Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/0068—General culture methods using substrates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
  • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
  • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
  • A61L27/48—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with macromolecular fillers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L27/507—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials for artificial blood vessels
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L27/56—Porous materials, e.g. foams or sponges
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L27/58—Materials at least partially resorbable by the body
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2533/00—Supports or coatings for cell culture, characterised by material
  • C12N2533/30—Synthetic polymers
  • C12N2533/40—Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers

Definitions

• the present invention relates to a substrate for culturing a cardiovascular tissue, which can regenerate a blood vessel at an extremely high efficiency by transplantation by cell seeding, as well as a method of producing a cardiovascular tissue for transplantation using the same, a method of regenerating a cardiovascular tissue, and a cardiovascular tissue for transplantation.
• an artificial blood vessel that is most frequently used in clinic is an artificial blood vessel using a non-absorbable polymer such as GORE-TEX.
• a non-absorbable polymer such as GORE-TEX.
• an artificial blood vessel using a non-absorbable polymer has a problem that an anti-coagulant or the like must be continuously administered since a foreign matter remains in a body over a long term after transplantation.
• an anti-coagulant or the like must be continuously administered since a foreign matter remains in a body over a long term after transplantation.
• Regeneration therapy is a trial attempting to seed cells constituting a tissue on a cell culturing substrate that is to be an anchorage, and transplant this, thereby, regenerating an autologous tissue.
• a skin M L. Cooper, L. F. Hansbrough, R. L. Spielvogel et al., Biomaterials, 12: 243-248, 1991
• a cartilage C. A. Vacanti, R. langer, et al., Plast. Reconstr. Surg, 88: 753-759, 1991.
• the present inventors developed a substrate for culturing a cardiovascular tissue in which a reinforcing material comprising a bioabsorbable polymer as a core material is incorporated into a foam comprising a bioabsorbable polymer (Japanese Kokai Publication 2001-78750).
• a foam becomes an anchorage which can adhere seeded cells firmly, and the reinforcing material plays a role of withstanding a blood flow to retain a strength after transplantation for a term until a blood vessel is regenerated, and also plays a role as a reinforcing material which withstands suturing.
• both of the foam and the reinforcing material comprise a bioabsorbable polymer, thereby, materials are absorbed after regeneration of a blood vessel, it becomes unnecessary to continuously use an anti-coagulant or the like. Furthermore, since a regenerated blood vessel is an autologous tissue, growth is also expected. Actually, the substrate for culturing a cardiovascular tissue is being confirmed that it is extremely significant also clinically. However, it goes without saying that a higher revascularization efficiency should be aimed for actual clinical application.
• a substrate for culturing a cardiovascular tissue seeded with cells When a substrate for culturing a cardiovascular tissue seeded with cells is transplanted, whether a blood vessel is regenerated or not depends on a sufficient amount of binding of seeded cells, and no occurrence of stenosis until a blood vessel is regenerated. Since cells are usually seeded in the state of a suspension in which the cells are suspended in a culturing solution or the like, a substrate for culturing cells is required to be flexible and have a high water absorption for highly efficient seeding.
• An object of the present invention is to provide a substrate for culturing a cardiovascular tissue which can realize both of a seeding efficiency and difficulty in collapse of a cell, and can regenerate a blood vessel at an extremely high efficacy by transplantation by cell seeding, as well as a method of producing a cardiovascular tissue for transplantation using the same, a method of regenerating a cardiovascular tissue, and a cardiovascular tissue for transplantation.
• the present invention 1 is a substrate for culturing a cardiovascular tissue, which is tubular, and comprises a foam comprising a bioabsorbable material reinforced with a reinforcing material comprising a bioabsorbable material, the foam comprising lactide (D, L, DL isomer)- ⁇ -caprolactone copolymer containing lactide (D, L, DL isomer) in a content of 50 to 54 mole % and ⁇ -caprolactone in a content of 50 to 46 mole %, and the reinforcing material being covered with the foam.
• a foam comprising a bioabsorbable material reinforced with a reinforcing material comprising a bioabsorbable material
• the foam comprising lactide (D, L, DL isomer)- ⁇ -caprolactone copolymer containing lactide (D, L, DL isomer) in a content of 50 to 54 mole % and ⁇ -caprolactone in a content
• the present invention 2 is a substrate for culturing a cardiovascular tissue, which is tubular, and comprises a foam comprising a bioabsorbable material reinforced with a reinforcing material comprising a bioabsorbable material, the foam having a thickness of 0.2 to 3.0 mm, the reinforcing material being situated at a center or on an outer plane, and an inner plane comprising the foam.
• the present invention 3 is a substrate for culturing a cardiovascular tissue, which is tubular, and comprises a foam comprising a bioabsorbable material reinforced with a reinforcing material comprising a bioabsorbable material, the reinforcing material comprising a bioabsorbable fiber coated with a bioabsorbable material, the reinforcing material being situated at a center or on an outer plane, and an inner plane comprising the foam.
• the present invention 4 is a substrate for culturing a cardiovascular tissue, which is tubular, and comprises a foam comprising a bioabsorbable material reinforced with a reinforcing material comprising a bioabsorbable material, the reinforcing material comprising a twisted yarn comprising a twisted bioabsorbable multifilament yarn, the reinforcing material being situated at a center or on an outer plane, and an inner plane comprising the foam.
• the present invention 5 is a substrate for culturing a cardiovascular tissue, which is tubular, and comprises a foam comprising a bioabsorbable material reinforced with a reinforcing material comprising a bioabsorbable material, and a reinforcing yarn comprising a bioabsorbable material, the reinforcing yarn and the reinforcing material being situated at a center or on an outer plane of the foam, and an inner plane comprising the foam.
• a substrate for culturing a cardiovascular tissue which is tubular, and comprises a foam comprising a bioabsorbable material reinforced with a reinforcing material comprising a bioabsorbable material
• a method of using a particular compositional ratio of lactide (D, L, DL isomer)- ⁇ -caprolactone copolymer as a material for the foam present invention 1
• a method of adopting a thickness of the foam in a specific range present invention 2
• a method of using a bioabsorbable fiber coated with a bioabsorbable material as a material for the reinforcing material present invention 3
• a method of using a reinforcing material comprising a twisted yarn present invention 4
• a method of further reinforcing with a reinforcing yarn comprising a bioabsorbable material present invention 5
• the substrate for culturing a cardiovascular tissue of the present invention is such that a foam comprising a bioabsorbable material is reinforced with a reinforcing material comprising a bioabsorbable material.
• the foam becomes an anchorage to which a seeded cell can adhere firmly, and the reinforcing material plays a role of withstanding a blood flow to retain a strength after transplantation for a term until a blood vessel is regenerated.
• both of the foam and the reinforcing material comprise a bioabsorbable polymer, thereby, materials are absorbed after regeneration of a blood vessel, it becomes unnecessary to continuously use an anti-coagulant or the like.
• a regenerated blood vessel is an autologous tissue, growth is also expected.
• examples of the cardiovascular tissue include a blood vessel, a heart valve, a pericardium and the like.
• a pore diameter of the foam is required to be such an extent that a seeded cell can be appropriately adhered and proliferated and, at the same time, upon transplantation as a cardiovascular tissue, little blood leakage occurs.
• a preferable lower limit is 5 ⁇ m
• a preferable upper limit is 100 ⁇ m.
• a more preferable lower limit is 10 ⁇ m
• a more preferable upper limit is 50 ⁇ m.
• an average pore diameter of the fine pore can be measured by previously known methods such as a mercury pressing-in method and an image analyzing method.
• the foam may be subjected to a hydrophilization treatment.
• a hydrophilization treatment when contacted with a cell suspension, the foam can rapidly absorb this, and a cell can be more effectively and more uniformly seeded.
• the hydrophilization treatment is not particularly limited, but examples include plasma treatment, glow discharge treatment, corona discharge treatment, ozone treatment, surface graft treatment, ultraviolet-ray irradiation treatment and the like. Among them, plasma treatment is preferable since a water absorption can be dramatically improved without changing an appearance of a substrate for an artificial blood vessel.
• the reinforcing material is not particularly limited as far as it has a higher strength than that of the foam, but examples include a fibrous body, a non-woven body, a film-form body and the like. Among them, a fibrous body comprising a bioabsorbable material such as a traverse knitted fabric, a longitudinal knitted fabric, a braid and a woven fabric is suitable.
• the foam and the reinforcing material are incorporated.
• a positional relationship between the foam and the reinforcing material is such that the reinforcing material is situated at a center or on an outer plane of a tubular body which is the substrate for culturing a cardiovascular tissue of the present invention, and an inner plane of the tubular body comprises the foam.
• the reinforcing material can sufficiently exert a role of retaining a strength, and can progress regeneration from an inner side of a blood vessel to perform early blood vessel regeneration.
• An inner diameter and a length of the tubular body which is the substrate for the culturing a cardiovascular tissue of the present invention may be selected in conformity with an objective blood vessel.
• a preferable lower limit of a thickness of the substrate for culturing a cardiovascular tissue of the present invention is 50 ⁇ m and a preferable upper limit is 5 mm.
• a thickness is less than 50 ⁇ m, a sufficient strength which can withstand a blood flow is not obtained, and suturing becomes difficult in some cases, and when the thickness exceeds 5 mm, a time for absorption becomes longer without limitation, and this may be cause for stenosis in some cases.
• bioabsorbable material constituting the foam, the reinforcing material and the reinforcing yarn examples include polyglycolic acid, polylactide (D, L, DL isomer), polycaprolactone, glycolic acid-lactide (D, L, DL isomer) copolymer, glycolic acid- ⁇ -caprolactone copolymer, lactide (D, L, DL isomer)- ⁇ -caprolactone copolymer, poly(p-dioxanone) and the like. These may be used alone, or two or more kinds thereof may be used in combination. Among these, the material is selected in every invention.
• the foam comprises lactide (D, L, DL isomer)- ⁇ -caprolactone copolymer containing lactide (D, L, DL isomer) in a content of 50 to 54 mole %, and ⁇ -caprolactone in a content of 50 to 46 mole %.
• lactide (D, L, DL isomer)- ⁇ -caprolactone copolymer having such a compositional ratio, flexibility and water absorbability by which a sufficient amount of the seeded cell number can be maintained, and a high compressive elastic modulus at compression of the tubular body by which stenosis does not occur can be both realized.
• a compositional ratio of the lactide (D, L, DL isomer)- ⁇ -caprolactone copolymer may be such that, by using only one kind of the copolymer, a compositional ratio of each component in the copolymer satisfies the above-described range, or by using a plurality of kinds of copolymers having different compositional ratios, a compositional ratio of each component as a whole of the plurality of kinds of copolymers satisfies the above-described range.
• the reinforcing material comprises at least one kind of compound selected from the group consisting of polyglycolic acid, polylactide (D, L, DL isomer), polycaprolactone, glycolic acid-lactide (D, L, DL isomer) copolymer, glycolic acid- ⁇ -caprolactone copolymer, lactide (D, L, DL isomer)- ⁇ -caprolactone copolymer and poly(p-dioxanone).
• the foam has a lower limit of a thickness of 0.2 mm, and an upper limit of 3.0 mm.
• the foam having such the thickness, flexibility and water absorbability by which a sufficient amount of a cell seeding number can be maintained, and a high compressive elastic modulus at compression of the tubular body which does not cause stenosis can be both realized.
• the thickness is less than 0.2 mm, a compressive elastic modulus at compression of the tubular body is low, and stenosis is easily caused in some cases, and when the thickness exceeds 3.0 mm, flexibility is lacked, a water absorption becomes low, and a sufficient amount of cells cannot be seeded.
• a method of adjusting a thickness of the foam is not particularly limited, but examples include a method of adjusting a concentration and an amount of a solution of a bioabsorbable material forming the foam when the substrate for culturing a cardiovascular tissue of the present invention is produced in a production process described later.
• the foam and the reinforcing material comprise at least one kind of compound selected from the group consisting of polyglycolic acid, polylactide (D, L, DL isomer), polycaprolactone, glycolic acid-lactide (D, L, DL isomer) copolymer, glycolic acid- ⁇ -caprolactone copolymer, lactide (D, L, DL isomer)- ⁇ -caprolactone copolymer and poly(p-dioxanone).
• the reinforcing material comprises a bioabsorbable fiber coated with a bioabsorbable material.
• a bioabsorbable fiber coated with a bioabsorbable material flexibility and water absorbability by which a sufficient amount of a cell seeding number can be maintained, and a high compressive elastic modulus at compression of the tubular body which does not cause stenosis can be both realized.
• the bioabsorbable fiber coated with the bioabsorbable material is not particularly limited, but a polyglycolic acid fiber coated with lactide (D, L, DL isomer)- ⁇ -caprolactone copolymer is suitable.
• the coating method is not particularly limited, but examples include a method of immersing the polyglycolic fiber in a solution of lactide (D, L, DL isomer)- ⁇ -caprolactone copolymer, pulling out the polyglycolic fiber from the solution, thereafter, drying it, and forming a reinforcing material, a method of forming a reinforcing material using a polyglycolic acid fiber, thereafter, immersing the material in a solution of lactide (D, L, DL isomer)- ⁇ -caprolactone copolymer, pulling out the polyglycolic fiber from the solution, and drying it, and the like.
• a method of immersing the polyglycolic fiber in a solution of lactide (D, L, DL isomer)- ⁇ -caprolactone copolymer pulling out the polyglycolic fiber from the solution, and drying it, and the like.
• the foam comprises at least one kind of compound selected from the group consisting of polyglycolic acid, polylactide (D, L, DL isomer), polycaprolactone, glycolic acid-lactide (D, L, DL isomer) copolymer, glycolic acid- ⁇ -caprolactone copolymer, lactide (D, L, DL isomer)- ⁇ -caprolactone copolymer and poly(p-dioxanone).
• the reinforcing material comprises a foam comprising a bioabsorbable material reinforced with a reinforcing material comprising a bioabsorbable material.
• Twisting of the twisted yarn is preferably such that S twisting is 350 T/m or more, and Z twisting is 220 T/m or more. Outside these ranges, the sufficient effect is not obtained in some cases.
• the foam and the reinforcing material comprise at least one kind of compound selected from the group consisting of polyglycolic acid, polylactide (D, L, DL isomer), polycaprolactone, glycolic acid-lactide (D, L, DL isomer) copolymer, glycolic acid- ⁇ -caprolactone copolymer, lactide (D, L, DL isomer)- ⁇ -caprolactone copolymer and poly(p-dioxanone).
• a composite comprising the foam and the reinforcing material is further reinforced with a reinforcing yarn comprising a bioabsorbable material.
• a reinforcing yarn comprising a bioabsorbable material.
• the reinforcing yarn is wound for a composite comprising the foam and the reinforcing material in a spiral form, a ring form or a X-shaped form.
• the reinforcing yarn comprises one kind of compound selected from the group consisting of poly-L-lactide, lactide (D, L, DL isomer)- ⁇ -caprolactone copolymer and glycolic acid- ⁇ -caprolactone copolymer.
• the method of producing the substrate for culturing a cardiovascular tissue of the present invention is not particularly limited, but includes a method of mounting the reinforcing material which has been prepared in advance, in a mold, pouring a solution of a bioabsorbable material for forming the foam into the mold, freezing and lyophilizing this (lyophilization process), a method of adhering a mixed solution of a water-soluble substance and a bioabsorbable material for forming the foam to the reinforcing material which has been prepared in advance, drying this, thereafter, washing off the water-soluble substance by washing with water (dissolution out process), and the like.
• foams having a variety of pore diameters can be prepared depending on a freezing temperature and a polymer concentration.
• a pore diameter of the foam can be controlled by adjusting particles of the water-soluble substance.
• the cell As a cell to be seeded, an almost common cell species is used in a cardiovascular tissue. That is, the cell is endothelial cell, bone marrow cell, smooth muscle cell, and fibroblast and, usually, a mixed cultured cell of these two or three kinds, or a monocyte component in bone marrow is seeded to perform tissue construction.
• a to C exemplify cell collection, culturing, seeding method upon preparation of a heart valve, a pericardium, and a blood vessel when a mixed cultured cell is used
• D exemplifies a method when a bone marrow monocyte component is used.
• a blood vessel tissue collected under complete cleanness is immersed in a cell culturing solution, and is washed using a phosphated saline in a clean bench. Then, a tissue is cut on a petri dish using a surgical knife according to simple explant technique. Fine tissue species having a size of about 1 to 2 mm 2 are evenly dispensed on a dish and, after about 20 minutes, a culturing solution is added after a tissue has adhered firmly to an underside of a dish.
• DMEM Dulbecco's modified Eagle medium
• antibiotic solution L-glutamine 29.2 mg/mL, penicillin G 1000 u/mL, streptomycin sulfate 10,000 ⁇ g/mL
• a blood vessel wall cell usually initiates to move from a tissue onto a dish after 5 to 7 days and, further after one week, a mixed cell colony is formed around a tissue species. After 2 to 3 weeks, a mixed cell forms the confluent state on a dish. Once the cell is in this state, the cell is immediately recovered with 0.25% trypsin, and is subcultured.
• Subculturing is performed, for example, on a 75 cm 2 culturing flask and, when this flask becomes almost confluent, about two million cells are obtained. Under the environment of 5% CO 2 and 21% O 2 , subculturing is continued, and culturing is usually continued until a cell number of around 10'10 6 is obtained. A culturing solution is exchanged every 4 to 5 days and, according to the result of pre-experiment, a cell doubling time is about 48 hours. In addition, counting of a cell number with time is performed according to a typical staining method with Trypan Blue.
• an endothelial cell is selected and separated from a mixed cell using FACS according to the following procedure. That is, for example, Dil-acetylated LDL (fluorescent dye marker) (hereinafter, referred to as Dil-Ac-LDL) of Biomedical Technology is added to a mixed cell culturing solution at a concentration of 1 ⁇ g/mL, and this is incubated for 24 hours. This marker is taken into a cell through a scavenger pathway peculiar to an endothelial cell or macrophage.
• Dil-acetylated LDL fluorescent dye marker
• a trypsin treatment is performed to make a mixed cell suspension, and this is sorted using a cell sorter (FACS machine: manufactured by Becton, Dickinson and Company).
• Cells are selected into Dil-Ac-LDL positive and negative based on its size and fluorescent emission. After separation, these are separately cultured, and culturing is continued until the number of an endothelial cell becomes 2,000,000.
• a first stage of constructing a tissue is in vitro cell seeding. Specifically, about 1,000,000/cm 2 Dil-Ac-LDL negative fibroblast is seeded on the substrate for culturing a cardiovascular tissue of the present invention. For 30 to 60 minutes immediately after fibroblast seeding, the cell is allowed to stand on a culturing dish in a clean bench and, thereafter, about 50 mL of a culturing solution is added. A culturing solution is fundamentally exchanged every day, seven days after, before one day of surgical transplantation, a cell suspension (about 2,000,000) of endothelial cells is further seeded, and conversion into a monolayer of endothelial cells is performed by this operation.
• a bone marrow is collected from iliac bone into a cylinder containing heparin for anti-coagulation using a bone marrow piercing needle by a clean procedure equivalent to a clean field at operation.
• a bone marrow is first applied to a filter in a clean bench, this is calmly injected in an upper part of a gradient solution (e.g. trade name “Ficoll”: manufactured by Pharmacia), and centrifuged. Thereafter, plasma components are separately fractionated under clean condition, and a monocyte layer is separated. In order to obtain only a cell mass of a monocyte layer, centrifugation is further performed to obtain a cell mass of a monocyte.
• a gradient solution e.g. trade name “Ficoll”: manufactured by Pharmacia
• a size of the obtained cell mass is adjusted to a size of the substrate for culturing a cardiovascular tissue to be seeded, diluted with an autoserum which has been appropriately fractionated, stirred, and is seeded on the substrate for culturing a cardiovascular tissue for utility.
• the substrate for culturing a cardiovascular tissue after cell seeding is stored in an incubator at 37° C., 5% carbon dioxide and a humidity of 100% in the state it is immersed in autoserum until immediately before transplantation, in order to retain a bone marrow monocyte cell.
• a method of producing a cardiovascular tissue for transplantation wherein a substrate surface is covered with a cell which comprises seeding a cell in vitro on the substrate for culturing a cardiovascular tissue of the present invention, and further culturing the cell in vitro is also one of the present invention.
• a method of regenerating a cardiovascular tissue which comprises seeding a cell in vitro on the substrate for culturing a cardiovascular tissue of the present invention, and further culturing the cell to regenerate a cardiovascular tissue in vitro is also one of the present inventions.
• An endothelialized cardiovascular tissue for transplantation which is obtained by seeding a cell in vitro on the substrate for culturing a cardiovascular tissue of the present invention, and further culturing them in vitro is also one of the present inventions.
• the cardiovascular tissue for transplantation of the present invention uses the substrate for culturing a cardiovascular tissue of the present invention which realizes both of a seeding efficiency and difficulty in collapse of a cell, it can regenerate a blood vessel at an extremely high efficiency by transplantation.
• a method of transplanting the cardiovascular tissue for transplantation of the present invention is not particularly limited, but previously known methods can be used.
• an anti-thrombus drug an anti-coagulant, an anti-platelet drug, a glucocorticoid drug (steroid drug), or a non-steroidal anti-inflammatory drug (NSAID) in combination
• a glucocorticoid drug affords the extremely high effect.
• the anti-thrombus drug is not particularly limited, but examples include aspirin and the like.
• the anti-coagulant is not particularly limited, but examples include heparin, warfarin, acenocoumarol, phenindione and the like.
• the anti-platelet drug is not particularly limited, but examples include cilostazol, aspirin, ticlopidine and the like.
• the glucocorticoid drug is not particularly limited, but examples include predonisolone, dexamethasone, cortisol, and the like.
• the non-steroidal anti-inflammatory drug is not particularly limited, but examples include aspirin, diclofenac, indometacin, ibuprofen, naproxen, and the like.
• a method of using the glucocorticoid drug (steroid drug) or the like in combination is not particularly limited, but previously known methods can be used.
• the glucocorticoid drug (steroid drug) is used in combination, it is contemplated that after transplantation of the cardiovascular tissue for transplantation of the present invention, a glucocorticoid drug (steroid drug) is orally administered for a certain term.
• the present invention provides a substrate for culturing a cardiovascular tissue, which can regenerate a blood vessel at an extremely high efficiency by transplantation by cell seeding, as well as a method of producing a cardiovascular tissue for transplantation using the same, a method of regenerating a cardiovascular tissue, and a cardiovascular tissue for transplantation.
• L-lactide- ⁇ -caprolactone copolymer (molar ratio 50:50) and L-lactide- ⁇ -caprolactone copolymer (molar ratio 75:25) were mixed at a proportion of 100:0, 90:10, 80:20 and 70:30 to prepare L-lactide- ⁇ -caprolactone copolymer in which L-lactide: ⁇ -caprolactone (molar ratio) is 50:50, 52.5:47.5, 55:45 and 57.5:42.5, and a 4 weight % dioxane solution of this was prepared.
• a plain fabric obtained by knitting a 140 denier polyglycolic acid yarn into a cylinder was mounted on a bar made of Teflon having an outer diameter of 12 mm, this was immersed in the L-lactide- ⁇ -caprolactone copolymer solution, frozen at ⁇ 80° C., and lyophilized at ⁇ 40° C. to 40° C. for 12 hours. Then, this was detached from the bar made of Teflon with inverting it, and mounted on the bar made of Teflon again, and the same procedure as that described above was performed to obtain a tubular substrate for culturing a cardiovascular tissue having a sandwich structure reinforced with a glycolic acid knitted fabric. A thickness of a foamed layer (sponge layer) was about 1.3 mm as expressed by a sum of both sides.
• the obtained substrate for culturing a cardiovascular tissue was evaluated in the following method. Results are shown in Table 1.
• the obtained tubular substrate for culturing a cardiovascular tissue was cut into a size of 1 cm, which was used as a sample, and a weight thereof was measured.
• the sample was immersed into a physiological saline, and the sample was pushed with a finger 15 times to expel bubbles in the sample. After water was slightly removed, a weight after absorbing water was measured. From the weights before and after absorbing water, a water absorption was calculated. When this value is large, it means that an absorption amount of a cell suspension is large.
• dioxane solutions having four kinds of concentrations of 2%, 4%, 6% and 8% were prepared.
• the cylindrical knitted fabric having the same construction as that of Experimental Example 1 was mounted on a bar made of Teflon having an outer diameter of 10 mm, this was immersed into L-lactide- ⁇ -caprolactone copolymer solution and, thereafter, according to the same condition and procedure as those of Experimental Example 1, a tubular substrate for culturing a cardiovascular tissue having a sandwich structure, which was reinforced with a glycolic acid knitted fabric, was obtained.
• a thickness thereof was 0.55 mm (2% concentration), 0.90 mm (4% concentration), 1.30 mm (6% concentration) and 2.10 mm (8% concentration), respectively.
• the obtained substrate for culturing a cardiovascular tissue was evaluated according to the same manner as that of Experimental Example 1.
• a non-coated polyglycolic acid fiber was used to prepare a reinforcing material and, according to the similar manner, a substrate for culturing a cardiovascular tissue was obtained.
• twisted yarns which is obtained by S-twisting a 140 denier multifilament yarn (35d/16 filaments) comprising polyglycolic acid one by one, bundling four yarns to make a bundling yarn, and further Z-twisting, three kinds of twisted yarns of low twisting (single yarn S twisting 120 T/m, bundling yarn Z twisting 75 T/m), intermediate twisting (single yarn S twisting 600 T/m, bundling yarn Z twisting 375 T/m) and high twisting (single yarn S twisting 1200 T/m, bundling yarn Z twisting 750 T/m) were obtained.
• Each of the twisted yarns was knitted into the same kitting structure as that of Experimental Example 1, it was mounted on the same bar made of Teflon as that of Experimental Example 1 and, thereafter, according to the same condition and procedure as those of Experimental Example 1, this was immersed into a 4 weight % dioxane solution of L-lactide- ⁇ -caprolactone copolymer (molar ratio 50:50) to obtain a tubular substrate for culturing a cardiovascular tissue of a sandwich structure, having a foam layer of a thickness of 0.9 mm.
• a 4 weight % dioxane solution of L-lactide- ⁇ -caprolactone copolymer (molar ratio 50:50) was prepared.
• a plain fabric obtained by knitting a 140 denier polyglycolic acid yarn into a cylinder was mounted on a bar made of Teflon having an outer diameter of 10 mm, and this was immersed into the L-lactide- ⁇ -caprolactone copolymer solution, frozen at ⁇ 80° C., and lyophilized at ⁇ 40° C. to 40° C. for 12 hours. Then, this was taken out from the bar made of Teflon with inverting it, and mounted again on the Teflon bar. On a surface thereof was wound a monofilament yarn (thickness, two kinds of 1-0 and 3-0) of L-lactide- ⁇ -caprolactone copolymer spirally at a pitch of 3 mm and 5 mm.
• the obtained substrate for culturing a cardiovascular tissue was evaluated according to the same manner as that of Experimental Example 1. Results are shown in Table 5.
• twisted yarns which is obtained by S-twisting a 140 denier multifilament yarn (35d/16 filaments) comprising polyglycolic acid one by one, bundling four yarns to make a bundling yarn, and further Z-twisting, a twisted yarn of low twisting (single yarn S twisting 120 T/m, bundling yarn Z twisting 75 T/m) was obtained.
• the obtained twisted yarn of low twisting was knitted into a cylinder of the same knitting structure as that of Experimental Example 1, it was mounted on the same bar made of Teflon as that of Experimental Example 1 and, thereafter, according to the same condition and procedure as those of Experimental Example 1, this was immersed into a 4 weight % dioxane solution of L-lactide- ⁇ -caprolactone copolymer (molar ratio 50:50) to obtain a tubular substrate for culturing a cardiovascular tissue of a sandwich structure, having a foam layer of a thickness of 0.9 mm.
• a bone marrow was taken from a head of femur and a head of ilium of a dog (beagle, weight about 10 kg) into a syringe containing heparin using a bone marrow piercing needle.
• the bone marrow was first applied to a filter in a clean bench, this was calmly injected in an upper part of a gradient solution (trade name “Ficoll”: manufactured by Pharmacia), and this was centrifuged. Thereafter, plasma components were separately fractionated under clean condition, and a monocyte layer was separated.
• centrifugation was further performed to obtain a cell mass of the monocyte.
• the obtained cell mass was seeded on a substrate for culturing a cardiovascular tissue which had been cut into a length of 3 cm, this was transplanted into inferior cava of the same dog, and a group to which 0.5 mg/kg prednisolone which is a glucocorticoid drug (steroid drug) was administered by mixing into a feed for one month after operation, and a group to which no drug was administered were made (3 animals in both groups).

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