WO2006137546A1 - Treatment method for preventing transplantation tissue with biological origin from calcification and tissue treated thereby - Google Patents

Treatment method for preventing transplantation tissue with biological origin from calcification and tissue treated thereby Download PDF

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Publication number
WO2006137546A1
WO2006137546A1 PCT/JP2006/312662 JP2006312662W WO2006137546A1 WO 2006137546 A1 WO2006137546 A1 WO 2006137546A1 JP 2006312662 W JP2006312662 W JP 2006312662W WO 2006137546 A1 WO2006137546 A1 WO 2006137546A1
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WIPO (PCT)
Prior art keywords
tissue
group
biological tissue
adhesion
biological
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PCT/JP2006/312662
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French (fr)
Japanese (ja)
Inventor
Satoshi Taketani
Eiichiro Uchimura
Masayuki Hara
Masakazu Furuta
Tadahiro Matsumoto
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Cardio Incorporated
Osaka Prefecture University Public Corporation
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Publication of WO2006137546A1 publication Critical patent/WO2006137546A1/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
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
    • 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
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/02—Treatment of implants to prevent calcification or mineralisation in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/10—X-ray therapy; Gamma-ray therapy; Particle-irradiation therapy

Definitions

  • the present invention relates to a method and system for strengthening an isolated biological tissue (untreated tissue), and a medicine and treatment method using a tissue, tissue graft, and the like prepared by such a strengthening method.
  • the present invention relates to a treatment method and a treated tissue for suppressing calcification of a living tissue-derived transplant tissue.
  • the present invention also relates to an anti-adhesion membrane for use at a site where there is a risk of adhesion, which is a thread and weave that has undergone surgery or treatment. Specifically, the present invention relates to the prevention of adhesions that occur between the surgical or surgical cut surface or defect site and the surrounding tissue.
  • a major obstacle in using foreign tissues for organ transplantation is immune rejection. Changes that occurred in allografts (or allografts) and xenografts were first described more than 90 years ago (Non-Patent Documents 1-2 and 4-5) ). Arterial graft rejection pathologically results in either graft expansion (leading to rupture) or occlusion. In the former case, it is said to be caused by the degradation of the extracellular matrix, while the latter is caused by proliferation of intravascular cells (Non-patent Document 6).
  • Non-Patent Document 7 reduced the immune response of the host
  • Non-Patent Document 8 attempted to reduce the antigenicity of the allograft or xenograft mainly by cross-linking
  • Non-patent document 9 attempted to reduce the antigenicity of the allograft or xenograft mainly by cross-linking
  • Cross-linking of the extracellular matrix decreases the antigenicity of the graft, but the biomechanical function (Non-patent Document 11; and Non-patent Document 12) changes and becomes sensitive to mineralization (Non-patent Document). 13).
  • Cardiovascular diseases including coronary and peripheral vascular diseases
  • are being treated by surgical replacement therapy the number of cases of cardiovascular disease is increasing around the world recently.
  • it is difficult to apply replacement therapy if the diameter is small, bypassed surgery uses autologous veins or arterial grafts (18-20).
  • Appropriate vascular prosthesis is needed for thin blood vessels, but development towards the development of artificial materials was made to reduce the use of autografts or allografts. Limb peripheral and coronary movement Neither is it necessary to construct small diameter arteries (less than 6mm)!
  • Non-patent Document 21 discloses that cross-linking techniques have also been investigated to stabilize the collagen-based structure of tissues and have been found to be an ideal procedure.
  • Non-Patent Document 22 As an alternative to natural tissue grafts, attempts have been made to produce a completely acellular tissue matrix. This production is lime By specifically removing cellular components that are thought to promote immunogenicity and elicit an immunological response. These decellularization techniques include removal of cellular components by chemical, enzymatic, and mechanical means. This treatment leaves a material that is also essentially composed of extracellular matrix component forces. These decellularized tissues retain their natural mechanical properties and promote regeneration.
  • Non-Patent Document 24 Non-Patent Document 25; Non-Patent Document.
  • Reference 26 Non-patent reference 27.
  • Removal of lipids from rabbit pericardium by treatment with either black mouth form Z methanol or sodium dodecyl sulfate (SDS) reduced tissue calcification in a rat model (28).
  • SDS sodium dodecyl sulfate
  • EPC endothelial progenitor cells
  • examples of the polymerization reaction include: A: a method in which a vinyl monomer, a crosslinking monomer, and a photopolymerization initiator are mixed and then UV irradiation; and B: a nucleophilic group such as an amino group or a thiol group. And a method of nucleophilic reaction between a polymer such as PEG and a polymer such as ⁇ ,
  • a polymer such as PEG
  • a polymer such as ⁇ ,
  • Patent Document 4 requires a free radical initiator (a polymerization initiator that generates free radicals). As a general rule, if these are left in the body, they may be toxic and may cause problems if transplanted in vivo.
  • Adhesion In a surgical operation or treatment, if an affected part, an organ or an organ is removed, or a damaged part is repaired, adhesion may occur at the part. Adhesion can be a major problem in surgery and treatment, as it can lead to organ dysfunction and can lead to inferior prognostic forces or death. In order to eliminate such inconveniences, it is necessary to isolate the cut surface or defect of the organ from other tissues that may come into contact with each other to prevent adhesion. In order to solve this problem, an anti-adhesion film using a synthetic polymer or biopolymer as a material has been developed (Non-Patent Documents 30 to 36).
  • Synthetic polymers that have been conventionally used for anti-adhesion membranes include polyethylene (Non-patent document 30), silicon (Non-patent document 31), cellulose (Non-patent document 32), polytetrafluoroethylene. Ren (for example, Goatex (registered trademark) (Japan Goatex Co., Ltd.), etc. These synthetic polymers cause excessive calcification and inflammatory reaction with low biocompatibility There is a problem.
  • biopolymers that have been used for adhesion-preventing membranes include collagen membranes (Patent Literature 5, Non-Patent Literatures 33 to 35) and hyaluronic acid (Patent Literature 6, Patent Literature 7 and Non-Patent Literature 36). )and so on.
  • These biopolymers 1) cannot completely prevent adhesion, and 2) natural collagen contains various cell adhesion sites, which may cause unnecessary cell migration or adhesion depending on the application. Inevitable! /, 3) Platelet adheres to cause platelet aggregation, and 4) Hageman factor (blood coagulation factor VII) is activated and blood coagulation is promoted. There are challenges.
  • an anti-adhesion membrane capable of preventing adhesion without causing adverse reactions such as calcification, inflammatory reaction, unnecessary cell migration or adhesion, platelet aggregation, and promotion of blood coagulation is required. It becomes.
  • Patent Document 1 JP 2002-543950 A
  • Patent Document 2 Japanese Patent Laid-Open No. 2001-78750
  • Patent Literature 3 Pamphlet of International Publication No. 89Z05371
  • Patent Document 4 International Publication No. 2005Z042044 Pamphlet
  • Patent Document 5 JP 2000-93497 A
  • Patent Document 6 JP 2000-271207 A
  • Patent Document 7 Japanese Unexamined Patent Publication No. 2000-210376
  • Non-Patent Document l Carrel A., 1907, J Exp Med 9: 226— 8
  • Non-Patent Document 2 Carrel A., 1912., J Exp Med 9: 389—92
  • Non-Patent Literature 3 Toshiharu Niioka, Yasuharu Imai, Kazuhiro Seo et al .; Development and application of cardiovascular materials by Tessh Engineering. Japan Cardiovascular Society 2000; 29: 38
  • Non-Patent Document 4 Calne RY., 1970, Transplant Proc 2: 550
  • Non-Patent Document 5 Auchinclossl988, Transplantation 46: 1
  • Non-Patent Document 6 Uretsky BF, Mulari S, Reddy S, et al., 1987, Circulatio n 76: 827-34
  • Non-Patent Document 7 Schmitz—RixenT, Megerman J, Colvin RB, Williams AM, Abbot W., 1988, J Vase Surg 7: 82—92
  • Non-Patent Document 8 Plissonnier D, et al., 1993, Arteriosclerosis Thromb 13: 112-9
  • Non-Patent Document 9 Rosenberg N, et al., 1956, Surg Forum 6: 242-6
  • Non-Patent Document 10 Dumont C, Pissonnier D, Michel JB., 1993, J Surg Res 54: 61-69
  • Non-patent literature ll Cosgrove DM, Lytle BW, Golding CC, et al., 1983, J T home CardiovascSurgery 64: 172-176
  • Non-Patent Document 12 BroomN, Christie GW., 1982, In: Cohn LH, Gallucci V, editors. Ardiac bioprostheses: Proceedings of the Second International al Symposium. New York: York Medical Books Pages 476—491
  • Non-Patent Document 13 Schoen FJ, Levy RJ, Piehler HR., Cardiovasc Pathology
  • Non-Patent Document 14 J Thorac Cardiovasc Surg 1998; 115; 536- 46
  • Non-Patent Document 15 Malone JM, Brendel K, Duhamel RC, Reinert RL., 1984, J Vase Surgl: 181-91
  • Non-Patent Document 16 Lalka SG, OelkerLM, and Malone JM, et al., 1989, An n Vase Surg 3: 108—17
  • Non-Patent Document 17 O'Brien MF, etal., 1999 (October), Seminars in Thorac and Cardiovasc Surg; 111 (4), Suppl 1: 194- 200
  • Non-Patent Document 18 Canver CC., 1995, Chest 1995; 108 1150-1155
  • Non-Patent Document 19 Barner HB., 1998, Ann Thorac Surg 66 (Suppl 5) S2— 5; discussion S25— 28
  • Non-Patent Document 20 Bhan A, GuptaV, Choudhary SK, etal., 1999, Ann Thor ac Surg 1999; 67 1631-1636
  • Non-Patent Document 21 Hilbert SL, Ferrans VJ, Jone M., 1988, Med Prog Tec hnol 89; 14, 115—163
  • Non-Patent Document 22 Rao KP, Shanthi C., 1999, Biomatrials Appl 13: 238-
  • Non-Patent Document 23 Grabenwoger M, Sider J, Fitzal F, et al., 1996, Ann T home Surg 62; 772—777
  • Non-Patent Document 24 Valente M, Bortolotti U, Thiene G,, 1985, Am J Patho 1 119, 12-21
  • Non-Patent Document 25 Maranto AR, Shoen FJ, 1988, ASAIO Trans 34, 827— 8 30
  • Non-Patent Document 26 Courtman DW, Pereira CA, Kashef V, McComb D, Lee-like Wilson GL, 1994, J Biomed Mater Res 28: 655—666
  • Non-Patent Document 27 Levy RJ, Schoen FJ, Anderson HC, et al., 1991, Bioma terials 12: 707-714
  • Non-Patent Document 28 Jorge—Herrero E, Fernandez P, Gutierrez MP, Castillo-Olivares JL, 1991, Biomaterials 12: 683— 689
  • Non-Patent Document 29 Sunjay Kaushal, Gilad E. Amiel, Kristine J. Guleserian, et al. 2001, Nature Medicine Vol. 9, 1035— 1040
  • Non-Patent Document 30 Stark, H. H., 1977, J. Bone & Joint Surg., 59 A, 908
  • Non-Patent Document 31 Helal, B., 1973, Hand, 5, 85—90
  • Non-Patent Document 32 Ashley, F. L. et al., 1959, Plast. Reconst. Surg., 23, 52 6-534
  • Non-patent document 33 Mitsutoshi Okui, Journal of Nikkeikai, 1989, 5, 1138—
  • Non-patent literature 34 Muggli, R. et al., Throm. Res., 1973, 3, 715-728
  • Non-patent literature 35 Wilner, GD et al., 1968, J. Clin. Invest., 47, 2608— 26
  • Non-Patent Document 36 Hiroshi Aso, 1958, Nichiga Hoho, 22, 310
  • an object of the present invention is to provide a tissue for transplantation that solves problems in transplantation such as calcification.
  • the present inventors have found that by including a biocompatible polymer in an untreated tissue and exposing it to ⁇ -ray irradiation, the tissue was unexpectedly strengthened and calcification was suppressed. The above issues have been solved. The inventors have also found an unexpectedly strong reinforcing effect by exposing the tissue to a biocompatible polymer and to gamma radiation. Conventionally, it has been shown by the present invention that force ⁇ -irradiation, which has been recognized that decellularization of a tissue derived from a living body is a key point for suppressing calcification, can achieve an effect more than decellularization.
  • the living body-derived tissue of the present invention can be used as, for example, a permanent artificial blood vessel. Thus, it can be said that the usefulness of the biological tissue of the present invention is extensive.
  • the strength of the biological tissue reinforced by the present invention was also significantly improved unexpectedly. Such an effect is a powerful and unexpected effect that could never be achieved with the prior art, and it was possible to provide such a reinforced biological tissue and tissue graft! / This is a significant advance in transplantation medicine.
  • the present invention provides the following.
  • a biological tissue containing a biocompatible polymer and receiving y-ray irradiation (Item 2) Item 2. The living tissue according to item 1, wherein the living tissue is not decellularized.
  • Item 2 The biological tissue according to Item 1, wherein the extracellular matrix component is at least partially crosslinked by a covalent bond.
  • Item 4 The living tissue according to Item 3, wherein the extracellular matrix component is selected from the group consisting of collagen, elastin, laminin, fibronectin, tenascin, glycosaminodarlican and proteodarican power.
  • Item 2 The biological tissue according to Item 1, wherein the biocompatible polymer coats the biological tissue.
  • Item 2 The biological tissue according to Item 1, wherein the biocompatible polymer is crosslinked to the biological tissue.
  • Item 2 The biological tissue according to Item 1, wherein the biocompatible polymer is biodegradable.
  • the biocompatible polymer may be polybulal alcohol (PVA), polybulurpyrrolidone (PV P), elastin, polyethylene glycol (PEG), gelatin, collagen, ⁇ -polyglutamic acid and a mixture of two or more thereof.
  • PVA polybulal alcohol
  • PV P polybulurpyrrolidone
  • PEG polyethylene glycol
  • gelatin collagen, ⁇ -polyglutamic acid and a mixture of two or more thereof.
  • Item 2 The biological tissue according to Item 1, comprising a polymer selected from the group.
  • Item 2 The biological tissue according to Item 1, wherein the biocompatible polymer comprises polyethylene glycol (PEG).
  • Item 12 The biological tissue according to Item 11, wherein the polyethylene glycol (PEG) is in a molecular weight range of 1,000 to 200,000.
  • PEG polyethylene glycol
  • the biological tissue according to Item 11 wherein the polyethylene glycol (PEG) is in the range of molecular weight 8,000 force to 50,000.
  • PEG polyethylene glycol
  • Item 2 The living tissue according to Item 1, wherein the living tissue has tissue strength that can be clinically applied.
  • Item 2 The biological tissue according to Item 1, wherein the biocompatible polymer is randomly crosslinked.
  • Item 2 The living tissue according to Item 1, wherein the living tissue is a membranous tissue, a valve-like tissue, or a tubular tissue.
  • Item 2 The living tissue according to Item 1, wherein the living tissue is a tissue selected from blood vessels, blood vessel-like tissues, heart valves, pericardium, dura mater, cornea and bone.
  • Item 2 The living tissue according to Item 1, wherein the living tissue is derived from a mammal.
  • a tissue graft for transplantation comprising the living tissue according to item 1.
  • Item 22 The tissue graft according to Item 21, wherein the tissue graft has a shape selected from the group consisting of a membrane shape, a tubular shape, and a valve shape.
  • a method for producing a tissue for transplantation comprising:
  • Item 24 The method according to Item 23, wherein the gamma irradiation is performed under conditions in which a chemical reaction occurs in the biocompatible polymer.
  • Item 24 The method according to Item 23, wherein the gamma irradiation is performed in a range of 0.5 to 240 hours.
  • the biocompatible polymer includes a polymer selected from the group consisting of polybulal alcohol, polybulurpyrrolidone, elastin, polyethylene glycol, gelatin, collagen, ⁇ -polyglutamic acid and a mixture of two or more thereof.
  • Item 3 The method according to Item 31, wherein the positive ethylene glycolate has a molecular weight in the range of 1,000 to 200,000.
  • Item 24 The method according to Item 23, wherein the biocompatible polymer is used at a concentration of 1% (w / v) to 50% (w / v).
  • the biological tissue is a tissue selected from the group consisting of blood vessels, blood vessel-like tissues, heart valves, pericardium, dura mater, corneas and bones.
  • Item 24 The method according to Item 23, wherein the biological tissue is derived from a mammal.
  • Item 24 The method according to Item 23, wherein the biological tissue is derived from human, ushi or pig. (Item 37)
  • the biological tissue is a tissue selected from the group consisting of blood vessels, blood vessel-like tissues, heart valves, pericardia, dura mater, corneas, and bones.
  • a method of producing a yarn and woven graft comprising:
  • the biological tissue is a tissue selected from the group consisting of blood vessels, blood vessel-like tissues, heart valves, pericardium, dura mater, corneas, and bones.
  • a method of treating or preventing a force requiring tissue or organ transplantation or a subject at risk is provided.
  • the biological tissue is a tissue selected from blood vessels, blood vessel-like tissues, heart valves, pericardia, dura mater, corneas and bones.
  • Item 52 The medicament according to Item 51, wherein the biological tissue is a tissue selected from blood vessels, blood vessel-like tissues, heart valves, pericardia, dura mater, corneas and bones.
  • the biological tissue is a tissue selected from blood vessels, blood vessel-like tissues, heart valves, pericardia, dura mater, corneas and bones.
  • Item 2 The biological tissue according to Item 1, further characterized by being chemically crosslinked.
  • the bond is a bond formed between groups selected from the group consisting of sulfhydryl group, aldehyde group, carbo group, sulfo group and -tro group, carbon-carbon bond, peptide bond, ether bond and ester bond strength.
  • the biological tissue according to Item 58 which is selected from the group consisting of a binding force selected from the group consisting of
  • the bifunctional molecular crosslinking agent is selected from the group consisting of daltaraldehyde, cyanimide, 1-ethyl-1- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), epoxy (poly (glycidyl ether)), 2 , 6 bis (4 azidobenzylidene) -4-methylcyclohexanone, 4, 4 'diazide diphenyl ether, 4, 4' diazide diphenyl sulfone, 4, 4 'diazide diphenyl acetone, 4, 4' diazide 59.
  • the biological tissue according to Item 58 which is selected from the group consisting of diphenylmethane, 1,4 bis ( ⁇ diazobenzyl), and 4- [ ⁇ azidosalicyamido] ptylamine.
  • the biological tissue is chemically treated with dartal aldehyde, exposed to polyethylene glycol (PEG), and irradiated with ⁇ rays to form crosslinks.
  • PEG polyethylene glycol
  • Item 2 The biological tissue according to Item 1, having at least one crosslink formed between non-chemical crosslinkable groups other than a carboxyl group, a hydroxyl group and an amino group.
  • Item 65 The biological tissue according to Item 64, wherein the non-chemical crosslinking group is selected from the group consisting of a sulfhydryl group, an aldehyde group, a carbonyl group, a sulfo group, and a -tro group force.
  • Item 2 The biological tissue according to Item 1, having at least one bridge formed by ⁇ -ray irradiation and formed by a bond selected from the group consisting of a carbon-carbon bond, an ether bond and an ester bond.
  • Item 2 The biological tissue according to Item 1, which has at least one crosslink formed by a new bond formed by cleavage of the skeleton.
  • Item 2 The biological tissue according to Item 1, comprising at least one non-chemical crosslink formed between amino acids having no free amino group and no carboxyl group in the side chain.
  • Item 2 The biological tissue according to Item 1, having at least one crosslink formed between polysaccharides having non-chemical crosslinkable groups other than a carboxyl group, a hydroxyl group and an amino group.
  • Item 70 The item 70, wherein the polysaccharide having a non-chemical crosslinking group has a bridge formed between at least one group selected from the group consisting of a sulfhydryl group, an aldehyde group, a carbonyl group, a sulfo group, and a -tro group. Living body tissue. (Item 72)
  • Item 2 The biological tissue according to Item 1, comprising at least one non-chemical crosslink other than bisepoxide crosslink, divinylsulfone crosslink, intramolecular esterification, glutaraldehyde crosslink, carpositimide crosslink and hydrazide crosslink.
  • Item 2 The biological tissue according to Item 1, which is used for preventing adhesion between tissues.
  • Item 2 The biological tissue according to Item 1, for use in vivo.
  • Item 2 The biological tissue according to Item 1, for use in vitro.
  • a method for producing an anti-adhesion membrane for transplantation comprising:
  • a method for producing an adhesion-preventing membrane for transplant according to item 76 comprising:
  • a method further comprising the step of chemically crosslinking the biological membrane with a bifunctional molecular crosslinking agent.
  • step D) is carried out between at least one selected from the group consisting of step ii) step-step ii) and step ii) step-step ii). .
  • the bond is a bond formed between a group selected from the group consisting of a sulfhydryl group, an aldehyde group, a carbo group, a sulfo group, and a -tro group, a carbon-carbon bond, and a peptide bond.
  • a bond strength selected from the group consisting of an ether bond and an ester bond strength.
  • bifunctional molecular crosslinking agent has a functional group selected from the group consisting of an amino group, a carboxyl group, and an aldehyde group.
  • the bifunctional molecular crosslinking agent is selected from the group consisting of daltaraldehyde, cyanimide, 1-ethyl-1- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), epoxy (poly (glycidyl ether)), 2 , 6 bis (4 azidobenzylidene) -4-methylcyclohexanone, 4, 4 'diazide diphenyl ether, 4, 4' diazide diphenyl sulfone, 4, 4 'diazide diphenyl acetone, 4, 4' diazide 80.
  • the method of item 79, wherein the compound is also selected from the group consisting of diphenylmethane, 1,4 bis ( ⁇ diazobenzyl), and 4- [ ⁇ azidosalicyamido] plutamine.
  • a method according to item 82, wherein the bifunctional molecular crosslinking agent is dartalaldehyde.
  • the biocompatible polymer includes a polymer selected from the group consisting of polybulal alcohol, polybulurpyrrolidone, elastin, polyethylene glycol, gelatin, collagen, ⁇ -polyglutamic acid, and a mixture of two or more thereof. 76. The method according to 76. (Item 85)
  • the biological membrane is collected from a membranous tissue, a valve-like tissue or a tubular tissue.
  • the biological membrane is collected from a blood vessel, a blood vessel-like tissue, a heart valve, a pericardium, a dura mater, a cornea, and a bone.
  • Item 7 The method according to Item 76, wherein in the step A), the adhesion-preventing membrane is derived from human, ushi or pig.
  • step B) the exposure of the biocompatible polymer to the biological membrane is performed at 24 ° C. under a condition of 12 to 24 hours.
  • step C) the intensity of ⁇ -ray is 50 keV, and the irradiation of ⁇ -ray is performed in a sealed container at a dose of 12-50 kGy and at a temperature of 24-37 ° C. . (Item 92)
  • An anti-adhesion membrane comprising a biocompatible polymer and having been irradiated with y-rays, wherein the anti-adhesion membrane is derived from a living body.
  • the adhesion-preventing film according to Item 93 which has at least one crosslink formed between non-chemical crosslinkable groups other than a carboxyl group, a hydroxyl group and an amino group.
  • the adhesion-preventing film according to Item 93 which has at least one cross-link formed by ⁇ -ray irradiation and formed by a bond selected from the group consisting of a carbon-carbon bond, an ether bond and an ester bond.
  • the adhesion-preventing membrane according to Item 93 which has at least one cross-link formed by a carbon-carbon bond formed by forming a skeleton and a new bond formed by cleavage of an ester bond.
  • the adhesion-preventing membrane according to Item 93 which has at least one non-chemical crosslink formed between amino acids having no free amino group and no carboxyl group in the side chain.
  • Item 99 The adhesion-preventing membrane according to Item 98, wherein the non-chemical crosslinking is a crosslinking between amino acids having a crosslinking point other than lysine and arginine.
  • the adhesion-preventing membrane according to Item 93 having at least one crosslink formed between polysaccharides having non-chemical crosslinkable groups other than carboxyl, hydroxyl and amino groups.
  • the item 100 wherein the polysaccharide having a non-chemical crosslinking group has a bridge formed between at least one group selected from the group consisting of a sulfhydryl group, an aldehyde group, a carbonyl group, a sulfo group, and a -tro group.
  • Anti-adhesion membrane is an organic radical that causes a sulfhydrylation to a sulfhydryl group, an aldehyde group, a carbonyl group, a sulfo group, and a -tro group.
  • Anti-adhesion membrane wherein the polysaccharide having a non-chemical crosslinking group has a bridge formed between at least one group selected from the group consisting of a sulfhydryl group, an aldehyde group, a carbonyl group, a sulfo group, and a -tro group.
  • the adhesion-preventing film according to Item 93 comprising at least one non-chemical crosslink other than bisepoxide crosslink, divinylsulfone crosslink, intramolecular esterification, glutaraldehyde crosslink, carpositimide crosslink and hydrazide crosslink.
  • the adhesion-preventing membrane according to Item 93 which has a bond selected from the group consisting of a bond formed between the groups to be bonded, a carbon-carbon bond, a peptide bond, an ether bond, and an ester bond strength.
  • adhesion prevention membrane according to Item 93, wherein the adhesion prevention membrane is selected from the group consisting of pleura, pericardium, brain dura mater, serosa, peritoneum and skin.
  • the anti-adhesion membrane is as follows:
  • the bio-derived membrane is chemically treated with dartalaldehyde, exposed to polyethylene glycol (PEG), and irradiated with ⁇ rays to form crosslinks.
  • PEG polyethylene glycol
  • a force that needs to prevent tissue adhesions or a method of treating or preventing a subject at risk comprising:
  • a biological tissue in which calcification is remarkably suppressed is provided.
  • ⁇ -ray irradiation was significantly suppressed compared to other methods such as UV irradiation and scientific treatment.
  • Main departure Ming also provides biological tissue that is reinforced, improved or maintained in strength. Therefore, the present invention has an effect of reinforcing a living body-derived tissue and reducing immune rejection.
  • the transplanted tissue treated by the above treatment method exhibits an advantageous effect that calcification is remarkably suppressed as compared with the transplanted biological tissue prepared by the conventional technique.
  • A Inhibition of cell invasion by PEG and other gels
  • B Suppression of immunogenicity by modification of components of biological tissue including extracellular matrix (ECM) with PEG
  • ECM extracellular matrix
  • the conventional polymerization reaction is a relatively controlled chemical reaction, whereas ⁇ -ray irradiation is considered to introduce cross-links between PEG molecules relatively randomly. It is thought that it leads to effective suppression.
  • FIG. 1 shows a state 2 months after the biocompatible molecular treatment and ⁇ -irradiation irradiation of the present invention after transplantation into a rat.
  • the top is polymerized with 25 kGy gamma rays.
  • the pericardium is shown below, and the pericardium polymerized with 50 kGy gamma rays is shown below.
  • the left shows the appearance in the living body and the right shows the extracted biological tissue. Cell invasion is observed with 25 kGy treatment. Remarkable infiltration of cells is observed with 50 kGy treatment.
  • FIG. 2 shows a HE-stained diagram of 2 months after the biocompatible molecular treatment and ⁇ -irradiation irradiation of the present invention after transplantation into a rat.
  • the ushi pericardium polymerized with 25 kGy gamma rays is shown above, and the ushi pericardium polymerized with 50 kGy gamma rays is shown below.
  • Each figure is an example showing photographs of various parts.
  • FIG. 3 shows von Kossa staining 2 months after transplantation of biologically-derived tissue into rats after biocompatible molecular treatment and ⁇ -irradiation according to the present invention.
  • the pericardium superposed with 25 kGy gamma rays is shown above, and the pericardium polymerized with 50 kGy gamma rays is shown below.
  • Each figure is an example showing photographs of various parts.
  • FIG. 4 shows the amount of calcification in the two months after transplantation of the biological tissue of the present invention to the rat after the biocompatible molecular treatment and ⁇ -irradiation. From the left, the graph shows the persimmon pericardium polymerized with 25 kGy gamma rays, the persimmon pericardium polymerized with 50 kGy gamma rays, and the persimmon pericardium treated with dartal aldehyde.
  • FIG. 5 shows data 2 months after transplantation for untreated urine pericardium.
  • Figure 6 shows two months after transplantation of dartalaldehyde-fixed + PEGylated ushi pericardium (upper right) and dartalaldehyde-fixed ushi pericardium (upper left) in the rat dorsal skin. The photograph at the time of subsequent extraction is shown. The lower panel shows photographs of the extracted dartalaldehyde fixed + PEGylated pericardium (right) and dartalaldehyde fixed pericardium (left).
  • FIG. 7 is a graph showing the results of calcium deposition in dartalaldehyde-fixed ushi pericardium and dartalaldehyde-fixed + PEGylated ushi pericardium two months after transplantation subcutaneously on the back of rats. is there.
  • calcium a calcification finding
  • regeneration refers to a phenomenon in which the remaining tissue grows and is restored when a part of the tissue of an individual is lost.
  • the extent and mode of regeneration varies.
  • Many human tissues have limited ability to regenerate, and complete regeneration cannot be expected if they are largely lost.
  • a tissue having a strong proliferative power different from the lost tissue proliferates, and incomplete regeneration may occur in which the tissue is regenerated incompletely and the function cannot be recovered.
  • the “cell” used in the present specification is defined in the same way as the broadest meaning used in the field, and is a structural unit of a tissue of a multicellular organism and is enclosed in a membrane structure that isolates the outside world. Rarely, an organism that has self-regenerative ability and has genetic information and its expression mechanism. Any cell can be targeted in the method of the present invention.
  • Main departure The number of “cells” used in the light can be counted through a light microscope. When counting through an optical microscope, count by counting the number of nuclei.
  • the tissue is sliced into tissue slices and stained with hematoxylin-eosin (HE) to separate extracellular matrix (eg, elastin or collagen) and cell-derived nuclei with a dye. This tissue section can be examined with an optical microscope, and the number of nuclei per specific area (for example, 200 ⁇ 200 ⁇ m) can be estimated and counted.
  • HE hematoxylin-eosin
  • Cells may be responsible for calcification and eliciting an immune response. Therefore, for tissue or organ transplantation, it is necessary to suppress cell-derived calcification and the induction of an immune response.
  • cell replacement refers to the invasion and replacement of another cell in the decellularized tissue in place of the original cell. " Preferably, in the present invention, cell replacement is performed by the transplanted host cells.
  • tissue refers to a cell population having the same function 'form in a cell organism. In multicellular organisms, the cells that make it up are usually separated, function specialized, and division of labor occurs. Therefore, a tissue cannot be a mere assembly of cells, and constitutes an organic cell group or a social cell group having a certain function and structure. Tissues include, but are not limited to, integument tissue, connective tissue, muscle tissue, vascular tissue, heart tissue, nerve tissue, and the like. The tissue targeted by the present invention may be any organ or tissue derived from an organism.
  • tissues targeted by the present invention include, but are not limited to, blood vessels, blood vessel-like tissues, heart valves, pericardium, dura mater, corneas and bone tissues.
  • biological tissue refers to a tissue from which vital force is also separated or a tissue processed therefrom, and unless otherwise specified, refers to a tissue that has not undergone decellularization treatment. Is done. In this specification, after taking out the vitality, it is especially decellularized. Yarns and weaves that do not exist are also called “untreated cells”. It is understood that this concept includes, for example, tissues that have been separated from the living body and those that have been exposed to a biocompatible polymer, as well as tissues that have been separated from the living body. The Such living tissue is used as a graft.
  • the "biological membrane” refers to a membrane separated from a living organism or one obtained by treating the membrane.
  • Examples of the biological membrane used in the present specification include, but are not limited to, membranes derived from blood vessels, blood vessel-like tissues, pericardium, dura mater, cornea, amniotic membrane, peritoneum, skin and the like.
  • film-like structure refers to a “planar structure” as well as a film-like structure.
  • membranous tissues include organ tissues such as pericardium, dura mater and cornea.
  • adheresion-preventing membrane refers to a membrane that prevents adhesion, and prevents, for example, adhesion that occurs between a cut surface or a defect site after surgery or treatment and its surrounding tissue. A film that stops.
  • organs or organs are used interchangeably, and a certain function of an organism is localized in a specific part of an individual, and the part. Refers to a structure that is morphologically independent.
  • the organs are made up of several tissues with a specific spatial arrangement, and the tissues also have a number of cellular forces.
  • Such organs or organs include organs or organs associated with the vascular system.
  • organs targeted by the present invention include ischemic organs (hearts with myocardial infarction, skeletal muscles with ischemia, etc.).
  • organs targeted by the present invention are blood vessels, blood vessel-like tissues, heart valves, pericardium, dura mater, cornea and bone. In another preferred embodiment, the organs targeted by the present invention are heart valves, pericardium and blood vessels.
  • Organ culture refers to culturing a part or the whole of an embryonic organ or a mature organ from which vital force has been removed in vitro while maintaining its structure, function and ability to separate. In contrast, in general tissue culture, dedifferentiation is more likely to occur because the main purpose is cell proliferation. Organ culture includes the watch glass method and the block petri dish. Method, support base method, sponge substrate method, Trowell method, etc., but are not limited thereto.
  • Protein 'nucleic acid' enzymes 45, 2188-2200 (2000) as described in large cell cultures eg holofiber method, fixed bed type, spin filter, sedimentation tank type, perfusion culture, circulating organ culture Methods such as circulation type organ culture may also be used.
  • Organs or tissues prepared by the decellularization method of the present invention are added to a culture medium, and various substances are added to the organ culture medium to adjust to structure, function and developmental differentiation, and to organ-to-organ interaction. Adjustment of the action and the like can be performed.
  • decellularization and “decellularization” refer to removal of tissue or organ force cells.
  • decellularization can be performed without damaging the structure and function of the original tissue or organ.
  • the decellularized tissue force also removes cytoplasmic components, cytosolic components, cytoskeleton and cell membrane components, but extracellular matrix (ECM) components (e.g. elastin, collagen (type I, type IV) Etc.), laminin, etc.) the cellular components necessary to preserve the structure of the tissue remain intact. Therefore, it is preferable that the decellularized tissue or organ has substantially the same properties as the untreated tissue or organ in terms of properties such as the shape, mechanical strength, elasticity, and flexibility of the tissue or organ.
  • ECM extracellular matrix
  • the extracellular matrix since the extracellular matrix is native after transplantation, it provides a suitable environment for the invasion, adhesion, proliferation, and expression and maintenance of cells on the recipient's side. It is preferable to replace it.
  • the degree of decellularization can be measured using the cell survival rate as an index.
  • the (untreated) living tissue of the present invention has not been subjected to such decellularization treatment.
  • the decellularized tissue preferably has MHC class I and sputum proteins removed.
  • MHC class I and II proteins can be confirmed by SDS PAGE and Z or Western plot analysis.
  • Other extracellular matrix proteins can also be confirmed by SDS PAGE and Z or Western blot analysis.
  • Structural proteins in cells can be evaluated by amino acid analysis (eg, Edman degradation method or automated analysis with peptide analyzer available from PE Biosystems).
  • amino acid analysis eg, Edman degradation method or automated analysis with peptide analyzer available from PE Biosystems.
  • the decellularization process E Can determine the impact on CM.
  • Lipids and phospholipids contained in cell membranes and cells can be analyzed using thin layer chromatography and HPLC.
  • Sugar chains for example, glycosaminodarlicans
  • agarose gel electrophoresis or the like. By this analysis, it is possible to analyze the presence of a-Gal in addition to the glycosaminodarican composition of the extracellular matrix.
  • the tissue has been decellularized! /, And the tissue has the characteristics of the above decellularization and can be determined by confirming this.
  • a tissue that has not been decellularized is decellularized in the present specification (1) with a cell survival rate of 30% or more as compared to a tissue (living tissue) that has been extracted.
  • cell survival rate refers to the ratio of viable cells remaining after decellularization of a tissue by the method of the present invention to cells originally present in the tissue.
  • the method of measuring the cell survival rate is typically a microscopic counting method using hematoxylin and eosin staining (H & E staining), and this method involves the analysis of nuclei dyed by HE staining. Use counting with an optical microscope.
  • this method comprises the following steps: dyeing the sample by HE staining; this sample!
  • the total amount of DNA contained in a cell in a tissue is generally proportional to the number of cells in the tissue.
  • Methods for measuring DNA are known in the art.
  • a fluorescent reagent such as Hoechst 33258 from Molecular Probes is used. It can be used to quantify the amount of DNA extracted from tissues. Specifically, the tissue is made into a sol by sonication, etc., which specifically binds to DNA components in the buffer and generates fluorescence. Hoechst 33258 (Ex (excitation wavelength) 356, EM (emission wavelength) 492 ), And the amount of DNA in the extract supernatant can be measured and quantified as fluorescence intensity. Therefore, the cell survival rate can be used for the difference from the decellularized tissue, or the cell survival rate can be used even by ⁇ -irradiation. In this case, a 30% cell survival rate can be mentioned as a guide.
  • immune response refers to a response due to a lack of immune tolerance between a graft and a host, such as hyperacute rejection (within a few minutes after transplantation) (such as j8-gal).
  • hyperacute rejection within a few minutes after transplantation
  • j8-gal a host that a host that a grafts have a lack of immune tolerance between a grafts and a host.
  • Antibody-induced immune reaction such as acute rejection (reaction due to cellular immunity about 7-21 days after transplantation), chronic rejection (rejection due to cellular immunity after 3 months), and the like.
  • whether or not to induce an immune response is determined with respect to infiltration of cells (immune system) into a transplanted tissue by staining including HE staining, immunostaining, and microscopic examination of a tissue section. It can be determined by conducting histopathological examination of the species and number.
  • calcification refers to the deposition of calcareous substances in a living organism.
  • whether or not to "calcify” in vivo can be determined by measuring the calcium concentration.
  • the transplanted tissue is taken out, the tissue section is dissolved by acid treatment or the like, and the solution is used. It can be measured and quantified with a trace element quantification device such as atomic absorbance.
  • in vivo or “in vivo” refers to the inside of a living body.
  • in vivo refers to the location where the target tissue or organ is to be placed.
  • in vitro refers to the state in which a portion of a living organism has been removed or released “in vitro” (eg, in a test tube) for various research purposes. Say. A term that contrasts with in vivo.
  • the term "ex vivo" refers to a series of cases where target cells for gene transfer are extracted from a subject, a therapeutic gene is introduced in vitro, and then returned to the same subject again. The operation is ex vivo.
  • the "function that a tissue has in a normal state” refers to a function that the tissue has in a normal state in a living body. Therefore, for example, in the case of a heart valve, since it has a function of preventing blood backflow from the ventricle to the atrium or pulmonary artery and aorta force atrium, the function that the heart valve has in the normal state is that A function that prevents the backflow of blood from the atria. According to the present invention, it has been shown that a special effect has been shown in a conventional manner in that even if ⁇ -irradiation is performed after exposure to a biocompatible polymer, substantial damage to the function of the normal state has been helped. .
  • extracellular matrix is also called “extracellular matrix” and refers to a substance existing between somatic cells regardless of whether they are epithelial cells or non-epithelial cells.
  • the extracellular matrix is responsible for the internal environmental organization required for the survival of all somatic cells, not just tissue support.
  • Extracellular matrices are generally produced from connective tissue cells, but some are also secreted from the cells themselves that possess a basement membrane, such as epithelial cells and endothelial cells.
  • the fiber component and the matrix that fills it are roughly divided, and the fiber component includes collagen fiber and elastic fiber.
  • the basic component of the substrate is glycosaminodarlican (acid mucopolysaccharide), most of which binds to non-collagenous proteins to form a polymer of proteodalican (acid mucopolysaccharide-protein complex).
  • glycoproteins such as laminin in the basement membrane, microfibrils around elastic fibers, fibers, and fibronectin on the cell surface are also included in the substrate.
  • the basic structure is the same even in specially differentiated tissues.For example, in hyaline cartilage, cartilage matrix containing a large amount of proteodarican is produced by soft osteoblasts, and in bone, bone matrix where calcification is caused by osteoblasts is produced. Produced.
  • extracellular matrix eg, elastin, collagen (eg, type I, type IV, etc.), laminin, etc.
  • extracellular matrix eg, elastin, collagen (eg, type I, type IV, etc.), laminin, etc.
  • elastin elastin, collagen (eg, type I, type IV, etc.), laminin, etc.) is substantially free from the state prior to the decellularization treatment of the present invention. It can be a feature.
  • tissue damage rate refers to a parameter indicating the function of a tissue or organ, V,,, how much the tissue or organ after treatment is damaged or damaged. It is an indicator of whether or not the tissue or organ can perform its original function.
  • tissue strength refers to a parameter indicating the function of a tissue or organ, and refers to the physical strength of the tissue or organ, generally by measuring the tensile strength. Can be determined. Such general tensile tests are well known.
  • the specimen is cut into 5 X 30 mm strips. (Basically cut the wall part of the base of the aorta so that it becomes longer in the long axis direction.); (2) Fix the specimen about 5 mm at both ends to the fixed part of the tensile tester. (Use ORIENTEC's TENSILON universal testing machine RTC-1150A); and (3) Start pulling and pulling to the breaking point at lcmZmin. The load at the breaking point is adopted as the strength. Here, the load at break and the elastic modulus are measured.
  • the strength of the living tissue of the present invention can usually be such that the maximum point load is at least about 8N or more, more preferably about 10N or more, and even more preferably about 14N or more. In conventional biological tissues or natural tissues (eg, arteries), the strength is about 7N, and decellularization treatment has little or no effect on enhancing tissue strength! In view of this, the ability to strengthen the tissue strength of the present invention is unexpected!
  • the living tissue of the present invention when viewed in terms of elastic modulus, usually has an elastic modulus of at least 1.2 MPa, preferably at least about 1.6 MPa.
  • the biological tissue of the invention may have an elastic modulus (for example, ⁇ , 0.8J3 ⁇ 4_h, 0.8-1 OMPa, etc.) that is inferior to that of a natural product as long as it can be used. .
  • the elongation can be measured with the tensile tester (TENSILLON ORIENTEC) used in the strength measurement. Specifically, a strip-shaped material having a width of 5 mm and a length of 30 mm can be loaded at a speed of 10 mmZ in the minor axis direction, and the load at break and the elastic modulus can be measured. Specifically, the elongation can be measured by measuring the length in each direction before and after the tension stimulus and dividing the length after the tension by the length before the tension and multiplying by 100. Normal, Elongation is measured in both the longitudinal and transverse directions. It is preferable that there is no variation in both the elongations, but the present invention is not limited to this.
  • the elongation-related properties are for example at least 120%, preferably 150%, but are not limited thereto.
  • the living tissue of the present invention may have elongation (eg, at least 50%) which is inferior to that of a natural product as long as it can withstand use.
  • tissue strength can be expressed by a stiffness parameter ( ⁇ value). The ⁇ value is calculated after creating the P-D (pressure-diameter) relationship.
  • Ps and Ds are standard values at lOOmmHg.
  • P and D The diameter (D) value at each P (pressure) is shown.
  • Both ends of a tubular tissue such as a blood vessel are fixed to a pipe-shaped unit, and the inner chamber and the outer chamber are filled in physiological saline. From this state, the outer diameter at the time of pressurization is monitored at the same time as pressure is applied to the inner chamber from the outer arm. The relationship between the pressure obtained by the measurement and the outer diameter is introduced into the above equation (1) to calculate the j8 value (Sonoda H, Takamizawa K., et al. J. Biomed. Matr. Res. 2001). : 266—276).
  • biocompatibility refers to the property of being non-toxic and non-immunologically refractory and therefore capable of existing without injuries in vivo.
  • biocompatible polymer refers to a polymer that is biocompatible, and specifically, does not cause toxicity even if it remains.
  • a test method such as a subcutaneous implantation test in a laboratory animal such as a rat is used in this specification. In this test method, the results of the subcutaneous implantation test
  • cytotoxicity tests For medical device materials, cytotoxicity tests, sensitization tests, irritation tests, implantation tests, genotoxicity tests are conducted in order to evaluate their toxicity and reasonably regulate their use in medical devices. There are test items such as tests, blood compatibility tests, systemic toxicity tests, etc., and individual test methods are defined by the guidelines of the Ministry of Health, Labor and Welfare, the US National Standards, the international industry standard ISO-10993, etc. .
  • biocompatible polymer examples include polyvinyl alcohol, polylactic acid, polyglycolic acid, polybutylpyrrolidone, polyethylene glycol, gelatin, collagen, ⁇ -polyglutamic acid, silicone, polyvinyl chloride, and polymethyl methacrylate.
  • some of them are chemically modified with amino groups, carboxyl groups, acetyl groups, etc., and are commercially available. It can also be used in the present invention.
  • biocompatible The functional polymer is biodegradable, but is not limited thereto.
  • polyethylene glycol refers to a polymer of ethylene glycol, which is also referred to as polyethylene (poly (ethylene oxide)), and is represented by HO— (C H CH O) —H.
  • Polyethylene glycol is commercially available from various companies.
  • PEG polyethylene glycol
  • PEG has an average molecular weight of between 10,000 and 100,000.
  • the PEG can have an average molecular weight of 2,000-50,000. More preferably, the PEG can have an average molecular weight of about 35,000.
  • it may be 1) 1,000-200000, (2) preferably 4,000 to 000 000, and (3) more preferably 8,000-50,000.
  • PEG can be synthesized as appropriate in order to achieve the desired properties. Such synthetic methods are well known in the art.
  • the average molecular weight and the molecular weight are expressed by Dalton.
  • the PEG used in the present invention preferably has a homogeneous molecule, but this is not essential and can be usually used if the average molecular weight is within a certain range.
  • chemical substances other than PEG for example, enzymes (for example, DNasel), enzyme inhibitors, etc., but not limited to them
  • the amount or concentration of PEG to be added can vary depending on the amount of the chemical present.
  • the concentration used for cell fusion or the like is higher, for example, a concentration of lgZml or more (100% wZw or more) is preferable, but not limited thereto.
  • polybulal alcohol refers to a polymer obtained by hydrolysis (exactly transesterification) of polyacetic acid bule with both poval and PVA! /,. It is expressed as a general formula — (CH 2 C (OH) H) —. -Lu alcohol exists as a monomer
  • polybulurpyrrolidone is represented by the general formula [—CH 2 CH (C H NO) —].
  • biodegradable refers to the property of being degraded in vivo or by the action of microorganisms when referring to a substance.
  • the biodegradable polymer can be decomposed into water, carbon dioxide, methane and the like, for example, by hydrolysis.
  • the method for determining whether or not biodegradability is related to bioabsorbability, which is part of biodegradability is from several days to several years for laboratory animals such as rat, usagi and inu.
  • methods such as embedding 'disintegration tests for several days to several years in sheet-like polymers in soil are used.
  • transplantation it is preferable to use animal studies.
  • phosphate buffered saline PBS
  • a dissolution test in an aqueous solution may be carried out using a buffer solution containing the hydrolase of the polymer (protease, glycosidase, lipase, esterase, etc.).
  • Biodegradable polymers include natural and synthetic polymers. Examples of natural polymers include proteins such as collagen and starch, and polysaccharides, and examples of synthetic polymers include aliphatic polyesters such as polyglycolic acid, polylactic acid, and polyethylene succinate. Is not limited to them. Polyvinyl alcohol can be recognized as biodegradable in the present specification because it exhibits weak biodegradability.
  • polymer is used in the same meaning as “molecule”, and the molecular weight is not particularly limited.
  • the upper limit of the molecular weight of the polymer used in the present invention is infinite in principle.
  • the molecular weight can be discussed (in the sense that molecular weight measurements such as dynamic light scattering, gel filtration, and centrifugal sedimentation can be applied), but molecular weights up to about 5 million can be used, but are not limited thereto.
  • biopolymer” and “biocompatible polymer” are synonymous with terms such as “biomaterial” and “medical polymer”, as is the case in convention and industry. used.
  • immersing refers to placing an object in a fluid (eg, a liquid).
  • a fluid eg, a liquid
  • the tissue to be treated is treated with a surfactant or micelle, which is in the state of an amphiphilic molecule (for example, a 1,2-epoxide polymer such as polyethylene glycol).
  • a surfactant or micelle which is in the state of an amphiphilic molecule (for example, a 1,2-epoxide polymer such as polyethylene glycol).
  • soaking preferably performed so that the tissue to be treated completely penetrates into the solution for treatment.
  • a physical treatment for example, ironing with a glass rod
  • exposing refers to bringing a certain object into contact with a certain factor (for example, a biocompatible polymer).
  • the “washing” step in the present invention refers to removing the solution from the tissue force treated by, for example, the step of immersing in a biocompatible polymer solution. Therefore, preferably, the washing step is performed using a liquid.
  • the treated tissue is intended to be used in a living body, so it is preferably washed with a physiologically acceptable liquid.
  • the washing step may be performed using PBS (phosphate buffered saline).
  • the washing solution may contain other drugs (eg, protease inhibitors) as needed, but such other drugs are preferably non-toxic and biocompatible.
  • chemical treatment broadly means that a certain object is treated (for example, by dipping) with a chemical substance.
  • chemical treatment refers to other steps (eg, DNase treatment) other than the step of immersing in a solution containing a biocompatible polymer, for example.
  • the chemical treatment when chemical treatment is performed in addition to the step of immersing in a polyethylene glycol-containing solution having an average molecular weight of 1,000 to 200,000, the chemical treatment has an average molecular weight of 1, 000-200,000
  • solutions other than polyethylene glycol-containing solutions eg, but not limited to treatment with DNasel solutions, dartal aldehyde solutions, polyethylene glycol solutions with other average molecular weights, other biocompatible polymer solutions, etc.
  • tissue strength when referring to a tissue means that the tissue strength of the tissue is improved.
  • the tissue strength is preferably at least 110%, more preferably at least 120%, even more preferably at least 150%, or at least 200% of the state prior to some reinforcement treatment.
  • Such tissue strength can be expressed herein as a maximum point load (eg, unit N), for example, in a tensile strength test.
  • the term “radical reaction” refers to a chemical reaction that occurs when an unpaired electron is generated and reacts with another molecule or residue.
  • radical reaction examples include the following:
  • ⁇ -ray refers to a wavelength shorter than about 0.001 nm and electromagnetic waves. In terms of energy, the photon is several hundred keV or higher. Usually, ⁇ -rays are emitted at the transition between energy levels of nuclei. Any source used as a ⁇ -ray source can be used as the source of ⁇ -rays, but 6G Co (conoleto 60) and 137 Cs (cesium 13 7) are preferred. This is because it is preferable for the treatment of living tissue. [0084] The amount of ⁇ -ray irradiation that is often displayed in units of kGy can be measured by measuring the absorbed dose. For such measurements, measurement devices such as a hollow ionization chamber, calorimeter, film dosimeter, PMMA dosimeter are used.
  • Examples of methods for confirming ⁇ -ray irradiation include the following.
  • Detection method 2 Tyrosine (tyrosine), which is a type of amino acid, when protein casein in milk is irradiated with gamma rays together with polysaccharides such as carboxymethylcellulose and PEG to produce a crosslinked thin film. ) Is dimerized to form a bityrosine structure, and since it emits fluorescence, it has been reported that cross-linking sites are quantified (J. Agric. Food. Chem. 4 6, 1618-1623, 1998). You can use the power S.
  • vacuum refers to a pressure of 1 ⁇ 10 ⁇ 1 Pa or less.
  • Under reduced pressure refers to a pressure in the range of 1 ⁇ 10 — 1 Pa to l ⁇ 10 5 Pa.
  • Atmosphere is used in the meaning usually used. Usually, the composition is 78% nitrogen, 21% oxygen, 1% argon, 0.03% carbon dioxide, and about 0.3% water vapor. The one that is used is used.
  • oxygen refers to an environment having a higher oxygen concentration than the atmosphere.
  • oxygen refers to the presence of substantially 100% oxygen.
  • in water refers to a reaction in a medium that also has only H 2 O power.
  • water tap water, distilled water, ion-exchanged water and the like are used, but are not limited thereto.
  • biocompatible molecule in which radical reaction is performed in the present specification, an amphiphilic molecule solution may be used, but other different ones may be used.
  • physiologically active substance refers to a substance that acts on cells or tissues. Bioactive substances include site force-in and growth factors.
  • the physiologically active substance may be naturally occurring or synthesized. Preferably, the physiologically active substance is one produced by a cell or one having a similar action.
  • the physiologically active substance may be in protein form or nucleic acid form or other form, but at the point of actual action, cyto force-in usually means the protein form.
  • site force-in is defined in the same manner as the broadest meaning used in the art, and refers to a physiologically active substance that is produced by cell force and acts on the same or different cells.
  • Site force-in is generally a protein or polypeptide, and controls immune response, regulation of endocrine system, regulation of nervous system, antitumor action, antiviral action, regulation of cell proliferation, regulation of cell differentiation Etc.
  • cytoforce-in can be in protein form or nucleic acid form or other form, but at the point of actual action, cytoforce-in usually means protein form.
  • growth factor or “cell growth factor” is used interchangeably herein and refers to a substance that promotes or regulates cell growth. Growth factors are also referred to as growth factors or growth factors. Growth factors are used in cell or tissue culture. In addition, it can be added to the medium to replace the action of the serum polymer substance. In addition to cell growth, many growth factors have been shown to function as regulators of sorting.
  • cytoforce-in includes hematopoietic factors such as interleukins, chemokines, and colony stimulating factors, tumor necrosis factors, and interferons.
  • Typical growth factors include platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF).
  • PDGF platelet-derived growth factor
  • EGF epidermal growth factor
  • FGF fibroblast growth factor
  • HGF hepatocyte growth factor
  • VEGF vascular endothelial growth factor
  • Physiologically active substances such as cytoforce-in and growth factors generally have a redundancy phenomenon, so even if it is a site-force-in or growth factor known by other names and functions, As long as it has the activity of the physiologically active substance used in the invention, it can be used in the present invention.
  • the cytodynamic force or the growth factor can be used in a preferred embodiment of the therapeutic method or medicament of the present invention as long as it has a preferable activity in the present specification.
  • differentiation refers to a process of development of a state of a part of an organism such as a cell, tissue or organ that forms a characteristic tissue or organ. “Differentiation” can be used for developmental biology and developmental biology! It has been used. Until a fertilized egg consisting of one cell divides and becomes an adult, an organism forms various tissues and organs. It is difficult to distinguish individual cells or groups of cells without any morphological or functional characteristics in the early stages of life, such as in cases where pre-division or division is not enough! Such a state is called “undifferentiated”. “Differentiation” also occurs at the organ level, where the cells that make up the organ develop into distinctive cells or groups of cells!
  • the differentiation of a cell means that the cell has a morphological or functional characteristic of any force that it did not have before the treatment.
  • a stem cell e.g., embryonic stem cell or tissue stem cell
  • the form or function is at least partially similar to the cell or tissue present in the heart valve. That the cell has.
  • graft As used herein, "graft”, “graft” and “tissue graft” are used interchangeably The same or different type of tissue or cell group that is to be inserted into a specific part of the body and becomes a part of it after insertion into the body.
  • the graft include, but are not limited to, an organ or a part of an organ, a blood vessel, a blood vessel-like tissue, a skin piece, a heart valve, a pericardium, a dura mater, a corneal bone fragment, and a tooth.
  • a graft includes anything that can be used to fill a defect in a part to make up for the defect.
  • the graft may include, but is not limited to, autograft, allograft (allograft), xenograft, depending on the type of donor. .
  • an autograft or autograft refers to a graft derived from an individual when referring to the individual.
  • autograft can include a graft from another genetically identical individual (eg, an identical twin) in a broad sense.
  • an allograft refers to a graft that is transplanted to another individual force even though it is the same species. Because of genetic differences, allogeneic transplants can elicit an immune response in the transplanted individual (recipient). Examples of such grafts include, but are not limited to, parent-derived grafts.
  • xenograft refers to a graft to be transplanted with xenogeneic force.
  • a graft from Ushi, pig is called a xenograft
  • a “recipient” is an individual who receives a graft or transplant and is also called a “host”.
  • an individual who provides a graft or transplant is called a “donor”.
  • Any graft can be used with the tissue processing technique of the present invention. This is because grafts (for example, tissues, organs, etc.) reinforced by the method of the present invention have reduced immune disorders to the extent that they are not therapeutically impaired. Therefore, the fact that it has become possible to use allografts or xenografts even in situations where only conventional autografts could be used, has been impossible to achieve with the prior art. This is one of the special effects of the present invention.
  • subject refers to an organism to which the treatment of the present invention is applied, and is also referred to as "patient".
  • the patient or subject may preferably be a human.
  • the cells used as necessary in the method or yarn-and-woven graft of the present invention may be derived from the same line (derived from the self (self)) or from the same type (derived from another individual (other family)). It can be of different origin. Autologous cells are preferred because rejection is considered, but they may be allogeneic if rejection is not a problem. In addition, those that cause rejection can be used by performing treatment to eliminate rejection as necessary.
  • Procedures for avoiding rejection are well known in the art, and are described, for example, in the New Surgery System, Heart Transplantation, Lung Transplantation Technical and Ethical Maintenance Capabilities (Revised 3rd Edition). Examples of such methods include methods such as the use of immunosuppressants and steroids.
  • Immunosuppressants to prevent rejection are currently “cyclosporine” (Sandyimyung Z Neoral), “Tacrolimus” (Prograf), “Azathioprine” (Imlan), “Steroid Hormone” (predonin, methylpredonin), “T There are “cell antibodies” (OKT3, ATG, etc.), and the method used in many facilities around the world as preventive immunosuppressive therapy is a combination of three drugs: “cyclosporine, azathioprine, and steroid hormone”.
  • the immunosuppressive agent is desirably administered at the same time as the medicament of the present invention, but it is not always necessary. Therefore, the immunosuppressive agent can be administered before or after the regenerative treatment method of the present invention as long as an immunosuppressive effect is achieved.
  • the cells used in the present invention may be cells derived from any organism (for example, vertebrates and invertebrates).
  • cells derived from vertebrates are used, and more preferably cells derived from mammals (for example, primates, rodents, etc.) are used. More preferably, cells derived from primates are used. Most preferably, human-derived cells are used.
  • Examples of the combination of a subject targeted by the present invention and a biological tissue include, for example, heart disease
  • ischemic heart disease transplantation into the heart, pericardial patch, dural transplantation during brain surgery, myocardial infarction, blood vessel transplantation in the lower limbs, upper limbs, fractures, bones to patients with bone defects
  • Transplantation transplantation of the cornea of the present invention to a patient having a damaged cornea, but not limited thereto.
  • the tissue targeted by the present invention may be any organ or organ of the organism, and the tissue targeted by the present invention may be derived from any kind of organism.
  • organisms targeted by the present invention include vertebrates and invertebrates.
  • the present invention is intended
  • the living organisms are mammals (eg, primates, rodents, etc.). More preferably, the organism targeted by the present invention is a primate. Most preferably, the present invention is directed to humans.
  • the force used in the right heart system mainly within the blood pressure range of the pulmonary artery pressure.
  • Use within the aortic pressure, or application to valve tissue, AC bypass arterial graft, chordal tissue, etc. Can be performed as appropriate (latest treatment of cardiovascular disease 2002-2003; Nanedo p 29, 2002).
  • the present invention provides a biological tissue characterized by containing a biocompatible polymer and receiving y-ray irradiation.
  • the biocompatible polymer may be contained in any state in the living tissue, but it is advantageous that it is preferably bonded by a covalent bond. Therefore, those skilled in the art can appropriately include the biosynthetic polymer in the living tissue using a technique well known in the art depending on the state to be included. Such binding may be achieved by gamma irradiation.
  • the biological tissue that should contain the biocompatible polymer is preferably one that has not undergone a decellularization treatment.
  • the present invention has the power of finding that, by including a biocompatible polymer in a tissue derived from a living body and receiving ⁇ -ray irradiation, calcification is remarkably reduced and tissue strength is increased unexpectedly. is there.
  • the biocompatible polymer of the present invention offers the possibility of being used in powerful applications where conventional calcification and strength problems cannot be applied.
  • other radical reactions e.g. UV irradiation, chemicals
  • gamma irradiation A radical reaction using a quality
  • a cross-linking agent such as dartal aldehyde
  • the effect recognized by the present invention as a result of a long-term transplantation experiment into an animal body (about 2 months for small animals such as rats and about 6 to 12 months for large animals such as pigs).
  • an effect that functional failure due to calcification does not occur This is an effect that is not recognized at all during treatment with dartalaldehyde.
  • a significant effect was also observed quantitatively.
  • the calcium content per lmg of transplanted pericardium was 1.6 ( ⁇ g / mg) when glutaraldehyde was treated. During the treatment by this method, it decreased to about 0.15 to 0.2 (g / mg).
  • the strength reinforcement obtained by the tissue treatment of the present invention is preferably, for example, 101% or more, more preferably 110% or more, more preferably, compared to the state without biocompatible polymer or ⁇ -ray irradiation. Preferably it may be 120% or more, more preferably 150% or more, and most preferably 200% or more.
  • the cause of calcification was removed by gamma irradiation.
  • the level is lower than the level that elicits an immune response in vivo. There must be.
  • the present invention also has an effect in that the tissue treated with the biocompatible polymer is remarkably reinforced.
  • the present invention provides a biological tissue characterized in that, in this embodiment, the extracellular matrix component is at least partially crosslinked by a covalent bond.
  • the extracellular matrix component can include, but is not limited to, collagen, elastin, laminin, fibronectin, tenascin, glycosaminodancan and proteoglycan.
  • the bridge is formed between two or more amino acids that do not have a free amino group or carboxyl group in the side chain.
  • the cross-linking is a carboxyl group, a hydroxyl group and an amino group. It is characterized in that it is formed in a group other than a group (for example, a sulfhydryl group).
  • Proteins derived from extracellular matrices such as collagen, elastin, laminin, fibronectin and tenascin, and polysaccharides derived from extracellular matrices such as glycosaminodarlicans (such as hyaluronic acid, chondroitin sulfate and heparan sulfate) and proteodalycan It is known that when ⁇ -ray irradiation is applied to Z-glycoprotein, a cross-linking reaction and a decomposition reaction can occur in a mixed manner.
  • glycosaminodarlicans such as hyaluronic acid, chondroitin sulfate and heparan sulfate
  • the present invention provides a biological tissue derived from ⁇ -irradiation treatment, characterized in that the eluted protein is substantially absent in this embodiment.
  • substantially free of eluted protein means that a protein extract is prepared from a decellularized tissue by a known method, and the protein is detected by a general quantitative method such as Bio-Rad Protein assay.
  • the theoretical value (protein weight Z biological tissue weight) for which the calibration curve force is also calculated is 0.5 mgZmg or less, more preferably 0.2 mgZmg or less, still more preferably 0.1 mgZmg or less. .
  • the cross-linking caused by ⁇ -irradiation and the chemical cross-linking do not have a free amino group or carboxyl group in the side chain! These amino acids are distinguished according to whether or not they happened! / In other words, if it occurs only between two or more amino acids having a free amino group or carboxyl group in the side chain, it is due to chemical cross-linking, and if it also occurs between other amino acids, it means that ⁇ It can be said that this is a cross-linking caused by irradiation.
  • a group other than a carboxyl group, a hydroxyl group or an amino group for example, the group is a sulfhydryl group, An aldehyde group, a force group, a sulfo group and a group selected from the group consisting of a sulfo group force
  • the cross-linking is a carbon-carbon bond, an ether bond and an ester bond force.
  • a bond selected from the group It is discriminated according to whether or not it is formed. In particular, It may be characterized in that a skeleton is formed and a bond such as an intercarbon bond or an ester bond is cleaved to form a bridge by a new bond.
  • non-chemical cross-linking refers to cross-linking other than chemical cross-linking.
  • non-chemical crosslinking group refers to a group that does not form a chemical bridge, such as a sulfhydryl group, an aldehyde group, a carbo group, a sulfo group, or a -tro group. The powers mentioned are not limited to these.
  • the irradiation amount of y-ray irradiation used in the present invention is usually 10 to 250 kGy. Although not wishing to be bound by theory, it is possible that degeneration such as the extracellular matrix to remain should occur above about 250 kGy. The force required to maintain tissue strength such as extracellular matrix Any irradiation dose that does not cause denaturation can be used even if the irradiation dose exceeds this upper limit. Although not wishing to be bound by theory, it is possible to use ⁇ -ray irradiation even if it is less than lOkGy.
  • Irradiation times below lOkGy can also be used, as long as the irradiation time is considered to be significantly longer, as long as significant promotion of suppression of tissue calcification is observed, these are within the scope of the present invention. It will be understood by those skilled in the art.
  • the irradiation amount of ⁇ rays or the like is less than lOOkGy. I don't want to be bound by theory, but in the range below lOOkGy, there is also a force with almost no significant decrease in tissue strength.
  • the lower limit is 40 kGy or more. It is advantageous.
  • an irradiation amount of 50 to 80 kGy is advantageously used.
  • an irradiation amount of 50 to 80 kGy is advantageously used.
  • the irradiation time of gamma rays used in the present invention varies depending on the total irradiation dose, but is usually about 0.5 hours to 10 days, preferably 1 hour. It is advantageous to be about 2 days. More preferably, it is advantageous for 3 to 8 hours.
  • longer irradiation times promote suppression of calcification, but conventionally, extracellular matrix proteins such as collagen and elastin, which have elastic properties, are cleaved. Alternatively, it is cross-linked and becomes brittle with respect to mechanical deformation such as bending and pulling, so that there is a background that the properties as a transplanted tissue deteriorate. Therefore, since suppression of calcification depends in part on the total irradiation dose, it can be reacted in a few hours using a strong ⁇ -ray source.
  • the radical reaction eg, gamma irradiation
  • the radical reaction is performed in vacuum, oxygen, nitrogen, air, water, amphiphilic molecule solution and combinations thereof. It is performed in an atmosphere selected from a group of forces.
  • the biocompatible polymer used in the present invention is treated to coat the tissue.
  • Such coating can be performed by techniques well known in the art. For example, a method of immersing a biological tissue in such a biocompatible polymer solution or a method of applying by spraying or the like. Such as, but not limited to.
  • the biocompatible polymer of the present invention is advantageously cross-linked to a living tissue.
  • cross-linking makes the tissue of living tissue more robust.
  • Such cross-linking may be achieved by gamma irradiation, but may be another means.
  • the biocompatible polymer used in the present invention contains a biocompatible polymer. It can be degradable. Examples of such biodegradable polymers include, but are not limited to, polyethylene glycol, polyvinyl alcohol, polydaricholic acid, polylactic acid, and the like. Polyethylene glycol polymer is a biodegradable polymer with hydrophilicity. As the ethylene oxide content in the polymer increases, the hydrophilicity increases and swells in water.
  • the biocompatible polymer used in the present invention comprises polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, elastin and a mixture of two or more thereof.
  • a polymer selected from the group may be included. More preferably, the biocompatible polymer used in the present invention comprises polyethylene glycol.
  • the polyethylene glycol used in a preferred embodiment of the present invention typically has an average molecular weight between 200 and 6,000.
  • PEGs include Nacalai tesqu e polyethvlene glycol (H (OCH CH) OH) # molecular weight (eg 200, 600, 1
  • ⁇ PEG has an average molecular weight between 1,000 and 200,000.
  • the PEG can have an average molecular weight between 4,000 and 100,000. More preferably, the PEG can have an average molecular weight between 8,000 and 50,000.
  • PEG is commercially available, but can be appropriately synthesized in order to achieve desired characteristics. Such synthesis methods are well known in the art. In the present specification, the average molecular weight and the molecular weight are expressed by Dalton.
  • the PEG used in the present invention is preferably homogeneous in molecular weight, but this is not essential, and it can usually be used if the average molecular weight is within a certain range.
  • chemicals other than PEG for example, including but not limited to enzymes (eg, DNasel), enzyme inhibitors, etc.
  • the amount or concentration of PEG to be added may vary depending on the amount of the chemical present.
  • the concentration used for cell fusion or the like is good, for example, a concentration of lgZml or more (100% wZw or more) is preferable, but not limited thereto.
  • PEG can be used at a concentration between 60% w / v and 100% wZv.
  • the polybutyl alcohol used in the embodiment has a molecular weight in the range of 500 to 200,000. More preferably, the polybutyl alcohol used has a molecular weight of 500-100,000, more preferably ⁇ is 500-10,000.
  • the polyvinylpyrrolidone used in a preferred embodiment of the present invention has a molecular weight in the range of 500-200,000. More preferably, the polybutylpyrrolidone used has a molecular weight of 1.000 to 100,000, and more preferably 10,000 to 50,000 (for example, 40,000).
  • the biocompatible polymer used in a preferred embodiment of the present invention is used at a concentration of 1% (w / v) to 50% (wZv). More preferably, the biocompatible polymer can be used in the range of 5% (wZv) to 30% (wZv), more preferably the biocompatible polymer is 10 (w / v) to 20 % (w / v) can be used.
  • the tissue or organ may not be damaged so as to hinder the function of the normal function of the tissue or organ. preferable. This effect was substantially not damaged by the ⁇ -ray irradiation of the present invention. Whether there is such a hindrance can be determined by confirming that the extracellular matrix of the yarn and fabric is not substantially denatured.
  • the tissue of the present invention can also be characterized by the substantial functional presence of the extracellular matrix. The presence of the extracellular matrix can be confirmed by staining with a specific marker. The function of the extracellular matrix can be performed by selecting an appropriate assembly according to each member.
  • the biological tissue of the present invention has a tissue strength that allows clinical application. Sufficiently strong tissue strength is an important property, especially when applying membranous tissue clinically. Tissue strength can generally be determined by measuring tensile strength (eg, breaking strength, stiffness, Young's modulus, etc.).
  • Tissue strength can generally be determined by measuring tensile strength (eg, breaking strength, stiffness, Young's modulus, etc.).
  • the living tissue of the present invention has at least about 75%, preferably about 80% or more, more preferably about 85% or more of the tissue strength of the normal tissue before treatment. More preferably, it may be about 90% or more, or it may have substantially the same tissue strength. 110% or more, 1 20% or more, 150% or more, 200% or more).
  • the tissue strength when the tissue is in an untreated state is effective (for example, in a natural state) before the tissue is treated (for example, ⁇ -irradiation and treatment with a biocompatible molecule of the present invention). It refers to the strength of the tissue. Sufficiently strong tissue strength is a desirable characteristic even when applied to, for example, valve-like tissue and tubular tissue.
  • the calcification of the living tissue of the present invention is markedly suppressed when transplanted.
  • the degree of this suppression is, for example, (1) a calcium amount per 0.5 mg ( ⁇ g / mg) of transplanted pericardium extracted as a result of a long-term (2 months) rat transplantation experiment.
  • the amount of calcium at the time of treatment according to the present method can be exemplified by the amount of calcium at the time of Z-daltaraldehyde treatment being 0.5 or less, but is not limited thereto.
  • a level that is hardly stained can be achieved when Von Kossa staining is performed on the transplanted pericardium.
  • the biological tissue of the present invention is characterized in that a biocompatible polymer is randomly crosslinked.
  • the biocompatible polymer is randomly crosslinked by the treatment of the tissue derived from the living body by 7-ray irradiation of the present invention. Increased suppression effect was achieved.
  • the tissue strength can be represented by a ⁇ value.
  • the method for calculating the ⁇ value was described in detail elsewhere in this specification.
  • the living tissue of the present invention has a tissue strength of 13 values of about 15 or more, preferably has a tissue strength of 13 values of about 18 or more, more preferably
  • the biological tissue of the present invention is at least about 75% or more, preferably about 80% or more, more preferably about 85% or more, more preferably about the j8 value that the tissue before treatment had.
  • the biological tissue of the present invention may be any tissue of the body (for example, a valve-like tissue, a tubular tissue, a membrane-like tissue, etc.) as long as the tissue is intended for clinical application.
  • the biological tissue of the present invention can be a tissue in which the physical structure of the tissue is required.
  • the biological tissue of the present invention can be a tissue derived from a blood vessel, blood vessel-like tissue, heart valve, pericardium, dura mater, cornea and bone strength selected organ.
  • the biological tissue of the present invention can be cardiovascular tissue, for example, from an organ selected from blood vessels, blood vessel-like tissue, heart valves and pericardium.
  • the living tissue of the present invention undergoes adhesion between the cut surface or defect and its surrounding tissue after excision of the affected area or repair of the damaged site in surgery or treatment. Can be used to prevent it from occurring.
  • the living tissue of the present invention has at least one crosslink formed between non-chemical crosslinkable groups other than a carboxyl group, a hydroxyl group and an amino group.
  • the non-chemical crosslinking group include, but are not limited to, a sulfhydryl group, an aldehyde group, a carbo group, a sulfo group, and a -tro group.
  • the biological tissue of the present invention has at least one cross-link formed by gamma irradiation and formed from a carbon-carbon bond, an ether bond or an ester bond.
  • the living tissue of the present invention has at least one cross-link formed by a new bond formed by cleavage of an intercarbon bond or ester bond forming a skeleton.
  • the biological tissue of the present invention has at least one bridge formed between amino acids without a free amino group and a carboxyl group in the side chain.
  • the bridge formed between the amino acids having no free amino group and carboxyl group in the side chain is a bridge between amino acids other than lysine and arginine.
  • the living tissue of the present invention has at least a cross-link formed between polysaccharides having non-chemical cross-linking groups other than a carboxyl group, a hydroxyl group and an amino group.
  • This polysaccharide having a non-chemical crosslinking group is composed of a sulfhydryl group, an aldehyde group. , Having a bridge formed between at least one group selected from the group consisting of a carbonyl group, a sulfo group, and a -tro group.
  • the biological tissue of the present invention undergoes non-chemical crosslinking other than bisepoxide crosslinking, dibutylsulfone crosslinking, intramolecular esterification, glutaraldehyde crosslinking, carbodiimide crosslinking, and hydrazide crosslinking. Have at least one.
  • the living tissue of the present invention has no free amino group or carboxyl group in the side chain, and a covalent bond, carboxyl group, hydroxyl group formed between two or more amino acids.
  • the biological tissue of the present invention may further contain a crosslink formed by bonding with a bifunctional molecular crosslinker. This bifunctional molecular crosslinking agent is preferably dartalaldehyde.
  • the biological membrane of the present invention is obtained by chemically treating the biological membrane with dartalaldehyde, exposing it to polyethylene glycol (PEG), and irradiating ⁇ rays to form a crosslink. Compared to those treated with dartalaldehyde, the tissue strength after ⁇ -irradiation is increased, or the tissue strength is the same as that treated with dartalaldehyde. And a PEG layer is formed on the surface of the biological membrane.
  • PEG polyethylene glycol
  • the present invention provides adhesion between the cut surface or defect and the surrounding tissue after excision of the affected part or repair of the damaged site in surgery or treatment.
  • An anti-adhesion membrane for preventing the above is provided.
  • the anti-adhesion membrane of the present invention can be prepared using, for example, the pleura, pericardium, cerebral dura mater, serosa, peritoneum or skin.
  • the adhesion-preventing film of the present invention can prevent adhesion when, for example, treatment is performed in the heart, lung, liver, brain, digestive organ, excision site of the gallbladder, or a damaged site.
  • the anti-adhesion membrane of the present invention can also be used ex vivo. As an ex vivo, it can be used, for example, to store tissue for transplantation.
  • the method of producing an adhesion-preventing membrane for transplantation according to the present invention comprises: A) a step of providing a biologically-derived membrane; B) a step of exposing the biologically-derived membrane to a biocompatible polymer ; And C ) Including a step of exposing the biological membrane to ⁇ -ray irradiation.
  • the method for producing an adhesion-preventing membrane for transplantation according to the present invention can further include the step of D) chemically crosslinking the biological membrane with a bifunctional molecular crosslinking agent. Step D) may be performed between step ii) step—step ii) and at least one selected from the group consisting of step ii) step-step ii).
  • the bond formed by the bifunctional molecular crosslinking agent used in the present invention is selected from the group consisting of a sulfhydryl group, an aldehyde group, a carbon group, a sulfo group, and a -tro group. It can be a bond formed between groups, a carbon-carbon bond, a peptide bond, an ether bond or an ester bond.
  • This bifunctional molecular crosslinker may have a group selected from the group consisting of an amino group, a carboxyl group and an aldehyde group.
  • bifunctional molecular crosslinkers include dartalaldehyde, cyanimide, 1-ethyl 3- (3 dimethylaminopropyl) carbodiimide hydrochloride (EDC), epoxy (poly (glycidyl ether)), 2 , 6 bis (4azidobenzylidene) -4-methylcyclohexanone, 4,4'diazidodiphenyl ether, 4,4'diazidodiphenylsulfone, 4,4'diazidodiphenylacetone, 4,4'diazidodiphenylmethane, 1, 4 bis ( ⁇ diazobenzyl) and 4— [p azidosalicyamido] ptyramine are included, but not limited to.
  • the bifunctional molecule is dartalaldehyde.
  • the biocompatible polymer may be selected from polyvinyl alcohol, polyvinyl pyrrolidone, elastin, polyethylene glycol, gelatin, collagen, ⁇ -polyglutamic acid and a mixture of two or more thereof.
  • the biocompatible polymer comprises polyethylene glycol.
  • the method for producing the anti-adhesion membrane of the present invention can be provided by removing the biological membrane from livestock including humans or ushi or pigs in step i).
  • the biocompatible polymer is exposed to the biological membrane under conditions of 24 hours to 37 ° C for 1 hour to 4 hours, or 24 ° C for 12 to 24 hours. Can be done.
  • the intensity of ⁇ -rays is 50 keV, and irradiation with ⁇ -rays can be carried out in a sealed container at a dose of 12 kGy to 10 OkGy under conditions of 24-37 ° C.
  • the irradiation of ⁇ rays has a dose of 12-50 kGy.
  • Gamma rays can be irradiated for 1-4 hours.
  • chemical crosslinking can occur at 24 ° C under conditions of 1 hour to 4 hours. Chemical crosslinking 1 It can occur even with time and conditions.
  • the present invention can provide an adhesion-preventing membrane derived from a living body.
  • the anti-adhesion membrane of the present invention contains a biocompatible polymer and is characterized by receiving ⁇ -ray irradiation.
  • the adhesion-preventing film of the present invention has at least one crosslink formed between non-chemical crosslinkable groups other than carboxyl group, hydroxyl group and amino group. Examples of the non-chemical crosslinking group include, but are not limited to, a sulfhydryl group, an aldehyde group, a carbo group, a sulfo group, and a -tro group.
  • the anti-adhesion membrane of the present invention has at least one cross-link formed by ⁇ -ray irradiation and formed from a carbon-carbon bond, an ether bond or an ester bond.
  • the adhesion-preventing film of the present invention has at least one cross-link formed by a new bond formed by cleavage of an inter-carbon bond or an ester bond forming a skeleton.
  • the anti-adhesion membrane of the present invention has at least one crosslink formed between amino acids having no free amino group and carboxyl group in the side chain.
  • the bridge formed between amino acids without a free amino group and carboxyl group in the side chain is a bridge between amino acids except for lysine and arginine.
  • the anti-adhesion membrane of the present invention has at least one crosslinking formed between polysaccharides having non-chemical crosslinking groups other than carboxyl groups, hydroxyl groups and amino groups.
  • This polysaccharide having a non-chemical crosslinking group has a bridge formed between at least one group selected from the group consisting of a sulfhydryl group, an aldehyde group, a carbonyl group, a sulfo group, and a -tro group.
  • the anti-adhesion membrane of the present invention comprises a non-chemical crosslink other than bisepoxide crosslinks, dibule sulfone crosslinks, intramolecular esterification, dartalaldehyde crosslinks, carpositimide crosslinks and hydrazide crosslinks Have at least one.
  • the anti-adhesion membrane of the present invention has a free amino group or a force lpoxyl group in the side chain.
  • the adhesion-preventing film may further contain a crosslink formed by bonding with a bifunctional molecular crosslinker. This bifunctional molecular crosslinker is preferably dartalaldehyde.
  • the anti-adhesion membrane of the present invention preferably contains polyethylene glycol.
  • the anti-adhesion membrane of the present invention is the pleura, pericardium, brain dura mater, serosa, peritoneum or skin, but is not limited thereto.
  • the anti-adhesion membrane is derived from a mammal. More preferably, it is derived from human, ushi or pig.
  • the anti-adhesion membrane of the present invention is: A) (1) The pericardium is sandwiched between thin plastic sheets, (2) The thickness is measured at three or more locations with an electronic caliper so that the sample does not deform. And (3) having a thickness of 0.5 1. Omm when measured by the process of calculating the average thickness of the sample; and B) (1) cutting the specimen into 5 x 30 mm strips. (2) The step of fixing the both ends of the specimen to about 5 mm to the fixed part of the tensile tester, and (3) The step of starting pulling to the breaking point at a speed of lcmZmin and measuring with a radiometer. The case is characterized by having an elastic modulus of 2-lOMPa.
  • the anti-adhesion membrane of the present invention comprises
  • Anti-adhesion rate (%) ⁇ number without adhesion Z (number with adhesion + number without adhesion) ⁇ X 100
  • the adhesion-preventing membrane of the present invention is obtained by chemically treating the biological membrane with dartalaldehyde, exposing it to polyethylene glycol (PEG), and irradiating ⁇ rays to form a crosslink. Compared to those treated with dartalaldehyde, the tissue strength after ⁇ -irradiation is increased, or the tissue strength is the same as that treated with dartalaldehyde. And a PEG layer is formed on the surface of the biological membrane.
  • PEG polyethylene glycol
  • the biological tissue of the present invention may be any biological tissue as long as it is suitable for the intended clinical application. Therefore, the tissue of the present invention may be a tissue derived from any organism (for example, vertebrate animals, invertebrates).
  • tissue from vertebrates is preferably used, and more preferably tissue from mammals (eg, primates, cloven-hoofed, odd-hoofed, rodents, etc.) Is used.
  • mammals eg, primates, cloven-hoofed, odd-hoofed, rodents, etc.
  • primate-derived tissue is used.
  • porcine-derived tissue is used. It is also a force that is similar in size to humans.
  • most preferably human-derived tissue is used.
  • the size of the living tissue or tissue graft of the present invention is preferably close to that of a human when its non-human size is used, and its physical characteristics are close to that of a human.
  • Preferred eg pig.
  • the anti-adhesion membrane of the present invention can be used both in vivo and in vitro.
  • the part to which the living tissue of the present invention is applied may be any part in the body. Therefore, it may be applied again to the part of the body from which the biological tissue is derived, or may be applied to other parts. As shown in the examples and the like, the present invention can achieve a desired effect (for example, regeneration, self-organization) even if a biological tissue is applied to any part regardless of whether or not to “revert”. Proven to achieve. Therefore, the present invention has great utility in principle that it can be used for any transplantation and regenerative surgery.
  • Examples of the site to which the living tissue of the present invention can be applied include, for example, skin, blood vessels, cornea, kidney, heart, liver, umbilical cord, intestine, nerve, lung, placenta, spleen, brain, peripheral limbs, retina, valve, epithelium.
  • Examples include, but are not limited to, tissue, connective tissue, muscle tissue, and nerve tissue.
  • the present invention provides a tissue graft containing the biological tissue of the present invention.
  • the biological tissue is seeded and cultured with recipient-derived cells to form a desired tissue structure.
  • the tissue graft of the present invention may be intended for transplantation into any tissue of the body as long as it is a tissue graft intended for clinical application.
  • the tissue draft of the present invention may be a tissue in which the physical structure of the tissue is required.
  • cells from the recipient can be provided to the above-described living tissue prior to transplantation.
  • the recipient-derived cells may be supplied internally from the host.
  • the tissue graft of the present invention may be a tissue graft of a tissue derived from a vessel, blood vessel-like tissue, heart valve, pericardium, dura mater, cornea and bone force selected organ.
  • the tissue graft of the present invention can be a tissue graft of cardiovascular tissue, such as a tissue graft derived from an organ selected from blood vessels, blood vessel-like tissues, heart valves and pericardium obtain.
  • the tissue graft of the present invention may contain any biological tissue as long as it is compatible with the intended clinical application. Therefore, the tissue graft of the present invention may be a tissue derived from any organism (eg, vertebrate, invertebrate).
  • tissue from vertebrates is preferably used, and more preferably, tissue from mammals (eg, primates, rodents, etc.) is used in the tissue graft of the present invention. Used.
  • tissues derived from primates are used in the tissue graft of the present invention.
  • porcine tissue is used for the tissue graft of the present invention. This is because the size is similar to humans.
  • most preferably human-derived tissue is used for the tissue graft of the present invention.
  • the recipient cells used in the yarn and woven graft of the present invention may be any cells as long as they are suitable for clinical application. Accordingly, examples of such cells include, but are not limited to, vascular endothelial cells, smooth muscle cells, fibroblasts, blood cells, progenitor cells and somatic stem cells that differentiate into these cells.
  • the cell may be a cell that can perform a desired function at a site to be transplanted.
  • the site to which the tissue graft of the present invention is applied may be any site in the body. Thus, it may be reapplied to the part of the body from which the tissue graft originates or may be applied to other parts. As shown in the examples, the present invention achieves the desired effect (for example, regeneration, self-organization) regardless of whether the tissue graft is applied regardless of whether it is “undoed” or not. Proven to do. Therefore, the present invention has great utility in principle that it can be used for any transplantation and regenerative surgery.
  • tissue graft of the present invention examples include skin, blood vessel, cornea, kidney, heart, liver, umbilical cord, intestine, nerve, lung, placenta, spleen, brain, peripheral extremity, retina, valve, Examples include, but are not limited to, epithelial tissue, connective tissue, muscle tissue, and nerve tissue.
  • the present invention provides membranous tissue grafts, valvular tissue grafts, tubular tissue drafts, and the like.
  • This tissue graft includes the biological tissue of the present invention.
  • the living body-derived tissue may be seeded with recipient-derived cells and cultured to form a desired tissue structure, but the cells may not be present. It is advantageous that there are preferably no cells other than autologous cells. This is because immune rejection is not caused.
  • the present invention relates to a method for producing a biologically derived yarn and weaving, comprising A) providing a biologically derived tissue; B) exposing the biologically derived tissue to a biocompatible polymer. And C) exposing the biological tissue to ⁇ -irradiation. Any biological tissue may be used.
  • the biocompatible polymer used is, for example, polybutanololone, polyvinylpyrrolidone, elastin, polyethylene glycol, gelatin, collagen, ⁇ -polyglutamic acid and mixtures of two or more thereof Etc.
  • polyethylene glycol is used.
  • the step of exposing to the biocompatible polymer used in the present invention includes performing ⁇ -ray irradiation so that the biocompatible polymer is crosslinked. .
  • the dose of gamma irradiation used in the present invention is in the range of 10 to 250 kGy. More preferably, the ⁇ -ray irradiation dose is in the range of 50 kGy to 100 kGy.
  • such an irradiation dose is appropriate for increasing the tissue strength. It is appropriate to set the upper limit of the dose that can maintain the tissue strength according to the tissue. From the viewpoint of tissue strength, there are cases where 60 kGy is stronger than lOOkGy, but it is not limited to this.
  • ⁇ -ray irradiation is usually performed in an environment selected from the group consisting of vacuum, oxygen, nitrogen, air, water, amphiphilic molecular solutions, and a combination force thereof. Power that can be broken Not limited to them. Preferably, it is performed in the atmosphere.
  • the gamma irradiation used in the present invention is performed in the range of 0.5 to 240 hours, more preferably 1 hour to 24 hours, and further preferably 3 to 8 hours.
  • a strong ⁇ -ray source may be used. I don't want to be bound by theory, but if it's too weak, it will take a long time and the organization itself can be damaged by other factors.
  • any biocompatible polymer may be used in the present invention.
  • the average molecular weight of PEG used is between 200 and 6,000, and preferably the average molecular weight of PEG is between 200 and 2,000.
  • the PEG used in the present invention can have various average molecular weights as described above, and a desired effect (for example, suppression of calcification) can be achieved by changing the treatment time (immersion time) according to the average molecular weight. Can do.
  • the average molecular weight of the PEG used in the present invention is between 1,000 and 200,000.
  • the average molecular weight of the PEG used in the present invention is between 4,000 and 100,000.
  • the average molecular weight of PEG used in the present invention is between 8,000 and 50,000.
  • the biocompatible polymer is exposed to the tissue under any conditions as long as the living tissue and the biocompatible polymer interact with each other. Good, but can usually be performed between 0 ° C and 42 ° C and may be performed at room temperature (between about 15 ° C and about 25 ° C) or at 37 ° C . This step may be performed at a temperature above 37 ° C.
  • the biocompatible polymer such as PEG may be present in the solution at any concentration, but is preferably present at a concentration of lgZml or more. . I do not want to be bound by theory, but ⁇ rays are expected to function as radical scavengers.
  • the concentration of the PEG solution may be any concentration, but should be about 1% to about 50%, about 10% to 30%. For example, about 20% is used, but is not limited thereto.
  • the biocompatible polymer for example, PEG
  • the biocompatible polymer may be dissolved in any solvent, but is preferably dissolved in an aqueous medium, more preferably physiological saline, PB. s, or an aqueous solution containing other salts.
  • PEG for example, molecular weight 1,000
  • the biocompatible polymer may be dissolved in any solvent, but is preferably dissolved in an aqueous medium, more preferably physiological saline, PB. s, or an aqueous solution containing other salts.
  • polymers that are biocompatible and can be used in pharmaceutical grades.
  • PEG (1) SUNBRIGHT—MEH series manufactured by NOF Corporation High purity methoxy PEG, etc. (http: ZZwww. Nof.
  • polysaccharides generally appear to react predominantly with degradation over crosslinking by gamma irradiation. Therefore, it is thought that it can be used more effectively when mixed with the above-mentioned crosslinkable polymer as a filler.
  • biocompatible polymers are, for example, those listed in the Japanese Pharmacopoeia (or corresponding pharmacopoeia in other countries), or the Ministry of Health, Labor and Welfare (or other applicable countries). It can be a polymer approved by the government.
  • the method of the present invention further comprises the step of washing the tissue immersed in the solution.
  • This washing step can be performed using any liquid that is physiologically compatible (eg, physiological saline, PBS (phosphate buffered saline), etc.).
  • the washing of the present invention is performed with PBS.
  • the cleaning solution is preferably sterilized. More preferably, the wash solution includes an antibiotic (eg, penicillin, cephalosporin, streptomycin, tetracycline, erythromycin, noncomomycin, ciprofloxacin, linezlid, etc.).
  • an antibiotic eg, penicillin, cephalosporin, streptomycin, tetracycline, erythromycin, noncomomycin, ciprofloxacin, linezlid, etc.
  • washing is done under any conditions However, it can usually be performed between 0 ° C and 42 ° C, and can be performed at room temperature (between about 15 ° C and about 25 ° C) and at 37 ° C. Also good. Washing may be performed at a temperature above 37 ° C. In the method of the present invention, the washing step may be performed for any period as long as the solution used in the treatment (for example, a solution containing a biocompatible polymer) is sufficiently removed. 0. Can be done for 5 days to 5 days. Preferably, in the washing step, the washing solution (eg, PBS) may be changed several times.
  • the washing solution eg, PBS
  • the tissue used in the method of the present invention may be any biological thread and weave as long as it is compatible with the intended clinical application. Therefore, the tissue used in the method of the present invention may be a tissue derived from any organism (for example, vertebrates and invertebrates).
  • the tissue used in the method of the present invention is preferably a vertebrate tissue, more preferably a mammal (eg, primate, rodent, etc.). Origin tissue is used.
  • the tissue used in the method of the present invention is more preferably a primate-derived tissue. If intended for human application, more preferably, tissue from pigs or sushi is used. It is also a force that is similar in size to humans. When intended for human use, most preferably human-derived tissue is used.
  • the tissue used in the method of the present invention may be any tissue of the body as long as it is intended for clinical application.
  • the tissue used in the methods of the invention may be a tissue that requires the physical structure or physical properties of the tissue. In order to maintain the physical structure or physical properties, only the structure such as the extracellular matrix is necessary, and the intracellular components such as the cytoplasmic component or the cell membrane component are necessary! /. Further, if necessary, the required cells can be separately provided to the tissue derived from the living body, or can be supplied inside the transplanted host cell.
  • the tissue used in the methods of the present invention may be a tissue derived from a blood vessel, blood vessel-like tissue, heart valve, pericardium, dura mater, cornea and bone strength.
  • the tissue used in the methods of the invention can be cardiovascular tissue, such as from an organ selected from blood vessels, blood vessel-like tissue, heart valves and pericardium.
  • the present invention may further include chemical treatment.
  • it can be a treatment with a bifunctional molecular crosslinking agent (eg, dartalaldehyde or a derivative thereof).
  • a bifunctional molecular crosslinking agent eg, dartalaldehyde or a derivative thereof.
  • the purpose of the treatment with the bifunctional molecular crosslinking agent is to increase the physical strength by chemically crosslinking ECM or protein components including cells in the tissue. Therefore, as long as it is used for this purpose, any bifunctional molecular crosslinking agent can be used.
  • bifunctional molecular crosslinkers those actually used for tissue fixation (valve grafting) are, for example, cyanimide, 1-ethyl 3- (3 dimethylaminopropyl) carbodiimide.
  • EDC Hydrochloride
  • epoxy poly (glycidinoreetherenole)
  • BAMC 2,6-bis (4 azidobenzylidene) -4-methylcyclohexanone
  • 4 ' diazide diphenyl ether
  • 4 ' include, but are not limited to, diazidodiphenylsulfone, 4,4'diazidodiphenylacetone, 4,4'diazidediphenylmethane, 1,4bis ( ⁇ diazobenzyl) and 4— [p azidosalithiomide] butyramine (see Biomaterials ( 2 000) 21: 2215—2231).
  • the chemical treatment may include treatment with a nuclease such as DNase, such as DNasel. Since treatment with such DNase can further remove DNA components that are undesirable for transplantation, this treatment with DNase is preferred.
  • DNase may be any DNase, but may preferably be DNasel.
  • DNA which is a charged polymer substance, can be removed by DNase treatment. Since DNA can elicit an immune response, further removal of DNA can provide additional benefits.
  • the treatment with a nuclease may be carried out under any conditions, but may be combined with a caloric pressure condition, a stirring condition, and the like.
  • concentration include lUZml to 10 OUZml, and examples include, but are not limited to, 50 to 75 UZml.
  • the nuclease treatment can be performed, for example, for about 0.5 to 5 days. Among these, the first few hours (for example, 1 to 24 hours) are applied under pressure conditions (for example, 100 to 1000 kPa (for example, 500 kPa). )). Then, it can also carry out on stirring (for example, 100-50 ORPM, Preferably it is 100-150 RPM) conditions.
  • the method for producing a biological tissue of the present invention may further include a step of seeding cells, but is not limited thereto.
  • the cells to be seeded can be appropriately selected by those skilled in the art depending on the situation, as described above in the present specification.
  • the present invention also provides a biological tissue obtained by the method of the present invention.
  • This organism-derived tissue may preferably have the above-mentioned cell survival rate and Z or tissue damage rate and Z or tissue strength.
  • the method of the present invention provides a completely new substance that provides a tissue derived from a living body having characteristics such as the suppression of forced calcification that cannot be provided by conventional methods. Provide benefits
  • the present invention provides a tissue regeneration method comprising: a) a biological tissue containing a biocompatible polymer and receiving ⁇ -irradiation in vivo. And b) incubating for a time sufficient for tissue regeneration to occur in the living body. If necessary, do not provide cells to living tissue, which may include a step of providing a physiologically active substance that induces differentiation of the cells and a step of supplying cells to Z or this living tissue. May be.
  • the physiologically active substance those necessary for maintaining or separating the target tissue are used.
  • the physiologically active substance may be derived from in vivo or derived from outside the living body.
  • physiologically active substance examples include, but are not limited to, HGF, VEGF, FGF, IGF, PDGF, and EGF.
  • the cell may be a vascular cell or a blood vessel-like cell. More preferably, the cell may be derived from the recipient.
  • the tissue may be a tissue selected from the group consisting of blood vessels, vascular-like tissue, heart valves, pericardium, dura mater, cornea and bone strength.
  • the tissue and the cell can be from the same host.
  • the tissue and the cell can be from an allogeneic host.
  • the tissue and the cell can be from a heterologous host.
  • a rejection may be considered, so the recipient and the cell are preferably from the same host, but if the rejection is not a problem, the same species It may be of different origin or different origin.
  • those that cause rejection can be used by performing treatment to eliminate rejection as necessary.
  • Procedures for avoiding rejection are well known in the art and are described, for example, in the New Surgery System, Heart Transplantation, Lung Transplantation Technical and Ethical Preparations for Implementation (Revised 3rd Edition). . Examples of such methods include methods such as the use of immunosuppressants and steroids.
  • Immunosuppressants that prevent rejection are currently “cyclosporine” (Sandemiyun Yoon Z Neoral), “Tacrolimus” (Prograf), “Azathioprine” (Imran), “Steroid Hormone” (Predonin, Methylpredonin), There are “T-cell antibodies” (OKT3, ATG, etc.), and the method used in many facilities around the world as a preventive immunosuppressive therapy is a combination of three drugs, “cyclosporine, azathioprine and steroid hormones”.
  • the immunosuppressive agent is not necessary in the application of the medicament of the present invention, but it is desirable that it be administered at the same time when administered. However, it is not always necessary. Therefore, when an immunosuppressive agent is used, the immunosuppressive agent can be administered before or after performing the regeneration method as long as the immunosuppressive effect is achieved.
  • the present invention relates to a method for producing a yarn and woven graft, comprising: i) a step of providing a living tissue containing a biocompatible polymer in vivo; And a step of incubating for a period of time sufficient for the differentiation of the cells to occur.
  • the living tissue of the present invention may or may not further have cells.
  • Such cells can be autologous, allogeneic, allogeneic or xenogeneic.
  • the cell can be a vascular cell or a blood vessel-like cell. More preferably, the cell may be derived from the recipient.
  • the tissue may be that of a tissue selected from the group consisting of blood vessels, blood vessel-like tissue, heart valves, pericardium, dura mater, cornea and bone.
  • this organization The cells may be from the same host.
  • the tissue and the cell can be from an allogeneic host.
  • the tissue and the cell can be from a heterologous host. If the recipient and the cell are allogeneic or xenogeneic, rejection may be possible, so the recipient and the cell are preferably from the same host, but if the rejection is not a problem allogeneic It can be of different origin or different origin. In addition, those that cause rejection can be used by performing treatment to eliminate rejection as necessary. Procedures for resolving rejection are detailed herein.
  • the method for producing the tissue graft of the present invention may further include the step of D) providing a physiologically active substance that induces differentiation of the cells.
  • the physiologically active substance can be a cytodynamic force having hematopoietic activity.
  • this site force-in can be HGF, VEGF, FGF, and the like.
  • the physiologically active substance may be autologous or exogenous. It should be noted that if the bioactive substance is autologous, the goal is achieved simply by passing a certain amount of time after transplantation.
  • the present invention provides a native tissue or tissue graft produced by the method of the present invention.
  • a biological tissue or tissue graft has unique features that are not present in terms of tissue strength and calcification inhibition rate.
  • the present invention provides a method for treating or preventing a force requiring tissue or organ transplantation or a subject at risk thereof, comprising A) a biocompatible polymer, and gamma rays Providing a biological tissue characterized by being irradiated, or a tissue graft containing the biological tissue; and i) transferring the biological tissue or tissue graft to a subject.
  • This biological tissue further contains cells as necessary. The cells may be autologous or allogeneic.
  • the tissue may be that of a tissue selected from the group consisting of blood vessels, blood vessel-like tissue, heart valves, pericardium, dura mater, cornea and bone.
  • the tissue can be from a subject. In another embodiment, the tissue can be from a host allogeneic to the subject. In another embodiment, the tissue can be from a host heterologous to the subject. .
  • this treatment z prevention method of the present invention since a tissue-derived tissue that is suppressed to a tissue damage rate that can withstand transplantation and is sufficiently suppressed in calcification is used, no rejection reaction occurs. . However, when rejection occurs in the force or when cells other than those derived from the recipient are used, when rejection occurs, treatment can be performed to eliminate the rejection as necessary. The procedure for resolving rejection is described in detail herein.
  • the tissue used in the method of treating or preventing of the present invention may be derived from a subject.
  • the tissue used in the method of treatment or prevention of the present invention may be tissue from any organism (eg, vertebrate, invertebrate).
  • tissue from spinal animals is used when humans are treated or prevented, and more preferably from mammals (eg, primates, rodents, etc.) when humans are treated or prevented Organization is used.
  • mammals eg, primates, rodents, etc.
  • tissue from pigs or sushi are used. Pigs or sushi are preferred because they have a size similar to humans.
  • humans are treated or prevented most preferably human-derived tissue can be used.
  • step B) can also be performed at a site where adhesion needs to be prevented.
  • the present invention provides a medicament for organ transplantation.
  • This medicine includes A) a biological tissue containing a biocompatible polymer and receiving ⁇ -ray irradiation, or a tissue graft containing the biological tissue.
  • the medicaments of the present invention include blood vessels, blood vessel-like tissues, heart valves, pericardia, dura mater, corneas and bone strength tissue derived from selected organs.
  • the medicament of the present invention may comprise cardiovascular tissue, for example, derived from an organ selected from blood vessels, blood vessel-like tissue, heart valves and pericardium.
  • the medicament of the present invention may contain any biological tissue as long as it is compatible with the intended clinical application. Preferably, it may contain materials approved by the applicable national supervisory authority. Therefore, the medicament of the present invention may include tissues derived from any organism (for example, vertebrates and invertebrates).
  • tissues derived from vertebrates are used, and more preferably, tissues derived from mammals (eg, primates, rodents, etc.) are used.
  • primate-derived tissue is preferably used.
  • a tissue derived from pig or ushi is used. This is because the size is similar to humans.
  • human-derived tissue is most preferably used. However, when using human-derived tissue, it may be necessary to clear ethical rules and issues.
  • the medicament, tissue graft and biological tissue of the present invention may further comprise a biocompatible material.
  • This biocompatible material is, for example, silicone, collagen, gelatin, glycolic acid 'lactic acid copolymer, ethylene vinyl acetate copolymer, polyurethane, polyethylene, polytetrafluoroethylene, polypropylene, polyacrylate, It may comprise at least one selected from the group consisting of polymetatalates. Silicone is preferred because it is easy to mold.
  • biodegradable polymers include collagen, gelatin, a-hydroxyluronic acids (eg, glycolic acid, lactic acid, hydroxybutyric acid, etc.), hydroxydicarboxylic acids (eg, malic acid), and hydroxytricarboxylic acids (eg, A polymer, a copolymer or a mixture thereof synthesized by non-catalytic dehydration polycondensation from one or more selected from the group consisting of cuenic acid, etc., poly-a cyanoacrylate, poly-amino acid (for example, poly ⁇ -base) Njiru L glutamic acid, etc.), and polyanhydrides of maleic anhydride copolymers (eg, styrene maleic acid copolymers).
  • a-hydroxyluronic acids eg, glycolic acid, lactic acid, hydroxybutyric acid, etc.
  • hydroxydicarboxylic acids eg, malic acid
  • hydroxytricarboxylic acids e
  • the form of polymerization may be random, block, or graft.
  • a-hydroxycarboxylic acids, hydroxydicarboxylic acids, and hydroxytricarboxylic acids have an optically active center in the molecule, they are of D-form, L-form, and DL-form. Either can be used.
  • a glycolic acid / lactic acid copolymer may be used.
  • the medicament, tissue graft and biological tissue of the present invention may further contain other drugs.
  • Such an agent can be any agent known in the pharmaceutical arts.
  • the medicament, tissue graft and biological tissue of the present invention may contain two or more kinds of other drugs.
  • examples of such drugs include Japanese pharmacopoeia, US pharmacopoeia, and pharmacies in other countries.
  • the ones listed in the latest version of the Such an agent may preferably have an effect on the organ of the organism.
  • examples of such agents include thrombolytic agents, vasodilators, and tissue activators.
  • the amount of physiologically active substances, other drugs and cells contained in the medicament, tissue graft and biological tissue of the present invention depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, It can be easily determined by those skilled in the art in consideration of gender, medical history, and the like.
  • the present invention provides a biological tissue or a biotissue containing the biocompatible polymer of the present invention for producing a medicament for organ transplantation, which has been subjected to y-ray irradiation.
  • a tissue graft comprising a biological tissue characterized in that it comprises a biocompatible polymer of the invention and has been subjected to y-ray irradiation.
  • tissue derived from an organ selected from blood vessels, blood vessel-like tissue, heart valves, pericardium, dura mater, cornea and bone may be used in the use of the present invention.
  • cardiovascular tissue can be used, for example, derived from an organ selected from blood vessels, blood vessel-like tissues, heart valves and pericardium.
  • any biological tissue can be used as long as it is suitable for the intended clinical application.
  • materials approved by the applicable national regulatory authority may be used.
  • tissue from any organism eg, vertebrate, invertebrate
  • vertebrate tissue is preferably used, and more preferably, mammal (eg, primate, rodent, etc.) tissue is used. It is done.
  • primate-derived tissue is more preferably used.
  • a tissue derived from butterfly or sushi is used. This is because the size is similar to humans.
  • human-derived tissue is most preferably used. However, when using human-derived tissue, it may be necessary to clear the 'Code of Ethics' issue.
  • the amount of the biologically-derived yarn and tissue, graft, and medicament used in the treatment or prevention method of the present invention is the use purpose, target disease (type, severity, etc.), subject's age, weight, sex, A person skilled in the art can easily determine the past history, the form or type of the physiologically active substance, the form or type of the tissue, and the like.
  • the frequency with which the method of the present invention is applied to a subject (or patient) also depends on the amount of biologically derived tissue, graft and medicine used per time, purpose of use, target disease (type, severity, etc.) ), And can be easily determined by those skilled in the art in consideration of the patient's age, weight, sex, medical history, course of treatment, and the like. Examples of the frequency include administration every day to once every several months (for example, once a week to once a month). It is preferable to administer once a week to once a month while monitoring the course.
  • the biological tissue, graft and Z or medicament of the present invention can be provided in the form of a kit comprising instructions for administering the biological tissue, graft and Z or medicament.
  • the above-mentioned instruction manual describes a word indicating an appropriate administration method of a tissue derived from a living body, a graft, and Z or a medicine. This instruction is prepared in accordance with the format prescribed by the national supervisory authority (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States) in the United States. It will be clearly stated that it has been approved.
  • package inserts which are usually provided in paper media, but are not limited thereto, such as electronic media (for example, homepages and emails provided on the Internet). It can also be provided in form.
  • the living tissue, tissue graft, and medicament of the present invention can be transplanted using techniques well known in the art (for surgery, standard non-standard science 9th edition (medical school)) See surgical procedures (P41—p66), heart (p349—p398), blood vessels (p399—428), etc.).
  • the biological tissue of the present invention can be used for vascular anastomosis, patch closure, artificial blood vessel replacement, artificial valve replacement, and the like. Therefore, a person skilled in the art can appropriately apply the living tissue, tissue graft and medicament of the present invention according to the disclosure of the present specification, depending on the situation to be treated.
  • the site to which the medicament of the present invention is applied may be any site in the body. Thus, it may be reapplied to the part of the body from which the medicine is derived, or it may be applied to other parts. As shown in the examples, the present invention achieves the desired effect (for example, regeneration, self-organization) regardless of whether the drug is applied regardless of whether it is ⁇ reverted '' or not. Proven to do. Therefore, the present invention has great utility in principle that it can be used for any transplantation surgery and regenerative surgery.
  • Examples of the site to which the medicament of the present invention can be applied include skin, blood vessel, cornea, kidney, heart, liver, umbilical cord, intestine, nerve, lung, placenta, spleen, brain, extremity, retina, valve, and epithelial tissue. Include, but are not limited to, connective tissue, muscle tissue, nerve tissue, and the like.
  • the present invention provides a method of treating or preventing a subject in need or at risk of needing to prevent tissue adhesions.
  • the method comprises the steps of A) providing an anti-adhesion membrane comprising a biocompatible polymer and receiving x-ray irradiation; and B) implanting the anti-adhesion membrane into a subject. .
  • Kanamycin lOOmgZl (SIGMA)
  • PEG molecular weight 8,000, 35,000, 50,000
  • Usika also removed the pericardium and treated it with polyethylene glycol as the biocompatible polymer. The protocol is shown below.
  • Freshly collected ushi pericardium is physiological saline containing PBS (Gibco BRL, Life Technologies Inc. Rockville, MD, USA) or PBS (this is PBS (-) in this example. Gibco BRL, Life Technologies Inc. Rockville, MD, USA) and washed away blood components.
  • Collagen structure has been shown to be affected by pH, ionic strength, solvent polarity, anionic surfactants, etc. (Ripamonti A, et al., 1980, Biopolymers 19: 965-975; And Xiao WH, Knight DP, Chapman JA., 1997, Biochi mica et Biophysica Acta. 1134: 327-337). It is also known that biomechanical properties change when the collagen structure changes. In order to avoid such problems, in this example, the tissue processing process was designed so as not to affect the matrix. The following experiments demonstrated that the matrix was not damaged at all by histological examination of collagen structure and tensile strength measurement.
  • Paraffin sections (3 ⁇ m thick) of vascular grafts were prepared and stained with hematoxylin-eosin to identify the extracellular matrix. Immunohistochemical staining was used to identify type IZIV collagen, a component of the basement membrane.
  • the aorta of SD rats male, 5 weeks old, Nippon Animal Co. Ltd. Tokyo, Japan
  • Frozen sections (5 m thick) were prepared, permeabilized with PBS (—) for 3 hours, and blocked with 1% BSA in PBS (—) for 1 hour at room temperature.
  • the section was then incubated with a primary antibody (anti-rat collagen antibody, Cosmo Bio, Tokyo, Japan), and a secondary antibody (anti-hidge Ig antibody; Cosmo Bio, Tokyo, Japan) conjugated with FITC. ). Images were obtained using a Zeiss LSM510 confocal microscope.
  • von Kossa staining was performed as follows. If necessary, deparaffinization (for example, with pure ethanol), washing with water (distilled water), and immersion in 25% silver nitrate solution (under indirect light) for 2 hours were performed. Then, it was washed with distilled water and immersed in 42% sodium thiosulfate (hypo) for 5 minutes. After that, it was washed with running water for 5 minutes, and then immersed in Schojeuteroto for 5 minutes. Then, it was washed with running water for 5 minutes, dehydrated, clarified and sealed.
  • the calcium concentration was measured with an atomic absorption photometer.
  • PEG-treated living tissue is a glass sample bottle with a plastic lid (diameter 5 cm)
  • X height was about 8cm
  • the lid was closed (inside the tissue + air), and ⁇ -ray irradiation was performed with a cobalt 60 radiation source.
  • the temperature was room temperature (room temperature)
  • the temperature slightly increased with the amount of heat generated by irradiation, and it seems that the 25 ° C force was between 40 ° C at the time of irradiation. Cleaning may or may not be performed before gamma irradiation.
  • ⁇ rays include, for example, suppression of calcification, direct cytotoxicity by ⁇ rays, and free radical cell damage caused by the adoption of ⁇ rays. Both actions are conceivable. In this case, it may be possible to act as a PEG cover.
  • Biocompatible polymer-treated biological tissue prepared subcutaneously on the back of SD rats was transplanted and sacrificed after 1 week and 2 months, and the degree of inflammatory cell infiltration was scored and evaluated.
  • Specimens transplanted subcutaneously in rats were collected 1 week and 2 months later and evaluated for calcification by von Kossa staining.
  • the tissue Ca concentration was measured with an atomic absorption spectrometer. The Ca concentration was heated and dissolved by putting the tissue in concentrated hydrochloric acid (or concentrated acid). Thereafter, atomic absorption measurement is performed. The solution was diluted and sprayed into a high-temperature plasma, and quantitative measurement was performed from the element-specific absorption wavelength (Ca is 422 nm) of the spectrum generated during combustion.
  • a portion of the aortic wall tissue obtained by the process of the present invention was transplanted into the descending aorta.
  • the results of this example are the results of subcutaneous implantation of ⁇ -irradiated rats (real image of HE staining; Fig. 1), HE staining (Fig. 2), Von Kossa staining (Fig. 3), and calcium determination (Fig. 4; untreated valve control).
  • the results of non-eating meals of the untreated valve control as a control valve are shown.
  • the worm After transplanting the forearm artery of y-irradiated treated tussus prepared in Example 1 into the femoral aorta, the worm is sacrificed 10 days later and the degree of inflammatory cell infiltration is compared.
  • the biological tissue of the present invention hardly shows an inflammatory reaction and the overall tissue structure is not damaged. Furthermore, by observing the transplanted tissue, it can be confirmed that the treated tissue is replaced with autologous cells.
  • the artificial pericardium prepared according to Example 1 is transplanted into the rat infarcted heart.
  • Acute myocardial infarction was induced as described in the literature (Weisman HF, Bush DE, Mannisi JA et al. Cellular mechanism of myocardial infarct expansion. Circulation. 1988; 78: 186-201). Briefly, rats (300 g, 8 weeks old) are anesthetized with sodium bentvalpital and positive pressure breathing is performed. In order to create a rat myocardial infarction model, the thorax is opened between the left 4th ribs and the left coronary artery is completely ligated with 8-0 polypropylene thread at a root force of 3 mm.
  • Recipient rats are anesthetized and the heart is exposed by opening the rib cage between the fifth left intercostals.
  • Cardiac function is measured with cardiac ultrasound (SONOS 5500, manufactured by Agilent Technologies) at 2 weeks, 4 weeks after transplantation, and 8 weeks after transplantation.
  • SONOS 5500 manufactured by Agilent Technologies
  • a 12-MHz transducer Using a 12-MHz transducer, a short-axis image is drawn from the left side at the position where the left ventricle shows the maximum diameter.
  • M-mode left ventricular end-diastolic diameter
  • LV Ds left ventricular end-systolic diameter
  • LAWTh left ventricular anterior wall thickness
  • the heart is removed 8 W later, cut with a short axis, placed in a 10% formaldehyde solution, and fixed with norafine.
  • the ⁇ -ray treated living body tissue of the present invention can be sufficiently applied to portions other than the site from which the tissue before treatment is derived.
  • Ushika can also remove the dura, treat with polyethylene glycol solution as described in Example 1, and irradiate with gamma rays.
  • this dura mater can be transplanted into Lewis rats and examined for inflammatory cell infiltration and calcification.
  • the method of the present invention shows the same excellent characteristics regardless of the tissue used.
  • biocompatible polymers in addition to polyethylene glycol, polyvinyl alcohol, polybutylpyrrolidone, ⁇ polyglutamic acid, gelatin
  • biocompatible polymers have the same conditions of ⁇ -irradiation (dense Using the same glass bottle and Conoret 60 radiation source with a degree of 10%, it is crosslinked by irradiation of about 60-lOOkGy). The strengthening effect can be confirmed. Specifically, the maximum point weight (N), maximum point extension (in mm), and elastic modulus (MPa) are measured.
  • the strength is measured with a TENSILLON ORIENTEC. Specifically, in this specification, (1) Cut the specimen into 5 x 30 mm strips. (Basically, cut the wall part of the main artery base so that it is longer in the long axis direction.); (2) Fix the specimen about 5 mm at both ends to the fixed part of the tensile tester. (Use ORIENTEC's TENSILON universal testing machine RTC-1150A); and (3) Start pulling and pulling to the breaking point at lcmZmin. Measure the load at break and elastic modulus by doing the following (Sh moka T, Mayer JE. Tissue engineering heart valve leaflets. Circulati on.
  • Example 2 we will confirm whether the same effect appears in biological tissues such as blood vessels, intestinal tract, pericardium, mesentery, cornea, retina, bone, and ureter.
  • biological tissues such as blood vessels, intestinal tract, pericardium, mesentery, cornea, retina, bone, and ureter.
  • biocompatible polymer treatment and ⁇ -irradiation treatment using polyethylene glycol, polybutyl alcohol, polybutyl pyrrolidone, ⁇ polyglutamic acid and gelatin.
  • living tissue prepared by various treatment methods was used for other molecular weights of PEG, PVA, polybulurpyrrolidone (PVP), polyacrylic acid, polyethylene oxide (PEO), polyfluoride.
  • PVP polybulurpyrrolidone
  • PEO polyethylene oxide
  • PVdF polyvinylidene fluoride
  • the pericardium was spread over a sterile pad and fixed with paperweight to prevent wrinkling.
  • the pericardium (about 15 cm X about 15 cm) was transferred to a container, and 500 ml of 0.625% dartalaldehyde solution (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was added until the pericardium soaked.
  • the mixture was stirred at 50 RPM for 30 minutes at room temperature using M ULTI Shaker MMS (Tokyo Rika Instrument Co., Ltd., Chuo-ku, Tokyo, Japan). Subsequently, the dartalaldehyde solution (200 ml) was changed and fixed at room temperature for 0.5 hour.
  • Ushi pericardium fixed with dartalaldehyde solution was immersed in a 20% PEG solution (molecular weight 35,000).
  • the pericardium was taken out from the immersion liquid and irradiated with 50 kGy of ⁇ rays in an airtight container. After irradiation, ushi pericardium was stored refrigerated in PBS.
  • Daltaraldehyde-fixed and PEGylated ushi pericardium is transplanted subcutaneously on the back of the rat, and sacrificed 2 months later to score and evaluate the degree of inflammatory cell infiltration. Specifically, the evaluation is performed by staining the frozen section of the tissue with hematoxylin & eosin, visualizing the cell nucleus, and counting the number of inflammatory cells. In this example, the control is compared with a persimmon pericardium fixed with dartalaldehyde as a control.
  • Specimens transplanted subcutaneously in rats were collected 2 months later, and calcification was assessed by von Kossa staining.
  • the Ca concentration in the tissue was measured with an atomic absorption spectrometer. The Ca concentration was heated and dissolved by putting the tissue in concentrated hydrochloric acid (or concentrated acid). Thereafter, atomic absorption measurement was performed. The solution was diluted and sprayed into high-temperature plasma, and quantitative measurement was performed from the element-specific absorption wavelength (Ca is 422 nm) of the spectrum generated during combustion.
  • Figure 6 shows a photograph of subcutaneously transplanted daltaraldehyde-fixed + PEG-treated ashi pericardium, and a photograph of subcutaneously transplanted pericardium with only dartalaldehyde fixed (control). Is shown in Fig. 6 (left). The amount of calcium attached to each new membrane 2 months after transplantation is shown in the table below and in Figure 7. Two months after subcutaneous implantation, no inflammatory cell infiltration, platelet aggregation, or blood clotting were observed at any dose (25 kGy, 50 kGy).
  • the amount of Ca per mg of ⁇ gZ pericardium was 110. 373 ⁇ 23. 563 (see the lower table below and Figure 7). It was confirmed that calcium deposition was progressing in the pericardium treated only with dartalaldehyde fixation.
  • 2 male pigs (LWD, 40 kg, Cary, Inc. (Osaka City, Japan)) are used. Insert a persimmon pericardium fixed with dartalaldehyde solution into the abdomen of the pig. Two months after the pericardium is inserted, the pigs are sacrificed and observed for adhesions.
  • Daltaraldehyde-fixed PEG-treated ushi pericardium is transplanted subcutaneously into the abdomen of pigs and sacrificed 2 months later to score and evaluate the degree of inflammatory cell infiltration. Specifically, the evaluation is performed by staining the frozen section of the tissue with hematoxylin & eosin, visualizing the cell nucleus, and counting the number of inflammatory cells. In this example, the control is compared with a persimmon pericardium fixed with dartalaldehyde as a control.
  • Specimens transplanted subcutaneously in pigs are collected 2 months later and assessed for calcification by von Kossa staining.
  • the Ca concentration in the tissue is measured with an atomic absorption spectrometer.
  • the Ca concentration is heated and dissolved by putting the tissue in concentrated hydrochloric acid (or concentrated acid). Thereafter, atomic absorption measurement is performed.
  • the solution is diluted, sprayed into high-temperature plasma, and quantitatively measured from the element-specific absorption wavelength (Ca is 422 nm) of the spectrum generated during combustion.
  • the results of subcutaneous implantation of dartalaldehyde-fixed + PEG-treated rabbit pericardium are evaluated as a result of HE staining, Von Kossa staining, and calcium quantification.
  • Glutaraldehyde-fixed pericardium is used as a control.
  • Two months after subcutaneous transplantation, inflammatory cell infiltration, platelet aggregation, blood coagulation, and calcification were not observed at any irradiation dose (25 kGy, 50 kGy), and it is clear that they have settled at the transplant destination.
  • dartalaldehyde is fixed, calcium deposition is progressing.
  • Example 12 Film thickness, elastic modulus and adhesion prevention rate of adhesion prevention film
  • the thickness, elastic modulus and anti-adhesion rate of the membrane are measured.
  • Glutaraldehyde-fixed + PEG-treated pericardium and glutaraldehyde-fixed control pericardium are sandwiched between thin plastic sheets. At least 3 thicknesses are measured with an electronic vernier caliper (Digital Caliper Modell 9971: Shinjo Measurement Co., Ltd. (Sanjoen, Niigata)) so that these ushi pericardiums do not deform. By calculating the average of the measured values, it is confirmed that the dartalaldehyde-fixed + PEG-treated pericardium typically has a thickness of 0.5-1.0 mm.
  • Glutaraldehyde fixation + PEG-treated ushi pericardium and glutaraldehyde fixation only Cut the control bush pericardium into 5 x 30 mm strips.
  • fix both ends of the specimen about 5 mm to the fixed part of the tensile tester.
  • the dartal aldehyde fixed + PEG-treated pericardium typically has an elastic modulus of 2-lOMPa.
  • Anti-adhesion rate (%) ⁇ number without adhesion Z (number with adhesion + number without adhesion) ⁇ X 100
  • the dartalaldehyde-fixed + PEG-treated ushi pericardium has an adhesion prevention rate of 80% or more, and has a higher anti-adhesion effect than the dartalaldehyde-fixed ushi pericardium.
  • Example 14 Strengthening effect and prevention of adhesion of biological tissue by other polymer
  • biocompatible polymer in addition to polyethylene glycol, polyvinyl alcohol, polybulurpyrrolidone , ⁇
  • the same experiment as in Example 9 is performed for polyglutamic acid and gelatin.
  • These biocompatible polymers have the same conditions of ⁇ -irradiation (dense Use the same glass bottle and Conoret 60 radiation source at a degree of 10%, and carry out the same experiment as in Examples 6 and 10 to 13 because it is crosslinked by irradiation of about 60-lOOkGy.
  • Example 15 Effect of inhibiting calcification and strengthening of living tissue by other polymer and effect of preventing adhesion
  • Example 16 Effect of inhibiting and strengthening calcification of living tissue by other cross-linking agents, and effect of preventing adhesion
  • cyanimide 1-ethyl-3- (3 dimethylaminopropyl) carbodiimide hydrochloride (EDC), epoxy (poly (glycidyl ether)), 2,6 bis (4 azidobenzylidene).
  • EDC 1-ethyl-3- (3 dimethylaminopropyl) carbodiimide hydrochloride
  • epoxy poly (glycidyl ether)
  • 2,6 bis 4, azidobenzylidene
  • 4-methylcyclohexanone BAMC
  • 4,4'diazidodiphenyl ether 4,4'diazidodiphenylsulfone
  • 4,4'diazidodiphenylacetone 4,4'diazidodiphenylmethane
  • 4- [ ⁇ -azidosalicyamido] plutamine as a crosslinker.
  • Example 17 Effect of preventing adhesion of biological tissue irradiated with ⁇ -rays after PEG treatment
  • a tissue derived from a living body ( ⁇ G-treated + gamma-irradiated biological tissue) (ussi aorta, aortic valve, pericardium) is prepared and used in the following experiments.
  • PEG-treated + gamma-irradiated living tissue is transplanted subcutaneously on the back of the rat, and sacrificed 2 months later to score and evaluate the degree of inflammatory cell infiltration. Specifically, the evaluation is performed by staining the frozen section of the tissue with hematoxylin and eosin, visualizing the cell nucleus, and counting the number of inflammatory cells. In this embodiment, as a control, the above-mentioned untreated valve and ushi live valve are compared and examined.
  • Specimens transplanted subcutaneously in rats are collected 2 months later and evaluated for calcification by von Kossa staining.
  • the Ca concentration in the tissue is measured with an atomic absorption spectrometer.
  • the Ca concentration is heated and dissolved by placing the tissue in concentrated hydrochloric acid (or concentrated acid). Thereafter, atomic absorption measurement is performed.
  • the solution is diluted and sprayed into high-temperature plasma, and quantitative measurement is performed from the element-specific absorption wavelength (Ca is 422 nm) of the spectrum generated during combustion.
  • PEG treatment + ⁇ -irradiated tissue when transplanted subcutaneously, 2 months after subcutaneous implantation No inflammatory cell infiltration, platelet aggregation, or blood clotting are observed at any dose.
  • PEG-treated + gamma-irradiated living tissue mild adhesion is observed in all samples, but all tissues can be removed almost before transplantation. Therefore, it can be confirmed that it has been established in the transplantation destination without causing calcium deposition.
  • the control tissue moderate or severe adhesion is observed in all samples, and it can be confirmed that calcification has progressed.
  • the PEG-treated + ⁇ -irradiated biological tissue prepared in Example 16 is used.
  • untreated valve and ushi live valve are used.
  • results of subcutaneous implantation of PEG-treated + ⁇ -irradiated living tissue are evaluated as results of HE staining, Von Kossa staining, and calcium quantification.
  • Two months after subcutaneous transplantation inflammatory cell infiltration, platelet aggregation, blood coagulation, and calcification were not observed at any irradiation dose, and it is clear that they were established at the transplant destination.
  • calcium deposition is progressing.
  • Example 19 Elasticity and anti-adhesion rate of PEG-treated + ⁇ -irradiated biological tissue
  • PEG-treated + gamma-irradiated living tissue and control tissue are transplanted to the back of the rat as the test site. Each is removed 2 months after transplantation. Count the number of samples that have adhered and the number of samples that have adhered, and use the following formula to calculate.
  • Anti-adhesion rate (%) ⁇ number without adhesion ⁇ (number with adhesion + no adhesion) ⁇ X 100
  • the tissue derived from PEG-treated + ⁇ -irradiated living body has an adhesion prevention rate of 80% or more, and has a higher adhesion prevention effect than the control tissue.
  • cyanimide 1-ethyl-3- (3 dimethylaminopropyl) carbodiimide hydrochloride (EDC), epoxy (poly (glycidyl ether)), 2,6 bis (4 azidobenzylidene).
  • EDC 1-ethyl-3- (3 dimethylaminopropyl) carbodiimide hydrochloride
  • epoxy poly (glycidyl ether)
  • 2,6 bis 4, azidobenzylidene
  • 4-methylcyclohexanone BAMC
  • 4,4'diazidodiphenyl ether 4,4'diazidodiphenylsulfone
  • 4,4'diazidodiphenylacetone 4,4'diazidodiphenylmethane
  • 4- [ ⁇ -azidosalicyamido] plutamine as a crosslinker.
  • an improved method of a technique has been established that significantly increases tissue strength and achieves remarkable suppression of calcification while suppressing the tissue damage rate to a level that can be clinically applied.
  • Biological tissues and grafts prepared by this technique are further strengthened. Therefore, such an organization is industrially useful.
  • an anti-adhesion membrane capable of preventing adhesion without causing adverse reactions such as calcification, inflammatory reaction, unnecessary cell migration or adhesion, platelet aggregation, and promotion of blood coagulation. It has been developed.
  • This anti-adhesion membrane exhibits an anti-adhesion ability non-specifically regardless of the cell type, and has an anti-adhesion effect equivalent to or higher than that of the prior art.
  • Such anti-adhesion membranes are industrially useful in the fields of surgery and treatment.

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Abstract

It is intended to provide a tissue for transplantation being free from troubles accompanying transplantation such as calcification. It is also intended to provide a membrane for preventing adhesion by which adhesion caused by a surgical operation or treatment can be prevented. It is found out that when a biocompatible polymer is added to an untreated tissue and then the tissue is exposed to Ϝ-ray irradiation, the tissue is unexpectedly strengthened and calcification is prevented, thereby solving the above problems. Although there has been recognized that decellularization is the key factor in preventing a tissue with a biological origin from calcification, it is indicated that Ϝ-ray irradiation is more efficacious than decellularization. Namely, a membrane for preventing adhesion, by which adhesion can be prevented without inducing troublesome reactions (for example, calcification, an inflammatory reaction, unnecessary cell migration or adhesion, platelet aggregation, acceleration of blood coagulation, etc.), is provided.

Description

明 細 書  Specification
生体由来移植用組織の石灰化を抑制するための処理方法および処理さ れた組織  Treatment method for suppressing calcification of tissue for transplantation from living body and treated tissue
技術分野  Technical field
[0001] 本発明は、単離された生体由来組織 (未処理組織)を強化するための方法および システム、ならびにそのような強化方法によって調製された組織、組織グラフトなどを 利用した医薬および治療方法に関する。具体的には、本発明は、生体由来移植用 組織の石灰化を抑制するための処理方法および処理された組織に関する。  [0001] The present invention relates to a method and system for strengthening an isolated biological tissue (untreated tissue), and a medicine and treatment method using a tissue, tissue graft, and the like prepared by such a strengthening method. About. Specifically, the present invention relates to a treatment method and a treated tissue for suppressing calcification of a living tissue-derived transplant tissue.
[0002] 本発明はまた、外科手術または処置を受けた糸且織であって、癒着の生じるおそれ 力 Sある部位において使用するための癒着防止膜に関する。具体的には、外科手術ま たは処置後の切断面または欠損部位とその周辺組織との間に生じる癒着の防止に 関する。  [0002] The present invention also relates to an anti-adhesion membrane for use at a site where there is a risk of adhesion, which is a thread and weave that has undergone surgery or treatment. Specifically, the present invention relates to the prevention of adhesions that occur between the surgical or surgical cut surface or defect site and the surrounding tissue.
背景技術  Background art
[0003] 臓器 (例えば、心臓、血管など)の移植に外来性組織を使用する際の主な障害は 免疫拒絶反応である。同種異系移植片（または同種移植片、 allograft)および異種 移植片（xenograft)で起こる変化が最初に記述されたのは 90年以上前のことである (非特許文献 1〜2および 4〜5)。動脈移植片の拒絶反応は、病理学的には移植片 の拡張 (破裂に至る）または閉塞のいずれかを招く。前者の場合、細胞外マトリクスの 分解により生じ、他方で、後者は血管内細胞の増殖により起こるといわれている（非 特許文献 6)。  [0003] A major obstacle in using foreign tissues for organ transplantation (eg, heart, blood vessel, etc.) is immune rejection. Changes that occurred in allografts (or allografts) and xenografts were first described more than 90 years ago (Non-Patent Documents 1-2 and 4-5) ). Arterial graft rejection pathologically results in either graft expansion (leading to rupture) or occlusion. In the former case, it is said to be caused by the degradation of the extracellular matrix, while the latter is caused by proliferation of intravascular cells (Non-patent Document 6).
[0004] 従来、これらの物質の拒絶反応の軽減を目指して 2つの戦略が採用されてきた。ひ とつは、宿主の免疫反応を低下させた (非特許文献 7 ;および非特許文献 8)。もうひ とつは主に架橋結合により同種移植片または異種移植片の抗原性の低下を図った（ 非特許文献 9；および非特許文献 10)。細胞外マトリクスの架橋結合は移植片の抗原 性を低下させるが、生体工学的機能 (非特許文献 11；および非特許文献 12)が変化 し、無機質化に感受性を示すようになる (非特許文献 13)。  [0004] Conventionally, two strategies have been adopted to reduce the rejection of these substances. One reduced the immune response of the host (Non-Patent Document 7; and Non-Patent Document 8). The other attempted to reduce the antigenicity of the allograft or xenograft mainly by cross-linking (Non-patent document 9; and Non-patent document 10). Cross-linking of the extracellular matrix decreases the antigenicity of the graft, but the biomechanical function (Non-patent Document 11; and Non-patent Document 12) changes and becomes sensitive to mineralization (Non-patent Document). 13).
[0005] 細胞外マトリクス中の細胞は、拒絶反応を引き起こし得る組織適合性クラス Iおよび I I抗原を有する。また、その他に宿主の免疫系が特定できる細胞由来の糖ィ匕タンパク 質があり、拒絶反応が引き起こされる。故に、このような物質を細胞外マトリクスから除 去すれば、拒絶反応を防ぐことができる。ただ、全ての抗原を完全に除去するのは非 常に困難であり実証が難しい。 Maloneら (非特許文献 15)および Lalkaら (非特許文 献 16)は、「細胞のな 、」動脈同種移植片（同種動物への移植片）を移植したマトリク スも免疫応答反応を刺激したが、一方で血管内増殖と内皮細胞の生着も認められた と報告した。つい最近では、 O' Brianらカ 脱細胞化したブタ組織は心血管移植に 応用が可能であり、ヒッジに植込んだ際には超急性拒絶反応を示さな力つたと報告し た (非特許文献 17)。 [0005] Cells in the extracellular matrix can cause histocompatibility classes I and I I have an antigen. In addition, there are cell-derived glycoproteins that can identify the host immune system and cause rejection. Therefore, rejection can be prevented by removing such substances from the extracellular matrix. However, complete removal of all antigens is very difficult and difficult to demonstrate. Malone et al. (Non-patent Document 15) and Lalka et al. (Non-patent Document 16) reported that a matrix transplanted with “no cell” arterial allografts (grafts into allogeneic animals) also stimulated immune response. However, he reported that intravascular proliferation and endothelial cell engraftment were also observed. More recently, O 'Brian et al. Reported that decellularized porcine tissue can be applied to cardiovascular transplantation and did not show hyperacute rejection when implanted in a hedge (non-patented). Reference 17).
[0006] 心血管疾患 (冠状動脈および末梢血管の疾患を含む）は、手術による置換療法（に より処置されている。心血管疾患の症例は、世界中でこのところ増加している。直径 力 、さな血管の場合は、置換療法を適用することは困難である。直径が小さい場合、 バイパス手術では、自己由来の静脈または動脈の移植片が使用される (非特許文献 18〜20)。静脈および動脈の移植片が現状ではもつともよい結果が得られている力 複雑な手術を必要とし、ある疾患の患者では適切な血管が得られな 、と 、つた欠点 も存在する。その結果、直径が細い血管に適切な人工血管が必要とされている。自 己移植片または同種異系移植片の使用を減少させるために、人工材料の開発に向 けた開発がなされた。しかし、人工材料の場合、四肢末梢および冠状動脈のバイパ ス移植手術に必要な細！、直径の動脈 (6mm未満）の構築に適切なものはな!/ヽ。他方 、心臓血管の場合、天然の組織移植片もまた臨床応用における生体材料としての可 能性が試されている。異種移植片および同種異系移植片の組織の使用は、代表的 には、化学的または物理的な処理が必要である（例えば、ダルタルアルデヒド固定)。 架橋技術は、組織のコラーゲンベースの構造を安定ィヒするのに手順も調べられ、理 想的な手順であることが見出された (非特許文献 21)。  [0006] Cardiovascular diseases (including coronary and peripheral vascular diseases) are being treated by surgical replacement therapy (the number of cases of cardiovascular disease is increasing around the world recently. In the case of small blood vessels, it is difficult to apply replacement therapy: if the diameter is small, bypassed surgery uses autologous veins or arterial grafts (18-20). The strength of venous and arterial grafts that currently gives the best results Requires complex surgery, and there are other disadvantages that patients with certain diseases cannot get the proper blood vessels. Appropriate vascular prosthesis is needed for thin blood vessels, but development towards the development of artificial materials was made to reduce the use of autografts or allografts. Limb peripheral and coronary movement Neither is it necessary to construct small diameter arteries (less than 6mm)! / ヽ, whereas in the case of cardiovascular, natural tissue grafts are also useful as biomaterials in clinical applications. The use of xenograft and allograft tissue typically requires chemical or physical treatment (eg, dartalaldehyde fixation). Cross-linking techniques have also been investigated to stabilize the collagen-based structure of tissues and have been found to be an ideal procedure (Non-patent Document 21).
[0007] しかし、移植には、長期的な視点力も石灰化という問題がある。ダルタルアルデヒド 処理の際の石灰化という有害な副作用は、生体人工心臓弁の欠陥の主な原因であ る (非特許文献 22;および非特許文献 23)。天然の組織移植片の別の方法として、完 全に無細胞とした組織マトリクスを生産することが試みられている。この生産は、石灰 化を促進し、免疫学的応答を惹起すると考えられる細胞成分を特異的に除去するこ とによる。これらの脱細胞化技術としては、化学的手段、酵素学的手段、および機械 的手段での細胞成分の除去が挙げられる。この処理によって、本質的に細胞外マトリ タス成分力も構成される材料が残る。これらの脱細胞化組織は、天然の機械的特性 を保持し、再生を促進する。再生は、宿主による新血管形成および再細胞化のプロ セスによって生じる。代表的な細胞抽出方法である界面活性剤処理は、生体材料移 植片として使用するために完全に脱細胞化された組織を作製する手段として実施さ れてきた。なぜなら、処理された組織内に残る細胞成分、脂質および残留界面活性 剤は、石灰化のような所望されな 、効果を促進し得るからである (非特許文献 24;非 特許文献 25;非特許文献 26;および非特許文献 27)。クロ口ホルム Zメタノールまた はドデシル硫酸ナトリウム（SDS)のいずれかでの処理によるゥシ心膜からの脂質除 去は、ラットモデルにおいて組織の石灰化を減少させた (非特許文献 28)。最近、 Su njay et al.は、脱細胞化血管移植片が内皮前駆体細胞 (EPC)により皮質ィ匕 (cort) したことを実証した。なぜなら、これらの移植片は、十分に保存された細胞外マトリクス および動脈を含む天然の血管に類似する機械的特性を有するからである (非特許文 献 29)。この研究者らは、末梢血から内皮前駆体細胞 (EPC)を単離し、そしてこの E PCを脱細胞化ゥシ回腸血管に播種した。この EPCを播種した脱細胞化移植片は、 1 30日目までにインビボで新生血管を発達させ、 NO媒介される血管弛緩が生じた。 石灰化を防止するための別の技術として、未処理組織において PEG等を in situ で uv照射または化学反応によって重合させることにより移植用生体組織の石灰化 作用を低減させる試みが最近公開された (WO2005/042044 =特許文献 4)。こ の公開公報では、重合反応の例としては、 A：ビニルモノマー、架橋モノマーおよび 光重合開始剤を混合した上で UV照射を行う方法や、 B：アミノ基ゃチオール基など の求核基を有する PEG等ポリマーと、《, |8—不飽和基を有する PEG等ポリマーとを 求核反応させる方法などが開示されている。この場合の効果として、初期の炎症反応 が抑制されること、および長期にわたって石灰化が抑制されることが開示されている。 しかし、炎症反応の抑制は、実際の治療現場を考慮した場合不十分であり、石灰化 の抑制もまた不十分でないようであり、この技術には欠点が存在する。 [0009] また、特許文献 4の方法では、 free radical initiator (フリーラジカルを発生する 重合開始剤）が必要である。一般論として、これらが残留したまま体内に移植されると 毒性などを示す可能性があり生体移植されると問題が発生する可能性が高い。 [0007] However, transplantation has a problem that long-term viewpoint power is calcification. The detrimental side effect of calcification during the treatment with dartalaldehyde is the main cause of defects in bioprosthetic heart valves (Non-Patent Document 22; and Non-Patent Document 23). As an alternative to natural tissue grafts, attempts have been made to produce a completely acellular tissue matrix. This production is lime By specifically removing cellular components that are thought to promote immunogenicity and elicit an immunological response. These decellularization techniques include removal of cellular components by chemical, enzymatic, and mechanical means. This treatment leaves a material that is also essentially composed of extracellular matrix component forces. These decellularized tissues retain their natural mechanical properties and promote regeneration. Regeneration occurs through the process of neovascularization and recellularization by the host. Surfactant treatment, a typical cell extraction method, has been implemented as a means of creating fully decellularized tissue for use as a biomaterial implant. This is because the cellular components, lipids and residual surfactant remaining in the treated tissue can promote the desired and undesired effects such as calcification (Non-Patent Document 24; Non-Patent Document 25; Non-Patent Document). Reference 26; and Non-patent reference 27). Removal of lipids from rabbit pericardium by treatment with either black mouth form Z methanol or sodium dodecyl sulfate (SDS) reduced tissue calcification in a rat model (28). Recently, Sunjay et al. Demonstrated that decellularized vascular grafts were cortated by endothelial progenitor cells (EPC). This is because these grafts have mechanical properties similar to natural blood vessels including well-preserved extracellular matrix and arteries (Non-patent Document 29). The researchers isolated endothelial progenitor cells (EPCs) from peripheral blood and seeded the EPCs into decellularized guinea-pig blood vessels. This decellularized graft seeded with EPC developed neovascularization in vivo by day 130, resulting in NO-mediated vascular relaxation. As another technique to prevent calcification, an attempt was recently made to reduce the calcification of living tissue for transplantation by polymerizing PEG etc. in situ by uv irradiation or chemical reaction in untreated tissues ( WO2005 / 042044 = Patent Document 4). In this publication, examples of the polymerization reaction include: A: a method in which a vinyl monomer, a crosslinking monomer, and a photopolymerization initiator are mixed and then UV irradiation; and B: a nucleophilic group such as an amino group or a thiol group. And a method of nucleophilic reaction between a polymer such as PEG and a polymer such as <<, | 8-unsaturated group. As an effect in this case, it is disclosed that the initial inflammatory reaction is suppressed and calcification is suppressed over a long period of time. However, suppression of the inflammatory response is inadequate when considering the actual treatment site, and suppression of calcification also appears to be inadequate, and this technique has drawbacks. [0009] In addition, the method of Patent Document 4 requires a free radical initiator (a polymerization initiator that generates free radicals). As a general rule, if these are left in the body, they may be toxic and may cause problems if transplanted in vivo.
[0010] 特許文献 4の 10頁にぉ ヽては、各種重合開始剤 (initiator)が残存したまま移植さ れた場合の有害性については、おおむね以下のようなものが考えられる。（1) therm alinitiators：温度が上がると周辺のタンパク質が変性し有害となる可能性がある。 (2 ) peroxy compounds：周辺のタンパク質などと徐々に反応して架橋などを起こし有 害となる可能性がある。（3) azocompounds :周辺のタンパク質などと徐々に反応し て架橋などを起こし有害となる可能性がある。 (4) photo initiators :光があたると周 辺のタンパク質などと架橋などを起こし有害となる可能性がある。 (5) redoxinitiator s：酸ィ匕還元反応によりラジカル反応を起こし、周辺のタンパク質などと架橋などを起 こし有害となる可能性がある。このように重合開始剤には種々の問題が存在し、石灰 化の危険性も残存する。  [0010] From page 10 of Patent Document 4, the following are generally considered as harmful effects when transplanted with various polymerization initiators remaining. (1) therm alinitiators: When temperature rises, neighboring proteins may denature and become harmful. (2) peroxy compounds: It reacts gradually with nearby proteins, causing cross-linking and other harmful effects. (3) Azocompounds: Reacts gradually with nearby proteins, causing cross-linking and other harmful effects. (4) photo initiators: When exposed to light, there is a possibility that it will cause cross-linking with the surrounding proteins, etc., and may be harmful. (5) redoxinitiator s: A radical reaction is caused by the acid-oxidation reduction reaction, which may cause harmful effects due to cross-linking with surrounding proteins. As described above, the polymerization initiator has various problems and the risk of calcification remains.
[0011] このように、当該分野において、石灰化の防止など、生体適合性、生体定着性等を 改善させるベぐ組織を強化させる必要がある情況も存在することから、さらなる移植 用組織の改善が当該分野において求められている。  [0011] As described above, there is a situation in the field where it is necessary to reinforce the tissue that improves biocompatibility, biofixation, etc., such as prevention of calcification. Is required in the field.
[0012] 外科手術または処置において、患部、または臓器もしくは器官の切除または損傷 部位の修復などを行うと、その部位に癒着を生じるおそれがある。癒着が生じると、臓 器の機能不全を招き、予後を悪化させる力または死に至るといった不都合が生じる 可能性があり、外科手術および処置において大きな問題となっている。そのような不 都合を解消するために、臓器の切断面または欠損部と、それらに接触するおそれが ある別の組織とを互いに隔離し、癒着を防止することが必要となっている。この課題を 解決するために、従来から、合成高分子、生体高分子を材料とした癒着防止膜が開 発されてきた (非特許文献 30〜36)。  [0012] In a surgical operation or treatment, if an affected part, an organ or an organ is removed, or a damaged part is repaired, adhesion may occur at the part. Adhesion can be a major problem in surgery and treatment, as it can lead to organ dysfunction and can lead to inferior prognostic forces or death. In order to eliminate such inconveniences, it is necessary to isolate the cut surface or defect of the organ from other tissues that may come into contact with each other to prevent adhesion. In order to solve this problem, an anti-adhesion film using a synthetic polymer or biopolymer as a material has been developed (Non-Patent Documents 30 to 36).
[0013] 従来、癒着防止膜に用いられてきた合成高分子には、ポリエチレン (非特許文献 3 0)、シリコン (非特許文献 31)、セルロース (非特許文献 32)、ポリテトラフルォロェチ レン (例えば、ゴァテックス (登録商標）（ジャパンゴァテックス株式会社)などがある。 これらの合成高分子は、生体適合性が低ぐ過度の石灰化および炎症反応が生じる という問題がある。 [0013] Synthetic polymers that have been conventionally used for anti-adhesion membranes include polyethylene (Non-patent document 30), silicon (Non-patent document 31), cellulose (Non-patent document 32), polytetrafluoroethylene. Ren (for example, Goatex (registered trademark) (Japan Goatex Co., Ltd.), etc. These synthetic polymers cause excessive calcification and inflammatory reaction with low biocompatibility There is a problem.
[0014] また、癒着防止膜に用いられてきた生体高分子には、コラーゲン膜 (特許文献 5、 非特許文献 33〜35)およびヒアルロン酸 (特許文献 6、特許文献 7および非特許文 献 36)などがある。これらの生体高分子には、 1)癒着を完全には防止できないこと、 2)天然のコラーゲンには種々の細胞接着部位が含まれているため、用途によっては 不必要な細胞の遊走または接着が避けられな!/、こと、 3)血小板が接着して血小板凝 集を引き起こすこと、および 4)ハーゲマン因子 (血液凝固第 VII因子)を活性ィ匕し、血 液凝固を促進することなど、多くの課題が存在する。  [0014] In addition, biopolymers that have been used for adhesion-preventing membranes include collagen membranes (Patent Literature 5, Non-Patent Literatures 33 to 35) and hyaluronic acid (Patent Literature 6, Patent Literature 7 and Non-Patent Literature 36). )and so on. These biopolymers 1) cannot completely prevent adhesion, and 2) natural collagen contains various cell adhesion sites, which may cause unnecessary cell migration or adhesion depending on the application. Inevitable! /, 3) Platelet adheres to cause platelet aggregation, and 4) Hageman factor (blood coagulation factor VII) is activated and blood coagulation is promoted. There are challenges.
[0015] したがって、石灰化、炎症反応、不必要な細胞の遊走または接着、血小板凝集、血 液凝固の促進などの不都合な反応を引き起こすことなぐ癒着を防止することが可能 な癒着防止膜が必要となって 、る。  [0015] Therefore, an anti-adhesion membrane capable of preventing adhesion without causing adverse reactions such as calcification, inflammatory reaction, unnecessary cell migration or adhesion, platelet aggregation, and promotion of blood coagulation is required. It becomes.
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特許文献 4:国際公開第 2005Z042044号パンフレット  Patent Document 4: International Publication No. 2005Z042044 Pamphlet
特許文献 5：特開 2000— 93497号公報  Patent Document 5: JP 2000-93497 A
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15 15
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発明の開示 Disclosure of the invention
発明が解決しょうとする課題 Problems to be solved by the invention
従来の技術によって調製された移植用組織は、石灰化などの面において、問題が 残存する。多くの研究者が移植用組織の物理学的特性および生物学的特性を含む 多数の有用性を認識している。しかし、現状で入手可能な移植用組織は、石灰化お よび免疫惹起反応のような欠点が存在し、さらなる改善が必要とされて 、る (Christin e E. Schmidt, Jennie M. Baier. , 2000, Biomaterials 21 : 2215- 2231) 。従って、本発明は、石灰化など、移植における問題を解決した移植用組織を提供 することを課題とする。 The transplanted tissue prepared by the conventional technique still has problems in terms of calcification. Many researchers recognize a number of utilities, including the physical and biological properties of the transplanted tissue. However, currently available transplant tissues are calcified. There are disadvantages such as immune-inducing reactions and further improvements are needed (Christin e E. Schmidt, Jennie M. Baier., 2000, Biomaterials 21: 2215-2231). Accordingly, an object of the present invention is to provide a tissue for transplantation that solves problems in transplantation such as calcification.
[0017] 従来の癒着防止膜は、石灰化、炎症反応、不必要な細胞の遊走または接着、血小 板凝集、血液凝固の促進を引き起こすという面において課題が残存する。従って、外 科手術または処置において、患部の切除または損傷部位の修復などを行った後、上 記のような生体にとって不都合な副作用を生じさせずに、その切断面または欠損部と 、その周辺組織との間で癒着が生じるのを防止するために使用する癒着防止膜を提 供することを課題とする。  [0017] Conventional anti-adhesion membranes still have problems in terms of causing calcification, inflammatory reaction, unnecessary cell migration or adhesion, blood platelet aggregation, and promotion of blood coagulation. Therefore, after performing excision of the affected part or repairing the damaged part in external surgery or treatment, the cut surface or the defect part and the surrounding tissue are produced without causing adverse side effects on the living body as described above. It is an object to provide an anti-adhesion film used to prevent adhesion between the two.
課題を解決するための手段  Means for solving the problem
[0018] 本発明者らは、未処理組織に生体適合性高分子を含ませ、これに γ線照射を暴露 させることによって、予想外に組織が強化され、石灰化が抑制されたことを見出し、上 記課題を解決した。本発明者らは、組織を、生体適合性高分子に曝し、かつ、 γ線 に暴露させることによって、予想外に強い補強効果もまた見出した。従来、生体由来 組織の脱細胞化が石灰化抑制のポイントであるとの認識があった力 γ線照射でも、 脱細胞化以上の効果を達成しうることが本発明によって示された。  [0018] The present inventors have found that by including a biocompatible polymer in an untreated tissue and exposing it to γ-ray irradiation, the tissue was unexpectedly strengthened and calcification was suppressed. The above issues have been solved. The inventors have also found an unexpectedly strong reinforcing effect by exposing the tissue to a biocompatible polymer and to gamma radiation. Conventionally, it has been shown by the present invention that force γ-irradiation, which has been recognized that decellularization of a tissue derived from a living body is a key point for suppressing calcification, can achieve an effect more than decellularization.
[0019] このようにして作られた生体由来組織は、石灰化が顕著に抑制されている。本発明 の生体由来組織は、例えば、永久に使える人工血管として使用することも可能である 。このように、本発明の生体由来組織の有用性は広汎にわたるといえる。  [0019] Calcification is remarkably suppressed in the biological tissue produced in this way. The living body-derived tissue of the present invention can be used as, for example, a permanent artificial blood vessel. Thus, it can be said that the usefulness of the biological tissue of the present invention is extensive.
[0020] し力も、本発明により補強された生体由来組織は、組織強度が予想外に顕著に改 善された。このような効果は、従来技術では決して達成することができな力つた予想 外の効果であり、このように補強された生体由来組織および組織グラフトを提供する ことができたと!/、う事実は、移植医学にぉ 、て格別の進歩をもたらすと 、える。  [0020] The strength of the biological tissue reinforced by the present invention was also significantly improved unexpectedly. Such an effect is a powerful and unexpected effect that could never be achieved with the prior art, and it was possible to provide such a reinforced biological tissue and tissue graft! / This is a significant advance in transplantation medicine.
[0021] 従って、本発明は、以下を提供する。  Accordingly, the present invention provides the following.
(項目 1)  (Item 1)
生体適合性高分子を含み、 y線照射を受けたことを特徴とする生体由来組織。 (項目 2) 前記生体由来組織は、脱細胞化処理を受けていないことを特徴とする、項目 1に記 載の生体由来組織。 A biological tissue containing a biocompatible polymer and receiving y-ray irradiation. (Item 2) Item 2. The living tissue according to item 1, wherein the living tissue is not decellularized.
(項目 3)  (Item 3)
細胞外マトリクス成分が少なくとも一部が共有結合で架橋されていることを特徴とする 、項目 1に記載の生体由来組織。 Item 2. The biological tissue according to Item 1, wherein the extracellular matrix component is at least partially crosslinked by a covalent bond.
(項目 4) (Item 4)
前記細胞外マトリックス成分は、コラーゲン、エラスチン、ラミニン、フイブロネクチン、 テネイシン、グリコサミノダリカンおよびプロテオダリカン力もなる群より選択される、項 目 3に記載の生体由来組織。 Item 4. The living tissue according to Item 3, wherein the extracellular matrix component is selected from the group consisting of collagen, elastin, laminin, fibronectin, tenascin, glycosaminodarlican and proteodarican power.
(項目 5) (Item 5)
前記生体適合性高分子は、前記生体由来組織をコーティングする、項目 1に記載の 生体由来組織。 Item 2. The biological tissue according to Item 1, wherein the biocompatible polymer coats the biological tissue.
(項目 6) (Item 6)
前記生体適合性高分子は、前記生体由来組織に架橋されている、項目 1に記載の 生体由来組織。 Item 2. The biological tissue according to Item 1, wherein the biocompatible polymer is crosslinked to the biological tissue.
(項目 7) (Item 7)
前記 γ線照射は、 10kGy〜250kGyの間の線量で提供される、項目 1に記載の生 体由来組織。 The organism-derived tissue according to Item 1, wherein the gamma irradiation is provided at a dose between 10 kGy and 250 kGy.
(項目 8) (Item 8)
前記 γ線照射は、 25kGy〜50kGyの間の線量で提供される、項目 1に記載の生体 由来組織。 The biological tissue according to Item 1, wherein the gamma irradiation is provided at a dose between 25 kGy and 50 kGy.
(項目 9) (Item 9)
前記生体適合性高分子は、生分解性である、項目 1に記載の生体由来組織。 Item 2. The biological tissue according to Item 1, wherein the biocompatible polymer is biodegradable.
(項目 10) (Item 10)
前記生体適合性高分子は、ポリビュルアルコール (PVA)、ポリビュルピロリドン (PV P)、エラスチン、ポリエチレングリコール（PEG)、ゼラチン、コラーゲン、 γ -ポリグル タミン酸およびそれらの 2つ以上の混合物力 なる群より選択される高分子を含む、 項目 1に記載の生体由来組織。 前記生体適合性高分子は、ポリエチレングリコール (PEG)を含む、項目 1に記載の 生体由来組織。 The biocompatible polymer may be polybulal alcohol (PVA), polybulurpyrrolidone (PV P), elastin, polyethylene glycol (PEG), gelatin, collagen, γ-polyglutamic acid and a mixture of two or more thereof. Item 2. The biological tissue according to Item 1, comprising a polymer selected from the group. Item 2. The biological tissue according to Item 1, wherein the biocompatible polymer comprises polyethylene glycol (PEG).
(項目 12) (Item 12)
前記ポリエチレングリコール（PEG)は、分子量 1, 000〜200, 000の範囲内にあ る、項目 11に記載の生体由来組織。 Item 12. The biological tissue according to Item 11, wherein the polyethylene glycol (PEG) is in a molecular weight range of 1,000 to 200,000.
(項目 13) (Item 13)
前記ポリエチレングリコール（PEG)は、分子量 8, 000力ら 50, 000の範囲内にある 、項目 11に記載の生体由来組織。 The biological tissue according to Item 11, wherein the polyethylene glycol (PEG) is in the range of molecular weight 8,000 force to 50,000.
(項目 14) (Item 14)
前記生体由来組織は、臨床適用することができる組織強度を有する、項目 1に記載 の生体由来組織。 Item 2. The living tissue according to Item 1, wherein the living tissue has tissue strength that can be clinically applied.
(項目 15) (Item 15)
前記生体由来組織は、移植したときに石灰化が顕著に抑制される、項目 1に記載の 生体由来組織。 2. The biological tissue according to item 1, wherein the biological tissue is markedly suppressed in calcification when transplanted.
(項目 16) (Item 16)
前記生体適合性高分子は、ランダムに架橋されていることを特徴とする、項目 1に記 載の生体由来組織。 Item 2. The biological tissue according to Item 1, wherein the biocompatible polymer is randomly crosslinked.
(項目 17) (Item 17)
前記生体由来組織は、膜状組織、弁状組織または管状組織である、項目 1に記載の 生体由来組織。 Item 2. The living tissue according to Item 1, wherein the living tissue is a membranous tissue, a valve-like tissue, or a tubular tissue.
(項目 18) (Item 18)
前記生体由来組織は、血管、血管様組織、心臓弁、心膜、硬膜、角膜および骨から 選択されるものの組織である、項目 1に記載の生体由来組織。 Item 2. The living tissue according to Item 1, wherein the living tissue is a tissue selected from blood vessels, blood vessel-like tissues, heart valves, pericardium, dura mater, cornea and bone.
(項目 19) (Item 19)
前記生体由来組織は、哺乳動物由来である、項目 1に記載の生体由来組織。 Item 2. The living tissue according to Item 1, wherein the living tissue is derived from a mammal.
(項目 20) (Item 20)
前記生体由来組織は、ヒト、ゥシまたはブタ由来である、項目 1に記載の生体由来組 織。 The living body-derived tissue according to item 1, wherein the living body-derived tissue is derived from a human, ushi or pig. Weaving.
(項目 21)  (Item 21)
項目 1に記載の生体由来組織を含む、移植用の組織グラフト。 A tissue graft for transplantation comprising the living tissue according to item 1.
(項目 22) (Item 22)
前記組織グラフトは、膜状、管状および弁状からなる群より選択される形状を有する、 項目 21に記載の組織グラフト。 Item 22. The tissue graft according to Item 21, wherein the tissue graft has a shape selected from the group consisting of a membrane shape, a tubular shape, and a valve shape.
(項目 23) (Item 23)
移植用の組織を生産する方法であって、 A method for producing a tissue for transplantation, comprising:
A)生体由来組織を提供する工程；  A) providing a biological tissue;
B)生体適合性高分子に該生体由来組織を曝す工程;および  B) exposing the biological tissue to a biocompatible polymer; and
C)該生体由来組織を γ線照射に曝す工程、  C) a step of exposing the biological tissue to gamma irradiation,
を包含する、方法。 Including the method.
(項目 24)  (Item 24)
前記 γ線照射は、該生体適合性高分子に化学反応が起こる条件下で行われる、項 目 23に記載の方法。 Item 24. The method according to Item 23, wherein the gamma irradiation is performed under conditions in which a chemical reaction occurs in the biocompatible polymer.
(項目 25) (Item 25)
前記 γ線照射は、 10kGy〜250kGyの間の線量で提供される、項目 23に記載の方 法。 24. The method of item 23, wherein the gamma irradiation is provided at a dose between 10 kGy and 250 kGy.
(項目 26)  (Item 26)
前記 γ線照射は、 25kGy〜50kGyの間の線量で提供される、項目 23に記載の方 法。 24. The method of item 23, wherein the gamma irradiation is provided at a dose between 25 kGy and 50 kGy.
(項目 27)  (Item 27)
前記 γ線照射は、真空、酸素中、窒素中、大気中、水中、両親媒性分子溶液中およ びそれらの組み合わせ力 なる群より選択される環境下で行われる、項目 23に記載 の方法。 24. The method according to item 23, wherein the γ-ray irradiation is performed in an environment selected from the group consisting of vacuum, oxygen, nitrogen, air, water, amphiphilic molecule solution, and a combination force thereof. .
(項目 28) (Item 28)
前記 γ線照射は、 0. 5〜240時間の範囲で行われる、項目 23に記載の方法。 (項目 29) 前記生体適合性高分子は、生分解性である、項目 23に記載の方法。 Item 24. The method according to Item 23, wherein the gamma irradiation is performed in a range of 0.5 to 240 hours. (Item 29) 24. The method of item 23, wherein the biocompatible polymer is biodegradable.
(項目 30) (Item 30)
前記生体適合性高分子は、ポリビュルアルコール、ポリビュルピロリドン、エラスチン 、ポリエチレングリコール、ゼラチン、コラーゲン、 γ -ポリグルタミン酸およびそれらの 2つ以上の混合物力 なる群より選択される高分子を含む、項目 23に記載の方法。 (項目 31) The biocompatible polymer includes a polymer selected from the group consisting of polybulal alcohol, polybulurpyrrolidone, elastin, polyethylene glycol, gelatin, collagen, γ-polyglutamic acid and a mixture of two or more thereof. The method according to 23. (Item 31)
前記生体適合性高分子は、ポリエチレングリコールを含む、項目 23に記載の方法。 (項目 32) 24. The method of item 23, wherein the biocompatible polymer comprises polyethylene glycol. (Item 32)
前記ポジエチレングリコーノレは、分子量 1, 000〜200, 000の範囲内にある、項目 3 1に記載の方法。 Item 3. The method according to Item 31, wherein the positive ethylene glycolate has a molecular weight in the range of 1,000 to 200,000.
(項目 33) (Item 33)
前記生体適合性高分子は、 1% (w/v)〜50% (w/v)の濃度で使用される、項目 2 3に記載の方法。 Item 24. The method according to Item 23, wherein the biocompatible polymer is used at a concentration of 1% (w / v) to 50% (w / v).
(項目 34) (Item 34)
前記生体由来組織は、血管、血管様組織、心臓弁、心膜、硬膜、角膜および骨から なる群より選択されるものの組織である、項目 23に記載の方法。 24. The method according to item 23, wherein the biological tissue is a tissue selected from the group consisting of blood vessels, blood vessel-like tissues, heart valves, pericardium, dura mater, corneas and bones.
(項目 35) (Item 35)
前記生体由来組織は、哺乳動物由来である、項目 23に記載の方法。 Item 24. The method according to Item 23, wherein the biological tissue is derived from a mammal.
(項目 36) (Item 36)
前記生体由来組織は、ヒト、ゥシまたはブタ由来である、項目 23に記載の方法。 (項目 37) Item 24. The method according to Item 23, wherein the biological tissue is derived from human, ushi or pig. (Item 37)
項目 27に記載の方法によって得られる、生体由来組織。 A biological tissue obtained by the method according to item 27.
(項目 38) (Item 38)
糸且織の再生方法であって、 A method for regenerating yarn and weaving,
a)生体適合性高分子を含み、 y線照射を受けたことを特徴とする生体由来組織を 、生体内に提供する工程;および  a) providing a living body-derived tissue containing a biocompatible polymer, characterized by being irradiated with y-rays, into the living body; and
b)該生体内で糸且織再生が生じるに十分な時間インキュベートする工程、 を包含する、方法。 (項目 39) b) incubating for a time sufficient to produce yarn and weave regeneration in the living body. (Item 39)
前記生体由来組織は、血管、血管様組織、心臓弁、心膜、硬膜、角膜および骨から なる群より選択されるものの組織である、項目 38に記載の方法。 40. The method according to Item 38, wherein the biological tissue is a tissue selected from the group consisting of blood vessels, blood vessel-like tissues, heart valves, pericardia, dura mater, corneas, and bones.
(項目 40) (Item 40)
糸且織グラフトを生産する方法であって、 A method of producing a yarn and woven graft comprising:
A)生体適合性高分子を含み、 y線照射を受けたことを特徴とする生体由来組織を 生体内に提供する工程；  A) providing a living body-derived tissue containing a biocompatible polymer and receiving y-ray irradiation in vivo;
B)該生体由来組織に該生体の自己細胞を侵入させる工程;および  B) allowing the living body's own cells to enter the living tissue; and
C)該細胞の分ィ匕が生じるに十分な時間インキュベートする工程、  C) incubating for a time sufficient for the cell to divide;
を包含する、方法。 Including the method.
(項目 41)  (Item 41)
前記生体由来組織は、血管、血管様組織、心臓弁、心膜、硬膜、角膜および骨から なる群より選択されるものの組織である、項目 40に記載の方法。 41. The method according to Item 40, wherein the biological tissue is a tissue selected from the group consisting of blood vessels, blood vessel-like tissues, heart valves, pericardium, dura mater, corneas, and bones.
(項目 42) (Item 42)
前記生体由来組織は、自己由来である、項目 40に記載の方法。 41. The method of item 40, wherein the biological tissue is autologous.
(項目 43) (Item 43)
前記脱細胞組織は、同種異系宿主由来である、項目 40に記載の方法。 41. The method of item 40, wherein the decellularized tissue is derived from an allogeneic host.
(項目 44) (Item 44)
前記脱細胞組織は、異種宿主由来である、項目 40に記載の方法。 41. The method of item 40, wherein the decellularized tissue is derived from a heterologous host.
(項目 45) (Item 45)
項目 40に記載の方法によって生産された、組織グラフト。 41. A tissue graft produced by the method of item 40.
(項目 46) (Item 46)
組織または臓器移植を必要とする力または該危険にある被験体の処置または予防の 方法であって、 A method of treating or preventing a force requiring tissue or organ transplantation or a subject at risk.
A)生体適合性高分子を含み、 y線照射を受けたことを特徴とする生体由来組織、 または該生体由来組織を含む組織グラフトを提供する工程;および  A) providing a biological tissue containing a biocompatible polymer and receiving y-ray irradiation, or a tissue graft containing the biological tissue; and
B)該生体由来組織または組織グラフトを被験体に移植する工程、  B) Transplanting the living tissue or tissue graft into a subject,
を包含する、方法。 (項目 47) Including the method. (Item 47)
前記生体由来組織は、前記被験体由来である、項目 46に記載の方法。 49. The method of item 46, wherein the biological tissue is derived from the subject.
(項目 48) (Item 48)
前記生体由来組織は、血管、血管様組織、心臓弁、心膜、硬膜、角膜および骨から 選択されるものの組織である、項目 46に記載の方法。 49. The method according to item 46, wherein the biological tissue is a tissue selected from blood vessels, blood vessel-like tissues, heart valves, pericardia, dura mater, corneas and bones.
(項目 49) (Item 49)
前記被験体は、哺乳動物である、項目 46に記載の方法。 49. The method of item 46, wherein the subject is a mammal.
(項目 50) (Item 50)
前記被験体は、ヒトである、項目 46に記載の方法。 49. The method of item 46, wherein the subject is a human.
(項目 51) (Item 51)
臓器移植のための医薬であって、 A medicine for organ transplantation,
A)生体適合性高分子を含み、 y線照射を受けたことを特徴とする生体由来組織、 または該生体由来組織を含む組織グラフト、  A) A biological tissue containing a biocompatible polymer and receiving y-ray irradiation, or a tissue graft containing the biological tissue,
を含む、医薬。 Containing a medicament.
(項目 52) (Item 52)
前記生体由来組織は、血管、血管様組織、心臓弁、心膜、硬膜、角膜および骨から 選択されるものの組織である、項目 51に記載の医薬。 Item 52. The medicament according to Item 51, wherein the biological tissue is a tissue selected from blood vessels, blood vessel-like tissues, heart valves, pericardia, dura mater, corneas and bones.
(項目 53) (Item 53)
前記生体由来組織は、哺乳動物由来である、項目 51に記載の医薬。 52. The medicament according to item 51, wherein the biological tissue is derived from a mammal.
(項目 54) (Item 54)
前記生体由来組織は、ヒト、ゥシまたはブタ由来である、項目 51に記載の医薬。 (項目 55) 52. The medicament according to item 51, wherein the biological tissue is derived from human, ushi or pig. (Item 55)
前記生体由来組織は、前記移植を必要とする被験体由来である、項目 51に記載の 医薬。 52. The medicament according to item 51, wherein the biological tissue is derived from a subject in need of the transplant.
(項目 56)  (Item 56)
生体適合性高分子を含み、 γ線照射を受けたことを特徴とする生体由来組織、また は該生体由来組織を含む組織グラフトの、臓器移植のための医薬を製造するための 使用。 (項目 57) Use of a biological tissue containing a biocompatible polymer and receiving γ-ray irradiation, or a tissue graft containing the biological tissue for producing a medicament for organ transplantation. (Item 57)
化学架橋されたことをさらなる特徴とする、項目 1に記載の生体由来組織。 Item 2. The biological tissue according to Item 1, further characterized by being chemically crosslinked.
(項目 58) (Item 58)
前記さらなる特徴は、二官能性分子架橋剤による結合を含む、項目 57に記載の生 体由来組織。 58. The organism-derived tissue according to item 57, wherein the further characteristic includes binding by a bifunctional molecular crosslinking agent.
(項目 59) (Item 59)
前記結合は、スルフヒドリル基、アルデヒド基、カルボ-ル基、スルホ基および-トロ基 からなる群より選択される基の間で形成される結合、炭素 炭素結合、ペプチド結合 、エーテル結合ならびにエステル結合力 なる群より選択される結合力 なる群より選 択される、項目 58に記載の生体由来組織。 The bond is a bond formed between groups selected from the group consisting of sulfhydryl group, aldehyde group, carbo group, sulfo group and -tro group, carbon-carbon bond, peptide bond, ether bond and ester bond strength. 59. The biological tissue according to Item 58, which is selected from the group consisting of a binding force selected from the group consisting of
(項目 60) (Item 60)
前記二官能性分子架橋剤が、アミノ基、カルボキシル基およびアルデヒド基力 なる 群より選択される官能基を有する、項目 58に記載の生体由来組織。 59. The biological tissue according to Item 58, wherein the bifunctional molecular crosslinking agent has a functional group selected from the group consisting of an amino group, a carboxyl group, and an aldehyde group.
(項目 61) (Item 61)
前記二官能性分子架橋剤が、ダルタルアルデヒド、シアンイミド (cyanimide)、 1ーェ チル一 3— (3—ジメチルァミノプロピル）カルボジイミドヒドロクロリド（EDC)、エポキシ (ポリ（グリシジルエーテル））、 2, 6 ビス（4 アジドベンジリデン）ー4ーメチルシクロ へキサノン、 4, 4'ジアジドジフエニルエーテル、 4, 4'ジアジドジフエニルスルホン、 4 , 4'ジアジドジフエニルアセトン、 4, 4'ジアジドジフエニルメタン、 1, 4 ビス（α ジ ァゾベンジル)および 4— [ρ アジドサリチアミド]プチルァミン力もなる群より選択さ れる、項目 58に記載の生体由来組織。 The bifunctional molecular crosslinking agent is selected from the group consisting of daltaraldehyde, cyanimide, 1-ethyl-1- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), epoxy (poly (glycidyl ether)), 2 , 6 bis (4 azidobenzylidene) -4-methylcyclohexanone, 4, 4 'diazide diphenyl ether, 4, 4' diazide diphenyl sulfone, 4, 4 'diazide diphenyl acetone, 4, 4' diazide 59. The biological tissue according to Item 58, which is selected from the group consisting of diphenylmethane, 1,4 bis (α diazobenzyl), and 4- [ρazidosalicyamido] ptylamine.
(項目 62) (Item 62)
前記二官能性分子が、ダルタルアルデヒドである、項目 61に記載の生体由来組織。 (項目 63) 62. The biological tissue according to item 61, wherein the bifunctional molecule is dartalaldehyde. (Item 63)
前記生体由来組織を、ダルタルアルデヒドにより化学的に処理し、ポリエチレングリコ ール (PEG)に曝露し、 γ線を照射して架橋を形成することにより、ダルタルアルデヒ ド処理をしたものに比べて γ線照射後の組織強度が上昇している力、またはグルタ ルアルデヒド処理をしたものと同等の組織強度を有する生体由来組織であって、 該生体由来組織は、 PEGを内包し、かつ該生体由来組織の表面には PEG層が形 成されている、項目 1に記載の生体由来組織。 The biological tissue is chemically treated with dartal aldehyde, exposed to polyethylene glycol (PEG), and irradiated with γ rays to form crosslinks. A tissue derived from a living body having a tissue strength equivalent to that of a force that increases tissue strength after irradiation or glutaraldehyde treatment, 2. The biological tissue according to item 1, wherein the biological tissue contains PEG, and a PEG layer is formed on the surface of the biological tissue.
(項目 64) (Item 64)
カルボキシル基、ヒドロキシル基およびアミノ基以外の非化学架橋基の間に形成され る架橋を少なくとも 1つ有する、項目 1に記載の生体由来組織。 Item 2. The biological tissue according to Item 1, having at least one crosslink formed between non-chemical crosslinkable groups other than a carboxyl group, a hydroxyl group and an amino group.
(項目 65) (Item 65)
前記非化学架橋基が、スルフヒドリル基、アルデヒド基、カルボニル基、スルホ基およ び-トロ基力 なる群より選択される、項目 64に記載の生体由来組織。 Item 65. The biological tissue according to Item 64, wherein the non-chemical crosslinking group is selected from the group consisting of a sulfhydryl group, an aldehyde group, a carbonyl group, a sulfo group, and a -tro group force.
(項目 66) (Item 66)
γ線照射により生じた、炭素 炭素結合、エーテル結合およびエステル結合からな る群より選択される結合により形成される架橋が少なくとも 1つ有する、項目 1に記載 の生体由来組織。 Item 2. The biological tissue according to Item 1, having at least one bridge formed by γ-ray irradiation and formed by a bond selected from the group consisting of a carbon-carbon bond, an ether bond and an ester bond.
(項目 67) (Item 67)
骨格が開裂して形成された新たな結合により形成された架橋を少なくとも 1つ有する 、項目 1に記載の生体由来組織。 Item 2. The biological tissue according to Item 1, which has at least one crosslink formed by a new bond formed by cleavage of the skeleton.
(項目 68) (Item 68)
側鎖に遊離のアミノ基およびカルボキシル基を有さないアミノ酸の間に形成された非 化学架橋を少なくとも 1つ有する、項目 1に記載の生体由来組織。 Item 2. The biological tissue according to Item 1, comprising at least one non-chemical crosslink formed between amino acids having no free amino group and no carboxyl group in the side chain.
(項目 69) (Item 69)
前記非化学架橋が、リジン、アルギニン以外を架橋点としたアミノ酸間の架橋である、 項目 68に記載の生体由来組織。 70. The biological tissue according to Item 68, wherein the non-chemical cross-linking is a cross-linking between amino acids having a cross-linking point other than lysine and arginine.
(項目 70) (Item 70)
カルボキシル基、ヒドロキシル基およびアミノ基以外の非化学架橋基を有する多糖の 間に形成された架橋を少なくとも 1つ有する、項目 1に記載の生体由来組織。 Item 2. The biological tissue according to Item 1, having at least one crosslink formed between polysaccharides having non-chemical crosslinkable groups other than a carboxyl group, a hydroxyl group and an amino group.
(項目 71) (Item 71)
前記非化学架橋基を有する多糖が、スルフヒドリル基、アルデヒド基、カルボニル基、 スルホ基および-トロ基力 なる群より選択される少なくとも 1つの基の間で形成され る架橋を有する、項目 70に記載の生体由来組織。 (項目 72) Item 70. The item 70, wherein the polysaccharide having a non-chemical crosslinking group has a bridge formed between at least one group selected from the group consisting of a sulfhydryl group, an aldehyde group, a carbonyl group, a sulfo group, and a -tro group. Living body tissue. (Item 72)
ビスエポキシド架橋、ジビニルスルフォン架橋、分子内エステル化、グルタルアルデヒ ド架橋、カルポジイミド架橋およびヒドラジド架橋以外の非化学架橋を少なくとも 1つ 有する、項目 1に記載の生体由来組織。 Item 2. The biological tissue according to Item 1, comprising at least one non-chemical crosslink other than bisepoxide crosslink, divinylsulfone crosslink, intramolecular esterification, glutaraldehyde crosslink, carpositimide crosslink and hydrazide crosslink.
(項目 73) (Item 73)
組織間の癒着を防止するために使用される、項目 1に記載の生体由来組織。 Item 2. The biological tissue according to Item 1, which is used for preventing adhesion between tissues.
(項目 74) (Item 74)
生体内において使用するための、項目 1に記載の生体由来組織。 Item 2. The biological tissue according to Item 1, for use in vivo.
(項目 75) (Item 75)
生体外において使用するための、項目 1に記載の生体由来組織。 Item 2. The biological tissue according to Item 1, for use in vitro.
(項目 76) (Item 76)
移植用の癒着防止膜を生産する方法であって、 A method for producing an anti-adhesion membrane for transplantation, comprising:
A)生体由来膜を提供する工程；  A) providing a biological membrane;
B)該生体由来膜を、生体適合性高分子に曝す工程；および  B) exposing the biological membrane to a biocompatible polymer; and
C)該生体由来膜を γ線照射に曝す工程、  C) exposing the biological membrane to γ-ray irradiation,
を包含する、方法。 Including the method.
(項目 77)  (Item 77)
項目 76に記載の移植用の癒着防止膜を生産する方法であって、 A method for producing an adhesion-preventing membrane for transplant according to item 76, comprising:
D)前記生体由来膜を二官能性分子架橋剤により化学架橋させる工程をさらに包 含する、方法。  D) A method further comprising the step of chemically crosslinking the biological membrane with a bifunctional molecular crosslinking agent.
(項目 78)  (Item 78)
前記 D)工程が、 Α)工程— Β)工程の間、および Β)工程— Α)工程の間からなる群よ り選択される少なくとも 1つの間にて実施される、項目 77に記載の方法。 78. A method according to item 77, wherein the step D) is carried out between at least one selected from the group consisting of step ii) step-step ii) and step ii) step-step ii). .
(項目 79) (Item 79)
前記化学架橋が、二官能性分子架橋剤による結合を含む、項目 77に記載の方法。 (項目 80) 80. A method according to item 77, wherein the chemical cross-linking includes bonding with a bifunctional molecular cross-linking agent. (Item 80)
前記結合は、スルフヒドリル基、アルデヒド基、カルボ-ル基、スルホ基および-トロ基 からなる群より選択される基の間で形成される結合、炭素 炭素結合、ペプチド結合 、エーテル結合ならびにエステル結合力 なる群より選択される結合力 なる群より選 択される、項目 79に記載の方法。 The bond is a bond formed between a group selected from the group consisting of a sulfhydryl group, an aldehyde group, a carbo group, a sulfo group, and a -tro group, a carbon-carbon bond, and a peptide bond. 80. The method according to Item 79, wherein the bond is selected from the group consisting of a bond strength selected from the group consisting of an ether bond and an ester bond strength.
(項目 81) (Item 81)
前記二官能性分子架橋剤が、アミノ基、カルボキシル基およびアルデヒド基力 なる 群より選択される官能基を有する、項目 79に記載の方法。 80. The method according to Item 79, wherein the bifunctional molecular crosslinking agent has a functional group selected from the group consisting of an amino group, a carboxyl group, and an aldehyde group.
(項目 82) (Item 82)
前記二官能性分子架橋剤が、ダルタルアルデヒド、シアンイミド (cyanimide)、 1ーェ チル一 3— (3—ジメチルァミノプロピル）カルボジイミドヒドロクロリド（EDC)、エポキシ (ポリ（グリシジルエーテル））、 2, 6 ビス（4 アジドベンジリデン）ー4ーメチルシクロ へキサノン、 4, 4'ジアジドジフエニルエーテル、 4, 4'ジアジドジフエニルスルホン、 4 , 4'ジアジドジフエニルアセトン、 4, 4'ジアジドジフエニルメタン、 1, 4 ビス（α ジ ァゾベンジル)および 4— [ρ アジドサリチアミド]プチルァミン力もなる群より選択さ れる、項目 79に記載の方法。 The bifunctional molecular crosslinking agent is selected from the group consisting of daltaraldehyde, cyanimide, 1-ethyl-1- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), epoxy (poly (glycidyl ether)), 2 , 6 bis (4 azidobenzylidene) -4-methylcyclohexanone, 4, 4 'diazide diphenyl ether, 4, 4' diazide diphenyl sulfone, 4, 4 'diazide diphenyl acetone, 4, 4' diazide 80. The method of item 79, wherein the compound is also selected from the group consisting of diphenylmethane, 1,4 bis (α diazobenzyl), and 4- [ρazidosalicyamido] plutamine.
(項目 83) (Item 83)
前記二官能性分子架橋剤が、ダルタルアルデヒドである、項目 82に記載の方法。 (項目 84) 83. A method according to item 82, wherein the bifunctional molecular crosslinking agent is dartalaldehyde. (Item 84)
前記生体適合性高分子は、ポリビュルアルコール、ポリビュルピロリドン、エラスチン 、ポリエチレングリコール、ゼラチン、コラーゲン、 γ -ポリグルタミン酸およびそれらの 2つ以上の混合物力 なる群より選択される高分子を含む、項目 76に記載の方法。 (項目 85) The biocompatible polymer includes a polymer selected from the group consisting of polybulal alcohol, polybulurpyrrolidone, elastin, polyethylene glycol, gelatin, collagen, γ-polyglutamic acid, and a mixture of two or more thereof. 76. The method according to 76. (Item 85)
前記生体適合性高分子は、ポリエチレングリコールを含む、項目 76に記載の方法。 (項目 86) 77. A method according to item 76, wherein the biocompatible polymer comprises polyethylene glycol. (Item 86)
前記 Α)工程において、前記生体由来膜は、膜状組織、弁状組織または管状組織か ら採取される、項目 76に記載の方法。 77. The method according to item 76, wherein in the step (ii), the biological membrane is collected from a membranous tissue, a valve-like tissue or a tubular tissue.
(項目 87) (Item 87)
前記 Α)工程において、前記生体由来膜は、血管、血管様組織、心臓弁、心膜、硬 膜、角膜および骨から採取される、項目 76に記載の方法。 77. The method according to item 76, wherein, in the step (ii), the biological membrane is collected from a blood vessel, a blood vessel-like tissue, a heart valve, a pericardium, a dura mater, a cornea, and a bone.
(項目 88) 前記 A)工程において、前記生体由来膜は、哺乳動物力も採取される、項目 76に記 載の方法。 (Item 88) 79. The method according to Item 76, wherein in the step A), the biological membrane is also collected from mammal strength.
(項目 89) (Item 89)
前記 A)工程において、前記癒着防止膜は、ヒト、ゥシまたはブタ由来である、項目 7 6に記載の方法。 Item 7. The method according to Item 76, wherein in the step A), the adhesion-preventing membrane is derived from human, ushi or pig.
(項目 90) (Item 90)
前記 B)工程において、前記生体由来膜に対する前記生体適合性高分子の曝露が 、 24°Cにて、 12— 24時間という条件下で実施される、項目 76に記載の方法。 77. The method according to item 76, wherein in step B), the exposure of the biocompatible polymer to the biological membrane is performed at 24 ° C. under a condition of 12 to 24 hours.
(項目 91) (Item 91)
前記 C)工程において、 γ線の強度は 50keVであり、 γ線の照射は、密閉容器中、 線量 12— 50kGyで、 24— 37°Cという条件下で実施される、項目 76に記載の方法。 (項目 92) 77. The method according to item 76, wherein, in step C), the intensity of γ-ray is 50 keV, and the irradiation of γ-ray is performed in a sealed container at a dose of 12-50 kGy and at a temperature of 24-37 ° C. . (Item 92)
前記化学架橋が、 24°Cにて、 1〜4時間という条件下で生じる、項目 77に記載の方 法。 80. A method according to item 77, wherein the chemical crosslinking occurs at 24 ° C. under a condition of 1 to 4 hours.
(項目 93)  (Item 93)
生体適合性高分子を含み、 y線照射を受けたことを特徴とする癒着防止膜であって 、該癒着防止膜は生体由来である、癒着防止膜。 An anti-adhesion membrane comprising a biocompatible polymer and having been irradiated with y-rays, wherein the anti-adhesion membrane is derived from a living body.
(項目 94) (Item 94)
カルボキシル基、ヒドロキシル基およびアミノ基以外の非化学架橋基の間に形成され る架橋を少なくとも 1つ有する、項目 93に記載の癒着防止膜。 94. The adhesion-preventing film according to Item 93, which has at least one crosslink formed between non-chemical crosslinkable groups other than a carboxyl group, a hydroxyl group and an amino group.
(項目 95) (Item 95)
前記非化学架橋基が、スルフヒドリル基、アルデヒド基、カルボニル基、スルホ基およ び-トロ基力 なる群より選択される、項目 94に記載の癒着防止膜。 95. The adhesion-preventing membrane according to Item 94, wherein the non-chemical crosslinking group is selected from the group consisting of a sulfhydryl group, an aldehyde group, a carbonyl group, a sulfo group, and a -tro group force.
(項目 96) (Item 96)
γ線照射により生じた、炭素 炭素結合、エーテル結合およびエステル結合からな る群より選択される結合により形成される架橋を少なくとも 1つ有する、項目 93に記載 の癒着防止膜。 94. The adhesion-preventing film according to Item 93, which has at least one cross-link formed by γ-ray irradiation and formed by a bond selected from the group consisting of a carbon-carbon bond, an ether bond and an ester bond.
(項目 97) 骨格を形成して ヽる炭素間結合、エステル結合が開裂して形成された新たな結合に より形成された架橋を少なくとも 1つ有する、項目 93に記載の癒着防止膜。 (Item 97) 94. The adhesion-preventing membrane according to Item 93, which has at least one cross-link formed by a carbon-carbon bond formed by forming a skeleton and a new bond formed by cleavage of an ester bond.
(項目 98) (Item 98)
側鎖に遊離のアミノ基およびカルボキシル基を有さないアミノ酸の間に形成された非 化学架橋を少なくとも 1つ有する、項目 93に記載の癒着防止膜。 94. The adhesion-preventing membrane according to Item 93, which has at least one non-chemical crosslink formed between amino acids having no free amino group and no carboxyl group in the side chain.
(項目 99) (Item 99)
前記非化学架橋が、リジン、アルギニン以外を架橋点としたアミノ酸間の架橋である、 項目 98に記載の癒着防止膜。 Item 99. The adhesion-preventing membrane according to Item 98, wherein the non-chemical crosslinking is a crosslinking between amino acids having a crosslinking point other than lysine and arginine.
(項巨 100) (Term 100)
カルボキシル基、ヒドロキシル基およびアミノ基以外の非化学架橋基を有する多糖の 間に形成された架橋を少なくとも 1つ有する、項目 93に記載の癒着防止膜。 94. The adhesion-preventing membrane according to Item 93, having at least one crosslink formed between polysaccharides having non-chemical crosslinkable groups other than carboxyl, hydroxyl and amino groups.
(項目 101) (Item 101)
前記非化学架橋基を有する多糖が、スルフヒドリル基、アルデヒド基、カルボニル基、 スルホ基および-トロ基力 なる群より選択される少なくとも 1つの基の間で形成され る架橋を有する、項目 100に記載の癒着防止膜。 101. The item 100, wherein the polysaccharide having a non-chemical crosslinking group has a bridge formed between at least one group selected from the group consisting of a sulfhydryl group, an aldehyde group, a carbonyl group, a sulfo group, and a -tro group. Anti-adhesion membrane.
(項目 102) (Item 102)
ビスエポキシド架橋、ジビニルスルフォン架橋、分子内エステル化、グルタルアルデヒ ド架橋、カルポジイミド架橋およびヒドラジド架橋以外の非化学架橋を少なくとも 1つ 有する、項目 93に記載の癒着防止膜。 94. The adhesion-preventing film according to Item 93, comprising at least one non-chemical crosslink other than bisepoxide crosslink, divinylsulfone crosslink, intramolecular esterification, glutaraldehyde crosslink, carpositimide crosslink and hydrazide crosslink.
(項目 103) (Item 103)
側鎖に遊離アミノ基またはカルボキシル基を有しない 2以上のアミノ酸の間に形成さ れる共有結合、カルボキシル基、ヒドロキシル基、アミノ基、スルフヒドリル基、アルデヒ ド基およびカルボ-ル基からなる群より選択される基の間で形成される結合、炭素 炭素結合、ペプチド結合、エーテル結合ならびにエステル結合力 なる群より選択さ れる結合を有する、項目 93に記載の癒着防止膜。 Selected from the group consisting of a covalent bond, carboxyl group, hydroxyl group, amino group, sulfhydryl group, aldehyde group and carbocycle group formed between two or more amino acids having no free amino group or carboxyl group in the side chain 94. The adhesion-preventing membrane according to Item 93, which has a bond selected from the group consisting of a bond formed between the groups to be bonded, a carbon-carbon bond, a peptide bond, an ether bond, and an ester bond strength.
(項目 104) (Item 104)
二官能性分子架橋剤による結合により形成される架橋をさらに含む、項目 93に記載 の癒着防止膜。 (項目 105) 94. The adhesion-preventing film according to Item 93, further comprising a crosslink formed by bonding with a bifunctional molecular crosslinker. (Item 105)
前記二官能性分子架橋剤が、ダルタルアルデヒドである、項目 104に記載の癒着防 止膜。 105. The adhesion-preventing film according to Item 104, wherein the bifunctional molecular crosslinking agent is dartalaldehyde.
(項目 106) (Item 106)
前記癒着防止膜が、ポリエチレングリコールを含む、項目 93に記載の癒着防止膜。 (項目 107) 94. The adhesion prevention film according to Item 93, wherein the adhesion prevention film contains polyethylene glycol. (Item 107)
前記癒着防止膜が、胸膜、心膜、脳硬膜、漿膜、腹膜および皮膚からなる群より選択 される、項目 93に記載癒着防止膜。 94. The adhesion prevention membrane according to Item 93, wherein the adhesion prevention membrane is selected from the group consisting of pleura, pericardium, brain dura mater, serosa, peritoneum and skin.
(項目 108) (Item 108)
前記癒着防止膜は、哺乳動物由来である、項目 93に記載癒着防止膜。 94. The adhesion prevention film according to Item 93, wherein the adhesion prevention film is derived from a mammal.
(項目 109) (Item 109)
前記癒着防止膜は、ヒト、ゥシまたはブタ由来である、項目 93に記載癒着防止膜。 (項目 110) 94. The adhesion prevention film according to Item 93, wherein the adhesion prevention film is derived from human, ushi or pig. (Item 110)
前記癒着防止膜は、以下： The anti-adhesion membrane is as follows:
A) A)
1)プラスチック製の薄いシートに心膜を挟み込む工程、  1) The process of sandwiching the pericardium in a thin plastic sheet,
2)サンプルが変形しないように電子ノギスにて 3ケ所以上の厚みを測定する工程、 および  2) A process of measuring the thickness of three or more locations with an electronic caliper so that the sample does not deform, and
3)該サンプルの厚みの平均を算出する工程により測定した場合、 0. 5- 1. Omm の厚みを有すること；ならびに  3) have a thickness of 0.5-1. Omm when measured by the process of calculating the average thickness of the sample; and
B)  B)
1)検体を 5 X 30mmの短冊状に切断する工程、  1) Cutting the specimen into 5 x 30mm strips,
2)引っ張り試験器の固定部分に該検体の両端約 5mmを固定する工程、および 2) fixing about 5 mm at both ends of the specimen to the fixed part of the tensile tester; and
3)引っ張り開始し破断点まで lcmZminの速度で引っ張り、レジオメーターにて測 定する工程により測定した場合、 2— lOMPaの弾性率を有すること 3) It has a modulus of elasticity of 2-lOMPa when it is measured by the process of measuring with a radiometer after pulling to the breaking point at a speed of lcmZmin.
を特徴とする、項目 93に記載の癒着防止膜。 94. The adhesion-preventing membrane according to item 93, wherein
(項目 111) (Item 111)
前記癒着防止膜は、試験部位に該癒着防止膜を貼付けた場合に、以下の式： 癒着防止率 (％) = {癒着なしの数 Z (癒着あり +癒着なしの数） } X loo により、 80%以上の癒着防止率を有する、項目 93に記載の癒着防止膜。 The adhesion preventing film has the following formula when the adhesion preventing film is attached to a test site: Anti-adhesion rate (%) = {number without adhesion Z (number with adhesion + number without adhesion)} The adhesion prevention film according to Item 93, which has an adhesion prevention rate of 80% or more due to X loo.
(項目 112)  (Item 112)
生体内において使用するための、項目 93に記載の癒着防止膜。  94. The adhesion-preventing membrane according to Item 93, for use in vivo.
(項目 113)  (Item 113)
生体外において使用するための、項目 93に記載の癒着防止膜。  94. The antiadhesion membrane according to Item 93, which is used in vitro.
(項目 114)  (Item 114)
前記生体由来膜を、ダルタルアルデヒドにより化学的に処理し、ポリエチレングリコー ル (PEG)に曝露し、 γ線を照射して架橋を形成することにより、ダルタルアルデヒド 処理をしたものに比べて γ線照射後の組織強度が上昇しているか、またはダルタル アルデヒド処理をしたものと同等の組織強度を有する生体由来膜であって、 該癒着防止は、 PEGを内包し、かつ該生体由来膜の表面には PEG層が形成されて いる、項目 93に記載の生体由来膜。  The bio-derived membrane is chemically treated with dartalaldehyde, exposed to polyethylene glycol (PEG), and irradiated with γ rays to form crosslinks. A bio-derived membrane having increased tissue strength after irradiation or having tissue strength equivalent to that treated with dartalaldehyde, wherein the adhesion prevention includes PEG and the surface of the bio-derived membrane 94. The biological membrane according to Item 93, wherein a PEG layer is formed on.
(項目 115)  (Item 115)
前記 B)工程が、癒着を防ぐ必要のある部位において実施される、項目 46に記載の 処置または予防の方法。  47. The treatment or prevention method according to item 46, wherein the step B) is performed at a site where adhesion needs to be prevented.
(項目 116)  (Item 116)
組織の癒着を防ぐ必要がある力または該危険にある被験体の処置または予防の方 法であって、  A force that needs to prevent tissue adhesions or a method of treating or preventing a subject at risk, comprising:
A)生体適合性高分子を含み、 γ線照射を受けたことを特徴とする癒着防止膜を提 供する工程;および  A) providing an anti-adhesion membrane comprising a biocompatible polymer and characterized by receiving γ-irradiation; and
Β)該癒着防止膜を被験体に移植する工程、  I) transplanting the anti-adhesion membrane to a subject;
を包含する、方法。  Including the method.
[0022] 従って、本発明のこれらおよび他の利点は、添付の図面を参照して、以下の詳細な 説明を読みかつ理解すれば、当業者には明白〖こなることが理解される。  [0022] Accordingly, it will be appreciated that these and other advantages of the present invention will be apparent to those of ordinary skill in the art upon reading and understanding the following detailed description with reference to the accompanying drawings.
発明の効果  The invention's effect
[0023] 石灰化が顕著に抑制された生体由来組織が提供される。石灰化の抑制は、 UV照 射、科学的処理などの他の方法に比べて、 γ線照射が顕著に抑制されていた。本発 明ではまた、強度が補強、改善または維持された生体由来組織が提供される。従つ て、本発明は、生体由来組織を補強し、免疫拒絶反応を低減させるという効果を奏 する。 [0023] A biological tissue in which calcification is remarkably suppressed is provided. In terms of suppression of calcification, γ-ray irradiation was significantly suppressed compared to other methods such as UV irradiation and scientific treatment. Main departure Ming also provides biological tissue that is reinforced, improved or maintained in strength. Therefore, the present invention has an effect of reinforcing a living body-derived tissue and reducing immune rejection.
[0024] 上記処理方法によって処理された移植用組織は、従来の技術によって調製された 移植用生体組織に比べて、石灰化が顕著に抑制されるという有利な効果を示す。  [0024] The transplanted tissue treated by the above treatment method exhibits an advantageous effect that calcification is remarkably suppressed as compared with the transplanted biological tissue prepared by the conventional technique.
[0025] 作用メカニズムについては、 A: PEG等のゲルィ匕による細胞浸潤の阻害、もしくは B ：細胞外マトリックス (ECM)を含む生体組織の成分が PEGにより修飾されることによ る免疫原生の抑制などが考えられる力 これに限定されない。  [0025] Regarding the mechanism of action: A: Inhibition of cell invasion by PEG and other gels, or B: Suppression of immunogenicity by modification of components of biological tissue including extracellular matrix (ECM) with PEG The power that can be considered is not limited to this.
[0026] γ線照射による重合反応が従来の重合反応に比べて有利な理由については、例 えば以下のようなものが予想される力 これに限定されない。  [0026] The reason why the polymerization reaction by γ-ray irradiation is more advantageous than the conventional polymerization reaction is not limited to this.
[0027] 従来の重合反応は比較的制御された化学反応であるのに対し、 γ線照射では比 較的ランダムに PEG分子間に架橋が導入されると考えられ、そのことが石灰化のより 効果的な抑制に繋がっていると考えられる。  [0027] The conventional polymerization reaction is a relatively controlled chemical reaction, whereas γ-ray irradiation is considered to introduce cross-links between PEG molecules relatively randomly. It is thought that it leads to effective suppression.
[0028] また、従来の free radical initiator (フリーラジカルを発生する重合開始剤）を利 用した処理における欠点、例えば、これらが残留したまま体内に移植されると毒性な どを示す可能性が本発明によって、克服された。本発明の方法は、 γ線照射による 照射誘導性重合開始反応 (radiation induced initiators)によるものであり、放 射線によりタンパク質などの高分子のラジカルができる力 比較的短時間で消滅する ため、ラジカルは全く残留しないようであり、これは、顕著な効果である。  [0028] In addition, there is a possibility that the treatment using a conventional free radical initiator (polymerization initiator that generates free radicals), for example, there is a possibility of showing toxicity when transplanted into the body with these remaining. Overcame by the invention. The method of the present invention is based on radiation induced initiators by γ-ray irradiation, and the force that can generate radicals of macromolecules such as proteins by radiation disappears in a relatively short time. There seems to be no residue at all, which is a significant effect.
[0029] 照射された生体組織中のラジカルの寿命については、ヒト真皮由来の移植用の脱 細胞化組織（商品名 Allorderm, Life Cell Corp. )をシンクロトロン放射光で照射 して、コラーゲンマトリクスに生じた超寿命のラジカルを EPRで測定したところ、一時 間程度以内でほぼ減衰したという報告がある（Radiation Research 163, 535— 543, 2005)。本発明でも同様の寿命であるようであることから、その悪影響を与え るラジカルは、実質的に問題がないという点で顕著な効果があると考えられる。  [0029] Regarding the lifetime of radicals in irradiated biological tissue, the decellularized tissue for transplantation derived from human dermis (trade name Allorderm, Life Cell Corp.) was irradiated with synchrotron radiation to the collagen matrix. When the generated long-lived radicals were measured by EPR, there was a report that they were almost attenuated within about one hour (Radiation Research 163, 535-543, 2005). Since it seems to have the same lifetime in the present invention, it is considered that radicals that exert an adverse effect have a remarkable effect in that there is substantially no problem.
図面の簡単な説明  Brief Description of Drawings
[0030] [図 1]図 1は、本発明の生体適合性分子処理および γ線照射後の生体由来組織のラ ットへの移植後 2ヶ月の様子を示す。本図では、上に 25kGyの γ線で重合させたゥ シ心膜を示し、下に 50kGyの γ線で重合させたゥシ心膜を示す。左は生体内の様 子および右は取り出した生体由来組織を示す。 25kGy処理では細胞の浸潤は薄 ヽ 力 50kGy処理では細胞の顕著な浸潤が認められる。 [0030] [Fig. 1] Fig. 1 shows a state 2 months after the biocompatible molecular treatment and γ-irradiation irradiation of the present invention after transplantation into a rat. In this figure, the top is polymerized with 25 kGy gamma rays. The pericardium is shown below, and the pericardium polymerized with 50 kGy gamma rays is shown below. The left shows the appearance in the living body and the right shows the extracted biological tissue. Cell invasion is observed with 25 kGy treatment. Remarkable infiltration of cells is observed with 50 kGy treatment.
[図 2]図 2は、本発明の生体適合性分子処理および γ線照射後の生体由来組織のラ ットへの移植後 2ヶ月の HE染色図を示す。本図では、上に 25kGyの γ線で重合させ たゥシ心膜を示し、下に 50kGyの γ線で重合させたゥシ心膜を示す。各図は種々の 部位での写真を示した例である。  [FIG. 2] FIG. 2 shows a HE-stained diagram of 2 months after the biocompatible molecular treatment and γ-irradiation irradiation of the present invention after transplantation into a rat. In this figure, the ushi pericardium polymerized with 25 kGy gamma rays is shown above, and the ushi pericardium polymerized with 50 kGy gamma rays is shown below. Each figure is an example showing photographs of various parts.
[図 3]図 3は、本発明の生体適合性分子処理および γ線照射後の生体由来組織のラ ットへの移植後 2ヶ月の von Kossa染色を示す。本図では、上に 25kGyの γ線で重 合させたゥシ心膜を示し、下に 50kGyの γ線で重合させたゥシ心膜を示す。各図は 種々の部位での写真を示した例である。  [FIG. 3] FIG. 3 shows von Kossa staining 2 months after transplantation of biologically-derived tissue into rats after biocompatible molecular treatment and γ-irradiation according to the present invention. In this figure, the pericardium superposed with 25 kGy gamma rays is shown above, and the pericardium polymerized with 50 kGy gamma rays is shown below. Each figure is an example showing photographs of various parts.
[図 4]図 4は、本発明の生体適合性分子処理および γ線照射後の生体由来組織のラ ットへの移植後 2ヶ月のカルシウム沈着量を示す。グラフでは、左から、 25kGyの γ線 で重合させたゥシ心膜、 50kGyの γ線で重合させたゥシ心膜およびダルタルアルデ ヒド処理したゥシ心膜を示す。  [FIG. 4] FIG. 4 shows the amount of calcification in the two months after transplantation of the biological tissue of the present invention to the rat after the biocompatible molecular treatment and γ-irradiation. From the left, the graph shows the persimmon pericardium polymerized with 25 kGy gamma rays, the persimmon pericardium polymerized with 50 kGy gamma rays, and the persimmon pericardium treated with dartal aldehyde.
[図 5]図 5は、未処理のゥシ心膜についての移植 2ヶ月後のデータを示す。  [FIG. 5] FIG. 5 shows data 2 months after transplantation for untreated urine pericardium.
[図 6]図 6は、ラット背部の皮下に、ダルタルアルデヒド固定 + PEG化処理ゥシ心膜（ 右上段)およびダルタルアルデヒド固定ゥシ心膜 (左上段)を移植して 2ヶ月経過後の 摘出時の写真を示す。下段は、摘出後のダルタルアルデヒド固定 +PEG化処理ゥシ 心膜 (右）、およびダルタルアルデヒド固定ゥシ心膜 (左）の写真を示す。  [Figure 6] Figure 6 shows two months after transplantation of dartalaldehyde-fixed + PEGylated ushi pericardium (upper right) and dartalaldehyde-fixed ushi pericardium (upper left) in the rat dorsal skin. The photograph at the time of subsequent extraction is shown. The lower panel shows photographs of the extracted dartalaldehyde fixed + PEGylated pericardium (right) and dartalaldehyde fixed pericardium (left).
[図 7]図 7は、ラット背部の皮下に移植した 2ヶ月後の、ダルタルアルデヒド固定ゥシ心 膜およびダルタルアルデヒド固定 +PEG化処理ゥシ心膜におけるカルシウム沈着の 結果を示す図である。ダルタルアルデヒド固定 +PEG化処理ゥシ心膜 (N= 5)では、 すべてのサンプルで石灰化の所見である Caは検出されなかった。ダルタルアルデヒ ド固定ゥシ心膜 (N = 6)では、すべてのサンプルにて石灰化の所見である Caは検出 された。  [FIG. 7] FIG. 7 is a graph showing the results of calcium deposition in dartalaldehyde-fixed ushi pericardium and dartalaldehyde-fixed + PEGylated ushi pericardium two months after transplantation subcutaneously on the back of rats. is there. In all samples, calcium, a calcification finding, was not detected in dartalaldehyde-fixed + PEGylated treated pericardium (N = 5). In dartal aldehyde fixed pericardium (N = 6), Ca, a calcification finding, was detected in all samples.
発明を実施するための最良の形態 BEST MODE FOR CARRYING OUT THE INVENTION
以下、本発明に関して、発明の実施の形態を説明する。本明細書の全体にわたり、 単数形の表現 (例えば、英語の場合は「a」、「an」、「the」など、独語の場合の「ein」、 「der」、「das」、「die」などおよびその格変化形、仏語の場合の「un」、「une」、「le」、 riajなど、スペイン語における「un」、「una」、「el」、「la」など、他の言語における対 応する冠詞、形容詞など）は、特に言及しない限り、その複数形の概念をも含むこと が理解されるべきである。また、本明細書において使用される用語は、特に言及しな い限り、当該分野で通常用いられる意味で用いられることが理解されるべきである。し たがって、他に定義されない限り、本明細書中で使用される全ての専門用語および 科学技術用語は、本発明の属する分野の当業者によって一般的に理解されるのと同 じ意味を有する。矛盾する場合、本明細書 (定義を含めて)が優先する。 Hereinafter, embodiments of the present invention will be described with respect to the present invention. Throughout this specification, Singular expressions (e.g. "a", "an", "the" for English, "ein", "der", "das", "die" for German, etc. “Un”, “une”, “le”, riaj in French, etc., “un”, “una”, “el”, “la” in Spanish, etc. Corresponding articles and adjectives in other languages ) Should be understood to include its plural concept unless otherwise stated. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. . In case of conflict, the present specification, including definitions, will control.
[0032] (用語の定義）  [0032] (Definition of terms)
以下に本明細書において特に使用される用語の定義を列挙する。  Listed below are definitions of terms particularly used in the present specification.
[0033] (再生医療）  [0033] (Regenerative medicine)
本明細書において使用される「再生」（regeneration)とは、個体の組織の一部が失 われた際に残った組織が増殖して復元される現象をいう。動物種間または同一個体 における組織種に応じて、再生のその程度および様式は変動する。ヒト組織の多くは その再生能が限られており、大きく失われると完全再生は望めない。大きな傷害では 、失われた組織とは異なる増殖力の強い組織が増殖し、不完全に組織が再生され機 能が回復できない状態で終わる不完全再生が起こり得る。この場合には、生体内吸 収性材料力もなる構造物を用いて、組織欠損部への増殖力の強 、組織の侵入を阻 止することで本来の組織が増殖できる空間を確保し、さらに細胞増殖因子を補充す ることで本来の組織の再生能力を高める再生医療が行われている。この例として、軟 骨、骨および末梢神経の再生医療がある。神経細胞および心筋は再生能力がない 力または著しく低いとこれまでは考えられてきた。近年、自己組織に由来する組織片 を用いた再生医療が注目されて 、る。  As used herein, “regeneration” refers to a phenomenon in which the remaining tissue grows and is restored when a part of the tissue of an individual is lost. Depending on the species of tissue between species or in the same individual, the extent and mode of regeneration varies. Many human tissues have limited ability to regenerate, and complete regeneration cannot be expected if they are largely lost. In a large injury, a tissue having a strong proliferative power different from the lost tissue proliferates, and incomplete regeneration may occur in which the tissue is regenerated incompletely and the function cannot be recovered. In this case, using a structure that also has a bioabsorbable material strength, it is possible to secure a space where the original tissue can proliferate by inhibiting the invasion of the tissue by increasing the ability to grow into the tissue defect, Regenerative medicine is being carried out that replenishes cell growth factors to enhance the regenerative capacity of the original tissue. Examples include regenerative medicine of soft bones, bones and peripheral nerves. It has previously been thought that neurons and myocardium are incapable of regeneration or are significantly less powerful. In recent years, regenerative medicine using tissue fragments derived from self-organization has attracted attention.
[0034] 本明細書において使用される「細胞」は、当該分野において用いられる最も広義の 意味と同様に定義され、多細胞生物の組織の構成単位であって、外界を隔離する膜 構造に包まれ、内部に自己再生能を備え、遺伝情報およびその発現機構を有する 生命体をいう。本発明の方法においては、どのような細胞でも対象とされ得る。本発 明で使用される「細胞」の数は、光学顕微鏡を通じて計数することができる。光学顕 微鏡を通じて計数する場合は、核の数を数えることにより計数を行う。当該組織を組 織切片スライスとし、へマトキシリン—ェォシン (HE)染色を行うことにより細胞外マトリ タス (例えば、エラスチンまたはコラーゲン)および細胞に由来する核を色素によって 染め分ける。この組織切片を光学顕微鏡にて検鏡し、特定の面積 (例えば、 200 X 200 μ m)あたりの核の数を細胞数と見積って計数することができる。 The “cell” used in the present specification is defined in the same way as the broadest meaning used in the field, and is a structural unit of a tissue of a multicellular organism and is enclosed in a membrane structure that isolates the outside world. Rarely, an organism that has self-regenerative ability and has genetic information and its expression mechanism. Any cell can be targeted in the method of the present invention. Main departure The number of “cells” used in the light can be counted through a light microscope. When counting through an optical microscope, count by counting the number of nuclei. The tissue is sliced into tissue slices and stained with hematoxylin-eosin (HE) to separate extracellular matrix (eg, elastin or collagen) and cell-derived nuclei with a dye. This tissue section can be examined with an optical microscope, and the number of nuclei per specific area (for example, 200 × 200 μm) can be estimated and counted.
[0035] 細胞は、石灰化および免疫反応惹起の原因となり得る。従って、組織または臓器の 移植のためには、細胞に由来する石灰化および免疫反応惹起を抑制する必要があ る。 [0035] Cells may be responsible for calcification and eliciting an immune response. Therefore, for tissue or organ transplantation, it is necessary to suppress cell-derived calcification and the induction of an immune response.
[0036] 本明細書にぉ 、て「細胞の置換」とは、脱細胞化された組織内で、もとあった細胞 に代わり、別の細胞が侵入し置き換わることをいい、「細胞の浸潤」ともいう。好ましく は、本発明では、細胞の置換は、移植された宿主の細胞によって行われる。  [0036] As used herein, "cell replacement" refers to the invasion and replacement of another cell in the decellularized tissue in place of the original cell. " Preferably, in the present invention, cell replacement is performed by the transplanted host cells.
[0037] 本明細書にぉ 、て「組織」（tissue)または「生体組織」とは、細胞生物にお!、て、同 一の機能'形態をもつ細胞集団をいう。多細胞生物では、通常それを構成する細胞 が分ィ匕し、機能が専能化し、分業化がおこる。従って、組織とは、細胞の単なる集合 体であり得ず、ある機能と構造を備えた有機的細胞集団、社会的細胞集団としての 組織が構成されることになる。組織としては、外皮組織、結合組織、筋組織、血管組 織、心臓組織、神経組織などが挙げられるがそれらに限定されない。本発明が対象 とする組織は、生物のどの臓器または器官由来の組織でもよい。本発明の好ましい 実施形態では、本発明が対象とする組織としては、血管、血管様組織、心臓弁、心 膜、硬膜、角膜および骨の組織が挙げられるがそれらに限定されない。本発明で例 示的に用いられるミセルイ匕して 、な 、状態の両親媒性分子は、細胞成分の抽出に類 似する機構で作用することから、原理的にはどの器官由来の組織でも本発明の方法 によって処理され得る。従って、本発明の生体適合性高分子での処理は、どのような 組織であっても対象とすることができることが理解される。  [0037] As used herein, "tissue" or "biological tissue" refers to a cell population having the same function 'form in a cell organism. In multicellular organisms, the cells that make it up are usually separated, function specialized, and division of labor occurs. Therefore, a tissue cannot be a mere assembly of cells, and constitutes an organic cell group or a social cell group having a certain function and structure. Tissues include, but are not limited to, integument tissue, connective tissue, muscle tissue, vascular tissue, heart tissue, nerve tissue, and the like. The tissue targeted by the present invention may be any organ or tissue derived from an organism. In a preferred embodiment of the present invention, tissues targeted by the present invention include, but are not limited to, blood vessels, blood vessel-like tissues, heart valves, pericardium, dura mater, corneas and bone tissues. The micelles used in the present invention, which are used in the present invention, act in a similar manner to the extraction of cellular components. It can be processed by the inventive method. Therefore, it is understood that the treatment with the biocompatible polymer of the present invention can be applied to any tissue.
[0038] 本明細書において「生体由来組織」とは、生体力も分離された組織またはその組織 を処理したものをいい、特に言及しない場合は、脱細胞化処理を経ていない組織を いうと理解される。本明細書では、生体力 取り出した後、特に脱細胞化処理をして いない糸且織を「未処理細胞」ともいう。そのような生体由来糸且織については、例えば、 生体から分離されたままの組織のほか、そのような組織を生体適合性高分子に暴露 させたものなどもこの概念に含まれることが理解される。このような生体由来組織は、 移植片として利用される。 [0038] In the present specification, "biological tissue" refers to a tissue from which vital force is also separated or a tissue processed therefrom, and unless otherwise specified, refers to a tissue that has not undergone decellularization treatment. Is done. In this specification, after taking out the vitality, it is especially decellularized. Yarns and weaves that do not exist are also called “untreated cells”. It is understood that this concept includes, for example, tissues that have been separated from the living body and those that have been exposed to a biocompatible polymer, as well as tissues that have been separated from the living body. The Such living tissue is used as a graft.
[0039] 本明細書において「生体由来膜」とは、生体から分離された膜またはその膜を処理 したものをいう。本明細書において使用される生体由来膜としては、例えば、血管、 血管様組織、心膜、硬膜、角膜、羊膜、腹膜、皮膚などに由来する膜が挙げられるが 、これらに限定されない。  [0039] In the present specification, the "biological membrane" refers to a membrane separated from a living organism or one obtained by treating the membrane. Examples of the biological membrane used in the present specification include, but are not limited to, membranes derived from blood vessels, blood vessel-like tissues, pericardium, dura mater, cornea, amniotic membrane, peritoneum, skin and the like.
[0040] 本明細書にお!、て「膜状組織」とは、「平面状組織」とも! 、、膜状の組織を 、う。膜 状組織には、心膜、硬膜、角膜などの器官の組織が挙げられる。  In the present specification, the term “film-like structure” refers to a “planar structure” as well as a film-like structure. Examples of membranous tissues include organ tissues such as pericardium, dura mater and cornea.
[0041] 本明細書において「癒着防止膜」とは、癒着を防止する膜をいい、例えば、外科手 術または処置後の切断面または欠損部位と、その周辺組織との間に生じる癒着を防 止する膜をいう。  [0041] As used herein, "adhesion-preventing membrane" refers to a membrane that prevents adhesion, and prevents, for example, adhesion that occurs between a cut surface or a defect site after surgery or treatment and its surrounding tissue. A film that stops.
[0042] 本明細書において「臓器」または「器官」（organ)とは、互換的に用いられ、生物個 体のある機能が個体内の特定の部分に局在して営まれ、かつその部分が形態的に 独立性をもっている構造体をいう。一般に多細胞生物（例えば、動物、植物）では器 官は特定の空間的配置をもつ 、くつかの組織からなり、組織は多数の細胞力もなる。 そのような臓器または器官としては、血管系に関連する臓器または器官が挙げられる 。 1つの実施形態では、本発明が対象とする器官は、虚血性の器官 (心筋梗塞を起こ した心臓、虚血を起こした骨格筋など）が挙げられる。 1つの好ましい実施形態では、 本発明が対象とする器官は、血管、血管様組織、心臓弁、心膜、硬膜、角膜および 骨である。別の好ましい実施形態では、本発明が対象とする器官は、心臓弁、心膜 および血管である。  [0042] In this specification, "organ" or "organ" is used interchangeably, and a certain function of an organism is localized in a specific part of an individual, and the part. Refers to a structure that is morphologically independent. In general, in multicellular organisms (eg animals, plants), the organs are made up of several tissues with a specific spatial arrangement, and the tissues also have a number of cellular forces. Such organs or organs include organs or organs associated with the vascular system. In one embodiment, organs targeted by the present invention include ischemic organs (hearts with myocardial infarction, skeletal muscles with ischemia, etc.). In one preferred embodiment, organs targeted by the present invention are blood vessels, blood vessel-like tissues, heart valves, pericardium, dura mater, cornea and bone. In another preferred embodiment, the organs targeted by the present invention are heart valves, pericardium and blood vessels.
[0043] 移植するための適切な移植片を調製するためには、必要に応じて器官培養をする ことが望ましくあり得る。器官培養とは、生体力も摘出した胚器官あるいは成熟器官の 一部あるいは全体を、その構造、機能および分ィ匕能を保持したまま、インビトロで培 養することをいう。これに対し、一般の組織培養では、細胞増殖を主たる目的とするた めに、むしろ脱分化を起こしやすい。器官培養としては、時計皿法、ブロックシャーレ 法、支持台法、スポンジ基質法、 Trowell法などがあるがこれらに限定されない。タン パク質 '核酸'酵素 45、 2188— 2200 (2000)に記載されるような大量細胞培養（ 例えば、ホロファイバ一法、固定床型、スピンフィルター、沈降槽型、灌流培養、循環 式器官培養法（circulation type organ culture)など）も用いられ得る。本発明 の脱細胞化方法によって調製された器官または組織を培養培地に 、れ、その器官 培養培地に種々の物質を加えることによって、構造、機能および発育分化への調整 、ならびに器官対器官の相互作用などの調節を行うことができる。 [0043] In order to prepare an appropriate graft for transplantation, it may be desirable to perform organ culture as needed. Organ culture refers to culturing a part or the whole of an embryonic organ or a mature organ from which vital force has been removed in vitro while maintaining its structure, function and ability to separate. In contrast, in general tissue culture, dedifferentiation is more likely to occur because the main purpose is cell proliferation. Organ culture includes the watch glass method and the block petri dish. Method, support base method, sponge substrate method, Trowell method, etc., but are not limited thereto. Protein 'nucleic acid' enzymes 45, 2188-2200 (2000) as described in large cell cultures (eg holofiber method, fixed bed type, spin filter, sedimentation tank type, perfusion culture, circulating organ culture Methods such as circulation type organ culture may also be used. Organs or tissues prepared by the decellularization method of the present invention are added to a culture medium, and various substances are added to the organ culture medium to adjust to structure, function and developmental differentiation, and to organ-to-organ interaction. Adjustment of the action and the like can be performed.
[0044] 本明細書にぉ 、て「脱細胞 (化）」および「脱細胞 (化)する」とは、組織または器官 力 細胞を除去することをいう。好ましくは、脱細胞化はもとの組織または器官の構造 および機能を損傷することなく行われ得る。脱細胞化が行われた組織力もは、細胞 質成分、細胞質ゾル成分、細胞骨格および細胞膜成分は除去されているが、細胞外 マトリクス (ECM)成分 (例えば、エラスチン、コラーゲン (I型、 IV型など）、ラミニンな ど）、組織の構造を保持するために必要な細胞の成分は未変性のまま保持されてい る。したがって、脱細胞化された組織または器官は、組織または器官の形状、力学的 強度、弾性、柔軟性などの諸性質は未処理の組織または器官と実質的に同等である ことが好ましい。また、移植後には細胞外マトリクスが未変性であるために、レシピエ ント側の細胞の侵入、接着、増殖、分化形質の発現、維持に好適な環境を提供し、 自己細胞により構成される組織に置き換わることが好ましい。脱細胞化の程度は、細 胞残存率を指標に測定することができる。  As used herein, “decellularization” and “decellularization” refer to removal of tissue or organ force cells. Preferably, decellularization can be performed without damaging the structure and function of the original tissue or organ. The decellularized tissue force also removes cytoplasmic components, cytosolic components, cytoskeleton and cell membrane components, but extracellular matrix (ECM) components (e.g. elastin, collagen (type I, type IV) Etc.), laminin, etc.) the cellular components necessary to preserve the structure of the tissue remain intact. Therefore, it is preferable that the decellularized tissue or organ has substantially the same properties as the untreated tissue or organ in terms of properties such as the shape, mechanical strength, elasticity, and flexibility of the tissue or organ. In addition, since the extracellular matrix is native after transplantation, it provides a suitable environment for the invasion, adhesion, proliferation, and expression and maintenance of cells on the recipient's side. It is preferable to replace it. The degree of decellularization can be measured using the cell survival rate as an index.
[0045] 従って、本発明の (未処理)生体由来組織は、このような脱細胞化処理を受けてい ないことが好ましい。  [0045] Therefore, it is preferable that the (untreated) living tissue of the present invention has not been subjected to such decellularization treatment.
[0046] 脱細胞化された組織は、 MHCクラス Iおよび Πタンパク質が除去されていることが好 ましい。このような MHCクラス Iおよび IIタンパク質は、 SDS PAGEおよび Zまたは ウェスタンプロット分析により確認することができる。他の細胞外マトリクスタンパク質も また、 SDS PAGEおよび Zまたはウェスタンブロット分析により確認することができる 。細胞中の構造タンパク質 (コラーゲン、エラスチンなど）はアミノ酸分析 (例えば、ェ ドマン分解法など、あるいは PE Biosystemsから入手可能なペプチド分析機器によ る自動分析など）により評価することができる。その結果により、脱細胞化プロセスの E CMへの影響を判断することができる。細胞膜および細胞中に含まれる脂質およびリ ン脂質は、薄層クロマトグラフィーおよび HPLCを用いて分析することができる。糖鎖 (例えば、グリコサミノダリカンなど）は、ァガロースゲル電気泳動などにより分析するこ とができる。この分析により、細胞外マトリクスのグリコサミノダリカン組成のほか、 a - Galなどの存在も分析することができる。 [0046] The decellularized tissue preferably has MHC class I and sputum proteins removed. Such MHC class I and II proteins can be confirmed by SDS PAGE and Z or Western plot analysis. Other extracellular matrix proteins can also be confirmed by SDS PAGE and Z or Western blot analysis. Structural proteins in cells (collagen, elastin, etc.) can be evaluated by amino acid analysis (eg, Edman degradation method or automated analysis with peptide analyzer available from PE Biosystems). As a result, the decellularization process E Can determine the impact on CM. Lipids and phospholipids contained in cell membranes and cells can be analyzed using thin layer chromatography and HPLC. Sugar chains (for example, glycosaminodarlicans) can be analyzed by agarose gel electrophoresis or the like. By this analysis, it is possible to analyze the presence of a-Gal in addition to the glycosaminodarican composition of the extracellular matrix.
[0047] 従って、脱細胞化されて!/、な 、組織は、上記脱細胞化による特徴を有して 、な 、こ とを確認することによって判定することができる。特に、脱細胞化されていない組織は 、本明細書において（1)生体力 取り出したば力りの組織 (生組織）に比較して細胞 残存率が 30%以上のものを脱細胞化されて 、な 、か、または（2)生体から取り出し たば力りの組織 (生組織）に比較して可溶性タンパク質の残存率が 50%以上である 力 または（3)生体力 取り出したば力りの組織 (生組織）に比較して DNAの残存率 が 50%以上であるものとする。  [0047] Therefore, the tissue has been decellularized! /, And the tissue has the characteristics of the above decellularization and can be determined by confirming this. In particular, in the present specification, a tissue that has not been decellularized is decellularized in the present specification (1) with a cell survival rate of 30% or more as compared to a tissue (living tissue) that has been extracted. ,,,, Or (2) the force that the residual rate of soluble protein is 50% or more compared to the tissue that is removed from the living body (live tissue) or (3) the force that is removed from the living body It is assumed that the residual rate of DNA is 50% or more compared to the tissue (living tissue).
[0048] 本明細書にぉ 、て「細胞残存率」とは、本発明の方法により組織の脱細胞化を行つ た後に残る生存細胞の、その組織にもともと存在していた細胞に対する比率をいう。 本明細書にぉ 、て細胞残存率を測定する方法は、へマトキシリンおよびェォシン染 色 (H&E染色）による顕微鏡による計数法が代表的に用いられ、この方法は、 HE染 色により染め分けた核を光学顕微鏡により検鏡により計数することを利用する。従つ て、この方法は例えば、以下の工程を包含する：サンプルを HE染色により染め分け る工程；このサンプルにお!/、て 100 m X 100 mの面積に存在する核の数（ =細 胞数)を計数する工程;必要に応じて計数を複数 (例えば、 8回)行い、その平均を取 る工程;コントロール (例えば、未処理の組織を 100%とする）に対する割合を算出す る工程を包含する。コントロールに対する割合は、例えば、未処理組織に対する残存 核の割合を細胞残存率とすることができる。従って、この場合、細胞残存率（％) = ( 処理組織中の核の数) Z (未処理組織中の核の数） X 100で算出することができる。 細胞残存率はまた、残存する DNA量を測定することによつても測定することができる 。細胞が有する DNAの量の組織における総量は、一般に組織における細胞数に比 例することが知られているからである。 DNAを測定する方法は、当該分野において 公知であり、例えば、 Molecular Probes社の Hoechst 33258などの蛍光試薬を 用いて組織より抽出した DNA量を定量することができる。具体的には、組織を破砕 超音波処理などでゾル化し、緩衝液中 DNA成分に特異的に結合し、かつ蛍光能を 発生させる Hoechst 33258 (Ex (励起波長） 356、 EM (発光波長） 492)を反応さ せ、抽出上清中の DNA量を蛍光強度として測定し、定量することができる。従って、 脱細胞化組織との違いに細胞残存率を用いることができ、あるいは γ線照射にっ 、 てもこの細胞残存率を使用することができる。その場合の目安は、細胞残存率 30% を挙げることができる。 [0048] As used herein, "cell survival rate" refers to the ratio of viable cells remaining after decellularization of a tissue by the method of the present invention to cells originally present in the tissue. Say. In this specification, the method of measuring the cell survival rate is typically a microscopic counting method using hematoxylin and eosin staining (H & E staining), and this method involves the analysis of nuclei dyed by HE staining. Use counting with an optical microscope. Thus, for example, this method comprises the following steps: dyeing the sample by HE staining; this sample! /, The number of nuclei present in an area of 100 m x 100 m (= cells) The number of counts (e.g., 8 times) and average the results; calculating the ratio to the control (e.g., 100% untreated tissue) Is included. As a ratio with respect to the control, for example, a ratio of remaining nuclei with respect to an untreated tissue can be defined as a cell remaining ratio. Therefore, in this case, the cell survival rate (%) = (number of nuclei in the treated tissue) Z (number of nuclei in the untreated tissue) × 100 can be calculated. Cell survival rate can also be measured by measuring the amount of DNA remaining. This is because it is known that the total amount of DNA contained in a cell in a tissue is generally proportional to the number of cells in the tissue. Methods for measuring DNA are known in the art. For example, a fluorescent reagent such as Hoechst 33258 from Molecular Probes is used. It can be used to quantify the amount of DNA extracted from tissues. Specifically, the tissue is made into a sol by sonication, etc., which specifically binds to DNA components in the buffer and generates fluorescence. Hoechst 33258 (Ex (excitation wavelength) 356, EM (emission wavelength) 492 ), And the amount of DNA in the extract supernatant can be measured and quantified as fluorescence intensity. Therefore, the cell survival rate can be used for the difference from the decellularized tissue, or the cell survival rate can be used even by γ-irradiation. In this case, a 30% cell survival rate can be mentioned as a guide.
[0049] 本明細書において「免疫反応」とは、移植片と宿主との間の免疫寛容の失調による 反応をいい、例えば、超急性拒絶反応 (移植後数分以内）（j8— Galなどの抗体によ る免疫反応）、急性拒絶反応 (移植後約 7〜21日の細胞性免疫による反応）、慢性拒 絶反応（3力月以降の細胞性免疫による拒絶反応)などが挙げられる。  [0049] As used herein, the term "immune response" refers to a response due to a lack of immune tolerance between a graft and a host, such as hyperacute rejection (within a few minutes after transplantation) (such as j8-gal). Antibody-induced immune reaction), acute rejection (reaction due to cellular immunity about 7-21 days after transplantation), chronic rejection (rejection due to cellular immunity after 3 months), and the like.
[0050] 本明細書にお!、て免疫反応を惹起するかどうかは、 HE染色などを含む染色、免疫 染色、組織切片の検鏡によって、移植組織中への細胞 (免疫系）浸潤について、そ の種、数などの病理組織学的検討を行うことにより判定することができる。  [0050] In the present specification, whether or not to induce an immune response is determined with respect to infiltration of cells (immune system) into a transplanted tissue by staining including HE staining, immunostaining, and microscopic examination of a tissue section. It can be determined by conducting histopathological examination of the species and number.
[0051] 本明細書において「石灰化」とは、生物体で石灰質が沈着することをいう。  [0051] As used herein, "calcification" refers to the deposition of calcareous substances in a living organism.
[0052] 本明細書において生体内で「石灰化する」かどうかは、カルシウム濃度を測定する ことによって判定することができ、移植組織を取り出し、酸処理などにより組織切片を 溶解させ、その溶液を原子吸光度などの微量元素定量装置により測定し、定量する ことができる。  [0052] In the present specification, whether or not to "calcify" in vivo can be determined by measuring the calcium concentration. The transplanted tissue is taken out, the tissue section is dissolved by acid treatment or the like, and the solution is used. It can be measured and quantified with a trace element quantification device such as atomic absorbance.
[0053] 本明細書において「生体内」または「インビボ」（in vivo)とは、生体の内部をいう。  As used herein, “in vivo” or “in vivo” refers to the inside of a living body.
特定の文脈において、「生体内」は、目的とする組織または器官が配置されるべき位 置をいう。  In a particular context, “in vivo” refers to the location where the target tissue or organ is to be placed.
[0054] 本明細書にぉ 、て「インビトロ」 (in vitro)とは、種々の研究目的のために生体の 一部分が「生体外に」（例えば、試験管内に)摘出または遊離されている状態をいう。 インビボと対照をなす用語である。  [0054] As used herein, "in vitro" refers to the state in which a portion of a living organism has been removed or released "in vitro" (eg, in a test tube) for various research purposes. Say. A term that contrasts with in vivo.
[0055] 本明細書にぉ 、て「ェキソビボ」とは、遺伝子導入を行うための標的細胞を被験体 より抽出し、インビトロで治療遺伝子を導入した後に、再び同一被験体に戻す場合、 一連の動作をェキソビボと 、う。 [0056] 本明細書にぉ 、てある組織の「正常状態で有する機能」とは、その組織が正常状 態で生体内で有する機能をいう。従って、例えば、心臓弁の場合、通常心室から心 房または肺動脈および大動脈力 心房へ血液の逆流を防ぐ機能を有することから、 心臓弁が正常状態で有する機能とは、心室から心房または肺動脈および大動脈から 心房へ血液の逆流を防ぐ機能をいう。本発明では、生体適合性高分子に暴露した後 γ線照射をしてもこの正常状態で有する機能に実質的な損傷は無力つたという点で 従来にな 、格別な効果が示されたと 、える。 [0055] As used herein, the term "ex vivo" refers to a series of cases where target cells for gene transfer are extracted from a subject, a therapeutic gene is introduced in vitro, and then returned to the same subject again. The operation is ex vivo. [0056] As used herein, the "function that a tissue has in a normal state" refers to a function that the tissue has in a normal state in a living body. Therefore, for example, in the case of a heart valve, since it has a function of preventing blood backflow from the ventricle to the atrium or pulmonary artery and aorta force atrium, the function that the heart valve has in the normal state is that A function that prevents the backflow of blood from the atria. According to the present invention, it has been shown that a special effect has been shown in a conventional manner in that even if γ-irradiation is performed after exposure to a biocompatible polymer, substantial damage to the function of the normal state has been helped. .
[0057] 本明細書において「細胞外マトリクス」 (ECM)とは「細胞外基質」とも呼ばれ、上皮 細胞、非上皮細胞を問わず体細胞（somatic cell)の間に存在する物質をいう。細 胞外マトリクスは、組織の支持だけでなぐすべての体細胞の生存に必要な内部環境 の構成に関与する。細胞外マトリクスは一般に、結合組織細胞から産生されるが、一 部は上皮細胞や内皮細胞のような基底膜を保有する細胞自身からも分泌される。線 維成分とその間を満たす基質とに大別され、線維成分としては膠原線維および弾性 線維がある。基質の基本構成成分はグリコサミノダリカン (酸性ムコ多糖)であり、その 大部分は非コラーゲン性タンパクと結合してプロテオダリカン (酸性ムコ多糖—タンパ ク複合体)の高分子を形成する。このほかに、基底膜のラミニン、弾性線維周囲のミク ロフイブリル（microfibril)、線維、細胞表面のフイブロネクチンなどの糖タンパクも基 質に含まれる。特殊に分化した組織でも基本構造は同一で、例えば硝子軟骨では軟 骨芽細胞によって特徴的に大量のプロテオダリカンを含む軟骨基質が産生され、骨 では骨芽細胞によって石灰沈着が起こる骨基質が産生される。本発明の 1つの実施 形態では、細胞外マトリクス (たとえば、エラスチン、コラーゲン (例えば、 I型、 IV型な ど）、ラミニンなど）は本発明の脱細胞化処理をする前の状態からは実質的に変化し て!、な 、ことが特徴であり得る。  In the present specification, “extracellular matrix” (ECM) is also called “extracellular matrix” and refers to a substance existing between somatic cells regardless of whether they are epithelial cells or non-epithelial cells. The extracellular matrix is responsible for the internal environmental organization required for the survival of all somatic cells, not just tissue support. Extracellular matrices are generally produced from connective tissue cells, but some are also secreted from the cells themselves that possess a basement membrane, such as epithelial cells and endothelial cells. The fiber component and the matrix that fills it are roughly divided, and the fiber component includes collagen fiber and elastic fiber. The basic component of the substrate is glycosaminodarlican (acid mucopolysaccharide), most of which binds to non-collagenous proteins to form a polymer of proteodalican (acid mucopolysaccharide-protein complex). In addition, glycoproteins such as laminin in the basement membrane, microfibrils around elastic fibers, fibers, and fibronectin on the cell surface are also included in the substrate. The basic structure is the same even in specially differentiated tissues.For example, in hyaline cartilage, cartilage matrix containing a large amount of proteodarican is produced by soft osteoblasts, and in bone, bone matrix where calcification is caused by osteoblasts is produced. Produced. In one embodiment of the present invention, extracellular matrix (eg, elastin, collagen (eg, type I, type IV, etc.), laminin, etc.) is substantially free from the state prior to the decellularization treatment of the present invention. It can be a feature.
[0058] 本明細書にぉ 、て「組織損傷率」とは、組織または器官の機能を示すパラメータを V、 、、処理後の組織または器官がどの程度損なわれ傷つ 、て 、るかの指標であり、 その組織または器官の本来の機能を発揮することができるかどうかの指標である。本 明細書において組織損傷率を測定する方法は、当該分野において公知であり、例え ば、エラスチン断裂部位を計数することによって判定することができる。本明細書にお いて用いられる方法では、一視野を 100 mX 100 mごとのユニットに区切り、ュ ニットを単位としてエラスチン断裂部位がある場合にカウントして算出した。一視野あ たり 24ユニットが存在した。 HE染色により組織切片における細胞外マトリクスの検鏡 により計数し、未処理組織を 0%となるように規定し、損傷率 =xZ24で算出する。こ の場合未処理を x=0として規定する。 As used herein, the term “tissue damage rate” refers to a parameter indicating the function of a tissue or organ, V,,, how much the tissue or organ after treatment is damaged or damaged. It is an indicator of whether or not the tissue or organ can perform its original function. The method for measuring the tissue damage rate in the present specification is known in the art, and can be determined, for example, by counting the elastin rupture sites. In this specification In this method, one field of view was divided into units of 100 m × 100 m, and the unit was used as a unit to count when there was an elastin rupture site. There were 24 units per field of view. Count by microscopic examination of extracellular matrix in tissue sections by HE staining, prescribe untreated tissue to be 0%, and calculate damage rate = xZ24. In this case, unprocessed is defined as x = 0.
[0059] 本明細書にぉ 、て「組織強度」とは、組織または器官の機能を示すパラメータを ヽ い、その組織または器官の物理的強度であり、一般に、引っ張り強さを測定すること により判定することができる。そのような一般的な引っ張り試験は周知である。  [0059] As used herein, the term "tissue strength" refers to a parameter indicating the function of a tissue or organ, and refers to the physical strength of the tissue or organ, generally by measuring the tensile strength. Can be determined. Such general tensile tests are well known.
[0060] 本明細書では、（1)検体を 5 X 30mmの短冊状に切る。（基本的に大動脈基部の w allの部分を長軸方向に長くなるように切る。 )； (2)引っ張り試験器の固定部分に検 体の両端約 5mmを固定する。（ORIENTEC社製 TENSILON万能試験機 RTC— 1150Aを使用）；および（3)引っ張り開始し破断点まで lcmZminの速度で引っ張る 。という作業を行うことによって、破断点における負荷を強度として採用する。ここでは 、破断点負荷および弾性率を測定する。  [0060] In the present specification, (1) the specimen is cut into 5 X 30 mm strips. (Basically cut the wall part of the base of the aorta so that it becomes longer in the long axis direction.); (2) Fix the specimen about 5 mm at both ends to the fixed part of the tensile tester. (Use ORIENTEC's TENSILON universal testing machine RTC-1150A); and (3) Start pulling and pulling to the breaking point at lcmZmin. The load at the breaking point is adopted as the strength. Here, the load at break and the elastic modulus are measured.
[0061] 本発明の生体由来組織が有する強度は、通常、最大点荷重が、少なくとも約 8N以 上、より好ましくは、約 10N以上、さらに好ましくは約 14N以上であり得る。従来の生 体由来組織あるいは天然の組織 (例えば、動脈)では、 7N程度であることが多力つた こと、および脱細胞化処理では、組織強度を増強する効果はないかあつてもほとんど な！、ことを考慮すれば、本発明の組織強度強化能力は予想外と!/、える。  [0061] The strength of the living tissue of the present invention can usually be such that the maximum point load is at least about 8N or more, more preferably about 10N or more, and even more preferably about 14N or more. In conventional biological tissues or natural tissues (eg, arteries), the strength is about 7N, and decellularization treatment has little or no effect on enhancing tissue strength! In view of this, the ability to strengthen the tissue strength of the present invention is unexpected!
[0062] 好ましい実施形態では、弾性率でみると、本発明の生体由来組織は、通常少なくと も 1.2MPa以上の弾性率を有し、好ましくは少なくとも約 1. 6MPaの弾性率を有する 力 本発明の生体由来組織は、使用に耐え得る限り、天然物よりも劣る弾性率 (例え ί 、 0. 8J¾_h, 0. 8〜1. OMPaなど）を ¾ "して!/ヽてもよ!ヽ。  [0062] In a preferred embodiment, when viewed in terms of elastic modulus, the living tissue of the present invention usually has an elastic modulus of at least 1.2 MPa, preferably at least about 1.6 MPa. The biological tissue of the invention may have an elastic modulus (for example, ί, 0.8J¾_h, 0.8-1 OMPa, etc.) that is inferior to that of a natural product as long as it can be used. .
[0063] 伸びの測定は、強度測定で使用した引張試験機 (TENSILLON ORIENTEC) で行うことができる。具体的には、幅 5mm長さ 30mmの短冊状素材を短軸方向に 10 mmZ分の速度で荷重負荷し、破断点負荷および弾性率を測定することができる。 具体的には、伸びの測定は、引張り刺激の前後での各方向の長さを測定し、引張り 後の長さを引張り前の長さで割り 100を乗ずることによって得ることができる。通常、 伸びは、縦方向と横方向とを両方測定する。この両方の伸びにばらつきがないほうが 好ましいがそれに限定されない。用途に応じて、伸びに関する特性は、例えば、少な くとも 120%、好ましくは 150%であることが好ましいが、それに限定されない。伸びに 関する特性についても、本発明の生体由来組織は、使用に耐え得る限り、天然物よ りも劣る伸び (例えば、少なくとも 50%など）を有して 、てもよ 、。 [0063] The elongation can be measured with the tensile tester (TENSILLON ORIENTEC) used in the strength measurement. Specifically, a strip-shaped material having a width of 5 mm and a length of 30 mm can be loaded at a speed of 10 mmZ in the minor axis direction, and the load at break and the elastic modulus can be measured. Specifically, the elongation can be measured by measuring the length in each direction before and after the tension stimulus and dividing the length after the tension by the length before the tension and multiplying by 100. Normal, Elongation is measured in both the longitudinal and transverse directions. It is preferable that there is no variation in both the elongations, but the present invention is not limited to this. Depending on the application, the elongation-related properties are for example at least 120%, preferably 150%, but are not limited thereto. Regarding the properties relating to elongation, the living tissue of the present invention may have elongation (eg, at least 50%) which is inferior to that of a natural product as long as it can withstand use.
[0064] 一般的な引っ張り試験によって得られたデータの解析により、破断強度、剛性率、 ヤング率などの種々のデータを得ることができ、そのような値もまた、本明細書におい て組織強度の指標として用いることができる。本明細書において管状組織の場合、 組織強度は、剛性パラメータ（ β値)で表現することができる。 β値は、 P— D (圧力― 直径)関係を作成した後、  [0064] By analyzing the data obtained by a general tensile test, various data such as breaking strength, rigidity, Young's modulus, etc. can be obtained. It can be used as an index. In the present specification, in the case of a tubular tissue, the tissue strength can be expressed by a stiffness parameter (β value). The β value is calculated after creating the P-D (pressure-diameter) relationship.
Ln (P/Ps) = β (D/Ds- 1) (1)  Ln (P / Ps) = β (D / Ds- 1) (1)
で算出することができる。 Psおよび Dsは、 lOOmmHgでの標準値を示す。 Pおよび D 各々の P (圧力）における直径 (D)の値を示す。  Can be calculated. Ps and Ds are standard values at lOOmmHg. P and D The diameter (D) value at each P (pressure) is shown.
[0065] 血管などの管状組織の両端をパイプ状のユニットに固定し、生理食塩水中に内室 および外室を満たす。この状態から、内室へ圧力を外武装置より加えていくと同時に 、その加圧時の外径をモニタリングする。その測定によって得られる圧力と、外径との 関係を上記（1)の式に導入して、 j8値を算出する（Sonoda H, Takamizawa K. , et al. J. Biomed. Matr. Res. 2001 : 266— 276)。  [0065] Both ends of a tubular tissue such as a blood vessel are fixed to a pipe-shaped unit, and the inner chamber and the outer chamber are filled in physiological saline. From this state, the outer diameter at the time of pressurization is monitored at the same time as pressure is applied to the inner chamber from the outer arm. The relationship between the pressure obtained by the measurement and the outer diameter is introduced into the above equation (1) to calculate the j8 value (Sonoda H, Takamizawa K., et al. J. Biomed. Matr. Res. 2001). : 266—276).
[0066] 本明細書にぉ 、て「生体適合性」とは、毒性および免疫学的拒絶能がな!、ために 生体内で障害なく存在することができる性質を 、う。  [0066] As used herein, "biocompatibility" refers to the property of being non-toxic and non-immunologically refractory and therefore capable of existing without injuries in vivo.
[0067] 本明細書にぉ 、て「生体適合性高分子」とは、生体適合性のょ 、高分子を 、い、具 体的には、残存しても毒性を生じないことをいう。ある高分子がそのような生体適合性 を有しているかどうかを判定する方法は、本明細書においては、ラット等の実験動物 皮下への埋植試験等の試験法を使用する。この試験法では、皮下埋植試験の結果 As used herein, the term “biocompatible polymer” refers to a polymer that is biocompatible, and specifically, does not cause toxicity even if it remains. As a method for determining whether a certain polymer has such a biocompatibility, a test method such as a subcutaneous implantation test in a laboratory animal such as a rat is used in this specification. In this test method, the results of the subcutaneous implantation test
、比較的急性の免疫反応やアレルギー反応等が起き、腫れたり、発赤もしくは発熱し たりする場合には肉眼的に生体適合性が低いことが解る。さらに人工血管を動物の 血管に移植した場合など、特定の患部に移植した場合には、数日力も数ケ月後に、 移植箇所を観察し、組織の生着の有無、移植組織周辺の炎症、癒着、血液凝固によ る血栓形成などの程度を観察して、組織適合性の判定を行う。この他、移植部位の 組織切片を作成してへマトキシリン'ェォシン染色その他の染色法にて細胞を染色' 観察し、組織適合性の低さの指標としては免疫系を担当する顆粒性の細胞が多く侵 入しているかどうか、もしくは従来組織と移植組織の間に両者を隔てる瘢痕組織の形 成が認められるか否かを判定する。また、特に生体由来組織の組織適合性の高さの 指標としては、血管内皮細胞、繊維芽細胞、平滑筋細胞など各種の細胞が従来組 織より侵入し、再細胞化が起きているかどうか (すなわち従来組織と移植組織の境界 が明確でな!、程度に移植組織が生着して ヽるかどうか）を判定する。 When a relatively acute immune reaction or allergic reaction occurs, and swelling, redness or fever occurs, it is understood that the biocompatibility is low. Furthermore, when an artificial blood vessel is transplanted into a blood vessel of an animal, for example, after several days or months of force, the transplanted site is observed, whether or not the tissue is engrafted, inflammation around the transplanted tissue, adhesion Blood coagulation Observe the degree of thrombus formation, etc., and determine histocompatibility. In addition, a tissue section of the transplantation site was prepared, and hematoxylin 'eosin staining and other staining methods were used to observe the cells'. As an indicator of poor tissue compatibility, granular cells responsible for the immune system were used. Determine whether there is a large amount of invasion, or whether there is formation of scar tissue separating the conventional tissue and the transplanted tissue. In particular, as an index of the high tissue compatibility of living tissue, whether various cells such as vascular endothelial cells, fibroblasts, and smooth muscle cells have invaded from conventional tissues and recellularization has occurred ( In other words, it is determined whether the boundary between the conventional tissue and the transplanted tissue is clear!
[0068] また、上記の組織や細胞の形状観察による判定以外にも、炎症反応の進行に起因 する血液中のサイト力インの濃度、異物として認識された移植組織に対する抗体の濃 度 (力価)、補体成分の濃度、異物埋入により誘導された薬物代謝酵素 (P450等)の 酵素量、等の生物化学的な定量値とその移植前後の変動の追跡をもって、組織適 合性の指標とする場合もある。  [0068] In addition to the above-described determination by observing the shape of tissues and cells, the concentration of cytodynamic force in blood caused by the progression of inflammatory reaction, the concentration of antibody against the transplanted tissue recognized as a foreign body (titer ), Biochemical quantitative values such as the concentration of complement components, the amount of enzyme of drug metabolizing enzymes (such as P450) induced by foreign substance implantation, and the tracking of changes before and after transplantation. In some cases.
[0069] 医療器具類の材料については、その毒性を評価して医療器具への使用を合理的 に規制する為に、細胞毒性試験、感作性試験、刺激性試験、埋植試験、遺伝毒性 試験、血液適合性試験、全身毒性試験、などの試験項目があり、厚生労働省のガイ ドライン、米国の National Standards,国際的産業基準の ISO-10993等により個別 の試験法が規定されて!、る。  [0069] For medical device materials, cytotoxicity tests, sensitization tests, irritation tests, implantation tests, genotoxicity tests are conducted in order to evaluate their toxicity and reasonably regulate their use in medical devices. There are test items such as tests, blood compatibility tests, systemic toxicity tests, etc., and individual test methods are defined by the guidelines of the Ministry of Health, Labor and Welfare, the US National Standards, the international industry standard ISO-10993, etc. .
[0070] 生体適合性高分子としては、例えば、ポリビニルアルコール、ポリ乳酸、ポリグリコー ル酸、ポリビュルピロリドン、ポリエチレングリコール、ゼラチン、コラーゲン、 γ -ポリグ ルタミン酸、シリコーン、ポリ塩化ビニル、ポリメタクリル酸メチル、ポリテトラフルォロェ チレン、ポリエステル、ポリプロピレン、ポリウレタン、セルロース、ポリスチレン、ナイ口 ン、ポリカーボネート、ピリサルホン、ポリアクリロニトリル、コラーゲン、ゼラチン、フイブ リン、キチン、キトサン、アルギン酸、デンプン、 γ —ポリグルタミン酸、ポリ力プロラクト ン、ポリヒドロキシ酪酸などが挙げられるがそれらに限定されない。ここで、ポリビュル アルコールの中には、未修飾のものの他に、アミノ基、カルボキシル基、ァセチル基 などにより若干の程度化学修飾されたものもあり、市販されており、これらの改変ポリ ビュルアルコールもまた本発明において使用することができる。好ましくは、生体適合 性高分子は、生分解性であるがそれに限定されない。 [0070] Examples of the biocompatible polymer include polyvinyl alcohol, polylactic acid, polyglycolic acid, polybutylpyrrolidone, polyethylene glycol, gelatin, collagen, γ-polyglutamic acid, silicone, polyvinyl chloride, and polymethyl methacrylate. , Polytetrafluoroethylene, Polyester, Polypropylene, Polyurethane, Cellulose, Polystyrene, Naiguan, Polycarbonate, Pyrisulfone, Polyacrylonitrile, Collagen, Gelatin, Fibrin, Chitin, Chitosan, Alginic acid, Starch, γ-Polyglutamic acid, Poly Examples include, but are not limited to, force prolacton and polyhydroxybutyric acid. Here, in addition to unmodified ones, some of them are chemically modified with amino groups, carboxyl groups, acetyl groups, etc., and are commercially available. It can also be used in the present invention. Preferably biocompatible The functional polymer is biodegradable, but is not limited thereto.
本明細書において「ポリエチレングリコール（PEG)」とは、エチレングリコールのポリ マーをいい、ポリエチレンォキシド（poly (ethylene oxide) )ともいわれ、 HO— (C H CH O) —Hで表される。ポリエチレングリコールは、種々の企業から市販されて In the present specification, “polyethylene glycol (PEG)” refers to a polymer of ethylene glycol, which is also referred to as polyethylene (poly (ethylene oxide)), and is represented by HO— (C H CH O) —H. Polyethylene glycol is commercially available from various companies.
2 2 η 2 2 η
おり、例えば、 Union Carbide (Carbowax) , Dow (Polyglycol E)、 Texaco C hemical)jeffox)、 Olin (PolyG)、 BASF Wyandotte (Pluracol E)、 Hodag、 I CI Americas (ATEG)、 Nacalai tesque、日本油脂などから市販されている。通 常、 PEGは、平均分子量が 200と 200, 000との間である。そのような PEGとしては、 Nacalai tesqueの polyethylene glycol (H (OCH CH ) OH) #分子量（例え For example, Union Carbide (Carbowax), Dow (Polyglycol E), Texaco Chemical) jeffox), Olin (PolyG), BASF Wyandotte (Pluracol E), Hodag, I CI Americas (ATEG), Nacalai tesque, Japanese fat and oil, etc. Commercially available. Usually, PEG has an average molecular weight between 200 and 200,000. Such PEGs include Nacalai tesque polyethylene glycol (H (OCH CH) OH) # molecular weight (eg
2 2 n  2 2 n
ば、 200、 600、 1, 000、 2, 000、 6, 000、 8, 000、 35, 000、 50, 000)のような製 品が挙げられる。本明細書では、通常平均分子量が 1 , 000-200, 000のものが使 用され、好ましくは PEGは、平均分子量が 10, 000と 100, 000との間である。別の 好ましい実施形態では、 PEGは、平均分子量が 2, 000-50, 000であり得る。より 好ましくは、 PEGは、平均分子量が約 35, 000であり得る。あるいは、 1) 1, 000-2 00, 000であり、（2)好ましくは、 4, 000〜風 000であり、（3)さらに好ましくは、 8 , 000-50, 000であり得る。このように PEGは市販されている力 所望の特性を達 成するために、適宜合成することもできる。そのような合成方法は当該分野において 周知である。なお、本明細書において平均分子量および分子量は、ダルトン（Dalto n)であらわす。本発明において使用される PEGは、分子が均質であることが好まし いが、そのことは必須ではなぐ平均分子量が一定範囲にあれば通常使用することが できる。本発明の組織を調製するために、 PEG以外の化学物質 (例えば、酵素 (例え ば DNaselなど）、酵素阻害剤などを含むがそれらに限定されない）を加えることがで きる。 PEG以外の化学物質を加えると、加えるべき PEGの量または濃度は、その化 学物質の存在量に応じて変動し得る。好ましくは、細胞融合などで用いられる濃度が よぐ例えば、 lgZml以上（100%wZw以上）の濃度が好ましいが、それに限定さ れない。あるいは、 PEGは、 60%wZv〜100%wZvの間の濃度で使用され得る。 ポリエチレングリコールは、本発明において、生体由来組織を調製した後に、その組 織を強化する方法にぉ 、ても用いることができる。 [0072] 本明細書にぉ 、てポリビュルアルコールとは、ポバール (poval)、 PVAとも!/、 、、ポ リ酢酸ビュルの加水分解 (正確にはエステル交換)によって得られる高分子をいう。一 般式—（CH C (OH) H) —として表される。 -ルアルコールは単量体としては存在し 200, 600, 1,000, 2,000, 6,000, 8,000, 35,000, 50,000). In the present specification, those having an average molecular weight of 1,000 to 200,000 are usually used, and preferably PEG has an average molecular weight of between 10,000 and 100,000. In another preferred embodiment, the PEG can have an average molecular weight of 2,000-50,000. More preferably, the PEG can have an average molecular weight of about 35,000. Alternatively, it may be 1) 1,000-200000, (2) preferably 4,000 to 000 000, and (3) more preferably 8,000-50,000. Thus, PEG can be synthesized as appropriate in order to achieve the desired properties. Such synthetic methods are well known in the art. In the present specification, the average molecular weight and the molecular weight are expressed by Dalton. The PEG used in the present invention preferably has a homogeneous molecule, but this is not essential and can be usually used if the average molecular weight is within a certain range. In order to prepare the tissue of the present invention, chemical substances other than PEG (for example, enzymes (for example, DNasel), enzyme inhibitors, etc., but not limited to them) can be added. When chemicals other than PEG are added, the amount or concentration of PEG to be added can vary depending on the amount of the chemical present. Preferably, the concentration used for cell fusion or the like is higher, for example, a concentration of lgZml or more (100% wZw or more) is preferable, but not limited thereto. Alternatively, PEG can be used at a concentration between 60% wZv and 100% wZv. In the present invention, polyethylene glycol can be used in a method for reinforcing a tissue after preparing a living tissue. [0072] In the present specification, polybulal alcohol refers to a polymer obtained by hydrolysis (exactly transesterification) of polyacetic acid bule with both poval and PVA! /,. It is expressed as a general formula — (CH 2 C (OH) H) —. -Lu alcohol exists as a monomer
2 n  2 n
な!、のでポリ酢酸ビュルの加水分解でつくることができる。細胞融合に用いられて ヽ る。  It can be made by hydrolysis of poly (acetate) butyl. Used for cell fusion.
[0073] 本明細書においてポリビュルピロリドンとは、一般式 [—CH CH(C H NO)-]として  [0073] In this specification, polybulurpyrrolidone is represented by the general formula [—CH 2 CH (C H NO) —].
2 4 6 n 表される水に可溶の高分子化合物であり、主に薬剤の賦形剤として用いられている。  2 4 6 n This is a water-soluble polymer compound, and is mainly used as a pharmaceutical excipient.
[0074] 本明細書において「生分解性」とは、物質について言及するとき、生体内で、あるい は微生物の作用により分解される性質をいう。生分解性の高分子は、例えば、加水 分解により、水、二酸化炭素、メタンなどに分解され得る。本明細書では、生分解性 であるかどうかを判定する方法は、生分解性の一部である生体吸収性に関しては、ラ ット、ゥサギ、ィヌなど実験動物への数日間から数年間にわたる埋植試験、微生物に よる分解の試験に関しては、シート状の高分子の土壌中での数日間から数年間にわ たる埋入 '崩壊試験などの方法を使用する。移植に関する場合、動物における試験 を使用することが好ましい。また、上記の試験方法に準ずるより簡便な代替試験法と しては、高分子の非酵素的分解についてはリン酸緩衝液生理食塩水（PBS)などの 各種緩衝液を、高分子の酵素分解については当該高分子の加水分解酵素 (プロテ ァーゼ、グリコシダーゼ、リパーゼ、エステラーゼ等）を添加した緩衝液を、それぞれ 用いて水溶液中での溶解試験を行うこともある。生分解性の高分子には、天然およ び合成高分子がある。天然高分子の例としては、コラーゲン、デンプンなどのタンパ ク質、多糖類が挙げられ、そして合成高分子の例としてはポリグリコール酸、ポリ乳酸 、ポリエチレンスクシナートなどの脂肪族ポリエステルが挙げられるがそれらに限定さ れない。ポリビニルアルコールは、生分解性については、弱い生分解性を示すことか ら、本明細書では、生分解性を有するものとして認識され得る。  As used herein, “biodegradable” refers to the property of being degraded in vivo or by the action of microorganisms when referring to a substance. The biodegradable polymer can be decomposed into water, carbon dioxide, methane and the like, for example, by hydrolysis. In this specification, the method for determining whether or not biodegradability is related to bioabsorbability, which is part of biodegradability, is from several days to several years for laboratory animals such as rat, usagi and inu. For various vegetation tests and microbial degradation tests, methods such as embedding 'disintegration tests for several days to several years in sheet-like polymers in soil are used. For transplantation, it is preferable to use animal studies. As a simpler alternative test method based on the above test method, for non-enzymatic degradation of polymers, various buffer solutions such as phosphate buffered saline (PBS) can be used. With regard to, a dissolution test in an aqueous solution may be carried out using a buffer solution containing the hydrolase of the polymer (protease, glycosidase, lipase, esterase, etc.). Biodegradable polymers include natural and synthetic polymers. Examples of natural polymers include proteins such as collagen and starch, and polysaccharides, and examples of synthetic polymers include aliphatic polyesters such as polyglycolic acid, polylactic acid, and polyethylene succinate. Is not limited to them. Polyvinyl alcohol can be recognized as biodegradable in the present specification because it exhibits weak biodegradability.
[0075] 本明細書において「高分子」は、「分子」と同様の意味で用いられ、特に分子量を限 定しない。高分子について分子量を限定して議論する場合、通常、分子量が 500以 上のものを指すがそれに限定されな 、。本発明にお 、て用いられる高分子の分子量 の上限は、原理的には無限大である力 本発明では、通常、溶液として取り扱い、分 子量を議論できる (動的光散乱、ゲル濾過、遠心分離器による沈降平衡などの分子 量測定が適用できるという意味で)のは分子量 500万程度までが使用され得るがそ れに限定されない。本明細書において、学会および業界での慣例と同様に、「生体 高分子」、「生体適合性高分子」という用語は、「バイオマテリアル」、「医療用高分子」 等の用語と同義語として使用される。 In the present specification, “polymer” is used in the same meaning as “molecule”, and the molecular weight is not particularly limited. When discussing a polymer with a limited molecular weight, it usually refers to a polymer with a molecular weight of 500 or more, but not limited to it. In the present invention, the upper limit of the molecular weight of the polymer used in the present invention is infinite in principle. The molecular weight can be discussed (in the sense that molecular weight measurements such as dynamic light scattering, gel filtration, and centrifugal sedimentation can be applied), but molecular weights up to about 5 million can be used, but are not limited thereto. In this specification, the term “biopolymer” and “biocompatible polymer” are synonymous with terms such as “biomaterial” and “medical polymer”, as is the case in convention and industry. used.
[0076] 本明細書にぉ 、て「浸す」とは、ある物体をある流体 (例えば、液体)の中に入れる ことをいう。本発明においては、処理すべき組織を、処理のために、界面活性剤、ま たはミセルイ匕して ヽな 、状態の両親媒性分子 (例えば、ポリエチレングリコールのよう な 1, 2—エポキシドポリマー)含有溶液に入れることを「浸す」という。従って、浸すェ 程は、好ましくは、処理すべき組織が処理のための溶液中に完全に浸透するように 行われる。また、浸す工程においては、好ましくは、除去すべき成分の除去を効率的 に行うために、物理的な処理 (例えば、ガラス棒でしごく）を行ってもよい。  As used herein, “immersing” refers to placing an object in a fluid (eg, a liquid). In the present invention, the tissue to be treated is treated with a surfactant or micelle, which is in the state of an amphiphilic molecule (for example, a 1,2-epoxide polymer such as polyethylene glycol). ) Putting in the solution is called “soaking”. Therefore, the soaking is preferably performed so that the tissue to be treated completely penetrates into the solution for treatment. Further, in the soaking step, preferably, a physical treatment (for example, ironing with a glass rod) may be performed in order to efficiently remove the component to be removed.
[0077] 本明細書において「曝す」とは、ある物体をある因子 (例えば、生体適合性高分子） に接触させることをいう。  In the present specification, “exposing” refers to bringing a certain object into contact with a certain factor (for example, a biocompatible polymer).
[0078] 本明細書において「洗浄する」工程は、本発明において、例えば、生体適合性高分 子溶液に浸す工程によって処理された組織力ゝらその溶液を取り除くことをいう。従つ て、好ましくは、洗浄する工程は、液体を用いて行われる。本発明においては、処理 された組織は、生体での使用が企図されることから、生理的に受容可能な液体で洗 浄されることが好ましい。 1つの好ましい実施形態では、洗浄する工程は、 PBS (リン 酸緩衝化生理食塩水）を用いて行われ得る。洗浄液には、必要に応じて他の薬剤（ 例えば、プロテアーゼ阻害剤）が含まれていてもよいが、そのような他の薬剤は、毒性 を示さず生体適合性であることが好まし 、。  In the present specification, the “washing” step in the present invention refers to removing the solution from the tissue force treated by, for example, the step of immersing in a biocompatible polymer solution. Therefore, preferably, the washing step is performed using a liquid. In the present invention, the treated tissue is intended to be used in a living body, so it is preferably washed with a physiologically acceptable liquid. In one preferred embodiment, the washing step may be performed using PBS (phosphate buffered saline). The washing solution may contain other drugs (eg, protease inhibitors) as needed, but such other drugs are preferably non-toxic and biocompatible.
[0079] 本明細書において「ィ匕学的処理」とは、広義には、ある物体をィ匕学物質で処理 (例 えば、浸すことによる）することをいう。ただし、本明細書において使用される場合、化 学的処理は、たとえば、生体適合性高分子を含む溶液に浸す工程以外の他の工程 (例えば、 DNァーゼの処理）を指す。従って、本発明の方法において、例えば、平均 分子量が 1, 000〜200, 000のポリエチレングリコール含有溶液に浸す工程の他に 化学的処理が施される場合、化学的処理は、平均分子量が 1, 000-200, 000の ポリエチレングリコール含有溶液以外の溶液 (例えば、 DNasel溶液での処理、ダル タルアルデヒド溶液、他の平均分子量を有するポリエチレングリコール溶液、他の生 体適合性高分子溶液などを含むがそれらに限定されない）に浸す工程を包含する。 [0079] In this specification, "chemical treatment" broadly means that a certain object is treated (for example, by dipping) with a chemical substance. However, as used herein, chemical treatment refers to other steps (eg, DNase treatment) other than the step of immersing in a solution containing a biocompatible polymer, for example. Therefore, in the method of the present invention, for example, when chemical treatment is performed in addition to the step of immersing in a polyethylene glycol-containing solution having an average molecular weight of 1,000 to 200,000, the chemical treatment has an average molecular weight of 1, 000-200,000 To solutions other than polyethylene glycol-containing solutions (eg, but not limited to treatment with DNasel solutions, dartal aldehyde solutions, polyethylene glycol solutions with other average molecular weights, other biocompatible polymer solutions, etc.) A step of soaking is included.
[0080] 本明細書にぉ 、て「補強する」とは、組織に関して言及するとき、その組織の組織 強度が改善されていることをいう。好ましくは、組織強度は、ある補強処理をする前の 状態の少なくとも 110%、より好ましくは少なくとも 120%、さらに好ましくは少なくとも 1 50%、あるいは少なくとも 200%となっていることが好ましい。そのような組織強度は、 本明細書では、例えば、引っ張り強度試験において、最大点荷重 (例えば、単位 N) で示すことができる。 As used herein, “reinforcing” when referring to a tissue means that the tissue strength of the tissue is improved. Preferably, the tissue strength is preferably at least 110%, more preferably at least 120%, even more preferably at least 150%, or at least 200% of the state prior to some reinforcement treatment. Such tissue strength can be expressed herein as a maximum point load (eg, unit N), for example, in a tensile strength test.
[0081] 本明細書にぉ 、て「ラジカル反応」とは、離基反応とも 、 、、不対電子が生じ、これ が他の分子や残基に反応して起きる化学反応を ヽぅ。  As used herein, the term “radical reaction” refers to a chemical reaction that occurs when an unpaired electron is generated and reacts with another molecule or residue.
[0082] ラジカル反応としては、例えば、以下が挙げられる： [0082] Examples of the radical reaction include the following:
1)安定な分子の結合が切れて不対電子ができる反応、  1) Reactions where stable molecular bonds break and unpaired electrons form,
たとえば C H→CH · +C H ·  For example, C H → CH · + C H ·
3 8 3 2 5  3 8 3 2 5
2)不対電子と安定な分子とが反応して他の分子と不対電子とができる反応、 たとえば CH - +CH COCH→CH +CH COCH ·  2) Reactions where unpaired electrons react with stable molecules to form unpaired electrons with other molecules, such as CH-+ CH COCH → CH + CH COCH
3 3 3 4 3 3  3 3 3 4 3 3
3)不対電子に安定な分子が反応して大きい不対電子をつくる反応、  3) Reaction in which stable molecules react with unpaired electrons to create large unpaired electrons,
たとえば CH · +C H→C H ·  For example, CH · + C H → C H ·
3 2 4 3 7  3 2 4 3 7
4)不対電子が分解して小さ!/、不対電子と分子とができる反応、  4) The unpaired electrons decompose and are small! /, The reaction that can form unpaired electrons and molecules,
たとえば n— C H -→C H +CH ·  For example, n— C H-→ C H + CH ·
3 7 2 4 3  3 7 2 4 3
5)不対電子どうしの反応、これには、  5) Reaction between unpaired electrons,
再結合反応 CH ' +CH ，→C H  Recombination reaction CH '+ CH, → C H
3 3 2 6  3 3 2 6
不均化反応 C H · +C H -→C H +C H。  Disproportionation reaction C H · + C H-→ C H + C H.
2 5 2 5 2 4 2 6  2 5 2 5 2 4 2 6
[0083] 本明細書にぉ 、て「 γ線」とは、波長が約 0. 001 nmよりも短!、電磁波を!、う。ェ ネルギ一で表わせば数 100 keV以上の光量子であることになる。通常、原子核の エネルギー準位間の遷移の際に γ線が放出される。 γ線の放出源としては、 γ線源 として使用される任意の線源が使用されるが、 6GCo (コノ レト 60)、 137Cs (セシウム 13 7)の線源が好ま U、。生体由来組織の処理に好ま 、からである。 [0084] γ線の照射量は、 kGy単位で表示することが多ぐ照射量は、吸収線量の測定によ り測定することができる。そのような測定には空洞電離箱、熱量計、フィルム線量計、 PMMA線量計、等の測定装置が用いられる。 In the present specification, the term “γ-ray” refers to a wavelength shorter than about 0.001 nm and electromagnetic waves. In terms of energy, the photon is several hundred keV or higher. Usually, γ-rays are emitted at the transition between energy levels of nuclei. Any source used as a γ-ray source can be used as the source of γ-rays, but 6G Co (conoleto 60) and 137 Cs (cesium 13 7) are preferred. This is because it is preferable for the treatment of living tissue. [0084] The amount of γ-ray irradiation that is often displayed in units of kGy can be measured by measuring the absorbed dose. For such measurements, measurement devices such as a hollow ionization chamber, calorimeter, film dosimeter, PMMA dosimeter are used.
[0085] 本明細書において、 γ線照射がなされた力どうかを組織上にて確認する方法として は、例えば、従って、 γ線照射によって生成した生成物の検索から、そこにどのような 不対電子が生成しかつ反応したかを知ることができる。ラジカルは、化学的方法およ び電子スペクトル、常磁性共鳴吸収などの測定法の進歩によって確認することができ る。きわめて寿命の短いラジカル (例えば、 · ΟΗ)についても閃光光分解法 (電子線 パルスラジオリシス法および閃光分光測定法)、剛体溶媒法、マトリクス単離法などに よって存在を確認することができる。ただし、遷移金属錯体の金属原子力 1個または 複数個の不対電子を持つ場合は、通常ラジカルとは呼ばな!/、。  [0085] In the present specification, as a method for confirming on a tissue whether or not a force has been subjected to γ-ray irradiation, for example, therefore, there is no unpairing from the search for a product generated by γ-ray irradiation. You can see if electrons are generated and reacted. Radicals can be identified by advances in chemical methods and methods such as electronic spectra and paramagnetic resonance absorption. The existence of extremely short-lived radicals (for example, ΟΗ) can also be confirmed by flash photolysis (electron beam pulse radiolysis and flash spectroscopy), rigid solvent method, matrix isolation, and so on. However, if you have one or more unpaired electrons in a transition metal complex, it is not usually called a radical! /.
[0086] γ線照射の確認方法としては、以下を挙げることができる。  [0086] Examples of methods for confirming γ-ray irradiation include the following.
[0087] (1) γ線照射により直接または間接作用（水分子の解裂による Η— radical OH— radical力ゝらの転移)を経て、タンパク質のアミノ酸残基や糖鎖上には、不対電子 (ラ ジカル）が生じる。ラジカルが再転移するといろいろな残基上を動き回ることになるが 、タンパク質ではエネルギー的に安定な芳香族アミノ酸 (チロシン、フエ二ルァラニン 、トリブトファン等)ゃ含硫アミノ酸 (システィン、メチォニン）にラジカルが集まりやすい 傾向があり、最終的には有る確率でポリペプチド鎖や糖鎖の架橋や切断を引き起こ すことが知られている。  [0087] (1) Directly or indirectly by γ-ray irradiation (transition of Η—radical OH—radical force caused by the cleavage of water molecules) and unpaired on amino acid residues and sugar chains of proteins. Electrons (radial) are generated. When radicals retransfer, they move around on various residues, but in proteins, aromatic amino acids (tyrosine, phenylalanine, tributophane, etc.) and sulfur-containing amino acids (cystine, methionine) collect radicals. It is known that it tends to be easy, and eventually causes cross-linking and cleavage of polypeptide chains and sugar chains with a certain probability.
[0088] (A)検知方法その 1：比較的長寿命の高分子ラジカルが組織中に残って 、れば、 常磁性共鳴吸収（EPR)などで確認できる (Radiation Researchl63, 535-543, 200 5)。  [0088] (A) Detection method # 1: If relatively long-lived polymer radicals remain in the tissue, they can be confirmed by paramagnetic resonance absorption (EPR) (Radiation Researchl 63, 535-543, 200 5 ).
[0089] (B)検知方法その 2：牛乳中のタンパク質カゼインを、多糖類のカルボキシメチルセ ルロース、 PEGなどとともに γ線照射して架橋薄膜を作製したときに、アミノ酸の一種 であるチロシン（tyrosine)が 2量体化して bityrosineという構造ができ、蛍光を発す るので架橋部位の定量になるという報告がなされており（J. Agric. Food. Chem. 4 6, 1618 - 1623, 1998)、これを使用すること力 Sできる。  [0089] (B) Detection method 2: Tyrosine (tyrosine), which is a type of amino acid, when protein casein in milk is irradiated with gamma rays together with polysaccharides such as carboxymethylcellulose and PEG to produce a crosslinked thin film. ) Is dimerized to form a bityrosine structure, and since it emits fluorescence, it has been reported that cross-linking sites are quantified (J. Agric. Food. Chem. 4 6, 1618-1623, 1998). You can use the power S.
[0090] 本明細書において「真空」とは、 l X 10_1Pa以下の圧力をいう。本明細書において「 減圧」下とは、 1 X 10_1Pa〜l X 105Paの範囲の圧力をいう。 In this specification, “vacuum” refers to a pressure of 1 × 10 −1 Pa or less. In this specification, “ “Under reduced pressure” refers to a pressure in the range of 1 × 10 — 1 Pa to l × 10 5 Pa.
[0091] 本明細書において「大気」とは、通常用いられる意味で用いられ、通常、組成として 窒素 78%、酸素 21%、アルゴン 1%、二酸ィ匕炭素 0.03%、水蒸気約 0.3%となってい るものが使用される。 [0091] In this specification, "atmosphere" is used in the meaning usually used. Usually, the composition is 78% nitrogen, 21% oxygen, 1% argon, 0.03% carbon dioxide, and about 0.3% water vapor. The one that is used is used.
[0092] 本明細書において「酸素中」とは、大気よりも酸素濃度が高い環境をさす。好ましく は、酸素中は、実質的に 100%の酸素の存在をいう。  In this specification, “in oxygen” refers to an environment having a higher oxygen concentration than the atmosphere. Preferably, oxygen refers to the presence of substantially 100% oxygen.
[0093] 本明細書において「水中」とは、実質的に H Oのみ力もなる媒体中における反応を [0093] As used herein, "in water" refers to a reaction in a medium that also has only H 2 O power.
2  2
いう。水としては、水道水の他、蒸留水、イオン交換水などが用いられるがそれらに限 定されない。  Say. As the water, tap water, distilled water, ion-exchanged water and the like are used, but are not limited thereto.
[0094] 本明細書にぉ ヽてラジカル反応が行われる「生体適合性分子」としては、両親媒性 分子溶液が用いられ得るが、それ以外の異なるものが用いられてもよ 、。  [0094] As the "biocompatible molecule" in which radical reaction is performed in the present specification, an amphiphilic molecule solution may be used, but other different ones may be used.
[0095] 本明細書において「生理活性物質」（physiologically active substance)とは、 細胞または組織に作用する物質をいう。生理活性物質には、サイト力インおよび増殖 因子が含まれる。生理活性物質は、天然に存在するものであっても、合成されたもの でもよい。好ましくは、生理活性物質は、細胞が産生するものまたはそれと同様の作 用を有するものである。本明細書では、生理活性物質はタンパク質形態または核酸 形態あるいは他の形態であり得るが、実際に作用する時点においては、サイト力イン は通常はタンパク質形態を意味する。  [0095] As used herein, "physiologically active substance" refers to a substance that acts on cells or tissues. Bioactive substances include site force-in and growth factors. The physiologically active substance may be naturally occurring or synthesized. Preferably, the physiologically active substance is one produced by a cell or one having a similar action. As used herein, the physiologically active substance may be in protein form or nucleic acid form or other form, but at the point of actual action, cyto force-in usually means the protein form.
[0096] 本明細書において使用される「サイト力イン」は、当該分野において用いられる最も 広義の意味と同様に定義され、細胞力 産生され同じまたは異なる細胞に作用する 生理活性物質をいう。サイト力インは、一般にタンパク質またはポリペプチドであり、免 疫応答の制禦作用、内分泌系の調節、神経系の調節、抗腫瘍作用、抗ウィルス作用 、細胞増殖の調節作用、細胞分化の調節作用などを有する。本明細書では、サイト 力インはタンパク質形態または核酸形態あるいは他の形態であり得るが、実際に作用 する時点においては、サイト力インは通常はタンパク質形態を意味する。  [0096] As used herein, "site force-in" is defined in the same manner as the broadest meaning used in the art, and refers to a physiologically active substance that is produced by cell force and acts on the same or different cells. Site force-in is generally a protein or polypeptide, and controls immune response, regulation of endocrine system, regulation of nervous system, antitumor action, antiviral action, regulation of cell proliferation, regulation of cell differentiation Etc. As used herein, cytoforce-in can be in protein form or nucleic acid form or other form, but at the point of actual action, cytoforce-in usually means protein form.
[0097] 本明細書において用いられる「増殖因子」または「細胞増殖因子」とは、本明細書で は互換的に用いられ、細胞の増殖を促進または制御する物質をいう。増殖因子は、 成長因子または発育因子ともいわれる。増殖因子は、細胞培養または組織培養にお いて、培地に添加されて血清高分子物質の作用を代替し得る。多くの増殖因子は、 細胞の増殖以外に、分ィ匕状態の制御因子としても機能することが判明している。 [0097] As used herein, "growth factor" or "cell growth factor" is used interchangeably herein and refers to a substance that promotes or regulates cell growth. Growth factors are also referred to as growth factors or growth factors. Growth factors are used in cell or tissue culture. In addition, it can be added to the medium to replace the action of the serum polymer substance. In addition to cell growth, many growth factors have been shown to function as regulators of sorting.
[0098] サイト力インには、代表的には、インターロイキン類、ケモカイン類、コロニー刺激因 子のような造血因子、腫瘍壊死因子、インターフェロン類が含まれる。増殖因子として は、代表的には、血小板由来増殖因子 (PDGF)、上皮増殖因子 (EGF)、線維芽細 胞増殖因子 (FGF)、肝実質細胞増殖因子 (HGF)、血管内皮増殖因子 (VEGF)の ような増殖活性を有するものが挙げられる。  [0098] Typically, cytoforce-in includes hematopoietic factors such as interleukins, chemokines, and colony stimulating factors, tumor necrosis factors, and interferons. Typical growth factors include platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF). And those having a proliferative activity such as
[0099] サイト力インおよび増殖因子などの生理活性物質は一般に、機能重複現象 (redund ancy)があることから、他の名称および機能で知られるサイト力インまたは増殖因子で あっても、本発明に使用される生理活性物質の活性を有する限り、本発明において 使用され得る。また、サイト力インまたは増殖因子は、本明細書における好ましい活 性を有してさえいれば、本発明の治療法または医薬の好ましい実施形態において使 用することができる。  [0099] Physiologically active substances such as cytoforce-in and growth factors generally have a redundancy phenomenon, so even if it is a site-force-in or growth factor known by other names and functions, As long as it has the activity of the physiologically active substance used in the invention, it can be used in the present invention. In addition, the cytodynamic force or the growth factor can be used in a preferred embodiment of the therapeutic method or medicament of the present invention as long as it has a preferable activity in the present specification.
[0100] 本明細書にぉ 、て「分化」とは、細胞、組織または器官のような生物の部分の状態 の発達過程であって、特徴のある組織または器官を形成する過程をいう。「分化」は、 王に発生学 (embryology)、発生生物' developmental biologyリなどにお！、て 使用されている。 1個の細胞からなる受精卵が***を行い成体になるまで、生物は種 々の組織および器官を形成する。***前または***が十分でな!、場合のような生物 の発生初期は、一つ一つの細胞や細胞群が何ら形態的または機能的特徴を示さず 区別することが困難である。このような状態を「未分化」であるという。「分化」は、器官 のレベルでも生じ、器官を構成する細胞が!/、ろ 、ろの違った特徴的な細胞または細 胞群へと発達する。これも器官形成における器官内での分化という。従って、本発明 における再生では、細胞の分化とは、その細胞が処理前には持っていな力つた何ら 力の形態的または機能的な特徴を有するようになることをいう。そのような例としては、 心臓弁の場合、細胞として幹細胞 (例えば、胚性幹細胞または組織幹細胞)を提供し たとき、心臓弁に存在する細胞または組織と少なくとも部分的に同様の形態または機 能をその細胞が有するようになることが挙げられる。  [0100] As used herein, "differentiation" refers to a process of development of a state of a part of an organism such as a cell, tissue or organ that forms a characteristic tissue or organ. “Differentiation” can be used for developmental biology and developmental biology! It has been used. Until a fertilized egg consisting of one cell divides and becomes an adult, an organism forms various tissues and organs. It is difficult to distinguish individual cells or groups of cells without any morphological or functional characteristics in the early stages of life, such as in cases where pre-division or division is not enough! Such a state is called “undifferentiated”. “Differentiation” also occurs at the organ level, where the cells that make up the organ develop into distinctive cells or groups of cells! This is also called differentiation within the organ in organ formation. Therefore, in the regeneration according to the present invention, the differentiation of a cell means that the cell has a morphological or functional characteristic of any force that it did not have before the treatment. For example, in the case of a heart valve, when a stem cell (e.g., embryonic stem cell or tissue stem cell) is provided as a cell, the form or function is at least partially similar to the cell or tissue present in the heart valve. That the cell has.
[0101] 本明細書において「移植片」、「グラフト」および「組織グラフト」は、交換可能に用い られ、身体の特定部位に挿入されるべき同種または異種の組織または細胞群であつ て、身体への挿入後その一部となるものをいう。移植片としては、例えば、臓器または 臓器の一部、血管、血管様組織、皮片、心臓弁、心膜、硬膜、角膜骨片、歯などが挙 げられるがそれらに限定されない。従って、移植片には、ある部分の欠損部に差し込 んで欠損を補うために用いられるものすべてが包含される。移植片としては、そのド ナー（donor)の種類によって、自己（自家)移植片（autograft) ,同種移植片（同種 異系移植片）（allograft)、異種移植片が挙げられるがそれらに限定されない。 [0101] As used herein, "graft", "graft" and "tissue graft" are used interchangeably The same or different type of tissue or cell group that is to be inserted into a specific part of the body and becomes a part of it after insertion into the body. Examples of the graft include, but are not limited to, an organ or a part of an organ, a blood vessel, a blood vessel-like tissue, a skin piece, a heart valve, a pericardium, a dura mater, a corneal bone fragment, and a tooth. Thus, a graft includes anything that can be used to fill a defect in a part to make up for the defect. The graft may include, but is not limited to, autograft, allograft (allograft), xenograft, depending on the type of donor. .
[0102] 本明細書において自己移植片または自家移植片とは、ある個体についていうとき、 その個体に由来する移植片をいう。本明細書において自己移植片というときは、広義 には遺伝的に同じ他個体 (例えば一卵性双生児)からの移植片をも含み得る。  [0102] As used herein, an autograft or autograft refers to a graft derived from an individual when referring to the individual. In this specification, the term “autograft” can include a graft from another genetically identical individual (eg, an identical twin) in a broad sense.
[0103] 本明細書において同種移植片（同種異系移植片）とは、同種であっても遺伝的に は異なる他個体力 移植される移植片をいう。遺伝的に異なることから、同種異系移 植片は、移植された個体 (レシピエント）において免疫反応を惹起し得る。そのような 移植片の例としては、親由来の移植片などが挙げられるがそれらに限定されない。  [0103] In the present specification, an allograft (allogeneic allograft) refers to a graft that is transplanted to another individual force even though it is the same species. Because of genetic differences, allogeneic transplants can elicit an immune response in the transplanted individual (recipient). Examples of such grafts include, but are not limited to, parent-derived grafts.
[0104] 本明細書にぉ 、て異種移植片とは、異種個体力 移植される移植片を 、う。従って 、例えば、ヒトがレシピエントである場合、ゥシ、ブタからの移植片は異種移植片という  [0104] As used herein, xenograft refers to a graft to be transplanted with xenogeneic force. Thus, for example, if a human is a recipient, a graft from Ushi, pig is called a xenograft
[0105] 本明細書にお！、て「レシピエント」（受容者）とは、移植片または移植体を受け取る 個体といい、「宿主」とも呼ばれる。これに対し、移植片または移植体を提供する個体 は、「ドナー」（供与者)という。 [0105] In this description! Thus, a “recipient” (recipient) is an individual who receives a graft or transplant and is also called a “host”. In contrast, an individual who provides a graft or transplant is called a “donor”.
[0106] 本発明の組織処理技術を用いれば、どのような移植片も使用することができる。な ぜなら、本発明の方法により補強された移植片 (例えば、組織、器官など）は、治療目 的に障害のない程度に免疫障害が減少するからである。従って、従来自己移植片に しか用いることができな力つた状況においても、同種異系移植片または異種移植片 を用いることが可能になったことは、従来技術では達成することができな力つた本発 明の格別の効果の一つといえる。  [0106] Any graft can be used with the tissue processing technique of the present invention. This is because grafts (for example, tissues, organs, etc.) reinforced by the method of the present invention have reduced immune disorders to the extent that they are not therapeutically impaired. Therefore, the fact that it has become possible to use allografts or xenografts even in situations where only conventional autografts could be used, has been impossible to achieve with the prior art. This is one of the special effects of the present invention.
[0107] 本明細書において「被験体」とは、本発明の処置が適用される生物をいい、「患者」 ともいわれる。患者または被験体は好ましくは、ヒトであり得る。 [0108] 本発明の方法または糸且織グラフトで必要に応じて使用される細胞は、同系由来（自 己（自家）由来)でも、同種異系由来 (他個体 (他家）由来)でも、異種由来でもよ 、。 拒絶反応が考えられることから、自己由来の細胞が好ましいが、拒絶反応が問題で ない場合同種異系由来であってもよい。また、拒絶反応を起こすものも必要に応じて 拒絶反応を解消する処置を行うことにより利用することができる。拒絶反応を回避す る手順は当該分野において公知であり、例えば、新外科学体系、心臓移植 '肺移植 技術的、倫理的整備力も実施に向けて (改訂第 3版）に記載されている。そのような 方法としては、例えば、免疫抑制剤、ステロイド剤の使用などの方法が挙げられる。 拒絶反応を予防する免疫抑制剤は、現在、「シクロスポリン」（サンデイミユン Zネオ一 ラル）、「タクロリムス」（プログラフ）、「ァザチォプリン」（イムラン）、「ステロイドホルモン 」（プレドニン、メチルプレドニン）、「T細胞抗体」（OKT3、 ATGなど）があり、予防的 免疫抑制療法として世界の多くの施設で行われている方法は、「シクロスポリン、ァザ チォプリン、ステロイドホルモン」の 3剤併用である。免疫抑制剤は、本発明の医薬と 同時期に投与されることが望ましいが、必ずしも必要ではない。従って、免疫抑制効 果が達成される限り免疫抑制剤は本発明の再生'治療方法の前または後にも投与さ れ得る。 [0107] As used herein, "subject" refers to an organism to which the treatment of the present invention is applied, and is also referred to as "patient". The patient or subject may preferably be a human. [0108] The cells used as necessary in the method or yarn-and-woven graft of the present invention may be derived from the same line (derived from the self (self)) or from the same type (derived from another individual (other family)). It can be of different origin. Autologous cells are preferred because rejection is considered, but they may be allogeneic if rejection is not a problem. In addition, those that cause rejection can be used by performing treatment to eliminate rejection as necessary. Procedures for avoiding rejection are well known in the art, and are described, for example, in the New Surgery System, Heart Transplantation, Lung Transplantation Technical and Ethical Maintenance Capabilities (Revised 3rd Edition). Examples of such methods include methods such as the use of immunosuppressants and steroids. Immunosuppressants to prevent rejection are currently "cyclosporine" (Sandyimyung Z Neoral), "Tacrolimus" (Prograf), "Azathioprine" (Imlan), "Steroid Hormone" (predonin, methylpredonin), "T There are “cell antibodies” (OKT3, ATG, etc.), and the method used in many facilities around the world as preventive immunosuppressive therapy is a combination of three drugs: “cyclosporine, azathioprine, and steroid hormone”. The immunosuppressive agent is desirably administered at the same time as the medicament of the present invention, but it is not always necessary. Therefore, the immunosuppressive agent can be administered before or after the regenerative treatment method of the present invention as long as an immunosuppressive effect is achieved.
[0109] 本発明で用いられる細胞は、どの生物（例えば、脊椎動物、無脊椎動物）由来の細 胞でもよい。好ましくは、脊椎動物由来の細胞が用いられ、より好ましくは、哺乳動物 (例えば、霊長類、齧歯類など）由来の細胞が用いられる。さらに好ましくは、霊長類 由来の細胞が用 、られる。最も好ましくはヒト由来の細胞が用 、られる。  [0109] The cells used in the present invention may be cells derived from any organism (for example, vertebrates and invertebrates). Preferably, cells derived from vertebrates are used, and more preferably cells derived from mammals (for example, primates, rodents, etc.) are used. More preferably, cells derived from primates are used. Most preferably, human-derived cells are used.
[0110] 本発明が対象とする被験体と、生体由来組織との組合せとしては、例えば、心疾患  [0110] Examples of the combination of a subject targeted by the present invention and a biological tissue include, for example, heart disease
(例えば、虚血性心疾患)を起こした心臓への移植、心膜パッチ、脳外科手術時の硬 膜移植、心筋梗塞、下肢、上肢などへの血管移植、骨折、骨欠損を有する患者への 骨の移植、損傷した角膜を有する患者に本発明の角膜を移植することなどが挙げら れるがそれらに限定されない。  (For example, ischemic heart disease) transplantation into the heart, pericardial patch, dural transplantation during brain surgery, myocardial infarction, blood vessel transplantation in the lower limbs, upper limbs, fractures, bones to patients with bone defects Transplantation, transplantation of the cornea of the present invention to a patient having a damaged cornea, but not limited thereto.
[0111] 本発明が対象とする組織は、生物のどの臓器または器官でもよぐまた、本発明が 対象とする組織は、どのような種類の生物由来であってもよい。本発明が対象とする 生物としては、脊椎動物または無脊椎動物が挙げられる。好ましくは、本発明が対象 とする生物は、哺乳動物 (例えば、霊長類、齧歯類など)である。より好ましくは、本発 明が対象とする生物は、霊長類である。最も好ましくは、本発明はヒトを対象とする。 [0111] The tissue targeted by the present invention may be any organ or organ of the organism, and the tissue targeted by the present invention may be derived from any kind of organism. Examples of organisms targeted by the present invention include vertebrates and invertebrates. Preferably, the present invention is intended The living organisms are mammals (eg, primates, rodents, etc.). More preferably, the organism targeted by the present invention is a primate. Most preferably, the present invention is directed to humans.
[0112] 自己細胞または組織を用いた血管再生療法が注目されており、組織を移植する方 法自体は当該分野において周知のとおり実施することができる。心臓血管外科領域 では多種の人工弁または人工血管などが用いられているが、その耐久性または抗凝 固療法の必要性とそれに付随する出血傾向、易感染性など多くの問題が挙げられて おり、再生医工学に期待が寄せられている。新岡らは肺動脈欠損症の 4歳の女児に 対して、患者の足の静脈より得られた血管平滑筋細胞を生体内吸収性高ポリマー上 に培養して再生血管を作製し、再生血管移植を行った (新岡俊治、今井康晴、瀬尾 和宏ほか;テツシュエンジニアリングによる心血管材料の開発、応用。 日心臓血管外 会誌 2000； 29： 38)。心血管系における組織工学（tissue engineering)では、移 植された細胞、構造物が血管内の血液に直接接触が可能で、移植直後から酸素、 栄養物の供給が得られ有利な条件であると考えられている。特に、自己細胞化 (細胞 置換)することの利点は以下のように多岐にわたる。  [0112] Revascularization therapy using autologous cells or tissues has attracted attention, and the method of transplanting tissues can be performed as is well known in the art. Various prosthetic valves or artificial blood vessels are used in the field of cardiovascular surgery, but there are many problems such as the need for durability or anticoagulant therapy, the associated bleeding tendency, and easy infection. , There is hope for regenerative medical engineering. Niioka et al. Produced a revascularized blood vessel by culturing vascular smooth muscle cells obtained from the veins of the patient's foot on a bioabsorbable high polymer for a 4-year-old girl with pulmonary artery deficiency. (Shunji Niioka, Yasuharu Imai, Kazuhiro Seo et al .; Development and application of cardiovascular materials by tissue engineering. Journal of the Japan Cardiovascular Society 2000; 29: 38). In tissue engineering in the cardiovascular system, the transplanted cells and structures can directly contact the blood in the blood vessels, providing oxygen and nutrient supply immediately after transplantation. It is considered. In particular, the advantages of self-cellization (cell replacement) are diverse as follows.
[0113] 1.拒絶反応の可能性を除去。  [0113] 1. Removed the possibility of rejection.
[0114] 2.ドナーを考慮する必要がない。  [0114] 2. There is no need to consider donors.
[0115] 3.生きた組織のため長い耐久性が期待できる。  [0115] 3. Long life expectancy due to living tissue.
[0116] 4.細胞が細胞外間質を完成させた時点で足場としてのポリマーが完全に生分解さ れ移植後長期的に異物が全く残存しない。  [0116] 4. When the cells complete the extracellular stroma, the polymer as a scaffold is completely biodegraded and no foreign matter remains in the long term after transplantation.
[0117] 5.最終的内皮で覆われるため抗血栓性にもすぐれており、移植後、抗凝固療法を 必要としない。 [0117] 5. Since it is covered with the final endothelium, it has excellent antithrombotic properties and does not require anticoagulant therapy after transplantation.
[0118] 6. 自己組織のため成長が期待できる、などが考えられる。  [0118] 6. Because of self-organization, growth can be expected.
[0119] 主に肺動脈圧程度の血圧範囲内で右心系において用いられている力 大動脈圧 内での使用、または弁組織、 ACバイパス用動脈グラフト、腱索組織などへの応用も 当業者が適宜おこなうことができる (循環器疾患の最新治療 2002— 2003；南江堂 p 29、 2002年発行）。  [0119] The force used in the right heart system mainly within the blood pressure range of the pulmonary artery pressure. Use within the aortic pressure, or application to valve tissue, AC bypass arterial graft, chordal tissue, etc. Can be performed as appropriate (latest treatment of cardiovascular disease 2002-2003; Nanedo p 29, 2002).
[0120] 人工弁の一般的使用は、当該分野において周知であり、本発明もまたこの周知事 項に基づいて実施することができる。例えば、ステントレス異種生体弁について知ら れている。異種生体弁では、ステントの存在で有効な弁口面積が小さくなり、また弁 葉の石灰化や変性が問題であった。最近、ブタ大動脈基部の形態を生かし、ステント を用いな!/、ステントレス異種生体弁が大動脈弁位の人工弁として注目されて 、る (Gr oss C et a Ann Thorac Surg 68 : 919, 1999)。ステント力 ^ないことで、/ Jヽ έ ヽサイズの弁を使用せざるを得な 、場合でも弁を介した圧較差が少なく、術後の左 心室肥大に対しても有効であると考えられている。また大動脈の基部の弾性が維持 され、弁尖に力かるストレスが少なくステント付き生体弁に比較して耐久性の向上も期 待できる。さらに、感染による心内膜炎、人工弁感染時にも使用が可能である。現在 欧米でのステントレス異種生体弁の中期術後成績は十分に満足できる報告がなされ ており、長期成績にも期待できる（GrossC et al、 AnnThorac Surg 68 : 919, 1999) o [0120] The general use of the artificial valve is well known in the art, and the present invention can also be implemented based on this well-known matter. For example, know about stentless heterogeneous valve It is. In the case of heterogeneous biological valves, the effective valve area is reduced due to the presence of the stent, and calcification and degeneration of the leaflets have been problems. Recently, taking advantage of the morphology of the porcine aortic root and not using a stent! / Stentless heterogeneous valve has been attracting attention as a prosthetic valve for aortic valve position (Gross C et a Ann Thorac Surg 68: 919, 1999) . Because there is no stent force, it is necessary to use a valve of / J ヽ έ 弁 size. Even in this case, the pressure difference through the valve is small, and it is considered effective for postoperative left ventricular hypertrophy. ing. In addition, the elasticity of the base of the aorta is maintained, so that stress applied to the valve leaflets is less, and durability can be expected to be improved compared to a biological valve with a stent. In addition, it can be used for endocarditis due to infection and infection of a prosthetic valve. The mid-term postoperative results of stentless heterogeneous flaps in Europe and the United States have been sufficiently satisfactory, and long-term results can be expected (GrossC et al, AnnThorac Surg 68: 919, 1999) o
[0121] (好ましい実施形態）  [0121] (Preferred embodiment)
以下に本発明の最良の形態を説明する。以下に提供される実施形態は、本発明の よりよい理解のために提供されるものであり、本発明の範囲は以下の記載に限定され るべきでないことが理解される。従って、当業者は、本明細書中の記載を参酌して、 本発明の範囲内で適宜改変を行うことができることは明らかである。  The best mode of the present invention will be described below. The embodiments provided below are provided for a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention with reference to the description in the present specification.
[0122] 1つの局面において、本発明は、生体適合性高分子を含み、 y線照射を受けたこ とを特徴とする生体由来組織を提供する。生体適合性高分子は、生体由来組織にど のような状態で含まれていても良いが、好ましくは、共有結合で結合されていることが 有利である。従って、当業者は、当該分野において周知の技術を用いて、含まれる べき状態に応じて、適宜、生態的合成高分子を生体由来組織に含ませることができ る。そのような結合は、 γ線照射によって達成されていてもよい。本発明において生 体適合性高分子が含まれるべき生体由来組織は、好ましくは脱細胞化処理を受けて いないものである。本発明は、生体適合性高分子を生体由来組織に含ませ、 γ線照 射を受けさせることによって、予想外に顕著に石灰化が減少し、組織強度を上昇させ ることを見出した力もである。従って、本発明の生体適合性高分子は、従来石灰化お よび強度の問題力も適用ができな力つた用途においても、用いることができる可能性 を提供する。特に、 γ線照射によって、他のラジカル反応 (例えば、 UV照射、化学物 質を使用したラジカル反応)またはダルタルアルデヒドなどの架橋剤によっては達成 できな力つた程度の石灰化の抑制が達成された。例えば、本発明によって認められ た効果の例としては、長期（ラット等の小動物では 2ヶ月程度であり、ブタ等の大動物 では 6〜12ヶ月程度である）の動物体内への移植実験の結果、石灰化による機能不 全が生じな 、効果などがある。これはダルタルアルデヒド処理時には全く認められな い効果である。定量的にも顕著な効果が観察された。例えば、一つの例では、長期（ 2ヶ月）ラット移植実験の結果、採り出した移植心膜 lmgあたりのカルシウム量が、グ ルタルアルデヒド処理時には 1. 6 ( μ g/mg)であったのが本件方法による処理時に は 0. 15〜0. 2 ( g/mg)程度にまで減少した。 [0122] In one aspect, the present invention provides a biological tissue characterized by containing a biocompatible polymer and receiving y-ray irradiation. The biocompatible polymer may be contained in any state in the living tissue, but it is advantageous that it is preferably bonded by a covalent bond. Therefore, those skilled in the art can appropriately include the biosynthetic polymer in the living tissue using a technique well known in the art depending on the state to be included. Such binding may be achieved by gamma irradiation. In the present invention, the biological tissue that should contain the biocompatible polymer is preferably one that has not undergone a decellularization treatment. The present invention has the power of finding that, by including a biocompatible polymer in a tissue derived from a living body and receiving γ-ray irradiation, calcification is remarkably reduced and tissue strength is increased unexpectedly. is there. Thus, the biocompatible polymer of the present invention offers the possibility of being used in powerful applications where conventional calcification and strength problems cannot be applied. In particular, other radical reactions (e.g. UV irradiation, chemicals) by gamma irradiation (A radical reaction using a quality) or a cross-linking agent such as dartal aldehyde, a powerful degree of calcification that could not be achieved was achieved. For example, as an example of the effect recognized by the present invention, as a result of a long-term transplantation experiment into an animal body (about 2 months for small animals such as rats and about 6 to 12 months for large animals such as pigs). In addition, there is an effect that functional failure due to calcification does not occur. This is an effect that is not recognized at all during treatment with dartalaldehyde. A significant effect was also observed quantitatively. For example, in one example, as a result of a long-term (2 months) rat transplantation experiment, the calcium content per lmg of transplanted pericardium was 1.6 (μg / mg) when glutaraldehyde was treated. During the treatment by this method, it decreased to about 0.15 to 0.2 (g / mg).
[0123] 本発明の組織処理によって得られる強度の補強は、好ましくは、生体適合性高分 子または γ線照射のない状態に比べて、例えば、 101%以上、さらに好ましくは 110 %以上、さらに好ましくは 120%以上、さらに好ましくは 150%以上、最も好ましくは 2 00%以上であり得る。 [0123] The strength reinforcement obtained by the tissue treatment of the present invention is preferably, for example, 101% or more, more preferably 110% or more, more preferably, compared to the state without biocompatible polymer or γ-ray irradiation. Preferably it may be 120% or more, more preferably 150% or more, and most preferably 200% or more.
[0124] 理論に束縛されることを望まないが、 γ線照射によって、石灰化の原因が取り除か れることが明らかになった。なお、自己由来以外の組織を移植する場合は、残存する 細胞が所望されない免疫反応を惹起する可能性があることから、本発明の生体由来 組織では、生体内において免疫反応を惹起するレベルより低くある必要がある。本発 明ではまた、生体適合性高分子の処理を受けた組織が、顕著に補強されるという点 にも 1つの効果がある。  [0124] Although not wishing to be bound by theory, it became clear that the cause of calcification was removed by gamma irradiation. When transplanting a tissue other than autologous tissue, the remaining cells may elicit an undesired immune response. Therefore, in the living tissue according to the present invention, the level is lower than the level that elicits an immune response in vivo. There must be. The present invention also has an effect in that the tissue treated with the biocompatible polymer is remarkably reinforced.
[0125] 好ま 、実施形態では、 γ線照射を受けた力どうかは、細胞外マトリクス成分が少 なくとも一部、好ましくは実質的に全部が共有結合で架橋されていること確認すること によって判定することができる。従って、本発明は、この実施形態において、細胞外 マトリクス成分が少なくとも一部が共有結合で架橋されていることを特徴とする、生体 由来組織を提供する。好ましくは、この細胞外マトリックス成分としては、コラーゲン、 エラスチン、ラミニン、フイブロネクチン、テネイシン、グリコサミノダンカンおよびプロテ ォグリカンを挙げることができるがそれらに限定されない。さらに好ましくは、この架橋 は、側鎖に遊離アミノ基またはカルボキシル基を有しない 2以上のアミノ酸の間に形 成される。この場合、好ましくは、架橋は、カルボキシル基、ヒドロキシル基およびアミ ノ基以外の基 (例えば、スルフヒドリル基）において形成されていることを特徴とする。 [0125] Preferably, in the embodiment, whether or not the force is γ-irradiated is determined by confirming that at least a part, preferably substantially all of the extracellular matrix component is covalently crosslinked. can do. Therefore, the present invention provides a biological tissue characterized in that, in this embodiment, the extracellular matrix component is at least partially crosslinked by a covalent bond. Preferably, the extracellular matrix component can include, but is not limited to, collagen, elastin, laminin, fibronectin, tenascin, glycosaminodancan and proteoglycan. More preferably, the bridge is formed between two or more amino acids that do not have a free amino group or carboxyl group in the side chain. In this case, preferably the cross-linking is a carboxyl group, a hydroxyl group and an amino group. It is characterized in that it is formed in a group other than a group (for example, a sulfhydryl group).
[0126] コラーゲン、エラスチン、ラミニン、フイブロネクチンおよびテネイシン等の細胞外マト リックス由来タンパク質、ならびにグリコサミノダリカン（ヒアルロン酸、コンドロイチン硫 酸およびへパラン硫酸等）およびプロテオダリカン等の細胞外マトリックス由来多糖 Z 糖タンパク質に対して γ線照射を行うと、架橋反応と分解反応とが混合して起こり得 ることが知られている。  [0126] Proteins derived from extracellular matrices such as collagen, elastin, laminin, fibronectin and tenascin, and polysaccharides derived from extracellular matrices such as glycosaminodarlicans (such as hyaluronic acid, chondroitin sulfate and heparan sulfate) and proteodalycan It is known that when γ-ray irradiation is applied to Z-glycoprotein, a cross-linking reaction and a decomposition reaction can occur in a mixed manner.
[0127] γ線照射を受けたかどうかは、一つの実施形態において、溶出タンパク質が実質 的に存在しないことにより確認することができる。従って、本発明は、この実施形態に おいて、溶出タンパク質が実質的に存在しないことを特徴とする、 γ線照射処理によ る生体由来組織を提供する。本明細書において「溶出タンパク質が実質的に存在し ない」とは、既知の方法で脱細胞組織からタンパク質抽出液を調整し、 Bio -Rad P rotein assay等の一般的な定量法でタンパク質を検出した場合、検量線力も計算さ れる理論値 (タンパク質重量 Z生体由来組織重量）が 0. 5mgZmg以下であり、より 好ましくは 0. 2mgZmg以下であり、さらに好ましくは 0. lmgZmg以下であることを いう。  [0127] In one embodiment, whether or not γ-irradiation has been received can be confirmed by the substantial absence of eluted protein. Therefore, the present invention provides a biological tissue derived from γ-irradiation treatment, characterized in that the eluted protein is substantially absent in this embodiment. In this specification, “substantially free of eluted protein” means that a protein extract is prepared from a decellularized tissue by a known method, and the protein is detected by a general quantitative method such as Bio-Rad Protein assay. In this case, the theoretical value (protein weight Z biological tissue weight) for which the calibration curve force is also calculated is 0.5 mgZmg or less, more preferably 0.2 mgZmg or less, still more preferably 0.1 mgZmg or less. .
[0128] 本発明において、細胞外マトリックス由来タンパク質に γ線照射を行う実験を行つ たところ、酸素と水が存覆する条件では、架橋反応が起こりやすいようであった。また 照射した試料はポリアクリルアミドゲル電気泳動（SDS— PAGE)に供しても、照射前 と異なりポリアクリルアミドゲルの中に入らなくなった。従って、溶出タンパク質は実質 的に存在していないことが明らかになった。  [0128] In the present invention, an experiment was performed in which extracellular matrix-derived protein was subjected to γ-ray irradiation. As a result, a cross-linking reaction seemed to occur easily under conditions where oxygen and water subsidized. In addition, the irradiated sample, when subjected to polyacrylamide gel electrophoresis (SDS-PAGE), did not enter the polyacrylamide gel, unlike before irradiation. Therefore, it was revealed that the eluted protein was substantially absent.
[0129] また、 y線照射によるタンパク質の架橋においてどのアミノ酸残基が架橋に関与す るかについては、いくつかの報告がある。そしてそれは化学架橋の場合に較べて特 異性が低いようである。  [0129] In addition, there are several reports on which amino acid residues are involved in cross-linking in protein cross-linking by y-ray irradiation. And it seems to be less specific than chemical crosslinking.
[0130] 化学架橋の場合、たとえば、ダルタルアルデヒド架橋では側鎖に遊離のアミノ基（ NH2)を持ったアミノ酸同士が架橋するので、必然的にリジン、アルギニン等が架橋 点となり得る。その他化学架橋の中には主にカルボキシル基をターゲットとするものも ある。よく知られている化学架橋の種類としては幾つかのものがある。例えば Nimni ME et al . , J. Biomed. Matr. Res. 21, 741— 771 (1987)に報告力 Sあるコラ 一ゲンの数種の架橋パターンの他、多くの架橋パターンの存在が知られて 、る。 [0130] In the case of chemical cross-linking, for example, in dartalaldehyde cross-linking, amino acids having a free amino group (NH2) in the side chain cross-link with each other, so that lysine, arginine or the like can inevitably be a cross-linking point. Some other chemical crosslinks mainly target carboxyl groups. There are several well-known types of chemical cross-linking. For example, Nimni ME et al., J. Biomed. Matr. Res. 21, 741—771 (1987) Many cross-linking patterns are known in addition to several types of cross-linking patterns.
[0131] 一方、 γ線照射において引き起こされる架橋は上記と同様の反応に加えて、多様 な反応が起こっているようである。また上記よりも多くの種類のアミノ酸が架橋点にな るようである。 J. H. Bowesetal . , Radiation Research 16, 211— 223 (1962) は、ゥシの真皮力も水溶性の成分を洗浄除去した試料について、酸素、窒素の存在 下で、乾燥状態および湿潤状態でそれぞれ γ線照射を行い、酸で全タンパク質を分 解後のアミノ酸の変化を定量したデータを報告している。その結果によると、グリシン やパリンなど、ほとんど減少しないアミノ酸もあるものの、それ以外のアミノ酸は少しず つ減少しており、その減少の程度は様々であった。このことは、 γ線によるタンパク質 問の架橋が比較的ランダムに様々なアミノ酸にお 、て起きて 、ることを示唆して!/、る といえる。 [0131] On the other hand, in addition to the reaction similar to the above, various reactions appear to occur in the cross-linking caused by γ-ray irradiation. Also, more types of amino acids than above appear to be cross-linking points. JH Bowesetal., Radiation Research 16, 211-223 (1962), γ-irradiated dry and wet samples, respectively, in the presence of oxygen and nitrogen, with regard to samples from which the water-soluble components of sushi were washed and removed. And reported data quantifying the amino acid changes after total protein digestion with acid. According to the results, some amino acids, such as glycine and parin, hardly decreased, but other amino acids decreased little by little, and the degree of the decrease varied. This suggests that cross-linking of proteins by γ-rays occurs relatively randomly on various amino acids! /.
[0132] すなわち、細胞外マトリックス由来タンパク質に限っていえば、 γ線照射において引 き起こされる架橋とィ匕学架橋とは、側鎖に遊離のアミノ基もしくはカルボキシル基を持 たな!/、二以上のアミノ酸の間にお!/、て起こって!/、るか否かによって峻別される。すな わち、側鎖に遊離のアミノ基もしくはカルボキシル基を有する二以上のアミノ酸の間 においてのみ起こっていればそれは化学架橋によるものであり、それ以外のアミノ酸 同士でも起こっていれば、それは γ線照射において引き起こされた架橋であると言え る。  [0132] In other words, as far as extracellular matrix-derived proteins are concerned, the cross-linking caused by γ-irradiation and the chemical cross-linking do not have a free amino group or carboxyl group in the side chain! These amino acids are distinguished according to whether or not they happened! / In other words, if it occurs only between two or more amino acids having a free amino group or carboxyl group in the side chain, it is due to chemical cross-linking, and if it also occurs between other amino acids, it means that γ It can be said that this is a cross-linking caused by irradiation.
[0133] また多糖にっ 、ては、種々の化学架橋（ビスエポキシドおよびジビニルスルフォン 架橋、分子内エステル化、ダルタルアルデヒド架橋、カルポジイミド架橋ならびにヒド ラジド架橋等）がカルボキシル基、ヒドロキシル基もしくはアミノ基をターゲットとするこ と力ら（Laurent, T. C. et al, , Acta Chem. Scand, 18, 274 - 275, (1964) ； Kuo, JW. et al . , Bioconjug Chem. (4) : 232— 4L (1991) ;Luo Y. et al . , J Control Release, 69 (1) ; 169— 84. (2000) )、カルボキシル基、ヒドロキシ ル基もしくはアミノ基を除く基 (例えば、前記基は、スルフヒドリル基、アルデヒド基、力 ルポ二ル基、スルホ基および-トロ基力 なる群より選択される基を含む）の間で起こ つているか、または前記架橋は、炭素 炭素結合、エーテル結合およびエステル結 合力ゝらなる群より選択される結合により形成されるカゝ否かによって峻別される。特に、 骨格を形成して 、る炭素間結合、エステル結合などの結合が開裂して新たな結合に よって架橋が形成されて ヽることが特徴であり得る。 [0133] For polysaccharides, various chemical crosslinks (bisepoxide and divinylsulfone crosslinks, intramolecular esterification, dartalaldehyde crosslinks, carpositimide crosslinks, hydrazide crosslinks, etc.) (Laurent, TC et al,, Acta Chem. Scand, 18, 274-275, (1964); Kuo, JW. Et al., Bioconjug Chem. (4): 232-4L ( 1991); Luo Y. et al., J Control Release, 69 (1); 169-84. (2000)), a group other than a carboxyl group, a hydroxyl group or an amino group (for example, the group is a sulfhydryl group, An aldehyde group, a force group, a sulfo group and a group selected from the group consisting of a sulfo group force), or the cross-linking is a carbon-carbon bond, an ether bond and an ester bond force. A bond selected from the group It is discriminated according to whether or not it is formed. In particular, It may be characterized in that a skeleton is formed and a bond such as an intercarbon bond or an ester bond is cleaved to form a bridge by a new bond.
[0134] (基の例）  [Example of group]
SH基 スルフヒドリル基  SH group Sulfhydryl group
CHO基 アルデヒド基  CHO group Aldehyde group
c=o基 カルボニル基  c = o group Carbonyl group
-SO H基 スルホ基  -SO H group Sulfo group
3  Three
-NO 2基 ニトロ基。  -NO 2 group Nitro group.
[0135] (結合の例示）  [0135] (Example of binding)
-C-C- CC結合 (炭素 炭素結合）  -C-C- CC bond (carbon-carbon bond)
-0- エーテル結合  -0- ether bond
— (c=o)o— エステル結合。  — (C = o) o— Ester bond.
[0136] 本明細書において使用される場合、「非化学架橋」とは、化学架橋以外による架橋 をいう。本明細書において使用される場合、「非化学架橋基」とは、化学架橋を形成 していない基をいい、例えば、スルフヒドリル基、アルデヒド基、カルボ-ル基、スルホ 基または-トロ基などが挙げられる力 これらに限定されない。  [0136] As used herein, "non-chemical cross-linking" refers to cross-linking other than chemical cross-linking. As used herein, “non-chemical crosslinking group” refers to a group that does not form a chemical bridge, such as a sulfhydryl group, an aldehyde group, a carbo group, a sulfo group, or a -tro group. The powers mentioned are not limited to these.
[0137] 1つの実施形態において、本発明において使用される y線照射の照射量は、通常 10〜250kGyである。理論に束縛されることを望まないが、約 250kGyを超えると、 残留すべき細胞外マトリクスなどの変性が生じ得るからである力 細胞外マトリクスな どの組織強度の維持に必要な成分の実質的な変性を生じないような照射量であれ ば、この上限を超えるような照射量であっても使用することができる。理論に束縛され ることを望まないが、 γ線照射量としては、 lOkGy以下の量であっても使用すること ができる。照射時間が著しく長くなることが考えられることが、組織の石灰化抑制の促 進が有意に認められる限り、 lOkGy以下の照射量もまた使用されることができ、これ らは本発明の範囲内にあることが当業者に理解される。  [0137] In one embodiment, the irradiation amount of y-ray irradiation used in the present invention is usually 10 to 250 kGy. Although not wishing to be bound by theory, it is possible that degeneration such as the extracellular matrix to remain should occur above about 250 kGy. The force required to maintain tissue strength such as extracellular matrix Any irradiation dose that does not cause denaturation can be used even if the irradiation dose exceeds this upper limit. Although not wishing to be bound by theory, it is possible to use γ-ray irradiation even if it is less than lOkGy. Irradiation times below lOkGy can also be used, as long as the irradiation time is considered to be significantly longer, as long as significant promotion of suppression of tissue calcification is observed, these are within the scope of the present invention. It will be understood by those skilled in the art.
[0138] 好ましくは、 γ線などの照射量は lOOkGy以下であることが有利である。理論に束 縛されることを望まないが、 lOOkGy以下の範囲では、著しい組織強度の低下がほと んど見られな力つた力もである。好ましくは下限としては、 40kGy以上の照射量を用 いることが有利である。 [0138] Preferably, the irradiation amount of γ rays or the like is less than lOOkGy. I don't want to be bound by theory, but in the range below lOOkGy, there is also a force with almost no significant decrease in tissue strength. Preferably, the lower limit is 40 kGy or more. It is advantageous.
[0139] より好ましくは、照射量としては、 50〜80kGyの照射量が有利に用いられる。理論 に束縛されることを望まないが、 γ線照射による遺伝子 DNA等の生体分子の切断と Ρ EGによる抽出操作の組み合わせにより石灰化の抑制が促進されるものの、組織の機 械強度を保つべき細胞外基質タンパク質には著し 、損傷を与えな 、と 、う理由があ るカゝらである。  [0139] More preferably, as the irradiation amount, an irradiation amount of 50 to 80 kGy is advantageously used. Although not wishing to be bound by theory, although the suppression of calcification is promoted by the combination of cutting of biomolecules such as gene DNA by γ-irradiation and extraction operation by EG, the mechanical strength of the tissue should be maintained There is a reason why extracellular matrix proteins are not markedly damaged.
[0140] 1つの実施形態において、本発明で用いられる γ線の照射時間は、総照射量に依 存して変動するが、通常、 0. 5時間〜 10日程度であり、好ましくは 1時間〜 2日程度 であることが有利である。より好ましくは、 3〜8時間であることが有利である。理論に 束縛されることを望まないが、より長い照射時間にすると、石灰化の抑制が促進され るが、従来は弾性に富んだ性質を有するコラーゲン、エラスチンなどの細胞外基質タ ンパク質が切断もしくは架橋され、折り曲げや引っ張りなどの力学的変形に対して脆 くなるので移植用組織としての性質が劣化するであるという背景があるからである。し たがって、石灰化の抑制は、総照射量に一部依存することから、強い γ線源を用い て数時間程度で反応させることができる。  [0140] In one embodiment, the irradiation time of gamma rays used in the present invention varies depending on the total irradiation dose, but is usually about 0.5 hours to 10 days, preferably 1 hour. It is advantageous to be about 2 days. More preferably, it is advantageous for 3 to 8 hours. Although not wishing to be bound by theory, longer irradiation times promote suppression of calcification, but conventionally, extracellular matrix proteins such as collagen and elastin, which have elastic properties, are cleaved. Alternatively, it is cross-linked and becomes brittle with respect to mechanical deformation such as bending and pulling, so that there is a background that the properties as a transplanted tissue deteriorate. Therefore, since suppression of calcification depends in part on the total irradiation dose, it can be reacted in a few hours using a strong γ-ray source.
[0141] 好ましい実施形態において、本発明において使用されるラジカル反応 (例えば、 γ 線照射）は、真空、酸素中、窒素中、大気中、水中、両親媒性分子溶液中およびそ れらの組み合わせ力 なる群より選択される雰囲気下で行われる。  [0141] In a preferred embodiment, the radical reaction (eg, gamma irradiation) used in the present invention is performed in vacuum, oxygen, nitrogen, air, water, amphiphilic molecule solution and combinations thereof. It is performed in an atmosphere selected from a group of forces.
[0142] 好ましい実施形態において、本発明において用いられる生体適合性高分子は、前 記組織をコーティングするように処理される。このようなコーティングは、当該分野に おいて周知の技術によって実施することができ、例えば、そのような生体適合性高分 子溶液中に生体由来組織を浸す方法、あるいは、スプレーなどで塗布する方法など が挙げられるがそれらに限定されな 、。  [0142] In a preferred embodiment, the biocompatible polymer used in the present invention is treated to coat the tissue. Such coating can be performed by techniques well known in the art. For example, a method of immersing a biological tissue in such a biocompatible polymer solution or a method of applying by spraying or the like. Such as, but not limited to.
[0143] さらに好ましい実施形態において、本発明の生体適合性高分子は、生体由来組織 に架橋されていることが有利である。理論に束縛されることを望まないが、架橋される ことによって、生体由来組織の組織性をより強固にするからである。そのような架橋は 、 γ線照射によって達成されていても良いが、別の手段であってもよい。  [0143] In a further preferred embodiment, the biocompatible polymer of the present invention is advantageously cross-linked to a living tissue. Although not wishing to be bound by theory, it is because cross-linking makes the tissue of living tissue more robust. Such cross-linking may be achieved by gamma irradiation, but may be another means.
[0144] 好ましい実施形態において、本発明において用いられる生体適合性高分子は、生 分解性であり得る。そのような生分解性高分子の例としては、例えば、ポリエチレング リコール、ポリビニルアルコール、ポリダリコール酸、ポリ乳酸などが挙げられるがそれ らに限定されない。ポリエチレングリコール系ポリマーは親水性が付与された生分解 性ポリマーである。ポリマー中のエチレンォキシド含量が増加するにしたがって親水 性が増し、水に膨潤する。 [0144] In a preferred embodiment, the biocompatible polymer used in the present invention contains a biocompatible polymer. It can be degradable. Examples of such biodegradable polymers include, but are not limited to, polyethylene glycol, polyvinyl alcohol, polydaricholic acid, polylactic acid, and the like. Polyethylene glycol polymer is a biodegradable polymer with hydrophilicity. As the ethylene oxide content in the polymer increases, the hydrophilicity increases and swells in water.
[0145] 別の好ま 、実施形態にぉ 、て、本発明にお 、て用いられる生体適合性高分子は 、ポリエチレングリコール、ポリビニルアルコール、ポリビニルピロリドン、エラスチンお よびそれらの 2つ以上の混合物からなる群より選択される高分子を含み得る。さらに 好ましくは、本発明において使用される生体適合性高分子は、ポリエチレングリコー ルを含む。  [0145] According to another preferred embodiment, the biocompatible polymer used in the present invention comprises polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, elastin and a mixture of two or more thereof. A polymer selected from the group may be included. More preferably, the biocompatible polymer used in the present invention comprises polyethylene glycol.
[0146] 本発明の好ましい実施形態において用いられるポリエチレングリコールは、通常、 平均分子量が 200と 6, 000との間である。そのような PEGとしては、 Nacalai tesqu eの polyethvlene glycol (H (OCH CH ) OH) #分子量（例えば、 200、 600、 1  [0146] The polyethylene glycol used in a preferred embodiment of the present invention typically has an average molecular weight between 200 and 6,000. Such PEGs include Nacalai tesqu e polyethvlene glycol (H (OCH CH) OH) # molecular weight (eg 200, 600, 1
2 2 n  2 2 n
, 000、 2, 000、 6, 000、 35, 000、 50, 000)のような製品力挙げられる。好まし < は PEGは、平均分子量が 1, 000と 200, 000との間である。別の好ましい実施形態 では、 PEGは、平均分子量が 4, 000と 100, 000との間であり得る。より好ましくは、 PEGは、平均分子量が 8, 000と 50, 000との間であり得る。このように PEGは巿販さ れているが、所望の特性を達成するために、適宜合成することもできる。そのような合 成方法は当該分野において周知である。なお、本明細書において平均分子量およ び分子量は、ダルトン (Dalton)であらわす。本発明において使用される PEGは、分 子が均質であることが好ましいが、そのことは必須ではなぐ平均分子量が一定範囲 にあれば通常使用することができる。本発明において石灰化の抑制を調製するため に、 PEG以外の化学物質 (例えば、酵素（例えば DNaselなど）、酵素阻害剤などを 含むがそれらに限定されな 、）を加えることができる。 PEG以外の化学物質を加える と、加えるべき PEGの量または濃度は、その化学物質の存在量に応じて変動し得る 。好ましくは、細胞融合などで用いられる濃度がよぐ例えば、 lgZml以上（100%w Zw以上）の濃度が好ましいが、それに限定されない。あるいは、 PEGは、 60%w/ v〜 100%wZvの間の濃度で使用され得る。 [0147] 本発明の好まし 、実施形態にぉ 、て用いられるポリビュルアルコールは、分子量 5 00〜200, 000の範囲内にある。より好ましくは、使用されるポリビュルアルコールは 、分子量 500〜100, 000であり、さらに好まし <は、 500〜10, 000である。 , 000, 2,000, 6,000, 35,000, 50,000). Preferably <PEG has an average molecular weight between 1,000 and 200,000. In another preferred embodiment, the PEG can have an average molecular weight between 4,000 and 100,000. More preferably, the PEG can have an average molecular weight between 8,000 and 50,000. As described above, PEG is commercially available, but can be appropriately synthesized in order to achieve desired characteristics. Such synthesis methods are well known in the art. In the present specification, the average molecular weight and the molecular weight are expressed by Dalton. The PEG used in the present invention is preferably homogeneous in molecular weight, but this is not essential, and it can usually be used if the average molecular weight is within a certain range. In the present invention, chemicals other than PEG (for example, including but not limited to enzymes (eg, DNasel), enzyme inhibitors, etc.) can be added to prepare calcification inhibition. When a chemical other than PEG is added, the amount or concentration of PEG to be added may vary depending on the amount of the chemical present. Preferably, the concentration used for cell fusion or the like is good, for example, a concentration of lgZml or more (100% wZw or more) is preferable, but not limited thereto. Alternatively, PEG can be used at a concentration between 60% w / v and 100% wZv. [0147] In the preferred embodiment of the present invention, the polybutyl alcohol used in the embodiment has a molecular weight in the range of 500 to 200,000. More preferably, the polybutyl alcohol used has a molecular weight of 500-100,000, more preferably <is 500-10,000.
[0148] 本発明の好ましい実施形態において用いられるポリビニルピロリドンは、分子量 50 0-200, 000の範囲内にある。より好ましくは、使用されるポリビュルピロリドンは、分 子量 1. 000〜100, 000であり、さらに好ましくは、 10, 000〜50, 000 (例えば、 40 , 000)である。  [0148] The polyvinylpyrrolidone used in a preferred embodiment of the present invention has a molecular weight in the range of 500-200,000. More preferably, the polybutylpyrrolidone used has a molecular weight of 1.000 to 100,000, and more preferably 10,000 to 50,000 (for example, 40,000).
[0149] 本発明の好ましい実施形態において使用される生体適合性高分子は、 1% (w/v )〜50% (wZv)の濃度で使用される。より好ましくは、その生体適合性高分子は、 5 % (wZv)〜30% (wZv)の範囲で使用され得、さらに好ましくは、その生体適合性 高分子は、 10 (w/v)〜20% (w/v)の範囲内で使用され得る。  [0149] The biocompatible polymer used in a preferred embodiment of the present invention is used at a concentration of 1% (w / v) to 50% (wZv). More preferably, the biocompatible polymer can be used in the range of 5% (wZv) to 30% (wZv), more preferably the biocompatible polymer is 10 (w / v) to 20 % (w / v) can be used.
[0150] 本発明の生体由来組織は、移植治療で使用されることから、その組織または器官 が正常状態において有していた機能を発揮するのに支障があるような損傷を受けて いないことが好ましい。この効果は、本発明の γ線照射によっても実質的に損傷を受 けな力つた。そのような支障があるかどうかは、その糸且織の細胞外マトリクスが実質的 に変性を受けていないことを確認することにより判定することができる。従って、本発 明の組織は、細胞外マトリクスが実質的に機能的に存在していることによつても特徴 付けられ得る。細胞外マトリクスの存在の確認は、特異的マーカーによる染色などに よって行うことができる。細胞外マトリクスの機能は、それぞれのメンバーによって適切 なアツセィを選択して実施することができる。  [0150] Since the living tissue of the present invention is used in transplantation treatment, the tissue or organ may not be damaged so as to hinder the function of the normal function of the tissue or organ. preferable. This effect was substantially not damaged by the γ-ray irradiation of the present invention. Whether there is such a hindrance can be determined by confirming that the extracellular matrix of the yarn and fabric is not substantially denatured. Thus, the tissue of the present invention can also be characterized by the substantial functional presence of the extracellular matrix. The presence of the extracellular matrix can be confirmed by staining with a specific marker. The function of the extracellular matrix can be performed by selecting an appropriate assembly according to each member.
[0151] 1つの実施形態において、本発明の生体由来組織は、臨床適用することができるよ うな組織強度を有する。十分に強い組織強度は、特に、膜状の組織を臨床適用する とき重要な特性である。組織強度は一般に、引っ張り強さ（例えば、破断強度、剛性 率、ヤング率など）を測定することによって判定することができる。ある実施形態にお いて、本発明の生体由来組織は、処理前の正常組織が有していた組織強度の少な くとも約 75%以上、好ましくは約 80%以上、より好ましくは約 85%以上、さらに好まし くは約 90%以上であり得、あるいは実質的に同じ組織強度を有していてもよぐ未処 理状態で正常組織力 Sもともと有していた組織強度以上の値 (例えば、 110%以上、 1 20%以上、 150%以上、 200%以上）を有していてもよい。ここで、組織が未処理状 態での組織強度とは、その組織の処理 (例えば、本発明の γ線照射および生体適合 性分子での処理)の前 (例えば、天然での状態)で有していた組織強度をいう。十分 に強い組織強度は、例えば、弁状組織、管状組織で適用する場合にも有することが 好ましい特性である。 [0151] In one embodiment, the biological tissue of the present invention has a tissue strength that allows clinical application. Sufficiently strong tissue strength is an important property, especially when applying membranous tissue clinically. Tissue strength can generally be determined by measuring tensile strength (eg, breaking strength, stiffness, Young's modulus, etc.). In certain embodiments, the living tissue of the present invention has at least about 75%, preferably about 80% or more, more preferably about 85% or more of the tissue strength of the normal tissue before treatment. More preferably, it may be about 90% or more, or it may have substantially the same tissue strength. 110% or more, 1 20% or more, 150% or more, 200% or more). Here, the tissue strength when the tissue is in an untreated state is effective (for example, in a natural state) before the tissue is treated (for example, γ-irradiation and treatment with a biocompatible molecule of the present invention). It refers to the strength of the tissue. Sufficiently strong tissue strength is a desirable characteristic even when applied to, for example, valve-like tissue and tubular tissue.
[0152] 1つの実施形態において、本発明の生体由来組織は、移植したときに石灰化が顕 著に抑制される。この抑制の程度としては、例えば、（1)長期（2ヶ月）ラット移植実験 の結果、採り出した移植心膜 lmgあたりのカルシウム量力 0. 5 ( μ g/mg)以下で ある。または本件方法による処理時の上記カルシウム量 Zダルタルアルデヒド処理時 の上記カルシウム量が 0. 5以下であることなどを挙げることができるがそれらに限定さ れない。あるいは、長期（2ヶ月）ラット移植実験の結果、採り出した移植心膜に対して Von Kossa染色をした際に、ほとんど染色されないレベルが達成され得る。  [0152] In one embodiment, the calcification of the living tissue of the present invention is markedly suppressed when transplanted. The degree of this suppression is, for example, (1) a calcium amount per 0.5 mg (μg / mg) of transplanted pericardium extracted as a result of a long-term (2 months) rat transplantation experiment. Alternatively, the amount of calcium at the time of treatment according to the present method can be exemplified by the amount of calcium at the time of Z-daltaraldehyde treatment being 0.5 or less, but is not limited thereto. Alternatively, as a result of long-term (2 months) rat transplantation experiments, a level that is hardly stained can be achieved when Von Kossa staining is performed on the transplanted pericardium.
[0153] 1つの実施形態において、本発明の生体由来組織は、生体適合性高分子がランダ ムに架橋されていることを特徴とする。理論に束縛されることを望まないが、本発明の 7線照射による生体由来組織の処理によって、生体適合性高分子がランダムに架橋 され、その結果組織強度が強化されていること、石灰化の抑制効果の増強が達成さ れた。  [0153] In one embodiment, the biological tissue of the present invention is characterized in that a biocompatible polymer is randomly crosslinked. Although not wishing to be bound by theory, the biocompatible polymer is randomly crosslinked by the treatment of the tissue derived from the living body by 7-ray irradiation of the present invention. Increased suppression effect was achieved.
[0154] 管状組織の場合、組織強度は、 β値で表すことができる。 β値の算出方法は、本 明細書の別の場所において詳述した。ある実施形態において、本発明の生体由来 組織は、約 15以上の 13値の組織強度を有し、好ましくは、約 18以上の 13値の組織 強度を有し、より好ましくは約 20以上の |8値の組織強度を有し、さらに好ましくは約 2 2以上の β値の組織強度を有する。別の実施形態において、本発明の生体由来組 織は、処理前の組織が有していた j8値の少なくとも約 75%以上、好ましくは約 80% 以上、より好ましくは約 85%以上、さらに好ましくは約 90%以上であり得、未処理状 態で正常組織がもともと有していた j8値以上の値を有していてもよい。ここで、組織が 未処理状態での特性 (例えば、 j8値）とは、その組織の処理 (例えば、本発明の γ線 処理または生体適合性高分子での処理)の前 (例えば、天然での状態)で有して!/ヽ た特性をいう。 [0155] 本発明の生体由来組織は、臨床適用が意図される組織であれば、身体のどのよう な組織 (例えば、弁状組織、管状組織、膜状組織など)であってもよい。ある実施形態 では、本発明の生体由来組織は、組織の物理的構造が必要とされる組織であり得る 。物理的構造を保持するためには、細胞の骨格などの構造のみが必要であり、細胞 質成分などの細胞内の成分は必ずしも必要ないからである。また、必要に応じて、必 要とされる細胞は、別途その生体由来組織に提供され得る力 または移植された宿 主から内的に供給され得る。ある実施形態では、本発明の生体由来組織は、血管、 血管様組織、心臓弁、心膜、硬膜、角膜および骨力 選択される器官に由来する組 織であり得る。別の実施形態では、本発明の生体由来組織は、心臓血管系の組織で あり得、例えば、血管、血管様組織、心臓弁および心膜から選択される器官に由来し 得る。ある実施形態では、本発明の生体由来組織は、外科手術または処置において 、患部の切除または損傷部位の修復などを行った後、その切断面または欠損部と、 その周辺組織との間で癒着が生じるのを防止するために使用され得る。 [0154] In the case of a tubular tissue, the tissue strength can be represented by a β value. The method for calculating the β value was described in detail elsewhere in this specification. In certain embodiments, the living tissue of the present invention has a tissue strength of 13 values of about 15 or more, preferably has a tissue strength of 13 values of about 18 or more, more preferably | It has a tissue strength of 8 values, more preferably a tissue strength of β value of about 22 or more. In another embodiment, the biological tissue of the present invention is at least about 75% or more, preferably about 80% or more, more preferably about 85% or more, more preferably about the j8 value that the tissue before treatment had. May be about 90% or higher, and may have a value of j8 or higher that normal tissue originally had in the untreated state. Here, a characteristic of a tissue in an untreated state (for example, j8 value) means that the tissue is treated (for example, the γ-ray treatment or the biocompatible polymer of the present invention) (for example, naturally). In the state of!). [0155] The biological tissue of the present invention may be any tissue of the body (for example, a valve-like tissue, a tubular tissue, a membrane-like tissue, etc.) as long as the tissue is intended for clinical application. In certain embodiments, the biological tissue of the present invention can be a tissue in which the physical structure of the tissue is required. This is because in order to maintain the physical structure, only a structure such as a cell skeleton is required, and intracellular components such as cytoplasmic components are not necessarily required. In addition, if necessary, the required cells can be supplied separately from the force that can be separately provided to the living tissue or from the transplanted host. In certain embodiments, the biological tissue of the present invention can be a tissue derived from a blood vessel, blood vessel-like tissue, heart valve, pericardium, dura mater, cornea and bone strength selected organ. In another embodiment, the biological tissue of the present invention can be cardiovascular tissue, for example, from an organ selected from blood vessels, blood vessel-like tissue, heart valves and pericardium. In one embodiment, the living tissue of the present invention undergoes adhesion between the cut surface or defect and its surrounding tissue after excision of the affected area or repair of the damaged site in surgery or treatment. Can be used to prevent it from occurring.
[0156] 本発明の生体由来組織は、カルボキシル基、ヒドロキシル基およびアミノ基以外の 非化学架橋基の間に形成される架橋を少なくとも 1つ有する。この非化学架橋基とし ては、スルフヒドリル基、アルデヒド基、カルボ-ル基、スルホ基または-トロ基が挙げ られる力 これらに限定されない。 1つの実施形態において、本発明の生体由来組織 は、 γ線照射により生じた、炭素 炭素結合、エーテル結合またはエステル結合より 形成される架橋を少なくとも 1つ有する。本発明の生体由来組織は、骨格を形成して Vヽる炭素間結合、エステル結合が開裂して形成された新たな結合により形成された 架橋を少なくとも 1つ有する。  [0156] The living tissue of the present invention has at least one crosslink formed between non-chemical crosslinkable groups other than a carboxyl group, a hydroxyl group and an amino group. Examples of the non-chemical crosslinking group include, but are not limited to, a sulfhydryl group, an aldehyde group, a carbo group, a sulfo group, and a -tro group. In one embodiment, the biological tissue of the present invention has at least one cross-link formed by gamma irradiation and formed from a carbon-carbon bond, an ether bond or an ester bond. The living tissue of the present invention has at least one cross-link formed by a new bond formed by cleavage of an intercarbon bond or ester bond forming a skeleton.
[0157] 1つの実施形態において、本発明の生体由来組織は、側鎖に遊離のアミノ基およ びカルボキシル基を有さな 、アミノ酸の間に形成された架橋を少なくとも 1つ有する。 この側鎖に遊離のアミノ基およびカルボキシル基を有さないアミノ酸の間に形成され た架橋は、リジン、アルギニン以外を架橋点としたアミノ酸間の架橋である。  [0157] In one embodiment, the biological tissue of the present invention has at least one bridge formed between amino acids without a free amino group and a carboxyl group in the side chain. The bridge formed between the amino acids having no free amino group and carboxyl group in the side chain is a bridge between amino acids other than lysine and arginine.
[0158] 1つの実施形態にお!ヽて、本発明の生体由来組織は、カルボキシル基、ヒドロキシ ル基およびアミノ基以外の非化学架橋基を有する多糖の間に形成された架橋を少な くとも 1つ有する。この非化学架橋基を有する多糖は、スルフヒドリル基、アルデヒド基 、カルボニル基、スルホ基および-トロ基力 なる群より選択される少なくとも 1つの基 の間で形成される架橋を有する。 [0158] According to one embodiment, the living tissue of the present invention has at least a cross-link formed between polysaccharides having non-chemical cross-linking groups other than a carboxyl group, a hydroxyl group and an amino group. Have one. This polysaccharide having a non-chemical crosslinking group is composed of a sulfhydryl group, an aldehyde group. , Having a bridge formed between at least one group selected from the group consisting of a carbonyl group, a sulfo group, and a -tro group.
[0159] 1つの実施形態にお!ヽて、本発明の生体由来組織は、ビスエポキシド架橋、ジビ- ルスルフォン架橋、分子内エステル化、グルタルアルデヒド架橋、カルボジイミド架橋 およびヒドラジド架橋以外の非化学架橋を少なくとも 1つ有する。  [0159] According to one embodiment, the biological tissue of the present invention undergoes non-chemical crosslinking other than bisepoxide crosslinking, dibutylsulfone crosslinking, intramolecular esterification, glutaraldehyde crosslinking, carbodiimide crosslinking, and hydrazide crosslinking. Have at least one.
[0160] 1つの実施形態において、本発明の生体由来組織は、側鎖に遊離アミノ基または カルボキシル基を有しな 、2以上のアミノ酸の間に形成される共有結合、カルボキシ ル基、ヒドロキシル基、アミノ基、スルフヒドリル基、アルデヒド基およびカルボ-ル基 からなる群より選択される基の間で形成される結合、炭素 炭素結合、ペプチド結合 、エーテル結合ならびにエステル結合力もなる群より選択される結合を有する。本発 明の生体由来組織は、二官能性分子架橋剤による結合により形成される架橋をさら に含んでいてもよい。この二官能性分子架橋剤は、好ましくは、ダルタルアルデヒドで ある。  [0160] In one embodiment, the living tissue of the present invention has no free amino group or carboxyl group in the side chain, and a covalent bond, carboxyl group, hydroxyl group formed between two or more amino acids. , A bond formed between a group selected from the group consisting of an amino group, a sulfhydryl group, an aldehyde group and a carbo group, a carbon carbon bond, a peptide bond, an ether bond, and a bond selected from the group that also has an ester bond strength Have The biological tissue of the present invention may further contain a crosslink formed by bonding with a bifunctional molecular crosslinker. This bifunctional molecular crosslinking agent is preferably dartalaldehyde.
[0161] 本発明の生体由来膜は、該生体由来膜をダルタルアルデヒドにより化学的に処理 し、ポリエチレングリコール (PEG)に曝露し、 γ線を照射して架橋を形成することによ り、ダルタルアルデヒド処理をしたものに比べて γ線照射後の組織強度が上昇してい るカゝ、またはダルタルアルデヒド処理をしたものと同等の組織強度を有し、該生体由 来膜は、 PEGを内包し、かつ該生体由来膜の表面には PEG層が形成されている。  [0161] The biological membrane of the present invention is obtained by chemically treating the biological membrane with dartalaldehyde, exposing it to polyethylene glycol (PEG), and irradiating γ rays to form a crosslink. Compared to those treated with dartalaldehyde, the tissue strength after γ-irradiation is increased, or the tissue strength is the same as that treated with dartalaldehyde. And a PEG layer is formed on the surface of the biological membrane.
[0162] 別の局面において、本発明は、外科手術または処置において、患部の切除または 損傷部位の修復などを行った後、その切断面または欠損部と、その周辺組織との間 で癒着が生じるのを防止するための癒着防止膜を提供する。本発明の癒着防止膜 は、例えば、胸膜、心膜、脳硬膜、漿膜、腹膜または皮膚などを用いて調製され得る 。本発明の癒着防止膜は、例えば、心臓、肺、肝臓、脳、消化器官、胆嚢の切除部 位または損傷部位において処置を行う場合に、癒着を防ぐことができる。本発明の癒 着防止膜は、生体外においても使用することができる。生体外としては、例えば、移 植用の組織を保存するために使用することができる。  [0162] In another aspect, the present invention provides adhesion between the cut surface or defect and the surrounding tissue after excision of the affected part or repair of the damaged site in surgery or treatment. An anti-adhesion membrane for preventing the above is provided. The anti-adhesion membrane of the present invention can be prepared using, for example, the pleura, pericardium, cerebral dura mater, serosa, peritoneum or skin. The adhesion-preventing film of the present invention can prevent adhesion when, for example, treatment is performed in the heart, lung, liver, brain, digestive organ, excision site of the gallbladder, or a damaged site. The anti-adhesion membrane of the present invention can also be used ex vivo. As an ex vivo, it can be used, for example, to store tissue for transplantation.
[0163] 別の局面において、本発明の移植用の癒着防止膜を生産する方法は、 A)生体由 来膜を提供する工程； B)該生体由来膜を、生体適合性高分子に曝す工程；および C )該生体由来膜を γ線照射に曝す工程を包含する。 1つの実施形態において、本発 明の移植用の癒着防止膜を生産する方法は、 D)前記生体由来膜を二官能性分子 架橋剤により化学架橋させる工程をさらに包含することもできる。 D)工程は、 Α)工程 — Β)工程の間、および Β)工程— Α)工程の間からなる群より選択される少なくとも 1 つの間にて実施され得る。別の実施形態において、本発明において使用されるニ官 能性分子架橋剤により形成される結合は、スルフヒドリル基、アルデヒド基、カルボ- ル基、スルホ基および-トロ基からなる群より選択される基の間で形成される結合、炭 素 炭素結合、ペプチド結合、エーテル結合またはエステル結合であり得る。この二 官能性分子架橋剤は、アミノ基、カルボキシル基およびアルデヒド基力 なる群より選 択される基を有し得る。例えば、二官能性分子架橋剤としては、ダルタルアルデヒド、 シアンイミド（cyanimide)、 1—ェチル 3— (3 ジメチルァミノプロピル）カルボジィ ミドヒドロクロリド（EDC)、エポキシ（ポリ（グリシジルエーテル））、 2, 6 ビス（4 アジ ドベンジリデン）ー4ーメチルシクロへキサノン、 4, 4'ジアジドジフエニルエーテル、 4 , 4'ジアジドジフ ニルスルホン、 4, 4'ジアジドジフ ニルアセトン、 4, 4'ジアジドジ フエ-ノレメタン、 1, 4 ビス（α ジァゾベンジル）および 4— [p アジドサリチアミド] プチルァミンが挙げられるが、これらに限定されない。好ましくは、二官能性分子は、 ダルタルアルデヒドである。本方法において使用される場合、生体適合性高分子は、 ポリビニルアルコール、ポリビニルピロリドン、エラスチン、ポリエチレングリコール、ゼ ラチン、コラーゲン、 γ -ポリグルタミン酸およびそれらの 2つ以上の混合物力 選択さ れ得る。好ましくは、生体適合性高分子は、ポリエチレングリコールを含む。 [0163] In another aspect, the method of producing an adhesion-preventing membrane for transplantation according to the present invention comprises: A) a step of providing a biologically-derived membrane; B) a step of exposing the biologically-derived membrane to a biocompatible polymer ; And C ) Including a step of exposing the biological membrane to γ-ray irradiation. In one embodiment, the method for producing an adhesion-preventing membrane for transplantation according to the present invention can further include the step of D) chemically crosslinking the biological membrane with a bifunctional molecular crosslinking agent. Step D) may be performed between step ii) step—step ii) and at least one selected from the group consisting of step ii) step-step ii). In another embodiment, the bond formed by the bifunctional molecular crosslinking agent used in the present invention is selected from the group consisting of a sulfhydryl group, an aldehyde group, a carbon group, a sulfo group, and a -tro group. It can be a bond formed between groups, a carbon-carbon bond, a peptide bond, an ether bond or an ester bond. This bifunctional molecular crosslinker may have a group selected from the group consisting of an amino group, a carboxyl group and an aldehyde group. For example, bifunctional molecular crosslinkers include dartalaldehyde, cyanimide, 1-ethyl 3- (3 dimethylaminopropyl) carbodiimide hydrochloride (EDC), epoxy (poly (glycidyl ether)), 2 , 6 bis (4azidobenzylidene) -4-methylcyclohexanone, 4,4'diazidodiphenyl ether, 4,4'diazidodiphenylsulfone, 4,4'diazidodiphenylacetone, 4,4'diazidodiphenylmethane, 1, 4 bis (α diazobenzyl) and 4— [p azidosalicyamido] ptyramine are included, but not limited to. Preferably the bifunctional molecule is dartalaldehyde. When used in the method, the biocompatible polymer may be selected from polyvinyl alcohol, polyvinyl pyrrolidone, elastin, polyethylene glycol, gelatin, collagen, γ-polyglutamic acid and a mixture of two or more thereof. Preferably, the biocompatible polymer comprises polyethylene glycol.
本発明の癒着防止膜を産生する方法は、 Α)工程において、生体由来膜は、ヒトま たはゥシもしくはブタを含む家畜より摘出することにより提供され得る。 Β)工程におい て、生体由来膜に対する前記生体適合性高分子の曝露は、 24°C〜37°Cにて 1時間 〜4時間、または 24°Cにて 12— 24時間という条件下で実施され得る。 C)工程にお いて、 γ線の強度は 50keVであり、 γ線の照射は、密閉容器中、線量 12kGy〜10 OkGyにて、 24— 37°Cという条件下で実施され得る。好ましくは、 γ線の照射は、線 量 12— 50kGyである。 γ線は 1—4時間にわたって照射され得る。 D)工程において 、化学架橋は、 24°Cにて、 1時間〜 4時間という条件下で生じ得る。化学架橋は、 1 時間と!/、う条件であっても生じ得る。 The method for producing the anti-adhesion membrane of the present invention can be provided by removing the biological membrane from livestock including humans or ushi or pigs in step i). I) In the process, the biocompatible polymer is exposed to the biological membrane under conditions of 24 hours to 37 ° C for 1 hour to 4 hours, or 24 ° C for 12 to 24 hours. Can be done. In step C), the intensity of γ-rays is 50 keV, and irradiation with γ-rays can be carried out in a sealed container at a dose of 12 kGy to 10 OkGy under conditions of 24-37 ° C. Preferably, the irradiation of γ rays has a dose of 12-50 kGy. Gamma rays can be irradiated for 1-4 hours. In step D), chemical crosslinking can occur at 24 ° C under conditions of 1 hour to 4 hours. Chemical crosslinking 1 It can occur even with time and conditions.
[0165] 1つの局面において、本発明は、生体由来の癒着防止膜を提供し得る。本発明の 癒着防止膜は、生体適合性高分子を含み、 γ線照射を受けたことを特徴とする。本 発明の癒着防止膜は、カルボキシル基、ヒドロキシル基およびアミノ基以外の非化学 架橋基の間に形成される架橋を少なくとも 1つ有する。この非化学架橋基としては、ス ルフヒドリル基、アルデヒド基、カルボ-ル基、スルホ基または-トロ基が挙げられるが 、これらに限定されない。 1つの実施形態において、本発明の癒着防止膜は、 γ線 照射により生じた、炭素 炭素結合、エーテル結合またはエステル結合より形成され る架橋を少なくとも 1つ有する。本発明の癒着防止膜は、骨格を形成している炭素間 結合、エステル結合が開裂して形成された新たな結合により形成された架橋を少なく とも 1つ有する。  [0165] In one aspect, the present invention can provide an adhesion-preventing membrane derived from a living body. The anti-adhesion membrane of the present invention contains a biocompatible polymer and is characterized by receiving γ-ray irradiation. The adhesion-preventing film of the present invention has at least one crosslink formed between non-chemical crosslinkable groups other than carboxyl group, hydroxyl group and amino group. Examples of the non-chemical crosslinking group include, but are not limited to, a sulfhydryl group, an aldehyde group, a carbo group, a sulfo group, and a -tro group. In one embodiment, the anti-adhesion membrane of the present invention has at least one cross-link formed by γ-ray irradiation and formed from a carbon-carbon bond, an ether bond or an ester bond. The adhesion-preventing film of the present invention has at least one cross-link formed by a new bond formed by cleavage of an inter-carbon bond or an ester bond forming a skeleton.
[0166] 1つの実施形態において、本発明の癒着防止膜は、側鎖に遊離のアミノ基および カルボキシル基を有さな ヽアミノ酸の間に形成された架橋を少なくとも 1つ有する。こ の側鎖に遊離のアミノ基およびカルボキシル基を有さな 、アミノ酸の間に形成された 架橋は、リジン、アルギニン以外を架橋点としたアミノ酸間の架橋である。  [0166] In one embodiment, the anti-adhesion membrane of the present invention has at least one crosslink formed between amino acids having no free amino group and carboxyl group in the side chain. The bridge formed between amino acids without a free amino group and carboxyl group in the side chain is a bridge between amino acids except for lysine and arginine.
[0167] 1つの実施形態にお!、て、本発明の癒着防止膜は、カルボキシル基、ヒドロキシル 基およびアミノ基以外の非化学架橋基を有する多糖の間に形成された架橋を少なく とも 1つ有する。この非化学架橋基を有する多糖は、スルフヒドリル基、アルデヒド基、 カルボニル基、スルホ基および-トロ基力 なる群より選択される少なくとも 1つの基 の間で形成される架橋を有する。  [0167] In one embodiment, the anti-adhesion membrane of the present invention has at least one crosslinking formed between polysaccharides having non-chemical crosslinking groups other than carboxyl groups, hydroxyl groups and amino groups. Have. This polysaccharide having a non-chemical crosslinking group has a bridge formed between at least one group selected from the group consisting of a sulfhydryl group, an aldehyde group, a carbonyl group, a sulfo group, and a -tro group.
[0168] 1つの実施形態にお!、て、本発明の癒着防止膜は、ビスエポキシド架橋、ジビュル スルフォン架橋、分子内エステル化、ダルタルアルデヒド架橋、カルポジイミド架橋お よびヒドラジド架橋以外の非化学架橋を少なくとも 1つ有する。  [0168] In one embodiment, the anti-adhesion membrane of the present invention comprises a non-chemical crosslink other than bisepoxide crosslinks, dibule sulfone crosslinks, intramolecular esterification, dartalaldehyde crosslinks, carpositimide crosslinks and hydrazide crosslinks Have at least one.
[0169] 1つの実施形態において、本発明の癒着防止膜は、側鎖に遊離アミノ基または力 ルポキシル基を有しな ヽ 2以上のアミノ酸の間に形成される共有結合、カルボキシル 基、ヒドロキシル基、アミノ基、スルフヒドリル基、アルデヒド基およびカルボ-ル基から なる群より選択される基の間で形成される結合、炭素 炭素結合、ペプチド結合、ェ 一テル結合ならびにエステル結合力 なる群より選択される結合を有する。本発明の 癒着防止膜は、二官能性分子架橋剤による結合により形成される架橋をさらに含ん でいてもよい。この二官能性分子架橋剤は、好ましくは、ダルタルアルデヒドである。 本発明の癒着防止膜は、好ましくは、ポリエチレングリコールを含む。 [0169] In one embodiment, the anti-adhesion membrane of the present invention has a free amino group or a force lpoxyl group in the side chain. 共有 A covalent bond formed between two or more amino acids, a carboxyl group, a hydroxyl group , An amino group, a sulfhydryl group, an aldehyde group, and a bond formed between groups selected from the group consisting of a carbocyclic group, a carbon-carbon bond, a peptide bond, an ether bond, and an ester bond force. Have a bond. Of the present invention The adhesion-preventing film may further contain a crosslink formed by bonding with a bifunctional molecular crosslinker. This bifunctional molecular crosslinker is preferably dartalaldehyde. The anti-adhesion membrane of the present invention preferably contains polyethylene glycol.
[0170] 本発明の癒着防止膜は、胸膜、心膜、脳硬膜、漿膜、腹膜または皮膚であるが、こ れらに限定されない。好ましくは、癒着防止膜は哺乳動物由来である。さらに好ましく は、ヒト、ゥシまたはブタ由来である。  [0170] The anti-adhesion membrane of the present invention is the pleura, pericardium, brain dura mater, serosa, peritoneum or skin, but is not limited thereto. Preferably, the anti-adhesion membrane is derived from a mammal. More preferably, it is derived from human, ushi or pig.
[0171] 本発明の癒着防止膜は、 A) (1)プラスチック製の薄いシートに心膜を挟み込むェ 程、（2)サンプルが変形しないように電子ノギスにて 3ケ所以上の厚みを測定するェ 程、および（3)該サンプルの厚みの平均を算出する工程により測定した場合、 0. 5 1. Ommの厚みを有すること；ならびに B) (1)検体を 5 X 30mmの短冊状に切断 する工程、 (2)引っ張り試験器の固定部分に該検体の両端約 5mmを固定する工程 、および（3)引っ張り開始し破断点まで lcmZminの速度で引っ張り、レジオメータ 一にて測定する工程により測定した場合、 2— lOMPaの弾性率を有することを特徴 とする。  [0171] The anti-adhesion membrane of the present invention is: A) (1) The pericardium is sandwiched between thin plastic sheets, (2) The thickness is measured at three or more locations with an electronic caliper so that the sample does not deform. And (3) having a thickness of 0.5 1. Omm when measured by the process of calculating the average thickness of the sample; and B) (1) cutting the specimen into 5 x 30 mm strips. (2) The step of fixing the both ends of the specimen to about 5 mm to the fixed part of the tensile tester, and (3) The step of starting pulling to the breaking point at a speed of lcmZmin and measuring with a radiometer. The case is characterized by having an elastic modulus of 2-lOMPa.
[0172] 別の実施形態において、本発明の癒着防止膜は、  [0172] In another embodiment, the anti-adhesion membrane of the present invention comprises
癒着防止率 (％) = {癒着なしの数 Z (癒着あり +癒着なしの数） } X 100  Anti-adhesion rate (%) = {number without adhesion Z (number with adhesion + number without adhesion)} X 100
により 80%以上の癒着防止率を有し得る。  May have an adhesion prevention rate of 80% or more.
[0173] 本発明の癒着防止膜は、該生体由来膜をダルタルアルデヒドにより化学的に処理 し、ポリエチレングリコール (PEG)に曝露し、 γ線を照射して架橋を形成することによ り、ダルタルアルデヒド処理をしたものに比べて γ線照射後の組織強度が上昇してい るカゝ、またはダルタルアルデヒド処理をしたものと同等の組織強度を有し、該癒着防 止膜は、 PEGを内包し、かつ該生体由来膜の表面には PEG層が形成されている。  [0173] The adhesion-preventing membrane of the present invention is obtained by chemically treating the biological membrane with dartalaldehyde, exposing it to polyethylene glycol (PEG), and irradiating γ rays to form a crosslink. Compared to those treated with dartalaldehyde, the tissue strength after γ-irradiation is increased, or the tissue strength is the same as that treated with dartalaldehyde. And a PEG layer is formed on the surface of the biological membrane.
[0174] 本発明の生体由来組織は、意図される臨床適用に適合していれば、どのような生 物由来の組織であってもよい。従って、本発明の組織は、どの生物（例えば、脊椎動 物、無脊椎動物）由来の組織でもあってもよい。ヒトへの適用が意図される場合、好ま しくは、脊椎動物由来の組織が用いられ、より好ましくは、哺乳動物 (例えば、霊長類 、偶蹄類、奇蹄類、齧歯類など）由来の組織が用いられる。ヒトへの適用が意図され る場合、さらに好ましくは、霊長類由来の組織が用いられる。ヒトへの適用が意図され る場合、さらに好ましくは、ブタ由来の組織が用いられる。大きさがヒトに類似している 力もである。ヒトへの適用が意図される場合、最も好ましくはヒト由来の組織が用いら れる。本発明の生体由来組織または組織グラフトのサイズは、ヒト以外のものを使用 する場合、ヒトのものに近いものであることが好ましぐその物理的特性がヒトのものに 近いものであることが好ましい (例えば、ブタ)。本発明の癒着防止膜は、生体内であ つても生体外であっても使用され得る。 [0174] The biological tissue of the present invention may be any biological tissue as long as it is suitable for the intended clinical application. Therefore, the tissue of the present invention may be a tissue derived from any organism (for example, vertebrate animals, invertebrates). When intended for human application, tissue from vertebrates is preferably used, and more preferably tissue from mammals (eg, primates, cloven-hoofed, odd-hoofed, rodents, etc.) Is used. If intended for human application, more preferably primate-derived tissue is used. Intended for human use More preferably, porcine-derived tissue is used. It is also a force that is similar in size to humans. When intended for human application, most preferably human-derived tissue is used. The size of the living tissue or tissue graft of the present invention is preferably close to that of a human when its non-human size is used, and its physical characteristics are close to that of a human. Preferred (eg pig). The anti-adhesion membrane of the present invention can be used both in vivo and in vitro.
[0175] 本発明において、本発明の生体由来組織が適用される部位は、身体中のどのよう な部位であってもよい。従って、生体由来組織が由来する身体の部分に再度適用さ れてもよぐあるいは、他の部分に適用されてもよい。実施例などで示されているよう に、「元に戻す」かどうかに拘わりなぐいずれの部分に生体由来組織を適用しても、 本発明は所望の効果 (例えば、再生、自己組織化)を達成することが証明された。従 つて、本発明は、原理的にはどのような移植手術、再生手術にも使用することができ るという多大なる有用性を有する。本発明の生体由来組織が適用され得る部位として は、例えば、皮膚、血管、角膜、腎臓、心臓、肝臓、臍帯、腸、神経、肺、胎盤、脾臓 、脳、四肢末梢、網膜、弁、上皮組織、結合組織、筋肉組織、神経組織などが挙げら れるがそれらに限定されない。  [0175] In the present invention, the part to which the living tissue of the present invention is applied may be any part in the body. Therefore, it may be applied again to the part of the body from which the biological tissue is derived, or may be applied to other parts. As shown in the examples and the like, the present invention can achieve a desired effect (for example, regeneration, self-organization) even if a biological tissue is applied to any part regardless of whether or not to “revert”. Proven to achieve. Therefore, the present invention has great utility in principle that it can be used for any transplantation and regenerative surgery. Examples of the site to which the living tissue of the present invention can be applied include, for example, skin, blood vessels, cornea, kidney, heart, liver, umbilical cord, intestine, nerve, lung, placenta, spleen, brain, peripheral limbs, retina, valve, epithelium. Examples include, but are not limited to, tissue, connective tissue, muscle tissue, and nerve tissue.
[0176] 別の局面において、本発明は、本発明の生体由来組織を含む組織グラフトを提供 する。この組織グラフトにおいて、上記生体由来組織には、レシピエント由来の細胞 が播種され、培養されて、所望の組織の構造が形成されていることが特徴である。本 発明の組織グラフトは、臨床適用が意図される組織の組織グラフトであれば、身体の どのような組織への移植を目的としてもよい。ある実施形態では、本発明の組織ダラ フトは、組織の物理的構造が必要とされる組織であり得る。物理的構造を保持するた めに、レシピエント由来の細胞が上述の生体由来組織に移植前に提供され得る。レ シピエント由来の細胞は、宿主から内的に供給されていてもよい。ある実施形態では 、本発明の組織グラフトは、血管、血管様組織、心臓弁、心膜、硬膜、角膜および骨 力 選択される器官に由来する組織の組織グラフトであり得る。別の実施形態では、 本発明の組織グラフトは、心臓血管系の組織の組織グラフトであり得、例えば、血管 、血管様組織、心臓弁および心膜から選択される器官に由来する組織グラフトであり 得る。 [0176] In another aspect, the present invention provides a tissue graft containing the biological tissue of the present invention. In this tissue graft, the biological tissue is seeded and cultured with recipient-derived cells to form a desired tissue structure. The tissue graft of the present invention may be intended for transplantation into any tissue of the body as long as it is a tissue graft intended for clinical application. In certain embodiments, the tissue draft of the present invention may be a tissue in which the physical structure of the tissue is required. In order to preserve the physical structure, cells from the recipient can be provided to the above-described living tissue prior to transplantation. The recipient-derived cells may be supplied internally from the host. In certain embodiments, the tissue graft of the present invention may be a tissue graft of a tissue derived from a vessel, blood vessel-like tissue, heart valve, pericardium, dura mater, cornea and bone force selected organ. In another embodiment, the tissue graft of the present invention can be a tissue graft of cardiovascular tissue, such as a tissue graft derived from an organ selected from blood vessels, blood vessel-like tissues, heart valves and pericardium obtain.
[0177] 本発明の組織グラフトは、意図される臨床適用に適合していれば、どのような生物 由来の組織を含んでいてもよい。従って、本発明の組織グラフトは、どの生物（例え ば、脊椎動物、無脊椎動物）由来の組織でもあってもよい。ヒトへの適用が意図される 場合、好ましくは、脊椎動物由来の組織が用いられ、より好ましくは、哺乳動物 (例え ば、霊長類、齧歯類など）由来の組織が本発明の組織グラフトに用いられる。ヒトへの 適用が意図される場合、さらに好ましくは、霊長類由来の組織が本発明の組織グラフ トに用いられる。ヒトへの適用が意図される場合、さらに好ましくは、ブタ由来の組織 が本発明の組織グラフトに用いられる。大きさがヒトに類似しているからである。ヒトへ の適用が意図される場合、最も好ましくはヒト由来の組織が本発明の組織グラフトに 用いられる。  [0177] The tissue graft of the present invention may contain any biological tissue as long as it is compatible with the intended clinical application. Therefore, the tissue graft of the present invention may be a tissue derived from any organism (eg, vertebrate, invertebrate). When intended for human application, tissue from vertebrates is preferably used, and more preferably, tissue from mammals (eg, primates, rodents, etc.) is used in the tissue graft of the present invention. Used. When intended for human application, more preferably, tissues derived from primates are used in the tissue graft of the present invention. If intended for human application, more preferably, porcine tissue is used for the tissue graft of the present invention. This is because the size is similar to humans. When intended for human application, most preferably human-derived tissue is used for the tissue graft of the present invention.
[0178] 本発明の糸且織グラフトに用いられるレシピエントの細胞は、臨床適用に適切であれ ば、どのような細胞であってもよい。従って、この細胞としては、血管内皮細胞、平滑 筋細胞、線維芽細胞、血球ならびにこれらに分化する前駆細胞および体性幹細胞な どが挙げられるがそれらに限定されない。好ましくは、この細胞は、移植される部位に おいて所望の機能を発揮することができる細胞であり得る。  [0178] The recipient cells used in the yarn and woven graft of the present invention may be any cells as long as they are suitable for clinical application. Accordingly, examples of such cells include, but are not limited to, vascular endothelial cells, smooth muscle cells, fibroblasts, blood cells, progenitor cells and somatic stem cells that differentiate into these cells. Preferably, the cell may be a cell that can perform a desired function at a site to be transplanted.
[0179] 本発明において、本発明の組織グラフトが適用される部位は、身体中のどのような 部位であってもよい。従って、組織グラフトが由来する身体の部分に再度適用されて もよぐあるいは、他の部分に適用されてもよい。実施例などで示されているように、「 元に戻す」かどうかに拘わりなぐいずれの部分に組織グラフトを適用しても、本発明 は所望の効果 (例えば、再生、自己組織化)を達成することが証明された。従って、本 発明は、原理的にはどのような移植手術、再生手術にも使用することができるという 多大なる有用性を有する。本発明の組織グラフトが適用され得る部位としては、例え ば、皮膚、血管、角膜、腎臓、心臓、肝臓、臍帯、腸、神経、肺、胎盤、脾臓、脳、四 肢末梢、網膜、弁、上皮組織、結合組織、筋肉組織、神経組織などが挙げられるが それらに限定されない。  [0179] In the present invention, the site to which the tissue graft of the present invention is applied may be any site in the body. Thus, it may be reapplied to the part of the body from which the tissue graft originates or may be applied to other parts. As shown in the examples, the present invention achieves the desired effect (for example, regeneration, self-organization) regardless of whether the tissue graft is applied regardless of whether it is “undoed” or not. Proven to do. Therefore, the present invention has great utility in principle that it can be used for any transplantation and regenerative surgery. Examples of the site to which the tissue graft of the present invention can be applied include skin, blood vessel, cornea, kidney, heart, liver, umbilical cord, intestine, nerve, lung, placenta, spleen, brain, peripheral extremity, retina, valve, Examples include, but are not limited to, epithelial tissue, connective tissue, muscle tissue, and nerve tissue.
[0180] 別の局面において、本発明は膜状組織グラフト、弁状組織グラフト、管状組織ダラ フトなどを提供する。この組織グラフトは、本発明の生体由来組織を含む。ここで、こ の生体由来組織には、レシピエント由来の細胞が播種され、培養されて、所望の組 織の構造が形成されていてもよいが、細胞はなくてもよい。好ましくは自己由来以外 の細胞がな 、ことが有利である。免疫拒絶反応を惹起しな 、からである。 [0180] In another aspect, the present invention provides membranous tissue grafts, valvular tissue grafts, tubular tissue drafts, and the like. This tissue graft includes the biological tissue of the present invention. Where The living body-derived tissue may be seeded with recipient-derived cells and cultured to form a desired tissue structure, but the cells may not be present. It is advantageous that there are preferably no cells other than autologous cells. This is because immune rejection is not caused.
[0181] 他の局面において、本発明は、生体由来糸且織を生産する方法であって、 A)生体由 来組織を提供する工程； B)生体適合性高分子に該生体由来組織を曝す工程；およ び C)該生体由来組織を γ線照射に曝す工程、を包含する、方法を提供する。生体 由来組織は、どのようなものを使用しても良い。  [0181] In another aspect, the present invention relates to a method for producing a biologically derived yarn and weaving, comprising A) providing a biologically derived tissue; B) exposing the biologically derived tissue to a biocompatible polymer. And C) exposing the biological tissue to γ-irradiation. Any biological tissue may be used.
[0182] 1つの実施形態において、使用される生体適合性高分子は、例えば、ポリビュルァ ノレコーノレ、ポリビニルピロリドン、エラスチン、ポリエチレングリコール、ゼラチン、コラ 一ゲン、 γ -ポリグルタミン酸およびそれらの 2つ以上の混合物等であり得る。好ましく は、ポリエチレングリコールが使用される。  [0182] In one embodiment, the biocompatible polymer used is, for example, polybutanololone, polyvinylpyrrolidone, elastin, polyethylene glycol, gelatin, collagen, γ-polyglutamic acid and mixtures of two or more thereof Etc. Preferably, polyethylene glycol is used.
[0183] 好ま 、実施形態にぉ 、て、本発明にお 、て用いられ生体適合性高分子に曝す 工程は、生体適合性高分子を、架橋するように γ線照射を行うことを包含する。  [0183] Preferably, according to the embodiment, the step of exposing to the biocompatible polymer used in the present invention includes performing γ-ray irradiation so that the biocompatible polymer is crosslinked. .
[0184] 1つの好ましい実施形態において、本発明において用いられる γ線照射の照射量 は、 10〜250kGyの範囲内である。より好ましくは、 γ線照射量は、 50kGy〜100k Gyの範囲内にある。理論に束縛されることを望まないが、このような照射量では、組 織強度を上昇させるのに適切であるからである。組織強度を維持することができる線 量の上限を組織に応じて設定することが適当である。組織強度の観点からは、 60kG yの方が lOOkGyよりもより強い強度が得られている場合も存在するがそれに限定さ れない。  [0184] In one preferred embodiment, the dose of gamma irradiation used in the present invention is in the range of 10 to 250 kGy. More preferably, the γ-ray irradiation dose is in the range of 50 kGy to 100 kGy. Although not wishing to be bound by theory, such an irradiation dose is appropriate for increasing the tissue strength. It is appropriate to set the upper limit of the dose that can maintain the tissue strength according to the tissue. From the viewpoint of tissue strength, there are cases where 60 kGy is stronger than lOOkGy, but it is not limited to this.
[0185] 本明細書において、 γ線照射は、通常、真空、酸素中、窒素中、大気中、水中、両 親媒性分子溶液中およびそれらの組み合わせ力 なる群より選択される環境下で行 われ得る力 それらに限定されない。好ましくは、大気中で行われる。  [0185] In the present specification, γ-ray irradiation is usually performed in an environment selected from the group consisting of vacuum, oxygen, nitrogen, air, water, amphiphilic molecular solutions, and a combination force thereof. Power that can be broken Not limited to them. Preferably, it is performed in the atmosphere.
[0186] 好ましい実施形態において、本発明において用いられる γ線照射は、 0. 5〜240 時間の範囲で行われ、より好ましくは 1時間〜 24時間、さらに好ましくは 3〜8時間で ある。理論に束縛されることを望まないが、強い γ線源を用いて単位時間当たりの線 量をあげれば照射時間が短縮可能であるが、強すぎる場合は組織に悪影響が出る 可能性があるからである。また、時間を長くするには、弱い γ線源を用いればよい。 理論に束縛されることを望まないが、弱すぎると、長時間係り、しかも、組織自体が別 の要因で損傷を受ける可能性があるからである。 [0186] In a preferred embodiment, the gamma irradiation used in the present invention is performed in the range of 0.5 to 240 hours, more preferably 1 hour to 24 hours, and further preferably 3 to 8 hours. Although not wishing to be bound by theory, it is possible to shorten the irradiation time by increasing the dose per unit time using a strong γ-ray source, but if it is too strong, the tissue may be adversely affected. It is. In order to lengthen the time, a weak γ-ray source may be used. I don't want to be bound by theory, but if it's too weak, it will take a long time and the organization itself can be damaged by other factors.
[0187] 本発明において用いられる生体適合性高分子は、どのようなものでもよい。 1つの 実施形態では、使用される PEGの平均分子量は、 200と 6, 000との間であり、好ま しくは PEGの平均分子量は、 200と 2, 000との間である。本発明において用いられ る PEGは、このように種々の平均分子量を持ち得、平均分子量に応じて処理時間（ 浸す時間）を変更して所望の効果 (例えば、石灰化の抑制）を達成することができる。 1つの好ましい実施形態では、本発明において用いられる PEGの平均分子量は、 1 , 000と 200, 000との間である。另 IJの好ましい実施形態では、本発明において用い られる PEGの平均分子量は、 4, 000と 100, 000との間である。別の実施形態では 、本発明において用いられる PEGの平均分子量は、 8, 000と 50, 000との間である 。あるいは、 1つの好ましい実施形態では、 35, 000の平均分子量を有する PEGを 用いることが好ましい。  [0187] Any biocompatible polymer may be used in the present invention. In one embodiment, the average molecular weight of PEG used is between 200 and 6,000, and preferably the average molecular weight of PEG is between 200 and 2,000. The PEG used in the present invention can have various average molecular weights as described above, and a desired effect (for example, suppression of calcification) can be achieved by changing the treatment time (immersion time) according to the average molecular weight. Can do. In one preferred embodiment, the average molecular weight of the PEG used in the present invention is between 1,000 and 200,000. In a preferred embodiment of additional IJ, the average molecular weight of the PEG used in the present invention is between 4,000 and 100,000. In another embodiment, the average molecular weight of PEG used in the present invention is between 8,000 and 50,000. Alternatively, in one preferred embodiment, it is preferred to use PEG having an average molecular weight of 35,000.
[0188] 本発明の方法において、上記生体適合性高分子を組織に曝すは、生体由来組織 と生体適合性高分子とが相互作用するような条件であればどのような条件で行われ てもよいが、通常 0°Cと 42°Cとの間で行われ得、室温 (約 15°Cと約 25°Cとの間）で行 われてもよぐ 37°Cで行われてもよい。この工程は、 37°Cを超える温度で行われても よい。  [0188] In the method of the present invention, the biocompatible polymer is exposed to the tissue under any conditions as long as the living tissue and the biocompatible polymer interact with each other. Good, but can usually be performed between 0 ° C and 42 ° C and may be performed at room temperature (between about 15 ° C and about 25 ° C) or at 37 ° C . This step may be performed at a temperature above 37 ° C.
[0189] 本発明の方法において、 PEGなどの生体適合性高分子は、溶液中にどのような濃 度で存在してもよいが、好ましくは、 lgZml以上の濃度で存在することが有利である 。理論に束縛されることは望まないが、 γ線はラジカルスカベンジャーとしての機能が 期待されるからである。あるいは、 γ線照射の効率を考慮した場合、 PEG溶液の濃 度は、どのような濃度のものであってもよいが、約 1%〜約 50%、約 10%〜30%であ ることが好ましぐ例えば、約 20%のものが使用されるがそれらに限定されない。  [0189] In the method of the present invention, the biocompatible polymer such as PEG may be present in the solution at any concentration, but is preferably present at a concentration of lgZml or more. . I do not want to be bound by theory, but γ rays are expected to function as radical scavengers. Alternatively, considering the efficiency of gamma irradiation, the concentration of the PEG solution may be any concentration, but should be about 1% to about 50%, about 10% to 30%. For example, about 20% is used, but is not limited thereto.
[0190] ただし、生体適合性高分子の原料試薬として PEG (例えば、分子量 1, 000)を使 用する場合、常温で固体であることから、水溶液とすることが好ましくあり得る。本発 明の方法において、生体適合性高分子 (例えば、 PEG)は、どのような溶媒に溶解し ていてもよいが、好ましくは、水性媒体に溶解され、より好ましくは、生理食塩水、 PB s、または他の塩類などが含有される水溶液などに溶解され得る。生体適合性高分 子は、生体適合性を有すると同時に医薬品グレードに用いることが可能な高分子を 使用することが好ましぐ例えば、 PEG, (1)日本油脂製 SUNBRIGHT— MEHシリ 一ズの高純度メトキシ PEG等（http:ZZwww. nof. co. jp/business/dds/pr oductOl. html) , (2)重松貿易（株）製ポリエチレングリコール系ポリマー（http:Z Z www. shigematsu— bio . com/j/ products/ ecology/ poly/hight. htm 1)を挙げることができるがそれらに限定されない。あるいは、（1) γ線で架橋してゲル になる合成高分子のうち、弁や心膜の補強という今回の目的に最も適していると期待 される好ましいものとしては PEG、 PVA、ポリビュルピロリドン（PVP)、ポリアクリル酸 、ポリエチレンォキシド (PEO)、ポリフッ化ビ-リデン (PVdF)等を挙げることができる がそれらに限定されない。（2) γ線で架橋してゲルになる合成高分子のうち、弁や心 膜の補強という今回の目的に適しているか可能性があるものとしては、ポリエチレン、 ポリプロピレン、ポリウレタン、天然ゴム (イソプレン単位を含む）および合成ゴム、なら びに各種ポリアミノ酸（ εポリリジン、 γポリグルタミン酸等）および各種タンパク質 (ゼ ラチン、コラーゲン、エラスチン、フイブリンアルブミン、ケラチン、カゼイン、フイブロイ ン等）が挙げられるがそれらに限定されない。 （3)多糖類は、一般に γ線照射により 架橋よりも分解優位的に反応するようである。従って、フイラ一として上記の架橋性ポ リマーと混ぜて使うと 、う使 、方ができると考えられる。 [0190] However, when PEG (for example, molecular weight 1,000) is used as a raw material reagent for the biocompatible polymer, it may be preferably an aqueous solution because it is solid at room temperature. In the method of the present invention, the biocompatible polymer (for example, PEG) may be dissolved in any solvent, but is preferably dissolved in an aqueous medium, more preferably physiological saline, PB. s, or an aqueous solution containing other salts. For biocompatible polymers, it is preferable to use polymers that are biocompatible and can be used in pharmaceutical grades. For example, PEG, (1) SUNBRIGHT—MEH series manufactured by NOF Corporation High purity methoxy PEG, etc. (http: ZZwww. Nof. Co.jp/business/dds/productOl.html), (2) Polyethylene glycol polymer (http: ZZ www.shigematsu-bio.com) manufactured by Shigematsu Trading Co., Ltd. /j/products/ecology/poly/hight.htm 1) can be mentioned, but not limited to them. Alternatively, (1) Among the synthetic polymers that are cross-linked with γ-rays to become gels, preferred ones that are expected to be most suitable for the current purpose of reinforcing valves and pericardium are PEG, PVA, and polybulurpyrrolidone. (PVP), polyacrylic acid, polyethylene oxide (PEO), polyvinylidene fluoride (PVdF) and the like can be mentioned, but not limited thereto. (2) Among the synthetic polymers that are cross-linked by γ-rays into gels, those that may be suitable for this purpose of reinforcing valves and pericardium are polyethylene, polypropylene, polyurethane, natural rubber (isoprene). Unit) and synthetic rubber, as well as various polyamino acids ( ε polylysine, γ polyglutamic acid, etc.) and various proteins (gelatin, collagen, elastin, fibrin albumin, keratin, casein, fibroin, etc.). It is not limited. (3) Polysaccharides generally appear to react predominantly with degradation over crosslinking by gamma irradiation. Therefore, it is thought that it can be used more effectively when mixed with the above-mentioned crosslinkable polymer as a filler.
[0191] そのような生体適合性高分子は、例えば、 日本薬局方 (あるいは、対応する他の国 における薬局方）に掲載されている力、または厚生労働省（あるいは、対応する他の 国における監督官庁）が認可した高分子であり得る。  [0191] Such biocompatible polymers are, for example, those listed in the Japanese Pharmacopoeia (or corresponding pharmacopoeia in other countries), or the Ministry of Health, Labor and Welfare (or other applicable countries). It can be a polymer approved by the government.
[0192] 好ましくは、本発明の方法は、溶液に浸された組織を洗浄する工程、をさらに包含 する。この洗浄工程は、生理的に適合する液体 (例えば、生理食塩水、 PBS (リン酸 緩衝ィ匕生理食塩水）など)であれば、どのような液体を用いても行うことができる。好ま しい実施形態では、本発明の上記洗浄は、 PBSで行われる。洗浄溶液は、滅菌され ていることが好ましい。さらに好ましくは、洗浄溶液は、抗生物質 (例えば、ペニシリン 、セファロスポリン、ストレプトマイシン、テトラサイクリン、エリスロマイシン、ノ ンコマイ シン、シプロフロキサシン、リネズリドなど）を含む。洗浄は、どのような条件で行われ てもよいが、通常 0°Cと 42°Cとの間で行われ得、室温 (約 15°Cと約 25°Cとの間）で行 われてもよぐ 37°Cで行われてもよい。洗浄は、 37°Cを超える温度で行われてもよい 。本発明の方法において、洗浄する工程は、処理に使用した溶液 (例えば、生体適 合性高分子を含む溶液)が十分に除去されれば、どのような期間で行われてもよいが 、通常 0. 5日間〜 5日間行われ得る。好ましくは、洗浄する工程において、洗浄溶液 (例えば、 PBS)は、数回交換されてもよい。 [0192] Preferably, the method of the present invention further comprises the step of washing the tissue immersed in the solution. This washing step can be performed using any liquid that is physiologically compatible (eg, physiological saline, PBS (phosphate buffered saline), etc.). In a preferred embodiment, the washing of the present invention is performed with PBS. The cleaning solution is preferably sterilized. More preferably, the wash solution includes an antibiotic (eg, penicillin, cephalosporin, streptomycin, tetracycline, erythromycin, noncomomycin, ciprofloxacin, linezlid, etc.). Cleaning is done under any conditions However, it can usually be performed between 0 ° C and 42 ° C, and can be performed at room temperature (between about 15 ° C and about 25 ° C) and at 37 ° C. Also good. Washing may be performed at a temperature above 37 ° C. In the method of the present invention, the washing step may be performed for any period as long as the solution used in the treatment (for example, a solution containing a biocompatible polymer) is sufficiently removed. 0. Can be done for 5 days to 5 days. Preferably, in the washing step, the washing solution (eg, PBS) may be changed several times.
[0193] 本発明の方法において使用される組織は、意図される臨床適用に適合していれば 、どのような生物由来の糸且織であってもよい。従って、本発明の方法において用いら れる組織は、どの生物（例えば、脊椎動物、無脊椎動物）由来の組織でもあってもよ い。ヒトへの適用が意図される場合、本発明の方法において用いられる組織としては 、好ましくは、脊椎動物由来の組織が用いられ、より好ましくは、哺乳動物（例えば、 霊長類、齧歯類など）由来の組織が用いられる。ヒトへの適用が意図される場合、本 発明の方法において用いられる組織としては、さらに好ましくは、霊長類由来の組織 が用いられる。ヒトへの適用が意図される場合、さらに好ましくは、ブタまたはゥシ由来 の組織が用いられる。大きさがヒトに類似している力もである。ヒトへの適用が意図さ れる場合、最も好ましくはヒト由来の組織が用 、られる。  [0193] The tissue used in the method of the present invention may be any biological thread and weave as long as it is compatible with the intended clinical application. Therefore, the tissue used in the method of the present invention may be a tissue derived from any organism (for example, vertebrates and invertebrates). When intended for human application, the tissue used in the method of the present invention is preferably a vertebrate tissue, more preferably a mammal (eg, primate, rodent, etc.). Origin tissue is used. When application to humans is intended, the tissue used in the method of the present invention is more preferably a primate-derived tissue. If intended for human application, more preferably, tissue from pigs or sushi is used. It is also a force that is similar in size to humans. When intended for human use, most preferably human-derived tissue is used.
[0194] 本発明の方法において用いられる組織は、臨床適用が意図される組織であれば、 身体のどのような組織であってもよい。ある実施形態では、本発明の方法において用 いられる組織は、組織の物理的構造または物性が必要とされる組織であり得る。物理 的構造または物性を保持するためには、細胞外マトリクスなどの構造のみが必要であ り、細胞質成分または細胞膜成分などの細胞内の成分は必ずしも必要な!/、からであ る。また、必要に応じて、必要とされる細胞は、別途その生体由来組織に提供され得 るか、または移植された宿主カゝら内的に供給され得る。ある実施形態では、本発明の 方法において用いられる組織は、血管、血管様組織、心臓弁、心膜、硬膜、角膜お よび骨力も選択される器官に由来する組織であり得る。別の実施形態では、本発明 の方法において用いられる組織は、心臓血管系の組織であり得、例えば、血管、血 管様組織、心臓弁および心膜から選択される器官に由来し得る。  [0194] The tissue used in the method of the present invention may be any tissue of the body as long as it is intended for clinical application. In certain embodiments, the tissue used in the methods of the invention may be a tissue that requires the physical structure or physical properties of the tissue. In order to maintain the physical structure or physical properties, only the structure such as the extracellular matrix is necessary, and the intracellular components such as the cytoplasmic component or the cell membrane component are necessary! /. Further, if necessary, the required cells can be separately provided to the tissue derived from the living body, or can be supplied inside the transplanted host cell. In certain embodiments, the tissue used in the methods of the present invention may be a tissue derived from a blood vessel, blood vessel-like tissue, heart valve, pericardium, dura mater, cornea and bone strength. In another embodiment, the tissue used in the methods of the invention can be cardiovascular tissue, such as from an organ selected from blood vessels, blood vessel-like tissue, heart valves and pericardium.
[0195] 1つの実施形態において、本発明は、さらに化学的処理を含んでいてもよぐ例え ば、二官能性分子架橋剤（例えば、ダルタルアルデヒドまたはその誘導体）による処 理であり得る。二官能性分子架橋剤による処理は、 ECMもしくは組織中の細胞を含 むタンパク質成分などをィ匕学的に架橋することにより物理的強度を増カロさせることを 目的とする。従って、この目的に使用するのであれば、二官能性分子架橋剤である 限りどのようなものでも使用することができる。そのような二官能性分子架橋剤のなか で実際に組織の固定 (弁グラフト）に使用されているものは、例えば、シアンイミド (cy animide)、 1—ェチル 3— (3 ジメチルァミノプロピル）カルボジイミドヒドロクロリド (EDC)、エポキシ（ポリ（グリシジノレエーテノレ） )または、 2, 6-ビス（4 アジドベンジ リデン）ー4ーメチルシクロへキサノン（BAMC)、 4, 4，ジアジドジフエ-ルエーテル、 4, 4'ジアジドジフ ニルスルホン、 4, 4'ジアジドジフ ニルアセトン、 4, 4'ジアジド ジフエ-ルメタン、 1, 4 ビス（α ジァゾベンジル）および 4— [p アジドサリチアミ ド]ブチルァミンが挙げられるがそれらに限定されない（参考として、 Biomaterials (2 000) 21 : 2215— 2231を参照のこと）。 [0195] In one embodiment, the present invention may further include chemical treatment. For example, it can be a treatment with a bifunctional molecular crosslinking agent (eg, dartalaldehyde or a derivative thereof). The purpose of the treatment with the bifunctional molecular crosslinking agent is to increase the physical strength by chemically crosslinking ECM or protein components including cells in the tissue. Therefore, as long as it is used for this purpose, any bifunctional molecular crosslinking agent can be used. Among such bifunctional molecular crosslinkers, those actually used for tissue fixation (valve grafting) are, for example, cyanimide, 1-ethyl 3- (3 dimethylaminopropyl) carbodiimide. Hydrochloride (EDC), epoxy (poly (glycidinoreetherenole)) or 2,6-bis (4 azidobenzylidene) -4-methylcyclohexanone (BAMC), 4, 4, diazide diphenyl ether, 4, 4 ' These include, but are not limited to, diazidodiphenylsulfone, 4,4'diazidodiphenylacetone, 4,4'diazidediphenylmethane, 1,4bis (α diazobenzyl) and 4— [p azidosalithiomide] butyramine (see Biomaterials ( 2 000) 21: 2215—2231).
[0196] 別の実施形態において、この化学的処理は、 DNaselのような DNaseなどのヌクレ ァーゼでの処理を包含し得る。そのような DNaseでの処理により、移植に好ましくな い DNA成分をさらに除去することができることから、この DNaseでの処理は、好まし い。そのような DNaseはどのような DNaseでもよいが、好ましくは DNaselであり得る。 DNase処理により荷電性高分子物質である DNAを除去することができる。 DNAは 免疫反応を誘起する可能性があることから、 DNAをさらに除去することによってさら なる利点を提供することができる。  [0196] In another embodiment, the chemical treatment may include treatment with a nuclease such as DNase, such as DNasel. Since treatment with such DNase can further remove DNA components that are undesirable for transplantation, this treatment with DNase is preferred. Such DNase may be any DNase, but may preferably be DNasel. DNA, which is a charged polymer substance, can be removed by DNase treatment. Since DNA can elicit an immune response, further removal of DNA can provide additional benefits.
[0197] ヌクレアーゼ（例えば、 DNase)での処理は、どのような条件で行ってもよいが、カロ 圧条件、攪拌条件などを組み合わせてもよい。濃度としては、例えば、 lUZml〜10 OUZmlなどが意図され、例示としては、例えば 50〜75UZmlが挙げられるがそれ らに限定されない。ヌクレアーゼ処理は、例えば、 0. 5日〜5日程度行うことができ、 このうち、最初の数時間（例えば、 1〜24時間）は、加圧条件（例えば、 100〜1000k Pa (例示として 500kPa) )で行うことが考えられる。その後、攪拌 (例えば、 100〜50 ORPM、好ましくは 100〜150RPM)条件で行うこともできる。ヌクレアーゼ処理の前 後は、上述の洗浄溶液を用いて保存することが好ま 、。 [0198] 別の好ましい実施形態において、本発明の生体由来組織の製造法では、細胞を 播種する工程をさらに包含していてもよいが、これに限定されない。播種する細胞に ついては、本明細書において上述されるように、情況に応じて当業者が適宜選択す ることがでさる。 [0197] The treatment with a nuclease (for example, DNase) may be carried out under any conditions, but may be combined with a caloric pressure condition, a stirring condition, and the like. Examples of the concentration include lUZml to 10 OUZml, and examples include, but are not limited to, 50 to 75 UZml. The nuclease treatment can be performed, for example, for about 0.5 to 5 days. Among these, the first few hours (for example, 1 to 24 hours) are applied under pressure conditions (for example, 100 to 1000 kPa (for example, 500 kPa). ))). Then, it can also carry out on stirring (for example, 100-50 ORPM, Preferably it is 100-150 RPM) conditions. Before and after nuclease treatment, it is preferable to store using the above washing solution. [0198] In another preferred embodiment, the method for producing a biological tissue of the present invention may further include a step of seeding cells, but is not limited thereto. The cells to be seeded can be appropriately selected by those skilled in the art depending on the situation, as described above in the present specification.
[0199] 本発明はまた、本発明の方法によって得られる生体由来組織を提供する。この生 体由来組織は、好ましくは、上述の細胞残存率および Zまたは組織損傷率および Z または組織強度を有し得る。本発明のラジカル反応を用いた処理方法が提供される 前は、上述の細胞残存率および Zまたは組織損傷率および Zまたは組織強度を有 する生体由来組織を提供することはできな力つたことから、本発明の方法は、従来の 方法では提供することができな力つた石灰化の抑制などの特性を有する生体由来組 織を提供するという全く新規の物質を提供するものであり、予想外の利点を提供する  [0199] The present invention also provides a biological tissue obtained by the method of the present invention. This organism-derived tissue may preferably have the above-mentioned cell survival rate and Z or tissue damage rate and Z or tissue strength. Before the treatment method using the radical reaction of the present invention was provided, it was impossible to provide a living tissue having the above-mentioned cell survival rate and Z or tissue damage rate and Z or tissue strength. The method of the present invention provides a completely new substance that provides a tissue derived from a living body having characteristics such as the suppression of forced calcification that cannot be provided by conventional methods. Provide benefits
[0200] 別の局面において、本発明は、組織の再生方法であって、 a)生体適合性高分子を 含み、 γ線照射を受けたことを特徴とする生体由来組織を、生体内に提供する工程; および b)該生体内で組織再生が生じるに十分な時間インキュベートする工程、を包 含する。必要に応じて、この細胞の分化を誘導する生理活性物質を提供する工程お よび Zまたはこの生体由来組織に細胞を提供する工程を含んでいてもよぐ生体由 来組織に細胞を提供しなくてもよい。好ましくは、生理活性物質は、目的の組織の維 持または分ィ匕に必要なものが使用される。生理活性物質は、生体内に由来するもの であっても、生体外に由来するものであってもよい。生体内に由来するものの場合は 、実質的には何ら操作をすることなぐ単に移植後一定時間が経過することによって、 適切な生理活性物質への暴露がなされることが理解される。生理活性物質としては、 例えば、 HGF、 VEGF、 FGF、 IGF、 PDGFおよび EGFが挙げられるがそれらに限 定されない。 [0200] In another aspect, the present invention provides a tissue regeneration method comprising: a) a biological tissue containing a biocompatible polymer and receiving γ-irradiation in vivo. And b) incubating for a time sufficient for tissue regeneration to occur in the living body. If necessary, do not provide cells to living tissue, which may include a step of providing a physiologically active substance that induces differentiation of the cells and a step of supplying cells to Z or this living tissue. May be. Preferably, as the physiologically active substance, those necessary for maintaining or separating the target tissue are used. The physiologically active substance may be derived from in vivo or derived from outside the living body. In the case of a substance derived from a living body, it is understood that exposure to an appropriate physiologically active substance is made by simply passing a certain time after transplantation without performing any manipulation. Examples of the physiologically active substance include, but are not limited to, HGF, VEGF, FGF, IGF, PDGF, and EGF.
[0201] 好ましくは、この細胞は、血管細胞または血管様細胞であり得る。より好ましくは、こ の細胞は、レシピエント由来であり得る。あるいは、好ましくは、この組織は、血管、血 管様組織、心臓弁、心膜、硬膜、角膜および骨力 なる群より選択されるものの組織 であり得る。好ましい実施形態では、この組織とこの細胞とは、同じ宿主由来であり得 る。別の実施形態では、この組織とこの細胞とは、同種異系宿主由来であり得る。別 の実施形態では、この組織とこの細胞とは、異種宿主由来であり得る。レシピエントと 細胞とが同種異系由来または異種由来である場合、拒絶反応が考えられることから、 レシピエントと細胞とは同じ宿主由来であることが好ましいが、拒絶反応が問題でな い場合同種異系由来または異種由来であってもよい。また、拒絶反応を起こすものも 必要に応じて拒絶反応を解消する処置を行うことにより利用することができる。拒絶 反応を回避する手順は当該分野において公知であり、例えば、新外科学体系、心臓 移植'肺移植 技術的、倫理的整備カゝら実施に向けて (改訂第 3版)に記載されてい る。そのような方法としては、例えば、免疫抑制剤、ステロイド剤の使用などの方法が 挙げられる。拒絶反応を予防する免疫抑制剤は、現在、「シクロスポリン」（サンデイミ ユン Zネオ一ラル）、「タクロリムス」（プログラフ）、「ァザチォプリン」（イムラン）、「ステ ロイドホルモン」（プレドニン、メチルプレドニン）、 「T細胞抗体」（OKT3、 ATGなど） があり、予防的免疫抑制療法として世界の多くの施設で行われている方法は、「シク ロスポリン、ァザチォプリンおよびステロイドホルモン」の 3剤併用である。本発明は、 石灰化を抑制したという点において顕著な効果を奏することから、免疫抑制剤は、本 発明の医薬の適用において必要ではないが、投与する場合は同時期に投与される ことが望ましいが、必ずしも必要ではない。従って、免疫抑制剤を利用する場合は、 免疫抑制効果が達成される限り免疫抑制剤は再生方法を行う前または後にも投与さ れ得る。 [0201] Preferably, the cell may be a vascular cell or a blood vessel-like cell. More preferably, the cell may be derived from the recipient. Alternatively, preferably the tissue may be a tissue selected from the group consisting of blood vessels, vascular-like tissue, heart valves, pericardium, dura mater, cornea and bone strength. In a preferred embodiment, the tissue and the cell can be from the same host. The In another embodiment, the tissue and the cell can be from an allogeneic host. In another embodiment, the tissue and the cell can be from a heterologous host. If the recipient and the cell are allogeneic or xenogeneic, a rejection may be considered, so the recipient and the cell are preferably from the same host, but if the rejection is not a problem, the same species It may be of different origin or different origin. In addition, those that cause rejection can be used by performing treatment to eliminate rejection as necessary. Procedures for avoiding rejection are well known in the art and are described, for example, in the New Surgery System, Heart Transplantation, Lung Transplantation Technical and Ethical Preparations for Implementation (Revised 3rd Edition). . Examples of such methods include methods such as the use of immunosuppressants and steroids. Immunosuppressants that prevent rejection are currently "cyclosporine" (Sandemiyun Yoon Z Neoral), "Tacrolimus" (Prograf), "Azathioprine" (Imran), "Steroid Hormone" (Predonin, Methylpredonin), There are “T-cell antibodies” (OKT3, ATG, etc.), and the method used in many facilities around the world as a preventive immunosuppressive therapy is a combination of three drugs, “cyclosporine, azathioprine and steroid hormones”. Since the present invention has a remarkable effect in that it suppresses calcification, the immunosuppressive agent is not necessary in the application of the medicament of the present invention, but it is desirable that it be administered at the same time when administered. However, it is not always necessary. Therefore, when an immunosuppressive agent is used, the immunosuppressive agent can be administered before or after performing the regeneration method as long as the immunosuppressive effect is achieved.
別の局面において、本発明は、糸且織グラフトを生産する方法であって、 Α)生体適 合性高分子を含む生体由来組織を生体内に提供する工程; Β)該生体由来組織に 該生体の自己細胞を侵入させる工程;および C)該細胞の分ィ匕が生じるに十分な時 間インキュベートする工程、を包含する。この場合、本発明の生体由来組織は、細胞 をさらに有していても、有していなくてもよい。そのような細胞は、自己由来、同種由 来、同種異系由来、異種由来のものであり得る。好ましくは、この細胞は、血管細胞ま たは血管様細胞であり得る。より好ましくは、この細胞は、レシピエント由来であり得る 。好ましくは、この組織は、血管、血管様組織、心臓弁、心膜、硬膜、角膜および骨か らなる群より選択されるものの組織であり得る。好ましい実施形態では、この組織とこ の細胞とは、同じ宿主由来であり得る。別の実施形態では、この組織とこの細胞とは、 同種異系宿主由来であり得る。別の実施形態では、この組織とこの細胞とは、異種宿 主由来であり得る。レシピエントと細胞とが同種異系由来または異種由来である場合 、拒絶反応が考えられることから、レシピエントと細胞とは同じ宿主由来であることが 好ましいが、拒絶反応が問題でない場合同種異系由来または異種由来であってもよ い。また、拒絶反応を起こすものも必要に応じて拒絶反応を解消する処置を行うこと により利用することができる。拒絶反応を解消する処置は、本明細書において詳述さ れている。 In another aspect, the present invention relates to a method for producing a yarn and woven graft, comprising: i) a step of providing a living tissue containing a biocompatible polymer in vivo; And a step of incubating for a period of time sufficient for the differentiation of the cells to occur. In this case, the living tissue of the present invention may or may not further have cells. Such cells can be autologous, allogeneic, allogeneic or xenogeneic. Preferably, the cell can be a vascular cell or a blood vessel-like cell. More preferably, the cell may be derived from the recipient. Preferably, the tissue may be that of a tissue selected from the group consisting of blood vessels, blood vessel-like tissue, heart valves, pericardium, dura mater, cornea and bone. In a preferred embodiment, this organization The cells may be from the same host. In another embodiment, the tissue and the cell can be from an allogeneic host. In another embodiment, the tissue and the cell can be from a heterologous host. If the recipient and the cell are allogeneic or xenogeneic, rejection may be possible, so the recipient and the cell are preferably from the same host, but if the rejection is not a problem allogeneic It can be of different origin or different origin. In addition, those that cause rejection can be used by performing treatment to eliminate rejection as necessary. Procedures for resolving rejection are detailed herein.
[0203] 好ま 、実施形態にぉ 、て、本発明の組織グラフトを生産する方法は、 D)上記細 胞の分化を誘導する生理活性物質を提供する工程、をさらに包含し得る。好ましくは 、この生理活性物質は、造血活性を有するサイト力インであり得る。組織グラフトを製 造する場合、このサイト力インは、 HGF、 VEGF、 FGFなどであり得る。生理活性物 質は、自己由来であっても、外来由来であってもよい。生理活性物質が自己由来の 場合は、単に移植後一定時間時間を経過させることによって目的が達成されることに 留意すべきである。  [0203] Preferably, according to the embodiment, the method for producing the tissue graft of the present invention may further include the step of D) providing a physiologically active substance that induces differentiation of the cells. Preferably, the physiologically active substance can be a cytodynamic force having hematopoietic activity. When producing a tissue graft, this site force-in can be HGF, VEGF, FGF, and the like. The physiologically active substance may be autologous or exogenous. It should be noted that if the bioactive substance is autologous, the goal is achieved simply by passing a certain amount of time after transplantation.
[0204] 別の局面において、本発明は、本発明の方法によって生産された、生来由来組織 または組織グラフトを提供する。このような生体由来組織または組織グラフトは、組織 強度および石灰化抑制率の点で従来存在しなカゝつた特徴を有する。  [0204] In another aspect, the present invention provides a native tissue or tissue graft produced by the method of the present invention. Such a biological tissue or tissue graft has unique features that are not present in terms of tissue strength and calcification inhibition rate.
[0205] 他の局面において、本発明は、組織または臓器移植を必要とする力または該危険 にある被験体の処置または予防の方法であって、 A)生体適合性高分子を含み、 γ 線照射を受けたことを特徴とする生体由来組織、または該生体由来組織を含む組織 グラフトを提供する工程;および Β)該生体由来組織または組織グラフトを被験体に移 植する工程、を包含する、方法を提供する。この生体由来組織は、必要に応じてさら に細胞を含む。この細胞は、自己由来であっても、同種異系由来であってもよい。好 ましくは、この組織は、血管、血管様組織、心臓弁、心膜、硬膜、角膜および骨からな る群より選択されるものの組織であり得る。好ましい実施形態では、この組織は、被験 体由来であり得る。別の実施形態では、この組織は、被験体と同種異系の宿主由来 であり得る。別の実施形態では、この組織は、被験体とは異種の宿主由来であり得る 。本発明のこの治療 z予防方法では、移植に耐え得る程度の組織損傷率に抑えら れ、かつ、石灰化抑制が十分に行われた生体由来組織が使用されることから、拒絶 反応は生じない。ただし、力りに拒絶反応が生じた場合、またはレシピエント由来以 外の細胞が用いられた場合、拒絶反応を起こしたときに、必要に応じて拒絶反応を 解消する処置を行うことができる。拒絶反応を解消する処置は、本明細書において詳 述されている。 1つの実施形態では、本発明の処置または予防する方法において使 用される組織は、被験体由来であってもよい。別の実施形態では、本発明の処置ま たは予防する方法において使用される組織は、どの生物（例えば、脊椎動物、無脊 椎動物）由来の組織でもよい。好ましくは、ヒトが処置または予防される場合、脊椎動 物由来の組織が用いられ、ヒトが処置または予防される場合、より好ましくは、哺乳動 物 (例えば、霊長類、齧歯類など）由来の組織が用いられる。ヒトが処置または予防さ れる場合、さらに好ましくは、霊長類由来の組織が用いられる。ヒトが処置または予防 される場合、別の好ましい実施形態では、ブタまたはゥシ由来の組織が用いられる。 ブタまたはゥシが好ましいのは、ヒトに類似した大きさを有するからである。ヒトが処置 または予防される場合、最も好ましくはヒト由来の組織が用いられ得る。本発明の方 法において、 B)工程は、癒着を防ぐ必要のある部位において実施されることもできる [0205] In another aspect, the present invention provides a method for treating or preventing a force requiring tissue or organ transplantation or a subject at risk thereof, comprising A) a biocompatible polymer, and gamma rays Providing a biological tissue characterized by being irradiated, or a tissue graft containing the biological tissue; and i) transferring the biological tissue or tissue graft to a subject. Provide a method. This biological tissue further contains cells as necessary. The cells may be autologous or allogeneic. Preferably, the tissue may be that of a tissue selected from the group consisting of blood vessels, blood vessel-like tissue, heart valves, pericardium, dura mater, cornea and bone. In preferred embodiments, the tissue can be from a subject. In another embodiment, the tissue can be from a host allogeneic to the subject. In another embodiment, the tissue can be from a host heterologous to the subject. . In this treatment z prevention method of the present invention, since a tissue-derived tissue that is suppressed to a tissue damage rate that can withstand transplantation and is sufficiently suppressed in calcification is used, no rejection reaction occurs. . However, when rejection occurs in the force or when cells other than those derived from the recipient are used, when rejection occurs, treatment can be performed to eliminate the rejection as necessary. The procedure for resolving rejection is described in detail herein. In one embodiment, the tissue used in the method of treating or preventing of the present invention may be derived from a subject. In another embodiment, the tissue used in the method of treatment or prevention of the present invention may be tissue from any organism (eg, vertebrate, invertebrate). Preferably, tissue from spinal animals is used when humans are treated or prevented, and more preferably from mammals (eg, primates, rodents, etc.) when humans are treated or prevented Organization is used. When humans are treated or prevented, more preferably primate-derived tissue is used. When humans are treated or prevented, in another preferred embodiment, tissues from pigs or sushi are used. Pigs or sushi are preferred because they have a size similar to humans. When humans are treated or prevented, most preferably human-derived tissue can be used. In the method of the present invention, step B) can also be performed at a site where adhesion needs to be prevented.
[0206] 別の局面において、本発明は、臓器移植のための医薬を提供する。この医薬は、 A )生体適合性高分子を含み、 γ線照射を受けたことを特徴とする生体由来組織、また は該生体由来組織を含む組織グラフト、を含む。 [0206] In another aspect, the present invention provides a medicament for organ transplantation. This medicine includes A) a biological tissue containing a biocompatible polymer and receiving γ-ray irradiation, or a tissue graft containing the biological tissue.
ある実施形態では、本発明の医薬は、血管、血管様組織、心臓弁、心膜、硬膜、角 膜および骨力 選択される器官に由来する組織を含む。別の実施形態では、本発明 の医薬は、心臓血管系の組織を含み得、例えば、血管、血管様組織、心臓弁および 心膜から選択される器官に由来するものを含み得る。  In certain embodiments, the medicaments of the present invention include blood vessels, blood vessel-like tissues, heart valves, pericardia, dura mater, corneas and bone strength tissue derived from selected organs. In another embodiment, the medicament of the present invention may comprise cardiovascular tissue, for example, derived from an organ selected from blood vessels, blood vessel-like tissue, heart valves and pericardium.
[0207] 本発明の医薬は、意図される臨床適用に適合していれば、どのような生物由来の 組織を含んでいてもよい。好ましくは、適用される国の監督官庁が認可した材料を含 み得る。従って、本発明の医薬は、どの生物 (例えば、脊椎動物、無脊椎動物）由来 の組織を含んで 、てもよ 、。本発明の医薬にぉ 、てヒトへの適用が意図される場合、 好ましくは、脊椎動物由来の組織が用いられ、より好ましくは、哺乳動物（例えば、霊 長類、齧歯類など）由来の組織が用いられる。本発明の医薬においてヒトへの適用が 意図される場合、さら〖こ好ましくは、霊長類由来の組織が用いられる。本発明の医薬 においてヒトへの適用が意図される場合、さらに好ましくは、ブタまたはゥシ由来の組 織が用いられる。大きさがヒトに類似しているからである。本発明の医薬においてヒト への適用が意図される場合、最も好ましくはヒト由来の組織が用いられる。ただし、ヒト 由来の組織を使用する場合は、倫理規定 ·問題をクリアしていることが必要とされ得る [0207] The medicament of the present invention may contain any biological tissue as long as it is compatible with the intended clinical application. Preferably, it may contain materials approved by the applicable national supervisory authority. Therefore, the medicament of the present invention may include tissues derived from any organism (for example, vertebrates and invertebrates). When the medicament of the present invention is intended to be applied to humans, Preferably, tissues derived from vertebrates are used, and more preferably, tissues derived from mammals (eg, primates, rodents, etc.) are used. When the medicament of the present invention is intended to be applied to humans, primate-derived tissue is preferably used. When the pharmaceutical of the present invention is intended to be applied to humans, more preferably, a tissue derived from pig or ushi is used. This is because the size is similar to humans. When the medicament of the present invention is intended to be applied to humans, human-derived tissue is most preferably used. However, when using human-derived tissue, it may be necessary to clear ethical rules and issues.
[0208] 本発明の医薬、組織グラフトおよび生体由来組織はまた、さらに生体親和性材料を 含み得る。この生体親和性材料は、例えば、シリコーン、コラーゲン、ゼラチン、グリコ ール酸 '乳酸の共重合体、エチレンビニル酢酸共重合体、ポリウレタン、ポリエチレン 、ポリテトラフルォロエチレン、ポリプロピレン、ポリアタリレート、ポリメタタリレートから なる群より選択される少なくとも 1つを含み得る。成型が容易であることからシリコーン が好ましい。生分解性高分子の例としては、コラーゲン、ゼラチン、 a—ヒロドキシ力 ルボン酸類（例えば、グリコール酸、乳酸、ヒドロキシ酪酸など）、ヒドロキシジカルボン 酸類 (例えば、リンゴ酸など）およびヒドロキシトリカルボン酸 (例えば、クェン酸など） からなる群より選択される 1種以上から無触媒脱水重縮合により合成された重合体、 共重合体またはこれらの混合物、ポリ a シァノアクリル酸エステル、ポリアミノ酸（ 例えば、ポリ γ—べンジルー L グルタミン酸など）、無水マレイン酸系共重合体（ 例えば、スチレン マレイン酸共重合体など）のポリ酸無水物などが挙げられる。重 合の形式は、ランダム、ブロック、グラフトのいずれでもよく、 aーヒドロキシカルボン酸 類、ヒドロキシジカルボン酸類、ヒドロキシトリカルボン酸類が分子内に光学活性中心 を有する場合、 D 体、 L 体、 DL 体のいずれでも用いることが可能である。ある 実施形態では、グリコール酸 ·乳酸の共重合体が使用され得る。 [0208] The medicament, tissue graft and biological tissue of the present invention may further comprise a biocompatible material. This biocompatible material is, for example, silicone, collagen, gelatin, glycolic acid 'lactic acid copolymer, ethylene vinyl acetate copolymer, polyurethane, polyethylene, polytetrafluoroethylene, polypropylene, polyacrylate, It may comprise at least one selected from the group consisting of polymetatalates. Silicone is preferred because it is easy to mold. Examples of biodegradable polymers include collagen, gelatin, a-hydroxyluronic acids (eg, glycolic acid, lactic acid, hydroxybutyric acid, etc.), hydroxydicarboxylic acids (eg, malic acid), and hydroxytricarboxylic acids (eg, A polymer, a copolymer or a mixture thereof synthesized by non-catalytic dehydration polycondensation from one or more selected from the group consisting of cuenic acid, etc., poly-a cyanoacrylate, poly-amino acid (for example, poly γ-base) Njiru L glutamic acid, etc.), and polyanhydrides of maleic anhydride copolymers (eg, styrene maleic acid copolymers). The form of polymerization may be random, block, or graft. When a-hydroxycarboxylic acids, hydroxydicarboxylic acids, and hydroxytricarboxylic acids have an optically active center in the molecule, they are of D-form, L-form, and DL-form. Either can be used. In some embodiments, a glycolic acid / lactic acid copolymer may be used.
[0209] 本発明の医薬、組織グラフトおよび生体由来組織は、さらに他の薬剤を含み得る。  [0209] The medicament, tissue graft and biological tissue of the present invention may further contain other drugs.
そのような薬剤は、薬学分野において公知の任意の薬剤であり得る。当然、本発明 の医薬、組織グラフトおよび生体由来組織は、 2種類以上の他の薬剤を含んでいて もよい。そのような薬剤としては、例えば、 日本薬局方、米国薬局方、他の国の薬局 方などの最新版において掲載されているものなどが挙げられる。そのような薬剤は、 好ましくは、生物の器官に対して効果を有するものであり得る。そのような薬剤として は、例えば、血栓溶解剤、血管拡張剤、組織賦活化剤が挙げられる。本発明の医薬 、組織グラフトおよび生体由来組織において含まれる生理活性物質、他の薬剤およ び細胞などの量は、使用目的、対象疾患 (種類、重篤度など）、患者の年齢、体重、 性別、既往歴などを考慮して、当業者が容易に決定することができる。 Such an agent can be any agent known in the pharmaceutical arts. Of course, the medicament, tissue graft and biological tissue of the present invention may contain two or more kinds of other drugs. Examples of such drugs include Japanese pharmacopoeia, US pharmacopoeia, and pharmacies in other countries. The ones listed in the latest version of the Such an agent may preferably have an effect on the organ of the organism. Examples of such agents include thrombolytic agents, vasodilators, and tissue activators. The amount of physiologically active substances, other drugs and cells contained in the medicament, tissue graft and biological tissue of the present invention depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, It can be easily determined by those skilled in the art in consideration of gender, medical history, and the like.
[0210] 別の局面において、本発明は、臓器移植のための医薬を製造するための、本発明 の生体適合性高分子を含み、 y線照射を受けたことを特徴とする生体由来組織また は本発明の生体適合性高分子を含み、 y線照射を受けたことを特徴とする生体由来 組織を含む組織グラフトの、使用に関する。ある実施形態では、本発明の使用にお いて、血管、血管様組織、心臓弁、心膜、硬膜、角膜および骨から選択される器官に 由来する組織が使用され得る。別の実施形態では、本発明の使用において、心臓血 管系の組織が使用され得、例えば、血管、血管様組織、心臓弁および心膜から選択 される器官に由来するものを使用され得る。  [0210] In another aspect, the present invention provides a biological tissue or a biotissue containing the biocompatible polymer of the present invention for producing a medicament for organ transplantation, which has been subjected to y-ray irradiation. Relates to the use of a tissue graft comprising a biological tissue characterized in that it comprises a biocompatible polymer of the invention and has been subjected to y-ray irradiation. In certain embodiments, tissue derived from an organ selected from blood vessels, blood vessel-like tissue, heart valves, pericardium, dura mater, cornea and bone may be used in the use of the present invention. In another embodiment, in the use of the present invention, cardiovascular tissue can be used, for example, derived from an organ selected from blood vessels, blood vessel-like tissues, heart valves and pericardium.
[0211] 本発明の使用において、意図される臨床適用に適合していれば、どのような生物由 来の組織でも使用することができる。好ましくは、適用される国の監督官庁が認可し た材料が使用され得る。従って、本発明の使用において、どの生物 (例えば、脊椎動 物、無脊椎動物）由来の組織が使用されてもよい。本発明の使用においてヒトへの適 用が意図される場合、好ましくは、脊椎動物由来の組織が用いられ、より好ましくは、 哺乳動物 (例えば、霊長類、齧歯類など）由来の組織が用いられる。本発明の使用に おいてヒトへの適用が意図される場合、さらに好ましくは、霊長類由来の組織が用い られる。本発明の医薬においてヒトへの適用が意図される場合、さらに好ましくは、ブ タまたはゥシ由来の組織が用いられる。大きさがヒトに類似しているからである。本発 明の使用においてヒトへの適用が意図される場合、最も好ましくはヒト由来の組織が 用いられる。ただし、ヒト由来の組織を使用する場合は、倫理規定'問題をクリアして いることが必要とされ得る。  [0211] In the use of the present invention, any biological tissue can be used as long as it is suitable for the intended clinical application. Preferably, materials approved by the applicable national regulatory authority may be used. Accordingly, tissue from any organism (eg, vertebrate, invertebrate) may be used in the use of the present invention. When intended for human use in the use of the present invention, vertebrate tissue is preferably used, and more preferably, mammal (eg, primate, rodent, etc.) tissue is used. It is done. When the use of the present invention is intended to be applied to humans, primate-derived tissue is more preferably used. When the pharmaceutical of the present invention is intended to be applied to humans, more preferably, a tissue derived from butterfly or sushi is used. This is because the size is similar to humans. When the use of the present invention is intended for human application, human-derived tissue is most preferably used. However, when using human-derived tissue, it may be necessary to clear the 'Code of Ethics' issue.
[0212] 本発明の生体由来組織、グラフトおよび医薬の使用は、通常は医師の監督のもと で行われるが、その国の監督官庁および法律が許容する場合は、医師の監督なしに 使用することができる。 [0212] The use of biological tissue, grafts and medicaments of the present invention is usually under the supervision of a physician, but without the supervision of a physician, if the national regulatory authority and law allow. Can be used.
[0213] 本発明の処置または予防方法において使用される生体由来糸且織、グラフトおよび 医薬の量は、使用目的、対象疾患 (種類、重篤度など)、被験体の年齢、体重、性別 、既往歴、生理活性物質の形態または種類、組織の形態または種類などを考慮して 、当業者が容易に決定することができる。  [0213] The amount of the biologically-derived yarn and tissue, graft, and medicament used in the treatment or prevention method of the present invention is the use purpose, target disease (type, severity, etc.), subject's age, weight, sex, A person skilled in the art can easily determine the past history, the form or type of the physiologically active substance, the form or type of the tissue, and the like.
[0214] 本発明の方法を被験体 (または患者）に対して施す頻度もまた、 1回あたりの生体由 来組織、グラフトおよび医薬の使用量、使用目的、対象疾患 (種類、重篤度など)、患 者の年齢、体重、性別、既往歴、および治療経過などを考慮して、当業者が容易に 決定することができる。頻度としては、例えば、毎日〜数ケ月に 1回（例えば、 1週間に 1回ー1ヶ月に 1回）の投与が挙げられる。 1週間〜 1ヶ月に 1回の投与を、経過を見 ながら施すことが好ましい。  [0214] The frequency with which the method of the present invention is applied to a subject (or patient) also depends on the amount of biologically derived tissue, graft and medicine used per time, purpose of use, target disease (type, severity, etc.) ), And can be easily determined by those skilled in the art in consideration of the patient's age, weight, sex, medical history, course of treatment, and the like. Examples of the frequency include administration every day to once every several months (for example, once a week to once a month). It is preferable to administer once a week to once a month while monitoring the course.
[0215] 本明細書では、必要に応じて当該分野で周知の分子生物学的手法、生化学的手 法、微生物学的手法が用いられる。そのような方法は、例えば、 Ausubel F. A.ら 編 (1988)、 Current Protocols in Molecular Biology、 Wiley、 NewYork；、 NY; Sambrook Jら (1987) Molecular Cloning : A Laboratory Manual, 2n d Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY、別冊実験医学「遺伝子導入 &発現解析実験法」羊土社、 1997などに記載され る。  [0215] In the present specification, molecular biological techniques, biochemical techniques, and microbiological techniques well known in the art are used as necessary. Such methods are described, for example, by Ausubel FA et al. (1988), Current Protocols in Molecular Biology, Wiley, New York ;, NY; Sambrook J et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor. It is described in Laboratory Press, Cold Spring Harbor, NY, a separate volume of experimental medicine, “Gene Transfer & Expression Analysis Experiment Method”, Yodosha, 1997, etc.
[0216] 別の局面において、本発明の生体由来組織、グラフトおよび Zまたは医薬は、この 生体由来組織、グラフトおよび Zまたは医薬を投与する指針を与える指示書を備え るキットという形態で提供され得る。ここで、上記指示書は、生体由来組織、グラフトお よび Zまたは医薬の適切な投与方法を指示する文言が記載されている。この指示書 は、本発明が実施される国の監督官庁 (例えば、 日本であれば厚生労働省、米国で あれば食品医薬品局 (FDA)など)が規定した様式に従って作成され、その監督官 庁により承認を受けた旨が明記される。指示書は、いわゆる添付文書 (package ins ert)であり、通常は紙媒体で提供されるが、それに限定されず、例えば、電子媒体（ 例えば、インターネットで提供されるホームページ、電子メール)のような形態でも提 供され得る。 [0217] 本発明の生体由来組織、組織グラフトおよび医薬は、当該分野において周知の技 術を用いて移植することができる (外科手術に関しては、標準外科学第 9版 (医学書 院)基本的外科手術手技 (P41— p66)、心臓 (p349— p398)、血管 (p399— 428) などを参照のこと)。本発明の生体由来組織は、血管吻合、パッチ閉鎖術、人工血管 置換術、人工弁置換術などに使用され得る。したがって、当業者は、処置する状況 に応じて、本明細書の開示に従って、本発明の生体由来組織、組織グラフトおよび 医薬を、適宜適用することができる。 [0216] In another aspect, the biological tissue, graft and Z or medicament of the present invention can be provided in the form of a kit comprising instructions for administering the biological tissue, graft and Z or medicament. . Here, the above-mentioned instruction manual describes a word indicating an appropriate administration method of a tissue derived from a living body, a graft, and Z or a medicine. This instruction is prepared in accordance with the format prescribed by the national supervisory authority (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States) in the United States. It will be clearly stated that it has been approved. Instructions are so-called package inserts (package inserts), which are usually provided in paper media, but are not limited thereto, such as electronic media (for example, homepages and emails provided on the Internet). It can also be provided in form. [0217] The living tissue, tissue graft, and medicament of the present invention can be transplanted using techniques well known in the art (for surgery, standard non-standard science 9th edition (medical school)) See surgical procedures (P41—p66), heart (p349—p398), blood vessels (p399—428), etc.). The biological tissue of the present invention can be used for vascular anastomosis, patch closure, artificial blood vessel replacement, artificial valve replacement, and the like. Therefore, a person skilled in the art can appropriately apply the living tissue, tissue graft and medicament of the present invention according to the disclosure of the present specification, depending on the situation to be treated.
[0218] 本発明において、本発明の医薬が適用される部位は、身体中のどのような部位で あってもよい。従って、医薬が由来する身体の部分に再度適用されてもよぐあるいは 、他の部分に適用されてもよい。実施例などで示されているように、「元に戻す」かどう 力に拘わりなぐいずれの部分に医薬を適用しても、本発明は所望の効果 (例えば、 再生、自己組織化)を達成することが証明された。従って、本発明は、原理的にはど のような移植手術、再生手術にも使用することができるという多大なる有用性を有する 。本発明の医薬が適用され得る部位としては、例えば、皮膚、血管、角膜、腎臓、心 臓、肝臓、臍帯、腸、神経、肺、胎盤、脾臓、脳、四肢末梢、網膜、弁、上皮組織、結 合組織、筋肉組織、神経組織などが挙げられるがそれらに限定されない。  [0218] In the present invention, the site to which the medicament of the present invention is applied may be any site in the body. Thus, it may be reapplied to the part of the body from which the medicine is derived, or it may be applied to other parts. As shown in the examples, the present invention achieves the desired effect (for example, regeneration, self-organization) regardless of whether the drug is applied regardless of whether it is `` reverted '' or not. Proven to do. Therefore, the present invention has great utility in principle that it can be used for any transplantation surgery and regenerative surgery. Examples of the site to which the medicament of the present invention can be applied include skin, blood vessel, cornea, kidney, heart, liver, umbilical cord, intestine, nerve, lung, placenta, spleen, brain, extremity, retina, valve, and epithelial tissue. Include, but are not limited to, connective tissue, muscle tissue, nerve tissue, and the like.
[0219] 別の局面において、本発明は、組織の癒着を防ぐ必要がある力または該危険にあ る被験体の処置または予防の方法を提供する。この方法は、 A)生体適合性高分子 を含み、 Ί線照射を受けたことを特徴とする癒着防止膜を提供する工程;および B) 該癒着防止膜を被験体に移植する工程を包含する。  [0219] In another aspect, the present invention provides a method of treating or preventing a subject in need or at risk of needing to prevent tissue adhesions. The method comprises the steps of A) providing an anti-adhesion membrane comprising a biocompatible polymer and receiving x-ray irradiation; and B) implanting the anti-adhesion membrane into a subject. .
[0220] 以下に、実施例に基づいて本発明を説明するが、以下の実施例は、例示の目的の みに提供される。従って、本発明の範囲は、上記発明の詳細な説明にも下記実施例 にも限定されるものではなぐ特許請求の範囲によってのみ限定される。  [0220] Hereinafter, the present invention will be described based on examples. However, the following examples are provided for illustrative purposes only. Accordingly, the scope of the present invention is limited only by the appended claims, which are not limited to the above detailed description of the invention nor the following examples.
実施例  Example
[0221] 以下に実施例を示して本発明をさらに詳しく説明するが、この発明は以下の例に限 定されるものではない。以下の実施例において用いられる試薬などは、例外を除き、 Sigma (St. Louis, USA)、和光純薬（大阪、 日本)など力も巿販されるものを用い た。また、動物実験は、大阪大学に規定される規準を遵守して行った。 [0222] (実施例 1) [0221] Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the following examples. The reagents used in the following examples were those with commercially available powers such as Sigma (St. Louis, USA), Wako Pure Chemical (Osaka, Japan), with the exception. Animal experiments were conducted in compliance with the standards stipulated by Osaka University. [0222] (Example 1)
(材料および方法）  (Materials and methods)
(材料）  (Material)
(1)チャージ溶液 (Charge solution)  (1) Charge solution
•PBS (-) (pH7. 4 ;0. 1M リン酸緩衝液（リン酸 2水素ナトリウム · 2水（リン酸 1ナト リウム） NaH PO · 2Η Ο 11. 84gおよびリン酸水素 2ナトリウム · 12水（リン酸 2ナト  • PBS (-) (pH7.4; 0. 1M phosphate buffer (sodium dihydrogen phosphate · 2 water (sodium 1 phosphate) NaH PO · 2Η 84 11.84 g and disodium hydrogen phosphate · 12 water (Nato phosphate
2 4 2  2 4 2
リウム） Na HPO · 12Η 0 116. Og ;作成法：およそ 1800mlの蒸留水をとり、リン酸  Li HP) Na HPO · 12Η 0 116. Og; Preparation method: Take approximately 1800 ml of distilled water and use phosphoric acid
2 4 2  2 4 2
2水素ナトリウムを溶かす (容易に溶ける)。リン酸水素 2ナトリウムを加える (難溶)。完 全に溶解したら蒸留水をカ卩えて全量で 2000mlにする。 ) 1000 mlに NaClを 9 g加 える）にて以下を調製した。  2 Dissolve sodium hydride (dissolves easily). Add disodium hydrogen phosphate (slightly soluble). When completely dissolved, add distilled water to make a total volume of 2000 ml. The following was prepared by adding 9 g of NaCl to 1000 ml).
20mM EDTA  20mM EDTA
プロテアーゼインヒビターカクテル（P2714、 Sigma -Aldrich Co. USA) 0. l%NaN  Protease inhibitor cocktail (P2714, Sigma-Aldrich Co. USA) 0. l% NaN
3  Three
100U/mlペニシリン  100U / ml penicillin
100 μ gZmlストレプトマイシン（1%)  100 μg Zml streptomycin (1%)
1. 25 /z gZmlアムホテリシン B (0. 5%)  1.25 / z gZml amphotericin B (0.5%)
カナマイシン lOOmgZl (SIGMA)  Kanamycin lOOmgZl (SIGMA)
(2)リンス溶液  (2) Rinse solution
•PBS (—） (pH7. 4)にて以下を調製した。  • The following was prepared with PBS (—) (pH 7.4).
0. l%NaN  0. l% NaN
3  Three
100U/mlペニシリン  100U / ml penicillin
100 μ gZmlストレプトマイシン（1%)  100 μg Zml streptomycin (1%)
1. 25 /z gZmlアムホテリシン B (0. 5%)  1.25 / z gZml amphotericin B (0.5%)
カナマイシン lOOmgZl  Kanamycin lOOmgZl
(3) PEG溶液  (3) PEG solution
•PEG (分子量 8, 000、 35, 000、 50, 000)を電子レンジにて溶解し、滅菌 で 20%に調整。  • PEG (molecular weight 8,000, 35,000, 50,000) is dissolved in a microwave oven and adjusted to 20% by sterilization.
[0223] ( y線照射適用のための生体適合性高分子を用いた処理） ゥシ生体組織 (大動脈、大動脈弁、心膜)を Hybrid (混合種） (Labo Products Co. Ltd. , Osaka, Japan)から、およびラット大動脈を SDラット（雄性、 5週齢、日本 動物、 Osaka、 Japan)から減菌条件下で調製した。動物使用の倫理に関しては、大 阪大学の動物実験の倫理に関する規定に従って行った。 [0223] (Treatment using biocompatible polymer for y-ray irradiation application) The living body tissue (aorta, aortic valve, pericardium) from Hybrid (mixed species) (Labo Products Co. Ltd., Osaka, Japan) and rat aorta from SD rat (male, 5 weeks old, Japanese animal, Osaka) ), Japan) under sterile conditions. Regarding the ethics of animal use, it was carried out in accordance with the Osaka University ethics regulations for animal experiments.
[0224] ゥシカも心膜を取り出し、生体適合性高分子としてポリエチレングリコールを用いて 処理を行った。以下にそのプロトコールを示す。  [0224] Usika also removed the pericardium and treated it with polyethylene glycol as the biocompatible polymer. The protocol is shown below.
[0225] (1)心膜の取り出し  [0225] (1) Removal of pericardium
新鮮に収集したゥシ心膜は、抗生物質（Gibco BRL, Life Technologies Inc . Rockville, MD, USA)を含む生理食塩水または PBS (これを本実施例において PBS (―)とする。 Gibco BRL, Life Technologies Inc. Rockville, MD, US A)に入れ、血液成分を洗浄除去した。  Freshly collected ushi pericardium is physiological saline containing PBS (Gibco BRL, Life Technologies Inc. Rockville, MD, USA) or PBS (this is PBS (-) in this example. Gibco BRL, Life Technologies Inc. Rockville, MD, USA) and washed away blood components.
[0226] (2)心膜加工  [0226] (2) Pericardial processing
1)クリーンベンチ内にてチャージ溶液に入って送られてきた心膜を滅菌バットに移し 、脂肪組織や厚い部分を取り除いた。  1) The pericardium sent in the charge solution in the clean bench was transferred to a sterilized vat to remove adipose tissue and thick parts.
2)使用する大きさ（現在 10cm X 5cm)にカットし、リンス溶液で数回、洗浄して使用 する（洗浄する際の溶液の量は心膜の量により違うが、例えば 3〜5枚で 10Lの溶液 を使用した)。  2) Cut to the size to be used (currently 10cm x 5cm) and wash with rinse solution several times. (The amount of the solution varies depending on the amount of pericardium. 10L solution was used).
[0227] (3) PEGの組織への浸潤  [0227] (3) Infiltration of PEG into tissues
1) 20%PEG溶液 20mlを滅菌シャーレに入れ、その中に心膜を浸す。浸した後コン ラージ棒又はローラーで心膜をしごき、溶液を圧入した。  1) Put 20ml of 20% PEG solution in a sterile petri dish and immerse the pericardium in it. After soaking, the pericardium was squeezed with a convergence rod or roller, and the solution was injected.
2)前記心膜を 20%PEG溶液とともにハイプリバッグに封入し、できるだけ空気を除 去した後、密閉した。  2) The pericardium was sealed in a high prebag together with a 20% PEG solution, and after removing air as much as possible, it was sealed.
3)前記バックに γ線を照射（25kGyまたは 50kGy)した。  3) The bag was irradiated with γ rays (25 kGy or 50 kGy).
4)照射終了後はそのまま 4°Cにて保存した。  4) After irradiation, it was stored at 4 ° C.
[0228] (コラーゲン構造） [0228] (Collagen structure)
コラーゲン構造には、 pH、イオン強度、溶剤の極性、陰イオン界面活性剤などが影 響を与え得ることが示されている（Ripamonti A, et al. , 1980, Biopolymers 1 9 : 965— 975 ;および Xiao WH, Knight DP, Chapman JA. , 1997, Biochi mica et Biophysica Acta. 1134 : 327— 337)。コラーゲン構造が変化すると生 体工学的特性が変化することも知られている。このような問題を回避するために、この 実施例では、組織処理プロセスを、マトリクスに影響がないように設計した。コラーゲ ン構造の組織学的試験および張力強度測定によりマトリクスが全く損なわれていない ことを以下の実験で実証した。 Collagen structure has been shown to be affected by pH, ionic strength, solvent polarity, anionic surfactants, etc. (Ripamonti A, et al., 1980, Biopolymers 19: 965-975; And Xiao WH, Knight DP, Chapman JA., 1997, Biochi mica et Biophysica Acta. 1134: 327-337). It is also known that biomechanical properties change when the collagen structure changes. In order to avoid such problems, in this example, the tissue processing process was designed so as not to affect the matrix. The following experiments demonstrated that the matrix was not damaged at all by histological examination of collagen structure and tensile strength measurement.
[0229] (組織学的検査）  [0229] (Histological examination)
血管移植片のパラフィン切片 (厚さ 3 μ m)を調製し、そしてへマトキシリン—ェオシ ン染色を行って、細胞外マトリクスの同定を行った。基底膜の成分である IZIV型コラ 一ゲンを同定するために、免疫組織化学的染色法を用いた。 SDラット (雄性、 5週齢 、 Nippon Animal Co. Ltd.東京、 日本）の大動脈を 4%パラホルムアルデヒドで 固定し、凍結保護した。凍結切片（5 m厚)を調製し、 PBS (—）で 3時間透過性にし 、そして PBS (—）中の 1%BSAで 1時間室温でブロッキングした。ついで、この切片 を一次抗体 (抗ラットコラーゲン抗体、コスモバイオ、東京、 日本）とともにインキュべ ートし、そして FITCと結合体ィ匕した二次抗体 (抗ヒッジ Ig抗体;コスモバイオ、東京、 日本）とインキュベートした。画像は、 Zeiss LSM510共焦点顕微鏡を用いて得た。  Paraffin sections (3 μm thick) of vascular grafts were prepared and stained with hematoxylin-eosin to identify the extracellular matrix. Immunohistochemical staining was used to identify type IZIV collagen, a component of the basement membrane. The aorta of SD rats (male, 5 weeks old, Nippon Animal Co. Ltd. Tokyo, Japan) was fixed with 4% paraformaldehyde and cryoprotected. Frozen sections (5 m thick) were prepared, permeabilized with PBS (—) for 3 hours, and blocked with 1% BSA in PBS (—) for 1 hour at room temperature. The section was then incubated with a primary antibody (anti-rat collagen antibody, Cosmo Bio, Tokyo, Japan), and a secondary antibody (anti-hidge Ig antibody; Cosmo Bio, Tokyo, Japan) conjugated with FITC. ). Images were obtained using a Zeiss LSM510 confocal microscope.
[0230] (von Kossa染色）  [0230] (von Kossa staining)
von Kossa染色は以下のとおり行った。必要に応じて脱パラフィン (例えば、純ェ タノールにて)、水洗 (蒸留水)を行い、 25% 硝酸銀液 (間接光下）に 2時間浸した。 その後、蒸留水水洗し、 42% チォ硫酸ナトリウム (ハイポ）に 5分浸した。その後、流 水水洗を 5分行い、次いでケルンェヒテロートに 5分浸した。その後、流水水洗を 5分 行い、脱水し、透徹し、封入した。  von Kossa staining was performed as follows. If necessary, deparaffinization (for example, with pure ethanol), washing with water (distilled water), and immersion in 25% silver nitrate solution (under indirect light) for 2 hours were performed. Then, it was washed with distilled water and immersed in 42% sodium thiosulfate (hypo) for 5 minutes. After that, it was washed with running water for 5 minutes, and then immersed in Kölnjeuteroto for 5 minutes. Then, it was washed with running water for 5 minutes, dehydrated, clarified and sealed.
[0231] (カルシウムの定量）  [0231] (Calculation of calcium)
1.サンプル（10 mg)を量りとつた。  1. A sample (10 mg) was weighed.
2. 6N HC1をサンプル mgあたり 50 1カロえた。  2. 50N of 6N HC1 was added per mg of sample.
3. 85°C、 48時間インキュベートした。  3. Incubated at 85 ° C for 48 hours.
4.等量の 6N NaOH 500 μ 1を加えた。  4. An equal volume of 500 μl 6N NaOH was added.
5.原子吸光光度計でカルシウム濃度を測定した。  5. The calcium concentration was measured with an atomic absorption photometer.
[0232] ( γ線照射） 本実施例では、 γ線照射は以下のように実施した。その手順を簡単に示す。 [0232] (Gamma irradiation) In the present example, γ-ray irradiation was performed as follows. The procedure is shown briefly.
[0233] PEG処理後の生体由来組織をプラスチック蓋付きのガラス製サンプル瓶 (径 5cm [0233] PEG-treated living tissue is a glass sample bottle with a plastic lid (diameter 5 cm)
X高さ 8cm程度）に入れ、蓋を閉めた状態（内部には組織 +空気)でコバルト 60線 源により γ線照射を行った。温度は常温 (室温）にて行ったが照射により生じる熱量 にて温度がやや上昇し、照射時には 25°C力も 40°Cの間であったようである。 γ線照 射する前に洗浄は行ってもよぐ行わなくても良い。  X height was about 8cm), and the lid was closed (inside the tissue + air), and γ-ray irradiation was performed with a cobalt 60 radiation source. Although the temperature was room temperature (room temperature), the temperature slightly increased with the amount of heat generated by irradiation, and it seems that the 25 ° C force was between 40 ° C at the time of irradiation. Cleaning may or may not be performed before gamma irradiation.
[0234] 理論の束縛されることは望まないが、 γ線の効果としては、例えば、石灰化の抑制 、 γ線により直接的細胞障害作用、 γ線の採用によって発生するフリーラジカルの細 胞障害作用の両方が考えられる。この場合 PEGカ^カベンジャーとして作用すること が考えられる。 [0234] Although we do not want to be bound by theory, the effects of γ rays include, for example, suppression of calcification, direct cytotoxicity by γ rays, and free radical cell damage caused by the adoption of γ rays. Both actions are conceivable. In this case, it may be possible to act as a PEG cover.
[0235] 次に、 γ線照射の種々の条件について検討した。  [0235] Next, various conditions of γ-ray irradiation were examined.
[0236] ( γ線照射量） [0236] (Gamma irradiation dose)
Ύ線照射量として、なし、 20kGy、 40kGy、 50kGy、 80kGy、 100kGy、 130kGy 、 250kGyを用いた。  None, 20 kGy, 40 kGy, 50 kGy, 80 kGy, 100 kGy, 130 kGy, 250 kGy were used as the X-ray irradiation dose.
[0237] (攪拌処理） [0237] (Agitation treatment)
上述の条件での攪拌の有無を組み合わせた。  The presence or absence of stirring under the above conditions was combined.
[0238] (周囲条件） [0238] (Ambient conditions)
大気中の他、水中、 PEG (平均分子量 35, 000の処理溶液と同様）のものを用いた  In addition to air, PEG (similar to treatment solution with an average molecular weight of 35,000) in water was used.
[0239] (対照実験） [0239] (Control experiment)
対照として、ダルタルアルデヒドィ匕学処理を行った（ γ線照射を行って 、な 、）生体 由来組織 (弁)およびゥシ生弁を含めた。  As controls, biological tissue (valves) and pupa biovalves that were treated with dartalaldehyde (treated with γ-rays) were included.
[0240] 以上のように調製した生体由来組織を、以下の実施例で評価した。 [0240] The living tissue prepared as described above was evaluated in the following Examples.
[0241] (実施例 2：生体組織内での反応の比較） [0241] (Example 2: Comparison of reaction in living tissue)
(方法）  (Method)
(免疫学的応答）  (Immunological response)
SDラットの背部皮下に作製した生体適合性高分子処理した生体由来組織を移植 し、 1週間および 2ヶ月後屠殺して、炎症細胞浸潤の程度をスコア化し評価した。具 体的には、評価は、糸且織の凍結切片に対してへマトキシリン &ェォシン染色を行い、 細胞の核を視覚化したうえで炎症細胞の数を数えることにより行った。本実施例にお いてコントロールとして上記未処理弁およびゥシ生弁と比較検討した。 Biocompatible polymer-treated biological tissue prepared subcutaneously on the back of SD rats was transplanted and sacrificed after 1 week and 2 months, and the degree of inflammatory cell infiltration was scored and evaluated. Ingredients Physically, the evaluation was performed by performing hematoxylin and eosin staining on a frozen section of yarn and tissue, visualizing the cell nucleus, and counting the number of inflammatory cells. In this example, as a control, comparison was made with the untreated valve and the ushi live valve.
[0242] (石灰化）  [0242] (Calcification)
ラット皮下に移植した検体を 1週間および 2力月後に採収し、 von Kossa染色で石 灰沈着を評価した。また、原子吸光分光計 (Atomic absorption spectrometry) で組織内 Ca濃度を測定した。 Ca濃度は、濃塩酸 (または濃酸）に組織を入れて、加 熱および溶解させた。その後、原子吸光測定を行う。その溶液を希釈し、高温プラズ マ中に噴霧し、燃焼時に発生するスペクトルの元素特異的吸収波長（Caは 422nm) より定量測定を行った。  Specimens transplanted subcutaneously in rats were collected 1 week and 2 months later and evaluated for calcification by von Kossa staining. In addition, the tissue Ca concentration was measured with an atomic absorption spectrometer. The Ca concentration was heated and dissolved by putting the tissue in concentrated hydrochloric acid (or concentrated acid). Thereafter, atomic absorption measurement is performed. The solution was diluted and sprayed into a high-temperature plasma, and quantitative measurement was performed from the element-specific absorption wavelength (Ca is 422 nm) of the spectrum generated during combustion.
[0243] SDラット（250g)の背部皮下に γ線照射処理ゥシ大動脈弁および他のコントロー ル弁の大動脈壁部分（10 X 10mm)を移植し 2力月後に検体を採収し、 Atomic ab sorption spectrometry (SPS7800： Seiko Instrument Inc. )で糸且織内 Ca濃 度（乾燥重量あたりの Ca含量）を測定した（Ozaki S. Pathophysiology of calci fication oi Dioprosthetic heart valves. Acta Biomedical し ovaniensia. 2001を参照のこと）。  [0243] Gamma-irradiated treated aortic valve and other control valve aortic wall (10 X 10mm) were transplanted subcutaneously in the back of SD rat (250g), and specimens were collected 2 months later. Atomic ab Measured Ca concentration (Ca content per dry weight) in yarn and fabric by sorption spectrometry (SPS7800: Seiko Instrument Inc.) (see Ozaki S. Pathophysiology of calci fication oi Dioprosthetic heart valves. Acta Biomedical and ovaniensia. 2001) )
[0244] (移植）  [0244] (Transplant)
本発明のプロセスで得られた大動脈壁組織の一部をィヌ下行大動脈に移植した。  A portion of the aortic wall tissue obtained by the process of the present invention was transplanted into the descending aorta.
[0245] (結果） [0245] (Result)
本実施例の結果を、 γ線照射処理ラット皮下移植結果 (HE染色の実像；図 1)、 H E染色（図 2)、 Von Kossa染色（図 3)およびカルシウム定量（図 4 ;未処理弁コント口 ールを含む）の結果として、ならびにコントロール弁としての未処理弁コントロールの 非会食結果 (HE染色の実像；図 5)を示す。  The results of this example are the results of subcutaneous implantation of γ-irradiated rats (real image of HE staining; Fig. 1), HE staining (Fig. 2), Von Kossa staining (Fig. 3), and calcium determination (Fig. 4; untreated valve control). The results of non-eating meals of the untreated valve control as a control valve (real image of HE staining; Fig. 5) are shown.
[0246] 皮下移植の結果の 2ヶ月後の結果（図 1〜3)に示すように、いずれの照射量（25k Gy、 50kGy)でも炎症細胞の浸潤は認められず、移植先に定着していることが明ら 力になった。他方、未処理のゥシ生弁は複数層の炎症細胞の浸潤が認められた（図 5)。他の架橋反応として、ダルタルアルデヒド処理をした場合 (示さず）、カルシウム 沈着が進んでいた（図 4を参照)。線量の比較をすると、 von Kossa染色で見た場合 、 25kGyの照射は、ほとんど石灰沈着を認めなカゝつた。 50kGyでもほとんど認められ なかったことから、両者とも、石灰沈着において顕著な効果が達成されたことが示さ れた。 HE染色でみた細胞の浸潤は、いずれの線量でも認められなかった。 [0246] As shown in the results 2 months after the results of subcutaneous transplantation (Figures 1 to 3), no infiltration of inflammatory cells was observed at any dose (25 kGy, 50 kGy), and the transplantation site had settled. It became clear that it was. On the other hand, the untreated wings were infiltrated with multiple layers of inflammatory cells (Fig. 5). As another cross-linking reaction, calcium deposition progressed when treated with dartalaldehyde (not shown) (see Figure 4). When comparing the doses, when seen with von Kossa staining The 25 kGy irradiation showed almost no calcification. Both were found to have achieved significant effects on calcification, as they were hardly observed at 50 kGy. No cellular infiltration as seen by HE staining was observed at any dose.
[0247] (実施例 3 :細胞置換の確認） [Example 3: Confirmation of cell replacement]
(各弁の生体組織での反応の比較）  (Comparison of reaction of each valve in living tissue)
ィヌ大腿大動脈に、実施例 1で作製した y線照射処理ゥシの前腕の動脈を移植し 、 10日後そのゥシを屠殺して、炎症細胞浸潤の程度を比較検討する。  After transplanting the forearm artery of y-irradiated treated tussus prepared in Example 1 into the femoral aorta, the worm is sacrificed 10 days later and the degree of inflammatory cell infiltration is compared.
[0248] (結果） [0248] (Result)
本発明の生体由来組織は、炎症反応はほとんど見られず、全体的な組織構造は損 なわれていないことが分かる。さら〖こ、移植組織を観察することによって、処理された 組織が自己細胞に置換されることを確認することができる。  It can be seen that the biological tissue of the present invention hardly shows an inflammatory reaction and the overall tissue structure is not damaged. Furthermore, by observing the transplanted tissue, it can be confirmed that the treated tissue is replaced with autologous cells.
[0249] (実施例 4：心筋梗塞ラットモデルでの検証）  [0249] (Example 4: Validation in a rat model of myocardial infarction)
次に、実施例 1に従って作製した人工心膜をラット梗塞心に移植する。  Next, the artificial pericardium prepared according to Example 1 is transplanted into the rat infarcted heart.
[0250] (心筋梗塞ラットモデル）  [0250] (Myocardial infarction rat model)
雄性 Lewis系統ラットを本実施例において用いた。 National Society for Med ical Research力 S作成した「Prmciples of Laboratory Animal Care」、およ ひnstitute of Laboratory Animal Resource力 S作成し National Intitute of Healtha^公表した「Guide for the Care and Use of Laboratory Ani malsj (NIH Publication No. 86— 23、 1985改訂）に遵つて、動物愛護精ネ申に 則った世話を動物に対して行う。  Male Lewis strain rats were used in this example. `` Prmciples of Laboratory Animal Care '' prepared by the National Society for Medical Research S and `` Guide for the Care and Use of Laboratory Ani malsj '' (NIH) prepared by the National Institute of Health Animal ^ In accordance with Publication No. 86—23, revised in 1985, the animals will be cared for in accordance with the animal welfare statement.
[0251] 急性心筋梗塞を文献（Weisman HF, Bush DE, Mannisi JA et al. Cellul ar mechanism of myocardial infarct expansion. Circulation. 1988 ; 78 : 186— 201)に記載されるように誘導した。簡潔には、ラット（300g、 8週齢)をベント バルピタールナトリウムで麻酔をし、陽圧式呼吸を行う。ラットの心筋梗塞モデルを作 製するために、左第四肋間で胸郭を開き、左冠動脈を根元力も 3mmの距離で、 8- 0ポリプロピレン糸で完全に結紮する。  [0251] Acute myocardial infarction was induced as described in the literature (Weisman HF, Bush DE, Mannisi JA et al. Cellular mechanism of myocardial infarct expansion. Circulation. 1988; 78: 186-201). Briefly, rats (300 g, 8 weeks old) are anesthetized with sodium bentvalpital and positive pressure breathing is performed. In order to create a rat myocardial infarction model, the thorax is opened between the left 4th ribs and the left coronary artery is completely ligated with 8-0 polypropylene thread at a root force of 3 mm.
[0252] (移植）  [0252] (Transplant)
レシピエントラットを麻酔し、左第五肋間で胸郭を開いて心臓を露出させる。このラッ トを心筋梗塞領域に投与した物質に従って 2群に分ける。 C群 (無治療群、 n= 5)と S 群 (脱細胞心膜移植群、 n= 5) 0脱細胞心膜は左前下降枝結紮 2週後に梗塞部位 に直接移植する。 Recipient rats are anesthetized and the heart is exposed by opening the rib cage between the fifth left intercostals. This rack In accordance with the substance administered to the myocardial infarction area, it is divided into two groups. Group C (untreated group, n = 5) and S group (decellularized pericardium transplanted group, n = 5) 0 decellularized pericardium is implanted directly into the infarcted area after left anterior descending branch ligation 2 weeks.
[0253] (ラット心臓の心機能の測定）  [0253] (Measurement of cardiac function of rat heart)
梗塞モデル作成 2週後、移植後 4週、 8週後に、心臓超音波（SONOS 5500, Ag ilent Technologies社製）にて心機能を測定する。 12— MHzの transducerを用 い、左側方より、左室が最大径を示す位置で、短軸像を描出する。 B— modeにて、 左室収縮末期面積（end systolic area)、 M— modeにて、左室拡張末期径（LV Dd)、左室収縮末期径 (LVDs)、左室前壁厚（LVAWTh)を測定し、 LVEF、 LVF Sを算出する。  Cardiac function is measured with cardiac ultrasound (SONOS 5500, manufactured by Agilent Technologies) at 2 weeks, 4 weeks after transplantation, and 8 weeks after transplantation. Using a 12-MHz transducer, a short-axis image is drawn from the left side at the position where the left ventricle shows the maximum diameter. In B-mode, left ventricular end systolic area (end systolic area), M-mode, left ventricular end-diastolic diameter (LV Dd), left ventricular end-systolic diameter (LVDs), left ventricular anterior wall thickness (LVAWTh) Measure LVEF and LVF S.
[0254] (組織学的分析）  [0254] (Histological analysis)
心筋細胞移植後、 8W後に心臓を摘出し、短軸にて切断し、 10%ホルムアルデヒド 溶液につけ、ノラフィン固定を行う。切片を作成し、へマトキシリンーェォジン染色、 マッソン—トリクローム染色を行う。また同時期の凍結切片を作製し、 FactorVin免 疫染色を行う。  After cardiomyocyte transplantation, the heart is removed 8 W later, cut with a short axis, placed in a 10% formaldehyde solution, and fixed with norafine. Prepare sections and perform hematoxylin-eosin staining and Masson-trichrome staining. In addition, prepare frozen sections at the same time and perform FactorVin immunostaining.
[0255] このように、本発明の γ線処理生体由来組織は、処理前の組織が由来する部位以 外の部分にも充分に適用できることが明らかになる。  [0255] As described above, it is clear that the γ-ray treated living body tissue of the present invention can be sufficiently applied to portions other than the site from which the tissue before treatment is derived.
[0256] (実施例 5：硬膜の処理） [0256] (Example 5: Treatment of dura mater)
次に、ゥシカも硬膜を取り出し、実施例 1に記載のようにポリエチレングリコール溶液 で処理し、 γ線照射することができる。  Next, Ushika can also remove the dura, treat with polyethylene glycol solution as described in Example 1, and irradiate with gamma rays.
[0257] この硬膜を、実施例 2に記載のように、 Lewisラットに移植し、炎症細胞の浸潤およ び石灰化について調べることができる。 [0257] As described in Example 2, this dura mater can be transplanted into Lewis rats and examined for inflammatory cell infiltration and calcification.
[0258] このように、本発明の方法は、どのような組織を用いても同様の優れた特性を示す [0258] Thus, the method of the present invention shows the same excellent characteristics regardless of the tissue used.
γ線照射処理を行うことができることがわかる。  It can be seen that γ-ray irradiation treatment can be performed.
[0259] (実施例 6 :他の高分子による生体由来組織の強化効果） [0259] (Example 6: Strengthening effect of biological tissue by other polymer)
次に、他の生体適合性高分子として、ポリエチレングリコール以外にも、ポリビニル アルコール、ポリビュルピロリドン、 Ί ポリグルタミン酸、ゼラチンについて、実施例Next, as other biocompatible polymers, in addition to polyethylene glycol, polyvinyl alcohol, polybutylpyrrolidone, Ίpolyglutamic acid, gelatin
1と同様の実験を行う。これらの生体適合性高分子は、同程度の γ線照射の条件 (濃 度 10%、同一のガラス瓶とコノ レト 60線源を利用して、 60— lOOkGy程度の照射） で架橋 *ゲルイ匕することから、以下のような実験を行うことによって、同様の生体由来 組織の強化効果を確認することができる。具体的には、最大点加重 (N)、最大点伸 び (mm表示）、弾性率 (MPa)を測定する。 Perform the same experiment as 1. These biocompatible polymers have the same conditions of γ-irradiation (dense Using the same glass bottle and Conoret 60 radiation source with a degree of 10%, it is crosslinked by irradiation of about 60-lOOkGy). The strengthening effect can be confirmed. Specifically, the maximum point weight (N), maximum point extension (in mm), and elastic modulus (MPa) are measured.
[0260] (組織強度） [0260] (Tissue strength)
引張り強度、弾性率および伸びを、引張り試験により観察する。その概要を以下に 示す。  Tensile strength, elastic modulus and elongation are observed by a tensile test. The outline is shown below.
[0261] 本実施例では、弓 I張試験機 (TENSILLON ORIENTEC)で強度測定する。具 体的には、 本明細書では、（1)検体を 5 X 30mmの短冊状に切る。（基本的に大動 脈基部の wallの部分を長軸方向に長くなるように切る。 )； (2)引っ張り試験器の固定 部分に検体の両端約 5mmを固定する。（ORIENTEC社製 TENSILON万能試験 機 RTC— 1150Aを使用）；および（3)引っ張り開始し破断点まで lcmZminの速度 で引っ張る。という作業を行うことによって、破断点負荷および弾性率を測定する（Sh moka T, Mayer JE. Tissue engineering heart valve leaflets. Circulati on. 1997 ; 96 (suppl II) :11— 102— II— 107 ; Shum— Tim D, Mayer JE. Tis sue engineering of autologus aorta using a new biodegradable poly mer. Ann Thorac Surg. 1999 ; 68 : 2298— 2304)。このような測定実験により 、ポリエチレングリコールのほ力 、ポリビニルアルコール、ポリビニルピロリドン、 Ίーポ リグルタミン酸、ゼラチンを用いても、同様の強化効果を確認することができる。 [0261] In this example, the strength is measured with a TENSILLON ORIENTEC. Specifically, in this specification, (1) Cut the specimen into 5 x 30 mm strips. (Basically, cut the wall part of the main artery base so that it is longer in the long axis direction.); (2) Fix the specimen about 5 mm at both ends to the fixed part of the tensile tester. (Use ORIENTEC's TENSILON universal testing machine RTC-1150A); and (3) Start pulling and pulling to the breaking point at lcmZmin. Measure the load at break and elastic modulus by doing the following (Sh moka T, Mayer JE. Tissue engineering heart valve leaflets. Circulati on. 1997; 96 (suppl II): 11—102—II—107; Shum — Tim D, Mayer JE. Tis sue engineering of autologus aorta using a new biodegradable polymer. Ann Thorac Surg. 1999; 68: 2298— 2304). Such measurement experiment, intensification of polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, I over Po Li glutamate, also be used gelatin, it is possible to confirm the same reinforcing effect.
[0262] (実施例 7 :他の高分子による石灰化抑制効果および強化効果） [0262] (Example 7: Calcification suppression effect and strengthening effect by other polymer)
次に、同様の効果が、血管、腸管、心膜、腸間膜、角膜、網膜、骨、尿管の生体由 来組織にも現れるかどうかを確認する。実施例 1と同様の実験を行う。これらをポリエ チレングリコール、ポリビュルアルコール、ポリビュルピロリドン、 Ί ポリグルタミン酸 およびゼラチンを用いて生体適合性高分子処理および γ線照射処理をする。この強 化生体由来組織について、実施例 2〜5と同様の実験を行うことによって、同様の生 体由来組織の強化効果を確認することができる。弁以外の血管、腸管、心膜、腸間 膜、角膜、網膜、骨、尿管の生体由来組織であっても、同様の石灰化抑制効果およ び強化効果を確認することができる。 [0263] (実施例 8 :最適パラメータの検討） Next, we will confirm whether the same effect appears in biological tissues such as blood vessels, intestinal tract, pericardium, mesentery, cornea, retina, bone, and ureter. The same experiment as in Example 1 is performed. These are subjected to biocompatible polymer treatment and γ-irradiation treatment using polyethylene glycol, polybutyl alcohol, polybutyl pyrrolidone, Ίpolyglutamic acid and gelatin. By conducting experiments similar to those in Examples 2 to 5 for this strengthened living body-derived tissue, the same effect of strengthening the living body tissue can be confirmed. Similar calcification-inhibiting and strengthening effects can be confirmed even in tissues derived from living bodies such as blood vessels other than valves, intestinal tract, pericardium, mesentery, cornea, retina, bone, and ureter. [0263] (Example 8: Examination of optimum parameters)
次に、上記実施例以外にも、種々の処理方法で作製した生体由来組織を、 PEGの 他の分子量のもの、 PVA、ポリビュルピロリドン（PVP)、ポリアクリル酸、ポリエチレン ォキシド (PEO)、ポリフッ化ビ-リデン (PVdF)を用いて強化したときの効果につ!ヽ て検証することができる。  Next, in addition to the examples described above, living tissue prepared by various treatment methods was used for other molecular weights of PEG, PVA, polybulurpyrrolidone (PVP), polyacrylic acid, polyethylene oxide (PEO), polyfluoride. The effect of strengthening with polyvinylidene fluoride (PVdF) can be verified.
[0264] (実施例 9：ダルタルアルデヒド固定および PEG化処理したゥシ心膜の調製） [0264] (Example 9: Preparation of ushi pericardium fixed with daltaraldehyde and treated with PEG)
実施例 1と同様の方法を使用して、 Hybrid (混合種）（Labo Products Co. Ltd . , Osaka, Japan)からゥシ心膜を取り出し、調製した。  Using the same method as in Example 1, Ushi pericardium was taken out from Hybrid (mixed species) (Labo Products Co. Ltd., Osaka, Japan) and prepared.
[0265] (1.グルタルアルデヒド固定） [0265] (1. Glutaraldehyde fixation)
クリーンベンチ内にて、滅菌パッドに心膜を広げ、しわができるのを防ぐために文鎮 で固定した。容器に心膜 (約 15cm X約 15cm)を移し、 0. 625%ダルタルアルデヒド 溶液 (和光純薬工業株式会社、大阪市、 日本) 500mlを心膜が浸るまで加えた。 M ULTI Shaker MMS (東京理化器械株式会社、東京都中央区、 日本)を用いて、 室温にて 30分間、 50RPMで撹拌した。次いで、ダルタルアルデヒド溶液（200ml) を交換し、室温、 0. 5時間にて固定した。  In a clean bench, the pericardium was spread over a sterile pad and fixed with paperweight to prevent wrinkling. The pericardium (about 15 cm X about 15 cm) was transferred to a container, and 500 ml of 0.625% dartalaldehyde solution (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was added until the pericardium soaked. The mixture was stirred at 50 RPM for 30 minutes at room temperature using M ULTI Shaker MMS (Tokyo Rika Instrument Co., Ltd., Chuo-ku, Tokyo, Japan). Subsequently, the dartalaldehyde solution (200 ml) was changed and fixed at room temperature for 0.5 hour.
[0266] (2. PEG化処理および γ線照射） [0266] (2. PEGylation and gamma irradiation)
ダルタルアルデヒド溶液で固定したゥシ心膜を、 20%PEG溶液 (分子量 35, 000) に一晚浸漬した。浸漬液から心膜を取り出し、気密容器中にて γ線を 50kGy照射し た。照射後、ゥシ心膜を、 PBS中にて冷蔵保存した。  Ushi pericardium fixed with dartalaldehyde solution was immersed in a 20% PEG solution (molecular weight 35,000). The pericardium was taken out from the immersion liquid and irradiated with 50 kGy of γ rays in an airtight container. After irradiation, ushi pericardium was stored refrigerated in PBS.
[0267] (実施例 10 :ラットへの移植実験） [0267] (Example 10: Rat transplantation experiment)
(1.ラット背部への移植）  (1. Transplantation to the back of rats)
レシピエントとして、雄性 7週齢の SDラット 12匹 (オリエンタル酵母工業株式会社（ 東京都板橋区、 日本)）を用いた。ラット背部に、ダルタルアルデヒド溶液で固定した ゥシ心膜を挿入した (N=6)。心膜を挿入して 2ヶ月後にラットを屠殺し、癒着につい て観察した。  As recipients, 12 male 7-week-old SD rats (Oriental Yeast Co., Ltd. (Itabashi, Tokyo, Japan)) were used. A persimmon pericardium fixed with dartalaldehyde solution was inserted into the back of the rat (N = 6). Two months after the pericardium was inserted, the rats were sacrificed and observed for adhesions.
[0268] (2.生体糸且織内での反応の比較） [0268] (2. Comparison of reaction in living body and weaving)
(方法）  (Method)
(免疫学的応答） ラット背部皮下に、ダルタルアルデヒド固定し、 PEG化処理したゥシ心膜を移植し、 2ヶ月後に屠殺して、炎症細胞浸潤の程度をスコア化し評価する。具体的には、評価 は、組織の凍結切片に対してへマトキシリン &ェォシン染色を行い、細胞の核を視覚 化したうえで炎症細胞の数を数えることにより行う。本実施例においてコントロールと してダルタルアルデヒドで固定したゥシ心膜と比較検討する。 (Immunological response) Daltaraldehyde-fixed and PEGylated ushi pericardium is transplanted subcutaneously on the back of the rat, and sacrificed 2 months later to score and evaluate the degree of inflammatory cell infiltration. Specifically, the evaluation is performed by staining the frozen section of the tissue with hematoxylin & eosin, visualizing the cell nucleus, and counting the number of inflammatory cells. In this example, the control is compared with a persimmon pericardium fixed with dartalaldehyde as a control.
[0269] (石灰化） [0269] (Calcification)
ラット皮下に移植した検体を 2力月後に採収し、 von Kossa染色で石灰沈着を評 価した。また、原子吸光分光計で組織内 Ca濃度を測定した。 Ca濃度は、濃塩酸 (ま たは濃酸）に組織を入れて、加熱および溶解させた。その後、原子吸光測定を行つ た。その溶液を希釈し、高温プラズマ中に噴霧し、燃焼時に発生するスペクトルの元 素特異的吸収波長（Caは 422nm)より定量測定を行った。  Specimens transplanted subcutaneously in rats were collected 2 months later, and calcification was assessed by von Kossa staining. In addition, the Ca concentration in the tissue was measured with an atomic absorption spectrometer. The Ca concentration was heated and dissolved by putting the tissue in concentrated hydrochloric acid (or concentrated acid). Thereafter, atomic absorption measurement was performed. The solution was diluted and sprayed into high-temperature plasma, and quantitative measurement was performed from the element-specific absorption wavelength (Ca is 422 nm) of the spectrum generated during combustion.
[0270] ラット背部皮下に、ダルタルアルデヒド固定 PEG処理し、 γ線照射したゥシ心膜 (グ ルタルアルデヒド固定 + PEG処理ゥシ心膜）およびグルタルアルデヒド固定のみのコ ントロールゥシ心膜 (ダルタルアルデヒド固定ゥシ心膜）（ 10 X 10mm)を移植し、 2力 月後に検体を採収し、 Atomic absorption spectrometry (SPS7800： Seiko I nstrument Inc. )で組織内 Ca濃度（乾燥重量あたりの Ca含量）を測定した（Ozak i S. Pathophysiology of calcification of Dioprosthetic heart valves. Acta Biomedical Lovaniensia. 2001を参照のこと）。  [0270] Daltaraldehyde-fixed PEG-treated and gamma-irradiated rabbit pericardium (glutaraldehyde-fixed + PEG-treated rabbit pericardium) and glutaraldehyde-fixed control pericardial pericardium (Dartal) Aldehyde-fixed rabbit pericardium (10 X 10mm) was transplanted, and a sample was collected 2 months later. Atomic absorption spectrometry (SPS7800: Seiko Instruments Inc.), the tissue Ca concentration (Ca content per dry weight) (See Ozak i S. Pathophysiology of calcification of Dioprosthetic heart valves. Acta Biomedical Lovaniensia. 2001).
[0271] (結果）  [0271] (Result)
ダルタルアルデヒド固定 +PEG処理ゥシ心膜を皮下に移植した場合の写真を図 6 ( 右）に示し、ダルタルアルデヒド固定のみ（コントロール）を行った心膜を皮下に移植し た場合の写真を図 6 (左）に示す。移植の 2ヶ月後に各々の新膜に付着したカルシゥ ム量を、以下の表および図 7に示す。皮下移植の 2ヶ月後、いずれの照射量（25kGy 、 50kGy)でも炎症細胞の浸潤、血小板凝集、血液凝固は認められない。  Figure 6 (right) shows a photograph of subcutaneously transplanted daltaraldehyde-fixed + PEG-treated ashi pericardium, and a photograph of subcutaneously transplanted pericardium with only dartalaldehyde fixed (control). Is shown in Fig. 6 (left). The amount of calcium attached to each new membrane 2 months after transplantation is shown in the table below and in Figure 7. Two months after subcutaneous implantation, no inflammatory cell infiltration, platelet aggregation, or blood clotting were observed at any dose (25 kGy, 50 kGy).
[0272] ダルタルアルデヒド固定 + PEG処理ゥシ心膜を移植した結果、移植した 6匹のうち 、傷口の開いたラット 1匹を除外した。残りのすべてのサンプルにおいて軽度の癒着 が観察された力 すべての心膜がほぼ移植前の状態にて摘出可能であった（図 6右 を参照）。 / gZ心膜 lmgあたりの Ca量は、 0. 699 ±0. 494であった（以下の表上 段および図 7を参照)。従って、カルシウム沈着を起こすことなく移植先に定着してい たことが確認できた。他方、ダルタルアルデヒド固定のみを行った場合、すべてのサ ンプルにおいて中度または重度の癒着が観察された（図 6左を参照)。 μ gZ心膜 1 mgあたりの Ca量は、 110. 373 ± 23. 563であった（以下の表下段および図 7を参 照）。ダルタルアルデヒド固定のみを行ったゥシ心膜では、カルシウム沈着が進んで いることが確認できた。 [0272] As a result of transplantation of dartalaldehyde-fixed + PEG-treated rabbit pericardium, one rat with an open wound was excluded from the six transplanted animals. Forces where mild adhesions were observed in all remaining samples All pericardium could be removed almost pre-transplanted (see right in Fig. 6). The amount of Ca per 1 mg / gZ pericardium was 0. 699 ± 0.494 (on the table below) Column and Figure 7). Therefore, it was confirmed that it had settled in the transplant destination without causing calcium deposition. On the other hand, when only dartalaldehyde fixation was performed, moderate or severe adhesions were observed in all samples (see left in Fig. 6). The amount of Ca per mg of μgZ pericardium was 110. 373 ± 23. 563 (see the lower table below and Figure 7). It was confirmed that calcium deposition was progressing in the pericardium treated only with dartalaldehyde fixation.
[表 1] [table 1]
(グリ ルァルデヒド固定 +P EG化処理したゥシ心膜における Ca量） (Ca content in the pericardium treated with glycaldehyde and PEG)
Ξ β g/心膜 1 mgあたりの Ξ β g per 1 mg pericardium
Cam mg  Cam mg
し 3里  3 villages
GA+PEG1 * 0.000 0.000  GA + PEG1 * 0.000 0.000
GA+PEG2 0.007 0.699  GA + PEG2 0.007 0.699
GA+PEG4 0.014 1.399  GA + PEG4 0.014 1.399
GA+PEG5 0.007 0.699  GA + PEG5 0.007 0.699
GA+PEG6 0.007 0.699  GA + PEG6 0.007 0.699
平均 0.007 0.699  Average 0.007 0.699
標準偏差 0.005 0.494  Standard deviation 0.005 0.494
* GA+PEG1は、傷口が開いていたため除外した。  * GA + PEG1 was excluded because the wound was open.
(グリ ルァルデヒド固定したゥシ心膜における Ca (Ca in the pericardium fixed with glycaldehyde
β g/心膜 1 mgあたりの  β g per 1 mg pericardium
Can mg  Can mg
し 3至  3 to
GA1 1.049 104.895  GA1 1.049 104.895
GA2 0.916 91.608  GA2 0.916 91.608
GA3 0.860 86.014  GA3 0.860 86.014
GA4 1.014 101.399  GA4 1.014 101.399
GA5 1.448 144.755  GA5 1.448 144.755
GA6 1.336 133.566  GA6 1.336 133.566
平均 1.104 1 10.373  Average 1.104 1 10.373
標準偏差 0.236 23.563 以上の結果より、ダルタルアルデヒド固定 +PEG化処理ゥシ心膜では、すべてのサ ンプルで石灰化はおこらず、ダルタルアルデヒド固定ゥシ心膜では、すべてのサンプ ルにて石灰化が生じた。このこと力ゝら、生体適合性高分子で処理すると、従来移植治 療にお 、て、使 、物にならな 、とされて 、たダルタルアルデヒド固定の人工組織を移 植治療に適したものへと変換することに成功したといえる。カロえて、本発明の移植用 組織は、癒着防止膜として使用可能であることも実証することができた。 Standard deviation 0.236 23.563 Based on the above results, all susceptibility was obtained with dartal aldehyde fixed + PEGylated pericardium. In the sample, calcification occurred in all samples in the dartalaldehyde-fixed rabbit pericardium. For this reason, when treated with biocompatible polymers, it has been said that it has not been used or used in conventional transplantation treatments, and artificial tissue fixed with dartalaldehyde is suitable for transplantation treatments. It can be said that it has succeeded in converting it into a thing. In fact, it was also possible to demonstrate that the tissue for transplantation of the present invention can be used as an anti-adhesion membrane.
[0274] (実施例 11：ブタへの移植）  [Example 11: Transplantation into pigs]
(1.ブタ腹部への移植）  (1. Transplantation to pig abdomen)
レシピエントとして、雄性ブタ 2頭 (LWD種、 40kg,株式会社ケアリー（大阪市、 日 本)）を用いる。ブタの腹部に、ダルタルアルデヒド溶液で固定したゥシ心膜を挿入す る。心膜を挿入して 2ヶ月後にブタを屠殺し、癒着について観察する。  As recipients, 2 male pigs (LWD, 40 kg, Cary, Inc. (Osaka City, Japan)) are used. Insert a persimmon pericardium fixed with dartalaldehyde solution into the abdomen of the pig. Two months after the pericardium is inserted, the pigs are sacrificed and observed for adhesions.
[0275] (2.生体糸且織内での反応の比較） [0275] (2. Comparison of reaction in living body and weaving)
(方法）  (Method)
(免疫学的応答）  (Immunological response)
ブタの腹部皮下に、ダルタルアルデヒド固定 PEG化処理したゥシ心膜を移植し、 2 ヶ月後に屠殺して、炎症細胞浸潤の程度をスコア化し評価する。具体的には、評価 は、組織の凍結切片に対してへマトキシリン &ェォシン染色を行い、細胞の核を視覚 化したうえで炎症細胞の数を数えることにより行う。本実施例においてコントロールと してダルタルアルデヒドで固定したゥシ心膜と比較検討する。  Daltaraldehyde-fixed PEG-treated ushi pericardium is transplanted subcutaneously into the abdomen of pigs and sacrificed 2 months later to score and evaluate the degree of inflammatory cell infiltration. Specifically, the evaluation is performed by staining the frozen section of the tissue with hematoxylin & eosin, visualizing the cell nucleus, and counting the number of inflammatory cells. In this example, the control is compared with a persimmon pericardium fixed with dartalaldehyde as a control.
[0276] (石灰化） [0276] (Calcification)
ブタ皮下に移植した検体を 2力月後に採収し、 von Kossa染色で石灰沈着を評価 する。また、原子吸光分光計で組織内 Ca濃度を測定する。 Ca濃度は、濃塩酸 (また は濃酸）に組織を入れて、加熱および溶解させる。その後、原子吸光測定を行う。そ の溶液を希釈し、高温プラズマ中に噴霧し、燃焼時に発生するスペクトルの元素特 異的吸収波長（Caは 422nm)より定量測定を行う。  Specimens transplanted subcutaneously in pigs are collected 2 months later and assessed for calcification by von Kossa staining. In addition, the Ca concentration in the tissue is measured with an atomic absorption spectrometer. The Ca concentration is heated and dissolved by putting the tissue in concentrated hydrochloric acid (or concentrated acid). Thereafter, atomic absorption measurement is performed. The solution is diluted, sprayed into high-temperature plasma, and quantitatively measured from the element-specific absorption wavelength (Ca is 422 nm) of the spectrum generated during combustion.
[0277] ブタ (40kg)の腹部皮下に、ダルタルアルデヒド固定 PEG処理し、 y線照射したゥ シ心膜 (ダルタルアルデヒド固定 + PEG処理ゥシ心膜）およびダルタルアルデヒド固 定のみのコントロールゥシ心膜（グルタルアルデヒド固定ゥシ心膜）（10 X 10mm)を 移植し、 2力月後に検体を採収し、 Atomic absorption spectrometry (SPS780 0 : Seiko Instrument Inc. )で組織内 Ca濃度（乾燥重量あたりの Ca含量）を測定 する (Ozaki S. Pathophysiology of calcification of bioprosthetic heart valves. Acta Biomedical Lovaniensia. 2001を参照のこと)。 [0277] Pig pericardium (daltaraldehyde-fixed + PEG-treated cush pericardium) treated with y-irradiated darthalaldehyde-fixed PEG treated subcutaneously in the abdomen of pig (40 kg) and daltaraldehyde-fixed control only Ushi pericardium (glutaraldehyde-fixed Ushi pericardium) (10 X 10mm) was transplanted, and after 2 months, specimens were collected and atomic absorption spectrometry (SPS780 0: Seiko Instrument Inc.) Measure the tissue Ca concentration (Ca content per dry weight) (see Ozaki S. Pathophysiology of calcification of bioprosthetic heart valves. Acta Biomedical Lovaniensia. 2001).
[0278] (結果） [0278] (Result)
ダルタルアルデヒド固定 + PEG処理ゥシ心膜を皮下に移植した結果を、 HE染色、 Von Kossa染色およびカルシウム定量の結果として評価する。グルタルアルデヒド 固定のみ心膜を、コントロールとする。皮下移植の 2ヶ月には、いずれの照射量（25k Gy、 50kGy)でも炎症細胞の浸潤、血小板凝集、血液凝固、カルシウム沈着は認め られず、移植先に定着していることが明らかである。他方、ダルタルアルデヒド固定を した場合には、カルシウム沈着が進んでいる。  The results of subcutaneous implantation of dartalaldehyde-fixed + PEG-treated rabbit pericardium are evaluated as a result of HE staining, Von Kossa staining, and calcium quantification. Glutaraldehyde-fixed pericardium is used as a control. Two months after subcutaneous transplantation, inflammatory cell infiltration, platelet aggregation, blood coagulation, and calcification were not observed at any irradiation dose (25 kGy, 50 kGy), and it is clear that they have settled at the transplant destination. On the other hand, when dartalaldehyde is fixed, calcium deposition is progressing.
[0279] 以上の結果より、ダルタルアルデヒド固定 +PEG化処理ゥシ心膜では、すべてのサ ンプルで石灰化はおこらず、ダルタルアルデヒド固定ゥシ心膜では、すべてのサンプ ルにて石灰化が生じる。このことから、生体適合性高分子で処理すると、従来移植治 療にお 、て、使 、物にならな 、とされて 、たダルタルアルデヒド固定の人工組織を移 植治療に適したものへと変換することに成功したといえる。カロえて、本発明の移植用 組織は、癒着防止膜として使用可能であることも実証することができる。  [0279] Based on the above results, calcification did not occur in all samples in the dartalaldehyde-fixed + PEGylated pericardium, whereas calcification occurred in all samples in the dartalaldehyde-fixed pericardium. Will occur. For this reason, when treated with biocompatible polymers, it has been said that it has not been used or used in conventional transplantation treatments, so that artificial tissue fixed with dartalaldehyde is suitable for transplantation treatments. It can be said that the conversion was successful. In addition, it can be demonstrated that the tissue for transplantation of the present invention can be used as an anti-adhesion membrane.
[0280] (実施例 12 :癒着防止膜の膜の厚み、弾性率および癒着防止率）  (Example 12: Film thickness, elastic modulus and adhesion prevention rate of adhesion prevention film)
実施例 9により調製したゥシ心膜について、膜の厚み、弾性率および癒着防止率を 測定する。  For the rabbit pericardium prepared according to Example 9, the thickness, elastic modulus and anti-adhesion rate of the membrane are measured.
[0281] (1.膜の厚み）  [0281] (1. Film thickness)
グルタルアルデヒド固定 + PEG処理ゥシ心膜およびグルタルアルデヒド固定のみ のコントロールゥシ心膜を、プラスチック製の薄いシートに挟み込む。これらのゥシ心 膜が変形しないように電子ノギス（Digital Caliper Modell 9971：シンヮ測定株式 会社 (新潟県三条巿)）にて 3ケ所以上の厚みを測定する。測定値の平均を計算する ことにより、ダルタルアルデヒド固定 +PEG処理ゥシ心膜は、代表的に、 0. 5 - 1. 0 mmの厚みを有することが確認される。  Glutaraldehyde-fixed + PEG-treated pericardium and glutaraldehyde-fixed control pericardium are sandwiched between thin plastic sheets. At least 3 thicknesses are measured with an electronic vernier caliper (Digital Caliper Modell 9971: Shinjo Measurement Co., Ltd. (Sanjoen, Niigata)) so that these ushi pericardiums do not deform. By calculating the average of the measured values, it is confirmed that the dartalaldehyde-fixed + PEG-treated pericardium typically has a thickness of 0.5-1.0 mm.
[0282] (2.膜の弾性力）  [0282] (2. Elastic force of membrane)
グルタルアルデヒド固定 + PEG処理ゥシ心膜およびグルタルアルデヒド固定のみ のコントロールゥシ心膜を、 5 X 30mmの短冊状に切断する。次いで、引っ張り試験 器の固定部分に該検体の両端約 5mmを固定する。引っ張り開始し破断点まで lcm Zminの速度で引っ張り、レジオメーターにて測定する。その結果、ダルタルアルデヒ ド固定 +PEG処理ゥシ心膜では、代表的に、 2— lOMPaの弾性率を有することが確 認される。 Glutaraldehyde fixation + PEG-treated ushi pericardium and glutaraldehyde fixation only Cut the control bush pericardium into 5 x 30 mm strips. Next, fix both ends of the specimen about 5 mm to the fixed part of the tensile tester. Start pulling and pull to the breaking point at a speed of lcm Zmin, and measure with a regiometer. As a result, it is confirmed that the dartal aldehyde fixed + PEG-treated pericardium typically has an elastic modulus of 2-lOMPa.
[0283] (3.癒着防止率） [0283] (3. Anti-adhesion rate)
試験部位に、ダルタルアルデヒド固定 +PEG処理ゥシ心膜およびダルタルアルデ ヒド固定のみのコントロールゥシ心膜を貼付けた場合に、どの程度の癒着が防止され るのかを測定する。  Measure how much adhesion will be prevented when the control pericardium with only dartalaldehyde-fixed + PEG-treated pericardium and dartal aldehyde fixed is attached to the test site.
[0284] これらのゥシ心膜を、試験部位としてラット背部に移植する。移植の 2力月後、移植し たゥシ心膜を摘出する。癒着したサンプル数と、癒着しな力つたサンプル数を数え、 以下の式により計算する。  [0284] These ushi pericardium are implanted into the back of the rat as the test site. Two months after transplantation, the transplanted persimmon pericardium is removed. Count the number of samples that have adhered and the number of samples that have adhered, and use the following formula to calculate.
癒着防止率 (％) = {癒着なしの数 Z (癒着あり +癒着なしの数） } X 100  Anti-adhesion rate (%) = {number without adhesion Z (number with adhesion + number without adhesion)} X 100
その結果、ダルタルアルデヒド固定 +PEG処理ゥシ心膜は 80%以上の癒着防止率 を有し、ダルタルアルデヒド固定ゥシ心膜に比べて高 、癒着防止効果を有することを 確認する。  As a result, it is confirmed that the dartalaldehyde-fixed + PEG-treated ushi pericardium has an adhesion prevention rate of 80% or more, and has a higher anti-adhesion effect than the dartalaldehyde-fixed ushi pericardium.
[0285] (実施例 13：生体外における癒着防止膜の癒着防止効果）  (Example 13: Adhesion prevention effect of adhesion prevention membrane in vitro)
グルタルアルデヒド固定 + PEG処理ゥシ心膜およびグルタルアルデヒド固定のみ のコントロールゥシ心膜を用いて、生体外における癒着防止効果を確認する。チヤ一 ジ溶液中にて、これらの心膜の上に、ラットから摘出した肝臓、腸管、筋肉組織をの せる。数日間インキュベートし、癒着したサンプル数と、癒着しな力つたサンプル数を 数え、上記の式により計算する。その結果、ダルタルアルデヒド固定 + PEG処理ゥシ 心膜は 80%以上の癒着防止率を有し、ダルタルアルデヒド固定ゥシ心膜に比べて高 V、癒着防止効果を有することを確認する。  Confirm the anti-adhesion effect in vitro using glutaraldehyde fixation + PEG-treated ushi pericardium and control ushi pericardium only with glutaraldehyde fixation. Place the liver, intestinal tract, and muscle tissue removed from the rat on the pericardium in a change solution. Incubate for several days, count the number of samples that have adhered, and the number of samples that have adhered, and calculate using the above formula. As a result, it was confirmed that dartalaldehyde-fixed + PEG-treated rabbit pericardium has an adhesion prevention rate of 80% or more, and has a higher V and adhesion prevention effect than dartalaldehyde-fixed rabbit pericardium.
[0286] (実施例 14 :他の高分子による生体由来組織の強化効果および癒着防止効果） 次に、他の生体適合性高分子として、ポリエチレングリコール以外にも、ポリビニル アルコール、ポリビュルピロリドン、 Ί ポリグルタミン酸、ゼラチンについて、実施例 9と同様の実験を行う。これらの生体適合性高分子は、同程度の γ線照射の条件 (濃 度 10%、同一のガラス瓶とコノ レト 60線源を利用して、 60— lOOkGy程度の照射） で架橋 'ゲルイ匕することから、同様の実施例 6および 10〜 13と同様の実験を行うこと によって、同様の生体由来組織の強化効果ならびに癒着防止効果を確認することが できる。 (Example 14: Strengthening effect and prevention of adhesion of biological tissue by other polymer) Next, as other biocompatible polymer, in addition to polyethylene glycol, polyvinyl alcohol, polybulurpyrrolidone , Ί The same experiment as in Example 9 is performed for polyglutamic acid and gelatin. These biocompatible polymers have the same conditions of γ-irradiation (dense Use the same glass bottle and Conoret 60 radiation source at a degree of 10%, and carry out the same experiment as in Examples 6 and 10 to 13 because it is crosslinked by irradiation of about 60-lOOkGy. Thus, it is possible to confirm the effect of strengthening the same living tissue and the effect of preventing adhesion.
[0287] (実施例 15 :他の高分子による生体由来組織の石灰化抑制効果および強化効果、 ならびに癒着防止効果）  [Example 15: Effect of inhibiting calcification and strengthening of living tissue by other polymer and effect of preventing adhesion)
次に、同様の効果が、血管、腸管または尿管由来の膜、および心膜、腸間膜、角膜 、網膜にも現れるかどうかを確認する。実施例 6および 9〜13と同様の実験を行う。こ れらをポリエチレングリコール、ポリビニルアルコール、ポリビニルピロリドン、 γ ポリ グルタミン酸およびゼラチンを用いて生体適合性高分子処理および γ線照射処理を する。この強化生体由来膜についても、同様の石灰化抑制効果および強化効果、な らびに癒着防止効果を確認することができる。  Next, we will check whether similar effects appear in blood vessels, intestinal or ureteral membranes, and in the pericardium, mesentery, cornea, and retina. Experiments similar to Examples 6 and 9-13 are performed. These are subjected to biocompatible polymer treatment and γ-irradiation treatment using polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, γ polyglutamic acid and gelatin. This reinforced biological membrane can also be confirmed to have the same calcification inhibiting effect, reinforcing effect, and adhesion preventing effect.
[0288] (実施例 16 :他の架橋剤による生体由来組織の石灰化抑制効果および強化効果、 ならびに癒着防止効果）  [Example 16: Effect of inhibiting and strengthening calcification of living tissue by other cross-linking agents, and effect of preventing adhesion)
次に、同様の効果が、シアンイミド（cyanimide)、 1—ェチル—3— (3 ジメチルァ ミノプロピル）カルボジイミドヒドロクロリド（EDC)、エポキシ（ポリ（グリシジルエーテル） )、 2, 6 ビス（4 アジドベンジリデン）一 4—メチルシクロへキサノン（BAMC)、 4, 4'ジアジドジフ ニルエーテル、 4, 4'ジアジドジフ ニルスルホン、 4, 4'ジアジドジ フエニルアセトン、 4, 4'ジアジドジフエニルメタン、 1, 4 ビス（α ジァゾベンジル） および 4—[ρ—アジドサリチアミド]プチルァミンを架橋剤として用いた場合にも現れ るかどうかを確認する。実施例 6および 9〜13と同様の実験を行う。これらをポリェチ レングリコール、ポリビュルアルコール、ポリビュルピロリドン、 Ί ポリグルタミン酸お よびゼラチンを用いて生体適合性高分子処理および γ線照射処理をする。この強化 生体由来膜についても、同様の石灰化抑制効果および強化効果、ならびに癒着防 止効果を確認することができる。 Next, the same effect can be obtained by using cyanimide, 1-ethyl-3- (3 dimethylaminopropyl) carbodiimide hydrochloride (EDC), epoxy (poly (glycidyl ether)), 2,6 bis (4 azidobenzylidene). 4-methylcyclohexanone (BAMC), 4,4'diazidodiphenyl ether, 4,4'diazidodiphenylsulfone, 4,4'diazidodiphenylacetone, 4,4'diazidodiphenylmethane, 1,4 bis (α diazobenzyl) And 4- [ρ-azidosalicyamido] plutamine as a crosslinker. Experiments similar to Examples 6 and 9-13 are performed. These are subjected to biocompatible polymer treatment and γ-irradiation treatment using polyethylene glycol, polybulal alcohol, polybulurpyrrolidone , Ίpolyglutamic acid and gelatin. For this reinforced biological membrane, the same calcification inhibiting effect, reinforcing effect, and adhesion prevention effect can be confirmed.
[0289] (実施例 17 : PEG処理後に γ線照射した生体由来組織の癒着防止効果）  [Example 17: Effect of preventing adhesion of biological tissue irradiated with γ-rays after PEG treatment]
(1.生体由来組織の調製）  (1. Preparation of biological tissue)
実施例 1と同様の方法を使用して、 PEG処理後に γ線照射した生体由来組織 (ΡΕ G処理 + γ線照射生体由来組織）（ゥシの大動脈、大動脈弁、心膜)を調製し、以下 の実験に使用する。 Using a method similar to that in Example 1, a tissue derived from a living body (ΡΕ G-treated + gamma-irradiated biological tissue) (ussi aorta, aortic valve, pericardium) is prepared and used in the following experiments.
[0290] (2.ラット背部への移植） [0290] (2. Implantation into the back of rats)
レシピエントとして、雄性 7週齢の SDラット 12匹 (オリエンタル酵母工業株式会社（ 東京都板橋区、 日本)）を用いる。ラット背部に、 PEG処理 + γ線照射生体由来組織 (大動脈、大動脈弁、心膜)を挿入する (Ν = 6)。この生体由来組織を挿入して 2ヶ月 後にラットを屠殺し、癒着について観察する。  As recipients, 12 male 7-week-old SD rats (Oriental Yeast Co., Ltd. (Itabashi, Tokyo, Japan)) are used. PEG-treated + gamma-irradiated biological tissue (aorta, aortic valve, pericardium) is inserted into the back of the rat (Ν = 6). Two months after inserting this tissue, the rats are sacrificed and observed for adhesions.
[0291] (3.生体糸且織内での反応の比較） [0291] (3. Comparison of reaction in living body and weaving)
(方法）  (Method)
(免疫学的応答）  (Immunological response)
ラット背部皮下に、 PEG処理 + γ線照射生体由来組織を移植し、 2ヶ月後に屠殺 して、炎症細胞浸潤の程度をスコア化し評価する。具体的には、評価は、組織の凍 結切片に対してへマトキシリン &ェォシン染色を行い、細胞の核を視覚化したうえで 炎症細胞の数を数えることにより行う。本実施例においてコントロールとして、上記未 処理弁およびゥシ生弁と比較検討する。  PEG-treated + gamma-irradiated living tissue is transplanted subcutaneously on the back of the rat, and sacrificed 2 months later to score and evaluate the degree of inflammatory cell infiltration. Specifically, the evaluation is performed by staining the frozen section of the tissue with hematoxylin and eosin, visualizing the cell nucleus, and counting the number of inflammatory cells. In this embodiment, as a control, the above-mentioned untreated valve and ushi live valve are compared and examined.
[0292] (石灰化） [0292] (Calcification)
ラット皮下に移植した検体を 2力月後に採収し、 von Kossa染色で石灰沈着を評 価する。また、原子吸光分光計で組織内 Ca濃度を測定する。 Ca濃度は、濃塩酸 (ま たは濃酸）に組織を入れて、加熱および溶解させる。その後、原子吸光測定を行う。 その溶液を希釈し、高温プラズマ中に噴霧し、燃焼時に発生するスペクトルの元素特 異的吸収波長（Caは 422nm)より定量測定を行う。  Specimens transplanted subcutaneously in rats are collected 2 months later and evaluated for calcification by von Kossa staining. In addition, the Ca concentration in the tissue is measured with an atomic absorption spectrometer. The Ca concentration is heated and dissolved by placing the tissue in concentrated hydrochloric acid (or concentrated acid). Thereafter, atomic absorption measurement is performed. The solution is diluted and sprayed into high-temperature plasma, and quantitative measurement is performed from the element-specific absorption wavelength (Ca is 422 nm) of the spectrum generated during combustion.
[0293] ラット背部皮下に、 PEG処理 + γ線照射生体由来組織およびコントロール組織を 移植し、 2力月後に検体を採収し、 Atomic absorption spectrometry (SPS780 0 : Seiko Instrument Inc. )で組織内 Ca濃度（乾燥重量あたりの Ca含量）を測定 する (Ozaki S. Pathophysiology of calcification of bioprosthetic heart valves. Acta Biomedical Lovaniensia. 2001を参照のこと)。  [0293] PEG-treated + gamma-irradiated living tissue and control tissue were transplanted subcutaneously in the back of the rat, and a sample was collected 2 months later. Atomic absorption spectrometry (SPS780 0: Seiko Instrument Inc.) Measure concentration (Ca content per dry weight) (see Ozaki S. Pathophysiology of calcification of bioprosthetic heart valves. Acta Biomedical Lovaniensia. 2001).
[0294] (結果）  [0294] (Result)
PEG処理 + γ線照射生体由来組織を皮下に移植した場合、皮下移植の 2ヶ月後 、いずれの照射量でも炎症細胞の浸潤、血小板凝集、血液凝固は認められない。 P EG処理 + γ線照射生体由来組織を移植した結果、すべてのサンプルにおいて軽 度の癒着が観察されるが、すべての組織がほぼ移植前の状態にて摘出可能である。 従って、カルシウム沈着を起こすことなく移植先に定着していたことが確認できる。他 方、コントロール組織では、すべてのサンプルにおいて中度または重度の癒着が観 察され、カルシウム沈着が進んで 、ることが確認できる。 PEG treatment + γ-irradiated tissue when transplanted subcutaneously, 2 months after subcutaneous implantation No inflammatory cell infiltration, platelet aggregation, or blood clotting are observed at any dose. As a result of transplantation of PEG-treated + gamma-irradiated living tissue, mild adhesion is observed in all samples, but all tissues can be removed almost before transplantation. Therefore, it can be confirmed that it has been established in the transplantation destination without causing calcium deposition. On the other hand, in the control tissue, moderate or severe adhesion is observed in all samples, and it can be confirmed that calcification has progressed.
[0295] 以上の結果より、 PEG処理 + y線照射生体由来組織では、すべてのサンプルで 石灰化はおこらず、コントロール組織では、すべてのサンプルにて石灰化が生じた。 このことから、 PEG処理 + γ線照射生体由来組織は癒着防止効果も有することが確 認できる。 [0295] From the above results, calcification occurred in all samples in the PEG-treated + y-ray-irradiated tissues, and all samples in the control tissues. From this, it can be confirmed that the PEG-treated + γ-irradiated living body-derived tissue also has an adhesion preventing effect.
[0296] (実施例 18.ブタ腹部への移植）  (Example 18. Transplantation into pig abdomen)
実施例 16で調製した PEG処理 + γ線照射生体由来組織を用いる。コントロールと して、未処理弁およびゥシ生弁を用いる。  The PEG-treated + γ-irradiated biological tissue prepared in Example 16 is used. As a control, untreated valve and ushi live valve are used.
レシピエントとして、雄性ブタ 2頭 (LWD種、 40kg,株式会社ケアリー（大阪市、 日本 ) )を用いる。ブタの腹部に、 PEG処理 + γ線照射生体由来組織を挿入する。この生 体由来組織を挿入して 2ヶ月後にブタを屠殺し、癒着について観察する。  As recipients, 2 male pigs (LWD, 40 kg, Cary, Inc. (Osaka City, Japan)) are used. PEG-treated + gamma-irradiated biological tissue is inserted into the abdomen of the pig. Two months after insertion of this organism-derived tissue, the pigs are sacrificed and observed for adhesions.
[0297] (5.生体糸且織内での反応の比較） [0297] (5. Comparison of reaction in living body and weaving)
(方法）  (Method)
免疫学的応答および石灰化についての実験は、実施例 16と同様の方法により行う [0298] (結果）  Experiments on immunological response and calcification are performed in the same way as in Example 16 [0298] (Results)
PEG処理 + γ線照射生体由来組織を皮下に移植した結果を、 HE染色、 Von K ossa染色およびカルシウム定量の結果として評価する。皮下移植の 2ヶ月には、いず れの照射量でも炎症細胞の浸潤、血小板凝集、血液凝固、カルシウム沈着は認めら れず、移植先に定着していることが明らかである。他方、コントロール組織では、カル シゥム沈着が進んでいる。  The results of subcutaneous implantation of PEG-treated + γ-irradiated living tissue are evaluated as results of HE staining, Von Kossa staining, and calcium quantification. Two months after subcutaneous transplantation, inflammatory cell infiltration, platelet aggregation, blood coagulation, and calcification were not observed at any irradiation dose, and it is clear that they were established at the transplant destination. On the other hand, in the control organization, calcium deposition is progressing.
[0299] 以上の結果より、 PEG処理 + y線照射生体由来組織では、すべてのサンプルで 石灰化はおこらず、コントロール組織では、すべてのサンプルにて石灰化が生じた。 このことから、 PEG処理 + γ線照射生体由来組織は癒着防止効果も有することが確 認できる。 [0299] From the above results, calcification occurred in all samples in the PEG-treated + y-ray-irradiated tissues, and all samples in the control tissues. From this, it can be confirmed that the tissue derived from PEG-treated + γ-irradiated organism also has an anti-adhesion effect.
[0300] (実施例 19： PEG処理 + γ線照射生体由来組織の弾性力および癒着防止率） 実施例 16および 17により調製した PEG処理 + γ線照射生体由来組織について、 弾性率および癒着防止率を測定する。  (Example 19: Elasticity and anti-adhesion rate of PEG-treated + γ-irradiated biological tissue) Regarding PEG-treated + γ-irradiated biological tissue prepared in Examples 16 and 17, the elastic modulus and anti-adhesion rate Measure.
[0301] (1.弾性力）  [0301] (1. Elastic force)
PEG処理 + γ線照射生体由来組織およびコントロール組織を、 5 X 30mmの短冊 状に切断する。次いで、引っ張り試験器の固定部分に該検体の両端約 5mmを固定 する。引っ張り開始し破断点まで lcmZminの速度で引っ張り、レジオメーターにて 測定する。その結果、 PEG処理 + γ線照射生体由来組織では、 2— lOMPaの弾性 率を有することが確認される。  Cut the PEG-treated + gamma-irradiated living tissue and control tissue into 5 X 30 mm strips. Next, about 5 mm of both ends of the specimen are fixed to the fixed part of the tensile tester. Start pulling and pulling to the breaking point at a speed of lcmZmin and measure with a regiometer. As a result, it is confirmed that the PEG-treated + γ-irradiated biological tissue has an elastic modulus of 2-lOMPa.
[0302] (2.癒着防止率）  [0302] (2. Adhesion prevention rate)
試験部位に、 PEG処理 + γ線照射生体由来組織およびコントロール組織を貼付 けた場合に、どの程度の癒着が防止されるの力を測定する。  Measure how much adhesion is prevented when PEG-treated + gamma-irradiated living tissue and control tissue are applied to the test site.
[0303] PEG処理 + γ線照射生体由来組織およびコントロール組織を、試験部位として、 ラット背部に移植する。移植の 2力月後、各々を摘出する。癒着したサンプル数と、癒 着しな力つたサンプル数を数え、以下の式により計算する。  [0303] PEG-treated + gamma-irradiated living tissue and control tissue are transplanted to the back of the rat as the test site. Each is removed 2 months after transplantation. Count the number of samples that have adhered and the number of samples that have adhered, and use the following formula to calculate.
癒着防止率 (％) = {癒着なしの数 Ζ (癒着あり +癒着なしの数） } X 100  Anti-adhesion rate (%) = {number without adhesion Ζ (number with adhesion + no adhesion)} X 100
その結果、 PEG処理 + γ線照射生体由来組織は 80%以上の癒着防止率を有し、 コントロール組織に比べて高い癒着防止効果を有することを確認する。  As a result, it is confirmed that the tissue derived from PEG-treated + γ-irradiated living body has an adhesion prevention rate of 80% or more, and has a higher adhesion prevention effect than the control tissue.
[0304] (実施例 20：生体外における癒着防止膜の癒着防止効果）  [0304] (Example 20: Anti-adhesion effect of anti-adhesion membrane in vitro)
PEG処理 + γ線照射生体由来組織およびコントロール組織を用いて、生体外に おける癒着防止効果を確認する。チャージ溶液中にて、これらの組織の上に、ラット から摘出した肝臓、腸管、筋肉組織をのせる。数日間インキュベートし、癒着したサン プル数と、癒着しな力つたサンプル数を数え、上記の式により計算する。その結果、 Ρ EG処理 + γ線照射生体由来組織は 80%以上の癒着防止率を有し、コントロール 組織に比べて高!、癒着防止効果を有することを確認する。  Use PEG-treated + gamma-irradiated living tissue and control tissue to confirm the anti-adhesion effect in vitro. In the charge solution, the liver, intestine, and muscle tissue removed from the rat are placed on these tissues. Incubate for several days, count the number of samples attached, and the number of samples with strong adhesion, and calculate according to the above formula. As a result, it was confirmed that the tissue derived from ΡEG treated + γ-irradiated organism had an adhesion prevention rate of 80% or more, higher than the control tissue, and had an adhesion prevention effect.
[0305] (実施例 21：他の高分子による生体由来組織の癒着防止効果） 次に、他の生体適合性高分子として、ポリエチレングリコール以外にも、ポリビニル アルコール、ポリビュルピロリドン、 Ί ポリグルタミン酸、ゼラチンについて、実施例(Example 21: Effect of preventing adhesion of biological tissue by other polymer) Next, as other biocompatible polymers, in addition to polyethylene glycol, polyvinyl alcohol, polybutylpyrrolidone, Ίpolyglutamic acid, gelatin
1と同様の実験を行う。これらの生体適合性高分子は、同程度の γ線照射の条件 (濃 度 10%、同一のガラス瓶とコノ レト 60線源を利用して、 60— lOOkGy程度の照射） で架橋 *ゲルイ匕することから、同様の実施例 10〜20と同様の実験を行うことによって 、同様の生体由来組織の癒着防止効果を確認することができる。 Perform the same experiment as 1. These biocompatible polymers cross-link under the same conditions of γ-irradiation (concentration: 10%, using the same glass bottle and correto 60 radiation source, irradiation of about 60-lOOkGy). Therefore, by performing the same experiment as in Examples 10 to 20, it is possible to confirm the same effect of preventing adhesion of living tissue.
[0306] (実施例 22 :他の高分子による生体由来組織の癒着防止効果） (Example 22: Effect of preventing adhesion of biological tissue by other polymer)
次に、同様の効果が、血管、腸管または尿管由来の膜、および心膜、腸間膜、角膜 、網膜にも現れるかどうかを確認する。実施例 1および 10〜20と同様の実験を行う。 これらをポリエチレングリコール、ポリビニルアルコール、ポリビニルピロリドン、 γ—ポ リグルタミン酸およびゼラチンを用いて生体適合性高分子処理および γ線照射処理 をする。この強化生体由来膜についても、同様の癒着防止効果を確認することがで きる。  Next, we will check whether similar effects appear in blood vessels, intestinal or ureteral membranes, and in the pericardium, mesentery, cornea, and retina. Experiments similar to Examples 1 and 10-20 are performed. These are subjected to biocompatible polymer treatment and γ-irradiation treatment using polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, γ-polyglutamic acid and gelatin. The same anti-adhesion effect can be confirmed for this reinforced biological membrane.
[0307] (実施例 23 :他の架橋剤による生体由来組織の癒着防止効果）  [0307] (Example 23: Effect of preventing adhesion of biological tissue by other crosslinking agent)
次に、同様の効果が、シアンイミド（cyanimide)、 1—ェチル—3— (3 ジメチルァ ミノプロピル）カルボジイミドヒドロクロリド（EDC)、エポキシ（ポリ（グリシジルエーテル） )、 2, 6 ビス（4 アジドベンジリデン）一 4—メチルシクロへキサノン（BAMC)、 4, 4'ジアジドジフ ニルエーテル、 4, 4'ジアジドジフ ニルスルホン、 4, 4'ジアジドジ フエニルアセトン、 4, 4'ジアジドジフエニルメタン、 1, 4 ビス（α ジァゾベンジル） および 4—[ρ—アジドサリチアミド]プチルァミンを架橋剤として用いた場合にも現れ るかどうかを確認する。実施例 1および 10〜20と同様の実験を行う。これらをポリェチ レングリコール、ポリビュルアルコール、ポリビュルピロリドン、 Ί ポリグルタミン酸お よびゼラチンを用いて生体適合性高分子処理および γ線照射処理をする。この強化 生体由来膜についても、同様の癒着防止効果を確認することができる。 Next, the same effect can be obtained by using cyanimide, 1-ethyl-3- (3 dimethylaminopropyl) carbodiimide hydrochloride (EDC), epoxy (poly (glycidyl ether)), 2,6 bis (4 azidobenzylidene). 4-methylcyclohexanone (BAMC), 4,4'diazidodiphenyl ether, 4,4'diazidodiphenylsulfone, 4,4'diazidodiphenylacetone, 4,4'diazidodiphenylmethane, 1,4 bis (α diazobenzyl) And 4- [ρ-azidosalicyamido] plutamine as a crosslinker. Experiments similar to Examples 1 and 10-20 are performed. These are subjected to biocompatible polymer treatment and γ-irradiation treatment using polyethylene glycol, polybulal alcohol, polybulurpyrrolidone , Ίpolyglutamic acid and gelatin. The same anti-adhesion effect can be confirmed for this reinforced biological membrane.
[0308] 以上のように、本発明の好ましい実施形態を用いて本発明を例示してきた力 本発 明は、この実施形態に限定して解釈されるべきものではない。本発明は、特許請求 の範囲によってのみその範囲が解釈されるべきであることが理解される。当業者は、 本発明の具体的な好ましい実施形態の記載から、本発明の記載および技術常識に 基づいて等価な範囲を実施することができることが理解される。本明細書において引 用した特許、特許出願および文献は、その内容自体が具体的に本明細書に記載さ れているのと同様にその内容が本明細書に対する参考として援用されるべきであるこ とが理解される。 [0308] As described above, the power of the present invention exemplified by the preferred embodiment of the present invention. The present invention should not be construed as being limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. Those skilled in the art will understand the description of the present invention and the common general knowledge from the description of specific preferred embodiments of the present invention. It is understood that equivalent ranges can be implemented on the basis. Patents, patent applications and literature references cited in this specification should be incorporated by reference as if the contents themselves were specifically described in the present specification. Is understood.
産業上の利用可能性 Industrial applicability
本発明により、組織損傷率を臨床適用可能な程度に抑えつつ、組織強度を顕著に 上昇させ、石灰化の顕著な抑制を達成する技術の改良法が確立された。この技術に よって調製された生体由来組織およびグラフトは、さらに強化されている。従って、こ のような組織は、産業上有用である。本発明により、石灰化、炎症反応、不必要な細 胞の遊走または接着、血小板凝集、血液凝固の促進などの不都合な反応を引き起こ すことなぐ癒着を防止することが可能な癒着防止膜が開発された。この癒着防止膜 は、細胞種にかかわらず非特異的に癒着防止能を発揮し、従来技術と同等またはそ れ以上の癒着防止効果を有する。このような癒着防止膜は、外科手術および処置の 分野において産業上有用である。  According to the present invention, an improved method of a technique has been established that significantly increases tissue strength and achieves remarkable suppression of calcification while suppressing the tissue damage rate to a level that can be clinically applied. Biological tissues and grafts prepared by this technique are further strengthened. Therefore, such an organization is industrially useful. According to the present invention, there is provided an anti-adhesion membrane capable of preventing adhesion without causing adverse reactions such as calcification, inflammatory reaction, unnecessary cell migration or adhesion, platelet aggregation, and promotion of blood coagulation. It has been developed. This anti-adhesion membrane exhibits an anti-adhesion ability non-specifically regardless of the cell type, and has an anti-adhesion effect equivalent to or higher than that of the prior art. Such anti-adhesion membranes are industrially useful in the fields of surgery and treatment.

Claims

請求の範囲 The scope of the claims
[I] 生体適合性高分子を含み、 γ線照射を受けたことを特徴とする生体由来組織。  [I] A biological tissue containing a biocompatible polymer and receiving γ-ray irradiation.
[2] 前記生体由来組織は、脱細胞化処理を受けて 、な 、ことを特徴とする、請求項 1に 記載の生体由来組織。  [2] The living tissue according to claim 1, wherein the living tissue is subjected to a decellularization treatment.
[3] 細胞外マトリクス成分が少なくとも一部が共有結合で架橋されていることを特徴とする [3] The extracellular matrix component is at least partially crosslinked by a covalent bond
、請求項 1に記載の生体由来組織。 The biological tissue according to claim 1.
[4] 前記細胞外マトリックス成分は、コラーゲン、エラスチン、ラミニン、フイブロネクチン、 テネイシン、グリコサミノダリカンおよびプロテオダリカン力もなる群より選択される、請 求項 3に記載の生体由来組織。 [4] The biological tissue according to claim 3, wherein the extracellular matrix component is selected from the group consisting of collagen, elastin, laminin, fibronectin, tenascin, glycosaminodarican and proteodarican.
[5] 前記生体適合性高分子は、前記生体由来組織をコーティングする、請求項 1に記載 の生体由来組織。 [5] The biological tissue according to claim 1, wherein the biocompatible polymer coats the biological tissue.
[6] 前記生体適合性高分子は、前記生体由来組織に架橋されている、請求項 1に記載 の生体由来組織。  6. The biological tissue according to claim 1, wherein the biocompatible polymer is crosslinked to the biological tissue.
[7] 前記 γ線照射は、 10kGy〜250kGyの間の線量で提供される、請求項 1に記載の 生体由来組織。  [7] The biological tissue according to claim 1, wherein the gamma irradiation is provided at a dose between 10 kGy and 250 kGy.
[8] 前記 γ線照射は、 25kGy〜50kGyの間の線量で提供される、請求項 1に記載の生 体由来組織。  [8] The biological tissue according to claim 1, wherein the γ-ray irradiation is provided at a dose between 25 kGy and 50 kGy.
[9] 前記生体適合性高分子は、生分解性である、請求項 1に記載の生体由来組織。  [9] The biological tissue according to claim 1, wherein the biocompatible polymer is biodegradable.
[10] 前記生体適合性高分子は、ポリビュルアルコール (PVA)、ポリビュルピロリドン (PV P)、エラスチン、ポリエチレングリコール（PEG)、ゼラチン、コラーゲン、 γ -ポリグル タミン酸およびそれらの 2つ以上の混合物力 なる群より選択される高分子を含む、 請求項 1に記載の生体由来組織。 [10] The biocompatible polymer may be polybulal alcohol (PVA), polybulurpyrrolidone (PV P), elastin, polyethylene glycol (PEG), gelatin, collagen, γ-polyglutamic acid, or two or more thereof. The biological tissue according to claim 1, comprising a polymer selected from the group consisting of mixture power.
[II] 前記生体適合性高分子は、ポリエチレングリコール (PEG)を含む、請求項 1に記載 の生体由来組織。  [II] The biological tissue according to claim 1, wherein the biocompatible polymer contains polyethylene glycol (PEG).
[12] 前記ポリエチレングリコール（PEG)は、分子量 1, 000〜200, 000の範囲内にあ る、請求項 11に記載の生体由来組織。  12. The biological tissue according to claim 11, wherein the polyethylene glycol (PEG) has a molecular weight in the range of 1,000 to 200,000.
[13] 前記ポリエチレングリコール（PEG)は、分子量 8, 000力ら 50, 000の範囲内にある[13] The polyethylene glycol (PEG) has a molecular weight in the range of 8,000 forces to 50,000.
、請求項 11に記載の生体由来組織。 The biological tissue according to claim 11.
[14] 前記生体由来糸且織は、臨床適用することができる糸且織強度を有する、請求項 1に記 載の生体由来組織。 14. The living body-derived tissue according to claim 1, wherein the living body-derived yarn and weaving has a thread and weaving strength that can be clinically applied.
[15] 前記生体由来組織は、移植したときに石灰化が顕著に抑制される、請求項 1に記載 の生体由来組織。  15. The biological tissue according to claim 1, wherein calcification is significantly suppressed when transplanted in the biological tissue.
[16] 前記生体適合性高分子は、ランダムに架橋されていることを特徴とする、請求項 1に 記載の生体由来組織。  16. The biological tissue according to claim 1, wherein the biocompatible polymer is randomly crosslinked.
[17] 前記生体由来組織は、膜状組織、弁状組織または管状組織である、請求項 1に記載 の生体由来組織。  17. The biological tissue according to claim 1, wherein the biological tissue is a membranous tissue, a valve-like tissue, or a tubular tissue.
[18] 前記生体由来組織は、血管、血管様組織、心臓弁、心膜、硬膜、角膜および骨から 選択されるものの組織である、請求項 1に記載の生体由来組織。  18. The biological tissue according to claim 1, wherein the biological tissue is a tissue selected from blood vessels, blood vessel-like tissues, heart valves, pericardia, dura mater, corneas and bones.
[19] 前記生体由来組織は、哺乳動物由来である、請求項 1に記載の生体由来組織。 [19] The biological tissue according to claim 1, wherein the biological tissue is derived from a mammal.
[20] 前記生体由来組織は、ヒト、ゥシまたはブタ由来である、請求項 1に記載の生体由来 組織。 [20] The biological tissue according to claim 1, wherein the biological tissue is derived from human, ushi or pig.
[21] 請求項 1に記載の生体由来組織を含む、移植用の組織グラフト。  [21] A tissue graft for transplantation, comprising the biological tissue according to claim 1.
[22] 前記組織グラフトは、膜状、管状および弁状からなる群より選択される形状を有する、 請求項 21に記載の組織グラフト。  22. The tissue graft according to claim 21, wherein the tissue graft has a shape selected from the group consisting of a membrane shape, a tubular shape, and a valve shape.
[23] 移植用の組織を生産する方法であって、 [23] A method of producing tissue for transplantation,
A)生体由来組織を提供する工程；  A) providing a biological tissue;
B)生体適合性高分子に該生体由来組織を曝す工程;および  B) exposing the biological tissue to a biocompatible polymer; and
C)該生体由来組織を γ線照射に曝す工程、  C) a step of exposing the biological tissue to gamma irradiation,
を包含する、方法。  Including the method.
[24] 前記 γ線照射は、該生体適合性高分子に化学反応が起こる条件下で行われる、請 求項 23に記載の方法。  [24] The method according to claim 23, wherein the γ-ray irradiation is performed under a condition in which a chemical reaction occurs in the biocompatible polymer.
[25] 前記 γ線照射は、 10kGy〜250kGyの間の線量で提供される、請求項 23に記載の 方法。  [25] The method of claim 23, wherein the gamma irradiation is provided at a dose between 10 kGy and 250 kGy.
[26] 前記 γ線照射は、 25kGy〜50kGyの間の線量で提供される、請求項 23に記載の 方法。  26. The method of claim 23, wherein the gamma irradiation is provided at a dose between 25 kGy and 50 kGy.
[27] 前記 γ線照射は、真空、酸素中、窒素中、大気中、水中、両親媒性分子溶液中およ びそれらの組み合わせ力 なる群より選択される環境下で行われる、請求項 23に記 載の方法。 [27] The γ-ray irradiation may be performed in vacuum, oxygen, nitrogen, air, water, amphiphilic molecule solution, and 24. The method according to claim 23, wherein the method is performed in an environment selected from the group consisting of:
[28] 前記 γ線照射は、 0. 5〜240時間の範囲で行われる、請求項 23に記載の方法。  [28] The method according to claim 23, wherein the gamma irradiation is performed in a range of 0.5 to 240 hours.
[29] 前記生体適合性高分子は、生分解性である、請求項 23に記載の方法。 [29] The method of claim 23, wherein the biocompatible polymer is biodegradable.
[30] 前記生体適合性高分子は、ポリビュルアルコール、ポリビュルピロリドン、エラスチン 、ポリエチレングリコール、ゼラチン、コラーゲン、 γ -ポリグルタミン酸およびそれらの 2つ以上の混合物力 なる群より選択される高分子を含む、請求項 23に記載の方法 [30] The biocompatible polymer may be a polymer selected from the group consisting of polybutanol, polypyrrole pyrrolidone, elastin, polyethylene glycol, gelatin, collagen, γ-polyglutamic acid and a mixture of two or more thereof. 24. The method of claim 23, comprising
[31] 前記生体適合性高分子は、ポリエチレングリコールを含む、請求項 23に記載の方法 [31] The method of claim 23, wherein the biocompatible polymer comprises polyethylene glycol.
[32] 前記ポリエチレングリコールは、分子量 1, 000〜200, 000の範囲内にある、請求項 31に記載の方法。 32. The method of claim 31, wherein the polyethylene glycol is in the molecular weight range of 1,000 to 200,000.
[33] 前記生体適合性高分子は、 1% (w/v)〜50% (w/v)の濃度で使用される、請求 項 23に記載の方法。  [33] The method of claim 23, wherein the biocompatible polymer is used at a concentration of 1% (w / v) to 50% (w / v).
[34] 前記生体由来組織は、血管、血管様組織、心臓弁、心膜、硬膜、角膜および骨から なる群より選択されるものの組織である、請求項 23に記載の方法。  [34] The method according to claim 23, wherein the biological tissue is a tissue selected from the group consisting of blood vessels, blood vessel-like tissues, heart valves, pericardia, dura mater, corneas, and bones.
[35] 前記生体由来組織は、哺乳動物由来である、請求項 23に記載の方法。 [35] The method according to claim 23, wherein the biological tissue is derived from a mammal.
[36] 前記生体由来組織は、ヒト、ゥシまたはブタ由来である、請求項 23に記載の方法。 [36] The method according to claim 23, wherein the biological tissue is derived from human, ushi or pig.
[37] 請求項 27に記載の方法によって得られる、生体由来組織。 [37] A biological tissue obtained by the method according to claim 27.
[38] 糸且織の再生方法であって、 [38] A method for regenerating yarn and weaving,
a)生体適合性高分子を含み、 y線照射を受けたことを特徴とする生体由来組織を 、生体内に提供する工程;および  a) providing a living body-derived tissue containing a biocompatible polymer, characterized by being irradiated with y-rays, into the living body; and
b)該生体内で糸且織再生が生じるに十分な時間インキュベートする工程、 を包含する、方法。  b) incubating for a time sufficient to produce yarn and weave regeneration in the living body.
[39] 前記生体由来組織は、血管、血管様組織、心臓弁、心膜、硬膜、角膜および骨から なる群より選択されるものの組織である、請求項 38に記載の方法。  [39] The method according to claim 38, wherein the biological tissue is a tissue selected from the group consisting of a blood vessel, a blood vessel-like tissue, a heart valve, a pericardium, a dura mater, a cornea, and a bone.
[40] 糸且織グラフトを生産する方法であって、 [40] A method of producing a yarn and woven graft comprising:
A)生体適合性高分子を含み、 y線照射を受けたことを特徴とする生体由来組織を 生体内に提供する工程； A) A biological tissue containing a biocompatible polymer and receiving y-ray irradiation Providing in vivo;
B)該生体由来組織に該生体の自己細胞を侵入させる工程;および  B) allowing the living body's own cells to enter the living tissue; and
C)該細胞の分ィ匕が生じるに十分な時間インキュベートする工程、  C) incubating for a time sufficient for the cell to divide;
を包含する、方法。  Including the method.
[41] 前記生体由来組織は、血管、血管様組織、心臓弁、心膜、硬膜、角膜および骨から なる群より選択されるものの組織である、請求項 40に記載の方法。  [41] The method according to claim 40, wherein the biological tissue is a tissue selected from the group consisting of a blood vessel, a blood vessel-like tissue, a heart valve, a pericardium, a dura mater, a cornea, and a bone.
[42] 前記生体由来組織は、自己由来である、請求項 40に記載の方法。 [42] The method according to claim 40, wherein the biological tissue is autologous.
[43] 前記脱細胞組織は、同種異系宿主由来である、請求項 40に記載の方法。 [43] The method of claim 40, wherein the decellularized tissue is derived from an allogeneic host.
[44] 前記脱細胞組織は、異種宿主由来である、請求項 40に記載の方法。 [44] The method of claim 40, wherein the decellularized tissue is derived from a heterologous host.
[45] 請求項 40に記載の方法によって生産された、組織グラフト。 45. A tissue graft produced by the method of claim 40.
[46] 組織または臓器移植を必要とする力または該危険にある被験体の処置または予防の 方法であって、  [46] A method of treating or preventing a force requiring tissue or organ transplantation or a subject at risk thereof,
A)生体適合性高分子を含み、 y線照射を受けたことを特徴とする生体由来組織、 または該生体由来組織を含む組織グラフトを提供する工程;および  A) providing a biological tissue containing a biocompatible polymer and receiving y-ray irradiation, or a tissue graft containing the biological tissue; and
B)該生体由来組織または組織グラフトを被験体に移植する工程、  B) Transplanting the living tissue or tissue graft into a subject,
を包含する、方法。  Including the method.
[47] 前記生体由来組織は、前記被験体由来である、請求項 46に記載の方法。  [47] The method of claim 46, wherein the biological tissue is derived from the subject.
[48] 前記生体由来組織は、血管、血管様組織、心臓弁、心膜、硬膜、角膜および骨から 選択されるものの組織である、請求項 46に記載の方法。  48. The method according to claim 46, wherein the biological tissue is a tissue selected from blood vessels, blood vessel-like tissues, heart valves, pericardia, dura mater, corneas and bones.
[49] 前記被験体は、哺乳動物である、請求項 46に記載の方法。 [49] The method of claim 46, wherein the subject is a mammal.
[50] 前記被験体は、ヒトである、請求項 46に記載の方法。 [50] The method of claim 46, wherein the subject is a human.
[51] 臓器移植のための医薬であって、 [51] a medicament for organ transplantation,
A)生体適合性高分子を含み、 y線照射を受けたことを特徴とする生体由来組織、 または該生体由来組織を含む組織グラフト、  A) A biological tissue containing a biocompatible polymer and receiving y-ray irradiation, or a tissue graft containing the biological tissue,
を含む、医薬。  Containing a medicament.
[52] 前記生体由来組織は、血管、血管様組織、心臓弁、心膜、硬膜、角膜および骨から 選択されるものの組織である、請求項 51に記載の医薬。  [52] The medicament according to claim 51, wherein the biological tissue is a tissue selected from blood vessels, blood vessel-like tissues, heart valves, pericardia, dura mater, corneas and bones.
[53] 前記生体由来組織は、哺乳動物由来である、請求項 51に記載の医薬。 [53] The medicament according to claim 51, wherein the biological tissue is derived from a mammal.
[54] 前記生体由来組織は、ヒト、ゥシまたはブタ由来である、請求項 51に記載の医薬。 [54] The medicament according to claim 51, wherein the tissue derived from a living body is derived from human, ushi or pig.
[55] 前記生体由来組織は、前記移植を必要とする被験体由来である、請求項 51に記載 の医薬。 [55] The medicament according to claim 51, wherein the biological tissue is derived from a subject in need of the transplant.
[56] 生体適合性高分子を含み、 y線照射を受けたことを特徴とする生体由来組織、また は該生体由来組織を含む組織グラフトの、臓器移植のための医薬を製造するための 使用。  [56] Use of a biological tissue containing a biocompatible polymer and receiving y-ray irradiation, or a tissue graft containing the biological tissue for producing a medicament for organ transplantation .
[57] 化学架橋されたことをさらなる特徴とする、請求項 1に記載の生体由来組織。  [57] The biological tissue according to claim 1, further characterized by being chemically crosslinked.
[58] 前記さらなる特徴は、二官能性分子架橋剤による結合を含む、請求項 57に記載の 生体由来組織。 [58] The biological tissue of claim 57, wherein the additional feature comprises binding by a bifunctional molecular crosslinker.
[59] 前記結合は、スルフヒドリル基、アルデヒド基、カルボ-ル基、スルホ基および-トロ基 からなる群より選択される基の間で形成される結合、炭素 炭素結合、ペプチド結合 、エーテル結合ならびにエステル結合力 なる群より選択される結合力 なる群より選 択される、請求項 58に記載の生体由来組織。  [59] The bond includes a bond formed between groups selected from the group consisting of a sulfhydryl group, an aldehyde group, a carbo group, a sulfo group, and a -tro group, a carbon-carbon bond, a peptide bond, an ether bond, and 59. The biological tissue according to claim 58, wherein the tissue is selected from the group consisting of binding forces selected from the group consisting of ester binding forces.
[60] 前記二官能性分子架橋剤が、アミノ基、カルボキシル基およびアルデヒド基力 なる 群より選択される官能基を有する、請求項 58に記載の生体由来組織。  [60] The biological tissue according to claim 58, wherein the bifunctional molecular crosslinking agent has a functional group selected from the group consisting of an amino group, a carboxyl group, and an aldehyde group.
[61] 前記二官能性分子架橋剤が、ダルタルアルデヒド、シアンイミド (cyanimide)、 1ーェ チル一 3— (3—ジメチルァミノプロピル）カルボジイミドヒドロクロリド（EDC)、エポキシ (ポリ（グリシジルエーテル））、 2, 6 ビス（4 アジドベンジリデン）ー4ーメチルシクロ へキサノン、 4, 4'ジアジドジフエニルエーテル、 4, 4'ジアジドジフエニルスルホン、 4 , 4'ジアジドジフエニルアセトン、 4, 4'ジアジドジフエニルメタン、 1, 4 ビス（α ジ ァゾベンジル)および 4— [ρ アジドサリチアミド]プチルァミン力もなる群より選択さ れる、請求項 58に記載の生体由来組織。  [61] The bifunctional molecular crosslinking agent is selected from the group consisting of dartalaldehyde, cyanimide, 1-ethyl-1- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), epoxy (poly (glycidyl ether)) ), 2, 6 bis (4azidobenzylidene) -4-methylcyclohexanone, 4,4'diazidodiphenyl ether, 4,4'diazidodiphenylsulfone, 4,4'diazidodiphenylacetone, 4, 4 59. The biological tissue according to claim 58, wherein the tissue is selected from the group consisting of 'diazidodiphenylmethane, 1,4 bis (α diazobenzyl) and 4- [ρ azidosalicyamido] ptylamine force.
[62] 前記二官能性分子が、ダルタルアルデヒドである、請求項 61に記載の生体由来組織  62. The biological tissue according to claim 61, wherein the bifunctional molecule is dartalaldehyde.
[63] 前記生体由来組織を、ダルタルアルデヒドにより化学的に処理し、ポリエチレングリコ ール (PEG)に曝露し、 γ線を照射して架橋を形成することにより、ダルタルアルデヒ ド処理をしたものに比べて γ線照射後の組織強度が上昇している力、またはグルタ ルアルデヒド処理をしたものと同等の組織強度を有する生体由来組織であって、 該生体由来組織は、 PEGを内包し、かつ該生体由来組織の表面には PEG層が形 成されている、請求項 1に記載の生体由来組織。 [63] The biological tissue is chemically treated with dartal aldehyde, exposed to polyethylene glycol (PEG), and irradiated with γ rays to form a crosslink, thereby forming a dartal aldehyde treatment. Compared to the force that increases the tissue strength after γ-irradiation or the tissue strength equivalent to that treated with glutaraldehyde, 2. The biological tissue according to claim 1, wherein the biological tissue contains PEG, and a PEG layer is formed on the surface of the biological tissue.
[64] カルボキシル基、ヒドロキシル基およびアミノ基以外の非化学架橋基の間に形成され る架橋を少なくとも 1つ有する、請求項 1に記載の生体由来組織。 64. The biological tissue according to claim 1, having at least one crosslink formed between non-chemical crosslinkable groups other than a carboxyl group, a hydroxyl group and an amino group.
[65] 前記非化学架橋基が、スルフヒドリル基、アルデヒド基、カルボニル基、スルホ基およ び-トロ基力 なる群より選択される、請求項 64に記載の生体由来組織。 [65] The biological tissue according to claim 64, wherein the non-chemical crosslinking group is selected from the group consisting of a sulfhydryl group, an aldehyde group, a carbonyl group, a sulfo group, and a -tro group force.
[66] γ線照射により生じた、炭素 炭素結合、エーテル結合およびエステル結合からな る群より選択される結合により形成される架橋が少なくとも 1つ有する、請求項 1に記 載の生体由来組織。 [66] The biological tissue according to claim 1, having at least one cross-link formed by γ-ray irradiation and formed by a bond selected from the group consisting of a carbon-carbon bond, an ether bond and an ester bond.
[67] 骨格が開裂して形成された新たな結合により形成された架橋を少なくとも 1つ有する [67] have at least one bridge formed by a new bond formed by cleavage of the skeleton
、請求項 1に記載の生体由来組織。 The biological tissue according to claim 1.
[68] 側鎖に遊離のアミノ基およびカルボキシル基を有さないアミノ酸の間に形成された非 化学架橋を少なくとも 1つ有する、請求項 1に記載の生体由来組織。 [68] The biological tissue according to claim 1, having at least one non-chemical bridge formed between amino acids having no free amino group and no carboxyl group in the side chain.
[69] 前記非化学架橋が、リジン、アルギニン以外を架橋点としたアミノ酸間の架橋である、 請求項 68に記載の生体由来組織。 69. The biological tissue according to claim 68, wherein the non-chemical cross-linking is a cross-linking between amino acids having a cross-linking point other than lysine and arginine.
[70] カルボキシル基、ヒドロキシル基およびアミノ基以外の非化学架橋基を有する多糖の 間に形成された架橋を少なくとも 1つ有する、請求項 1に記載の生体由来組織。 [70] The biological tissue according to claim 1, having at least one crosslink formed between polysaccharides having a non-chemical crosslinkable group other than a carboxyl group, a hydroxyl group, and an amino group.
[71] 前記非化学架橋基を有する多糖が、スルフヒドリル基、アルデヒド基、カルボニル基、 スルホ基および-トロ基力 なる群より選択される少なくとも 1つの基の間で形成され る架橋を有する、請求項 70に記載の生体由来組織。 [71] The polysaccharide having a non-chemical crosslinking group has a bridge formed between at least one group selected from the group consisting of a sulfhydryl group, an aldehyde group, a carbonyl group, a sulfo group, and a -tro group. Item 70. The biological tissue according to Item 70.
[72] ビスエポキシド架橋、ジビニルスルフォン架橋、分子内エステル化、グルタルアルデヒ ド架橋、カルポジイミド架橋およびヒドラジド架橋以外の非化学架橋を少なくとも 1つ 有する、請求項 1に記載の生体由来組織。 [72] The biological tissue according to claim 1, having at least one non-chemical crosslink other than bisepoxide crosslink, divinylsulfone crosslink, intramolecular esterification, glutaraldehyde crosslink, carpositimide crosslink and hydrazide crosslink.
[73] 組織間の癒着を防止するために使用される、請求項 1に記載の生体由来組織。 [73] The biological tissue according to claim 1, which is used for preventing adhesion between tissues.
[74] 生体内において使用するための、請求項 1に記載の生体由来組織。 [74] The biological tissue according to claim 1, for use in vivo.
[75] 生体外において使用するための、請求項 1に記載の生体由来組織。 [75] The biological tissue according to claim 1, for use in vitro.
[76] 移植用の癒着防止膜を生産する方法であって、 [76] A method of producing an anti-adhesion membrane for transplantation comprising:
Α)生体由来膜を提供する工程； B)該生体由来膜を、生体適合性高分子に曝す工程；および I) a step of providing a biological membrane; B) exposing the biological membrane to a biocompatible polymer; and
C)該生体由来膜を γ線照射に曝す工程、  C) exposing the biological membrane to γ-ray irradiation,
を包含する、方法。  Including the method.
[77] 請求項 76に記載の移植用の癒着防止膜を生産する方法であって、  [77] A method for producing the anti-adhesion membrane for transplantation according to claim 76,
D)前記生体由来膜を二官能性分子架橋剤により化学架橋させる工程をさらに包 含する、方法。  D) A method further comprising the step of chemically crosslinking the biological membrane with a bifunctional molecular crosslinking agent.
[78] 前記 D)工程が、 Α)工程— Β)工程の間、および Β)工程— Α)工程の間からなる群よ り選択される少なくとも 1つの間にて実施される、請求項 77に記載の方法。  [78] The step D) is carried out during at least one selected from the group consisting of Α) step—Β) step and Β) step—Α) step. The method described in 1.
[79] 前記化学架橋が、二官能性分子架橋剤による結合を含む、請求項 77に記載の方法 [79] The method of claim 77, wherein the chemical cross-linking comprises binding by a bifunctional molecular cross-linking agent.
[80] 前記結合は、スルフヒドリル基、アルデヒド基、カルボ-ル基、スルホ基および-トロ基 からなる群より選択される基の間で形成される結合、炭素 炭素結合、ペプチド結合 、エーテル結合ならびにエステル結合力 なる群より選択される結合力 なる群より選 択される、請求項 79に記載の方法。 [80] The bond includes a bond formed between groups selected from the group consisting of a sulfhydryl group, an aldehyde group, a carbo group, a sulfo group, and a -tro group, a carbon-carbon bond, a peptide bond, an ether bond, and 80. The method of claim 79, wherein the method is selected from the group consisting of bond strengths selected from the group consisting of ester bond strengths.
[81] 前記二官能性分子架橋剤が、アミノ基、カルボキシル基およびアルデヒド基力 なる 群より選択される官能基を有する、請求項 79に記載の方法。  81. The method according to claim 79, wherein the bifunctional molecular crosslinking agent has a functional group selected from the group consisting of an amino group, a carboxyl group, and an aldehyde group.
[82] 前記二官能性分子架橋剤が、ダルタルアルデヒド、シアンイミド (cyanimide)、 1ーェ チル一 3— (3—ジメチルァミノプロピル）カルボジイミドヒドロクロリド（EDC)、エポキシ (ポリ（グリシジルエーテル））、 2, 6 ビス（4 アジドベンジリデン）ー4ーメチルシクロ へキサノン、 4, 4'ジアジドジフエニルエーテル、 4, 4'ジアジドジフエニルスルホン、 4 , 4'ジアジドジフエニルアセトン、 4, 4'ジアジドジフエニルメタン、 1, 4 ビス（α ジ ァゾベンジル)および 4— [ρ アジドサリチアミド]プチルァミン力もなる群より選択さ れる、請求項 79に記載の方法。  [82] The bifunctional molecular crosslinking agent is selected from the group consisting of dartalaldehyde, cyanimide, 1-ethyl-1- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), epoxy (poly (glycidyl ether)) ), 2, 6 bis (4azidobenzylidene) -4-methylcyclohexanone, 4,4'diazidodiphenyl ether, 4,4'diazidodiphenylsulfone, 4,4'diazidodiphenylacetone, 4, 4 80. The method of claim 79, wherein the method is also selected from the group consisting of: diazidodiphenylmethane, 1,4 bis (α diazobenzyl) and 4- [ρazidosalicyamido] ptylamine power.
[83] 前記二官能性分子架橋剤が、ダルタルアルデヒドである、請求項 82に記載の方法。  [83] The method of claim 82, wherein the bifunctional molecular crosslinking agent is dartalaldehyde.
[84] 前記生体適合性高分子は、ポリビュルアルコール、ポリビュルピロリドン、エラスチン 、ポリエチレングリコール、ゼラチン、コラーゲン、 γ -ポリグルタミン酸およびそれらの 2つ以上の混合物力 なる群より選択される高分子を含む、請求項 76に記載の方法 [84] The biocompatible polymer may be a polymer selected from the group consisting of polybulal alcohol, polybulurpyrrolidone, elastin, polyethylene glycol, gelatin, collagen, γ-polyglutamic acid and a mixture of two or more thereof. 77. The method of claim 76, comprising
[85] 前記生体適合性高分子は、ポリエチレングリコールを含む、請求項 76に記載の方法 [85] The method of claim 76, wherein the biocompatible polymer comprises polyethylene glycol.
[86] 前記 A)工程において、前記生体由来膜は、膜状組織、弁状組織または管状組織か ら採取される、請求項 76に記載の方法。 [86] The method according to claim 76, wherein in the step A), the biological membrane is collected from a membranous tissue, a valve-like tissue or a tubular tissue.
[87] 前記 A)工程において、前記生体由来膜は、血管、血管様組織、心臓弁、心膜、硬 膜、角膜および骨から採取される、請求項 76に記載の方法。 [87] The method according to claim 76, wherein in the step A), the biological membrane is collected from a blood vessel, a blood vessel-like tissue, a heart valve, a pericardium, a dura mater, a cornea, and a bone.
[88] 前記 A)工程において、前記生体由来膜は、哺乳動物力 採取される、請求項 76に 記載の方法。 [88] The method according to claim 76, wherein in the step A), the biological membrane is collected by mammalian force.
[89] 前記 A)工程にぉ 、て、前記癒着防止膜は、ヒト、ゥシまたはブタ由来である、請求項 76に記載の方法。  [89] The method according to claim 76, wherein, in the step A), the anti-adhesion film is derived from human, ushi or pig.
[90] 前記 B)工程において、前記生体由来膜に対する前記生体適合性高分子の曝露が 、 24°Cにて、 12— 24時間という条件下で実施される、請求項 76に記載の方法。  [90] The method according to claim 76, wherein in the step B), the exposure of the biocompatible polymer to the biological membrane is performed at 24 ° C. under a condition of 12 to 24 hours.
[91] 前記 C)工程において、 γ線の強度は 50keVであり、 γ線の照射は、密閉容器中、 線量 12— 50kGyで、 24— 37°Cという条件下で実施される、請求項 76に記載の方 法。  [91] In the step C), the intensity of the γ-ray is 50 keV, and the irradiation of the γ-ray is performed in a sealed container at a dose of 12 to 50 kGy under a condition of 24 to 37 ° C. The method described in.
[92] 前記化学架橋が、 24°Cにて、 1〜4時間という条件下で生じる、請求項 77に記載の 方法。  [92] The method of claim 77, wherein the chemical crosslinking occurs at 24 ° C under conditions of 1 to 4 hours.
[93] 生体適合性高分子を含み、 γ線照射を受けたことを特徴とする癒着防止膜であって [93] An anti-adhesion membrane characterized by containing a biocompatible polymer and receiving γ-ray irradiation.
、該癒着防止膜は生体由来である、癒着防止膜。 The anti-adhesion membrane is derived from a living body.
[94] カルボキシル基、ヒドロキシル基およびアミノ基以外の非化学架橋基の間に形成され る架橋を少なくとも 1つ有する、請求項 93に記載の癒着防止膜。 [94] The adhesion-preventing membrane according to [93], which has at least one crosslink formed between non-chemical crosslinkable groups other than a carboxyl group, a hydroxyl group, and an amino group.
[95] 前記非化学架橋基が、スルフヒドリル基、アルデヒド基、カルボニル基、スルホ基およ び-トロ基力もなる群より選択される、請求項 94に記載の癒着防止膜。 [95] The adhesion-preventing membrane according to item 94, wherein the non-chemical crosslinking group is selected from the group consisting of a sulfhydryl group, an aldehyde group, a carbonyl group, a sulfo group, and a -tro group force.
[96] γ線照射により生じた、炭素 炭素結合、エーテル結合およびエステル結合からな る群より選択される結合により形成される架橋を少なくとも 1つ有する、請求項 93に記 載の癒着防止膜。 [96] The adhesion-preventing film according to [93], which has at least one cross-link formed by γ-ray irradiation and formed by a bond selected from the group consisting of a carbon-carbon bond, an ether bond and an ester bond.
[97] 骨格を形成して ヽる炭素間結合、エステル結合が開裂して形成された新たな結合に より形成された架橋を少なくとも 1つ有する、請求項 93に記載の癒着防止膜。 [97] The adhesion-preventing membrane according to [93], which has at least one cross-link formed by a new bond formed by cleavage of an intercarbon bond and an ester bond formed by forming a skeleton.
[98] 側鎖に遊離のアミノ基およびカルボキシル基を有さないアミノ酸の間に形成された非 化学架橋を少なくとも 1つ有する、請求項 93に記載の癒着防止膜。 [98] The adhesion-preventing membrane according to item 93, which has at least one non-chemical crosslink formed between amino acids having no free amino group and no carboxyl group in the side chain.
[99] 前記非化学架橋が、リジン、アルギニン以外を架橋点としたアミノ酸間の架橋である、 請求項 98に記載の癒着防止膜。 99. The adhesion-preventing membrane according to claim 98, wherein the non-chemical cross-linking is a cross-linking between amino acids having cross-linking points other than lysine and arginine.
[100] カルボキシル基、ヒドロキシル基およびアミノ基以外の非化学架橋基を有する多糖の 間に形成された架橋を少なくとも 1つ有する、請求項 93に記載の癒着防止膜。 [100] The adhesion-preventing membrane according to [93], which has at least one crosslink formed between polysaccharides having non-chemical crosslinkable groups other than a carboxyl group, a hydroxyl group, and an amino group.
[101] 前記非化学架橋基を有する多糖が、スルフヒドリル基、アルデヒド基、カルボニル基、 スルホ基および-トロ基力 なる群より選択される少なくとも 1つの基の間で形成され る架橋を有する、請求項 100に記載の癒着防止膜。 [101] The polysaccharide having a non-chemical crosslinking group has a bridge formed between at least one group selected from the group consisting of a sulfhydryl group, an aldehyde group, a carbonyl group, a sulfo group, and a -tro group. Item 100. The adhesion-preventing membrane according to Item 100.
[102] ビスエポキシド架橋、ジビニルスルフォン架橋、分子内エステル化、グルタルアルデヒ ド架橋、カルポジイミド架橋およびヒドラジド架橋以外の非化学架橋を少なくとも 1つ 有する、請求項 93に記載の癒着防止膜。 [102] The adhesion-preventing membrane according to [93], which has at least one non-chemical crosslink other than a bisepoxide crosslink, divinylsulfone crosslink, intramolecular esterification, glutaraldehyde crosslink, carpositimide crosslink and hydrazide crosslink.
[103] 側鎖に遊離アミノ基またはカルボキシル基を有しない 2以上のアミノ酸の間に形成さ れる共有結合、カルボキシル基、ヒドロキシル基、アミノ基、スルフヒドリル基、アルデヒ ド基およびカルボ-ル基からなる群より選択される基の間で形成される結合、炭素 炭素結合、ペプチド結合、エーテル結合ならびにエステル結合力 なる群より選択さ れる結合を有する、請求項 93に記載の癒着防止膜。 [103] Consists of covalent bond, carboxyl group, hydroxyl group, amino group, sulfhydryl group, aldehyde group, and carbocycle group formed between two or more amino acids having no free amino group or carboxyl group in the side chain 94. The adhesion-preventing membrane according to claim 93, which has a bond selected from the group consisting of a bond formed between groups selected from the group, a carbon-carbon bond, a peptide bond, an ether bond, and an ester bond strength.
[104] 二官能性分子架橋剤による結合により形成される架橋をさらに含む、請求項 93に記 載の癒着防止膜。 [104] The adhesion-preventing membrane according to [93], further comprising a crosslink formed by bonding with a bifunctional molecular crosslinker.
[105] 前記二官能性分子架橋剤が、ダルタルアルデヒドである、請求項 104に記載の癒着 防止膜。  [105] The adhesion-preventing membrane according to claim 104, wherein the bifunctional molecular crosslinking agent is dartalaldehyde.
[106] 前記癒着防止膜が、ポリエチレングリコールを含む、請求項 93に記載の癒着防止膜  [106] The adhesion-preventing film according to claim 93, wherein the adhesion-preventing film comprises polyethylene glycol.
[107] 前記癒着防止膜が、胸膜、心膜、脳硬膜、漿膜、腹膜および皮膚からなる群より選択 される、請求項 93に記載癒着防止膜。 [107] The adhesion-preventing membrane according to item 93, wherein the adhesion-preventing membrane is selected from the group consisting of the pleura, pericardium, brain dura mater, serosa, peritoneum and skin.
[108] 前記癒着防止膜は、哺乳動物由来である、請求項 93に記載癒着防止膜。 108. The adhesion prevention film according to claim 93, wherein the adhesion prevention film is derived from a mammal.
[109] 前記癒着防止膜は、ヒト、ゥシまたはブタ由来である、請求項 93に記載癒着防止膜。 [109] The adhesion-preventing film according to Claim 93, wherein the adhesion-preventing film is derived from human, ushi or pig.
[110] 前記癒着防止膜は、以下： A) [110] The adhesion-preventing film includes the following: A)
1)プラスチック製の薄いシートに心膜を挟み込む工程、  1) The process of sandwiching the pericardium in a thin plastic sheet,
2)サンプルが変形しないように電子ノギスにて 3ケ所以上の厚みを測定する工程、 および  2) A process of measuring the thickness of three or more locations with an electronic caliper so that the sample does not deform, and
3)該サンプルの厚みの平均を算出する工程により測定した場合、 0. 5- 1. Omm の厚みを有すること；ならびに  3) have a thickness of 0.5-1. Omm when measured by the process of calculating the average thickness of the sample; and
B)  B)
1)検体を 5 X 30mmの短冊状に切断する工程、  1) Cutting the specimen into 5 x 30mm strips,
2)引っ張り試験器の固定部分に該検体の両端約 5mmを固定する工程、および 2) fixing about 5 mm at both ends of the specimen to the fixed part of the tensile tester; and
3)引っ張り開始し破断点まで lcmZminの速度で引っ張り、レジオメーターにて測 定する工程により測定した場合、 2— lOMPaの弾性率を有すること 3) It has a modulus of elasticity of 2-lOMPa when it is measured by the process of measuring with a radiometer after pulling to the breaking point at a speed of lcmZmin.
を特徴とする、請求項 93に記載の癒着防止膜。  94. The adhesion-preventing membrane according to claim 93, wherein
[111] 前記癒着防止膜は、試験部位に該癒着防止膜を貼付けた場合に、以下の式： 癒着防止率 (％) = {癒着なしの数 Z (癒着あり +癒着なしの数） } X 100 [111] When the anti-adhesion film is affixed to the test site, the following formula: Adhesion prevention rate (%) = {number without adhesion Z (number with adhesion + number without adhesion)} X 100
により、 80%以上の癒着防止率を有する、請求項 93に記載の癒着防止膜。  94. The adhesion-preventing membrane according to claim 93, having an adhesion-preventing rate of 80% or more.
[112] 生体内において使用するための、請求項 93に記載の癒着防止膜。 [112] The adhesion-preventing membrane according to claim 93, for use in vivo.
[113] 生体外において使用するための、請求項 93に記載の癒着防止膜。 [113] The adhesion-preventing membrane according to claim 93, which is used in vitro.
[114] 前記生体由来膜を、ダルタルアルデヒドにより化学的に処理し、ポリエチレングリコー ル (PEG)に曝露し、 γ線を照射して架橋を形成することにより、ダルタルアルデヒド 処理をしたものに比べて γ線照射後の組織強度が上昇しているか、またはダルタル アルデヒド処理をしたものと同等の組織強度を有する生体由来膜であって、 該癒着防止は、 PEGを内包し、かつ該生体由来膜の表面には PEG層が形成されて いる、請求項 93に記載の生体由来膜。 [114] The biological membrane was chemically treated with dartalaldehyde, exposed to polyethylene glycol (PEG), and irradiated with γ rays to form a crosslink, thereby forming a dartalaldehyde-treated one. Compared with a tissue-derived membrane having increased tissue strength after γ-irradiation or having a tissue strength equivalent to that treated with dartalaldehyde, the adhesion prevention includes PEG and is derived from the organism 94. The biological membrane according to claim 93, wherein a PEG layer is formed on the surface of the membrane.
[115] 前記 B)工程が、癒着を防ぐ必要のある部位において実施される、請求項 46に記載 の処置または予防の方法。 [115] The method of treatment or prevention according to claim 46, wherein the step B) is performed at a site where adhesion needs to be prevented.
[116] 組織の癒着を防ぐ必要がある力または該危険にある被験体の処置または予防の方 法であって、 [116] A force that needs to prevent tissue adhesions or a method of treating or preventing a subject at risk, comprising:
A)生体適合性高分子を含み、 γ線照射を受けたことを特徴とする癒着防止膜を提 供する工程;および A) Providing anti-adhesion membranes that contain biocompatible polymers and are characterized by receiving gamma irradiation Providing steps; and
B)該癒着防止膜を被験体に移植する工程、 を包含する、方法。  B) Transplanting the adhesion-preventing membrane into a subject.
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