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WO2006137546A1 - Méthode de traitement pour empêcher la calcification de tissu de transplantation d’origine biologique et tissu ainsi traité - Google Patents

Méthode de traitement pour empêcher la calcification de tissu de transplantation d’origine biologique et tissu ainsi traité 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
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/JP2006/312662
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English (en)
Japanese (ja)
Inventor
Satoshi Taketani
Eiichiro Uchimura
Masayuki Hara
Masakazu Furuta
Tadahiro Matsumoto
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cardio Inc
Osaka Metropolitan University
Original Assignee
Osaka Prefecture University PUC
Cardio Inc
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Publication date
Application filed by Osaka Prefecture University PUC, Cardio Inc filed Critical Osaka Prefecture University PUC
Publication of WO2006137546A1 publication Critical patent/WO2006137546A1/fr
Anticipated expiration legal-status Critical
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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

L’invention permet d’obtenir un tissu pour transplantation sans les problèmes qui accompagnent la transplantation tels que la calcification. Elle permet aussi d’obtenir une membrane pour empêcher l’adhésion grâce à laquelle les adhésions causées par une intervention ou traitement chirurgical peuvent être évitées. Il a été découvert que, lorsqu’un polymère biocompatible est ajouté à un tissu non traité et que par la suite le tissu est exposé aux rayons Ϝ, le tissu est fortifié de façon inattendue et la calcification est empêchée, résolvant ainsi les problèmes précités. Bien qu’il ait été reconnu que la décellularisation est le facteur clé pour empêcher un tissu d’origine biologique de se calcifier, il est indiqué que l'irradiation aux rayons Ϝ est plus efficace que la décellularisation. À savoir, une membrane est obtenue pour empêcher l’adhésion, grâce à laquelle des adhésions peuvent être empêchées sans induire de réaction gênante (par exemple : calcification, réaction inflammatoire, migration ou adhésion cellulaire inutile, agrégat de plaquettes, accélération de la coagulation du sang, etc.).
PCT/JP2006/312662 2005-06-23 2006-06-23 Méthode de traitement pour empêcher la calcification de tissu de transplantation d’origine biologique et tissu ainsi traité Ceased WO2006137546A1 (fr)

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CN113730656A (zh) * 2021-08-04 2021-12-03 中南大学湘雅二医院 一种生物瓣膜材料及其制备方法
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