JP2008228744A - Treatment method for preventing transplantation tissue with biological origin from calcification and tissue treated thereby - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a tissue for transplantation being free from troubles accompanying transplantation such as calcification. <P>SOLUTION: When a biocompatible polymer is added to an untreated tissue and then the tissue is exposed to γ-ray irradiation, the tissue is unexpectedly strengthened and calcification is prevented, thereby solving the above problems. Although there has been recognized that decellularization is the key factor in preventing a tissue with a biological origin from calcification, it is indicated that γ-ray irradiation is more efficacious than decellularization. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method and system for strengthening an isolated biological tissue (untreated tissue), and a medicine and a treatment method using a tissue, tissue graft and the like prepared by such a strengthening method. Specifically, the present invention relates to a treatment method and a treated tissue for suppressing calcification of a living tissue-derived transplantation tissue.

The main obstacle to using foreign tissue for transplantation of organs (eg, heart, blood vessels, etc.) is immune rejection. The 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 degradation of the extracellular matrix, while the latter is caused by proliferation of intravascular cells (Non-patent Document 6).
Conventionally, two strategies have been adopted with the aim of reducing rejection of these substances. One reduced the host immune response (Non-Patent Document 7; and Non-Patent Document 8). The other attempted to reduce the antigenicity of the allograft or xenograft mainly by cross-linking (Non-patent document 9; and Non-patent document 10). Cross-linking of the extracellular matrix decreases the antigenicity of the graft, but the biomechanical function (Non-patent Document 11; and Non-patent Document 12) changes and becomes sensitive to mineralization (Non-patent Document). 13).

  Cells in the extracellular matrix have histocompatibility class I and II antigens that can cause rejection. In addition, there are cell-derived glycated proteins that can identify the host immune system, which causes rejection. Therefore, rejection of such substances can be prevented by removing them from the extracellular matrix. However, complete removal of all antigens is very difficult and difficult to demonstrate. Malone et al. (Non-patent Document 15) and Lalka et al. (Non-patent Document 16) showed that a matrix transplanted with “cell-free” arterial allografts (grafts into allogeneic animals) also stimulated an immune response. Reported intravascular proliferation and endothelial cell engraftment. More recently, O'Brian et al. Reported that decellularized porcine tissue was applicable to cardiovascular transplantation and did not show hyperacute rejection when implanted in sheep (Non-Patent Document 17). ).

  Cardiovascular disease (including coronary and peripheral vascular disease) has been treated with surgical replacement therapy (the number of cases of cardiovascular disease has increased recently around the world. For small diameter vessels Replacement therapy is difficult to apply: If the diameter is small, bypass surgery uses autologous veins or arterial grafts (18-20). Although the best results are currently available, there are drawbacks such as requiring complicated surgery and being unable to obtain appropriate blood vessels in patients with certain diseases, and as a result, artificial blood vessels suitable for blood vessels with small diameters. To reduce the use of autografts or allografts, developments have been made towards the development of artificial materials, but in the case of artificial materials, peripheral limbs and coronary arteries have been developed. None of the narrow diameter arteries (less than 6 mm) needed for ipas transplant surgery are suitable, whereas in the case of cardiovascular, natural tissue grafts are also being tested as potential biomaterials in clinical applications. The use of xenograft and allograft tissue typically requires chemical or physical treatment (eg, glutaraldehyde fixation). The procedure was also investigated to stabilize the structure and found to be an ideal procedure (Non-patent Document 21).

  However, transplantation has a problem of calcification from a long-term viewpoint. The detrimental side effect of calcification during glutaraldehyde treatment is a major cause of bioprosthetic heart valve defects (Non-Patent Document 22; and Non-Patent Document 23). As an alternative to natural tissue grafts, attempts have been made to produce a completely acellular tissue matrix. This production is by specifically removing cellular components thought to promote calcification and elicit an immunological response. These decellularization techniques include removal of cellular components by chemical, enzymatic, and mechanical means. This treatment leaves a material consisting essentially of extracellular matrix components. These decellularized tissues retain their natural mechanical properties and promote regeneration. Regeneration occurs by the process of neovascularization and recellularization by the host. Surfactant treatment, which is a typical cell extraction method, has been implemented as a means of creating fully decellularized tissue for use as a biomaterial graft. This is because cellular components, lipids, and residual surfactant remaining in the treated tissue can promote undesirable effects such as calcification (Non-patent Document 24; Non-patent Document 25; Non-patent Document 26). And Non-Patent Document 27). Lipid removal from bovine pericardium by treatment with either chloroform / methanol or sodium dodecyl sulfate (SDS) reduced tissue calcification in a rat model (28). Recently, Sunjay et al. Demonstrated that decellularized vascular grafts were cortated by endothelial progenitor cells (EPC). This is because these grafts have mechanical properties similar to natural blood vessels including well-preserved extracellular matrix and arteries (Non-patent Document 29). The researchers isolated endothelial progenitor cells (EPC) from peripheral blood and seeded the EPCs into decellularized bovine ileal vessels. This decellularized graft seeded with EPC developed neovascularization in vivo by day 130, resulting in NO-mediated vascular relaxation.

  As another technique for preventing calcification, an attempt to reduce the calcification action of living tissue for transplantation by polymerizing PEG etc. in situ by UV irradiation or chemical reaction in an untreated tissue was recently published ( WO2005 / 042044 = Patent Document 4). In this publication, examples of the polymerization reaction include: A: a method in which a vinyl monomer, a crosslinking monomer and a photopolymerization initiator are mixed and then UV irradiation; and B: a nucleophilic group such as an amino group or a thiol group. A method of nucleophilic reaction between a polymer such as PEG and a polymer such as PEG having an α, β-unsaturated group is disclosed. As an effect in this case, it is disclosed that the initial inflammatory reaction is suppressed and calcification is suppressed over a long period of time. However, suppression of the inflammatory response is inadequate when considering the actual treatment site, and suppression of calcification also appears to be inadequate, and this technique has drawbacks.

  Further, the method of Patent Document 4 requires a free radical initiator (a polymerization initiator that generates free radicals). As a general rule, if they are transplanted in the body while remaining, there is a possibility of showing toxicity and the like, and there is a high possibility that problems will occur when transplanted in vivo.

  On page 10 of Patent Document 4, the following are generally considered to be harmful when transplanted with various polymerization initiators remaining. (1) thermalinitiators: When the temperature rises, neighboring proteins may denature and become harmful. (2) Peroxy compounds: There is a possibility that it will be harmful by gradually reacting with surrounding proteins and causing crosslinking. (3) Azocompounds: Reacts gradually with nearby proteins, causing cross-linking and other harmful effects. (4) Photo initiators: When exposed to light, there is a possibility that it may cause harmful effects such as cross-linking with surrounding proteins. (5) redoxinitiators: A radical reaction is caused by an oxidation-reduction reaction, and there is a possibility that it may cause harmful effects such as cross-linking with surrounding proteins. As described above, the polymerization initiator has various problems and the risk of calcification remains.

Thus, in this field, there are situations where it is necessary to strengthen the tissue in order to improve biocompatibility, biofixation, etc., such as prevention of calcification. Is sought after.
JP 2002-543950 A JP 2001-78750 WO89 / 05371 WO2005 / 042044 CarrelA., 1907, J Exp Med 9: 226-8 CarrelA., 1912., J Exp Med 9: 389-92 Toshiharu Niioka, Yasuharu Imai, Kazuhiro Seo and others; Development and application of cardiovascular materials by tissue engineering. Japan Cardiovascular Journal 2000; 29: 38 CalneRY., 1970, Transplant Proc 2: 550 Auchincloss1988, Transplantation 46: 1 UretskyBF, Mulari S, Reddy S, et al., 1987, Circulation 76: 827-34 Schmitz-RixenT, Megerman J, Colvin RB, Williams AM, Abbot W., 1988, J Vasc Surg 7: 82-92 Plissonnier D, et al., 1993, Arteriosclerosis Thromb 13: 112-9 Rosenberg N, et al., 1956, Surg Forum 6: 242-6 Dumont C, Pissonnier D, Michel JB., 1993, J Surg Res 54: 61-69 Cosgrove DM, Lytle BW, Golding CC, et al., 1983, J Thorac CardiovascSurgery 64: 172-176 BroomN, Christie GW., 1982, In: Cohn LH, Gallucci V, editors.Cardiac bioprostheses: Proceedingsof the Second International Symposium.New York: York Medical Books Pages 476-491 SchoenFJ, Levy RJ, Piehler HR., Cardiovasc Pathology 1992; 1: 29-52 J ThoracCardiovasc Surg 1998; 115; 536-46 Malone JM, BrendelK, Duhamel RC, Reinert RL., 1984, J Vasc Surg 1: 181-91 Lalka SG, OelkerLM, and Malone JM, et al., 1989, Ann Vasc Surg 3: 108-17 O'Brien MF, etal., 1999 (October), Seminars in Thorac and Cardiovasc Surg; 111 (4), Suppl 1: 194-200 CanverCC., 1995, Chest 1995; 108 1150-1155 BarnerHB., 1998, Ann Thorac Surg 66 (Suppl 5) S2-5; discussion S25-28 (Bhan A, GuptaV, Choudhary SK, etal., 1999, Ann Thorac Surg 1999; 67 1631-1636) Hilbert SL, Ferrans VJ, Jone M., 1988, Med Prog Technol 89; 14,115-163 Rao KP, Shanthi C., 1999, Biomatrials Appl 13: 238-268 Grabenwoger M, Sider J, Fitzal F, et al., 1996, Ann Thorac Surg 62; 772-777 Valente M, Bortolotti U, Thiene G ,, 1985, Am J Pathol 119,12-21 Maranto AR, Shoen FJ, 1988, ASAIO Trans 34,827-830 Courtman DW, Pereira CA, Kashef V, McComb D, Lee JM, Wilson GL, 1994, J BiomedMater Res 28: 655-666 Levy RJ, Schoen FJ, Anderson HC, et al., 1991, Biomaterials 12: 707-714 Jorge-Herrero E, Fernandez P, Gutierrez MP, Castillo-Olivares JL, 1991, Biomaterials 12: 683-689 Sunjay Kaushal, Gilad E. Amiel, Kristine J. Guleserian, et al. 2001, Nature Medicine Vol. 9, 1035-1040

  The transplantable tissue prepared by the conventional technique still has problems in terms of calcification. Many researchers recognize a number of utilities, including the physical and biological properties of the transplanted tissue. However, currently available transplanted tissues have drawbacks such as calcification and immune elicitation and require further improvement (Christine E. Schmidt, Jennie M. Baier., 2000, Biomaterials 21 : 2215-2231). Therefore, 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 γ-ray irradiation, the tissue was unexpectedly strengthened and calcification was suppressed, and the above problem Solved. The inventors have also found an unexpectedly strong reinforcing effect by exposing the tissue to biocompatible polymers and to gamma radiation. Conventionally, it has been recognized that decellularization of a living tissue is a point for suppressing calcification, but it has been shown by the present invention that an effect more than decellularization can be achieved even by γ-ray irradiation.

  Calcification is remarkably suppressed in the living tissue produced in this way. The living body-derived tissue of the present invention can be used, for example, as an artificial blood vessel that can be used permanently. Thus, it can be said that the usefulness of the biological tissue of the present invention is extensive.

  Moreover, the tissue strength of the living tissue reinforced by the present invention is remarkably improved unexpectedly. Such an effect is an unexpected effect that could never be achieved by the prior art, and the fact that it was possible to provide such a reinforced biological tissue and tissue graft was the result of transplantation medicine. It can be said that it will bring about exceptional progress.

Accordingly, the present invention provides the following.
(1) A biological tissue containing a biocompatible polymer and receiving γ-ray irradiation.
(2) The living body tissue according to Item 1, wherein the living body tissue has not undergone a decellularization treatment (3) The extracellular matrix component is at least partially crosslinked by a covalent bond The living body-derived tissue according to item 1, wherein
(4) The biological tissue according to item 3, wherein the extracellular matrix component is selected from the group consisting of collagen, elastin, laminin, fibronectin, tenascin, glycosaminoglycan and proteoglycan.
(5) The living body-derived tissue according to item 1, wherein the biocompatible polymer coats the living body-derived tissue.
(6) The biological tissue according to item 1, wherein the biocompatible polymer is crosslinked to the biological tissue.
(7) The living body tissue according to Item 1, wherein the γ-ray irradiation is provided at a dose between 10 kG and 250 kG.
(8) The living body tissue according to Item 1, wherein the γ-ray irradiation is provided at a dose between 25 kG and 50 kG.
(9) The living body tissue according to Item 1, wherein the biocompatible polymer is biodegradable.
(10) The biocompatible polymer is a group consisting of polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), elastin, polyethylene glycol (PEG), gelatin, collagen, γ-polyglutamic acid and a mixture of two or more thereof. Item 2. The living body tissue according to Item 1, comprising a polymer selected from the group selected from the group.
(11) The living body tissue according to Item 1, wherein the biocompatible polymer contains polyethylene glycol (PEG).
(12) The living body tissue according to Item 11, wherein the polyethylene glycol (PEG) is in a molecular weight range of 1000 to 200,000.
(13) The biological tissue according to item 11, wherein the polyethylene glycol (PEG) is in the range of molecular weight 8,000 to 50,000.
(14) The living body-derived tissue according to item 1, wherein the living body-derived tissue has a tissue strength that can be clinically applied.
(15) The living body tissue according to item 1, wherein the living body tissue is remarkably suppressed in calcification when transplanted.
(16) The biological tissue according to item 1, wherein the biocompatible polymer is crosslinked at random.
(17) The living body tissue according to item 1, wherein the living body tissue is a membranous tissue, a valve-like tissue or a tubular tissue.
(18) The biological tissue according to item 1, wherein the biological tissue is a tissue selected from blood vessels, blood vessel-like tissues, heart valves, pericardia, dura mater, corneas and bones.
(19) The living body tissue according to Item 1, wherein the living body tissue is derived from a mammal.
(20) The living body-derived tissue according to item 1, wherein the living body-derived tissue is derived from a human, cow or pig.
(21) A tissue graft for transplantation including the living tissue according to item 1.
(22) The tissue graft according to item 21, wherein the tissue graft has a shape selected from the group consisting of a membrane, a tube, and a valve.
(23) A method for producing a tissue for transplantation,
A) providing a biological tissue;
B) exposing the biological tissue to the biocompatible polymer; and C) exposing the biological tissue to γ-irradiation;
Including the method.
(24) The method according to item 23, wherein the γ-irradiation is performed under conditions in which a chemical reaction occurs in the biocompatible polymer.
(25) The method according to item 23, wherein the gamma irradiation is provided at a dose between 10 kG and 250 kG.
(26) The method according to item 23, wherein the γ-ray irradiation is provided at a dose between 25 kG and 50 kG.
(27) The γ-ray irradiation is performed in an environment selected from the group consisting of vacuum, oxygen, nitrogen, air, water, amphiphilic molecule solution, and combinations thereof. Method.
(28) The method according to Item 23, wherein the γ-ray irradiation is performed in a range of 0.5 to 240 hours.
(29) A method according to item 23, wherein the biocompatible polymer is biodegradable.
(30) The biocompatible polymer is selected from the group consisting of polyvinyl alcohol, polyvinyl pyrrolidone, elastin, polyethylene glycol, gelatin, collagen, γ-polyglutamic acid and a mixture of two or more thereof. 24. A method according to item 23, comprising a polymer.
(31) A method according to item 23, wherein the biocompatible polymer comprises polyethylene glycol.
(32) The method according to item 31, wherein the polyethylene glycol is in the molecular weight range of 1000 to 200,000.
(33) The method according to item 23, wherein the biocompatible polymer is used at a concentration of 1% (w / v) to 50% (w / v).
(34) The method according to item 23, wherein the biological tissue is a tissue selected from the group consisting of blood vessels, blood vessel-like tissues, heart valves, pericardia, dura mater, corneas and bones.
(35) A method according to item 23, wherein the biological tissue is derived from a mammal.
(36) A method according to item 23, wherein the biological tissue is derived from human, cow or pig.
(37) A biological tissue obtained by the method according to item 27.
(38) A tissue regeneration method,
a) providing a living body-derived tissue containing a biocompatible polymer and receiving γ-ray irradiation into the living body; and b) incubating for a time sufficient to cause tissue regeneration in the living body. Process,
Including the method.
(39) A method according to item 38, wherein the biological tissue is a tissue selected from the group consisting of blood vessels, blood vessel-like tissues, heart valves, pericardia, dura mater, corneas and bones.
(40) A method of producing a tissue graft,
A) a step of providing a living body-derived tissue containing a biocompatible polymer and receiving γ-ray irradiation in vivo;
B) allowing the living body's own cells to invade the living body tissue; and C) incubating the cell for a time sufficient to cause differentiation.
Including the method.
(41) A method according to item 40, wherein the biological tissue is a tissue selected from the group consisting of blood vessels, blood vessel-like tissues, heart valves, pericardia, dura mater, corneas and bones.
(42) A method according to item 40, wherein the biological tissue is self-derived.
(43) A method according to item 40, wherein the decellularized tissue is derived from an allogeneic host.
(44) A method according to item 40, wherein the decellularized tissue is derived from a heterologous host.
(45) A tissue graft produced by the method according to item 40.
(46) A method for the treatment or prophylaxis of a subject who requires a tissue or organ transplant or is at risk.
A) a step of providing a biological tissue containing a biocompatible polymer and receiving γ-ray irradiation, or a tissue graft containing the biological tissue; and B) the biological tissue or tissue graft. Transplanting to a subject;
Including the method.
(47) A method according to item 46, wherein the biological tissue is derived from the subject.
(48) A method according to item 46, wherein the biological tissue is a tissue selected from blood vessels, blood vessel-like tissues, heart valves, pericardia, dura mater, corneas and bones.
(49) A method according to item 46, wherein the subject is a mammal.
(50) A method according to item 46, wherein the subject is a human.
(51) A medicine for organ transplantation,
A) A biological tissue containing a biocompatible polymer and receiving γ-ray irradiation, or a tissue graft containing the biological tissue,
Containing a medicament.
(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.
(53) The medicament according to item 51, wherein the biological tissue is derived from a mammal.
(54) The medicament according to item 51, wherein the living body-derived tissue is derived from human, cow or pig.
(55) The medicament according to item 51, wherein the biological tissue is derived from a subject in need of the transplantation.
(56) Use of a biological tissue containing a biocompatible polymer and receiving γ-ray irradiation or a tissue graft containing the biological tissue for producing a medicament for organ transplantation.

  Accordingly, it is understood that these and other advantages of the present invention will be apparent to those of ordinary skill in the art upon reading and understanding the following detailed description with reference to the accompanying drawings.

  A biological tissue in which calcification is significantly suppressed is provided. As for the suppression of calcification, γ-ray irradiation was remarkably suppressed as compared with other methods such as UV irradiation and scientific treatment. The present invention also provides a living tissue having a reinforced, improved or maintained 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.

  Regarding the mechanism of action, A: inhibition of cell invasion by gelation of PEG or the like, or B: suppression of immunogenicity by modifying a component of a biological tissue containing extracellular matrix (ECM) with PEG, etc. However, the present invention is not limited to this.

  The reason why the polymerization reaction by γ-ray irradiation is more advantageous than the conventional polymerization reaction is expected to be as follows, for example, but is not limited thereto.

  While conventional polymerization reactions are relatively controlled chemical reactions, gamma irradiation is considered to introduce cross-links between PEG molecules relatively randomly, which is a more effective suppression of calcification. It is thought that it is connected to.

  In addition, the present invention overcomes the disadvantages of treatments using conventional free radical initiators, such as the possibility of toxicity when transplanted into the body while remaining in the body. It was. The method of the present invention is based on radiation-induced polymerization initiators (radiation induced initiators) by γ-irradiation. Radiation of macromolecules such as proteins is generated by radiation, but the radical disappears in a relatively short time. There seems to be no residue at all, which is a significant effect.

  Regarding the lifetime of radicals in irradiated biological tissue, the decellularized tissue for transplantation derived from human dermis (trade name Allorderm, Life Cell Corp.) was irradiated with synchrotron radiation, and the ultra-saturation generated in the collagen matrix. When the lifetime radicals were measured by EPR, there was a report that they were almost attenuated within about one hour (RadiationResearch 163, 535-543, 2005). Since it seems to have the same lifetime in the present invention, it is considered that radicals that exert an adverse effect have a remarkable effect in that there is substantially no problem.

  Hereinafter, embodiments of the present invention will be described with respect to the present invention. Throughout this specification, singular representations (e.g., "a", "an", "the" for English, "ein", "der", "das", "die" for German) Etc. and their case changes, such as “un”, “une”, “le”, “la” in French, etc., “un”, “una”, “el”, “la”, etc. in Spanish It is to be understood that the corresponding articles, adjectives, etc. in a language also include the plural concept unless otherwise stated. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

(Definition of terms)
Listed below are definitions of terms particularly used in the present specification.

(Regenerative medicine)
As used herein, “regeneration” refers to a phenomenon in which when a part of an individual's tissue is lost, the remaining tissue grows and is restored. Depending on the tissue species between animal species or in the same individual, the degree and mode of regeneration varies. Many human tissues have limited regenerative ability, and complete regeneration cannot be expected if they are largely lost. In a large injury, a tissue with strong proliferative power different from the lost tissue proliferates, and incomplete regeneration may occur in which the tissue is regenerated incompletely and the function cannot be recovered. In this case, a structure made of a bioabsorbable material is used to prevent the invasion of a tissue having a strong proliferation ability into the tissue defect portion, thereby securing a space in which the original tissue can grow, and further, a cell growth factor. Regenerative medicine is performed to replenish the natural tissue and enhance the regenerative capacity of the original tissue. Examples of this include regenerative medicine of cartilage, bone and peripheral nerves. It has previously been thought that nerve cells and myocardium are incapable of regeneration or are extremely low. In recent years, regenerative medicine using a tissue piece derived from a self tissue has been attracting attention.

  A “cell” as used herein is defined in the same way as the broadest meaning used in the art, and is a structural unit of a tissue of a multicellular organism, surrounded by a membrane structure that isolates the outside world, Refers to a living organism having self-regenerative ability and having genetic information and its expression mechanism. Any cell can be targeted in the method of the present invention. The number of “cells” used in the present invention can be counted through an optical microscope. When counting through an optical microscope, counting is performed by counting the number of nuclei. The tissue is used as a tissue slice, and hematoxylin-eosin (HE) staining is performed, whereby the extracellular matrix (for example, elastin or collagen) and the nucleus derived from the cells are dyed with a dye. This tissue section can be examined with an optical microscope, and the number of nuclei per specific area (for example, 200 μm × 200 μm) can be estimated and counted.

  Cells can cause mineralization and elicit an immune response. Therefore, for tissue or organ transplantation, it is necessary to suppress cell-derived calcification and the induction of an immune response.

  In the present specification, “cell replacement” means that another cell invades and replaces the original cell in the decellularized tissue, and is also referred to as “cell infiltration”. Preferably, in the present invention, cell replacement is performed by the transplanted host cells.

  As used herein, “tissue” or “biological tissue” refers to a cell population having the same function / form in a cell organism. In multicellular organisms, the cells that make up it usually differentiate, function becomes specialized, and division of labor occurs. Therefore, a tissue cannot be a simple aggregate of cells, and constitutes an organic cell group or a social cell group having a certain function and structure. Examples of tissues include, but are not limited to, skin tissue, connective tissue, muscle tissue, vascular tissue, heart tissue, nerve tissue, and the like. The tissue targeted by the present invention may be any organ or tissue derived from an organism. In a preferred embodiment of the present invention, tissues targeted by the present invention include, but are not limited to, blood vessels, blood vessel-like tissues, heart valves, pericardium, dura mater, corneas and bone tissues. Since the non-micellar amphipathic molecules used in the present invention are exemplified by a mechanism similar to the extraction of cellular components, in principle, any organ-derived tissue is treated by the method of the present invention. Can be done. Therefore, it is understood that the treatment with the biocompatible polymer of the present invention can be applied to any tissue.

  In the present specification, the “biological tissue” refers to a tissue separated from a living body or a processed tissue thereof, and unless otherwise specified, is understood to refer to a tissue that has not undergone decellularization treatment. In the present specification, a tissue that has not been specifically decellularized after removal from a living body is also referred to as an “untreated cell”. With respect to such biological tissue, it is understood that this concept includes, for example, a tissue that has been separated from a biological body and a tissue in which such a tissue is exposed to a biocompatible polymer. Such a biological tissue is used as a graft.

  In the present specification, the “membrane structure” is also referred to as “planar structure” and refers to a film structure. Examples of membranous tissues include organ tissues such as pericardium, dura mater and cornea.

  In the present specification, the term “organ” or “organ” is used interchangeably, and a certain function of an organism individual is localized and operates in a specific part in the individual, and the part is morphologically A structure with independence. In general, in a multicellular organism (eg, animal, plant), an organ is composed of several tissues having a specific spatial arrangement, and the tissue is composed of many cells. Such organs or organs include organs or organs associated with the vascular system. In one embodiment, organs targeted by the present invention include ischemic organs (hearts with myocardial infarction, skeletal muscles with ischemia, etc.). In one preferred embodiment, organs targeted by the present invention are blood vessels, blood vessel-like tissues, heart valves, pericardium, dura mater, cornea and bone. In another preferred embodiment, organs targeted by the present invention are heart valves, pericardium and blood vessels.

  In order to prepare a suitable graft for transplantation, it may be desirable to do organ culture as needed. Organ culture refers to culturing a part or the whole of an embryonic organ or mature organ extracted from a living body in vitro while maintaining its structure, function and differentiation ability. On the other hand, in general tissue culture, since cell proliferation is the main purpose, dedifferentiation is more likely to occur. Examples of organ culture include, but are not limited to, the watch glass method, the block petri dish method, the support platform method, the sponge substrate method, and the Trowell method. Protein / nucleic acid / enzyme 45, 2188-2200 (2000), such as mass cell culture (for example, holofiber method, fixed bed type, spin filter, sedimentation tank type, perfusion culture, circulation organ culture method (circulation) type organ culture), etc.) may also be used. Organs or tissues prepared by the decellularization method of the present invention are placed in a culture medium, and various substances are added to the organ culture medium to adjust to structure, function and developmental differentiation, and organ-to-organ interaction. Etc. can be adjusted.

  As used herein, “decellularization” and “decellularization” refer to removal of cells from a tissue or organ. Preferably, decellularization can be performed without damaging the structure and function of the original tissue or organ. Although cytoplasmic components, cytosolic components, cytoskeletons and cell membrane components have been removed from the decellularized tissue, extracellular matrix (ECM) components (eg, elastin, collagen (type I, type IV, etc.) ), Laminin, etc.), the cellular components necessary to maintain the structure of the tissue are retained intact. Accordingly, it is preferred that the decellularized tissue or organ is substantially equivalent to the untreated tissue or organ in terms of properties such as the shape, mechanical strength, elasticity, and flexibility of the tissue or organ. In addition, since the extracellular matrix is native after transplantation, it provides a suitable environment for invasion, adhesion, proliferation, expression of differentiation traits, 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.

  Therefore, it is preferable that the (untreated) biological tissue of the present invention has not undergone such decellularization treatment.

  The decellularized tissue preferably has MHC class I and II proteins removed. Such MHC class I and II proteins can be confirmed by SDS PAGE and / or Western blot analysis. Other extracellular matrix proteins can also be confirmed by SDS PAGE and / or Western blot analysis. The structural proteins (collagen, elastin, etc.) in the cells can be evaluated by amino acid analysis (for example, Edman degradation method, automatic analysis using a peptide analyzer available from PE Biosystems, etc.). As a result, the influence of the decellularization process on the ECM can be determined. Lipids and phospholipids contained in cell membranes and cells can be analyzed using thin layer chromatography and HPLC. Sugar chains (such as glycosaminoglycans) can be analyzed by agarose gel electrophoresis or the like. By this analysis, the presence of α-Gal and the like can be analyzed in addition to the glycosaminoglycan composition of the extracellular matrix.

  Therefore, a tissue that has not been decellularized can be determined by confirming that it has no characteristics due to the decellularization. In particular, the tissue that has not been decellularized in this specification is (1) a cell that has a cell survival rate of 30% or more compared to a tissue that has just been removed from a living body (live tissue), Or (2) the residual rate of soluble protein is 50% or more compared to tissue (live tissue) just taken out from the living body, or (3) DNA compared to tissue (live tissue) just taken out from the living body It is assumed that the residual ratio of is 50% or more.

  As used herein, “cell survival rate” refers to the ratio of viable cells remaining after decellularization of a tissue by the method of the present invention to cells originally present in the tissue. As a method for measuring the cell survival rate in this specification, a microscopic counting method using hematoxylin and eosin staining (H & E staining) is typically used. This method is performed by examining a nucleus dyed by HE staining with an optical microscope. Use counting. Therefore, this method includes, for example, the following steps: a step of dyeing a sample by HE staining; a step of counting the number of nuclei (= cell number) present in an area of 100 mm × 100 mm in this sample; Counting a plurality of times (for example, 8 times) and taking an average thereof; and calculating a ratio with respect to a control (for example, setting untreated tissue as 100%). The ratio with respect to the control can be, for example, the ratio of the remaining nuclei to the untreated tissue as the cell remaining ratio. Therefore, in this case, the cell residual ratio (%) = (number of nuclei in the treated tissue) / (number of nuclei in the untreated tissue) × 100 can be calculated. The cell survival rate can also be measured by measuring the amount of remaining DNA. This is because it is known that the total amount of DNA contained in cells in a tissue is generally proportional to the number of cells in the tissue. Methods for measuring DNA are known in the art, and for example, the amount of DNA extracted from tissue can be quantified using a fluorescent reagent such as Hoechst 33258 from Molecular Probes. Specifically, Hoechst 33258 (Ex (excitation wavelength) 356, EM (emission wavelength) 492) that forms a sol by pulverization sonication or the like, specifically binds to a DNA component in a buffer solution, and generates fluorescence ability. ), 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 this cell survival rate can also be used for γ-irradiation. In that case, as a guide, a cell residual rate of 30% can be mentioned.

  As used herein, “immune reaction” refers to a reaction due to impaired immune tolerance between a graft and a host. For example, hyperacute rejection (within several minutes after transplantation) (immunization with an antibody such as β-Gal) Reaction), acute rejection (reaction due to cellular immunity about 7 to 21 days after transplantation), chronic rejection (rejection due to cellular immunity after 3 months) and the like.

  In the present specification, whether or not to induce an immune response is determined based on the pathology of the species, number, etc. of cells (immune system) infiltration into a transplanted tissue by staining including HE staining, immunostaining, and microscopic examination of tissue sections. It can be determined by histological examination.

  As used herein, “calcification” refers to the deposition of calcareous substances in a living organism.

  In this specification, whether or not to “calcify” in vivo can be determined by measuring the calcium concentration. The transplanted tissue is taken out, the tissue section is dissolved by acid treatment or the like, and the solution is subjected to atomic absorption or the like. It is possible to measure and quantify with a trace element quantification apparatus.

  As used herein, “in vivo” or “in vivo” refers to the inside of a living body. In a particular context, “in vivo” refers to the location where the target tissue or organ is to be placed.

  As used herein, “in vitro” refers to a state in which a part of a living body is removed or released “in vitro” (eg, in a test tube) for various research purposes. A term that contrasts with in vivo.

  In the present specification, “ex vivo” refers to a series of operations ex vivo when a target cell for gene transfer is extracted from a subject, a therapeutic gene is introduced in vitro, and then returned to the same subject again.

  In this specification, 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, it normally has a function to prevent blood backflow from the ventricle to the atrium or pulmonary artery and aorta to the atrium. A function that prevents the backflow of blood to the atria. In the present invention, it can be said that an exceptional effect not shown in the past has been shown in that even if γ-irradiation after exposure to a biocompatible polymer was performed, there was no substantial damage to the function of this normal state.

  In the present specification, “extracellular matrix” (ECM) is also called “extracellular matrix” and refers to a substance existing between somatic cells regardless of whether they are epithelial cells or non-epithelial cells. The extracellular matrix is responsible not only for tissue support, but also for the construction of the internal environment necessary for the survival of all somatic cells. The extracellular matrix is 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 is roughly classified into a fiber component and a matrix filling the space, and the fiber component includes collagen fiber and elastic fiber. The basic component of the substrate is glycosaminoglycan (acid mucopolysaccharide), most of which binds to non-collagenous protein to form a polymer of proteoglycan (acid mucopolysaccharide-protein complex). In addition, laminin of the basement membrane, microfibril around the elastic fiber, fiber, and glycoprotein such as fibronectin on the cell surface are included in the substrate. The basic structure is the same in specially differentiated tissues, for example, hyaline cartilage produces chondrocytes that contain a large amount of proteoglycan, characteristically by chondroblasts, and bone produces bone matrix that causes calcification by osteoblasts. . In one embodiment of the present invention, the extracellular matrix (eg, elastin, collagen (eg, type I, type IV, etc.), laminin, etc.) is substantially from the state prior to the decellularization treatment of the present invention. It may be characteristic that it has not changed.

  As used herein, “tissue damage rate” refers to a parameter indicating the function of a tissue or organ, and is an index of how much the tissue or organ after treatment is damaged or damaged, and the original function of the tissue or organ. Is an indicator of whether or not The method for measuring the tissue damage rate in the present specification is known in the art, and can be determined, for example, by counting elastin rupture sites. In the method used in the present specification, one visual field is divided into units of 100 μm × 100 μm, and calculation is performed by counting when there is an elastin rupture site in units. There were 24 units per field of view. Counting is performed by microscopic examination of the extracellular matrix in the tissue section by HE staining, the untreated tissue is defined to be 0%, and the damage rate is calculated as x / 24. In this case, unprocessed is defined as x = 0.

  As used herein, “tissue strength” refers to a parameter indicating the function of a tissue or organ, and is a physical strength of the tissue or organ, and can generally be determined by measuring tensile strength. Such general tensile tests are well known.

  In the present specification, (1) a specimen is cut into a 5 × 30 mm strip. (Basically cut the wall portion of the aortic base so as to be longer in the long axis direction); (2) Fix both ends of the specimen about 5 mm to the fixed part of the tensile tester. (Use TENSILON universal testing machine RTC-1150A manufactured by ORIENTEC); and (3) Start pulling and pull to the breaking point at a speed of 1 cm / min. By doing this work, the load at the breaking point is strengthened and adopted. Here, the load at break and the elastic modulus are measured.

  The strength of the living tissue of the present invention can be such that the maximum point load is usually at least about 8N or more, more preferably about 10N or more, and even more preferably about 14N or more. Considering that in conventional biological tissue or natural tissue (eg, artery), it was often about 7N, and that decellularization treatment has little or no effect of enhancing tissue strength. Then, the tissue strength strengthening ability of the present invention is unexpected.

  In a preferred embodiment, when viewed in terms of elastic modulus, the living tissue of the present invention usually has an elastic modulus of at least 1.2 MPa, and preferably has an elastic modulus of at least about 1.6 MPa. The derived tissue may have an elastic modulus (for example, 0.8 or more, 0.8 to 1.0 MPa, etc.) inferior to that of a natural product as long as it can withstand use.

  The measurement of elongation can be performed with a tensile tester (TENSILLON ORIENTEC) used for strength measurement. Specifically, a strip material having a width of 5 mm and a length of 30 mm can be loaded at a speed of 10 mm / min in the minor axis direction, and the load at break and the elastic modulus can be measured. Specifically, the measurement of elongation can be obtained 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. Usually, the elongation is measured both in the vertical direction and in the horizontal direction. It is preferable that there is no variation in both the elongations, but the present invention is not limited to this. Depending on the application, the elongation-related properties are preferably at least 120%, preferably 150%, for example, but are not limited thereto. As for the properties related to elongation, the living tissue of the present invention may have elongation (for example, at least 50%, etc.) inferior to that of a natural product as long as it can withstand use.

By analyzing data obtained by a general tensile test, various data such as breaking strength, rigidity, Young's modulus, etc. can be obtained, and such values are also used as an index of tissue strength in this specification. be able to. In the present specification, in the case of a tubular tissue, the tissue strength can be expressed by a stiffness parameter (β value). After creating the P-D (pressure-diameter) relationship,
Ln (P / Ps) = β (D / Ds−1) (1)
Can be calculated. Ps and Ds indicate standard values at 100 mmHg. The value of the diameter (D) in P (pressure) of P and D 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, pressure is applied to the inner chamber from the armor device, and at the same time, the outer diameter at the time of pressurization is monitored. The relationship between the pressure obtained by the measurement and the outer diameter is introduced into the formula (1) above, and the β value is calculated (Sonoda H, Takamzawa K., et al. J. Biomed. Matr. Res. 2001). : 266-276).

  As used herein, “biocompatibility” refers to the property of being able to exist without any damage in vivo due to lack of toxicity and immunological rejection ability.

  In the present specification, “biocompatible polymer” refers to a polymer having good biocompatibility, and specifically means that it does not cause toxicity even if it remains. In this specification, a method for determining whether a certain polymer has such biocompatibility uses a test method such as an implantation test under the skin of a laboratory animal such as a rat. In this test method, as a result of the subcutaneous implantation test, it is understood that the biocompatibility is low macroscopically when a relatively acute immune reaction, allergic reaction, etc. occurs, and swelling, redness or fever occurs. In addition, when transplanted to a specific affected area, such as when an artificial blood vessel is transplanted into a blood vessel of an animal, after several days to several months, observe the transplanted site, whether or not the tissue has been engrafted, inflammation around the transplanted tissue, adhesion The tissue compatibility is determined by observing the degree of thrombus formation due to blood coagulation. In addition, a tissue section of the transplant site is prepared, and the cells are stained and observed with hematoxylin / eosin staining and other staining methods, and many granular cells in charge of the immune system are used as an indicator of poor tissue compatibility. It is determined whether or not the invasion has occurred, or the formation of scar tissue that separates both the conventional tissue and the transplanted tissue is observed. In particular, as an index of the high tissue compatibility of a tissue derived from a living body, various cells such as vascular endothelial cells, fibroblasts, and smooth muscle cells have invaded from conventional tissues, and whether recellularization has occurred (that is, Whether or not the transplanted tissue is engrafted to such an extent that the boundary between the conventional tissue and the transplanted tissue is not clear)

  In addition to the above-described determination by observing the shape of tissues and cells, the concentration of cytokines in the blood due to the progression of the inflammatory reaction, the concentration of antibody against the transplanted tissue recognized as a foreign body (titer), the complement component In some cases, the biocompatibility quantitative value such as the concentration, the amount of a drug-metabolizing enzyme (such as P450) induced by foreign substance implantation, and the tracking of changes before and after transplantation may be used as an index of tissue compatibility.

  For medical device materials, cytotoxicity test, sensitization test, irritation test, implantation test, genotoxicity test, blood test, in order to evaluate its toxicity and rationally regulate its use in medical devices There are test items such as compatibility test, systemic toxicity test, etc. Individual test methods are defined by the Ministry of Health, Labor and Welfare guidelines, US National Standards, international industry standard ISO-10993, etc.

  Examples of the biocompatible polymer include polyvinyl alcohol, polylactic acid, polyglycolic acid, polyvinyl pyrrolidone, polyethylene glycol, gelatin, collagen, γ-polyglutamic acid, silicone, polyvinyl chloride, polymethyl methacrylate, polytetrafluoroethylene. Polyester, polypropylene, polyurethane, cellulose, polystyrene, nylon, polycarbonate, pyrisulfone, polyacrylonitrile, collagen, gelatin, fibrin, chitin, chitosan, alginic acid, starch, γ-polyglutamic acid, polycaprolactone, polyhydroxybutyric acid, etc. It is not limited to them. Here, in addition to unmodified polyvinyl alcohols, some are chemically modified to some extent by amino groups, carboxyl groups, acetyl groups, etc., and are commercially available. Can be used in the present invention. Preferably, the biocompatible polymer is biodegradable but not limited thereto.

In this specification, “polyethylene glycol (PEG)” refers to a polymer of ethylene glycol, which is also referred to as polyethylene (polyethylene oxide), and is represented by HO— (CH ₂ CH ₂ O) _n —H. Polyethylene glycol is commercially available from various companies, for example, Union Carbide (Carbowax), Dow (Polyglycol E), Texaco Chemical (Jeffox), Olin (PolyG), BASF Wyandotte (Pluracol E), IC ATEG), Nacalai tesque, Japanese fats and oils and the like. Usually, PEG has an average molecular weight between 200 and 200,000. Such PEGs include products such as Nacalai tesque's polyethylene glycol (H (OCH ₂ CH ₂ ) _n OH) # molecular weight (eg, 200, 600, 1000, 2000, 6000, 8000, 35000, 50000). It is done. In the present specification, those having an average molecular weight of 1,000 to 200,000 are usually used, and preferably PEG has an average molecular weight of between 10,000 and 100,000. In another preferred embodiment, the PEG can have an average molecular weight of 2000 to 50000. More preferably, the PEG can have an average molecular weight of about 35000. Alternatively, it may be 1) 1,000 to 200,000, (2) preferably 4,000 to 100,000, and (3) more preferably 8,000 to 50,000. Thus, although PEG is marketed, in order to achieve a desired characteristic, it can also synthesize | combine suitably. Such synthetic methods are well known in the art. In the present specification, the average molecular weight and the molecular weight are represented by Dalton. The PEG used in the present invention preferably has a homogeneous molecule, but this is not essential and can be used usually if the average molecular weight is within a certain range. To prepare the tissue of the present invention, chemical substances other than PEG (for example, including but not limited to enzymes (such as DNase I), enzyme inhibitors, etc.) can be added. When chemicals other than PEG are added, the amount or concentration of PEG to be added may vary depending on the amount of chemical present. Preferably, the concentration used for cell fusion or the like is good. For example, a concentration of 1 g / ml or more (100% w / w or more) is preferable, but not limited thereto. Alternatively, PEG can be used at a concentration between 60% w / v and 100% w / v. In the present invention, polyethylene glycol can also be used in a method for strengthening a tissue after preparing a living tissue.

In this specification, polyvinyl alcohol is also called poval or PVA, and refers to a polymer obtained by hydrolysis (exactly transesterification) of polyvinyl acetate. Formula chromatography _{(CH 2 C (OH) H} ) is represented as _n-. Since nyl alcohol does not exist as a monomer, it can be produced by hydrolysis of polyvinyl acetate. Used for cell fusion.

In the present specification, polyvinylpyrrolidone is a water-soluble polymer compound represented by the general formula [—CH ₂ CH (C ₄ H ₆ NO) —] _n , and is mainly used as an excipient for a drug. It has been.

  As used herein, “biodegradable” refers to the property of being degraded in vivo or by the action of microorganisms when referring to a substance. A biodegradable polymer can be decomposed into water, carbon dioxide, methane, and the like, for example, by hydrolysis. In this specification, the method for determining whether or not biodegradable is performed for several days to several years in a laboratory animal such as a rat, rabbit, or dog with respect to bioabsorbability, which is part of biodegradability. For testing and microbial degradation tests, methods such as embedding / disintegration tests for several days or years in soil of sheet-like polymers are used. Where transplantation is concerned, it is preferred to use studies in animals. In addition, as a simpler alternative test method based on the above test method, various buffer solutions such as phosphate buffer saline (PBS) are used for non-enzymatic degradation of polymers, and enzymatic degradation of polymers is used. In some cases, a dissolution test in an aqueous solution is performed using a buffer solution to which a hydrolase of the polymer (protease, glycosidase, lipase, esterase, etc.) is added. Biodegradable polymers include natural and synthetic polymers. Examples of natural polymers include proteins such as collagen and starch, and polysaccharides. Examples of synthetic polymers include aliphatic polyesters such as polyglycolic acid, polylactic acid, and polyethylene succinate. It is not limited to. Polyvinyl alcohol can be recognized as having biodegradability in the present specification because it exhibits weak biodegradability.

  In the present specification, “polymer” is used in the same meaning as “molecule”, and the molecular weight is not particularly limited. When discussing a polymer with a molecular weight limited, the molecular weight is usually 500 or more, but not limited thereto. The upper limit of the molecular weight of the polymer used in the present invention is infinite in principle, but in the present invention, it is usually handled as a solution and the molecular weight can be discussed (by dynamic light scattering, gel filtration, centrifuge). In the sense that molecular weight measurements such as sedimentation equilibrium can be applied) molecular weights up to about 5 million can be used, but are not limited thereto. In the present specification, the term “biopolymer” and “biocompatible polymer” are synonymous with terms such as “biomaterial” and “medical polymer”, as is the case with conventions in academia and industry. used.

  In the present specification, “immersing” refers to putting a certain object in a certain fluid (for example, a liquid). In the present invention, the tissue to be treated is placed in a solution containing a surfactant or non-micellar amphiphilic molecule (eg, 1,2-epoxide polymer such as polyethylene glycol) for treatment. This is called “soaking”. Thus, the soaking step is preferably performed so that the tissue to be treated completely penetrates into the solution for treatment. In the step of immersing, preferably, a physical treatment (for example, ironing with a glass rod) may be performed in order to efficiently remove the component to be removed.

  As used herein, “exposing” refers to bringing a certain object into contact with a certain factor (for example, a biocompatible polymer).

  In the present specification, the “washing” step in the present invention refers to removing the solution from the tissue treated by the step of immersing in a biocompatible polymer solution, for example. Therefore, preferably, the washing step is performed using a liquid. In the present invention, the treated tissue is intended to be used in a living body, and is preferably washed with a physiologically acceptable liquid. In one preferred embodiment, the washing step may be performed using PBS (phosphate buffered saline). The cleaning solution may contain other drugs (for example, protease inhibitors) as necessary, but such other drugs are preferably non-toxic and biocompatible.

  In this specification, “chemical treatment” refers to treatment of an object with a chemical substance (for example, by dipping) in a broad sense. However, as used herein, chemical treatment refers to other steps (for example, DNase treatment) other than the step of immersing in a solution containing a biocompatible polymer. Therefore, in the method of the present invention, for example, when chemical treatment is performed in addition to the step of immersing in a 00 polyethylene glycol-containing solution having an average molecular weight of 1000 to 2000, the chemical treatment is performed on polyethylene having an average molecular weight of 1000 to 200000. Soaking in a solution other than a glycol-containing solution (eg, treatment with DNase I solution, glutaraldehyde solution, polyethylene glycol solution with other average molecular weight, other biocompatible polymer solution, etc., but not limited thereto) Include.

  In this specification, “reinforcing” means that the tissue strength of the tissue is improved when referring to the tissue. Preferably, the tissue strength is preferably at least 110%, more preferably at least 120%, even more preferably at least 150%, or at least 200% of the state prior to some reinforcement treatment. Such tissue strength can be indicated herein by, for example, a maximum point load (eg, unit N) in a tensile strength test.

  In this specification, the “radical reaction” is also referred to as a group leaving reaction, and refers to a chemical reaction that occurs when an unpaired electron is generated and reacts with another molecule or residue.

Examples of radical reactions include the following:
1) Reaction in which stable molecular bonds are broken and unpaired electrons are formed,
For example, C ₃ H ₈ → CH ₃ · + C ₂ H ₅ ·
2) Reaction in which an unpaired electron reacts with a stable molecule to form an unpaired electron with another molecule. For example, CH ₃ + CH ₃ COCH ₃ → CH ₄ + CH ₃ COCH ₃
3) Reaction in which stable molecules react with unpaired electrons to create large unpaired electrons,
For example, CH ₃ + C ₂ H ₄ → C ₃ H ₇
4) Reaction in which unpaired electrons decompose to form small unpaired electrons and molecules,
For example, n-C ₃ H ₇ · → C ₂ H ₄ + CH ₃ ·
5) Reaction between unpaired electrons,
Recombination reaction CH ₃・ + CH ₃・ → C ₂ H ₆
Disproportionation reaction _{C 2 H 5 · + C 2} H 5 · → C 2 H 4 + C 2 H 6.

As used herein, “γ-ray” refers to electromagnetic waves having a wavelength shorter than about 0.001 nm. In terms of energy, it is a photon of several hundred keV or more. Normally, gamma rays are emitted during transitions between nuclear energy levels. As the γ-ray emission source, an arbitrary radiation source used as a γ-ray source is used, but a radiation source of ⁶⁰ Co (cobalt 60) or ¹³⁷ Cs (cesium 137) is preferable. This is because it is preferable for the treatment of biological tissue.

  The irradiation amount of γ rays is often displayed in units of kGy, and the irradiation amount can be measured by measuring the absorbed dose. For such measurement, measurement devices such as a hollow ionization chamber, a calorimeter, a film dosimeter, and a PMMA dosimeter are used.

  In this specification, as a method for confirming on a tissue whether or not γ-ray irradiation has been performed, for example, therefore, what unpaired electrons are generated from a search for a product generated by γ-ray irradiation. And you can know if you reacted. Radicals can be identified by advances in chemical methods and measurement methods such as electronic spectra and paramagnetic resonance absorption. The existence of radicals having a very short lifetime (for example, .OH) can also be confirmed by flash photolysis (electron beam pulse radiolysis and flash spectroscopy), rigid solvent method, matrix isolation, and the like. However, when the metal atom of the transition metal complex has one or more unpaired electrons, it is not usually called a radical.

  Examples of the method for confirming γ-ray irradiation include the following.

  (1) Unpaired electrons (radicals) are formed on amino acid residues and sugar chains of proteins through direct or indirect action (transfer from H-radical OH-radical by water molecule cleavage) by γ-ray irradiation. Arise. When radicals retransfer, they move around on various residues, but proteins tend to collect radicals on energetically stable aromatic amino acids (tyrosine, phenylalanine, tryptophan, etc.) and sulfur-containing amino acids (cysteine, methionine). In the end, it is known to cause crosslinking and cleavage of polypeptide chains and sugar chains with a certain probability. .

  (A) Detection method # 1: If a relatively long-lived polymer radical remains in the tissue, it can be confirmed by paramagnetic resonance absorption (EPR) or the like (RadiationResearch 163, 535-543, 2005).

  (B) Detection method # 2: When protein casein in milk is irradiated with gamma rays together with polysaccharides such as carboxymethylcellulose and PEG to produce a crosslinked thin film, tyrosine, a kind of amino acid, is a dimer. It has been reported that it has a structure called bityrosine and emits fluorescence, so that the cross-linking site is quantified (J. Agric. Food. Chem. 46, 1618-1623, 1998), which can be used.

In this specification, “vacuum” refers to a pressure of 1 × 10 ⁻¹ Pa or less. In this specification, “under reduced pressure” refers to a pressure in the range of 1 × 10 ⁻¹ Pa to ¹ × 10 ⁵ Pa.

  In this specification, the term “atmosphere” is used in the meaning that is usually used. Usually, the composition is 78% nitrogen, 21% oxygen, 1% argon, 0.03% carbon dioxide, and about 0.3% water vapor. Is done.

  In this specification, “in oxygen” refers to an environment having a higher oxygen concentration than the atmosphere. Preferably, oxygen refers to the presence of substantially 100% oxygen.

As used herein, “in water” refers to a reaction in a medium consisting essentially of H ₂ O. As the water, tap water, distilled water, ion-exchanged water and the like are used, but are not limited thereto.

  As the “biocompatible molecule” in which a radical reaction is performed in the present specification, an amphiphilic molecule solution may be used, but other different ones may be used.

  As used herein, “physiologically active substance” refers to a substance that acts on cells or tissues. Bioactive substances include cytokines 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 the same action. As used herein, a physiologically active substance can be in protein form or nucleic acid form or other form, but at the point of action, cytokine usually means protein form.

  As used herein, “cytokine” is defined in the same manner as the broadest meaning used in the art, and refers to a physiologically active substance produced from a cell and acting on the same or different cells. Cytokines are generally proteins or polypeptides, and have a suppressive action on the immune response, regulation of the endocrine system, regulation of the nervous system, antitumor action, antiviral action, regulation of cell proliferation, regulation of cell differentiation, etc. . As used herein, cytokines can be in protein or nucleic acid form or other forms, but at the point of action, cytokines usually mean protein forms.

  As used herein, “growth factor” or “cell growth factor” is used interchangeably herein and refers to a substance that promotes or regulates cell growth. Growth factors are also referred to as growth factors or growth factors. Growth factors can be added to the medium in cell or tissue culture to replace the action of serum macromolecules. Many growth factors have been found to function as regulators of the differentiation state in addition to cell growth.

  Cytokines typically include interleukins, chemokines, hematopoietic factors such as 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). Those having a proliferative activity such as

  Since biologically active substances such as cytokines and growth factors generally have a redundancy of function, even cytokines or growth factors known by other names and functions can be used for the physiologically active substances used in the present invention. As long as it has activity, it can be used in the present invention. Cytokines or growth factors can also be used in preferred embodiments of the therapeutic methods or medicaments of the present invention as long as they have the preferred activity herein.

  As used herein, “differentiation” refers to a process of development of a state of a part of an organism such as a cell, tissue or organ, which forms a characteristic tissue or organ. “Differentiation” is mainly used in embryology, developmental biology and the like. Until a fertilized egg consisting of one cell divides and becomes an adult, an organism forms various tissues and organs. In the early stages of development, such as before division or when division is not sufficient, each cell or group of cells does not show any morphological or functional characteristics and is difficult to distinguish. Such a state is called “undifferentiated”. “Differentiation” also occurs at the organ level, and the cells that make up the organ develop into a variety of distinctive cells or groups of cells. This is also called differentiation within the organ in organ formation. Therefore, in the regeneration in the present invention, cell differentiation means that the cell has some morphological or functional characteristics that it did not have before treatment. For example, in the case of a heart valve, when a stem cell (eg, an embryonic stem cell or tissue stem cell) is provided as a cell, its shape or function is at least partially similar to that of the cell or tissue present in the heart valve. It is mentioned that a cell comes to have.

  As used herein, “graft”, “graft”, and “tissue graft” are used interchangeably and are the same or different groups of tissues or cells that are to be inserted into a specific part of the body and inserted into the body. It will be a part of it later. Examples of the graft include, but are not limited to, an organ or a part of an organ, a blood vessel, a blood vessel-like tissue, a skin piece, a heart valve, a pericardium, a dura mater, a corneal bone piece, and a tooth. Thus, a graft includes everything that is used to fill a defect in a part and make up for the defect. Examples of the graft include, but are not limited to, an autograft, an allograft (allograft), and a xenograft depending on the type of donor.

  As used herein, an autograft or an autograft refers to a graft derived from an individual when referring to the individual. In this specification, the term “autograft” may include a graft from another genetically identical individual (for example, an identical twin).

  In this specification, an allograft (allogeneic allograft) refers to a graft transplanted from another individual that is genetically different even if it is the same species. Due to genetic differences, allogeneic grafts can elicit an immune response in the transplanted individual (recipient). Examples of such grafts include, but are not limited to, parent-derived grafts.

  As used herein, xenograft refers to a graft transplanted from a xenogeneic individual. Thus, for example, when a human is the recipient, a graft from a cow or pig is called a xenograft.

  As used herein, a “recipient” (recipient) is an individual that receives a graft or transplant and is also referred to as a “host”. In contrast, an individual that provides a graft or transplant is referred to as a “donor”.

  Any implant can be used with the tissue processing technique of the present invention. This is because grafts reinforced by the method of the present invention (eg, tissues, organs, etc.) have reduced immune disorders to the extent that they are not impaired for therapeutic purposes. Therefore, the present invention, which was not able to be achieved by the prior art, has made it possible to use allografts or xenografts even in a situation that could only be used for conventional autografts. This is one of the special effects.

  As used herein, “subject” refers to an organism to which the treatment of the present invention is applied, and is also referred to as a “patient”. The patient or subject may preferably be a human.

  The cells used as necessary in the method or tissue graft of the present invention may be derived from the same line (derived from the self (autologous)), derived from the same species (derived from another individual (external)), or derived from different species. Self-derived cells are preferred because rejection is considered, but they may be allogeneic if rejection is not a problem. Moreover, what causes rejection reaction can also be utilized by performing the treatment which eliminates rejection reaction as needed. Procedures for avoiding rejection are well known in the art and are described, for example, in the New Surgery System, Heart and Lung Transplant Technical and Ethical Preparations to 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" (Sandimune / Neoral), "Tacrolimus" (Prograf), "Azathioprine" (Imlan), "Steroid Hormone" (Predonin, Methylpredonin), "T cells There are “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, 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, as long as the immunosuppressive effect is achieved, the immunosuppressive agent can be administered before or after the regeneration / treatment method of the present invention.

  The cells used in the present invention may be cells derived from any organism (for example, vertebrates and invertebrates). Preferably, cells derived from vertebrates are used, and more preferably cells derived from mammals (for example, primates, rodents, etc.) are used. More preferably, cells derived from primates are used. Most preferably, human-derived cells are used.

  Examples of the combination of the subject targeted by the present invention and the living body-derived tissue include, for example, transplantation into the heart in which heart disease (for example, ischemic heart disease) has occurred, pericardial patch, and dura mater transplantation during brain surgery. , Transplantation of blood vessels to myocardial infarction, lower limbs, upper limbs, fractures, transplantation of bones to patients with bone defects, transplantation of corneas of the present invention to patients with damaged corneas, etc. .

  The tissue targeted by the present invention may be any organ or organ of an organism, and the tissue targeted by the present invention may be derived from any type of organism. Examples of organisms targeted by the present invention include vertebrates and invertebrates. Preferably, the organism targeted by the present invention is a mammal (eg, primate, rodent, etc.). More preferably, the organism targeted by the present invention is a primate. Most preferably, the present invention is directed to humans.

  Revascularization therapy using autologous cells or tissues has attracted attention, and the method of transplanting tissues can be performed as is well known in the art. Various prosthetic valves or artificial blood vessels are used in the field of cardiovascular surgery, but there are many problems such as durability or the necessity of anticoagulant therapy and the accompanying bleeding tendency, easy infection, Expectations are placed on regenerative medical engineering. Niioka et al. Cultured a vascular smooth muscle cell obtained from a patient's foot vein on a bioabsorbable high polymer for a 4-year-old girl with pulmonary artery deficiency. (Toshiharu Niioka, Yasuharu Imai, Kazuhiro Seo et al .; Development and application of cardiovascular materials by Tesh Engineering. Journal of the Japan Cardiovascular Society 2000; 29:38). In tissue engineering in the cardiovascular system, transplanted cells and structures can directly contact the blood in the blood vessels, and oxygen and nutrient supply can be obtained immediately after transplantation. It has been. In particular, the advantages of self-cellization (cell replacement) are diverse as follows.

  1. Eliminate the possibility of rejection.

  2. There is no need to consider donors.

  3. Long durability can be expected because it is a living tissue.

  4). When the cells complete the extracellular stroma, the polymer as a scaffold is completely biodegraded and no foreign matter remains in the long term after transplantation.

  5). Since it is covered with the final endothelium, it has excellent antithrombogenicity and does not require anticoagulation therapy after transplantation.

  6). It is possible that growth can be expected due to self-organization.

  Although it is used in the right heart system mainly within the blood pressure range of the pulmonary artery pressure, those skilled in the art appropriately use it within the aortic pressure or apply it to valve tissue, AC bypass arterial graft, chordal tissue, etc. (Latest treatment of cardiovascular disease 2002-2003; Nankodo p29, issued in 2002).

  The general use of prosthetic valves is well known in the art, and the present invention can also be implemented based on this well known matter. For example, a stentless heterogeneous valve is known. In the heterogeneous biological valve, the effective valve area is reduced by the presence of the stent, and calcification and degeneration of the leaflets have been problems. Recently, a stentless heterogeneous valve that does not use a stent, taking advantage of the morphology of the porcine aortic root, has attracted attention as a prosthetic valve for the aortic valve position (Gross C et al, Ann Thorac Surg 68: 919, 1999). Due to the absence of a stent, even when a small-sized valve must be used, the pressure difference through the valve is small, and it is considered effective for post-operative left ventricular hypertrophy. In addition, the elasticity of the base of the aorta is maintained, and the stress applied to the leaflets is less, and an improvement in durability can be expected as compared with a biological valve with a stent. In addition, it can be used for endocarditis due to infection and infection of a prosthetic valve. Currently, the mid-stage postoperative results of stentless heterogeneous flaps in Europe and the United States have been satisfactorily reported, and long-term results can be expected (GrossC et al, AnnThorac Surg 68: 919,1999).

(Preferred embodiment)
The best mode of the present invention will be described below. The embodiments provided below are provided for a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention with reference to the description in the present specification.

  In one aspect, the present invention provides a biological tissue containing a biocompatible polymer and receiving γ-ray irradiation. The biocompatible polymer may be contained in any state in the living tissue, but it is advantageous that it is preferably covalently bonded. Therefore, those skilled in the art can appropriately include the biosynthetic polymer in the tissue derived from the living body according to the state to be included, using techniques well known in the art. Such binding may be achieved by gamma irradiation. In the present invention, the biological tissue that should contain the biocompatible polymer is preferably one that has not undergone decellularization treatment. In the present invention, it has been found that by including a biocompatible polymer in a tissue derived from a living body and subjecting it to γ-ray irradiation, calcification is unexpectedly significantly reduced and the tissue strength is increased. Therefore, the biocompatible polymer of the present invention provides a possibility that it can be used even in applications that could not be conventionally applied due to problems of calcification and strength. In particular, γ-ray irradiation has achieved suppression of calcification to a degree that could not be achieved by other radical reactions (for example, UV irradiation, radical reactions using chemical substances) or cross-linking agents such as glutaraldehyde. For example, as an example of the effect recognized by the present invention, as a result of transplantation into a long-term animal body (about 2 months for small animals such as rats and about 6 to 12 months for large animals such as pigs) There is an effect that dysfunction due to calcification does not occur. This is an effect that is not recognized at all during the treatment with glutaraldehyde. A significant effect was also observed quantitatively. For example, in one example, the amount of calcium per 1 mg of transplanted pericardium extracted as a result of a long-term (2 months) rat transplantation experiment was 1.6 (μg / mg) at the time of glutaraldehyde treatment. Sometimes it decreased to 0.15-0.2 (μg / 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, and still more preferably 120%, compared to a state without biocompatible polymer or γ-ray irradiation. More preferably, it may be 150% or more, and most preferably 200% or more.

  Without wishing to be bound by theory, it has become clear that γ-irradiation eliminates the cause of calcification. When transplanting a tissue other than autologous tissue, the remaining cells may elicit an undesired immune response. Therefore, in the biogenic tissue of the present invention, the level is lower than the level that elicits an immune response in vivo. There must be. The present invention also has an effect in that the tissue subjected to the treatment with the biocompatible polymer is remarkably reinforced.

  In a preferred embodiment, whether γ-irradiation has been received can be determined by confirming that the extracellular matrix components are at least partially, preferably substantially all, crosslinked covalently. Accordingly, the present invention provides a biological tissue characterized in that, in this embodiment, the extracellular matrix component is at least partially crosslinked by a covalent bond. Preferably, the extracellular matrix component can include, but is not limited to, collagen, elastin, laminin, fibronectin, tenascin, glycosaminoguncan and proteoglycan. More preferably, the bridge is formed between two or more amino acids that do not have a free amino group or carboxyl group in the side chain. In this case, preferably, the cross-linking is formed in a group other than a carboxyl group, a hydroxyl group and an amino group (for example, a sulfhydryl group).

  Gamma rays for extracellular matrix-derived proteins such as collagen, elastin, laminin, fibronectin and tenascin, and polysaccharides / glycoproteins derived from extracellular matrices such as glycosaminoglycans (such as hyaluronic acid, chondroitin sulfate and heparan sulfate) and proteoglycans It is known that when irradiated, a cross-linking reaction and a decomposition reaction can occur in a mixed manner.

  In one embodiment, it can be confirmed whether or not the eluted protein is substantially not present. Therefore, the present invention provides a biological tissue derived from γ-irradiation treatment, characterized in that, in this embodiment, the eluted protein is substantially absent. In the present specification, “substantially free of eluted protein” means that a protein extract is prepared from a decellularized tissue by a known method, and the protein is detected by a general quantitative method such as Bio-Rad Proteinassay, The theoretical value (protein weight / biological tissue weight) calculated from the calibration curve is 0.5 mg / mg or less, more preferably 0.2 mg / mg or less, and even more preferably 0.1 mg / mg or less. That means.

  In the present invention, an experiment was performed in which extracellular matrix-derived protein was subjected to γ-ray irradiation. As a result, a cross-linking reaction was likely to occur under conditions where oxygen and water existed. Moreover, even if the irradiated sample was subjected to polyacrylamide gel electrophoresis (SDS-PAGE), it did not enter the polyacrylamide gel unlike before irradiation. Therefore, it was revealed that the eluted protein was substantially absent.

  In addition, there are several reports regarding which amino acid residues are involved in cross-linking in protein cross-linking by γ-ray irradiation. And it seems to be less specific than in the case of chemical crosslinking.

  In the case of chemical cross-linking, for example, in glutaraldehyde cross-linking, amino acids having a free amino group (one NH 2) in the side chain are cross-linked, so that lysine, arginine and the like can inevitably become cross-linking points. Other chemical crosslinks mainly target carboxyl groups. There are several well-known types of chemical cross-linking. For example, Nimni ME et a1. , J .; Biomed. Matr. Res. 21, 741-771 (1987), in addition to several types of cross-linking patterns of collagen, the existence of many cross-linking patterns is known.

  On the other hand, the cross-linking caused by γ-ray irradiation seems to cause various reactions in addition to the same reaction as described above. Also, more types of amino acids than above appear to be cross-linking points. J. et al. H. Boweseta1. , Radiation Research 16, 211-223 (1962) irradiates a sample obtained by washing and removing water-soluble components from bovine dermis in the presence of oxygen and nitrogen in a dry state and a wet state, respectively. Reported data quantifying amino acid changes after degradation of all proteins. According to the results, there were some amino acids such as glycine and valine that hardly decreased, but the other amino acids decreased little by little, and the degree of the reduction varied. This suggests that protein cross-linking by γ rays occurs in various amino acids relatively randomly.

  In other words, if limited to extracellular matrix-derived proteins, whether cross-linking and chemical cross-linking caused by γ-irradiation occur between two or more amino acids that do not have a free amino group or carboxyl group in the side chain. It is distinguished by. That is, 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 crosslinking, and if it occurs also between other amino acids, it is It can be said that this is an induced cross-linking.

  For polysaccharides, various chemical crosslinks (bisepoxide and divinylsulfone crosslinks, intramolecular esterification, glutaraldehyde crosslinks, carbodiimide crosslinks, hydrazide crosslinks, etc.) target carboxyl groups, hydroxyl groups, or amino groups (Laurent). , TC et al, Acta Chem. Scand, 18, 274-275, (1964); Kuo, JW. Et al., Bioconjug Chem. (4): 232-4L (1991); al., J Control Release, 69 (1); 169-84. (2000)), a group other than a carboxyl group, a hydroxyl group or an amino group (for example, the group includes a sulfhydryl group, an aldehyde group, a carbonyl group, a sulfo group). Whether the bridge is formed by a bond selected from the group consisting of a carbon-carbon bond, an ether bond and an ester bond. It is distinguished by whether or not. In particular, it may be characterized in that a bond such as an intercarbon bond or an ester bond forming a skeleton is cleaved to form a bridge by a new bond.

(Example of group)
-SH group Sulfhydryl group
-CHO group Aldehyde group
C = O group Carbonyl group
—SO ₃ H group, sulfo group
-NO ₂ group Nitro group.

(Example of binding)
-C-C- CC bond (carbon-carbon bond)
-O- ether bond
-(C = O) O- ester bond.

  In one embodiment, the dose of gamma irradiation used in the present invention is typically 10 to 250 kGy. Although not wishing to be bound by theory, if it exceeds about 250 kGy, it may cause degeneration of the extracellular matrix to remain, but the substantial components necessary for maintaining tissue strength such as the extracellular matrix. If the irradiation dose is such that no significant denaturation occurs, even an irradiation dose that exceeds this upper limit can be used. Although not wishing to be bound by theory, the amount of γ-ray irradiation can be 10 kGy or less. Irradiation times of 10 kGy or less can also be used as long as the irradiation time can be considered to be significantly longer, as long as significant promotion of tissue calcification inhibition is observed, and these are within the scope of the present invention. It will be understood by those skilled in the art.

  Preferably, the dose of γ rays and the like is advantageously 100 kGy or less. Although not wishing to be bound by theory, there was almost no significant decrease in tissue strength in the range of 100 kGy or less. It is advantageous to use an irradiation dose of 40 kGy or more as the lower limit.

  More preferably, an irradiation amount of 50 to 80 kGy is advantageously used as the irradiation amount. Although not wishing to be bound by theory, although the suppression of calcification is promoted by the combination of cleaving of biomolecules such as gene DNA by γ-irradiation and extraction operation with PEG, extracellular that should maintain the mechanical strength of the tissue This is because there is a reason that the substrate protein is not significantly damaged.

  In one embodiment, the irradiation time of γ rays used in the present invention varies depending on the total irradiation amount, but is usually about 0.5 hour to 10 days, preferably about 1 hour to 2 days. It is advantageous that More preferably, it is advantageous that it is 3 to 8 hours. Although not wishing to be bound by theory, longer irradiation times promote suppression of calcification, but conventionally, extracellular matrix proteins such as collagen and elastin, which have elastic properties, are cleaved or crosslinked. This is because the nature of the tissue for transplantation deteriorates because it becomes brittle against mechanical deformation such as bending and pulling. Therefore, since suppression of calcification partially depends on the total irradiation amount, it can be reacted in a few hours using a strong γ-ray source.

  In a preferred embodiment, the radical reaction (eg, gamma irradiation) used in the present invention is selected from the group consisting of vacuum, oxygen, nitrogen, air, water, amphiphilic molecule solution and combinations thereof. Performed in an atmosphere.

  In a preferred embodiment, the biocompatible polymer used in the present invention is treated to coat the tissue. Such coating can be performed by techniques well known in the art, and examples thereof include a method of immersing a biological tissue in such a biocompatible polymer solution, or a method of applying by spraying or the like. But not limited to them.

  In a further preferred embodiment, the biocompatible polymer of the present invention is advantageously cross-linked to a living tissue. Although not wishing to be bound by theory, it is because cross-linking makes the tissue of the living body tissue more robust. Such cross-linking may be achieved by gamma irradiation, but may be another means.

  In a preferred embodiment, the biocompatible polymer used in the present invention can be biodegradable. Examples of such biodegradable polymers include, but are not limited to, polyethylene glycol, polyvinyl alcohol, polyglycolic acid, polylactic acid, and the like. The polyethylene glycol-based polymer is a biodegradable polymer imparted with hydrophilicity. As the ethylene oxide content in the polymer increases, the hydrophilicity increases and swells in water.

  In another preferred embodiment, the biocompatible polymer used in the present invention is selected from a group selected from the group consisting of polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, elastin and mixtures of two or more thereof. It can contain molecules. More preferably, the biocompatible polymer used in the present invention comprises polyethylene glycol.

The polyethylene glycol used in preferred embodiments of the present invention typically has an average molecular weight between 200 and 6000. Such PEGs include products such as Nacalai tesque's polyethylene glycol (H (OCH ₂ CH ₂ ) _n OH) # molecular weight (eg, 200, 600, 1000, 2000, 6000, 35000, 50000). Preferably PEG has an average molecular weight between 1000 and 200000. In another preferred embodiment, the PEG can have an average molecular weight between 4000 and 100,000. More preferably, the PEG can have an average molecular weight between 8,000 and 50,000. Thus, although PEG is marketed, in order to achieve a desired characteristic, it can also synthesize | combine suitably. Such synthetic methods are well known in the art. In the present specification, the average molecular weight and the molecular weight are represented by Dalton. The PEG used in the present invention preferably has a homogeneous molecule, but this is not essential and can be used usually if the average molecular weight is within a certain range. In order to prepare the suppression of calcification in the present invention, chemical substances other than PEG (for example, including but not limited to enzymes (such as DNase I), enzyme inhibitors, etc.) can be added. When chemicals other than PEG are added, the amount or concentration of PEG to be added may vary depending on the amount of chemical present. Preferably, the concentration used for cell fusion or the like is good. For example, a concentration of 1 g / ml or more (100% w / w or more) is preferable, but not limited thereto. Alternatively, PEG can be used at a concentration between 60% w / v and 100% w / v.

  The polyvinyl alcohol used in a preferred embodiment of the present invention has a molecular weight in the range of 500-200,000. More preferably, the polyvinyl alcohol used has a molecular weight of 500 to 100,000, and more preferably 500 to 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 polyvinylpyrrolidone 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% (w / v). More preferably, the biocompatible polymer may be used in the range of 5% (w / v) to 30% (w / v), more preferably the biocompatible polymer is 10 (w / v). v) to 20% (w / v).

  Since the living body-derived tissue of the present invention is used in transplantation treatment, it is preferable that the tissue or organ is not damaged so as to hinder the function that the tissue or organ had in a normal state. This effect was not substantially damaged even by the gamma irradiation of the present invention. Whether there is such a hindrance can be determined by confirming that the extracellular matrix of the tissue is not substantially denatured. Thus, the tissue of the present invention can also be characterized by the substantial functional presence of the extracellular matrix. The presence of the extracellular matrix can be confirmed by staining with a specific marker. The function of the extracellular matrix can be performed by selecting an appropriate assay for each member.

  In one embodiment, the living tissue of the present invention has a tissue strength that can be clinically applied. Sufficiently strong tissue strength is an important property, especially when clinically applying membranous tissue. Tissue strength can generally be determined by measuring tensile strength (eg, breaking strength, stiffness, Young's modulus, etc.). In certain embodiments, the living tissue of the present invention is at least about 75% or more, preferably about 80% or more, more preferably about 85% or more, more preferably, the tissue strength of the normal tissue before treatment. It may be about 90% or more, or may have substantially the same tissue strength, and is a value higher than the tissue strength that the normal tissue originally had in the untreated state (eg, 110% or more, 120% or more , 150% or more, 200% or more). Here, the tissue strength when the tissue is in an untreated state has (for example, a natural state) before the treatment of the tissue (for example, treatment with γ-ray irradiation and 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.

  In one embodiment, calcification is remarkably suppressed when the living tissue of the present invention is transplanted. The degree of this suppression is, for example, (1) The amount of calcium per 1 mg of transplanted pericardium extracted as a result of a long-term (2 months) rat transplantation experiment is 0.5 (μg / mg) or less. Alternatively, the amount of calcium during the treatment by the present method / the amount of calcium during the glutaraldehyde treatment may be 0.5 or less, but not limited thereto. Alternatively, as a result of a long-term (2 months) rat transplantation experiment, when VonKossa staining is performed on the transplanted pericardium, a level that is hardly stained can be achieved.

  In one embodiment, the biological tissue of the present invention is characterized in that a biocompatible polymer is randomly crosslinked. Although not wishing to be bound by theory, the biocompatible polymer is randomly cross-linked by the treatment of biological tissue by γ-ray irradiation of the present invention, resulting in enhanced tissue strength, An enhancement of the inhibitory effect was achieved.

  In the case of a tubular tissue, the tissue strength can be represented by a β value. The method for calculating the β value was described in detail elsewhere in this specification. In certain embodiments, the biological tissue of the present invention has a tissue strength of β value of about 15 or more, preferably has a tissue strength of β value of about 18 or more, more preferably about 20 or more. A tissue strength of a value, more preferably a tissue strength of a β value of about 22 or greater. In another embodiment, the biological tissue of the present invention has at least about 75%, preferably about 80% or more, more preferably about 85% or more, more preferably, the β value of the tissue before treatment. It may be about 90% or more, and may have a value equal to or higher than the β value that the normal tissue originally had in the untreated state. Here, a characteristic of a tissue in an untreated state (for example, β value) means that the tissue is treated (for example, naturally treated with γ-ray treatment or biocompatible polymer of the present invention) (for example, naturally ) State).

  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 it is a tissue intended for clinical application. In certain embodiments, the biological tissue of the present invention can be a tissue in which the physical structure of the tissue is required. This is because in order to maintain the physical structure, only a structure such as a cell skeleton is necessary, and intracellular components such as cytoplasmic components are not necessarily required. In addition, if necessary, the required cells can be separately provided to the living tissue or supplied internally from the transplanted host. In certain embodiments, the biological tissue of the present invention can be tissue derived from an organ selected from blood vessels, blood vessel-like tissues, heart valves, pericardium, dura mater, cornea and bone. In another embodiment, the living 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 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, invertebrate). When intended for human application, tissue from vertebrates is preferably used, and more preferably, tissue from mammals (eg, primates, artiopods, odd-hoofs, rodents, etc.) is used. Used. If intended for human application, more preferably primate-derived tissue is used. If intended for human application, more preferably porcine tissue is used. This is because the size is similar to humans. When intended for human application, most preferably human-derived tissue is used. In the case of using a non-human tissue, the size of the biological tissue or tissue graft of the present invention is preferably close to that of human, and its physical characteristics are preferably close to that of human. (Eg pig).

  In the present invention, the site to which the living tissue of the present invention is applied may be any site 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) regardless of whether to “revert” or not, regardless of whether or not a biological tissue is applied to any part. Proved to achieve. Therefore, the present invention has great utility that it can be used in principle for any transplantation and regenerative surgery. Examples of the site to which the biological tissue of the present invention can be applied include, for example, skin, blood vessel, cornea, kidney, heart, liver, umbilical cord, intestine, nerve, lung, placenta, pancreas, brain, extremity, retina, valve, epithelium. Examples include, but are not limited to, tissue, connective tissue, muscle tissue, and nerve tissue.

  In another aspect, the present invention provides a tissue graft comprising the biological tissue of the present invention. The tissue graft is characterized in that the biological tissue is seeded with a recipient-derived cell and cultured to form a desired tissue structure. The tissue graft of the present invention may be intended for transplantation into any tissue of the body as long as it is a tissue graft intended for clinical application. In certain embodiments, the tissue graft of the present invention can be a tissue in which the physical structure of the tissue is required. In order to retain the physical structure, cells from the recipient can be provided to the above-described living tissue prior to transplantation. The recipient-derived cells may be supplied internally from the host. In certain embodiments, the tissue graft of the present invention can be a tissue graft of tissue derived from an organ selected from blood vessels, blood vessel-like tissues, heart valves, pericardium, dura mater, cornea and bone. In another embodiment, the tissue graft of the present invention can be a tissue graft of cardiovascular tissue, such as a tissue graft derived from an organ selected from blood vessels, blood vessel-like tissues, heart valves and pericardium obtain.

  The tissue graft of the present invention may comprise any biological tissue that is compatible with the intended clinical application. Therefore, the tissue graft of the present invention may be a tissue derived from any organism (eg, vertebrate, invertebrate). When intended for human application, preferably vertebrate tissue is used, more preferably mammal (eg, primate, rodent, etc.) tissue is used for the tissue graft of the present invention. It is done. When intended for human application, more preferably primate-derived tissue is used for the tissue graft of the present invention. If intended for human application, more preferably porcine tissue is used for the tissue graft of the present invention. This is because the size is similar to humans. When intended for human application, most preferably human-derived tissue is used for the tissue graft of the present invention.

  The recipient cell used for the tissue graft of the present invention may be any cell as long as it is suitable for clinical application. Accordingly, examples of the cells include, but are not limited to, vascular endothelial cells, smooth muscle cells, fibroblasts, blood cells, progenitor cells and somatic stem cells that differentiate into these cells. Preferably, the cell may be a cell that can perform a desired function at a site to be transplanted.

  In the present invention, the site to which the tissue graft of the present invention is applied may be any site in the body. Thus, it may be reapplied to the part of the body from which the tissue graft originates, or it may be applied to other parts. As shown in the examples and the like, the present invention achieves a desired effect (for example, regeneration, self-organization) regardless of whether or not “undo” is applied, regardless of whether the tissue graft is applied to any portion. Proven to achieve. Therefore, the present invention has great utility that it can be used in principle for any transplantation and regenerative surgery. Examples of the site to which the tissue graft of the present invention can be applied include skin, blood vessel, cornea, kidney, heart, liver, umbilical cord, intestine, nerve, lung, placenta, pancreas, brain, extremity, retina, valve, and epithelial tissue. , Connective tissue, muscle tissue, nerve tissue and the like.

  In another aspect, the present invention provides membranous tissue grafts, valvular tissue grafts, tubular tissue grafts, and the like. This tissue graft includes the biological tissue of the present invention. Here, in this living body-derived tissue, cells derived from the recipient may be seeded and cultured to form a desired tissue structure, but there may be no cells. Preferably there are no cells other than autologous. This is because immune rejection is not caused.

  In another aspect, the present invention is a method for producing a biological tissue, comprising A) providing the biological tissue; B) exposing the biological tissue to a biocompatible polymer; and C) Exposing a biological tissue to γ-irradiation. Any living body-derived tissue may be used.

  In one embodiment, the biocompatible polymer used can be, for example, polyvinyl alcohol, polyvinyl pyrrolidone, elastin, polyethylene glycol, gelatin, collagen, γ-polyglutamic acid and mixtures of two or more thereof. Preferably, polyethylene glycol is used.

  In a preferred embodiment, the step of exposing to a biocompatible polymer used in the present invention includes performing gamma irradiation to crosslink the biocompatible polymer.

  In one preferred embodiment, the dose of gamma irradiation used in the present invention is in the range of 10 to 250 kGy. More preferably, the amount of γ-ray irradiation is in the range of 50 kGy to 100 kGy. Although not wishing to be bound by theory, such an irradiation dose is appropriate for increasing the tissue strength. It is appropriate to set the upper limit of the dose that can maintain the tissue strength according to the tissue. From the viewpoint of tissue strength, 60 kGy may be stronger than 100 kGy, but is not limited thereto.

  In the present specification, γ-ray irradiation can be usually performed in an environment selected from the group consisting of vacuum, oxygen, nitrogen, air, water, amphiphilic molecule solution, and combinations thereof, It is not limited to them. Preferably, it is performed in the atmosphere.

  In a preferred embodiment, the γ-ray irradiation used in the present invention is performed in the range of 0.5 to 240 hours, more preferably 1 hour to 24 hours, and further preferably 3 to 8 hours. Although not wishing to be bound by theory, the irradiation time can be shortened by increasing the dose per unit time using a strong gamma ray source, but if it is too strong, the tissue may be adversely affected. Because. In order to lengthen the time, a weak γ-ray source may be used. I don't want to be bound by theory, but if it is too weak, it will take a long time and the tissue itself can be damaged by other factors.

  Any biocompatible polymer may be used in the present invention. In one embodiment, the average molecular weight of PEG used is between 200 and 6000, preferably the average molecular weight of PEG is between 200 and 2000. 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. it can. In one preferred embodiment, the average molecular weight of the PEG used in the present invention is between 1000 and 200000. In another preferred embodiment, the average molecular weight of the PEG used in the present invention is between 4000 and 100,000. In another embodiment, the average molecular weight of PEG used in the present invention is between 8000 and 50000. Alternatively, in one preferred embodiment, it is preferred to use PEG having an average molecular weight of 35000.

  In the method of the present invention, the biocompatible polymer is exposed to the tissue under any conditions as long as the living tissue and the biocompatible polymer interact with each other. Typically, it can be performed between 0 ° C. and 42 ° C., may be performed at room temperature (between about 15 ° C. and about 25 ° C.), and may be performed at 37 ° C. This step may be performed at a temperature above 37 ° C.

  In the method of the present invention, the biocompatible polymer such as PEG may be present in the solution at any concentration, but is preferably present at a concentration of 1 g / ml or more. Although not wishing to be bound by theory, γ rays are expected to function as radical scavengers. Alternatively, when considering the efficiency of γ-ray irradiation, the concentration of the PEG solution may be any concentration, but is preferably about 1% to about 50%, preferably about 10% to 30%. For example, about 20% is used, but not limited thereto.

  However, when PEG (for example, molecular weight 1000) is used as a raw material reagent for the biocompatible polymer, it may be preferably an aqueous solution because it is solid at ordinary temperature. In the method of the present invention, the biocompatible polymer (eg, PEG) may be dissolved in any solvent, but is preferably dissolved in an aqueous medium, more preferably physiological saline, PBS, Alternatively, it can be dissolved in an aqueous solution containing other salts. The biocompatible polymer is preferably a polymer having biocompatibility and capable of being used for pharmaceutical grades, such as PEG, (1) high purity methoxy PEG of SUNBRIGHT_MEH series manufactured by NOF Corporation ( http://www.nof.co.jp/business/dds/product01.html), (2) Polyethylene glycol polymer manufactured by Shigematsu Trading Co., Ltd. (http://www.shigematsu-bio.com/J/products /ecology/poly/hight.html), but not limited to. Alternatively, (1) Among the synthetic polymers that are cross-linked with γ rays to become gels, preferable ones that are expected to be most suitable for the purpose of reinforcing the valve or pericardium are PEG, PVA, polyvinylpyrrolidone ( PVP), polyacrylic acid, polyethylene oxide (PEO), polyvinylidene fluoride (PVdF) and the like can be mentioned, but not limited thereto. (2) Among the synthetic polymers that become gels by cross-linking with γ-rays, polyethylene, polypropylene, polyurethane, natural rubber (isoprene) may be suitable for this purpose of reinforcing valves and pericardium Unit) and synthetic rubber, and various polyamino acids (ε polylysine, γ polyglutamic acid, etc.) and various proteins (gelatin, collagen, elastin, fibrin albumin, keratin, casein, fibroin, etc.), but are not limited thereto. (3) Polysaccharides generally appear to react predominantly with degradation over crosslinking by gamma irradiation. Therefore, it is considered that the filler can be used by mixing with the crosslinkable polymer.

  Such biocompatible polymers are, for example, listed in the Japanese Pharmacopeia (or corresponding pharmacopoeia in other countries) or approved by the Ministry of Health, Labor and Welfare (or the corresponding regulatory authorities in other countries). Polymer.

  Preferably, the method of the present invention further comprises the step of washing the tissue immersed in the solution. This washing step can be performed using any liquid that is physiologically compatible (eg, physiological saline, PBS (phosphate buffered physiological saline), etc.). In a preferred embodiment, the washing of the present invention is performed with PBS. The cleaning solution is preferably sterilized. More preferably, the wash solution comprises an antibiotic (eg, penicillin, cephalosporin, streptomycin, tetracycline, erythromycin, vancomycin, ciprofloxacin, linezlid, etc.). Washing may be performed under any conditions, 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.). It may be performed at ° C. Washing may be performed at a temperature above 37 ° C. In the method of the present invention, the washing step may be performed in any period as long as the solution used in the treatment (for example, a solution containing a biocompatible polymer) is sufficiently removed, but is usually 0. Can be performed for 5 to 5 days. Preferably, in the washing step, the washing solution (eg, PBS) may be changed several times.

  The tissue used in the methods of the present invention may be any biological tissue that 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 (eg, vertebrate, invertebrate). When intended for human use, the tissue used in the method of the present invention is preferably a vertebrate tissue, more preferably a mammal (eg, primate, rodent, etc.). Origin tissue is used. When application to humans is intended, the tissue used in the method of the present invention is more preferably a primate-derived tissue. If intended for human application, more preferably, tissues derived from porcine or bovine are used. This is because the size is similar to humans. When intended for human application, 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. In certain embodiments, the tissue used in the methods of the present invention may be a tissue that requires the physical structure or physical properties of the tissue. This is because in order to maintain the physical structure or physical properties, only a structure such as an extracellular matrix is necessary, and an intracellular component such as a cytoplasmic component or a cell membrane component is not necessarily required. In addition, if necessary, the required cells can be separately provided to the living tissue or supplied internally from the transplanted host. In certain embodiments, the tissue used in the methods of the invention may be tissue derived from an organ selected from blood vessels, blood vessel-like tissues, heart valves, pericardia, dura mater, corneas and bones. In another embodiment, the tissue used in the methods of the invention can be cardiovascular tissue, such as from an organ selected from blood vessels, blood vessel-like tissue, heart valves and pericardium.

In one embodiment, the present invention may further comprise a chemical treatment, for example treatment with a bifunctional molecular crosslinker (eg glutaraldehyde or a derivative thereof). The treatment with the bifunctional molecular cross-linking agent aims to increase physical strength by chemically cross-linking protein components including cells in the ECM or tissue. Therefore, as long as it is used for this purpose, any bifunctional molecular crosslinking agent can be used. Among such bifunctional molecular crosslinkers, those actually used for tissue fixation (valve graft) are, for example, cyanimide, 1-ethyl-3- (3-dimethylaminopropyl) Examples include, but are not limited to, carbodiimide hydrochloride (EDC), epoxy (poly (glycidyl ether)) or photocrosslinker PhotoMix ^™ (Sulzer Carbomedics Co. Ltd.) (for reference, Biomaterials (2000) 21: 2215-2231). checking).

  In another embodiment, the chemical treatment can include treatment with a nuclease such as DNase, such as DNaseI. Since treatment with such DNase can further remove DNA components that are undesirable for transplantation, this treatment with DNase is preferred. Such DNase may be any DNase but is preferably DNaseI. DNA, which is a charged polymer substance, can be removed by DNase treatment. Since DNA can elicit an immune response, further removal of the DNA can provide additional advantages.

  The treatment with a nuclease (for example, DNase) may be performed under any conditions, but may be combined with a pressurizing condition, a stirring condition, and the like. Examples of the concentration include 1 U / ml to 100 U / ml, and examples include, but are not limited to, 50 to 75 U / ml. 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, 100-500 RPM, Preferably it is 100-150 RPM) conditions. Pre- and post-nuclease treatment is preferably stored using the above-described washing solution.

  In another preferred embodiment, the method for producing a biological tissue of the present invention may further include a step of seeding cells, but is not limited thereto. The cells to be seeded can be appropriately selected by those skilled in the art depending on the situation, as described above in the present specification.

  The present invention also provides a biological tissue obtained by the method of the present invention. This biological tissue can preferably have the above-mentioned cell survival rate and / or tissue damage rate and / or tissue strength. Before the treatment method using the radical reaction of the present invention was provided, it was not possible to provide a living tissue having the above-mentioned cell survival rate and / or tissue damage rate and / or tissue strength. The inventive method provides an entirely new material that provides a biologically derived tissue with properties such as inhibition of calcification that could not be provided by conventional methods, and provides unexpected advantages. .

  In another aspect, the present invention relates to a method for regenerating a tissue, comprising: a) providing a living body tissue containing a biocompatible polymer and receiving gamma irradiation in a living body; And b) incubating for a time sufficient for tissue regeneration to occur in the organism. If necessary, the method may include a step of providing a physiologically active substance that induces differentiation of the cell and / or a step of providing the cell to the living tissue, or may not provide the cell to the living tissue. Good. Preferably, the physiologically active substance used for maintenance or differentiation of the target tissue is used. The physiologically active substance may be derived from in vivo or derived from outside the living body. In the case of a substance derived from a living body, it is understood that exposure to an appropriate physiologically active substance can be performed simply by passing a certain period of time after transplantation without performing any operation. Examples of the physiologically active substance include, but are not limited to, HGF, VEGF, FGF, IGF, PDGF, and EGF.

  Preferably, the cell can be a vascular cell or a vascular-like cell. More preferably, the cell may be from a recipient. Alternatively, preferably, the tissue may be a tissue selected from the group consisting of blood vessels, blood vessel-like tissue, heart valves, pericardium, dura mater, cornea and bone. In a preferred embodiment, the tissue and the cell can be from the same host. In another embodiment, the tissue and the cell can be from an allogeneic host. In another embodiment, the tissue and the cell can be from a heterologous host. If the recipient and cell are allogeneic or xenogeneic, rejection may be considered, so the recipient and cell are preferably from the same host, but if rejection is not a problem allogeneic It may be derived from different origins or different origins. Moreover, what causes rejection reaction can also be utilized by performing the treatment which eliminates rejection reaction as needed. Procedures for avoiding rejection are well known in the art and are described, for example, in the New Surgery System, Heart and Lung Transplant Technical and Ethical Preparations to 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" (Sandimune / Neoral), "Tacrolimus" (Prograf), "Azathioprine" (Imlan), "Steroid Hormone" (Predonin, Methylpredonin), "T cells There are “antibodies” (OKT3, ATG, etc.), and the method used in many facilities around the world as a preventive immunosuppressive therapy is a combination of three drugs “cyclosporine, azathioprine and steroid hormones”. Since the present invention exerts a remarkable effect in that it suppresses calcification, an 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 using an immunosuppressive agent, the immunosuppressive agent can be administered before or after performing the regeneration method as long as the immunosuppressive effect is achieved.

In another aspect, the present invention is a method for producing a tissue graft, comprising: A) providing a living tissue containing a biocompatible polymer in vivo; B) the living body's self Invading the cells; and C) incubating for a time sufficient for the differentiation of the cells to occur;
Is included. In this case, the biological tissue of the present invention may or may not further have cells. Such cells can be autologous, allogeneic, allogeneic, or xenogeneic. Preferably, the cell can be a vascular cell or a vascular-like cell. More preferably, the cell may be from a recipient. Preferably, the tissue may be that of a blood vessel, a blood vessel-like tissue, one selected from the group consisting of a heart valve, pericardium, dura mater, cornea and bone. In a preferred embodiment, the tissue and the cell can be from the same host. In another embodiment, the tissue and the cell can be from an allogeneic host. In another embodiment, the tissue and the cell can be from a heterologous host. If the recipient and cell are allogeneic or xenogeneic, rejection may be considered, so the recipient and cell are preferably from the same host, but if rejection is not a problem allogeneic It may be derived from different origins or different origins. Moreover, what causes rejection reaction can also be utilized by performing the treatment which eliminates rejection reaction as needed. Treatments that resolve rejection are detailed herein.

  In a preferred embodiment, the method for producing a tissue graft of the present invention may further comprise D) providing a physiologically active substance that induces the differentiation of the cells. Preferably, the physiologically active substance can be a cytokine having hematopoietic activity. When producing a tissue graft, the cytokine 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.

  In another aspect, the present invention provides a native tissue or tissue graft produced by the method of the present invention. Such biological tissue or tissue graft has characteristics that did not exist in the past in terms of tissue strength and calcification inhibition rate.

  In another aspect, the present invention is a method of treating or preventing a subject in need of or at risk for tissue or organ transplantation, comprising A) a biocompatible polymer and receiving gamma irradiation. Providing a tissue derived from a living body, or a tissue graft containing the living tissue, and B) transplanting the living tissue or tissue graft into a subject. . This living body-derived tissue further includes cells as necessary. The cells may be autologous or allogeneic. Preferably, the tissue may be that of a blood vessel, a blood vessel-like tissue, one selected from the group consisting of a heart valve, pericardium, dura mater, cornea and bone. In preferred embodiments, the tissue can be from a subject. In another embodiment, the tissue can be from a host allogeneic to the subject. In another embodiment, the tissue can be from a host heterologous to the subject. In this treatment / 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 scale, or when cells other than the recipient are used, when rejection occurs, treatment to eliminate the rejection can be performed as necessary. Treatments that resolve rejection are detailed herein. In one embodiment, the tissue used in the method of treating or preventing of the present invention may be derived from a subject. In another embodiment, the tissue used in the treatment or prevention method of the present invention may be tissue from any organism (eg, vertebrate, invertebrate). Preferably, tissue from vertebrates is used when humans are treated or prevented, and more preferably tissue from mammals (eg, primates, rodents, etc.) when humans are treated or prevented Is used. When humans are treated or prevented, more preferably primate-derived tissue is used. When humans are treated or prevented, in another preferred embodiment, tissue from pigs or cattle is used. Pigs or cows are preferred because they have a size similar to humans. When humans are treated or prevented, most preferably human-derived tissue can be used.

In another aspect, the present invention provides a medicament for organ transplantation. This medicine includes A) a biological tissue containing a biocompatible polymer and receiving γ-ray irradiation, or a tissue graft containing the biological tissue.
In certain embodiments, the medicament of the present invention comprises tissue derived from an organ selected from blood vessels, blood vessel-like tissues, heart valves, pericardium, dura mater, cornea and bone. In another embodiment, the medicament of the present invention may comprise cardiovascular tissue, for example, derived from an organ selected from blood vessels, blood vessel-like tissue, heart valves and pericardium.

  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 include materials approved by the applicable national regulatory authority. Therefore, the medicament of the present invention may contain tissue derived from any organism (for example, vertebrates and invertebrates). When the pharmaceutical of the present invention is intended to be applied to humans, tissue derived from vertebrates is preferably used, and more preferably tissue derived from mammals (eg, primates, rodents, etc.). . When the medicament of the present invention is intended to be applied to humans, a primate-derived tissue is more preferably used. If the medicament of the present invention is intended for human application, more preferably, a tissue derived from porcine or bovine is used. This is because the size is similar to humans. When the medicament of the present invention is intended for human application, human-derived tissue is most preferably used. However, when using human-derived tissue, it may be necessary to clear ethical rules and problems.

  The medicament, tissue graft and biological tissue of the present invention may also further comprise a biocompatible material. This biocompatible material is, for example, a group consisting of silicone, collagen, gelatin, glycolic acid / lactic acid copolymer, ethylene vinyl acetic acid copolymer, polyurethane, polyethylene, polytetrafluoroethylene, polypropylene, polyacrylate, and polymethacrylate. It may include at least one more selected. Silicone is preferred because it is easy to mold. Examples of biodegradable polymers include collagen, gelatin, α-hydroxycarboxylic acids (eg, glycolic acid, lactic acid, hydroxybutyric acid, etc.), hydroxydicarboxylic acids (eg, malic acid, etc.) and hydroxytricarboxylic acids (eg, , Citric acid, etc.) from one or more selected from the group consisting of polymers, copolymers or mixtures thereof, poly-α-cyanoacrylates, polyamino acids (for example, Poly-γ-benzyl-L-glutamic acid and the like), and polyanhydrides of maleic anhydride copolymers (for example, styrene-maleic acid copolymer and the like). The form of polymerization may be random, block, or graft. When α-hydroxycarboxylic acids, hydroxydicarboxylic acids, and hydroxytricarboxylic acids have an optically active center in the molecule, D-form, L-form, and DL-form Any of these can be used. In some embodiments, a copolymer of glycolic acid / lactic acid 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. Of course, the medicament, tissue graft and biological tissue of the present invention may contain two or more other drugs. Examples of such drugs include those listed in the latest editions of the Japanese Pharmacopoeia, the US Pharmacopoeia, the pharmacopoeia of other countries, and the like. Such an agent may preferably have an effect on the organs of the organism. Examples of such agents include thrombolytic agents, vasodilators, and tissue activators. The amounts of physiologically active substances, other drugs and cells contained in the medicament, tissue graft and biological tissue of the present invention are the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, It can be easily determined by those skilled in the art in consideration of past medical history and the like.

  In another aspect, the present invention relates to a biological tissue comprising the biocompatible polymer of the present invention for producing a medicament for organ transplantation, which has been subjected to γ-irradiation, or The present invention relates to the use of a tissue graft containing a biologically derived tissue characterized by containing a biocompatible polymer and receiving γ-ray irradiation. In certain embodiments, tissue from an organ selected from blood vessels, blood vessel-like tissues, heart valves, pericardium, dura mater, cornea and bone may be used in the use of the present invention. In another embodiment, in the use of the present invention, cardiovascular tissue can be used, for example, derived from an organ selected from blood vessels, blood vessel-like tissue, heart valves and pericardium.

  In the use of the present invention, any biological tissue can be used as long as it is compatible with the intended clinical application. Preferably, materials approved by the applicable national regulatory authorities may be used. Accordingly, tissue from any organism (eg, vertebrates, invertebrates) may be used in the use of the present invention. When the use of the present invention is intended to be applied to humans, tissues derived from vertebrates are preferably used, and more preferably tissues derived from mammals (eg, primates, rodents, etc.) are used. . If the use of the present invention is intended for human application, more preferably primate-derived tissue is used. If the medicament of the present invention is intended for human application, more preferably, a tissue derived from porcine or bovine is used. This is because the size is similar to humans. When the use of the present invention is intended for human application, human-derived tissue is most preferably used. However, when using human-derived tissue, it may be necessary to clear ethical rules and problems.

  The use of biological tissue, grafts and medicaments of the present invention is usually under the supervision of a physician, but can be used without the supervision of a physician if the national supervisory authority and law allow. .

  The amount of biological tissue, graft, and medicament used in the treatment or prevention method of the present invention is the purpose of use, target disease (type, severity, etc.), subject's age, weight, sex, medical history, physiological activity. A person skilled in the art can easily determine in consideration of the form or type of a substance, the form or type of a 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 living tissue, graft and medicine used per time, purpose of use, target disease (type, severity, etc.), patient's A person skilled in the art can easily determine the age, weight, sex, medical history, treatment course, 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 observing the course.

  In the present specification, molecular biological techniques, biochemical techniques, and microbiological techniques well known in the art are used as necessary. Such methods are described, for example, by Ausubel FA et al. (1988), Current Protocols in Molecular Biology, Wiley, New York, NY; Sambrook J et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, It is described in Cold Spring Harbor, NY, a separate volume of experimental medicine "Gene transfer & expression analysis experiment method", Yodosha, 1997, etc.

  In another aspect, the biological tissue, graft and / or medicament of the present invention can be provided in the form of a kit with instructions giving instructions for administering the biological tissue, graft and / or medicament. Here, the instruction manual describes a word indicating an appropriate administration method of a tissue derived from a living body, a graft and / 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, etc.) in the country where the present invention is implemented, and is approved by the supervisory authority. It is clearly stated that it has been received. The instructions are so-called package inserts and are usually provided in a paper medium, but are not limited thereto, and are in a form such as an electronic medium (for example, a homepage or an e-mail provided on the Internet). But it can be provided.

  The living tissue, tissue graft and medicament of the present invention can be transplanted using techniques well known in the art (for surgical procedures, nonstandard science 9th edition (medical bookstore), basic 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, those skilled in the art can appropriately apply the biological tissue, tissue graft, and medicament of the present invention according to the disclosure of the present specification, depending on the situation to be treated.

  In the present invention, the site to which the medicament of the present invention is applied may be any site in the body. Thus, it may be reapplied to the part of the body from which the medicament is derived, or it may be applied to other parts. As shown in the examples and the like, the present invention achieves a desired effect (for example, regeneration, self-organization) regardless of whether it is “reverted” or not, regardless of whether it is applied to any part. Proven to do. Therefore, the present invention has great utility that it can be used in principle for any transplantation 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, pancreas, brain, extremity, retina, valve, epithelial tissue, Examples include, but are not limited to, connective tissue, muscle tissue, and nerve tissue.

  The present invention will be described below based on examples, but the following examples are provided for illustrative purposes only. Accordingly, the scope of the present invention is not limited to the detailed description of the invention nor the following examples, but is limited only by the scope of the claims.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples. The reagents used in the following examples were those commercially available from Sigma (St. Louis, USA), Wako Pure Chemical (Osaka, Japan), etc., with the exception. Animal experiments were conducted in compliance with the standards stipulated by Osaka University.

Example 1
(Materials and methods)
(material)
(1) Charge solution
PBS (-) (pH 7.4; 0.1 M phosphate buffer (sodium dihydrogen phosphate, 2 water (monosodium phosphate) NaH ₂ PO ₄ · 2H ₂ 0 11.84 g and disodium hydrogen phosphate • 12 water (disodium phosphate) Na ₂ HPO ₄ · 12H ₂ 0 116.0 g; Preparation method: Take approximately 1800 ml of distilled water to dissolve sodium dihydrogen phosphate (dissolve easily) Hydrogen phosphate 2 Sodium was added (slightly soluble.) When completely dissolved, distilled water was added to make a total volume of 2000 ml.) 9 g of NaCl was added to 1000 ml) to prepare the following.
20 mM EDTA, Protease Inhibitors cocktail
0.1% NaN ₃
100 U / ml penicillin 100 μg / ml streptomycin (1%)
1.25 μg / ml amphotericin B (0.5%)
Kanamycin 100mg / l (SIGMA)
(2) The following was prepared using a rinse solution bath / PBS (−) (pH 7.4).
0.1% NaN ₃
100 U / ml penicillin 100 μg / ml
Streptomycin (1%)
1.25 μg / ml amphotericin B (0.5%)
Kanamycin 100mg / l
(3) PEG solution / PEG (molecular weight 8,000, 35,000, 50,000) is dissolved in a microwave oven and adjusted to 20% with sterile Milli Q.

(Treatment using biocompatible polymer for gamma irradiation application)
Bovine biological tissue (aorta, aortic valve, pericardium) was obtained from Hybrid (mixed species) (Labo Products Co. Ltd., Osaka, Japan), and rat aorta was treated with SD rats (male, 5 weeks old, Japanese animals, Osaka, From Japan) under sterile conditions. The ethics of animal use was in accordance with the Osaka University animal experiment ethics regulations.

  The pericardium was removed from the cow and treated with polyethylene glycol as the biocompatible polymer. The protocol is shown below.

(1) Removal of pericardium Freshly collected bovine pericardium is prepared by using physiological saline or PBS containing antibiotics (Gibco BRL, Life Technologies Inc. Rockville, MD, USA) (this is PBS (−) in this example). (Gibco BRL, Life Technologies Inc. Rockville, MD, USA) and washed away blood components.

(2) Pericardium processing 1) The pericardium sent in the charge solution in the clean bench was transferred to a sterilized vat to remove adipose tissue and thick parts.
2) Cut to the size to be used (currently 10cm x 5cm) and wash several times with rinse solution. (The amount of the solution at the time of washing varies depending on the amount of pericardium. Solution was used).

(3) Infiltration of PEG into tissue 1) 20 ml of 20% PEG solution is placed in a sterile petri dish, and the pericardium is immersed therein. After soaking, the pericardium was squeezed with a large rod or roller, and the solution was injected.
2) The pericardium was sealed in a hybrid bag together with a 20% PEG solution, and after removing air as much as possible, it was sealed.
3) The bag was irradiated with γ rays (25 kGy or 50 kGy).
4) After the irradiation, it was stored as it was at 4 ° C.

(Collagen structure)
It has been shown that the collagen structure can 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, Biochimica 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 experiment demonstrated that the matrix was not damaged at all by histological examination of collagen structure and tensile strength measurement.

(Histological examination)
Paraffin sections (thickness 3 μm) of vascular grafts were prepared and stained with hematoxylin-eosin to identify the extracellular matrix. An immunohistochemical staining method was used to identify type I / IV collagen, which is a component of the basement membrane. The aorta of SD rats (male, 5 weeks old, Nippon Animal Co. Ltd. Tokyo, Japan) was fixed with 4% paraformaldehyde and cryoprotected. Cryosections (5 μm thick) were prepared, permeabilized with PBS (−) for 3 hours, and blocked with 1% BSA in PBS (−) for 1 hour at room temperature. The sections were then incubated with a primary antibody (anti-rat collagen antibody, Cosmo Bio, Tokyo, Japan) and incubated with a secondary antibody conjugated with FITC (anti-sheep Ig antibody; Cosmo Bio, Tokyo, Japan). . Images were obtained using a Zeiss LSM510 confocal microscope.

(Von Kossa staining)
von Kossa staining was performed as follows. If necessary, deparaffinization (for example, with pure ethanol), washing with water (distilled water) was performed, and immersed in a 25% silver nitrate solution (under indirect light) for 2 hours. Thereafter, it was washed with distilled water and immersed in 42% 2% sodium thiosulfate (hypo) for 5 minutes. Thereafter, it was washed with running water for 5 minutes and then immersed in Cologne heteroto for 5 minutes. Thereafter, it was washed with running water for 5 minutes, dehydrated, clarified and sealed.

(Quantification of calcium)
1. A sample (10 mg) was weighed.
2.6 μl HCl was added at 50 μl per mg sample.
3. Incubated at 85 ° C for 48 hours.
4). An equal volume of 500 μl of 6N NaOH was added.
5. The calcium concentration was measured with an atomic absorption photometer.

(Gamma irradiation)
In this example, γ-ray irradiation was performed as follows. The procedure is shown briefly.

  Place the biological tissue after PEG treatment in a glass sample bottle with a plastic lid (diameter 5 cm x height 8 cm), and close the lid (tissue + air inside). Went. Although the temperature was room temperature (room temperature), the temperature slightly increased due to the amount of heat generated by irradiation, and it seemed to be between 25 ° C. and 40 ° C. at the time of irradiation. Cleaning may or may not be performed before γ-ray irradiation.

  Although not wishing to be bound by theory, the effects of γ rays include, for example, both suppression of calcification, direct cytotoxicity by γ rays, and cytotoxicity of free radicals generated by the adoption of γ rays. Conceivable. In this case, PEG may act as a scavenger.

  Next, various conditions of γ-ray irradiation were examined.

(Γ-ray dose)
None, 20 kGy, 40 kGy, 50 kGy, 80 kGy, 100 kGy, 130 kGy, 250 kGy were used as the γ-ray irradiation dose.

(Agitation treatment)
The presence or absence of stirring under the above conditions was combined.

(Ambient conditions)
In addition to air, PEG (similar to a treatment solution having an average molecular weight of 35,000) in water was used.

(Control experiment)
As controls, biological tissue (valve) and bovine raw valve that had been treated with glutaraldehyde chemistry (no γ-irradiation) were included.

(Comparison of reactions in living tissue)
(Method)
(Immunological response)
A biocompatible polymer-treated living tissue prepared subcutaneously on the back of an SD rat was transplanted and sacrificed after 1 week and 2 months, and the degree of inflammatory cell infiltration was scored and evaluated. In this example, as a control, comparison was made with the untreated valve and bovine raw valve.

(Calcification)
Specimens implanted subcutaneously in rats were collected after 1 week and 2 months, and calcification was evaluated by von Kossa staining. In addition, the tissue Ca concentration was 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 was diluted and sprayed into high-temperature plasma, and quantitative measurement was performed from the element-specific absorption wavelength (Ca is 393.366 nm) of the spectrum generated during combustion.

  An aortic wall portion (10 × 10 mm) of a γ-irradiated bovine aortic valve and other control valves was transplanted subcutaneously to the back of an SD rat (250 g), and a specimen was collected 2 months later. Atomic absorption spectroscopy (SPS7800: Seiko Instrument) Inc.) was measured for tissue Ca concentration (Ca content per dry weight) (see Ozaki S. Pathology of biosynthesis of bioprosthetic heart vales. Acta Biomedical. 1).

(Transplant)
A portion of the aortic wall tissue obtained by the process of the present invention was transplanted into the canine descending aorta.

(result)
The results of this Example are the results of subcutaneous implantation of rats treated with γ-rays (real image of HE staining; FIG. 1), HE staining (FIG. 2), Von Kossa staining (FIG. 3), and calcium determination (FIG. 4; untreated valve control). As a result, the non-eatening result of the untreated valve control as a control valve (real image of HE staining; FIG. 5) is shown.

As shown in the results (FIGS. 1 to 3) two months after the result of subcutaneous transplantation, infiltration of inflammatory cells was not observed at any irradiation dose (25 kGy, 50 kGy), and it was clearly established at the transplant destination. Became. On the other hand, multiple layers of inflammatory cells were infiltrated in the untreated bovine live flap (FIG. 5 ) . As another cross-linking reaction, calcium deposition progressed when glutaraldehyde treatment was performed (not shown) (see FIG. 4). When the doses were compared, irradiation with 25 kGy showed almost no lime deposition when viewed with von Kossa staining. Both were found to have achieved a significant effect on calcification, as they were hardly observed at 50 kGy. Cell infiltration as seen by HE staining was not observed at any dose.

(Example 3) Confirmation of cell replacement Comparison of responses of each valve in living tissue The canine femoral aorta was transplanted with the forearm artery of the γ-irradiated bovine produced in Example 1, and 10 days later, the bovine was sacrificed. Compare the degree of inflammatory cell infiltration.

(result)
It can be seen that the biological tissue of the present invention hardly shows an inflammatory reaction and the entire tissue structure is not damaged. Furthermore, by observing the transplanted tissue, it can be confirmed that the treated tissue is replaced with autologous cells.

(Example 4: Validation in rat model of myocardial infarction)
Next, the artificial pericardium prepared according to Example 1 is transplanted into the rat infarcted heart.

(Myocardial infarction rat model)
Male Lewis strain rats were used in this example. National Society for Medical Research was created by the "Principles of Laboratory Animal Care", and the Institute of Laboratory Animal Resource has announced the creation and National Intitute of Health "Guide for the Care and Use of Laboratory Animals" (NIH Publication No.86- 23, 1985 revision), animals are cared for in accordance with the spirit of animal welfare.

  Acute myocardial infarction was induced as described in the literature (Weisman HF, Bush DE, Mannisi JA et al. Cellular mechanism of myocardial infusion expansion. Circulation. 1988; 78: 186-201). Briefly, rats (300 g, 8 weeks old) are anesthetized with sodium pentobarbital and positive pressure breathing is performed. To create a rat myocardial infarction model, the thorax is opened between the left fourth ribs and the left coronary artery is completely ligated with 8-0 polypropylene thread at a distance of 3 mm from the root.

(Transplant)
Recipient rats are anesthetized and the heart is exposed by opening the rib cage between the fifth left intercostals. The rats are divided into two groups according to the substance administered to the myocardial infarction region. Group C (no treatment group, n = 5) and group S (decellularized pericardium transplant group, n = 5). The decellularized pericardium is transplanted directly to the infarct site 2 weeks after ligation of the left anterior descending branch.

(Measurement of cardiac function of rat heart)
Cardiac function is measured with cardiac ultrasound (SONOS 5500, manufactured by Agilent Technologies) 2 weeks after the creation of the infarct model, 4 weeks and 8 weeks after transplantation. Using a 12-MHz transducer, a short-axis image is drawn from the left side at the position where the left ventricle shows the maximum diameter. In B-mode, the left ventricular end systolic area (end systolic area), M-mode, left ventricular end-diastolic diameter (LVDd), left ventricular end-systolic diameter (LVDs), left ventricular anterior wall thickness (LVATh) Measure LVEF and LVFS.

(Histological analysis)
After cardiomyocyte transplantation, the heart is removed 8 W later, cut with a short axis, attached to a 10% formaldehyde solution, and fixed with paraffin. A section is prepared, and hematoxylin-eosin staining and Masson-trichrome staining are performed. A frozen section at the same time is prepared, and Factor VIII immunostaining is performed.

  Thus, it becomes clear that the γ-ray treated living body tissue of the present invention can be sufficiently applied to portions other than the site from which the tissue before treatment is derived.

(Example 5: Treatment of dura mater)
Next, the dura mater can be removed from the cow, treated with a polyethylene glycol solution as described in Example 1, and irradiated with γ rays.

  This dura mater can be implanted into Lewis rats as described in Example 2 and examined for inflammatory cell infiltration and calcification.

  Thus, it can be seen that the method of the present invention can perform γ-ray irradiation treatment showing the same excellent characteristics regardless of the tissue used.

(Example 6: Other polymer)
Next, as other biocompatible polymers, in addition to polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, γ-polyglutamic acid, and gelatin are subjected to the same experiment as in Example 1. These biocompatible polymers are crosslinked and gelled under the same conditions of γ-ray irradiation (concentration 10%, irradiation of about 60-100 kGy using the same glass bottle and cobalt 60 radiation source). By conducting the same experiment as in Example 1, it is possible to confirm the effect of strengthening the same biological tissue. Specifically, by measuring the maximum point load (N), maximum point elongation (in mm), and elastic modulus (MPa) as described in Example 1, in addition to polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, The same reinforcing effect can be confirmed by using γ-polyglutamic acid and gelatin.

(Example 7)
Next, it is confirmed whether the same effect appears also in the living body tissue of a blood vessel, an intestinal tract, a pericardium, a mesentery, a cornea, a retina, a bone, and a ureter. The same experiment as in Example 1 is performed. These are subjected to biocompatible polymer treatment and γ-irradiation treatment using polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, γ-polyglutamic acid and gelatin. About this reinforcement | strengthening biological origin tissue, the reinforcement effect of the same biological origin tissue can be confirmed by performing the same experiment as Examples 2-5. The same calcification-inhibiting effect and reinforcing effect can be confirmed even in living tissue derived from blood vessels other than valves, intestinal tract, pericardium, mesentery, cornea, retina, bone, and ureter.

(Example 8: Examination of optimum parameters)
Next, in addition to the above examples, biological tissue prepared by various treatment methods were used for other molecular weights of PEG, PVA, polyvinyl pyrrolidone (PVP), polyacrylic acid, polyethylene oxide (PEO), polyfluorinated. The effect of strengthening with vinylidene (PVdF) can be verified.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  According to the present invention, an improved method of technology 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 useful in industry.

FIG. 1 shows a state two months after the transplantation of a biological tissue derived from the biocompatible molecular treatment and γ-irradiation of the present invention into a rat. In this figure, the bovine pericardium polymerized with 25 kGy gamma rays is shown on the top, and the bovine pericardium polymerized with 50 kGy gamma rays on the bottom. The left shows the state in the living body, and the right shows the extracted living tissue. The cell infiltration is thin with 25 kGy treatment, but significant cell infiltration is observed with 50 kGy treatment. FIG. 2 shows a HE-stained image of 2 months after transplantation of a biological tissue derived from the biocompatible molecule treatment and γ-irradiation of the present invention into a rat. In this figure, the bovine pericardium polymerized with 25 kGy gamma rays is shown on the top, and the bovine pericardium polymerized with 50 kGy gamma rays on the bottom. Each figure is an example showing photographs at various sites. FIG. 3 shows von Kossa staining for 2 months after transplantation of biologically derived tissue after biotreatment and γ-irradiation of the present invention into rats. In this figure, the bovine pericardium polymerized with 25 kGy gamma rays is shown on the top, and the bovine pericardium polymerized with 50 kGy gamma rays on the bottom. Each figure is an example showing photographs at various sites. FIG. 4 shows the amount of calcium deposition 2 months after the transplantation of the living body-derived tissue to the rat after the biocompatible molecular treatment and γ-ray irradiation of the present invention. In the graph, from the left, bovine pericardium polymerized with 25 kGy γ-ray, bovine pericardium polymerized with 50 kGy γ-ray, and bovine pericardium treated with glutaraldehyde are shown. FIG. 5 shows 2-month transplantation data for untreated bovine pericardium.

Claims (56)

A biological tissue comprising a biocompatible polymer and receiving γ-ray irradiation. The living body-derived tissue according to claim 1, wherein the living body-derived tissue has not undergone a decellularization treatment. The biological tissue according to claim 1, wherein the extracellular matrix component is at least partially crosslinked by a covalent bond. The living tissue according to claim 3, wherein the extracellular matrix component is selected from the group consisting of collagen, elastin, laminin, fibronectin, tenascin, glycosaminoglycan and proteoglycan. The biological tissue according to claim 1, wherein the biocompatible polymer coats the biological tissue. The biological tissue according to claim 1, wherein the biocompatible polymer is crosslinked to the biological tissue. The biological tissue according to claim 1, wherein the gamma irradiation is provided at a dose between 10 kG and 250 kG. The biological tissue according to claim 1, wherein the gamma irradiation is provided at a dose between 25 kG and 50 kG. The biological tissue according to claim 1, wherein the biocompatible polymer is biodegradable. The biocompatible polymer is selected from the group consisting of polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), elastin, polyethylene glycol (PEG), gelatin, collagen, γ-polyglutamic acid and mixtures of two or more thereof. The living body-derived tissue according to claim 1, comprising a polymer selected from the group. The biological tissue according to claim 1, wherein the biocompatible polymer comprises polyethylene glycol (PEG). The biological tissue according to claim 11, wherein the polyethylene glycol (PEG) has a molecular weight in the range of 1000 to 200,000. The biological tissue according to claim 11, wherein the polyethylene glycol (PEG) has a molecular weight in the range of 8,000 to 50,000. The biological tissue according to claim 1, wherein the biological tissue has a tissue strength that can be clinically applied. The living body-derived tissue according to claim 1, wherein the living body-derived tissue is remarkably suppressed in calcification when transplanted. The biological tissue according to claim 1, wherein the biocompatible polymer is crosslinked at random. The biological tissue according to claim 1, wherein the biological tissue is a membranous tissue, a valve-like tissue, or a tubular tissue. The biological tissue according to claim 1, wherein the biological tissue is a tissue selected from blood vessels, blood vessel-like tissues, heart valves, pericardium, dura mater, corneas and bones. The living body-derived tissue according to claim 1, wherein the living body-derived tissue is derived from a mammal. The living body-derived tissue according to claim 1, wherein the living body-derived tissue is derived from human, cow or pig. A tissue graft for transplantation comprising the living tissue according to claim 1. The tissue graft of claim 21, wherein the tissue graft has a shape selected from the group consisting of a membrane, a tube, and a valve. A method for producing a tissue for transplantation, comprising:
A) providing a biological tissue;
B) exposing the biological tissue to a biocompatible polymer; and C) exposing the biological tissue to gamma irradiation.
Including the method. The method according to claim 23, wherein the gamma irradiation is performed under a condition in which a chemical reaction occurs in the biocompatible polymer. 24. The method of claim 23, wherein the gamma irradiation is provided at a dose between 10 kG and 250 kG. 24. The method of claim 23, wherein the gamma irradiation is provided at a dose between 25 kG and 50 kG. 24. The method of claim 23, wherein the gamma irradiation is performed in an environment selected from the group consisting of vacuum, oxygen, nitrogen, air, water, amphiphilic molecule solution, and combinations thereof. The method according to claim 23, wherein the gamma irradiation is performed in a range of 0.5 to 240 hours. 24. The method of claim 23, wherein the biocompatible polymer is biodegradable. The biocompatible polymer is a polymer selected from the group consisting of polyvinyl alcohol, polyvinyl pyrrolidone, elastin, polyethylene glycol, gelatin, collagen, γ-polyglutamic acid and a mixture of two or more thereof. 24. The method of claim 23, comprising. 24. The method of claim 23, wherein the biocompatible polymer comprises polyethylene glycol. 32. The method of claim 31, wherein the polyethylene glycol is in the molecular weight range of 1000-200,000. 24. The method of claim 23, wherein the biocompatible polymer is used at a concentration of 1% (w / v) to 50% (w / v). 24. The method of claim 23, wherein the biological tissue is tissue selected from the group consisting of blood vessels, blood vessel-like tissue, heart valves, pericardium, dura mater, cornea and bone. 24. The method of claim 23, wherein the biological tissue is derived from a mammal. 24. The method of claim 23, wherein the biological tissue is from human, bovine or porcine. A biological tissue obtained by the method according to claim 27. An organizational regeneration method,
a) providing a living body tissue containing a biocompatible polymer and receiving γ-irradiation in vivo; and b) incubating for a time sufficient for tissue regeneration to occur in the living body. Process,
Including the method. 39. The method of claim 38, wherein the biological tissue is tissue selected from the group consisting of blood vessels, blood vessel-like tissue, heart valves, pericardium, dura mater, cornea and bone. A method for producing a tissue graft comprising:
A) a step of providing a living body-derived tissue containing a biocompatible polymer and receiving γ-ray irradiation in vivo;
B) allowing the living body's own cells to enter the living tissue; and C) incubating for a time sufficient for the cells to differentiate.
Including the method. 41. The method of claim 40, wherein the biological tissue is a tissue selected from the group consisting of blood vessels, blood vessel-like tissue, heart valves, pericardium, dura mater, cornea and bone. 41. The method of claim 40, wherein the biological tissue is autologous. 41. The method of claim 40, wherein the decellularized tissue is from an allogeneic host. 41. The method of claim 40, wherein the decellularized tissue is from a heterologous host. 41. A tissue graft produced by the method of claim 40. A method of treating or preventing a subject in need or at risk of a tissue or organ transplant comprising:
A) A step of providing a biological tissue containing a biocompatible polymer and receiving γ-ray irradiation, or a tissue graft containing the biological tissue; and B) the biological tissue or tissue graft. Transplanting to a subject;
Including the method. 47. The method of claim 46, wherein the biological tissue is derived from the subject. 47. The method of claim 46, wherein the biological tissue is tissue selected from blood vessels, blood vessel-like tissue, heart valves, pericardium, dura mater, cornea and bone. 48. The method of claim 46, wherein the subject is a mammal. 48. The method of claim 46, wherein the subject is a human. A medicine for organ transplantation,
A) A biological tissue containing a biocompatible polymer and receiving γ-ray irradiation, or a tissue graft containing the biological tissue,
Containing a medicament. 52. The medicament according to claim 51, wherein the biological tissue is a tissue selected from blood vessels, blood vessel-like tissues, heart valves, pericardium, dura mater, corneas and bones. 52. The medicament according to claim 51, wherein the biological tissue is derived from a mammal. 52. The medicament according to claim 51, wherein the biological tissue is derived from human, cow or pig. 52. The medicament according to claim 51, wherein the biological tissue is derived from a subject in need of the transplant. Use of a biological tissue containing a biocompatible polymer and receiving γ-irradiation, or a tissue graft containing the biological tissue for producing a medicament for organ transplantation.