特に制限されない。得られた絹布を前記熱可塑性樹脂製芯棒等に巻き付けて管状に形成すればよい。 In addition, if silk fibroin fibers are knit knitted to form a tubular structure, it can be made into a tubular structure that is highly elastic, flexible, excellent in tensile strength, and difficult to fray at the cut end. 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.
　本発明のコーティング用組成物で管状構造物をコーティングする方法は、特に限定されるものでないが、上記コーティング液に管状構造物を浸漬した後、凍結、熱処理し、次いで乾燥するのが好ましい。 The method for 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.
　浸漬処理は、特に制限されないが、常温、常圧下或いは減圧下で行われる。浸漬時間は、５～３０分間、特に１０～２０分間が好ましい。 The immersion treatment is not particularly limited, but is performed at room temperature, normal pressure, or reduced pressure. The immersion time is preferably 5 to 30 minutes, particularly 10 to 20 minutes.
　浸漬後、－２０℃～－１９６℃、好ましくは－５０℃～－１００℃で、０．５分～２時間凍結させる。凍結処理後、さらに凍結真空乾燥してもよい。凍結真空乾燥をする場合、絹フィブロインと水性ポリウレタン系樹脂を水に溶解乃至分散させたコーティング液を用いるのが好ましく、これにより、絹フィブロインが不溶化し微細な多孔質構造となって、より弾力性屈曲性に優れた管状構造物が得られる。 After immersion, freeze at −20 ° C. to −196 ° C., preferably −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.
　熱処理は、１００℃以上の温度で行うことが好ましく、１２０℃以上がより好ましい。熱処理は、オートクレーブ等の密閉加圧装置を用いてもよい。オートクレーブを用いる場合、１２０℃以上で２０～４０分間が好ましい。 The heat treatment is preferably performed at a temperature of 100 ° C. or higher, 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 at 120 ° C. or higher for 20 to 40 minutes.
　乾燥処理は、例えば、天然乾燥、加熱乾燥、真空乾燥等の方法が挙げられる。乾燥温度は、１５℃～１５０℃、好ましくは５０℃～８０℃で、時間は３０分～１時間するのが好ましい。
　このようにして、管状構造物の最内壁及び／又は最外壁がコーティングされる。 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.
　本発明において、管状構造物に形成されるコーティング層は、単層でもよいが、二層や三層、四層等の多層のコーティング層を有するコーティング管状構造物としてもよい。
　コーティング層の厚さは、特に限定されないが、最内壁部は０．００５～０．５ｍｍ、最外壁部は０．１～１．０ｍｍが好ましい。 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.
　本発明のコーティング管状構造物は、管状構造物の最内壁、最外壁のどちらか一方だけが、絹フィブロインとポリウレタン系樹脂を含有するコーティング用組成物でコーティングされていてもよく、両方がコーティングされていてもよい。血液の漏洩を防ぐ点、引っ張り強度・弾力性向上の点からは、管状構造物の最内壁が該コーティング用組成物でコーティングされているのが好ましい。この場合、他方は任意のコーティング剤でコーティングされていてもよい。このような他のコーティング剤として、一般的なコーティング剤、例えば、ポリウレタン系樹脂、ゼラチン等が挙げられる。 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 coating composition containing silk fibroin and polyurethane resin, and both are coated. It 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 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.
　このようにして得られる本発明のコーティング管状構造物は、例えば人工血管、人工気管、ステントグラフト、その他動物の生体の管状構造物の代用品として用いることができる。特に、絹の優れた生体適合性及び抗血栓性から、直径５ｍｍ以下、好ましくは１～５ｍｍ以下の小口径人工血管に好適である。 The thus obtained coated tubular structure of the present invention can be used, for example, as a substitute for an artificial blood vessel, an artificial trachea, a stent graft, and other animal biological tubular structures. Particularly, because of the excellent biocompatibility and antithrombotic properties 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.
　以下、本発明について実施例をあげて具体的に説明するが、本発明はこれらによって何等限定されるものではない。 Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.
製造例１
＜家蚕絹フィブロイン溶解物の調製＞
　家蚕繭を鋏で細かく切断（約２ｍｍ×１０ｍｍ程度）又は家蚕繭を繰糸し、定法により精練して、フィブロインを覆うタンパク質（セリシン）やその他脂肪分等を除去した絹フィブロイン繊維を得た。
　次いで、この絹フィブロイン繊維を９Ｍ臭化リチウム水溶液に１５ｗ／ｖ％となるように溶解した。この水溶液を、セルロース透析膜（ＶＩＳＫＡＳＥＳＥＬＥＳ　ＣＯＡＰ社製　Ｓｅａｍｌｅｓｓ　Ｃｅｌｌｕｌｏｓｅ　Ｔｕｂｉｎｇ　３６／３２）を用いて、３日間純水で透析を行い、塩化リチウムを取り除き、さらに遠心分離にて、溶け残りやゴミ等を除去し、家蚕絹フィブロイン溶解物を得た。家蚕絹フィブロイン溶解物中の絹フィブロイン濃度は３～８ｗ／ｖ％であった。 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%.
製造例２
＜家蚕絹人工血管の作成＞
１．組み機を用いた小口径絹グラフトの作製
　人工血管の作製には、組紐作製装置（１６打）（（株）コクブンリミテッド製）を用いた。ボビンには、上記定法により精練した絹フィブロイン繊維を巻いた。同機の中心部分に、外径１～５ｍｍの塩化ビニル、または、ポリテトラフルオロエチレン製芯棒を装着し、芯棒の周囲に絹フィブロイン繊維を組み、家蚕絹人工血管（内径１．５～５ｍｍφ）を得た。 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. A vinyl chloride or polytetrafluoroethylene core rod with an outer diameter of 1 to 5 mm is attached to the center of the machine, and silk fibroin fibers are assembled around the core rod, and a silkworm artificial blood vessel with an inner diameter of 1.5 to 5 mmφ )
製造例３
＜コーティング用組成物の調製＞
　製造例１で得た家蚕絹フィブロイン溶解物（ＳＩ）と水分散性ポリウレタン（ＰＵ、ネオステッカー１００ｃ、日華化学（株）製、ポリウレタン濃度３０ｗ／ｖ％）を体積比で１：１０、１０：１０、１：４、１：２の割合でそれぞれ混合して、コーティング用組成物（１）、（２）、（３）及び（４）を得た。
　さらに、前記水分散性ポリウレタンのみを用いたコーティング用組成物（５）と、家蚕絹フィブロイン溶解物のみを用いたコーティング用組成物（６）を得た。 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.
実施例１
＜家蚕絹人工血管のコーティング＞
　コーティング用組成物（５）中に、芯棒を装着したままの家蚕絹人工血管（内径４ｍｍφ）を常圧下で１０分浸漬した後、液体窒素に１分間浸漬して凍結させた。この操作をもう一度繰り返した。次いで、凍結させた家蚕絹人工血管を１２０℃、２０分間のオートクレーブ処理した後、芯棒から分離し、５０℃に設定した乾燥機中にて１時間乾燥することで家蚕絹人工血管の外壁を二層にコーティングしたコーティング家蚕絹人工血管を得た。
　その後、シリンジを利用して家蚕絹人工血管の内壁にコーティング用組成物（１）を６ｍＬ流した後、液体窒素に１分間浸漬して凍結させた。この操作をもう一度繰り返した。
　次いで、室温で３０分乾燥した後、１２０℃に設定した乾燥機中にて２時間乾燥して家蚕絹人工血管の内壁を二層にコーティングしたコーティング家蚕絹人工血管を得た。コーティング条件を表１に示す（以下、同じ）。 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).
比較例１
＜家蚕絹人工血管のコーティング＞
　実施例１と同様にして、家蚕絹人工血管の最外壁を、コーティング用組成物（５）で二層にコーティングしたコーティング家蚕絹人工血管を得た。
　その後、コーティング用組成物（１）をコーティング用組成物（６）に代えた以外は実施例１と同様にして家蚕絹人工血管の内壁を二層にコーティングしたコーティング家蚕絹人工血管を得た。 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).
実施例２～３並びに比較例２～３
＜家蚕絹人工血管のコーティング＞
　図１に示すように、コーティング用組成物（５）中に、芯棒を装着したままの家蚕絹人工血管（内径４ｍｍφ）を常圧下で１０分間浸漬した後、液体窒素に１分間浸漬して凍結させた。－８０℃の冷凍庫に１時間保存し、凍結真空乾燥にて１２時間処理を行なった。１０分間の浸漬から凍結乾燥までを２回繰り返した。次いで、該家蚕絹人工血管を１２０℃、２０分間のオートクレーブ処理した後、純水に１５分間浸漬して芯棒から分離し、５０℃に設定した乾燥機中にて１時間乾燥することで家蚕絹人工血管の外壁をニ層にコーティングしたコーティング家蚕絹人工血管を得た。
　その後、シリンジを利用して家蚕絹人工血管の内壁に、コーティング用組成物（１）、（２）、（５）又は（６）をそれぞれ６ｍＬ流し、液体窒素に１分間浸漬して凍結させた。－８０℃の冷凍庫に１時間保存し、凍結真空乾燥にて１２時間処理を行なった。それから、１２０℃、２０分間のオートクレーブ処理を施した。
　次いで、家蚕絹人工血管を５０℃の純水に浸漬、１２時間放置する操作を、水を替えて６回繰り返した。次いで、５０℃で乾燥して、家蚕絹人工血管の最内壁を単層コーティングしたコーティング家蚕絹人工血管を得た。 Examples 2 to 3 and Comparative Examples 2 to 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 was stored in a freezer at −80 ° C. for 1 hour, and treated by freeze vacuum drying for 12 hours. 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 was stored in a freezer at −80 ° C. for 1 hour, and treated by freeze vacuum drying for 12 hours. 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.
実施例４
＜家蚕絹人工血管のコーティング＞
　コーティング用組成物（５）中に、芯棒を装着したままの家蚕絹人工血管（内径４ｍｍφ）を常圧下で１０分間浸漬した後、液体窒素に１分間浸漬して凍結させた。－８０℃の冷凍庫に１時間保存し、凍結真空乾燥にて１２時間処理を行なった。次いで、該家蚕絹人工血管を１２０℃、２０分間のオートクレーブ処理した後、純水に１５分間浸漬して芯棒から分離し、５０℃に設定した乾燥機中にて１時間乾燥することで家蚕絹人工血管の最外壁を単層コーティングしたコーティング家蚕絹人工血管を得た。
　その後、シリンジを利用して家蚕絹人工血管の内壁に、コーティング用組成物（１）を６ｍＬ流し、実施例２と同様にして家蚕絹人工血管の最内壁を単層コーティングしたコーティング家蚕絹人工血管を得た。 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 was stored in a freezer at −80 ° C. for 1 hour, and treated by freeze vacuum drying for 12 hours. 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.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
試験例１　形態観察試験
　実施例１～４並びに比較例１～３で得られた各コーティング家蚕絹人工血管の形態観察を走査型電子顕微鏡（（株）キーエンス製ＶＥ７８００）を用いて行った。
　図２に示すように、各コーティング家蚕絹人工血管の外表面、内表面、断面は、管状構造物に沿って微細な多孔質で覆われた構造を形成していた。特に、真空乾燥の操作をコーティングの途中に入れることによって、殆ど、穴の認められない人工血管を作製することができた。他方、絹フィブロインのみで人工血管の内壁をコーティングしたもの（比較例１）は、大きな穴が開いていた。 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.
試験例２　透水率の測定
　実施例１～３並びに比較例１で得た各コーティング家蚕絹人工血管の側壁からの漏水量を定量的に測定するため、ＩＳＯ７１９８のガイダンスに沿って試験を行った。各コーティング家蚕絹人工血管の側壁に、１２０ｍｍＨｇの水圧がかかるように水を落下させ、人工血管を通過して固定治具から放出された１分間単位面積あたりの漏水量（透水率）を測定した。５回試験を行い、その平均値を求めた。結果を図３に示す。
　漏水量は、実施例１で９０．６４(ｍＬ/ｃｍ2/ｍｉｎ）、実施例２で３．１２(ｍＬ/ｃｍ2/ｍｉｎ）、実施例３で０．２４(ｍＬ/ｃｍ2/ｍｉｎ）、実施例４で７．３２(ｍＬ/ｃｍ2/ｍｉｎ）であった。真空乾燥の操作をコーティングの途中に入れることによって、漏水量は大幅に減少した。他方、比較例１で６４７．２(ｍＬ/ｃｍ2/ｍｉｎ）であった。
　なお、市販の人工血管でプレクロッティング(前血処理)が必要ないダクロン製人工血管の透水率は公表値１～１０ｍＬ/ｃｍ2/ｍｉｎである。 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, tests were conducted in accordance with ISO 7198 guidance. 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.
試験例３　圧縮弾性率の測定
　実施例２～３並びに比較例２～３で得た各コーティング家蚕絹人工血管を０．５cｍの長さにカットして、圧縮力を周軸方向にかけた。人工血管の直径を１０％減少させるまでの圧縮弾性率（Ｎ／ｍｍ2）（直径の変化を横軸に、圧縮力を縦軸としてプロットし、その傾きから求める）を求めた。５回試験を行い、その平均値を求めた。結果を図４に示す。
　図４より、実施例２及び３で得たコーティング家蚕絹人工血管の弾性率は、絹フィブロインのみで人工血管の内壁をコーティングしたもの（比較例２）と比べて、周軸方向の柔軟性が向上したことが確認された。 Test Example 3 Measurement of Compression Elastic Modulus Each coated rabbit silk artificial blood vessel obtained in Examples 2 to 3 and Comparative Examples 2 to 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.
試験例４　吻合糸保持強度の測定
　実施例２～３並びに比較例２～３で得た各コーティング家蚕絹人工血管を、ＩＳＯ７１９８のガイダンスに沿って試験を行った。５回試験を行い、その平均値を求めた。結果を図５に示す。
　図５より、実施例２及び３で得たコーティング家蚕絹人工血管は、絹フィブロインのみで人工血管の内壁をコーティングしたもの（比較例２）と同等の吻合糸保持強度を有していることが確認された。 Test Example 4 Measurement of anastomotic thread retention 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.
実施例５
＜家蚕絹人工血管のコーティング＞
　コーティング用組成物（５）中に、芯棒を装着したままの家蚕絹人工血管（内径４ｍｍφ）を常圧下で１０分間浸漬した後、液体窒素に１分間浸漬して凍結させた。－８０℃の冷凍庫に１時間保存し、凍結真空乾燥にて１２時間処理を行なった。コーティング用組成物（３）、（４）、（２）の順に代えて、１０分間の浸漬から凍結真空乾燥までを４回繰り返した。次いで、該家蚕絹人工血管を１２０℃、２０分間のオートクレーブ処理した後、純水に１５分間浸漬して芯棒から分離し、５０℃に設定した乾燥機中にて１時間乾燥することで家蚕絹人工血管の外壁を四層にコーティングしたコーティング家蚕絹人工血管を得た。 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 was stored in a freezer at −80 ° C. for 1 hour, and treated by freeze vacuum drying for 12 hours. 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.
試験例５　イヌへの移植試験
＜術式＞
　イヌ（ビーグル犬、雌性、１歳齢）への麻酔はアトロピン（０．１ｍｌ／ｋｇ、アトロピン注）、ミダゾラム（０．０４ｍｌ／ｋｇ、ミダゾラム注１０ｍｇ「サンド」）、ブトルファノール（０．０４ｍｌ／ｌｇ、ベトルファール注）の前処置後、プロポフォール（ベトルファール）を用いて導入、挿管し、麻酔の維持はイソフルランを用いて行った。術野を刈毛後、アルコール、クロルヘキシジンを用いて消毒し、ドレーピングした。血管にアプローチする前にヘパリン（１００ＩＵ／ｋｇ、ヘパリン注）を静脈内投与し、活性化凝固時間（ＡＣＴ）が２００以上になっていることを確認後、血管への操作を開始した。
　術中は２０分おきにＡＣＴを確認し、２００以下になっていることが確認された場合にはヘパリンを追加投与した。
　頚部正中切開にてアプローチ後、右側総頸動脈を約５ｃｍにわたり剥離し、絹糸にて支持した。ブルドッグ鉗子にて剥離した血管の両端を鉗圧して、血行を遮断後、約３ｃｍの頸動脈を鋏にて切除し、切除した血管を置き換える形で実施例５作製したコーティング家蚕絹人工血管を移植した。人工血管の縫合は手術用ルーペを用いた拡大視野下にて行い、血管縫合は９ｍｍ、３／８の丸針付きの７／０の太さのポリフッ化ビニリデンモノフィラメント（ＰＲＥＭＩＯ，２６ＳＺ０５ＡＧ，Ｐｅｔｅｒｓ　ＳＵＲＧＩＣＡＬ，Ｆｒａｎｃｅ）を用いて１０－１２カ所の結節縫合にて端々吻合を実施した。縫合後は血液の漏れがないことを確認し、漏れが確認された部位に関しては、Ｚ縫合を追加して出血のコントロールを行った。出血のコントロールが確認された後に皮下組織、および真皮を３／０の吸収糸（グリコライド・ジオキサノン・トリメチレンカーボネート、バイオシン１／２丸針　針長２２ｍｍ）を用いて縫合閉鎖した。 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 vessel sutures were performed under an enlarged field of view using a surgical loupe, and blood vessel sutures were 9 mm, 3/8 round needles and 7/0 thickness polyvinylidene fluoride monofilaments (PREMIO, 26SZ05AG, Peters SURGICAL, End-to-end anastomosis was performed using 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).
＜術後管理＞
　術後は一般的な術創の消毒、全身的な抗生物質の投与に加え、抗凝固療法を行った。低分子ヘパリン（ダルテパリンナトリウム注射液、ヘパクロン注５０００、エール薬品）の皮下投与２５００ＩＵ／ｄａｙ　ＢＩＤを２１日間、Ａｓｐｉｒｉｎ（０，５ｍｇ／ｋｇＢＩＤ）の投与を継続して実施した。 <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).
＜移植血管のエコーによる評価＞
　移植血管の開存性と血管内腔の血流の評価を行うために、超音波画像診断装置（Ａｌｏｋａ　ＰｒｏＳｏｕｎｄＳＳＤ－α１０）を用いた観察を経時的に行った。測定は動物の体動の影響を除外するために、プロポフォールの静脈内微量投与下における鎮静下で実施した。使用した電子リニア探蝕子は浅部表在用のＵＳＴ－５５２４－７．５であり、収縮期最大流速（Ｐｅａｋ　ｓｙｓｔｏｌｉｃ　ｖｅｌｏｃｉｔｙ：ＰＳＶ）、拡張末期流速（Ｅｎｄ－ｄｉａｓｔｏｌｉｃ　ｖｅｌｏｃｉｔｙ：ＥＤＶ）、拍動指数（Ｐｕｌｓａｔｉｌｉｔｙ　ｉｎｄｅｘ：ＰＩ）、抵抗係数（ｒｅｓｉｓｔａｎｃｅ　ｉｎｄｅｘ：ＲＩ）、収縮期加速時間（Ａｃｃｅｌｅｒａｔｉｏｎ　ｔｉｍｅ：ＡＴ）、人工血管内径の測定を人工血管の前後を含めて計７カ所（１．総頸動脈近位部、２．近位吻合部、３．人工血管近位部、４．人工血管中部、５．人工血管遠位部、６．遠位吻合部、７．総頸動脈遠位部）で行った。同時にカラードプラ、ｅ－ｆｌｏｗ、ソナゾイドを用いた超音波造形検査も駆使し、乱流、狭窄、プラークの形成についても評価した。 <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.
＜結果＞
　植え込み時におけるトラブルはなく、順調に移植手術は終了し、術後の動物の一般状態には問題がなかった。エコーによる評価の結果は、術後２週間で観察された像では乱流、狭窄、プラークの形成も認められず、良好な血流が保たれていた。各種エコーパラメータによる評価においても、術前と術後２週間では各パラメータ値の変動は認められず、術後の狭窄や閉塞が認められなかっただけでなく、術前の生来の血管壁と類似したパラメータ値が、植え込み後２週間の人工血管においても確認された（図６）。
　血管において、血流は拍動に合わせて間欠的に流れ、またその際に血管壁は伸展を繰り返すことによって生理的な血流を作り出している。このように、高い生体適合性を有しつつも、生来の血管壁に近い物理学的性質を有する人工血管は、より生理的な環境を提供することで血管の機能を維持することが可能であると考えられた。 <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 (12)

  1. 　絹フィブロインとポリウレタン系樹脂を含有する管状構造物のコーティング用組成物。 A composition for coating a tubular structure containing silk fibroin and polyurethane resin.
  2. 　絹フィブロインとポリウレタン系樹脂を水、有機溶媒又はこれらの混合物に分散又は溶解したものである、請求項１記載の管状構造物のコーティング用組成物。 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.
  3. 　有機溶媒が、ヘキサフロロイソプロパノール及び／又はヘキサフロロアセトンである、請求項２記載の管状構造物のコーティング用組成物。 The composition for coating a tubular structure according to claim 2, wherein the organic solvent is hexafluoroisopropanol and / or hexafluoroacetone.
  4. 　ポリウレタン系樹脂が、水性ポリウレタン系樹脂である、請求項１記載の管状構造物のコーティング用組成物。 The composition for coating a tubular structure according to claim 1, wherein the polyurethane resin is an aqueous polyurethane resin.
  5. 　管状構造物が、絹フィブロイン繊維が編、組、織及び絡から選ばれる１又は２以上の方法により巻かれてなる管状構造物である、請求項１～４のいずれか１項記載の管状構造物のコーティング用組成物。 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.
  6. 　絹フィブロインとポリウレタン系樹脂を含有する管状構造物のコーティング用組成物により管状構造物をコーティングする、コーティング管状構造物の製造方法。 A method for producing a coated tubular structure, wherein the tubular structure is coated with a composition for coating a tubular structure containing silk fibroin and polyurethane resin.
  7. 　管状構造物のコーティング用組成物が、絹フィブロインとポリウレタン系樹脂を水、有機溶媒又はこれらの混合物に分散又は溶解したものである、請求項６記載のコーティング管状構造物の製造方法。 The method for producing a coated tubular structure according to claim 6, wherein the coating composition for the tubular structure is obtained by dispersing or dissolving silk fibroin and polyurethane resin in water, an organic solvent or a mixture thereof.
  8. 　有機溶媒が、ヘキサフロロイソプロパノール及び／又はヘキサフロロアセトンである、請求項７記載のコーティング管状構造物の製造方法。 The method for producing a coated tubular structure according to claim 7, wherein the organic solvent is hexafluoroisopropanol and / or hexafluoroacetone.
  9. 　ポリウレタン系樹脂が、水性ポリウレタン系樹脂である、請求項６記載のコーティング管状構造物の製造方法。 The method for producing a coated tubular structure according to claim 6, wherein the polyurethane resin is an aqueous polyurethane resin.
  10. 　コーティングを、絹フィブロインとポリウレタン系樹脂を水、有機溶媒又はこれらの混合物に分散又は溶解したコーティング溶液に管状構造物を浸漬した後、凍結、熱処理し、次いで乾燥することにより行う、請求項６記載のコーティング管状構造物の製造方法。 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, and then freezing, heat-treating, and drying. A method for producing a coated tubular structure.
  11. 　管状構造物が、絹フィブロイン繊維が編、組、織及び絡から選ばれる１又は２以上の方法により巻かれてなる管状構造物である、請求項６～１０のいずれか１項記載のコーティング管状構造物の製造方法。 The coated tubular body according to any one of claims 6 to 10, 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. Manufacturing method of structure.
  12. 　請求項６～１１のいずれか１項記載の製造方法により得られたコーティング管状構造物。 A coated tubular structure obtained by the production method according to any one of claims 6 to 11.
PCT/JP2011/069691 2010-12-28 2011-08-31 Method for manufacturing artificial blood vessel WO2012090553A1 (en)

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