Method for producing decellularized material for transplantation and graft composition made of biocompatible material containing said material

Abstract

The object is to provide a method for producing a decellularized material for transplantation that can be used for a graft composition and minimizes the number of sutures made from threads. To achieve the above-mentioned objectives, the present invention provides a method for producing a decellularized material for transplantation, which comprises the steps of (a) harvesting a blood vessel having branches from a vertebrate (donor), (b) decellularizing the blood vessel, and (c) adhering and closing the excised branched portion by a protein denaturing treatment. Furthermore, the present invention also provides a graft composition comprising a biocompatible material including the decellularized material for transplantation.

Description

[Technical field]

[0001] The present invention relates to a method for producing a decellularized material that can be used for transplantation, and a graft composition comprising a biocompatible material containing said material. [Background technology]

[0002] When transplanting a graft derived from another person's living tissue, the problem is that the recipient's tissue may reject the graft. The development of artificial tissue is expected to solve this problem. Various polymers have been tried as materials, but the compatibility of these materials with living tissue is low, and they may cause detachment or infection at the junction between the graft and the living tissue. Therefore, in order to improve compatibility with living tissue, a technology has been developed in which decellularized living tissue, which is the supporting tissue remaining after cells have been removed from living tissue, is used as a graft.

[0003] The properties required for a decellularized material include (i) strength as a graft, (ii) removal of DNA in the decellularized tissue that causes rejection, and (iii) ease of infiltration of autologous cells after transplantation. In order to obtain a decellularized material that satisfies these requirements, various manufacturing methods have been considered. For example, a method using a surfactant (see, for example, Patent Documents 1 and 2), a method using an enzyme (see, for example, Patent Document 3), a method using an oxidizing agent (see, for example, Patent Document 4), a method using high hydrostatic pressure treatment (see, for example, Patent Documents 5 to 7), a method using a freeze-thaw treatment (see, for example, Patent Documents 8 to 9), a method using a hypertonic electrolyte solution (see, for example, Patent Document 10), etc. are known. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 60-501540 [Patent Document 2] Special Publication No. 2003-518981 [Patent Document 3] Special Publication No. 2002-507907 [Patent Document 4] Special Publication No. 2003-525062 [Patent Document 5] JP 2004-094552 A [Patent Document 6] International Publication No. 2008 / 111530 [Patent Document 7] Special Publication No. 2013-502275 [Patent Document 8] JP 2005-185507 A [Patent Document 9] JP 2005-211480 A [Patent Document 10] JP 2010-221012 A [Non-patent literature]

[0005] [Non-Patent Document 1] JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A, OCT 2015 Vol.103A, 10 Summary of the Invention [Problem to be solved by the invention]

[0006] In general, in order to maintain the functionality of decellularized tissue, it is important that the surface state is maintained, that is, that morphological damage to the tissue is minimized (e.g., Non-Patent Document 1), and even relatively slight damage can affect the infiltration of autologous cells. Therefore, the present inventors performed processing by suturing, which is thought to cause less damage to decellularized tissue, and found that this hinders the infiltration of autologous cells. In light of these results, it is considered important that graft compositions derived from biological tissues have the minimum number of sutures necessary. [Means for solving the problem]

[0007] Specifically, when decellularized tissue obtained from a blood vessel having branches (branched blood vessels) is used as a graft composition, it is necessary to cut off the branched portion and close it. It has been found that when the cut portion of the branch is closed by protein denaturation treatment rather than by suturing, the infiltration of autologous cells into the decellularized tissue is not hindered and tissue at the transplant site is regenerated.

[0008] That is, the present invention provides: (a) harvesting a branched blood vessel from a vertebrate (donor); (b) decellularizing the blood vessel; and (c) A process of gluing and closing the excised branched parts by protein denaturation treatment. The present invention provides a method for producing a decellularized material for transplantation, comprising the steps of:

[0009] Furthermore, the method for producing a decellularized material for transplantation of the present invention may further comprise the step of excising the branched portion of the blood vessel harvested in step (a).

[0010] The present invention also provides a graft composition comprising a biocompatible material including a decellularized transplant material that is made from a blood vessel from a vertebrate (donor) whose branches have been cut off and has at least one cut branch site that has been closed by protein denaturation. Effect of the Invention

[0011] The manufacturing method of the present invention can provide a decellularized material for transplantation for a graft composition that is prone to infiltration of autologous cells after transplantation. Furthermore, a graft composition that contains at least a portion of the decellularized material for transplantation obtained by the manufacturing method of the present invention can provide a decellularized material for transplantation after transplantation. To Autologous cells Soaking Because it is easily moisturized, it can function as normal biological tissue even after transplantation. [Brief description of the drawings]

[0012] [Figure 1]FIG. 1 shows a schematic diagram of a blood vessel having a branch. In (i), 1 indicates a blood vessel having a branch, 2 indicates a branched portion (branched blood vessel), and the dashed line indicates an example of a position where the branched portion is excised. This dashed line is preferably 1 mm to 2 mm away from the blood vessel. In another embodiment, the dashed line may be 5 mm to several cm away from the blood vessel. In (ii), 3 indicates a protein denatured portion. [Diagram 2] 2 is a diagram showing an internal thoracic artery fragment harvested from a pig and in which a branched portion was removed in the Examples. In the following Examples, the internal thoracic artery fragment (3) in the figure was used as a comparative example. [Diagram 3] In the figure, (α) is an enlarged photograph of a cross section of a harvested porcine internal thoracic artery piece stained with hematoxylin and eosin. Also, (β) is an enlarged photograph of a cross section of a harvested porcine internal thoracic artery piece decellularized and stained with hematoxylin and eosin, which is a decellularized material for transplantation. It can be confirmed that no nucleic acid is present and that the material has been decellularized. Each scale bar indicates 1000 μm. In the following examples, the internal thoracic artery piece in the figure (3) was used as a comparative example. [Figure 4] Figure 4 shows enlarged photographs of sections stained with hematoxylin-ession (HE stain) or Elastica van Gieson (EVG stain) 3 months after transplantation of a decellularized porcine internal thoracic artery fragment, a decellularized material for transplantation, into a rabbit. In the comparative example, the small white circle pointed to by the arrow in the circled area is a trace of the proline thread. The proline thread passed perpendicular to the paper. It can be seen that no cell infiltration is observed around the area where the proline thread passed. Each scale bar indicates 500 μm. [Diagram 5] The left image is an enlarged photograph of a section of a branch portion stained with HE of a decellularized porcine internal thoracic artery fragment, which is the decellularized transplantation material of the present invention, before transplantation. The right image is an enlarged photograph of a section of a branch portion stained with HE one month after transplantation. Cell infiltration is observed after transplantation. Each scale bar indicates 500 μm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The method for producing the decellularized material for transplantation of the present invention will now be described. In the present invention, a blood vessel having branches is harvested from a vertebrate (donor). Harvesting here means separating a blood vessel having branches from a donor. Also, "blood vessel having branches" means a blood vessel that had branches in the donor, and includes blood vessels whose branches have been cut off at the time of harvesting.

[0014] In the present invention, the vertebrate is not particularly limited, but since blood vessels are preferably easily available, animals other than humans are preferred, and in particular, mammalian livestock and avian livestock are preferred. Examples of mammalian livestock include cows, horses, camels, llamas, donkeys, yaks, sheep, pigs, goats, deer, alpacas, dogs, raccoon dogs, weasels, foxes, cats, rabbits, hamsters, guinea pigs, rats, squirrels, and raccoons. Examples of avian livestock include parakeets, parrots, chickens, ducks, turkeys, geese, guinea fowls, pheasants, ostriches, emus, and quails. Among these, pigs, rabbits, and cows are preferred because of their stable availability.

[0015] In the method for producing a decellularized material for transplantation of the present invention, a blood vessel having a branch is used. Here, a blood vessel having a branch refers to a blood vessel having at least one branch portion (branch blood vessel) as shown in FIG. Because blood vessels with a branched structure have a complex shape, they are considered to be unsuitable for processing even if decellularized, and because they have a branched structure, they have rarely been used as blood vessel transplants. According to the present invention, the branched parts of the blood vessels are adhered and closed, making them usable as unbranched blood vessels. It is extremely significant that blood vessels with branches, which have been difficult to use in the past, can be used as graft compositions.

[0016] Specific examples of blood vessels having branches include the internal thoracic artery, abdominal wall artery, gastroepiploic artery, carotid artery, radial artery, intercostal artery, muscular phrenic artery, femoral artery, deep femoral artery, aorta, ulnar artery, brachial artery, anterior tibial artery, posterior tibial artery, mesenteric artery, splenic artery, internal thoracic vein, anterior intercostal vein, azygos vein, hemiazygos vein, jugular vein, intestinal vein, femoral vein, saphenous vein, mesenteric vein, splenic vein, etc. Considering the physical properties (elongation, biocompatibility, strength, etc.) as a graft composition, the artery is preferred, and the internal thoracic artery is more preferred.

[0017] For blood vessels having branches, a vertebrate is anesthetized or sacrificed, and a part of the body such as the chest, abdomen, or legs is incised to excise the blood vessels having branches to be collected. The anesthesia and sacrifice methods can be the same as those conventionally used by those skilled in the art. When cutting open a part of the body of a vertebrate, scalpels, scissors, and the like that are normally used in animal experiments and surgical procedures can be used.

[0018] Similarly, when collecting a blood vessel having branches, scalpels, scissors, etc. that are commonly used in animal experiments and surgical procedures can be used. When resecting a blood vessel, it is preferable to use an ultrasonic scalpel or an electric scalpel. These can cut the blood vessel while coagulating the blood by applying ultrasonic vibration or high-frequency current to the area where the blood vessel is to be cut. In particular, it is preferable to use an ultrasonic scalpel because the degree of denaturation of proteins in the cut area is moderate. In this specification, the terms "ultrasonic scalpel" and "ultrasonic vibration scalpel" are used synonymously.

[0019] Examples of electric scalpels that can be used in the present invention include the Bio Series manufactured by Elbe and the SHAPPER Series manufactured by Senko Medical Industries Co., Ltd. Examples of ultrasonic scalpels that can be used in the present invention include the Sonopet UST-2001 manufactured by Stryker Medtech Co., Ltd. and the Harmonic Scalpel manufactured by Ethicon Endosurgery Co., Ltd.

[0020] The blood vessel collected in step (a) may have its branches already excised, or may have its branches remaining at a sufficient length (e.g., 5 mm to several cm). If the branches remain at a sufficient length, they are appropriately excised. The length of the branched portion where the protein denaturation treatment is performed is preferably 1 mm to 10 mm away from the branched portion of the blood vessel, more preferably 2 mm to 7 mm away, and most preferably 3 mm to 5 mm away. By performing the protein denaturation treatment within the above range, the effects of the denaturation treatment when using the decellularized material for transplantation of the present invention (such as changes in physical properties, lack of obstruction to blood flow, and antithrombotic properties) can be minimized, and reorganization can be promoted.

[0021] The blood vessels harvested from the vertebrate are subjected to a decellularization process. Decellularization treatments include surfactant treatment (Singelyn JM, et al., Biomaterials, 2009, 30, 5409-5416;Singelyn JM,, et al., J. Am. Coll. Cardiol., 2012, 59, 751-763;Sonya B., et al., Sci. Transl. Med., 2013, 5, 173ra25), enzyme treatment, osmotic pressure treatment, freeze-thaw treatment, oxidizing agent treatment, and high hydrostatic pressure treatment (Sasaki S., et al., Mol. Vis., 2009, 15, 2022-2028;Yoshihide H., et al., Biomaterials, 2010, 31, 3941-3949;Seiichi F., et al., Biomaterials, 2010, 31, 3590-3595; Negishi J., et al., J. Artif. Organs, 2011, 14, 223-231; Japanese Patent No. 4092397; Republished Publication No. 2008-111530; JP 2009-50297 A) and combinations thereof are exemplified, but decellularization treatment by high hydrostatic pressure treatment is preferred.

[0022] The pressure to be applied during high hydrostatic pressure treatment may be selected appropriately according to the donor animal species and blood vessel type, as long as the pressure is sufficient to destroy cells and pathogens derived from the vertebrate donor. Examples of hydrostatic pressure include 2 to 1,500 MPa. When the applied hydrostatic pressure is higher than 50 MPa, decellularization from the blood vessels is sufficiently performed. Therefore, the pressure is preferably 50 to 1,500 MPa, more preferably 80 to 1,300 MPa, even more preferably 90 to 1,200 MPa, and most preferably 95 to 1,100 MPa.

[0023] Examples of media used for applying high hydrostatic pressure include water, physiological saline, buffer solutions, propylene glycol or an aqueous solution thereof, glycerin or an aqueous solution thereof, and aqueous saccharide solutions. Examples of buffer solutions include acetate buffer solutions, phosphate buffer solutions, citrate buffer solutions, borate buffer solutions, tartrate buffer solutions, Tris buffer solutions, HEPES buffer solutions, and MES buffer solutions. Examples of saccharides in aqueous saccharide solutions include erythrose, xylose, arabinose, allose, talose, glucose, mannose, galactose, erythritol, xylitol, mannitol, sorbitol, galactitol, sucrose, lactose, maltose, trehalose, dextran, alginic acid, and hyaluronic acid.

[0024] The temperature of the medium for the high hydrostatic pressure treatment is not particularly limited as long as it does not generate ice and does not cause thermal damage to the tissue. Since the decellularization treatment is carried out smoothly and has little effect on the tissue, the temperature is preferably 0 to 45°C, more preferably 4 to 40°C, even more preferably 10 to 37°C, and most preferably 15 to 35°C.

[0025] If the high hydrostatic pressure treatment time is too short, the decellularization process will not be sufficient, and if it is too long, it will result in a waste of energy, so the treatment time is preferably 5 minutes to 12 hours, more preferably 7 minutes to 5 hours, and even more preferably 10 minutes to 3 hours.

[0026] The adequacy of decellularization can be confirmed by histological staining (hematoxylin-eosin staining) or quantification of remaining DNA.

[0027] In blood vessels to which high hydrostatic pressure has been applied, cells in the tissue are destroyed, and these cellular components are removed by a cleaning solution. The cleaning solution may be the same as the medium used to apply high hydrostatic pressure, or may be a different cleaning solution, or a combination of multiple types of cleaning solutions may be used. The cleaning solution preferably contains a nuclease, an organic solvent, or a chelating agent. The nuclease can improve the removal efficiency of nucleic acid components from blood vessels to which hydrostatic pressure has been applied, and the organic solvent can improve the removal efficiency of lipids, respectively, and the chelating agent can inactivate calcium ions and magnesium ions in the decellularized tissue, thereby preventing calcification when the decellularized tissue is applied to an affected area.

[0028] In the present invention, the portion where the branch has been excised is closed by subjecting it to a protein denaturing treatment. The means for denaturing the protein is not particularly limited, but it is preferable to use an ultrasonic scalpel or an electric scalpel from the viewpoints of ease and efficiency of the work and minimizing damage to the tissue, and it is preferable to use an ultrasonic scalpel from the viewpoints of minimizing damage to the tissue and ensuring the closure of the branch stump.

[0029] The ultrasonic scalpel has a structure in which the cutting edge mechanically vibrates ultrasonically, and frictional heat is generated near the biological tissue with which the cutting edge comes into contact, denaturing the proteins inside the biological tissue. The part of the branched blood vessel that was cut and opened is resected, and the protein is denatured, causing it to bond and close. In addition, the above phrase "the portion where the branches have been removed is closed by subjecting it to protein denaturation treatment" also includes the case where "removal of the branches" and "closing the portion by subjecting it to protein denaturation treatment" are carried out simultaneously.

[0030] The frequency when performing the treatment using ultrasonic waves as described above is preferably 20 kHz to 100 kHz, more preferably about 30 kHz to about 60 kHz. The output is preferably 50 to 500 mA, more preferably 100 to 400 mA, and even more preferably 200 to 300 mA (when using 100 VAC).

[0031] The time for which ultrasonic treatment is carried out varies depending on the frequency and output and is not particularly limited, but is preferably 0.1 seconds to 10 minutes, more preferably 1 second to 5 minutes, and further preferably 3 to 60 seconds.

[0032] Next, the present invention (a) harvesting a branched blood vessel from a vertebrate (donor); (b) decellularizing the blood vessel; and (c) A process of gluing and closing the excised branched parts by protein denaturation treatment. The order of each step in the method for producing a decellularized material for transplantation, including the steps described above, will be described.

[0033] In this embodiment, the steps (a), (b) and (c) are not limited to the above order. For example, in addition to carrying out steps (a), (b) and (c) in this order, step (c) may be carried out after step (a), and then step (b). In another embodiment, the branched portion of a blood vessel having branches may be excised before harvesting from a donor vertebrate, and the portion from which the branch has been excised may be subjected to a protein denaturation treatment and harvested from the donor, after which decellularization in step (b) may be carried out. In other words, embodiments including the steps (c), (a) and (b) in this order are also encompassed by the present invention.

[0034] In another embodiment, when the branched portion of the blood vessel having branches collected in step (a) is present with a sufficient length (for example, the length of the branched portion is 5 mm to several cm), the method may further include a step (d) of excising the branched portion. There is no particular limitation on when to perform this step (d). For example, it is possible to carry out the steps in the order of (a), (d), (b) and (c), or in the order of (a), (b), (d) and (c). In the present invention, in terms of being able to close the branched blood vessels without overlooking them and being able to work more efficiently, it is more preferable to simultaneously perform "removal of the branches" and "closing the blood vessels by protein denaturation treatment" in step (c) and to perform steps (c) and (a) consecutively. Even in this case, step (c) may be performed again before and after step (b).

[0035] The present invention also provides a graft composition comprising a biocompatible material including a decellularized transplant material that is made from a blood vessel from a vertebrate (donor) whose branches have been excised and has at least one excised branch site that has been closed by protein denaturation.

[0036] The vertebrate in the graft composition of the present invention is not particularly limited, but since blood vessels are preferably easily available, it is preferable that the vertebrate is an animal other than human, and in particular, livestock mammals and livestock birds are preferable. The livestock mammals and livestock birds used in the method for producing a decellularized material for transplantation of the present invention are the same as the livestock listed as specific examples of livestock mammals and livestock birds used in the method for producing a decellularized material for transplantation of the present invention. Among these, pigs, rabbits, and cows are preferable in terms of stable availability.

[0037] In the graft composition of the present invention, a "blood vessel from which branches have been removed" refers to a blood vessel obtained by removing the branched portion from a blood vessel having branches used to produce the decellularized material for transplantation of the present invention. As the blood vessel having branches, the same blood vessels as those used in the above-mentioned production method can be used, and in consideration of the physical properties (elongation, biocompatibility, strength, etc.) as a graft composition, an artery is preferred, and the internal thoracic artery is more preferred.

[0038] The decellularized transplant material used in the graft composition of the present invention has a protein denatured portion. Specifically, the resected portion of the branch portion is in an open tube state, and the open tube portion can be sealed by carrying out a protein denaturation treatment. The specific method of protein denaturation is not particularly limited, as in the case of the above-mentioned decellularized material for transplantation, but an ultrasonic scalpel or an electric scalpel can be used. It is preferable to use an ultrasonic scalpel because it causes less damage to the tissue.

[0039] In the graft composition of the present invention, the conditions regarding the ultrasonic frequency and treatment time for denaturing proteins are the same as the conditions regarding the ultrasonic frequency and treatment time in the above-mentioned method for producing a decellularized material for transplantation.

[0040] For the decellularized transplant material used in the graft composition of the present invention, the decellularization treatment can be the same as the decellularization treatment described above, and it is preferable to carry out the decellularization treatment by high hydrostatic pressure treatment. The pressure during the high hydrostatic pressure treatment may be such that cells and pathogens derived from a vertebrate donor are destroyed, and the treatment may be carried out under the same conditions as the above-mentioned hydrostatic pressure conditions. Examples of the medium used for applying the high hydrostatic pressure include water, physiological saline, a buffer solution, propylene glycol or an aqueous solution thereof, glycerin or an aqueous solution thereof, and an aqueous saccharide solution. Specific examples of each solvent include the same solvents as those listed above.

[0041] The graft composition of the present invention is composed of specific biocompatible materials, where "biocompatible" means that the graft is accepted by the recipient tissue and does not induce toxicity or significant immune rejection.

[0042] There are no particular limitations on the biocompatible material so long as it is solid and does not have fluidity, and examples of the biocompatible material include non-resorbable polymers, absorbable polymers, metals, glasses, and ceramics.

[0043] The non-resorbable polymers include, but are not limited to, polyethylene, polyethylene terephthalate, polybutylene, polybutylene terephthalate, polypropylene, acrylic, polyamide-imide, polyetheretherketone, polyaryletherketone, polycarbonate, polyamide, polyvinyl fluoride, polyvinylidene fluoride, polymethyl methacrylate, and combinations and equivalents thereof.

[0044] The absorbable polymers may be synthetic or natural polymers, and include polyamino acids, polyamides, fatty acid polyesters, and natural polymers include collagen, elastin, hyaluronic acid, laminin, and gelatin, keratin, chondroitin sulfate, and decellularized tissue.

[0045] Examples of the metals include tantalum, tantalum alloys, stainless steel, titanium, titanium alloys, cobalt-chromium alloys, and the like. Biocompatible metals that have been conventionally used in medical devices and the like can be used.

[0046] Such glasses or ceramics include, but are not limited to, phosphates such as tetracalcium phosphate, alpha- and beta-tricalcium phosphate, octacalcium phosphate, hydroxyapatite, substituted apatite, monetite, metaphosphates, pyrophosphates, phosphate glasses, calcium and magnesium carbonates, sulfates, and oxides, and combinations thereof.

[0047] The graft composition of the present invention is made of a biocompatible material that at least partially contains a specific decellularized material for transplantation. Here, "at least partially" means that the decellularized material for transplantation may be present in one or more of the biocompatible material. It is more preferable that the biocompatible material is made of only the decellularized material, since this allows more effective infiltration of autologous cells.

[0048] The graft composition of the present invention is capable of functioning as a part of living tissue after transplantation, in particular as a vascular substitute for transplantation. EXAMPLES

[0049] 1. Preparation of Decellularized Porcine Internal Thoracic Artery Harvesting of porcine internal thoracic arteries (processing of branched blood vessels) The thorax of a 3-4 month old edible pig (SPF (specific pathogen free) pig, female, weighing approximately 50 kg) was opened under anesthesia to expose the internal thoracic artery. The ultrasonic scalpel (Harmonic Scalpel II, Harmonic SYNERGY, Ethicon Endosurgery) was set at 55,500 Hz and output level 2, and the internal thoracic artery was dissected (harvested) from the chest wall while contacting and coagulating the branching blood vessels. Four internal mammary arteries were harvested from two pigs.

[0050] 2. Observation of the porcine internal thoracic artery The length and inner diameter of the four collected porcine internal thoracic arteries were measured. (1) Length: 140 mm, inner diameter: 3.2 mm, (2) Length: 135 mm, inner diameter: 2.6 mm, (3) Length: 95 mm, inner diameter: 2.9 mm, (4) Length: 105 mm, inner diameter: 2.8 mm. The obtained porcine internal thoracic arteries (1) to (4) are shown in Figure 2. Their cross-sectional views (HE staining) are shown in Figure 3.

[0051] 3. Decellularization of porcine internal thoracic arteries [Reagents used] ·DNs solution Otsuka saline solution containing DNase I (Roche, Grade II) 50 mg / L and MgCl2·6H2O (Wako, special grade reagent) 2.55 g / L ·EtOH solution A solution of 80% by volume of official ethanol and Otsuka saline. Citric acid solution A solution prepared by adding trisodium citrate dihydrate (Calbiochem, Molecular Biology Grade) and anhydrous citric acid (Wako, special grade) to Otsuka saline to adjust the pH to 7.4.

[0052] The above porcine internal thoracic artery was treated with an ultra-high hydrostatic pressure device (Dr. Chef, manufactured by Kobe Steel, Ltd.) using water as a medium. The ultra-high hydrostatic pressure treatment was performed under the following conditions: applied pressure 600 MPa, pressure rise time 9 minutes, pressure maintenance time 120 minutes, pressure drop time 9 minutes, and pressure medium temperature 30°C. After ultra-high pressure treatment, the tissue was washed with 50 mg / L DNase I solution for 4 days, EtOH solution for 3 days, and citric acid solution for 4 days to complete decellularization.

[0053] Decellularization was performed by staining with hematoxylin and eosin (hereinafter sometimes referred to as HE staining) and confirming under a microscope that there was no nucleic acid in the HE-stained section image. HE staining is a method that uses two dyes, hematoxylin and eosin, to stain cell nuclei and other tissues and components separately. Hematoxylin stains nuclei blue, and eosin stains cytoplasm, fibers, and red blood cells pink.

[0054] 4. Pressure resistance measurement After decellularization, one end of each porcine internal thoracic artery was clamped and saline was pumped from the other side using a 20 mL syringe to measure the pressure resistance. The pressure resistance of the decellularized transplant materials using porcine internal thoracic arteries (1), (2), and (4), excluding (3), was 150 mmHg or more. In addition, in the case where part of the branch was not blocked (3), leakage of saline was confirmed. The pressure resistance was measured by pumping saline from a 20 mL syringe into the porcine internal thoracic artery at a rate of 3 mL / min using a syringe pump (YSP-101), and measuring the pressure at that time with a digital pressure gauge (KDM30). In the following experiments, (1), (2), and (4) are examples, and (3) is a comparative example.

[0055] 5. Suturing of blood vessels in comparison (3) For the decellularized material for transplantation, the excised branch blood vessel where saline leakage was confirmed was ligated with 6-0 proline thread. The ligation was performed by passing the suture through the adventitia of the blood vessel, and then tying up the tubular part of the branch from the outside.

[0056] 6. Long-term transplantation of decellularized porcine internal thoracic artery (graft composition composed of decellularized materials for transplantation) into rabbit aorta ·Transplant method Pentobarbital sodium hydrochloride (trade name: Somnopentyl (registered trademark) 0.2 ml), saline (1.8 ml / rabbit), and medetomidine hydrochloride (trade name: Dolbene injection 1 ml) were subcutaneously injected into a rabbit (breed: Japanese white rabbit, 5 months old, male) weighing 3.70-4.00 kg. After confirming immobilization, the abdominal skin was shaved. Isoflurane was inhaled as an exhalation anesthesia (2% at induction, 1% at surgery), and the rabbit was restrained on a restraining table, after which the skin was disinfected with gauze soaked in a povidone-iodine preparation. Subsequently, saline 3-fold diluted lidocaine injection (trade name: Xylocaine (registered trademark)) was injected subcutaneously into the abdomen, and an intravenous line was inserted into the ear vein to administer Solita (registered trademark) T-3 intravenously. Using an electric scalpel and scissors, the skin and peritoneum were incised to expose the abdominal aorta, and 1000 units of heparin (1000 units / mL heparin 1mL) was administered intravenously through the infusion line. After confirming that 3 minutes had passed since the heparin administration, the central side of the abdominal aorta was blocked. After inserting a shunt tube into the abdominal aorta, the blood flow was released. While securing blood flow with the shunt tube, the decellularized porcine internal thoracic artery and the central part of the abdominal aorta were anastomosed using an end-to-side anastomosis method using 8-0 proline thread. Next, the decellularized porcine internal thoracic artery and the peripheral part of the abdominal aorta were anastomosed in the same manner to create a bypass vessel. In addition, the abdominal aorta between the central anastomosis and the peripheral anastomosis was ligated with 6-0 proline thread to direct blood flow to the bypass vessel. After transplantation of the decellularized porcine internal thoracic artery, the peritoneum was sutured with 4-0 PDS thread, the muscle layer with 2-0 PDS thread, and the skin with 4-0 PDS thread to close the abdomen. 0.5 mL of Baytril (registered trademark) was administered as an antibiotic, and atipamezole hydrochloride (trade name: Atipame injection 1 mL) was injected subcutaneously in the thigh as a medetomidine hydrochloride antagonist. After confirming that the rabbit was awake, the rabbit was transferred to a breeding cage.

[0057] ·autopsy Rabbits were autopsied one or three months after transplantation of the decellularized porcine internal thoracic artery. The rabbits were not removed from the infusion line, and the abdominal aorta (transplanted decellularized porcine internal thoracic artery) was exposed in the same manner as at the time of transplantation. Then, vascular patency evaluation and thrombus formation evaluation were performed. The vascular patency evaluation was performed by cutting the peripheral side of the abdominal aorta and judging whether or not blood was released. Blood release was confirmed in all samples, indicating that the blood vessels were patent. After the vascular patency evaluation, sodium pentobarbital (Somnopentyl (registered trademark) 5 mL) was injected into the ear vein and sacrificed. After arresting the heart and breathing, the decellularized porcine internal thoracic artery was removed. The inside of the decellularized porcine internal thoracic artery was washed with saline, then cut in the longitudinal direction and visually observed for thrombus formation. There was no adhesion of thrombus to the inner surface of the decellularized porcine internal thoracic artery, indicating that it has high antithrombotic properties. The term "patent" refers to the state in which the lumen of the blood vessel is not blocked and remains hollow.

[0058] -Pathological specimen preparation Pathological specimens of the transplanted decellularized porcine internal thoracic artery were prepared by Shin Histoscience Institute, Inc. At this time, HE-stained sections and Elastica van Gieson staining (hereinafter sometimes referred to as EVG staining) were performed on the decellularized porcine internal thoracic artery in the area where the tissue structure of the branched blood vessel part occluded with an ultrasonic scalpel or 6-0 proline thread could be confirmed, and EVG-stained sections were prepared. Specifically, continuous sections were prepared in the longitudinal direction of the blood vessel at a thickness of approximately 5 μm, and sections were prepared in the area where the tissue structure of the branched blood vessel part occluded with an ultrasonic scalpel or 6-0 proline thread could be confirmed.

[0059] EVG staining is a staining method for identifying elastic fibers, and can stain elastin in connective tissue black-purple and collagen reddish-purple.

[0060] The HE stained image and EVG stained image (3 months) of the example sample and the HE stained image and EVG stained image (3 months) of the comparative sample are shown below (Figure 4). In the example sample, it can be seen that cells have infiltrated into the decellularized porcine internal thoracic artery tissue part stained black purple by EVG staining. On the other hand, in the comparative sample, no cell infiltration was observed in the decellularized porcine internal thoracic artery tissue part stained black purple by EVG staining. Furthermore, in the example sample, one month after transplantation, the steps of the branching blood vessels were found to be covered with rabbit cells (FIG. 5).

[0061] 7. From block meat derived from distributed ration pigs, the internal thoracic arteries were dissected (harvested) from the chest wall while contacting and coagulating the branched blood vessels with an ultrasonic scalpel (EES generator, Harmonic SYNERGY, Harmonic FOCUS+, <using blue handpiece>, Ethicon Endosurgery) set at 55,500 Hz, output level 5 and 2. The coagulation of the branched blood vessels was performed at a position 3-5 mm away from the branched part of the blood vessel, and five internal thoracic arteries were dissected (harvested). After decellularization treatment was performed by the method described in "3" above, the pressure resistance was measured by the method described in "4" above. As a result, it was confirmed that the pressure resistance of all five samples was 250 mmHg or more. [Explanation of symbols]

[0062] 1: Blood vessel with branches, 2: Branched portion (branched blood vessel), 3: Protein denaturation portion

Claims

1. (a) harvesting a blood vessel having branches from a non-human vertebrate (donor); (b) decellularizing the blood vessel; and (c) A step of gluing and closing the portions from which the branches have been removed by protein denaturation treatment. A method for producing a decellularized material for transplantation, comprising: The protein denaturation treatment is an ultrasonic vibration treatment, A method for producing a decellularized material for transplantation, comprising the steps of: (a) measuring the pressure resistance of the blood vessel by closing the portion where the branch has been removed; and (c) confirming that the pressure resistance is 150 mmHg or more and therefore that there is no leakage from the closed portion.

2. The method for producing a decellularized material for transplantation according to claim 1 , wherein the decellularization in step (b) comprises ultra-high hydrostatic pressure treatment.

3. A graft composition comprising a biocompatible material including a decellularized material for transplantation, the decellularized material comprising a blood vessel from a vertebrate (donor) in which a branch has been removed, the blood vessel having at least one branch-removed section that has been closed by protein denaturation caused by ultrasonic vibration, and having a pressure resistance strength of 150 mmHg or more.

4. The graft composition of claim 3 , wherein the blood vessel comprises an internal mammary artery.

5. A graft composition comprising decellularized tissue of a blood vessel including a porcine internal thoracic artery, having a branch excision section that has been closed by protein denaturation using ultrasonic vibration, and having a pressure resistance strength of 150 mmHg or more.

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