US20080176206A1

US20080176206A1 - Cardiovascular tissue culture substrate

Info

Publication number: US20080176206A1
Application number: US11/769,080
Authority: US
Inventors: Toshiharu Shinoka; Goki Matsumura; Yoshito Ikada; Shojiro Matsuda; Yuki Sakamoto; Tsuguyoshi Taira
Original assignee: Gunze Ltd
Current assignee: Gunze Ltd
Priority date: 2007-01-18
Filing date: 2007-06-27
Publication date: 2008-07-24
Also published as: EP2103683B1; JP2013150909A; JP5986535B2; US20100055781A1; WO2008088042A1; EP2103683A1; US8372433B2; ATE535265T1; CN101568637B; JPWO2008088042A1; EP2103683A4; CN101568637A

Abstract

The substrate for culturing a cardiovascular tissue of the present invention 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)-ε-caprolactone copolymer containing lactide (D, L, DL isomer) in a content of 50 to 54 mole % and ε-caprolactone in a content of 50 to 46 mole %, and the reinforcing material being covered with the foam.

Description

CROSS-REFERENCE OF RELATED APPLICATION

The present application is an application claiming the priority of a first-filed application of Japanese Patent Application No. 2007-009469, which was filed on Jan. 18, 2007.

BACKGROUND OF THE INVENTION

1. Technical Field
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.
By quoting the application, the entire contents of the application are incorporated.
2. Explanation of Background Art
Currently, 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. Such the artificial blood vessel can exert physical property extremely close to that of a blood vessel, and brings a fixed result in reconstitution of a short-term blood vessel. However, 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. In addition, there is also a problem that, when used in infants, it becomes necessary to perform operation again as infants are grown.
On the other hand, in recent years, a tissue regenerating method by so-called regeneration therapy has been tried. 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. Regarding the regeneration therapy, many study examples are reported in a variety of tissues including, for example, a skin (M L. Cooper, L. F. Hansbrough, R. L. Spielvogel et al., Biomaterials, 12: 243-248, 1991) and a cartilage (C. A. Vacanti, R. langer, et al., Plast. Reconstr. Surg, 88: 753-759, 1991).
In order to apply such the regeneration therapy to revascularization, 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). In this substrate for culturing a cardiovascular tissue, 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. Since 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.
The entire contents of (M L. Cooper, L. F. Hansbrough, R. L. Spielvogel et al., Biomaterials, 12: 243-248, 1991) and (C. A. Vacanti, R. langer, et al., Plast. Reconstr. Surg, 88: 753-759, 1991), and Japanese Kokai Publication 2001-78750 are incorporated herein by quoting the application.

DISCLOSURE OF THE INVENTION

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. On the other hand, in order that stenosis does not occur, possession of such a mechanical strength that a tubular body is collapsed with difficulty, that is, exertion of a high compressive elastic modulus and maintenance of an aperture are required when the tubular body is compressed. As described above, flexibility and high water absorbability and a high compressive elastic modulus are in a relationship of trade-off, and are objects which are difficult to realize both of them.
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)-ε-caprolactone copolymer containing lactide (D, L, DL isomer) in a content of 50 to 54 mole % and ε-caprolactone in a content of 50 to 46 mole %, and the reinforcing material being covered with the foam.
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.
The present invention will be described in detailed below.
The present inventors intensively studied and, as a result, found that, in 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, by a method of using a particular compositional ratio of lactide (D, L, DL isomer)-ε-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), and a method of further reinforcing with a reinforcing yarn comprising a bioabsorbable material (present invention 5), both of a seeding efficiency and difficulty in collapse of a cell are realized, and a cell is seeded and transplanted, thereby, a substrate for culturing a cardiovascular tissue which can regenerate a blood vessel at an extremely high efficiency is obtained, which resulted in completion of the present invention.
In the present description, the inventions are conveniently described as present inventions 1 to 5, but these may be implemented alone, or may be used in combination thereof.
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. By adopting such a structure, 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. In addition, since 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.
In this respect, in the present description, 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. Specifically, a preferable lower limit is 5 μm, and a preferable upper limit is 100 μm. When the pore diameter is less than 5 μm, a seeded cell cannot enter a pore of the foam, and a sufficient seeding efficiency may not obtained in some cases and, when the pore diameter exceeds 100 μm, upon transplantation, blood leakage may occur in some cases. A more preferable lower limit is 10 μm, and a more preferable upper limit is 50 μm.
In addition, 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. By the 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.
In the substrate for culturing a cardiovascular tissue of the present invention, it is preferable that 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. By such a structure, 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 μm and a preferable upper limit is 5 mm. When the thickness is less than 50 μm, 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.
Examples of the bioabsorbable material constituting the foam, the reinforcing material and the reinforcing yarn include polyglycolic acid, polylactide (D, L, DL isomer), polycaprolactone, glycolic acid-lactide (D, L, DL isomer) copolymer, glycolic acid-ε-caprolactone copolymer, lactide (D, L, DL isomer)-ε-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.
In the substrate for culturing a cardiovascular tissue of the present invention 1, the foam comprises lactide (D, L, DL isomer)-ε-caprolactone copolymer containing lactide (D, L, DL isomer) in a content of 50 to 54 mole %, and ε-caprolactone in a content of 50 to 46 mole %. By using the lactide (D, L, DL isomer)-ε-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. When a content of lactide (D, L, DL isomer) is less than 50 mole % (when a content of ε-caprolactone exceeds 50 mole %), a compressive elastic modulus at compression of the tubular body is low, and stenosis is easily caused in some cases and, when a content of lactide (D, L, DL isomer) exceeds 54 mole % (a content of ε-caprolactone is less than 46 mole %), flexibility is lacked, a water absorption becomes low, and a sufficient amount of cells cannot be seeded. In the present description, a compositional ratio of the lactide (D, L, DL isomer)-ε-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.
In the substrate for culturing a cardiovascular tissue of the present invention 1, 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-ε-caprolactone copolymer, lactide (D, L, DL isomer)-ε-caprolactone copolymer and poly(p-dioxanone).
In the substrate for culturing a cardiovascular tissue of the present invention 2, the foam has a lower limit of a thickness of 0.2 mm, and an upper limit of 3.0 mm. By using 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. When 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.
In the substrate for culturing a cardiovascular tissue of the present invention 2, 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.
In the substrate for culturing a cardiovascular tissue of the present invention 2, 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-ε-caprolactone copolymer, lactide (D, L, DL isomer)-ε-caprolactone copolymer and poly(p-dioxanone).
In the substrate for culturing a cardiovascular tissue of the present invention 3, the reinforcing material comprises a bioabsorbable fiber coated with a bioabsorbable material. By using such 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)-ε-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)-ε-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)-ε-caprolactone copolymer, pulling out the polyglycolic fiber from the solution, and drying it, and the like.
In the material for culturing a cardiovascular tissue of the present invention 3, 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-ε-caprolactone copolymer, lactide (D, L, DL isomer)-ε-caprolactone copolymer and poly(p-dioxanone).
In the substrate for culturing a cardiovascular tissue of the present invention 4, the reinforcing material comprises a foam comprising a bioabsorbable material reinforced with a reinforcing material comprising a bioabsorbable material. By using such a twisted yarn, 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.
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.
In the substrate for culturing a cardiovascular tissue of the present invention 4, 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-ε-caprolactone copolymer, lactide (D, L, DL isomer)-ε-caprolactone copolymer and poly(p-dioxanone).
In the substrate for culturing a cardiovascular tissue of the present invention 5, a composite comprising the foam and the reinforcing material is further reinforced with a reinforcing yarn comprising a bioabsorbable material. By reinforcing with the reinforcing yarn, a seeding efficiency and difficulty in collapse of a cell are both realized, and a cell is seeded and transplanted, thereby, a blood vessel can be regenerated at an extremely high efficiency. The reinforcing yarn may be situated at a center of a foam, or may be situated on an outermost plane.
It is preferable that 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. By disposition of the reinforcing yarn in such an aspect, the resulting substrate for culturing a cardiovascular tissue becomes more difficult to be collapsed.
In the substrate for culturing a cardiovascular tissue of the present invention 5, it is preferable that the reinforcing yarn comprises one kind of compound selected from the group consisting of poly-L-lactide, lactide (D, L, DL isomer)-ε-caprolactone copolymer and glycolic acid-ε-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. In the lyophilization process, foams having a variety of pore diameters can be prepared depending on a freezing temperature and a polymer concentration. In the dissolution out process, a pore diameter of the foam can be controlled by adjusting particles of the water-soluble substance.
Then, a method for seeding cells on the substrate for culturing a cardiovascular tissue of the present invention will be explained.
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.
Conditions for culturing a cell used and a seeding method are shown below. The following 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, and D exemplifies a method when a bone marrow monocyte component is used.
A. Cell Isolation, Cell Culturing, Increase in Cell Number
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 mm²are 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. As the culturing solution, for example, Dulbecco's modified Eagle medium (DMEM) supplemented with 10% bovine fetal serum and 1% antibiotic solution (L-glutamine 29.2 mg/mL, penicillin G 1000 u/mL, streptomycin sulfate 10,000 μg/mL) is used. 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 cm²culturing flask and, when this flask becomes almost confluent, about two million cells are obtained. Under the environment of 5% CO₂and 21% O₂, subculturing is continued, and culturing is usually continued until a cell number of around 10'10⁶is 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.

B. Cell Sequestering, Endothelial Cell Purification

At a stage when a mixed cell reaches confluent, and a certain extent of a cell number is obtained, 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 μg/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. After 24 hours, 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.

C. Tissue Construction

A first stage of constructing a tissue is in vitro cell seeding. Specifically, about 1,000,000/cm²Dil-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.

D. Collection and Seeding of Monocyte

As a method of collecting a monocyte, first, on an operation day, after sedation and painkilling are obtained by general anesthesia, 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. In order to remove a bone strip component, a fat component and a blood coagulating component from the obtained bone marrow, 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 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.
Since 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. By using 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, higher effects can be obtained. Among them, a glucocorticoid drug (steroid 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 (steroid drug) is not particularly limited, but examples include predonisolone, dexamethasone, cortisol, and the like.
The non-steroidal anti-inflammatory drug (NSAID) 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. For example, when 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.
Accordingly, 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.

BEST MODE FOR CARRYING OUT THE INVENTION

Then, best embodiments for carrying out the present invention will be explained in the following.

EXPERIMENTAL EXAMPLE 1

L-lactide-ε-caprolactone copolymer (molar ratio 50:50) and L-lactide-ε-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-ε-caprolactone copolymer in which L-lactide:ε-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-ε-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.

(1) Compression Modulus Test

Regarding the obtained tubular substrate for culturing a cardiovascular tissue, a necessary strength for compressing a diameter to ½ was obtained. When this value is large, it means that the substrate has a high aperture diameter retaining force on stenosis.

(2) Stretch Test

Regarding the obtained tubular substrate for culturing a cardiovascular tissue, a maximum point strength when stretched in an outer diameter direction was obtained. When this value is great, it means that a strength on beat is great.

(3) Water Absorbing Test

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.

TABLE 1

		Maximum
		point
		strength	Water
L-lactide:caprolactone	Force necessary for	at stretching	absorption
(molar ratio)	½ compression (g)	(N)	(%)

50:50	7	72	380
52.5:47.5	12	65	275
55:45	14	68	205
57.5:42.5	23	86	150

EXPERIMENTAL EXAMPLE 2

Regarding L-lactide-ε-caprolactone copolymer (molar ratio 50:50), 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-ε-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.
When a part of only a foam part of the obtained substrate for culturing a cardiovascular tissue was taken and measured, 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.

TABLE 2

	Force necessary for
Foam thickness (mm)	½ compression (g)	Water absorption (%)

0.55	2	205
0.90	7	400
1.30	19	360
2.10	44	320

EXPERIMENTAL EXAMPLE 3

4 weight % dioxane solution of L-lactide-ε-caprolactone copolymer (molar ratio 50:50), and a 4 weight % dioxane solution of L-lactide-ε-caprolactone copolymer (molar ratio 75:25) were prepared. Into each of these solutions was immersed a polyglycolic acid fiber of 140 denier, and the fiber was slowly taken out, and dried to obtain a polyglycolic acid fiber coated with L-lactide-ε-caprolactone copolymer. The obtained coated polyglycolic acid fiber was used to make a cylindrical knitted fabric having the same knitting structure as that of Experimental Example 1, to obtain a reinforcing material.
This 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, a tubular substrate for culturing a cardiovascular tissue of a sandwich structure, having a foam layer of a thickness of 0.9 mm, wherein the foam layer has a reinforcing material knitted with the coated polyglycolic acid knitted fabric, was obtained.
As a control, 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.
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 3.

TABLE 3

		Maximum
		point
		strength at	Water
Coating	Force necessary for	stretching	absorption
(L-lactide:caprolactone)	½ compression (g)	(N)	(%)

Nothing	7	72	380
50:50	12	100	370
75:25	19	120	350

EXPERIMENTAL EXAMPLE 4

In 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-ε-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.
The obtsinrf substrate for culturing a cardiovascular tissue evaluated according to the same manner as that of Experimental Example 1. Results are shown in Table 4.

TABLE 4

	Force necessary for
Twisted yarn	½ compression (g)	Water absorption (N)

Low twisting	7	370
Intermediate twisting	20	400
High twisting	26	410

EXPERIMENTAL EXAMPLE 5

A 4 weight % dioxane solution of L-lactide-ε-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-ε-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-ε-caprolactone copolymer spirally at a pitch of 3 mm and 5 mm. This was immersed into the 4 weight % dioxane solution of the L-lactide-ε-caprolactone copolymer (molar ratio 50:50) for 30 seconds, frozen at −80° C., and lyophilized at −40° C. to 40° C. for 12 hours to obtain a substrate for culturing a cardiovascular tissue of a sandwich structure, having a foam layer of a thickness of 0.9 mm. As a control, according to the same manner except that a monofilament yarn was not wound, a substrate for culturing a cardiovascular tissue was obtained.
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.

TABLE 5

	Force necessary for
Reinforcing yarn	½ compression (g)	Water absorption (%)

1-0 yran/3 mm pitch	204	236
1-0 yarn/5 mm pitch	127	284
3-0 yarn/3 mm pitch	112	300
3-0 yarn/5 mm pitch	60	318
No reinforcing yarn	7	353

REFERENCE EXAMPLE

In 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-ε-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. In order to remove a bone strip component, a fat component, and a blood coagulating component from the resulting bone marrow, 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. In order to obtain only a cell mass of the monocyte layer, 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).
As a result, in the prednisolone-administered group, increase in leukocyte could be suppressed, and an inflammatory reaction could be suppressed.

Claims

1. 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)-ε-caprolactone copolymer containing lactide (D, L, DL isomer) in a content of 50 to 54 mole % and ε-caprolactone in a content of 50 to 46 mole %,

and the reinforcing material being covered with the foam.

2. A substrate for culturing a cardiovascular tissue,

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.

3. A substrate for culturing a cardiovascular tissue,

the reinforcing material comprising a bioabsorbable fiber coated with a bioabsorbable material,

4. The substrate for culturing a cardiovascular tissue according to claim 3,

wherein the reinforcing material comprises a polyglycolic acid fiber coated with lactide (D, L, DL isomer)-ε-caprolactone copolymer.

5. A substrate for culturing a cardiovascular tissue,

the reinforcing material comprising a twisted yarn comprising a twisted bioabsorbable multifilament yarn,

6. 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.

7. The substrate for culturing a cardiovascular tissue according to claim 6,

wherein the reinforcing yarn comprises at least one kind of compound selected from the group consisting of poly-L-lactide, lactide (D, L, DL isomer)-ε-caprolactone copolymer and glycolic acid-ε-caprolactone copolymer.

8. The substrate for culturing a cardiovascular tissue according to claim 6,

wherein the reinforcing yarn is wound in a spiral form, a ring form, or a X-shaped form.

9. 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 according to claim 1, 2, 3, 4, 5, 6, 7 or 8, and further culturing the cell in vitro.

10. A method of regenerating a cardiovascular tissue,

which comprises seeding a cell in vitro on the substrate for culturing a cardiovascular tissue according to claim 1, 2, 3, 4, 5, 6, 7 or 8, and further culturing the cell to regenerate a cardiovascular tissue in vitro.

11. A cardiovascular tissue for transplantation,

which is obtained by seeding an endothelial cell, a marrow cell, a smooth muscle cell or a fibroblast in vitro on the substrate for culturing a cardiovascular tissue according to claim 1, 2, 3, 4, 5, 6, 7 or 8, and further culturing them in vitro.