CA2197375C - Artificial blood vessel - Google Patents
Artificial blood vessel Download PDFInfo
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- CA2197375C CA2197375C CA002197375A CA2197375A CA2197375C CA 2197375 C CA2197375 C CA 2197375C CA 002197375 A CA002197375 A CA 002197375A CA 2197375 A CA2197375 A CA 2197375A CA 2197375 C CA2197375 C CA 2197375C
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- tube
- immobilized
- artificial blood
- eptfe
- heparin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
- A61L33/0005—Use of materials characterised by their function or physical properties
- A61L33/0011—Anticoagulant, e.g. heparin, platelet aggregation inhibitor, fibrinolytic agent, other than enzymes, attached to the substrate
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/04—Hollow or tubular parts of organs, e.g. bladders, tracheae, bronchi or bile ducts
- A61F2/06—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
- A61L27/28—Materials for coating prostheses
- A61L27/34—Macromolecular materials
-
- 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
- A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
- A61L33/0005—Use of materials characterised by their function or physical properties
- A61L33/0011—Anticoagulant, e.g. heparin, platelet aggregation inhibitor, fibrinolytic agent, other than enzymes, attached to the substrate
- A61L33/0029—Anticoagulant, e.g. heparin, platelet aggregation inhibitor, fibrinolytic agent, other than enzymes, attached to the substrate using an intermediate layer of polymer
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical & Material Sciences (AREA)
- Epidemiology (AREA)
- Transplantation (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Medicinal Chemistry (AREA)
- Dermatology (AREA)
- Vascular Medicine (AREA)
- Surgery (AREA)
- Hematology (AREA)
- Cardiology (AREA)
- Heart & Thoracic Surgery (AREA)
- Biomedical Technology (AREA)
- Pulmonology (AREA)
- Gastroenterology & Hepatology (AREA)
- Materials For Medical Uses (AREA)
- Prostheses (AREA)
Abstract
The present invention is directed to an artificial blood vessel made of a tube formed from expanded polytetrafluoroethylene comprising fibrils and nodes connecting fibrils, in which the inner surface of said tube and the surfaces of fibrils and nodes constituting pores in a layer portion extending from the inner surface of said tube to a depth of at least 5% of the wall thickness in a radial direction from said inner surface are made hydrophilic, and a tissue-inducing substance which is immobilized on said inner surface and said surfaces of fibrils and nodes constituting pores which are made hydrophilic. The artificial blood vessel of the present invention has a good patency ratio over a long period of time.
Description
2197~7~
M
ARTIFICIAL BLOOD VESSEL
The present invention relates to an artificial blood vessel. In particular, the present invention relates to an artificial blood vessel which is preferably used as a substitute blood vessel for replacing small-diameter blood vessels such as coronary arteries, peripheral blood vessels and the like.
Tubes of woven or knitted fabric of polyester fibers and expanded polytetrafluoroethylene (hereinafter referred to as "EPTFE") are used as artificial blood vessels. The EPTFE tubes are practically used in a smaller diameter range rather than the l0 polyester tubes since PTFE itself has excellent anti-thrombotic properties, and the pore structure of EPTFE tube comprising fibrils and nodes which is obtained by expanding PTFE tube has excellent biocompatibility.
However, EPTFE does not always have satisfactory anti-15 thrombotic properties, and the EPTFE tubes do not have a sufficient patency ratio when they have an inner diameter of 5 mm or less, in particular 4 mm or less. To remove this drawback of the EPTFE
tube, the following methods have been proposed:
(1 ) improvement of anti-thrombotic properties of 2o materials of artificial blood vessels;
M
ARTIFICIAL BLOOD VESSEL
The present invention relates to an artificial blood vessel. In particular, the present invention relates to an artificial blood vessel which is preferably used as a substitute blood vessel for replacing small-diameter blood vessels such as coronary arteries, peripheral blood vessels and the like.
Tubes of woven or knitted fabric of polyester fibers and expanded polytetrafluoroethylene (hereinafter referred to as "EPTFE") are used as artificial blood vessels. The EPTFE tubes are practically used in a smaller diameter range rather than the l0 polyester tubes since PTFE itself has excellent anti-thrombotic properties, and the pore structure of EPTFE tube comprising fibrils and nodes which is obtained by expanding PTFE tube has excellent biocompatibility.
However, EPTFE does not always have satisfactory anti-15 thrombotic properties, and the EPTFE tubes do not have a sufficient patency ratio when they have an inner diameter of 5 mm or less, in particular 4 mm or less. To remove this drawback of the EPTFE
tube, the following methods have been proposed:
(1 ) improvement of anti-thrombotic properties of 2o materials of artificial blood vessels;
(2) growing or seeding anti-thrombotic tissues on inner surfaces of artificial blood vessels; and
(3) accelerating the formation of tissues on inner . 219375 " _ - surfaces of artificial blood vessels after grafting the artificial blood vessels.
Concretely, as method (1), developments of anti-thrombotic polymer materials having a microphase separation structure or anti-thrombotic agent-immobilized materials have been discussed (see Noishiki et al, Trans. A. S. A. I. O., 23, 253 (1977), JP-A-58-180162 and JP-A-63-119773). Such anti-thrombotic materials can prevent the formation of thrombus temporarily after grafting, but thrombus eventually forms and occludes 1 0 the blood vessels at some time after grafting.
As method (2), a method for seeding vascular endo-thelial cells on the inner walls of artificial blood vessels has been proposed. However, the provision of human vascular endothelial cells is difficult, and the collection and culturing of such cells 1 5 require several weeks. Therefore, this method has not been used in practice (see Takagi et al, JINKOZOKI (Japanese Journal of Artificial Organs), 17, 679 (1988) and JP-A-1-170466).
As method (3), artificial blood vessels on which endothelial cell-adhesion or growth materials are coated or 20 covalently bonded have been proposed.
An artificial blood vessel is known, which sustainingly releases heparin for imparting anti-thrombotic properties to the blood vessel. For example, an artificial blood vessel to which heparin is immobilized using a material containing a quaternary 25 ammonium salt or protamine has been proposed (see JP-A-58-180162 and JP-A-63-119773).
An artificial blood vessel on which the endothelial cell-adhesion material and optionally the endothelial cell-growth .. . - 3 -- . material are coated is proposed, but the formation of endothelial cells is not accelerated and the patency does not improve (C. H.
Lundgren et al, Trans. A. S. A. I. O., 32, 346 (1986), and Glysler et al, SURGERY, 112, 244 (1992)).
An artificial blood vessel to which an endothelial cell-adhesion material is chemically immobilized is proposed (see JP-A-5-269198). In addition, an article reports that a material to which the endothelial cell-adhesion material and endothelial cell-growth material are both covalently bonded facilitated the formation of 1 0 endothelial cells in vivo . However, an artificial blood vessel made of such material loses its effects after grafting, and its patency is unsatisfactory (see J. Biomed. Mater. Res., 27, 901 (1993), etc.).
Furthermore, an artificial blood vessel which is coated with an endothelial cell-adhesion material such as collagen or 1 5 gelatin and heparin has been proposed for suppressing the formation of thrombus prior to the formation of endothelial cells. However, neither the formation of endothelial cells nor the suppression of the thrombus formation is insufficient, and no satisfactory patency is achieved (see JP-A-63-46169).
20 A required amount of heparin must be released in a requisite period after grafting for achieving a high patency ratio. In particular, a required amount and requisite period for releasing heparin vary with the conditions of living bodies such as the blood flow and inner diameters of blood vessels to be replaced. Therefore, 25 it is necessary to design a heparin-immobilized artificial blood vessel which satisfy the releasing amount and period of heparin according to respective artificial blood .vessels.
However, it is difficult for the conventional heparin-
Concretely, as method (1), developments of anti-thrombotic polymer materials having a microphase separation structure or anti-thrombotic agent-immobilized materials have been discussed (see Noishiki et al, Trans. A. S. A. I. O., 23, 253 (1977), JP-A-58-180162 and JP-A-63-119773). Such anti-thrombotic materials can prevent the formation of thrombus temporarily after grafting, but thrombus eventually forms and occludes 1 0 the blood vessels at some time after grafting.
As method (2), a method for seeding vascular endo-thelial cells on the inner walls of artificial blood vessels has been proposed. However, the provision of human vascular endothelial cells is difficult, and the collection and culturing of such cells 1 5 require several weeks. Therefore, this method has not been used in practice (see Takagi et al, JINKOZOKI (Japanese Journal of Artificial Organs), 17, 679 (1988) and JP-A-1-170466).
As method (3), artificial blood vessels on which endothelial cell-adhesion or growth materials are coated or 20 covalently bonded have been proposed.
An artificial blood vessel is known, which sustainingly releases heparin for imparting anti-thrombotic properties to the blood vessel. For example, an artificial blood vessel to which heparin is immobilized using a material containing a quaternary 25 ammonium salt or protamine has been proposed (see JP-A-58-180162 and JP-A-63-119773).
An artificial blood vessel on which the endothelial cell-adhesion material and optionally the endothelial cell-growth .. . - 3 -- . material are coated is proposed, but the formation of endothelial cells is not accelerated and the patency does not improve (C. H.
Lundgren et al, Trans. A. S. A. I. O., 32, 346 (1986), and Glysler et al, SURGERY, 112, 244 (1992)).
An artificial blood vessel to which an endothelial cell-adhesion material is chemically immobilized is proposed (see JP-A-5-269198). In addition, an article reports that a material to which the endothelial cell-adhesion material and endothelial cell-growth material are both covalently bonded facilitated the formation of 1 0 endothelial cells in vivo . However, an artificial blood vessel made of such material loses its effects after grafting, and its patency is unsatisfactory (see J. Biomed. Mater. Res., 27, 901 (1993), etc.).
Furthermore, an artificial blood vessel which is coated with an endothelial cell-adhesion material such as collagen or 1 5 gelatin and heparin has been proposed for suppressing the formation of thrombus prior to the formation of endothelial cells. However, neither the formation of endothelial cells nor the suppression of the thrombus formation is insufficient, and no satisfactory patency is achieved (see JP-A-63-46169).
20 A required amount of heparin must be released in a requisite period after grafting for achieving a high patency ratio. In particular, a required amount and requisite period for releasing heparin vary with the conditions of living bodies such as the blood flow and inner diameters of blood vessels to be replaced. Therefore, 25 it is necessary to design a heparin-immobilized artificial blood vessel which satisfy the releasing amount and period of heparin according to respective artificial blood .vessels.
However, it is difficult for the conventional heparin-
4 immobilized artificial blood vessels to control the amount of immobilized heparin and the rate of sustained release of heparin since heparin is ionically bonded to the quaternary ammonium salt or protamine which has been coated on the surface of the artificial blood vessel. Therefore, it is difficult to sustainingly release heparin in a sufficient amount for maintaining the patency of artificial blood vessel in a requisite period, and a sufficient patency ratio is not attained.
An object of the present invention is to provide an artificial blood vessel which has a high patency ratio after grafting.
Another object of the present invention is to provide an artificial blood vessel which can facilitate the growth of endothelial cells while suppressing the formation of thrombus after grafting.
Accordingly, the present invention provides an artificial blood vessel comprising: a tube formed from expanded polytetrafluoroethylene comprising fibrils and nodes connecting fibrils, wherein said tube has an inner surface and an outer surface, in which the inner surface of said tube and the surfaces of fibrils and nodes comprise a hydrophilic layer extending to a depth of between 5 % and 96% of the wall thickness of said tube in a radial direction from the inner surface toward the outer surface of said tube, and a tissue-inducing substance which is immobilized on said inner surface and said surfaces of fibrils and nodes of the hydrophilic layer.
In a preferred embodiment, the artificial blood vessel of the present invention further comprises an anti-thrombotic _ - 5 -_ substance immobilized on at least a part of a surface of said tube ~.
which will be in contact with the blood in use.
The properties required for the artificial blood vessels vary with parts of living bodies in which the artificial blood vessels are implanted. On the one hand, anti-thrombotic properties should be enhanced for the parts where blood flow rate is small such as peripheral blood vessels since thrombus tends to form in such parts. Therefore, an anti-thrombotic substance is immobilized on the surfaces of the artificial blood vessels for preventing the 1 0 formation of thrombus. On the other hand, the covering of the surfaces of the artificial blood vessels with the endothelial cells should be facilitated by immobilizing a tissue-inducing substance for the parts where blood flow rate is large since the formation of cell tissues is delayed while the thrombus is less formed.
1 5 In the drawings:
Fig. 1 is a graph showing the sustained release of heparin from the heparin-immobilized EPTFE artificial blood vessels In general, the mean fibril length of EPTFE tube, that is, the mean length of the fibrils between nodes, is between 20 and 200 2 5 p.m, the porosity of EPTFE tube is at least 50 %, preferably at least 70 %, and the wall thickness of the tube is between 300 and 1000 pm for adjusting the wall thickness of the artificial blood vessel to that of the living blood vessel.
prepared in Examples 19 through 24, and Fig. 2 is a graph showing the sustained release of 20 heparin from the heparin-immobilized EPTFE artificial blood vessels used in Comparative Examples 8, 9 and 10.
The EPTFE tube preferably has a mean fibril length of at least 60 pm on a side of the tube which will be in contact with the blood in use. More preferably, the EPTFE tube has a multilayer --structure having the mean fibril length of at least 60 pm on a side of the tube which will be in contact with the blood in use and at least one layer in which a fibril length of EPTFE tube is 40 pm or less.
When the EPTFE tube has the hydrophilic layer portion extending from its inner surface to the limited depth in the radial 1 0 direction of the tube from the inner surface, hydrophilic groups can be chemically introduced into the tube wall by the following method:
Firstly, the porous wall of the EPTFE tube is masked to a certain depth from the outer surface of the tube with a material, which is not decomposed or denatured by a defluorination reaction, can be removed from the tube without damaging or denaturing the subsequently immobilized materials and has affinity to PTFE, such as paraffin, and the unmasked portion of the EPTFE
tube is defluorinated by chemical treatment with alkali metal compounds, or by physical treatment such as irradiation with 'y-ray or electron beams, corona discharge or glow discharge.
Then, functional groups are introduced in the unmasked portion by treating the defluorinated portion with compounds having carboxyl, hydroxyl, amino or epoxy groups.
Finally, the masking material is removed.
2 5 The patency ratio can be increased when the thickness of the wall which has not been masked is between 5 % and 100 %, preferably 5 % and less than 96 %, more preferably between 50 and 90 %, of the total wall thickness.
i~2197~~5 i The wall thickness which is not masked can be adjusted -~ by controlling a depth to which molten paraffin penetrates through the change of a temperature gradient in the tube wall by flowing a cooling medium such as water through the hole of the tube, or filling all the pores in the tube wall with paraffin and then extracting paraffin on the inner wall side.
When the tube wall is not masked, the entire wall is treated, and the functional groups are introduced onto all the surfaces of the pores which present in the tube wall as well as the inner and to outer surfaces of the tube.
Among the defluorination methods, the chemical treatment with the alkali metals is more preferred than physical treatment by irradiation with y ray or electron beams or corona or glow discharge.
The irradiation with y ray decomposes PTFE to deeper parts of the crystals of PTFE. Therefore the molecular weight of PTFE decreases and the strength of the EPTFE tube deteriorates g reatly.
The glow discharge can treat only the outer and inner surfaces of the tube but may not treat surfaces of pores inside the 2o tube wall, that is the surfaces of fibrils which constitute the pores in EPTFE.
The treatment with the alkali metal compounds can defluorinate the whole surfaces of the pores of EPTFE tube uniformly only to a depth of about several angstroms from the 2197~7~
- surfaces of fibrils and nodes constituting the pores , and does not w decompose PTFE. Therefore, the strength of EPTFE tube does not decrease.
Since the inner and outer surfaces of the tube and also the surfaces of the pores inside the tube wall are defluorinated, proteins and peptides such as the tissue-inducing substances and anti-thrombotic substances can be covalently bonded to all of these surfaces.
Examples of the alkali metal compounds are methyl-lithium, n-butyllithium, tert.-butyllithium, sodium-naphthalene, sodium-benzophenone, vinyllithium and the like. They are used in the form of a solution. Among them, sodium-naphthalene and sodium-benzophenone will form a dark brown layer on the EPTFE
surfaces after treatment and cannot treat EPTFE uniformly. Thus, methyllithium, n-butyllithium and tert.-butyllithium are preferable for the production of the artificial blood vessel of the present invention. Methyllithium, n-butyllithium and tert.-butyllithium should be used together with a chelating agent such as hexamethyl-phosphoric triamide or N,N,N',N'-tetramethylenediamine, since they 2 0 have a weak activity for withdrawing the fluorine atom from EPTFE.
Examples of the compounds having the hydroxyl, carboxyl, epoxy or amino group are glycerol (meth)acrylate, 2-hydroxyethyl (meth)acrylate, 2-hydroxypropyl (meth)acrylate, polyethylene glycol (meth)acrylate, glycidyl (meth)acrylate, (meth)acrylic acid, allylamine, 2-aminoethyl (meth)acrylate, acrylamide and the like. Alternatively, an .acid anhydride such as malefic anhydride is added and then hydrolyzed.
The tissue-inducing substances are immobilized onto the r i ~~I9'~~35 - _ g _ inner surface, pore surfaces and optionally outer surface of the EPTFE tube which have been made hydrophilic, by simply applying them or chemically bonding them to the already introduced functional groups. The anti-thrombotic substance is immobilized onto the surfaces which will be in contact with blood in use. The chemical bonding of the substances to the functional groups is preferable when the substances do not lose their activities by the chemical bonding. A method for immobilizing the substances is selected according to the type of already introduced functional 1 0 groups, and preferably a method which does not inactivate the substances is selected.
For example, the substances are covalently bonded with the hydroxyl, carboxyl and amino groups through the dehydration-condensation, or with the epoxy group through the addition reaction.
The substances are directly bonded with the hydroxyl group through the dehydration-condensation in the presence of carbodiimide as a catalyst. Alternatively, an eliminating group such as a trifluoro-methanesulfonyl group is introduced into the hydroxyl group for improving the reactivity and then such hydroxyl group is reacted 2 0 with the amino group of the substances. The substances are directly bonded with the carboxyl group using a dehydration condensation catalyst such as carbodiimide. Alternatively, the carboxyl group is reacted with N-hydroxysuccinimide to introduce an active ester group in the carboxyl group and increase the reactivity and then the activated ester group is reacted with the amino group of the substances.
In particular, esters such as N-hydroxysuccinimide esters of carboxylic acids and sulfonate esters such as trifluoro-f ~19?3?5 » - 10 -ethanesulfonate ester of the hydroxyl group are preferred as the functional groups for immobilizing the tissue-inducing substance by the covalent bond. That is, the tissue-inducing substance is reacted with these functional groups which have been formed by graft polymerization using the alkali metal compound. Then, the tissue-inducing moiety is covalently bonded with these functional groups by replacing the ester moiety or sulfonate ester moiety with the tissue-inducing moiety of the tissue-inducing substance. Thereby, the tissue-inducing effect after the grafting of the artificial blood vessel is improved, and the high patency ratio is obtained.
The method for immobilizing the tissue-inducing substance through the covalent bond after introducing the carboxylic acid group and converting it to the N-hydroxysuccinimide ester will be explained more in detail.
1 5 An EPTFE tube is dipped in a solution of an alkali metal compound such as methyllithium in, for example, diethyl ether under a nitrogen or argon atmosphere and hexamethylphosphoric triamide is added to the solution. Then, the solution containing the tube is kept standing for at least 30 minutes at 0°C or lower (e.g. -10°C) to withdraw fluorine atoms from PTFE. After removing the solution, a solution of (meth)acrylic acid in an solvent such as tetrahydrofuran or water is added and reacted at an elevated temperature, for example, between 60°C and 80°C for at least 10 hours. After the reaction, excessive (meth)acrylic acid and non-grafted poly(meth)acrylic acid are washed off to obtain the grafted polymer of (meth)acrylic acid.
Then, the EPTFE tube on which poly(meth)acrylic acid has been grafted is dipped in a solution of N-hydroxysuccinimide and r~
~ 2197375 dicyclohexylcarbodiimide in a solvent such as 1,4-dioxane at 0° or lower for at least 12 hours to form N-hydroxysuccinimide ester.
After that, the EPTFE tube is dipped in a solution of the tissue-inducing substance in a phosphate buffer, the pH of which solution is adjusted to 11 to covalently bond the tissue-inducing substance.
Preferred examples of the tissue-inducing substance are cell-adhesion materials and cell-growth materials, since these materials achieve the best tissue-inducing functions. They may be used independently or in combination.
Examples of the cell-adhesion materials are collagen, gelatin, laminin, fibronectin and the like. Among them, fibronectin is preferred.
Examples of the cell-growth materials are TGF-a., transferrin, insulin, ECGF (endothelial cell growth factor), BPE
(bovine pituitary extract), PDGF (platelet-derived growth factor), FGF (fibroblast growth factor) and the like. Among them, TGF-a, transferrin, insulin and FGF are preferred.
Examples of the anti-thrombotic substances are anti-coagulants (e.g. hirudin, heparin, etc.), plasminogen activators (e.g.
t-PA, urokinase, etc.), fibrinolytic enzymes (e.g. plasmin, subtilisin, etc.), anti-platelet-aggregation agents (e.g. prostacyclin, aspirin, etc.) and the like.
The immobilized amount of the tissue-inducing substance is usually between 50 and 500 p.g, preferably between 100 and 250 pg per 1 cm of the tube.
The immobilized amount of the anti-thrombotic substance is usually between 300 and 2000 pg, or between 60 and ~ ~~g737~
400 units for heparin, preferably between 400 and 750 pg or between 80 and 150 units for heparin, per 1 cm of the tube.
In a preferred embodiment, the anti-thrombotic substance is ionically immobilized onto a polyamine or its salt which is covalently bonded to EPTFE tube through a polymer which has been graft polymerized, on the surface of the tube which will be in contact with the blood in use. Such ionic immobilization of the anti-thrombotic substance enables sustained release of the anti-thrombotic substance, and achieves the high patency ratio.
1 0 The polyamine herein used is intended to mean a synthetic polymer comprising amino groups as repeating units.
Preferred examples of the polyamine are polyallylamine, polyvinylamine, polyethyleneimine and their salts, and also mixtures of these polymers and/or salts.
In a preferable case, the amino groups of the polyamine are partially acetylated. ~ Preferably up to 90 %, more preferably 20 to 70 % of the amino groups are acetylated. The acetylation of the amino groups controls the sustained release rate of the anti-throm-botic substance such as heparin. That is, the sustained release rate depends on the degree of acetylation of the amino groups. When the degree of acetylation is higher, the anti-thrombotic substance is released in an earlier stage. When the degree of acetylation is lower, the anti-thrombotic substance is released over a longer period. As a result, the adjustment of the degree of acetylation enables the artificial blood vessels to release the necessary amount of the anti-thrombotic substance in the blood over the required period, and maintains the patency of the artificial blood vessel over a long period.
__, ~ ~1973~5 The polyamine can be covalently bonded to the surface of the EPTFE tube by reacting the amino group of the polyamine with functional groups (e.g. carboxyl, aldehyde, hydroxyl groups, etc.) --which have been introduced on the surface of the EPTFE tube and can covalently bond with the amino groups of the polymer. Such functional groups can be introduced in analogous methods to those explained above.
An example of the method for covalently bonding the polyamine to the functional group formed on the EPTFE surfaces will 1 0 be explained using the carboxyl group as the functional group by way of example:
On the surfaces of the EPTFE tube, (meth)acrylic acid is graft polymerized by the same method as described above.
Then, the amino group of the polyamine is covalently bonded with the carboxyl group through the dehydration-condensation. The dehydration reaction may be performed using a carbodiimide such as 1-ethyl-3-(dimethylaminopropyl)carbodiimide.
Alternatively, the carboxyl group is converted to an ester group such as the N-hydroxysuccinimide ester to increase the reactivity, 2 0 and then reacted with the polyamine.
The amino groups of the polyamine may be acetylated prior to or after the covalent bonding of the polyamine to the surface of the EPTFE tube.
The acetylation can be performed by any known method, for example, by reacting the polyamine with acetic acid in the presence of 1-ethyl-3-(dimethylaminopropyl)-carbodiimide. The degree of acetylation can be controlled 'by adjusting the acetylation time.
r yp ~-219737 ~
The molecular weight of the polyamine is usually between 1000 and 100,000 in terms of a weight average molecular weight which is measured on a 3.5 M solution of the polyamine in saline by the sedimentation equilibrium method. When the molecular weight is less than 1000, the amount of the immobilized anti-thrombotic substance tends to decrease. When the molecular weight exceeds 100,000, the efficiency for immobilizing the anti-thrombotic substance to EPTFE tube tends to decrease.
The anti-thrombotic substance is ionically immobilized 1 0 to the surfaces of the EPTFE tube by dipping the EPTFE tube carrying the covalently bonded polyamine in the solution of the anti-thrombotic substance.
In another embodiment, the anti-thrombotic substance may be immobilized only to the nodes of EPTFE on the surface of the 1 5 tube which will be in contact with the blood in use. To this end, the EPTFE tube preferably has a mean distance between nodes, that is, a mean fibril length of at least 60 pm on a side of the tube which will be in contact with the blood in use. More preferably, the EPTFE tube has a multilayer structure having the mean fibril length of at least 2 0 60 pm on a side of the tube which will be in contact with the blood in use and at least one layer in which a fibril length of EPTFE tube is between 20 and 40 Vim.
The mean fibril length means an average distance between the nodes which connect the fibrils of the EPTFE tube and 25 is measured using a scanning electron microscope (SEM). That is, the fibril lengths are measured in three fields of view at thirty points in each field of view in the SEM photograph and averaged.
The artificial blood vessel in which the tissue-inducing ----... r ~~
~~~°~3~~
- substance is immobilized onto all the surfaces of the EPTFE tube including the pore surfaces, and the . anti-thrombotic substance is immobilized onto the inner surface of the tube can be produced by one of the following methods:
In the first method, only the tissue-inducing substance is immobilized onto all the surface of the first EPTFE tube, while the tissue-inducing substance and the anti-thrombotic substance are immobilized onto all the surfaces of the second EPTFE tube.
Then, the first EPTFE tube is laminated over the outer surface of the 1 0 second EPTFE tube.
In the second method, methacrylic acid is graft polymerized on all the surfaces of the EPTFE tube. Prior to the bonding of polyamine or its salt, all the portions of the EPTFE tube except the inner surface are masked with a masking material such 1 5 ' as paraffin. Then, the polyamine or its salt is bonded to the unmasked inner surface, and the masking material is removed.
Finally, the tissue-inducing substance and the anti-thrombotic substance are bonded. Thus, the anti-thrombotic substance is immobilized only onto the inner surface of the EPTFE tube to which 20 the polyamine or its salt has been bonded, while the tissue-inducing substrate is immobilized onto all the surfaces of the EPTFE tube.
Preferably, the nodes exposed on the inner surface of the EPTFE tube which will be in contact with the blood occupies 5 to 50 of the whole inner surface area to effectively attain both of the 25 anti-thrombotic and tissue-inducing effects.
The anti-thrombotic substance can be immobilized to the nodes of the inner surface of EPTFE tube as follows:
The EPTFE tube to which the tissue-inducing substance 2,~9737'~
has been immobilized as explained above is compressed in the longi-tudinal direction, that is, the direction along the axis of the tube, and the fibrils of EPTFE tube are warped. By this warping of the fibrils, the nodes project from the inner surface of the tube. Under such condition of the tube, a solution of the anti-thrombotic substance is passed through the inside of the tube, and the anti-thrombotic substance is immobilized only on the nodes. After that, the tube is relaxed.
,~..__ 219~~?~
_ _ 17 _ - EXAMPLES
The present invention will be explained further in detail by the following Examples, in which a fibril length means a mean fibril length measured using a scanning electron microscope.
Example 1 Each of an EPTFE tube having an inner diameter of 2 mm, an outer diameter of 2.5 mm and a fibril length of 60 p.m (Tube 1 ) and an EPTFE tube having an inner diameter of 2.5 mm, an outer diameter of 3 mm and a fibril length of 30 p.m (Tube 2) was dipped in a mixture of a 1.4 M solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added and reacted at 80°C for 10 hours. After this, excessive acrylic acid and engrafted polymer of acrylic acid were washed off, and an acrylic acid-grafted EPTFE tube was obtained. The grafted amounts of acrylic acid were about 80 pg and about 120 pg per 1 cm of the tube for Tubes 1 and 2, respectively, when calculated from the weight change of the tubes.
2 0 Each tube was dipped in a solution of N-hydroxy-succinimide (2.1 g) and dicyclohexylcarbodiimide (1.9 g) in 1,4-dioxane (100 ml) at 0°C for 12 hours, and acrylic acid was changed to the N-hydroxysuccinimide ester. The esterified tube was dipped in a solution of fibronectin (30 mg) in a phosphate buffer (pH of 1 1 ) (100 ml), and fibronectin was immobilized through the covalent bond to the ester groups. The engrafted polymer of meth of fibronectin were about 45 pg and about 70 ug per 1 cm of the tube for Tubes 1 and 2, respectively when calculated from the weight r s 219~~7~
change.
Next, the tube was dipped in a solution of transferrin in a phosphate buffer (pH of 11) (100 ml), and transferrin was immobilized through the covalent bond. The immobilized amount of transferrin was about 35 p.g and about 60 p.g per 1 cm of the tube for Tubes 1 and 2, respectively when calculated from the weight change.
Then, Tube 1 was dipped in a 0.05 % aqueous solution of heparin (available from SANOFI) for 10 minutes and freeze dried, and the heparin-immobilized EPTFE tube was obtained. The immobilized amount of heparin was about 200 p.g per 1 cm of the tube when calculated from the weight change.
Finally, a stainless steel rod having a diameter of 2 mm was inserted in Tube 1. Then, Tube 2 was laminated over Tube 1 with close contact, and an artificial blood vessel was obtained.
Example 2 Each of an EPTFE tube having an inner diameter of 2 mm, an outer diameter of 2.5 mm and a fibril length of 60 p.m (Tube 1 ) and an EPTFE tube having an inner diameter of 2.5 mm, an outer diameter of 3 mm and a fibril length of 30 pm (Tube 2) was dipped in a mixture of a 1.4 M solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C in an argon atmosphere for 30 minutes. After discarding the mixture, a solu-tion of acrylic acid (1 g) in water (20 ml) was added and reacted at 80°C for 10 hours. After this, excessive acrylic acid and ungrafted polymer of acrylic acid were washed off, and an acrylic acid-grafted EPTFE tube was obtained. The grafted amounts of acrylic acid were about 80 pg and about 120 ~g per 1 cm of the tube for 2197~~~' _ _ _ 19 _ - Tubes 1 and 2, respectively when calculated from the weight change of the tubes.
Each tube was dipped in a solution of N-hydroxy-succinimide (2.1 g) and dicyclohexylcarbodiimide (1.9 g) in 1,4-dioxane (100 ml) at 0°C for 12 hours, and acrylic acid was changed to the N-hydroxysuccinimide ester. The esterified tube was dipped in a solution of fibronectin (30 mg) in a phosphate buffer (pH of 1 1 ) (100 ml), and fibronectin was immobilized through the covalent bond to the ester groups. The immobilized amount of fibronectin 1 0 was about 45 pg and about 70 1.,~.g per 1 cm of the tube for Tubes 1 and 2, respectively when calculated from the weight change.
Then, Tube 1 was dipped in a 0.05 % aqueous solution of heparin (available from SANOFI) for 10 minutes and freeze dried, and the heparin-immobilized EPTFE tube was obtained. The 1 5 immobilized amount of heparin was about 200 p.g per 1 cm of the tube when calculated from the weight change.
Finally, a stainless steel rod having a diameter of 2 mm was inserted in Tube 1. Then, Tube 2 was laminated over Tube 1 with close contact, and an artificial blood vessel was obtained.
20 Example 3 A stainless steel rod having a diameter of 2 mm was inserted in an EPTFE tube having an inner diameter of 2 mm, an outer diameter of 2.5 mm and a fibril length of 60 um (Tube 1 ).
Then, an EPTFE tube having an inner diameter of 2.5 mm, an outer 25 diameter of 3 mm and a fibril length of 30 pm (Tube 2) was laminated over Tube 1 with close contact and heated at 340°C for 20 minutes to adhere the tubes, and a laminated tube was obtained.
The laminated tube was dipped in a mixture of a 1.4 M
., 21g737~
' ' - 20 -solution of methyllithium in ethyl ether (20 ml) and hexamethyl-phosphoric amide (2 ml) at -10°C in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added and reacted at 80°C for 10 hours.
After this, excessive acrylic acid and ungrafted polymer of acrylic acid were washed off, and an acrylic acid-grafted EPTFE tube was obtained. The grafted amount of acrylic acid was about 190 ~g per 1 cm of the tube when calculated from the weight change of the tube.
The tube was dipped in a solution of N-hydroxy-succinimide (2.1 g) and dicyclohexylcarbodiimide (1.9 g) in 1,4-dioxane (100 ml) at 0°C for 12 hours, and acrylic acid was changed to the N-hydroxysuccinimide ester. The esterified tube was dipped in a solution of fibronectin (30 mg) in a phosphate buffer (pH of 1 1 ) (100 ml), and fibronectin was immobilized through the covalent bond to the ester groups. The immobilized amount of fibronectin was about 110 p.g per 1 cm of the tube when calculated from the weight change.
Example 4 A stainless steel rod having a diameter of 2 mm was inserted in an EPTFE tube having an inner diameter of 2 mm, an outer diameter of 2.5 mm and a fibril length of 60 gm (Tube 1 ).
Then, an EPTFE tube having an inner diameter of 2.5 mm, an outer diameter of 3 mm and a fibril length of 30 p,m (Tube 2) was laminated over Tube 1 with close contact and heated at 340°C for 20 minutes to adhere the tubes, and a laminated tube was obtained.
The laminated tube was dipped in a mixture of a 1.4 M
solution of methyllithium in ethyl ether (20 ml) and _ _ _ 21 _ hexamethylphosphoric amide (2 ml) at -10°C in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added and reacted at 80°C for 10 hours. After this, excessive acrylic acid and ungrafted polymer of acrylic acid were washed off, and an acrylic acid-grafted EPTFE
tube was obtained. The grafted amount of acrylic acid was about 190 p.g per 1 cm of the tube when calculated from the weight change of the tube.
The tube was dipped in a solution of N-hydroxy-1 0 succinimide (2.1 g) and dicyclohexylcarbodiimide (1.9 g) in 1,4-dioxane (100 ml) at 0°C for 12 hours, and acrylic acid was changed to the N-hydroxysuccinimide ester. The esterified tube was dipped in a solution of transferrin (30 mg) in a phosphate buffer (pH of 11 ) (100 ml), and transferrin was immobilized through the covalent bond to the ester groups. The immobilized amount of transferrin was about 93 p.g per 1 cm of the tube when calculated from the weight change.
Example 5 A stainless steel rod having a diameter of 2 mm was inserted in an EPTFE tube having an inner diameter of 2 mm, an outer diameter of 2.5 mm and a fibril length of 60 pm (Tube 1 ).
Then, an EPTFE tube having an inner diameter of 2.5 mm, an outer diameter of 3 mm and a fibril length of 30 pm (Tube 2) was laminated over Tube 1 with close contact and heated at 340°C for 20 minutes to adhere the tubes, and a laminated tube was obtained.
The laminated tube was dipped in a mixture of a 1.4 M
solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added and reacted at 80°C for 10 hours. After this, excessive acrylic acid and ungrafted polymer of acrylic acid were washed off, and an acrylic acid-grafted EPTFE
tube was obtained. The grafted amount of acrylic acid was about 190 pg per 1 cm of the tube when calculated from the weight change of the tube.
The tube was dipped in a solution of N-hydroxy-succinimide (2.1 g) and dicyclohexylcarbodiimide (1.9 g) in 1,4-dioxane (100 ml) at 0°C for 12 hours, and acrylic acid was changed to the N-hydroxysuccinimide ester. The. esterified tube was dipped in a solution of fibronectin (30 mg) in a phosphate buffer (pH of 1 1 ) (100 ml), and fibronectin was immobilized through the covalent bond to the ester groups. The immobilized amount of fibronectin 1 5 was about 110 p.g per 1 cm of the tube when calculated from the weight change.
Then, the tube was dipped in a solution of transferrin (30 mg) in a phosphate buffer (pH of 11 ) (100 ml), and transferrin was immobilized through the covalent bond to the ester groups. The immobilized amount of transferrin was about 93 pg per 1 cm of the tube when calculated from the weight change.
Example 6 A stainless steel rod having a diameter of 2 mm was inserted in an EPTFE tube having an inner diameter of 2 mm, an outer diameter of 2.5 mm and a fibril length of 60 pm (Tube 1).
Then, an EPTFE tube having an inner diameter of 2.5 mm, an outer diameter of 3 mm and a fibril length of 30 pm (Tube 2) was laminated over Tube 1 with close contact and heated at 340°C for r r 20 minutes to adhere the tubes, and a laminated tube was obtained.
The laminated tube was dipped in a mixture of a 1.4 M
solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added and reacted at 80°C for 10 hours. After this, excessive acrylic acid and ungrafted polymer of acrylic acid were washed off, and an acrylic acid-grafted EPTFE
tube was obtained. The grafted amount of acrylic acid was about 190 pg per 1 cm of the tube when calculated from the weight change of the tube.
The tube was dipped in a solution of N-hydroxy-succinimide (2.1 g) and dicyclohexylcarbadiimide (1.9 g) in 1,4-dioxane (100 ml) at 0°C for 12 hours, and acrylic acid was changed to the N-hydroxysuccinimide ester. The esterified tube was dipped in a solution of fibronectin (30 mg) in a phosphate buffer (pH of 1 1 ) (100 ml), and fibronectin was immobilized through the covalent bond to the ester groups. The immobilized amount of fibronectin was about 110 pg per 1 cm of the tube when calculated from the weight change.
Finally, the tube was dipped in a 0.02 % aqueous solution of heparin (available from SANOFI) for 10 minutes and freeze dried, and the heparin-immobilized EPTFE tube was obtained. The immobilized amount of heparin was about 200 pg per 1 cm of the tube when calculated from the weight change.
Example 7 .
An EPTFE tube having an inner diameter of 2 mm, an outer diameter of 3 mm and a fibril length of 60 ~m was dipped in a ~ 2'~9~375 - - mixture of a 1.4 M solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added and reacted at 80°C for 10 hours. After this, excessive acrylic acid and ungrafted polymer of acrylic acid were washed off, and an acrylic acid-grafted EPTFE tube was obtained. The grafted amount of acrylic acid was about 120 p.g per 1 cm of the tube when calculated from the weight change of the tube.
1 0 The tube was dipped in a solution of N-hydroxy-succinimide (2.1 g) and dicyclohexylcarbodiimide (1.9 g) in 1,4-dioxane (100 ml) at 0°C for 12 hours, and acrylic acid was changed to the N-hydroxysuccinimide ester. The esterified tube was dipped in a solution of fibronectin (30 mg) in a phosphate buffer (pH of 11 ) (100 ml), and fibronectin was immobilized through the covalent bond to the ester groups. The immobilized amount of fibronectin was about 90 pg per 1 cm of the tube when calculated from the weight change.
Example 8 Each of an EPTFE tube having an inner diameter of 2 mm, an outer diameter of 2.5 mm and a fibril length of 60 pm (Tube 1) and an EPTFE tube having an inner diameter of 2.5 mm, an outer diameter of 3 mm and a fibril length of 60 pm (Tube 2) was dipped in a mixture of a 1.4 M solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added and reacted at 80°C for 10 hours. After this, excessive acrylic acid and 219737 ~
i - - ungrafted polymer of acrylic acid were washed off, and an acrylic acid-grafted EPTFE tube was obtained. The grafted amounts of acrylic acid were about 80 pg and about 90 pg per 1 cm of the tube for Tubes 1 and 2, respectively when calculated from the weight change of the tubes Each of the acrylic acid-grafted EPTFE tubes was dipped in a solution of N-hydroxysuccinimide (2.1 g) and dicyclohexyl-carbodiimide (1.9 g) in 1,4-dioxane (100 ml) at 0°C for 12 hours, and acrylic acid was changed to the N-hydroxysuccinimide ester.
The esterified tube was dipped in a solution of fibronectin (30 mg) in a phosphate buffer (pH of 11) (100 ml), and fibronectin was immobilized through the covalent bond to the ester groups. The immobilized amount of fibronectin was about 45 gg and about 50 p.g per 1 cm of the tube for Tubes 1 and 2, respectively when calculated from the weight change.
The tube 1 was dipped in a 0.05 % aqueous solution of heparin (available from SANOFI) for 10 minutes and freeze dried, and the heparin-immobilized EPTFE tube was obtained. The immobilized amount of heparin was about 200 pg per 1 cm of the tube when calculated from the weight change.
Finally, a stainless steel rod having a diameter of 2 mm was inserted in Tube 1. Then, Tube 2 was laminated over Tube 1 with close contact, and an artificial blood vessel was obtained.
Example 9 Each of an EPTFE tube having an inner diameter of 2 mm, an outer diameter of 2.5 mm and a fibril length of 30 p.m (Tube 1 ) and an EPTFE tube having an inner diameter of 2.5 mm, an outer diameter of 3 mm and a fibril length of 30 ~m (Tube, 2) was dipped g,Z~g~375 i in a mixture of a 1.4 M solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added and reacted at 80°C for 10 hours. After this, excessive acrylic acid and ungrafted polymer of acrylic acid were washed off, and an acrylic acid-grafted EPTFE tube was obtained. The grafted amounts of acrylic acid were about 100 ~g and about 120 pg per 1 cm of the tube for Tubes 1 and 2, respectively when calculated from the 1 0 weight change of the tubes.
Then, each of the acrylic acid-grafted EPTFE tubes was dipped in a solution of N-hydroxysuccinimide (2.1 g) and dicyclohexylcarbodiimide (1.9 g) in 1,4-dioxane (100 ml) at 0°C for t n 0. .. . t :.-. ..I h n r, r, r, .-1 +r, + A 1 h r rc, w nni o tc. ttvurs, and acrjim aWu--vv as ~mcm~cu -w ~i'~e nv°m~dmn~fs-~.i..w.W~m~d~.
ester. The esterified tube was dipped in a solution of fibronectin (30 mg) in a phosphate buffer (pH of 11) (100 ml), and fibronectin was immobilized through the covalent bond to the ester groups. The immobilized amount of fibronectin was about 50 pg and about 70 pg per 1 cm of the tube for Tubes 1 and 2, respectively when calculated from the weight change.
The tube 1 was dipped in a 0.05 % aqueous solution of heparin (available from SANOFI) for 10 minutes and freeze dried, and the heparin-immobilized EPTFE tube was obtained. The immobilized amount of heparin was about 200 pg per 1 cm of the 2 5 tube when calculated from the weight change.
Finally, a stainless steel rod having a diameter of 2 mm was inserted in Tube 1. Then, Tube 2 was laminated over Tube 1 with close contact, and an artificial blood vessel was obtained.
~19737~
i _ _ _ 27 _ Comparative Example 1 An EPTFE tube having an inner diameter of 2 mm, an outer diameter of 3 mm and a fibril length of 30 p.m was used as an artificial blood vessel.
Comparative Example 2 An EPTFE tube having an inner diameter of 2 mm, an outer diameter of 3 mm and a fibril length of 60 ~m was used as an artificial blood vessel.
Comparative Example 3 .
1 0 An EPTFE tube having an inner diameter of 2 mm, an outer diameter of 2.5 mm and a fibril length of 60 ~m (Tube 1 ) and an EPTFE tube having an inner diameter of 2.5 mm, an outer diameter of 3 mm and a fibril length of 30 ~.m (Tube 2) were provided.
A stainless steel rod having a diameter of 2 mm was 1 5 inserted in Tube 1. Then, Tube 2 was laminated over Tube 1 with close contact and heated at 340°C for 20 minutes to cause the two tubes to adhere to each other.
Example 10 An EPTFE tube having an inner diameter of 2 mm, an 20 outer diameter of 3 mm and a fibril length of 30 ~.m was dipped in a mixture of a 1.4 M solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added and 25 reacted at 80°C for 10 hours. After this, excessive acrylic acid and ungrafted polymer of acrylic acid were washed off, and an acrylic acid-grafted EPTFE tube was obtained. 'The grafted amount of acrylic acid was about 210 pg per 1 cm of the tube when calculated i _ - 28 _ _ ~ from the weight change of the tubes.
The tube was dipped in a solution of N-hydroxy-succinimide (2.1 g) and dicyclohexylcarbodiimide (1.9 g) in 1,4-dioxane (100 ml) at 0°C for 12 hours, and acrylic acid was changed to the N-hydroxysuccinimide ester. The esterified tube was dipped in a solution of fibronectin (30 mg) in a phosphate buffer (pH of 11 ) (100 ml), and fibronectin was immobilized through the covalent bond to the ester groups. The immobilized amount of fibronectin was about 110 p.g per 1 cm of the tube when calculated from the weight change.
Example 11 A stainless steel rod having a diameter of 2 mm was inserted in an EPTFE tube having an inner diameter of 2 mm, an outer diameter of 2.5 mm and a fibril length of 60 p.m (Tube 1 ).
1 5 Then, an EPTFE tube having an inner diameter of 2.5 mm, an outer diameter of 3 mm and a fibril length of 30 wm (Tube 2) was laminated over Tube 1 with close contact and heated at 340°C for 20 minutes to adhere the tubes to each other, and a laminated tube was obtained.
The laminated tube was dipped in a mixture of a 1.4 M
solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added and reacted at 80°C for 10 hours. After this, excessive acrylic acid and ungrafted polymer of acrylic acid were washed off, and an acrylic acid-grafted EPTFE
tube was obtained. The grafted amount of acrylic acid was about 190 pg per 1 cm of the tube when calculated from the weight change of the tube.
~_ 21 97375 _ _ _ 29 -_ ' Then, the tube was dipped in an aqueous solution containing 0.05 % of fibronectin (derived from bovine plasma, manufactured by NIPPON HAM) and 0.5 % of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, pH of which had been adjusted to 5 with 1 N hydrochloric acid, for 24 hours, removed from the solution and washed with water, and the fibronectin-immobilized EPTFE tube was obtained. The immobilized amount of fibronectin was about 110 p.g per 1 cm of the tube.
Finally, the tube was dipped in an aqueous solution containing 0.05 % of transferrin and 0.5 % of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, pH of which had been adjusted to 5 with 1 N hydrochloric acid, for 24 hours, removed from the solution and washed with water, and the transferrin-immobilized EPTFE tube was obtained. The immobilized amount of transferrin 1 5 was about 95 p.g per 1 cm of the tube.
Ten artificial blood vessels (each having a length of 3 cm) prepared in each of Examples 1 to 11 and Comparative Examples 1 to 3 were implanted in carotid arteries of ten rabbits. The results are shown in Table 1.
The patency ratio means the ratio of the number of artificial blood vessels which maintained blood flow after the specified time to the number of tested artificial blood vessels (ten blood vessels).
The artificial blood vessels which had the fibril length 2 5 of 60 ~m on the surface which is in contact with blood and the fibril length of 30 arm in other parts, and to which both fibronectin and heparin were immobilized had excellent patency ratios. In particular, those having heparin immobilized in the inner layer had 2~ q737~
_ - 30 -the patency ratio of 100 % (10/10).
The surface area which was covered with endothelial cells was measured with each artificial blood vessel. The artificial -blood vessels to which fibronectin and/or transferrin were immobilized using N-hydroxysucciniimide (active ester method) had the high covered surface area. In particular, the artificial blood vessels to which both fibronectin and transferrin were immobilized had the highest covered surface area.
Many of the artificial blood vessels which did not contain the layer having the fibril length of 30 p.m were warped after implantation and/or adhered heavily to surrounding tissues.
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~21g7375 Example 12 (Reference) A quantity of paraffin was impregnated to a depth of about 50 um from the outer surface of an EPTFE tube having an inner diameter of 2 mm, an outer diameter of 3 mm, a length of 20 cm, a fibril length of 30 pm and a porosity of 75 %. Then, the tube was dipped in a mixture of a 1.4 M solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C in an , argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added and reacted at 80°C for 10 hours. After this, excessive acrylic acid, ungrafted polymer of acrylic acid and paraffin were washed off, and an acrylic acid-grafted EPTFE tube was obtained. The grafted amount of acrylic acid was about 235 pg per 1 cm of the tube when calculated from the weight change of the tubes.
Example 13 (Reference) A quantity of paraffin was impregnated to a depth of about 100 pm from the outer surface of an EPTFE tube having an inner diameter of 2 mm, an outer diameter of 3 mm, a length of 20 , cm, a fibril length of 30 p.m and a porosity of 75 %. Then, the tube was dipped in a mixture of a 1.4 M solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C
in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added and reacted at 80°C for 10 hours. After this, excessive acrylic acid, ungrafted polymer of acrylic acid and paraffin were washed off, and an acrylic acid-grafted EPTFE tube was obtained. The grafted amount of acrylic acid was about 195 pg per 1 cm of the tube when calculated from the weight change of the tubes.
~ 2197~7~
_ - 33 -Example 14 (Reference) A quantity of paraffin was impregnated to a depth of about 300 pm from the outer surface of an EPTFE tube having an inner diameter of 2 mm, an outer diameter of 3 mm, a length of 20 cm, a fibril length of 30 p.m and a porosity of 75 %. Then, the tube was dipped in a mixture of a 1.4 M solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C
in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added 1 0 and reacted at 80°C for 10 hours. After this, excessive acrylic acid, engrafted polymer of acrylic acid and paraffin were washed off, and an acrylic acid-grafted EPTFE tube was obtained. The grafted amount of acrylic acid was about 90 pg per 1 cm of the tube when calculated from the weight change of the tubes.
Example 15 (Reference) A quantity of paraffin was impregnated to a depth of about 480 p.m from the outer surface of an EPTFE tube having an inner diameter of 2 mm, an outer diameter of 3 mm, a length of 20 cm, a fibril length of 30 pm and a porosity of 75 %. Then, the tube was dipped in a mixture of a 1.4 M solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C
in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added and reacted at 80°C for 10 hours. After this, excessive acrylic acid, engrafted polymer of acrylic acid and paraffin were washed off, and an acrylic acid-grafted EPTFE tube was obtained. The grafted amount of acrylic acid was about 20 ug per 1 cm of the tube when calculated from the weight change of the tubes.
~ 219'375 -_ - 34 -Example 16 The acrylic acid-grafted EPTFE tube produced in Example 14 was dipped in a 0.05 % aqueous solution of heparin (available -from SANOFI) for 10 minutes and freeze dried, and the heparin-immobilized EPTFE tube was obtained. The immobilized amount of heparin was about 550 p.g per 1 cm of the tube when calculated from the weight change.
Example 17 The acrylic acid-grafted EPTFE tube prepared in Example 1 0 14 was dipped in an aqueous solution containing 0.05 % of fibronectin (derived from bovine plasma, manufactured by NIPPON
HAM) and 0.5 % of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, pH of which had been adjusted to 5 with 1 N hydrochloric acid, for 24 hours and washed with water, and the fibronectin-immobilized 1 5 EPTFE tube was obtained. The immobilized amount of fibronectin was about 160 p.g per 1 cm of the tube.
Example 18 The fibronectin-immobilized EPTFE tube prepared in Example 17 was dipped in a 0.5 % aqueous solution of heparin 20 (available from SANOFI) for 10 minutes and freeze dried, and the heparin-immobilized EPTFE tube was obtained. The immobilized amount of heparin was about 550 pg per 1 cm of the tube when calculated from the weight change.
Comparative Example 4 2 5 An EPTFE tube having an inner diameter of 2 mm, an outer diameter of 3 mm, a length of 20 cm, a fibril length of 30 pm and a porosity of 75 % was used as an -artificial blood vessel without any treatment.
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_ - 35 -Comparative Example 5 An EPTFE tube having an inner diameter of 2 mm, an outer diameter of 3 mm, a length of 20 cm, a fibril length of 30 pm and a porosity of 75 % was dipped in a mixture of a 1.4 M solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added and reacted at 80°C for 10 hours. After this, excessive acrylic acid and ungrafted polymer of acrylic acid were washed off, and an acrylic acid-grafted EPTFE tube was obtained.
The grafted amount of acrylic acid was about 250 pg per 1 cm of the tube when calculated from the weight change of the tubes.
Comparative Example 6 The acrylic acid-grafter EPTFE tube prepared in the 1 5 same manner as Comparative Example 5 was dipped in a 0.5 aqueous solution of heparin (available from SANOFI) for 10 minutes and freeze dried, and the heparin-immobilized EPTFE tube was obtained. The immobilized amount of heparin was about 920 ~g per 1 cm of the tube when calculated from the weight change.
Comparative Example 7 A quantity of paraffin was impregnated to a depth of about 20 ~,m from the outer surface of an EPTFE tube having an inner diameter of 2 mm, an outer diameter of 3 mm, a length of 20 cm, a fibril length of 30 ~m and a porosity of 75 %. Then, the tube was dipped in a mixture of a 1.4 M solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added and i~~1~~3~5 _ - _ - 36 -reacted at 80°C for 10 hours. After this, excessive acrylic acid, ungrafted polymer of acrylic acid and paraffin were washed off, and an acrylic acid-grafted EPTFE tube was obtained. The grafted amount of acrylic acid was about 250 pg per 1 cm of the tube when calculated from the weight change of the tubes.
The patency ratios of the artificial blood vessels produced in Examples 12-18 and Comparative Examples 4-7 were evaluated in the same way as in Examples 1-11. The results are shown in Table 2.
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_ _ _ 38 Example 19 An EPTFE tube having an inner diameter of 4 mm, an outer diameter of 5 mm and a length of 30 mm was dipped in a mixture of a 1.4 M solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at 0°C in a nitrogen atmos-phere for 30 minutes. After discarding the mixture, a solution of methacrylic acid (3 g) in tetrahydrofuran (20 ml) was added and reacted at 60°C for 10 hours. After this, excessive methacrylic acid and ungrafted polymer of methacrylic acid were washed off, and a methacrylic acid-grafted EPTFE tube was obtained. The grafted amount of methacrylic acid was about 132 p.g per 1 cm of the tube when calculated from the weight change of the tube.
Polyallylamine having an acetylation rate of 0 mole was reacted with the obtained methacrylic acid-grafted EPTFE tube in the presence of 1-ethyl-3-(dimethylaminopropyl)carbodiimide, and immobilized to the tube through the covalent bond. The immobilized amount of polyallylamine was about 162 p.g per cm when calculated from the weight change of the tube.
The tube was dipped in a 10 % aqueous solution of heparin for 2 hours for ionically bonding heparin to the tube, and the heparin-immobilized EPTFE tube was obtained. The immobilized amount of heparin was about 62 UNIT per 1 cm of the tube.
The sustained releasability of heparin from the obtained heparin-immobilized EPTFE tube was evaluated in vitro.
A 2 M (mol/I) aqueous solution of sodium chloride was flowed through the heparin=immobilized EPTFE tube at a flow rate of 10 ml/min. with a peristaltic pump, and the remaining amount of heparin was measured after a specific period. The results are _ . - 39 -- shown in Fig. 1.
Ten heparin-immobilized EPTFE artificial blood vessels were implanted in carotid arteries of ten dogs, and patency ratio after one and four weeks was checked. The results are shown in Table 3.
Example 20 Methacrylic acid was graft polymerized on the EPTFE
tube in an amount of 132 p.g/cm in the same manner as in Example 19. Then, polyallylamine having an acetylation rate of 30 mole was immobilized through covalent bond in an amount of 152 p.g/cm, and further heparin was immobilized through ion bond in an amount of 56 UNIT/cm in the same manner as in Example 19.
The sustained releasability of heparin from the obtained heparin-immobilized EPTFE tube was evaluated in vitro in the same 1 5 method as in Example 19. The results are shown in Fig. 1.
Ten heparin-immobilized EPTFE artificial blood vessels were implanted in carotid arteries of ten dogs, and patency ratio after one and four weeks was checked. The results are shown in Table 3.
2 0 Example 21 Methacrylic acid was graft polymerized on the EPTFE
tube in an amount of 132 p.g/cm in the same manner as in Example 19. Then, polyallylamine having an acetylation rate of 60 mole was immobilized through covalent bond in an amount of 138 p.g/cm, 25 and further heparin was immobilized through ion bond in an amount of 48 UNIT/cm in the same manner as in Example 19.
The sustained releasability of heparin from the obtained heparin-immobilized EPTFE tube was evaluated in vi r in the same ~ 21 ~ 735 . ~ method as in Example 19. The results are shown in Fig. 1.
' Ten heparin-immobilized EPTFE artificial blood vessels were implanted in carotid arteries of ten dogs, and patency ratio after one and four weeks was checked. The results are shown in Table 3.
Example 22 Methacrylic acid was graft polymerized on the EPTFE
tube in an amount of 232 pg/cm in the same manner as in Example 19 except that 6 g of methacrylic acid was used. Then, polyallyl-amine having an acetylation rate of 0 mole % was immobilized through covalent bond in an amount of 353 p.g/cm, and further heparin was immobilized through ion bond in an amount of 105 UNIT/cm in the same manner as in Example 19.
The sustained releasability of heparin from the obtained 1 5 heparin-immobilized EPTFE tube was evaluated in vitro in the same method as in Example 19. The results are shown in Fig. 1.
Ten heparin-immobilized EPTFE artificial blood vessels were implanted in carotid arteries of ten dogs, and patency ratio after one and four weeks was checked. The results are shown in 2 0 Table 3.
Example 23 Methacrylic acid was graft polymerized on the EPTFE
tube in an amount of 232 p,g/cm in the same manner as in Example 19 except that 6 g of methacrylic acid was used. Then, 2 5 polyallylamine having an acetylation rate of 30 mole % was immobilized through covalent bond in an amount of 348 pg/cm, and further heparin was immobilized through 'ion bond in an amount of 96 UNIT/cm in the same manner as in Example 19.
The sustained releasability of heparin from the obtained heparin-immobilized EPTFE tube was evaluated in vitro in the same method as in Example 19. The results are shown in Fig. 1. -Ten heparin-immobilized EPTFE artificial blood vessels were implanted in carotid arteries of ten dogs, and patency ratio after one and four weeks was checked. The results are shown in Table 3.
Example 24 Methacrylic acid was graft polymerized on the EPTFE
1 0 tube in an amount of 232 pg/cm in the same manner as in Example 19 except that 6 g of methacrylic acid was used. Then, polyallylamine having an acetylation rate of 60 mole % was immobilized through covalent bond in an amount of 339 p,g/cm, and further heparin was immobilized through ion bond in an amount of 1 5 88 UNIT/cm in the same manner as in Example 19.
The sustained releasability of heparin from the obtained heparin-immobilized EPTFE tube was evaluated in vitro in the same method as in Example 19. The results are shown in Fig. 1.
Ten heparin-immobilized EPTFE artificial blood vessels 2 0 were implanted in carotid arteries of ten dogs, and patency ratio after one and four weeks was checked. The results are shown in Table 3.
~omt~arative Exam Ip a 8 An EPTFE tube having an inner diameter of 4 mm, an 2 5 outer diameter of 5 mm and a length of 30 mm was dipped successively in ethanol, and water and maintained in a 0.1 solution of protamine for one hour. After this, the tube was dipped in a 1 % aqueous solution of glutaraldehyde.
~.21~7~~~
The tube was dipped in a 10 % aqueous solution of heparin for 2 hours for ionically bonding heparin to the tube, and the heparin-immobilized EPTFE tube was obtained.
The ungrafted polymer of meth of protamine and heparin were about 120 ~g/cm and 31 UNIT/cm, respectively.
The sustained releasability of heparin from the obtained heparin-immobilized EPTFE tube was evaluated in vi ro in the same method as in Example 19. The results are shown in Fig. 2.
Ten heparin-immobilized EPTFE artificial blood vessels were implanted in carotid arteries of ten dogs, and patency ratio after one and four weeks was checked. The results are shown in Table 3.
Comparative Example 9 An EPTFE tube having an inner diameter of 4 mm, an 1 5 outer diameter of 5 mm and a length of 30 mm was dipped successively in ethanol, water and then a 0.2 % solution of protamine for one hour. After this, the tube was dipped in a 1 aqueous solution of glutaraldehyde.
The tube was dipped in a 10 % aqueous solution of 2 0 heparin for 2 hours for ionically bonding heparin to the tube, and the heparin-immobilized EPTFE tube was obtained.
The ungrafted polymer of meth of protamine and heparin were about 220 pg/cm and 51 UNIT/cm, respectively.
The sustained releasability of heparin from the obtained 25 heparin-immobilized EPTFE tube was evaluated in vitro in the same method as in Example 19. The results are shown in Fig. 2.
Ten heparin-immobilized EPTFE artificial blood vessels were implanted in carotid arteries of ten dogs, and patency ratio A _ r 299737 _ - 43 -after one and four weeks was checked. The results are shown in Table 3.
Comparative Example 10 An EPTFE tube having an inner diameter of 4 mm, an outer diameter of 5 mm and a length of 30 mm was dipped successively in ethanol, water and then a 0.3 % solution of protamine for one hour. After this, the tube was dipped in a 1 aqueous solution of glutaraldehyde.
The tube was dipped in a 10 % aqueous solution of heparin for 2 hours for ionically bonding heparin to the tube, and the heparin-immobilized EPTFE tube was obtained.
The amounts of the immobilized protamine and heparin were about 320 p.g/cm and 53 UNIT/cm, respectively.
The sustained releasability of heparin from the obtained 1 5 heparin-immobilized EPTFE tube was evaluated in vitro in the same method as in Example 19. The results are shown in Fig. 2.
Ten heparin-immobilized EPTFE artificial blood vessels were implanted in carotid arteries of ten dogs, and patency ratio after one and four weeks was checked. The results are shown in 2 0 Table 3.
21 9 ~37~
- _ - 44 -Table 3 Ex. No. Patency ratio after One week One month Ex. 19 6/10 6/10 Ex.20 7/10 7/10 Ex.21 10/10 7/10 Ex.22 7/10 7/10 Ex. 23 1 0 / 1 1 0 / 1 Ex. 24 1 0 / 1 9/10 C. Ex. 6/10 3/10 C. Ex. 7/10 3/10 C. Ex. 7/10 3/10 Example 25 An EPTFE tube having an inner diameter of 4 mm, an
An object of the present invention is to provide an artificial blood vessel which has a high patency ratio after grafting.
Another object of the present invention is to provide an artificial blood vessel which can facilitate the growth of endothelial cells while suppressing the formation of thrombus after grafting.
Accordingly, the present invention provides an artificial blood vessel comprising: a tube formed from expanded polytetrafluoroethylene comprising fibrils and nodes connecting fibrils, wherein said tube has an inner surface and an outer surface, in which the inner surface of said tube and the surfaces of fibrils and nodes comprise a hydrophilic layer extending to a depth of between 5 % and 96% of the wall thickness of said tube in a radial direction from the inner surface toward the outer surface of said tube, and a tissue-inducing substance which is immobilized on said inner surface and said surfaces of fibrils and nodes of the hydrophilic layer.
In a preferred embodiment, the artificial blood vessel of the present invention further comprises an anti-thrombotic _ - 5 -_ substance immobilized on at least a part of a surface of said tube ~.
which will be in contact with the blood in use.
The properties required for the artificial blood vessels vary with parts of living bodies in which the artificial blood vessels are implanted. On the one hand, anti-thrombotic properties should be enhanced for the parts where blood flow rate is small such as peripheral blood vessels since thrombus tends to form in such parts. Therefore, an anti-thrombotic substance is immobilized on the surfaces of the artificial blood vessels for preventing the 1 0 formation of thrombus. On the other hand, the covering of the surfaces of the artificial blood vessels with the endothelial cells should be facilitated by immobilizing a tissue-inducing substance for the parts where blood flow rate is large since the formation of cell tissues is delayed while the thrombus is less formed.
1 5 In the drawings:
Fig. 1 is a graph showing the sustained release of heparin from the heparin-immobilized EPTFE artificial blood vessels In general, the mean fibril length of EPTFE tube, that is, the mean length of the fibrils between nodes, is between 20 and 200 2 5 p.m, the porosity of EPTFE tube is at least 50 %, preferably at least 70 %, and the wall thickness of the tube is between 300 and 1000 pm for adjusting the wall thickness of the artificial blood vessel to that of the living blood vessel.
prepared in Examples 19 through 24, and Fig. 2 is a graph showing the sustained release of 20 heparin from the heparin-immobilized EPTFE artificial blood vessels used in Comparative Examples 8, 9 and 10.
The EPTFE tube preferably has a mean fibril length of at least 60 pm on a side of the tube which will be in contact with the blood in use. More preferably, the EPTFE tube has a multilayer --structure having the mean fibril length of at least 60 pm on a side of the tube which will be in contact with the blood in use and at least one layer in which a fibril length of EPTFE tube is 40 pm or less.
When the EPTFE tube has the hydrophilic layer portion extending from its inner surface to the limited depth in the radial 1 0 direction of the tube from the inner surface, hydrophilic groups can be chemically introduced into the tube wall by the following method:
Firstly, the porous wall of the EPTFE tube is masked to a certain depth from the outer surface of the tube with a material, which is not decomposed or denatured by a defluorination reaction, can be removed from the tube without damaging or denaturing the subsequently immobilized materials and has affinity to PTFE, such as paraffin, and the unmasked portion of the EPTFE
tube is defluorinated by chemical treatment with alkali metal compounds, or by physical treatment such as irradiation with 'y-ray or electron beams, corona discharge or glow discharge.
Then, functional groups are introduced in the unmasked portion by treating the defluorinated portion with compounds having carboxyl, hydroxyl, amino or epoxy groups.
Finally, the masking material is removed.
2 5 The patency ratio can be increased when the thickness of the wall which has not been masked is between 5 % and 100 %, preferably 5 % and less than 96 %, more preferably between 50 and 90 %, of the total wall thickness.
i~2197~~5 i The wall thickness which is not masked can be adjusted -~ by controlling a depth to which molten paraffin penetrates through the change of a temperature gradient in the tube wall by flowing a cooling medium such as water through the hole of the tube, or filling all the pores in the tube wall with paraffin and then extracting paraffin on the inner wall side.
When the tube wall is not masked, the entire wall is treated, and the functional groups are introduced onto all the surfaces of the pores which present in the tube wall as well as the inner and to outer surfaces of the tube.
Among the defluorination methods, the chemical treatment with the alkali metals is more preferred than physical treatment by irradiation with y ray or electron beams or corona or glow discharge.
The irradiation with y ray decomposes PTFE to deeper parts of the crystals of PTFE. Therefore the molecular weight of PTFE decreases and the strength of the EPTFE tube deteriorates g reatly.
The glow discharge can treat only the outer and inner surfaces of the tube but may not treat surfaces of pores inside the 2o tube wall, that is the surfaces of fibrils which constitute the pores in EPTFE.
The treatment with the alkali metal compounds can defluorinate the whole surfaces of the pores of EPTFE tube uniformly only to a depth of about several angstroms from the 2197~7~
- surfaces of fibrils and nodes constituting the pores , and does not w decompose PTFE. Therefore, the strength of EPTFE tube does not decrease.
Since the inner and outer surfaces of the tube and also the surfaces of the pores inside the tube wall are defluorinated, proteins and peptides such as the tissue-inducing substances and anti-thrombotic substances can be covalently bonded to all of these surfaces.
Examples of the alkali metal compounds are methyl-lithium, n-butyllithium, tert.-butyllithium, sodium-naphthalene, sodium-benzophenone, vinyllithium and the like. They are used in the form of a solution. Among them, sodium-naphthalene and sodium-benzophenone will form a dark brown layer on the EPTFE
surfaces after treatment and cannot treat EPTFE uniformly. Thus, methyllithium, n-butyllithium and tert.-butyllithium are preferable for the production of the artificial blood vessel of the present invention. Methyllithium, n-butyllithium and tert.-butyllithium should be used together with a chelating agent such as hexamethyl-phosphoric triamide or N,N,N',N'-tetramethylenediamine, since they 2 0 have a weak activity for withdrawing the fluorine atom from EPTFE.
Examples of the compounds having the hydroxyl, carboxyl, epoxy or amino group are glycerol (meth)acrylate, 2-hydroxyethyl (meth)acrylate, 2-hydroxypropyl (meth)acrylate, polyethylene glycol (meth)acrylate, glycidyl (meth)acrylate, (meth)acrylic acid, allylamine, 2-aminoethyl (meth)acrylate, acrylamide and the like. Alternatively, an .acid anhydride such as malefic anhydride is added and then hydrolyzed.
The tissue-inducing substances are immobilized onto the r i ~~I9'~~35 - _ g _ inner surface, pore surfaces and optionally outer surface of the EPTFE tube which have been made hydrophilic, by simply applying them or chemically bonding them to the already introduced functional groups. The anti-thrombotic substance is immobilized onto the surfaces which will be in contact with blood in use. The chemical bonding of the substances to the functional groups is preferable when the substances do not lose their activities by the chemical bonding. A method for immobilizing the substances is selected according to the type of already introduced functional 1 0 groups, and preferably a method which does not inactivate the substances is selected.
For example, the substances are covalently bonded with the hydroxyl, carboxyl and amino groups through the dehydration-condensation, or with the epoxy group through the addition reaction.
The substances are directly bonded with the hydroxyl group through the dehydration-condensation in the presence of carbodiimide as a catalyst. Alternatively, an eliminating group such as a trifluoro-methanesulfonyl group is introduced into the hydroxyl group for improving the reactivity and then such hydroxyl group is reacted 2 0 with the amino group of the substances. The substances are directly bonded with the carboxyl group using a dehydration condensation catalyst such as carbodiimide. Alternatively, the carboxyl group is reacted with N-hydroxysuccinimide to introduce an active ester group in the carboxyl group and increase the reactivity and then the activated ester group is reacted with the amino group of the substances.
In particular, esters such as N-hydroxysuccinimide esters of carboxylic acids and sulfonate esters such as trifluoro-f ~19?3?5 » - 10 -ethanesulfonate ester of the hydroxyl group are preferred as the functional groups for immobilizing the tissue-inducing substance by the covalent bond. That is, the tissue-inducing substance is reacted with these functional groups which have been formed by graft polymerization using the alkali metal compound. Then, the tissue-inducing moiety is covalently bonded with these functional groups by replacing the ester moiety or sulfonate ester moiety with the tissue-inducing moiety of the tissue-inducing substance. Thereby, the tissue-inducing effect after the grafting of the artificial blood vessel is improved, and the high patency ratio is obtained.
The method for immobilizing the tissue-inducing substance through the covalent bond after introducing the carboxylic acid group and converting it to the N-hydroxysuccinimide ester will be explained more in detail.
1 5 An EPTFE tube is dipped in a solution of an alkali metal compound such as methyllithium in, for example, diethyl ether under a nitrogen or argon atmosphere and hexamethylphosphoric triamide is added to the solution. Then, the solution containing the tube is kept standing for at least 30 minutes at 0°C or lower (e.g. -10°C) to withdraw fluorine atoms from PTFE. After removing the solution, a solution of (meth)acrylic acid in an solvent such as tetrahydrofuran or water is added and reacted at an elevated temperature, for example, between 60°C and 80°C for at least 10 hours. After the reaction, excessive (meth)acrylic acid and non-grafted poly(meth)acrylic acid are washed off to obtain the grafted polymer of (meth)acrylic acid.
Then, the EPTFE tube on which poly(meth)acrylic acid has been grafted is dipped in a solution of N-hydroxysuccinimide and r~
~ 2197375 dicyclohexylcarbodiimide in a solvent such as 1,4-dioxane at 0° or lower for at least 12 hours to form N-hydroxysuccinimide ester.
After that, the EPTFE tube is dipped in a solution of the tissue-inducing substance in a phosphate buffer, the pH of which solution is adjusted to 11 to covalently bond the tissue-inducing substance.
Preferred examples of the tissue-inducing substance are cell-adhesion materials and cell-growth materials, since these materials achieve the best tissue-inducing functions. They may be used independently or in combination.
Examples of the cell-adhesion materials are collagen, gelatin, laminin, fibronectin and the like. Among them, fibronectin is preferred.
Examples of the cell-growth materials are TGF-a., transferrin, insulin, ECGF (endothelial cell growth factor), BPE
(bovine pituitary extract), PDGF (platelet-derived growth factor), FGF (fibroblast growth factor) and the like. Among them, TGF-a, transferrin, insulin and FGF are preferred.
Examples of the anti-thrombotic substances are anti-coagulants (e.g. hirudin, heparin, etc.), plasminogen activators (e.g.
t-PA, urokinase, etc.), fibrinolytic enzymes (e.g. plasmin, subtilisin, etc.), anti-platelet-aggregation agents (e.g. prostacyclin, aspirin, etc.) and the like.
The immobilized amount of the tissue-inducing substance is usually between 50 and 500 p.g, preferably between 100 and 250 pg per 1 cm of the tube.
The immobilized amount of the anti-thrombotic substance is usually between 300 and 2000 pg, or between 60 and ~ ~~g737~
400 units for heparin, preferably between 400 and 750 pg or between 80 and 150 units for heparin, per 1 cm of the tube.
In a preferred embodiment, the anti-thrombotic substance is ionically immobilized onto a polyamine or its salt which is covalently bonded to EPTFE tube through a polymer which has been graft polymerized, on the surface of the tube which will be in contact with the blood in use. Such ionic immobilization of the anti-thrombotic substance enables sustained release of the anti-thrombotic substance, and achieves the high patency ratio.
1 0 The polyamine herein used is intended to mean a synthetic polymer comprising amino groups as repeating units.
Preferred examples of the polyamine are polyallylamine, polyvinylamine, polyethyleneimine and their salts, and also mixtures of these polymers and/or salts.
In a preferable case, the amino groups of the polyamine are partially acetylated. ~ Preferably up to 90 %, more preferably 20 to 70 % of the amino groups are acetylated. The acetylation of the amino groups controls the sustained release rate of the anti-throm-botic substance such as heparin. That is, the sustained release rate depends on the degree of acetylation of the amino groups. When the degree of acetylation is higher, the anti-thrombotic substance is released in an earlier stage. When the degree of acetylation is lower, the anti-thrombotic substance is released over a longer period. As a result, the adjustment of the degree of acetylation enables the artificial blood vessels to release the necessary amount of the anti-thrombotic substance in the blood over the required period, and maintains the patency of the artificial blood vessel over a long period.
__, ~ ~1973~5 The polyamine can be covalently bonded to the surface of the EPTFE tube by reacting the amino group of the polyamine with functional groups (e.g. carboxyl, aldehyde, hydroxyl groups, etc.) --which have been introduced on the surface of the EPTFE tube and can covalently bond with the amino groups of the polymer. Such functional groups can be introduced in analogous methods to those explained above.
An example of the method for covalently bonding the polyamine to the functional group formed on the EPTFE surfaces will 1 0 be explained using the carboxyl group as the functional group by way of example:
On the surfaces of the EPTFE tube, (meth)acrylic acid is graft polymerized by the same method as described above.
Then, the amino group of the polyamine is covalently bonded with the carboxyl group through the dehydration-condensation. The dehydration reaction may be performed using a carbodiimide such as 1-ethyl-3-(dimethylaminopropyl)carbodiimide.
Alternatively, the carboxyl group is converted to an ester group such as the N-hydroxysuccinimide ester to increase the reactivity, 2 0 and then reacted with the polyamine.
The amino groups of the polyamine may be acetylated prior to or after the covalent bonding of the polyamine to the surface of the EPTFE tube.
The acetylation can be performed by any known method, for example, by reacting the polyamine with acetic acid in the presence of 1-ethyl-3-(dimethylaminopropyl)-carbodiimide. The degree of acetylation can be controlled 'by adjusting the acetylation time.
r yp ~-219737 ~
The molecular weight of the polyamine is usually between 1000 and 100,000 in terms of a weight average molecular weight which is measured on a 3.5 M solution of the polyamine in saline by the sedimentation equilibrium method. When the molecular weight is less than 1000, the amount of the immobilized anti-thrombotic substance tends to decrease. When the molecular weight exceeds 100,000, the efficiency for immobilizing the anti-thrombotic substance to EPTFE tube tends to decrease.
The anti-thrombotic substance is ionically immobilized 1 0 to the surfaces of the EPTFE tube by dipping the EPTFE tube carrying the covalently bonded polyamine in the solution of the anti-thrombotic substance.
In another embodiment, the anti-thrombotic substance may be immobilized only to the nodes of EPTFE on the surface of the 1 5 tube which will be in contact with the blood in use. To this end, the EPTFE tube preferably has a mean distance between nodes, that is, a mean fibril length of at least 60 pm on a side of the tube which will be in contact with the blood in use. More preferably, the EPTFE tube has a multilayer structure having the mean fibril length of at least 2 0 60 pm on a side of the tube which will be in contact with the blood in use and at least one layer in which a fibril length of EPTFE tube is between 20 and 40 Vim.
The mean fibril length means an average distance between the nodes which connect the fibrils of the EPTFE tube and 25 is measured using a scanning electron microscope (SEM). That is, the fibril lengths are measured in three fields of view at thirty points in each field of view in the SEM photograph and averaged.
The artificial blood vessel in which the tissue-inducing ----... r ~~
~~~°~3~~
- substance is immobilized onto all the surfaces of the EPTFE tube including the pore surfaces, and the . anti-thrombotic substance is immobilized onto the inner surface of the tube can be produced by one of the following methods:
In the first method, only the tissue-inducing substance is immobilized onto all the surface of the first EPTFE tube, while the tissue-inducing substance and the anti-thrombotic substance are immobilized onto all the surfaces of the second EPTFE tube.
Then, the first EPTFE tube is laminated over the outer surface of the 1 0 second EPTFE tube.
In the second method, methacrylic acid is graft polymerized on all the surfaces of the EPTFE tube. Prior to the bonding of polyamine or its salt, all the portions of the EPTFE tube except the inner surface are masked with a masking material such 1 5 ' as paraffin. Then, the polyamine or its salt is bonded to the unmasked inner surface, and the masking material is removed.
Finally, the tissue-inducing substance and the anti-thrombotic substance are bonded. Thus, the anti-thrombotic substance is immobilized only onto the inner surface of the EPTFE tube to which 20 the polyamine or its salt has been bonded, while the tissue-inducing substrate is immobilized onto all the surfaces of the EPTFE tube.
Preferably, the nodes exposed on the inner surface of the EPTFE tube which will be in contact with the blood occupies 5 to 50 of the whole inner surface area to effectively attain both of the 25 anti-thrombotic and tissue-inducing effects.
The anti-thrombotic substance can be immobilized to the nodes of the inner surface of EPTFE tube as follows:
The EPTFE tube to which the tissue-inducing substance 2,~9737'~
has been immobilized as explained above is compressed in the longi-tudinal direction, that is, the direction along the axis of the tube, and the fibrils of EPTFE tube are warped. By this warping of the fibrils, the nodes project from the inner surface of the tube. Under such condition of the tube, a solution of the anti-thrombotic substance is passed through the inside of the tube, and the anti-thrombotic substance is immobilized only on the nodes. After that, the tube is relaxed.
,~..__ 219~~?~
_ _ 17 _ - EXAMPLES
The present invention will be explained further in detail by the following Examples, in which a fibril length means a mean fibril length measured using a scanning electron microscope.
Example 1 Each of an EPTFE tube having an inner diameter of 2 mm, an outer diameter of 2.5 mm and a fibril length of 60 p.m (Tube 1 ) and an EPTFE tube having an inner diameter of 2.5 mm, an outer diameter of 3 mm and a fibril length of 30 p.m (Tube 2) was dipped in a mixture of a 1.4 M solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added and reacted at 80°C for 10 hours. After this, excessive acrylic acid and engrafted polymer of acrylic acid were washed off, and an acrylic acid-grafted EPTFE tube was obtained. The grafted amounts of acrylic acid were about 80 pg and about 120 pg per 1 cm of the tube for Tubes 1 and 2, respectively, when calculated from the weight change of the tubes.
2 0 Each tube was dipped in a solution of N-hydroxy-succinimide (2.1 g) and dicyclohexylcarbodiimide (1.9 g) in 1,4-dioxane (100 ml) at 0°C for 12 hours, and acrylic acid was changed to the N-hydroxysuccinimide ester. The esterified tube was dipped in a solution of fibronectin (30 mg) in a phosphate buffer (pH of 1 1 ) (100 ml), and fibronectin was immobilized through the covalent bond to the ester groups. The engrafted polymer of meth of fibronectin were about 45 pg and about 70 ug per 1 cm of the tube for Tubes 1 and 2, respectively when calculated from the weight r s 219~~7~
change.
Next, the tube was dipped in a solution of transferrin in a phosphate buffer (pH of 11) (100 ml), and transferrin was immobilized through the covalent bond. The immobilized amount of transferrin was about 35 p.g and about 60 p.g per 1 cm of the tube for Tubes 1 and 2, respectively when calculated from the weight change.
Then, Tube 1 was dipped in a 0.05 % aqueous solution of heparin (available from SANOFI) for 10 minutes and freeze dried, and the heparin-immobilized EPTFE tube was obtained. The immobilized amount of heparin was about 200 p.g per 1 cm of the tube when calculated from the weight change.
Finally, a stainless steel rod having a diameter of 2 mm was inserted in Tube 1. Then, Tube 2 was laminated over Tube 1 with close contact, and an artificial blood vessel was obtained.
Example 2 Each of an EPTFE tube having an inner diameter of 2 mm, an outer diameter of 2.5 mm and a fibril length of 60 p.m (Tube 1 ) and an EPTFE tube having an inner diameter of 2.5 mm, an outer diameter of 3 mm and a fibril length of 30 pm (Tube 2) was dipped in a mixture of a 1.4 M solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C in an argon atmosphere for 30 minutes. After discarding the mixture, a solu-tion of acrylic acid (1 g) in water (20 ml) was added and reacted at 80°C for 10 hours. After this, excessive acrylic acid and ungrafted polymer of acrylic acid were washed off, and an acrylic acid-grafted EPTFE tube was obtained. The grafted amounts of acrylic acid were about 80 pg and about 120 ~g per 1 cm of the tube for 2197~~~' _ _ _ 19 _ - Tubes 1 and 2, respectively when calculated from the weight change of the tubes.
Each tube was dipped in a solution of N-hydroxy-succinimide (2.1 g) and dicyclohexylcarbodiimide (1.9 g) in 1,4-dioxane (100 ml) at 0°C for 12 hours, and acrylic acid was changed to the N-hydroxysuccinimide ester. The esterified tube was dipped in a solution of fibronectin (30 mg) in a phosphate buffer (pH of 1 1 ) (100 ml), and fibronectin was immobilized through the covalent bond to the ester groups. The immobilized amount of fibronectin 1 0 was about 45 pg and about 70 1.,~.g per 1 cm of the tube for Tubes 1 and 2, respectively when calculated from the weight change.
Then, Tube 1 was dipped in a 0.05 % aqueous solution of heparin (available from SANOFI) for 10 minutes and freeze dried, and the heparin-immobilized EPTFE tube was obtained. The 1 5 immobilized amount of heparin was about 200 p.g per 1 cm of the tube when calculated from the weight change.
Finally, a stainless steel rod having a diameter of 2 mm was inserted in Tube 1. Then, Tube 2 was laminated over Tube 1 with close contact, and an artificial blood vessel was obtained.
20 Example 3 A stainless steel rod having a diameter of 2 mm was inserted in an EPTFE tube having an inner diameter of 2 mm, an outer diameter of 2.5 mm and a fibril length of 60 um (Tube 1 ).
Then, an EPTFE tube having an inner diameter of 2.5 mm, an outer 25 diameter of 3 mm and a fibril length of 30 pm (Tube 2) was laminated over Tube 1 with close contact and heated at 340°C for 20 minutes to adhere the tubes, and a laminated tube was obtained.
The laminated tube was dipped in a mixture of a 1.4 M
., 21g737~
' ' - 20 -solution of methyllithium in ethyl ether (20 ml) and hexamethyl-phosphoric amide (2 ml) at -10°C in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added and reacted at 80°C for 10 hours.
After this, excessive acrylic acid and ungrafted polymer of acrylic acid were washed off, and an acrylic acid-grafted EPTFE tube was obtained. The grafted amount of acrylic acid was about 190 ~g per 1 cm of the tube when calculated from the weight change of the tube.
The tube was dipped in a solution of N-hydroxy-succinimide (2.1 g) and dicyclohexylcarbodiimide (1.9 g) in 1,4-dioxane (100 ml) at 0°C for 12 hours, and acrylic acid was changed to the N-hydroxysuccinimide ester. The esterified tube was dipped in a solution of fibronectin (30 mg) in a phosphate buffer (pH of 1 1 ) (100 ml), and fibronectin was immobilized through the covalent bond to the ester groups. The immobilized amount of fibronectin was about 110 p.g per 1 cm of the tube when calculated from the weight change.
Example 4 A stainless steel rod having a diameter of 2 mm was inserted in an EPTFE tube having an inner diameter of 2 mm, an outer diameter of 2.5 mm and a fibril length of 60 gm (Tube 1 ).
Then, an EPTFE tube having an inner diameter of 2.5 mm, an outer diameter of 3 mm and a fibril length of 30 p,m (Tube 2) was laminated over Tube 1 with close contact and heated at 340°C for 20 minutes to adhere the tubes, and a laminated tube was obtained.
The laminated tube was dipped in a mixture of a 1.4 M
solution of methyllithium in ethyl ether (20 ml) and _ _ _ 21 _ hexamethylphosphoric amide (2 ml) at -10°C in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added and reacted at 80°C for 10 hours. After this, excessive acrylic acid and ungrafted polymer of acrylic acid were washed off, and an acrylic acid-grafted EPTFE
tube was obtained. The grafted amount of acrylic acid was about 190 p.g per 1 cm of the tube when calculated from the weight change of the tube.
The tube was dipped in a solution of N-hydroxy-1 0 succinimide (2.1 g) and dicyclohexylcarbodiimide (1.9 g) in 1,4-dioxane (100 ml) at 0°C for 12 hours, and acrylic acid was changed to the N-hydroxysuccinimide ester. The esterified tube was dipped in a solution of transferrin (30 mg) in a phosphate buffer (pH of 11 ) (100 ml), and transferrin was immobilized through the covalent bond to the ester groups. The immobilized amount of transferrin was about 93 p.g per 1 cm of the tube when calculated from the weight change.
Example 5 A stainless steel rod having a diameter of 2 mm was inserted in an EPTFE tube having an inner diameter of 2 mm, an outer diameter of 2.5 mm and a fibril length of 60 pm (Tube 1 ).
Then, an EPTFE tube having an inner diameter of 2.5 mm, an outer diameter of 3 mm and a fibril length of 30 pm (Tube 2) was laminated over Tube 1 with close contact and heated at 340°C for 20 minutes to adhere the tubes, and a laminated tube was obtained.
The laminated tube was dipped in a mixture of a 1.4 M
solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added and reacted at 80°C for 10 hours. After this, excessive acrylic acid and ungrafted polymer of acrylic acid were washed off, and an acrylic acid-grafted EPTFE
tube was obtained. The grafted amount of acrylic acid was about 190 pg per 1 cm of the tube when calculated from the weight change of the tube.
The tube was dipped in a solution of N-hydroxy-succinimide (2.1 g) and dicyclohexylcarbodiimide (1.9 g) in 1,4-dioxane (100 ml) at 0°C for 12 hours, and acrylic acid was changed to the N-hydroxysuccinimide ester. The. esterified tube was dipped in a solution of fibronectin (30 mg) in a phosphate buffer (pH of 1 1 ) (100 ml), and fibronectin was immobilized through the covalent bond to the ester groups. The immobilized amount of fibronectin 1 5 was about 110 p.g per 1 cm of the tube when calculated from the weight change.
Then, the tube was dipped in a solution of transferrin (30 mg) in a phosphate buffer (pH of 11 ) (100 ml), and transferrin was immobilized through the covalent bond to the ester groups. The immobilized amount of transferrin was about 93 pg per 1 cm of the tube when calculated from the weight change.
Example 6 A stainless steel rod having a diameter of 2 mm was inserted in an EPTFE tube having an inner diameter of 2 mm, an outer diameter of 2.5 mm and a fibril length of 60 pm (Tube 1).
Then, an EPTFE tube having an inner diameter of 2.5 mm, an outer diameter of 3 mm and a fibril length of 30 pm (Tube 2) was laminated over Tube 1 with close contact and heated at 340°C for r r 20 minutes to adhere the tubes, and a laminated tube was obtained.
The laminated tube was dipped in a mixture of a 1.4 M
solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added and reacted at 80°C for 10 hours. After this, excessive acrylic acid and ungrafted polymer of acrylic acid were washed off, and an acrylic acid-grafted EPTFE
tube was obtained. The grafted amount of acrylic acid was about 190 pg per 1 cm of the tube when calculated from the weight change of the tube.
The tube was dipped in a solution of N-hydroxy-succinimide (2.1 g) and dicyclohexylcarbadiimide (1.9 g) in 1,4-dioxane (100 ml) at 0°C for 12 hours, and acrylic acid was changed to the N-hydroxysuccinimide ester. The esterified tube was dipped in a solution of fibronectin (30 mg) in a phosphate buffer (pH of 1 1 ) (100 ml), and fibronectin was immobilized through the covalent bond to the ester groups. The immobilized amount of fibronectin was about 110 pg per 1 cm of the tube when calculated from the weight change.
Finally, the tube was dipped in a 0.02 % aqueous solution of heparin (available from SANOFI) for 10 minutes and freeze dried, and the heparin-immobilized EPTFE tube was obtained. The immobilized amount of heparin was about 200 pg per 1 cm of the tube when calculated from the weight change.
Example 7 .
An EPTFE tube having an inner diameter of 2 mm, an outer diameter of 3 mm and a fibril length of 60 ~m was dipped in a ~ 2'~9~375 - - mixture of a 1.4 M solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added and reacted at 80°C for 10 hours. After this, excessive acrylic acid and ungrafted polymer of acrylic acid were washed off, and an acrylic acid-grafted EPTFE tube was obtained. The grafted amount of acrylic acid was about 120 p.g per 1 cm of the tube when calculated from the weight change of the tube.
1 0 The tube was dipped in a solution of N-hydroxy-succinimide (2.1 g) and dicyclohexylcarbodiimide (1.9 g) in 1,4-dioxane (100 ml) at 0°C for 12 hours, and acrylic acid was changed to the N-hydroxysuccinimide ester. The esterified tube was dipped in a solution of fibronectin (30 mg) in a phosphate buffer (pH of 11 ) (100 ml), and fibronectin was immobilized through the covalent bond to the ester groups. The immobilized amount of fibronectin was about 90 pg per 1 cm of the tube when calculated from the weight change.
Example 8 Each of an EPTFE tube having an inner diameter of 2 mm, an outer diameter of 2.5 mm and a fibril length of 60 pm (Tube 1) and an EPTFE tube having an inner diameter of 2.5 mm, an outer diameter of 3 mm and a fibril length of 60 pm (Tube 2) was dipped in a mixture of a 1.4 M solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added and reacted at 80°C for 10 hours. After this, excessive acrylic acid and 219737 ~
i - - ungrafted polymer of acrylic acid were washed off, and an acrylic acid-grafted EPTFE tube was obtained. The grafted amounts of acrylic acid were about 80 pg and about 90 pg per 1 cm of the tube for Tubes 1 and 2, respectively when calculated from the weight change of the tubes Each of the acrylic acid-grafted EPTFE tubes was dipped in a solution of N-hydroxysuccinimide (2.1 g) and dicyclohexyl-carbodiimide (1.9 g) in 1,4-dioxane (100 ml) at 0°C for 12 hours, and acrylic acid was changed to the N-hydroxysuccinimide ester.
The esterified tube was dipped in a solution of fibronectin (30 mg) in a phosphate buffer (pH of 11) (100 ml), and fibronectin was immobilized through the covalent bond to the ester groups. The immobilized amount of fibronectin was about 45 gg and about 50 p.g per 1 cm of the tube for Tubes 1 and 2, respectively when calculated from the weight change.
The tube 1 was dipped in a 0.05 % aqueous solution of heparin (available from SANOFI) for 10 minutes and freeze dried, and the heparin-immobilized EPTFE tube was obtained. The immobilized amount of heparin was about 200 pg per 1 cm of the tube when calculated from the weight change.
Finally, a stainless steel rod having a diameter of 2 mm was inserted in Tube 1. Then, Tube 2 was laminated over Tube 1 with close contact, and an artificial blood vessel was obtained.
Example 9 Each of an EPTFE tube having an inner diameter of 2 mm, an outer diameter of 2.5 mm and a fibril length of 30 p.m (Tube 1 ) and an EPTFE tube having an inner diameter of 2.5 mm, an outer diameter of 3 mm and a fibril length of 30 ~m (Tube, 2) was dipped g,Z~g~375 i in a mixture of a 1.4 M solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added and reacted at 80°C for 10 hours. After this, excessive acrylic acid and ungrafted polymer of acrylic acid were washed off, and an acrylic acid-grafted EPTFE tube was obtained. The grafted amounts of acrylic acid were about 100 ~g and about 120 pg per 1 cm of the tube for Tubes 1 and 2, respectively when calculated from the 1 0 weight change of the tubes.
Then, each of the acrylic acid-grafted EPTFE tubes was dipped in a solution of N-hydroxysuccinimide (2.1 g) and dicyclohexylcarbodiimide (1.9 g) in 1,4-dioxane (100 ml) at 0°C for t n 0. .. . t :.-. ..I h n r, r, r, .-1 +r, + A 1 h r rc, w nni o tc. ttvurs, and acrjim aWu--vv as ~mcm~cu -w ~i'~e nv°m~dmn~fs-~.i..w.W~m~d~.
ester. The esterified tube was dipped in a solution of fibronectin (30 mg) in a phosphate buffer (pH of 11) (100 ml), and fibronectin was immobilized through the covalent bond to the ester groups. The immobilized amount of fibronectin was about 50 pg and about 70 pg per 1 cm of the tube for Tubes 1 and 2, respectively when calculated from the weight change.
The tube 1 was dipped in a 0.05 % aqueous solution of heparin (available from SANOFI) for 10 minutes and freeze dried, and the heparin-immobilized EPTFE tube was obtained. The immobilized amount of heparin was about 200 pg per 1 cm of the 2 5 tube when calculated from the weight change.
Finally, a stainless steel rod having a diameter of 2 mm was inserted in Tube 1. Then, Tube 2 was laminated over Tube 1 with close contact, and an artificial blood vessel was obtained.
~19737~
i _ _ _ 27 _ Comparative Example 1 An EPTFE tube having an inner diameter of 2 mm, an outer diameter of 3 mm and a fibril length of 30 p.m was used as an artificial blood vessel.
Comparative Example 2 An EPTFE tube having an inner diameter of 2 mm, an outer diameter of 3 mm and a fibril length of 60 ~m was used as an artificial blood vessel.
Comparative Example 3 .
1 0 An EPTFE tube having an inner diameter of 2 mm, an outer diameter of 2.5 mm and a fibril length of 60 ~m (Tube 1 ) and an EPTFE tube having an inner diameter of 2.5 mm, an outer diameter of 3 mm and a fibril length of 30 ~.m (Tube 2) were provided.
A stainless steel rod having a diameter of 2 mm was 1 5 inserted in Tube 1. Then, Tube 2 was laminated over Tube 1 with close contact and heated at 340°C for 20 minutes to cause the two tubes to adhere to each other.
Example 10 An EPTFE tube having an inner diameter of 2 mm, an 20 outer diameter of 3 mm and a fibril length of 30 ~.m was dipped in a mixture of a 1.4 M solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added and 25 reacted at 80°C for 10 hours. After this, excessive acrylic acid and ungrafted polymer of acrylic acid were washed off, and an acrylic acid-grafted EPTFE tube was obtained. 'The grafted amount of acrylic acid was about 210 pg per 1 cm of the tube when calculated i _ - 28 _ _ ~ from the weight change of the tubes.
The tube was dipped in a solution of N-hydroxy-succinimide (2.1 g) and dicyclohexylcarbodiimide (1.9 g) in 1,4-dioxane (100 ml) at 0°C for 12 hours, and acrylic acid was changed to the N-hydroxysuccinimide ester. The esterified tube was dipped in a solution of fibronectin (30 mg) in a phosphate buffer (pH of 11 ) (100 ml), and fibronectin was immobilized through the covalent bond to the ester groups. The immobilized amount of fibronectin was about 110 p.g per 1 cm of the tube when calculated from the weight change.
Example 11 A stainless steel rod having a diameter of 2 mm was inserted in an EPTFE tube having an inner diameter of 2 mm, an outer diameter of 2.5 mm and a fibril length of 60 p.m (Tube 1 ).
1 5 Then, an EPTFE tube having an inner diameter of 2.5 mm, an outer diameter of 3 mm and a fibril length of 30 wm (Tube 2) was laminated over Tube 1 with close contact and heated at 340°C for 20 minutes to adhere the tubes to each other, and a laminated tube was obtained.
The laminated tube was dipped in a mixture of a 1.4 M
solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added and reacted at 80°C for 10 hours. After this, excessive acrylic acid and ungrafted polymer of acrylic acid were washed off, and an acrylic acid-grafted EPTFE
tube was obtained. The grafted amount of acrylic acid was about 190 pg per 1 cm of the tube when calculated from the weight change of the tube.
~_ 21 97375 _ _ _ 29 -_ ' Then, the tube was dipped in an aqueous solution containing 0.05 % of fibronectin (derived from bovine plasma, manufactured by NIPPON HAM) and 0.5 % of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, pH of which had been adjusted to 5 with 1 N hydrochloric acid, for 24 hours, removed from the solution and washed with water, and the fibronectin-immobilized EPTFE tube was obtained. The immobilized amount of fibronectin was about 110 p.g per 1 cm of the tube.
Finally, the tube was dipped in an aqueous solution containing 0.05 % of transferrin and 0.5 % of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, pH of which had been adjusted to 5 with 1 N hydrochloric acid, for 24 hours, removed from the solution and washed with water, and the transferrin-immobilized EPTFE tube was obtained. The immobilized amount of transferrin 1 5 was about 95 p.g per 1 cm of the tube.
Ten artificial blood vessels (each having a length of 3 cm) prepared in each of Examples 1 to 11 and Comparative Examples 1 to 3 were implanted in carotid arteries of ten rabbits. The results are shown in Table 1.
The patency ratio means the ratio of the number of artificial blood vessels which maintained blood flow after the specified time to the number of tested artificial blood vessels (ten blood vessels).
The artificial blood vessels which had the fibril length 2 5 of 60 ~m on the surface which is in contact with blood and the fibril length of 30 arm in other parts, and to which both fibronectin and heparin were immobilized had excellent patency ratios. In particular, those having heparin immobilized in the inner layer had 2~ q737~
_ - 30 -the patency ratio of 100 % (10/10).
The surface area which was covered with endothelial cells was measured with each artificial blood vessel. The artificial -blood vessels to which fibronectin and/or transferrin were immobilized using N-hydroxysucciniimide (active ester method) had the high covered surface area. In particular, the artificial blood vessels to which both fibronectin and transferrin were immobilized had the highest covered surface area.
Many of the artificial blood vessels which did not contain the layer having the fibril length of 30 p.m were warped after implantation and/or adhered heavily to surrounding tissues.
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~21g7375 Example 12 (Reference) A quantity of paraffin was impregnated to a depth of about 50 um from the outer surface of an EPTFE tube having an inner diameter of 2 mm, an outer diameter of 3 mm, a length of 20 cm, a fibril length of 30 pm and a porosity of 75 %. Then, the tube was dipped in a mixture of a 1.4 M solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C in an , argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added and reacted at 80°C for 10 hours. After this, excessive acrylic acid, ungrafted polymer of acrylic acid and paraffin were washed off, and an acrylic acid-grafted EPTFE tube was obtained. The grafted amount of acrylic acid was about 235 pg per 1 cm of the tube when calculated from the weight change of the tubes.
Example 13 (Reference) A quantity of paraffin was impregnated to a depth of about 100 pm from the outer surface of an EPTFE tube having an inner diameter of 2 mm, an outer diameter of 3 mm, a length of 20 , cm, a fibril length of 30 p.m and a porosity of 75 %. Then, the tube was dipped in a mixture of a 1.4 M solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C
in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added and reacted at 80°C for 10 hours. After this, excessive acrylic acid, ungrafted polymer of acrylic acid and paraffin were washed off, and an acrylic acid-grafted EPTFE tube was obtained. The grafted amount of acrylic acid was about 195 pg per 1 cm of the tube when calculated from the weight change of the tubes.
~ 2197~7~
_ - 33 -Example 14 (Reference) A quantity of paraffin was impregnated to a depth of about 300 pm from the outer surface of an EPTFE tube having an inner diameter of 2 mm, an outer diameter of 3 mm, a length of 20 cm, a fibril length of 30 p.m and a porosity of 75 %. Then, the tube was dipped in a mixture of a 1.4 M solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C
in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added 1 0 and reacted at 80°C for 10 hours. After this, excessive acrylic acid, engrafted polymer of acrylic acid and paraffin were washed off, and an acrylic acid-grafted EPTFE tube was obtained. The grafted amount of acrylic acid was about 90 pg per 1 cm of the tube when calculated from the weight change of the tubes.
Example 15 (Reference) A quantity of paraffin was impregnated to a depth of about 480 p.m from the outer surface of an EPTFE tube having an inner diameter of 2 mm, an outer diameter of 3 mm, a length of 20 cm, a fibril length of 30 pm and a porosity of 75 %. Then, the tube was dipped in a mixture of a 1.4 M solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C
in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added and reacted at 80°C for 10 hours. After this, excessive acrylic acid, engrafted polymer of acrylic acid and paraffin were washed off, and an acrylic acid-grafted EPTFE tube was obtained. The grafted amount of acrylic acid was about 20 ug per 1 cm of the tube when calculated from the weight change of the tubes.
~ 219'375 -_ - 34 -Example 16 The acrylic acid-grafted EPTFE tube produced in Example 14 was dipped in a 0.05 % aqueous solution of heparin (available -from SANOFI) for 10 minutes and freeze dried, and the heparin-immobilized EPTFE tube was obtained. The immobilized amount of heparin was about 550 p.g per 1 cm of the tube when calculated from the weight change.
Example 17 The acrylic acid-grafted EPTFE tube prepared in Example 1 0 14 was dipped in an aqueous solution containing 0.05 % of fibronectin (derived from bovine plasma, manufactured by NIPPON
HAM) and 0.5 % of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, pH of which had been adjusted to 5 with 1 N hydrochloric acid, for 24 hours and washed with water, and the fibronectin-immobilized 1 5 EPTFE tube was obtained. The immobilized amount of fibronectin was about 160 p.g per 1 cm of the tube.
Example 18 The fibronectin-immobilized EPTFE tube prepared in Example 17 was dipped in a 0.5 % aqueous solution of heparin 20 (available from SANOFI) for 10 minutes and freeze dried, and the heparin-immobilized EPTFE tube was obtained. The immobilized amount of heparin was about 550 pg per 1 cm of the tube when calculated from the weight change.
Comparative Example 4 2 5 An EPTFE tube having an inner diameter of 2 mm, an outer diameter of 3 mm, a length of 20 cm, a fibril length of 30 pm and a porosity of 75 % was used as an -artificial blood vessel without any treatment.
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_ - 35 -Comparative Example 5 An EPTFE tube having an inner diameter of 2 mm, an outer diameter of 3 mm, a length of 20 cm, a fibril length of 30 pm and a porosity of 75 % was dipped in a mixture of a 1.4 M solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added and reacted at 80°C for 10 hours. After this, excessive acrylic acid and ungrafted polymer of acrylic acid were washed off, and an acrylic acid-grafted EPTFE tube was obtained.
The grafted amount of acrylic acid was about 250 pg per 1 cm of the tube when calculated from the weight change of the tubes.
Comparative Example 6 The acrylic acid-grafter EPTFE tube prepared in the 1 5 same manner as Comparative Example 5 was dipped in a 0.5 aqueous solution of heparin (available from SANOFI) for 10 minutes and freeze dried, and the heparin-immobilized EPTFE tube was obtained. The immobilized amount of heparin was about 920 ~g per 1 cm of the tube when calculated from the weight change.
Comparative Example 7 A quantity of paraffin was impregnated to a depth of about 20 ~,m from the outer surface of an EPTFE tube having an inner diameter of 2 mm, an outer diameter of 3 mm, a length of 20 cm, a fibril length of 30 ~m and a porosity of 75 %. Then, the tube was dipped in a mixture of a 1.4 M solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of acrylic acid (1 g) in water (20 ml) was added and i~~1~~3~5 _ - _ - 36 -reacted at 80°C for 10 hours. After this, excessive acrylic acid, ungrafted polymer of acrylic acid and paraffin were washed off, and an acrylic acid-grafted EPTFE tube was obtained. The grafted amount of acrylic acid was about 250 pg per 1 cm of the tube when calculated from the weight change of the tubes.
The patency ratios of the artificial blood vessels produced in Examples 12-18 and Comparative Examples 4-7 were evaluated in the same way as in Examples 1-11. The results are shown in Table 2.
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_ _ _ 38 Example 19 An EPTFE tube having an inner diameter of 4 mm, an outer diameter of 5 mm and a length of 30 mm was dipped in a mixture of a 1.4 M solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at 0°C in a nitrogen atmos-phere for 30 minutes. After discarding the mixture, a solution of methacrylic acid (3 g) in tetrahydrofuran (20 ml) was added and reacted at 60°C for 10 hours. After this, excessive methacrylic acid and ungrafted polymer of methacrylic acid were washed off, and a methacrylic acid-grafted EPTFE tube was obtained. The grafted amount of methacrylic acid was about 132 p.g per 1 cm of the tube when calculated from the weight change of the tube.
Polyallylamine having an acetylation rate of 0 mole was reacted with the obtained methacrylic acid-grafted EPTFE tube in the presence of 1-ethyl-3-(dimethylaminopropyl)carbodiimide, and immobilized to the tube through the covalent bond. The immobilized amount of polyallylamine was about 162 p.g per cm when calculated from the weight change of the tube.
The tube was dipped in a 10 % aqueous solution of heparin for 2 hours for ionically bonding heparin to the tube, and the heparin-immobilized EPTFE tube was obtained. The immobilized amount of heparin was about 62 UNIT per 1 cm of the tube.
The sustained releasability of heparin from the obtained heparin-immobilized EPTFE tube was evaluated in vitro.
A 2 M (mol/I) aqueous solution of sodium chloride was flowed through the heparin=immobilized EPTFE tube at a flow rate of 10 ml/min. with a peristaltic pump, and the remaining amount of heparin was measured after a specific period. The results are _ . - 39 -- shown in Fig. 1.
Ten heparin-immobilized EPTFE artificial blood vessels were implanted in carotid arteries of ten dogs, and patency ratio after one and four weeks was checked. The results are shown in Table 3.
Example 20 Methacrylic acid was graft polymerized on the EPTFE
tube in an amount of 132 p.g/cm in the same manner as in Example 19. Then, polyallylamine having an acetylation rate of 30 mole was immobilized through covalent bond in an amount of 152 p.g/cm, and further heparin was immobilized through ion bond in an amount of 56 UNIT/cm in the same manner as in Example 19.
The sustained releasability of heparin from the obtained heparin-immobilized EPTFE tube was evaluated in vitro in the same 1 5 method as in Example 19. The results are shown in Fig. 1.
Ten heparin-immobilized EPTFE artificial blood vessels were implanted in carotid arteries of ten dogs, and patency ratio after one and four weeks was checked. The results are shown in Table 3.
2 0 Example 21 Methacrylic acid was graft polymerized on the EPTFE
tube in an amount of 132 p.g/cm in the same manner as in Example 19. Then, polyallylamine having an acetylation rate of 60 mole was immobilized through covalent bond in an amount of 138 p.g/cm, 25 and further heparin was immobilized through ion bond in an amount of 48 UNIT/cm in the same manner as in Example 19.
The sustained releasability of heparin from the obtained heparin-immobilized EPTFE tube was evaluated in vi r in the same ~ 21 ~ 735 . ~ method as in Example 19. The results are shown in Fig. 1.
' Ten heparin-immobilized EPTFE artificial blood vessels were implanted in carotid arteries of ten dogs, and patency ratio after one and four weeks was checked. The results are shown in Table 3.
Example 22 Methacrylic acid was graft polymerized on the EPTFE
tube in an amount of 232 pg/cm in the same manner as in Example 19 except that 6 g of methacrylic acid was used. Then, polyallyl-amine having an acetylation rate of 0 mole % was immobilized through covalent bond in an amount of 353 p.g/cm, and further heparin was immobilized through ion bond in an amount of 105 UNIT/cm in the same manner as in Example 19.
The sustained releasability of heparin from the obtained 1 5 heparin-immobilized EPTFE tube was evaluated in vitro in the same method as in Example 19. The results are shown in Fig. 1.
Ten heparin-immobilized EPTFE artificial blood vessels were implanted in carotid arteries of ten dogs, and patency ratio after one and four weeks was checked. The results are shown in 2 0 Table 3.
Example 23 Methacrylic acid was graft polymerized on the EPTFE
tube in an amount of 232 p,g/cm in the same manner as in Example 19 except that 6 g of methacrylic acid was used. Then, 2 5 polyallylamine having an acetylation rate of 30 mole % was immobilized through covalent bond in an amount of 348 pg/cm, and further heparin was immobilized through 'ion bond in an amount of 96 UNIT/cm in the same manner as in Example 19.
The sustained releasability of heparin from the obtained heparin-immobilized EPTFE tube was evaluated in vitro in the same method as in Example 19. The results are shown in Fig. 1. -Ten heparin-immobilized EPTFE artificial blood vessels were implanted in carotid arteries of ten dogs, and patency ratio after one and four weeks was checked. The results are shown in Table 3.
Example 24 Methacrylic acid was graft polymerized on the EPTFE
1 0 tube in an amount of 232 pg/cm in the same manner as in Example 19 except that 6 g of methacrylic acid was used. Then, polyallylamine having an acetylation rate of 60 mole % was immobilized through covalent bond in an amount of 339 p,g/cm, and further heparin was immobilized through ion bond in an amount of 1 5 88 UNIT/cm in the same manner as in Example 19.
The sustained releasability of heparin from the obtained heparin-immobilized EPTFE tube was evaluated in vitro in the same method as in Example 19. The results are shown in Fig. 1.
Ten heparin-immobilized EPTFE artificial blood vessels 2 0 were implanted in carotid arteries of ten dogs, and patency ratio after one and four weeks was checked. The results are shown in Table 3.
~omt~arative Exam Ip a 8 An EPTFE tube having an inner diameter of 4 mm, an 2 5 outer diameter of 5 mm and a length of 30 mm was dipped successively in ethanol, and water and maintained in a 0.1 solution of protamine for one hour. After this, the tube was dipped in a 1 % aqueous solution of glutaraldehyde.
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The tube was dipped in a 10 % aqueous solution of heparin for 2 hours for ionically bonding heparin to the tube, and the heparin-immobilized EPTFE tube was obtained.
The ungrafted polymer of meth of protamine and heparin were about 120 ~g/cm and 31 UNIT/cm, respectively.
The sustained releasability of heparin from the obtained heparin-immobilized EPTFE tube was evaluated in vi ro in the same method as in Example 19. The results are shown in Fig. 2.
Ten heparin-immobilized EPTFE artificial blood vessels were implanted in carotid arteries of ten dogs, and patency ratio after one and four weeks was checked. The results are shown in Table 3.
Comparative Example 9 An EPTFE tube having an inner diameter of 4 mm, an 1 5 outer diameter of 5 mm and a length of 30 mm was dipped successively in ethanol, water and then a 0.2 % solution of protamine for one hour. After this, the tube was dipped in a 1 aqueous solution of glutaraldehyde.
The tube was dipped in a 10 % aqueous solution of 2 0 heparin for 2 hours for ionically bonding heparin to the tube, and the heparin-immobilized EPTFE tube was obtained.
The ungrafted polymer of meth of protamine and heparin were about 220 pg/cm and 51 UNIT/cm, respectively.
The sustained releasability of heparin from the obtained 25 heparin-immobilized EPTFE tube was evaluated in vitro in the same method as in Example 19. The results are shown in Fig. 2.
Ten heparin-immobilized EPTFE artificial blood vessels were implanted in carotid arteries of ten dogs, and patency ratio A _ r 299737 _ - 43 -after one and four weeks was checked. The results are shown in Table 3.
Comparative Example 10 An EPTFE tube having an inner diameter of 4 mm, an outer diameter of 5 mm and a length of 30 mm was dipped successively in ethanol, water and then a 0.3 % solution of protamine for one hour. After this, the tube was dipped in a 1 aqueous solution of glutaraldehyde.
The tube was dipped in a 10 % aqueous solution of heparin for 2 hours for ionically bonding heparin to the tube, and the heparin-immobilized EPTFE tube was obtained.
The amounts of the immobilized protamine and heparin were about 320 p.g/cm and 53 UNIT/cm, respectively.
The sustained releasability of heparin from the obtained 1 5 heparin-immobilized EPTFE tube was evaluated in vitro in the same method as in Example 19. The results are shown in Fig. 2.
Ten heparin-immobilized EPTFE artificial blood vessels were implanted in carotid arteries of ten dogs, and patency ratio after one and four weeks was checked. The results are shown in 2 0 Table 3.
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- _ - 44 -Table 3 Ex. No. Patency ratio after One week One month Ex. 19 6/10 6/10 Ex.20 7/10 7/10 Ex.21 10/10 7/10 Ex.22 7/10 7/10 Ex. 23 1 0 / 1 1 0 / 1 Ex. 24 1 0 / 1 9/10 C. Ex. 6/10 3/10 C. Ex. 7/10 3/10 C. Ex. 7/10 3/10 Example 25 An EPTFE tube having an inner diameter of 4 mm, an
5 outer diameter of 5 mm and a length of 1 m was dipped in a mixture of a 1.4 M solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at 0°C in a nitrogen atmosphere for 30 minutes. After discarding the mixture, a solution of methacrylic acid (5 g) in tetrahydrofuran (20 ml) was added and 10 reacted at 60°C for 10 hours. After this, excessive methacrylic acid and ungrafted polymer of methacrylic acid were washed off, and a meth acrylic acid-grafted EPTFE tube was obtained. The grafted amount of methacrylic acid was about 182 gg per 1 cm of the tube when calculated from the weight change of the tube.
Next, the methacrylic acid-grafted EPTFE tube was dipped in a solution of ~N-hydroxysuccinimide (2.1 g) and dicyclohexylcarbodiimide (1.9 g) in 1,4-dioxane (100 ml) at 0°C for 12 hours, and acrylic acid was changed to the N-hydroxysuccinimide ester.
2 0 The esterified tube was dipped in a solution of ?9 - - fibronectin (30 mg) and transferrin (30 mg) in a phosphate buffer (pH of 11 ) (100 ml),, and fibronectin and transferrin were immobilized through covalent bond to the ester groups. The ungrafted polymer of meth of fibronectin and transferrin were about 122 ~g and 95 ug per 1 cm of the tube, respectively.
Ten artificial blood vessels (each 3 cm in length) were implanted in carotid arteries of ten dogs. The patency ratio was 8/10 (80%) and the ratio of the surface area covered with endothelial cells was 95 % after four weeks.
Example 26 Fibronectin and insulin were immobilized onto the EPTFE
tube in amounts of 132 p,g/cm and 102 p.g/cm, respectively in the same manner as in Example 25 except that insulin was used in place of transferrin.
1 5 Ten artificial blood vessels were implanted in carotid arteries of ten dogs. The patency ratio was 9/10 (90%) and the ratio of the surface area covered with endothelial cells was 92 % after four weeks.
Example 27 Fibronectin and TGF-a were immobilized onto the EPTFE
tube in amounts of 118 p.g/cm and 89 pg/cm, respectively in the same manner as in Example 25 except that TGF-a was used in place of transferrin.
Ten artificial blood vessels were implanted in carotid 2 5 arteries of ten dogs. The patency ratio was 8/10 (80%) and the ratio of the surface area covered with endothelial cells was 95 % after four weeks.
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Example 28 - ' Fibronectin and FGF were immobilized onto the EPTFE
tube in the amounts of 120 Erg/cm and 90 ~ug/cm, respectively in the same manner as in Example 25 except that FGF was used in place of transferrin.
Ten artificial blood vessels were implanted in carotid arteries of ten dogs. The patency ratio was 9/10 (90%) and the ratio of the surface area covered with endothelial cells was 89 % after four weeks.
Example 29 a 2-Hydroxyethyl methacrylate was graft polymerized on EPTFE tube in an amount of 246 ~rg/cm in the same manner as in Example 25 except that 2-hydroxyethyl methacrylate was used in place of methacrylic acid. Then, the EPTFE tube was dipped in solution of 2,2,2-trifluoromethanesulfonyl chloride in dichloromethane for forming 2,2,2-trifluoromethanesulfonate ester.
The esterified tube was dipped in a solution of fibronectin (30 mg) and transferrin (30 mg) in a phosphate buffer (pH of 11) (100 ml), and fibronectin and transferrin were immobilized through covalent bond in amounts of 128 dug and 89 ,vg per 1 cm of the tube, respectively.
Ten artificial blood vessels were implanted in carotid arteries of ten dogs. The patency ratio was 8/10 (80%) and the ratio of the surFace area covered with endothelial cells was 95 % after four weeks.
Example 30 Methacrylic acid was graft polymerized on the EPTFE tube in an amount of 185 ,vg/cm in the same manner as in Example 25.
Then, the tube was dipped in a solution of fibronectin (30 mg) ~,2'1973~5 _ - 47 -and transferrin (30 mg) in a phosphate buffer (100 ml) at pH of 1.5 for 12 hours in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (30 mg), and fibronectin and transferrin were immobilized through covalent bond in amounts of 120 pg and 108 pg per 1 cm of the tube, respectively.
Ten artificial blood vessels were implanted in carotid arteries of ten dogs. The patency ratio was 6/10 (60%) and the ratio of the surface area covered with endothelial cells was 50 % after four weeks.
1 0 Examl l~ a 31 Methacrylic acid was graft polymerized on the EPTFE
tube in an amount of 178 p.g/cm in the same manner as in Example 25. Next, the methacrylic acid-grafted EPTFE tube was dipped in a solution of N-hydroxysuccinimide (2.1 g) and dicyclohexyl-carbodiimide (1.9 g) in 1,4-dioxane (100 ml) at 0°C for 12 hours, and meth acrylic acid was changed to the N-hydroxysuccinimide ester.
The esterified tube was dipped in a solution of fibronectin (30 mg) in a phosphate buffer (pH of 11) (100 ml), and fibronectin was immobilized through covalent bond to the ester groups. The immobilized amount of fibronectin was about 122 pg per 1 cm of the tube.
Ten artificial blood vessels were implanted in carotid arteries of ten dogs. The patency ratio was 3/10 (30%) and the ratio of the surface area covered with endothelia( cells was 70 % after four weeks.
Example 32 Transferrin was immobilized to the esterified tube 21 ~ 737g through the covalent bond in an amount of 108 gg/cm in the same manner as in Example 30 except that transferrin alone was used.
Ten artificial blood vessels were implanted in carotid arteries of ten dogs. The patency ratio was 3/10 (30%) and the ratio of the surface area covered with endothelial cells was 70 % after four weeks.
Comparative Example 11 The same EPTFE tube as used in Example 25 was dipped in an aqueous solution of fibronectin (30 mg) and transferrin (30 mg) for coating fibronectin and transferrin in amounts of 89 ~g/cm and 102 ~.g/cm, respectively, followed by dipping in a 1 % aqueous solution of glutaraldehyde to crosslink them.
Ten artificial blood vessels were implanted in carotid arteries of ten dogs. The patency ratio was 2/10 (20%) and the ratio of the surface area covered with endothelial cells was 40 % after four weeks.
Example 33 An EPTFE tube having an inner diameter of 4 mm, an outer diameter of 5 mm and a fibril length of 30 ~m was dipped in a mixture of a 1.4 M solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of methacrylic acid (5 g) in water (20 ml) was added and reacted at 80°C for 10 hours. After this, excessive meth acrylic acid and ungrafted polymer of methacrylic acid were washed off, and a meth acrylic acid-grafted EPTFE tube, was obtained. The grafted amount of methacrylic acid was about 220 pg per 1 cm of the tube when calculated from the weight change of the tubes.
- _ _ 4g _ The tube was dipped in a solution of N-hydroxy-succinimide 2.1 ( g) and dicyclohexylcarbodiimide (1.9 g) in 1,4-dioxane (100 ml) at 0°C for 12 hours, and methacrylic acid was -changed to the N-hydroxysuccinimide ester. The esterified tube was dipped in a solution of fibronectin (30 mg) in a phosphate buffer (pH
of 11) (100 ml), and fibronectin was immobilized through covalent bond to the ester groups. The immobilized amount of fibronectin was about 205 p.g per 1 cm of the tube when calculated from the weight change.
1 0 Next, the tube was compressed in the expanded direction, that is, the longitudinal direction of the tube, and fibrils between the nodes were warped so that only the nodes were exposed to the bore of the tube. Then, a 5 % solution of polyallylamine (molecu,lar weight of about 100,000) in phosphate buffer (pH = 11 ) flowed through the bore of the tube for 12 hours while maintaining the compressed state of the tube, and polyallylamine was immobilized onto the nodes appearing on the inner wall surface of the tube through covalent bonds. During this treatment, the polyallylamine solution did not exude onto the outside surface of 2 0 the tube.
After this, a 5 % aqueous solution of heparin (available from SANOFI) flowed through the bore of the tube for 1 hour while maintaining the compressed state of the tube. Then, the tube was freed from compression and washed with water, followed by freeze drying. The immobilized amount of heparin was 17 UNIT/cm.
Only the nodes of the EPTFE were dyed when the tube was dipped in a methylene blue solution: This indicated that heparin was immobilized to these nodes.
Example 34 An EPTFE tube having an inner diameter of 4 mm, an outer diameter of 5 mm and a fibril length of 60 p.m was treated w with methyllithium and then methacrylic acid was graft polymerized on the EPTFE tube in the same manner as in Example 33. The grafted amount of methacrylic acid was about 195 pg per cm of the tube.
The methacrylic acid-grafted EPTFE tube was treated with N-hydroxysuccinimide and then fibronectin in the same manner as in Example 33. The immobilized amount of fibronectiri was 198 p.g/cm.
Next, the fibronectin-immobilized EPTFE tube was treated with polyallylamine and then heparin in the same manner as in Example 33. The immobilized amount of heparin was 14 UNIT/cm, and heparin was immobilized only on the nodes which appeared on the inner surface of the tube.
Example 35 An EPTFE tube, which had an inner diameter of 4 mm, an outer diameter of 5 mm and a fibril length of 60 ~m in a thickness of 250 p.m from the inner surface of the tube and a fibril length of wm in another part of the tube, was treated with methyllithium and then methacrylic acid was graft polymerized on the EPTFE tube in the same manner as in Example 33. The grafted amount of methacrylic acid was about 231 pg per 1 cm of the tube.
25 The methacrylic acid-grafted EPTFE tube was treated with N-hydroxysuccinimide and then fibronectin in the same manner as in Example 33. The immobilized amount of fibronectin was 211 pg/cm.
_ _ 51 _ Next, the fibronectin-immobilized EPTFE tube was treated with polyallylamine and then heparin in the same manner as in Example 33. The immobilized amount of hanarin vemo , ~
UNIT/cm, and heparin was immobilized only on the nodes which appeared on the inner surface of the tube.
Example 36 The methacrylic acid-grafted EPTFE tube produced in the same manner as in Example 35 was treated with N-hydroxy-succinimide in the same manner as in Example 33, and then dipped in a solution of fibronectin (15 mg) and transferrin (15 mg) in phosphate buffer (pH = 11 ) (100 ml). The immobilized amounts of fibronectin and transferrin were 121 pg/cm and 118 p.g/cm, respectively.
Next, the fibronectin/transferrin-immobilized EPTFE
tube was treated with polyallylamine and then heparin in the same manners as in Example 33. The immobilized amount of heparin was 16 UNIT/cm, and heparin was immobilized only on the nodes which appeared on the inner surface of the tube.
Example 37 The methacrylic acid-grafted EPTFE tube produced in the same manner as in Example 35 was dipped in a solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (30 mg), fibronectin (15 mg) and transferrin (15 mg) in distilled water (100 ml), pH of which was adjusted to 5 with 1 N hydrochloric acid, for 24 hours. The immobilized amounts of fibronectin and transferrin were 131 ug/cm and 120 p.g/cm, respectively.
Next, the fibronectin/transferrin-immobilized EPTFE
tube was treated with polyallylamine and then heparin in the same I
= - 52 -manner as in Example 33. The immobilized amount of heparin was 16 UNIT/cm, and heparin was immobilized only on the nodes which appeared on the inner surface of the tube.
Comparative Example 12 An EPTFE tube having an inner diameter of 4 mm, an outer diameter of 5 mm and a fibril length of 30 p.m was used as an artificial blood vessel.
Example 38 The methacrylic acid-grafted EPTFE tube prepared in the 1 0 same manner as in Example 33 was treated with N-hydroxysuccinimide and then fibronectin in the same manner as in Example 33. The immobilized amount of fibronectin was 192 p.g/cm.
Next, the fibronectin-immobilized EPTFE tube in the uncompressed state was dipped in a 5 % solution of polyallylamine in phosphate buffer (pH = 11) for 12 hours, and Ipolyallylamine was immobilized on the inner and outer surfaces and also the pore surfaces of the tube. Then, the tube was dipped in a 10 % aqueous solution of heparin (available from SANOFI) for 1 hour and washed with water, followed by freeze drying. The immobilized amount of heparin was 85 UNIT/cm, and heparin was immobilized on all the surfaces of the tube, that is, the inner and outer surfaces and pore surfaces of the tube.
Example 39 The methacrylic acid-grafted EPTFE tube prepared in the same manner as in Example 33 was treated with N-hydroxy-succinimide in the same manner as in Example 33, and then dipped in a solution of fibronectin (15 mg) and transferrin (15 mg) in S3 ' 2 ~ ~ 7 ~ 7 _ phosphate buffer (pH = 11 ) (100 ml). The immobilized amounts of fibronectin and transferrin were 118 pg/cm and 109 pg/cm, respectively.
Next, the fibronectin/transferrin-immobilized EPTFE
tube in the uncompressed state was dipped in a 5 % solution of polyallylamine in phosphate buffer (pH = 11 ) for 12 hours, and polyallylamine was immobilized on the inner and outer surfaces and also the pore surfaces of the tube. Then, the tube was dipped in a % aqueous solution of heparin (available from SANOFI) for 1 hour 10 and washed with water followed by freeze drying. The immobilized amount of heparin was 83 UNIT/cm, and heparin was immobilized on all the surfaces of the tube, that is, the inner and outer surfaces and pore surfaces of the tube.
Exam Ip a 40 1 5 An EPTFE tube having an inner diameter of 4 mm, an outer diameter of 5 mm and a fibril length of 30 pm was treated with methyllithium and then methacrylic acid was graft polymerized on the EPTFE tube in the same manners as in Example 33 except that the amount of methacrylic acid was changed from 5 g 2 0 to 3 g. The grafted amount of methacrylic acid was about 220 p.g per 1 cm of the tube when calculated from the weight change.
The methacrylic acid-grafted EPTFE tube was treated with N-hydroxysuccinimide and then fibronectin in the same manner as in Example 33. The immobilized amount of fibronectin 2 5 was 192 ug/cm.
Comparative Example 13 _ An EPTFE tube, which had an~ inner diameter of 4 mm, an outer diameter of 5 mm and a fibril length of 60 pm in a.thickness of 250 ~m from the inner wall surface of the tube and a fibril length of 30 ~.m in other part of the tube, was used as an artificial blood vessel.
Ten artificial blood vessels {each having an inner diameter of 4 mm and a length of 4 cm) produced in each of Examples 33 to 40 and Comparative Examples 12 and 13 were implanted in carotid arteries of ten dogs. The patency ratio after one week and one month was checked, and the ratio of the surface area coverea witn endothelial cells was evaluated. The results are 1 0 shown in Table 4.
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Next, the methacrylic acid-grafted EPTFE tube was dipped in a solution of ~N-hydroxysuccinimide (2.1 g) and dicyclohexylcarbodiimide (1.9 g) in 1,4-dioxane (100 ml) at 0°C for 12 hours, and acrylic acid was changed to the N-hydroxysuccinimide ester.
2 0 The esterified tube was dipped in a solution of ?9 - - fibronectin (30 mg) and transferrin (30 mg) in a phosphate buffer (pH of 11 ) (100 ml),, and fibronectin and transferrin were immobilized through covalent bond to the ester groups. The ungrafted polymer of meth of fibronectin and transferrin were about 122 ~g and 95 ug per 1 cm of the tube, respectively.
Ten artificial blood vessels (each 3 cm in length) were implanted in carotid arteries of ten dogs. The patency ratio was 8/10 (80%) and the ratio of the surface area covered with endothelial cells was 95 % after four weeks.
Example 26 Fibronectin and insulin were immobilized onto the EPTFE
tube in amounts of 132 p,g/cm and 102 p.g/cm, respectively in the same manner as in Example 25 except that insulin was used in place of transferrin.
1 5 Ten artificial blood vessels were implanted in carotid arteries of ten dogs. The patency ratio was 9/10 (90%) and the ratio of the surface area covered with endothelial cells was 92 % after four weeks.
Example 27 Fibronectin and TGF-a were immobilized onto the EPTFE
tube in amounts of 118 p.g/cm and 89 pg/cm, respectively in the same manner as in Example 25 except that TGF-a was used in place of transferrin.
Ten artificial blood vessels were implanted in carotid 2 5 arteries of ten dogs. The patency ratio was 8/10 (80%) and the ratio of the surface area covered with endothelial cells was 95 % after four weeks.
i '2~ 9~'~7~
Example 28 - ' Fibronectin and FGF were immobilized onto the EPTFE
tube in the amounts of 120 Erg/cm and 90 ~ug/cm, respectively in the same manner as in Example 25 except that FGF was used in place of transferrin.
Ten artificial blood vessels were implanted in carotid arteries of ten dogs. The patency ratio was 9/10 (90%) and the ratio of the surface area covered with endothelial cells was 89 % after four weeks.
Example 29 a 2-Hydroxyethyl methacrylate was graft polymerized on EPTFE tube in an amount of 246 ~rg/cm in the same manner as in Example 25 except that 2-hydroxyethyl methacrylate was used in place of methacrylic acid. Then, the EPTFE tube was dipped in solution of 2,2,2-trifluoromethanesulfonyl chloride in dichloromethane for forming 2,2,2-trifluoromethanesulfonate ester.
The esterified tube was dipped in a solution of fibronectin (30 mg) and transferrin (30 mg) in a phosphate buffer (pH of 11) (100 ml), and fibronectin and transferrin were immobilized through covalent bond in amounts of 128 dug and 89 ,vg per 1 cm of the tube, respectively.
Ten artificial blood vessels were implanted in carotid arteries of ten dogs. The patency ratio was 8/10 (80%) and the ratio of the surFace area covered with endothelial cells was 95 % after four weeks.
Example 30 Methacrylic acid was graft polymerized on the EPTFE tube in an amount of 185 ,vg/cm in the same manner as in Example 25.
Then, the tube was dipped in a solution of fibronectin (30 mg) ~,2'1973~5 _ - 47 -and transferrin (30 mg) in a phosphate buffer (100 ml) at pH of 1.5 for 12 hours in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (30 mg), and fibronectin and transferrin were immobilized through covalent bond in amounts of 120 pg and 108 pg per 1 cm of the tube, respectively.
Ten artificial blood vessels were implanted in carotid arteries of ten dogs. The patency ratio was 6/10 (60%) and the ratio of the surface area covered with endothelial cells was 50 % after four weeks.
1 0 Examl l~ a 31 Methacrylic acid was graft polymerized on the EPTFE
tube in an amount of 178 p.g/cm in the same manner as in Example 25. Next, the methacrylic acid-grafted EPTFE tube was dipped in a solution of N-hydroxysuccinimide (2.1 g) and dicyclohexyl-carbodiimide (1.9 g) in 1,4-dioxane (100 ml) at 0°C for 12 hours, and meth acrylic acid was changed to the N-hydroxysuccinimide ester.
The esterified tube was dipped in a solution of fibronectin (30 mg) in a phosphate buffer (pH of 11) (100 ml), and fibronectin was immobilized through covalent bond to the ester groups. The immobilized amount of fibronectin was about 122 pg per 1 cm of the tube.
Ten artificial blood vessels were implanted in carotid arteries of ten dogs. The patency ratio was 3/10 (30%) and the ratio of the surface area covered with endothelia( cells was 70 % after four weeks.
Example 32 Transferrin was immobilized to the esterified tube 21 ~ 737g through the covalent bond in an amount of 108 gg/cm in the same manner as in Example 30 except that transferrin alone was used.
Ten artificial blood vessels were implanted in carotid arteries of ten dogs. The patency ratio was 3/10 (30%) and the ratio of the surface area covered with endothelial cells was 70 % after four weeks.
Comparative Example 11 The same EPTFE tube as used in Example 25 was dipped in an aqueous solution of fibronectin (30 mg) and transferrin (30 mg) for coating fibronectin and transferrin in amounts of 89 ~g/cm and 102 ~.g/cm, respectively, followed by dipping in a 1 % aqueous solution of glutaraldehyde to crosslink them.
Ten artificial blood vessels were implanted in carotid arteries of ten dogs. The patency ratio was 2/10 (20%) and the ratio of the surface area covered with endothelial cells was 40 % after four weeks.
Example 33 An EPTFE tube having an inner diameter of 4 mm, an outer diameter of 5 mm and a fibril length of 30 ~m was dipped in a mixture of a 1.4 M solution of methyllithium in ethyl ether (20 ml) and hexamethylphosphoric amide (2 ml) at -10°C in an argon atmosphere for 30 minutes. After discarding the mixture, a solution of methacrylic acid (5 g) in water (20 ml) was added and reacted at 80°C for 10 hours. After this, excessive meth acrylic acid and ungrafted polymer of methacrylic acid were washed off, and a meth acrylic acid-grafted EPTFE tube, was obtained. The grafted amount of methacrylic acid was about 220 pg per 1 cm of the tube when calculated from the weight change of the tubes.
- _ _ 4g _ The tube was dipped in a solution of N-hydroxy-succinimide 2.1 ( g) and dicyclohexylcarbodiimide (1.9 g) in 1,4-dioxane (100 ml) at 0°C for 12 hours, and methacrylic acid was -changed to the N-hydroxysuccinimide ester. The esterified tube was dipped in a solution of fibronectin (30 mg) in a phosphate buffer (pH
of 11) (100 ml), and fibronectin was immobilized through covalent bond to the ester groups. The immobilized amount of fibronectin was about 205 p.g per 1 cm of the tube when calculated from the weight change.
1 0 Next, the tube was compressed in the expanded direction, that is, the longitudinal direction of the tube, and fibrils between the nodes were warped so that only the nodes were exposed to the bore of the tube. Then, a 5 % solution of polyallylamine (molecu,lar weight of about 100,000) in phosphate buffer (pH = 11 ) flowed through the bore of the tube for 12 hours while maintaining the compressed state of the tube, and polyallylamine was immobilized onto the nodes appearing on the inner wall surface of the tube through covalent bonds. During this treatment, the polyallylamine solution did not exude onto the outside surface of 2 0 the tube.
After this, a 5 % aqueous solution of heparin (available from SANOFI) flowed through the bore of the tube for 1 hour while maintaining the compressed state of the tube. Then, the tube was freed from compression and washed with water, followed by freeze drying. The immobilized amount of heparin was 17 UNIT/cm.
Only the nodes of the EPTFE were dyed when the tube was dipped in a methylene blue solution: This indicated that heparin was immobilized to these nodes.
Example 34 An EPTFE tube having an inner diameter of 4 mm, an outer diameter of 5 mm and a fibril length of 60 p.m was treated w with methyllithium and then methacrylic acid was graft polymerized on the EPTFE tube in the same manner as in Example 33. The grafted amount of methacrylic acid was about 195 pg per cm of the tube.
The methacrylic acid-grafted EPTFE tube was treated with N-hydroxysuccinimide and then fibronectin in the same manner as in Example 33. The immobilized amount of fibronectiri was 198 p.g/cm.
Next, the fibronectin-immobilized EPTFE tube was treated with polyallylamine and then heparin in the same manner as in Example 33. The immobilized amount of heparin was 14 UNIT/cm, and heparin was immobilized only on the nodes which appeared on the inner surface of the tube.
Example 35 An EPTFE tube, which had an inner diameter of 4 mm, an outer diameter of 5 mm and a fibril length of 60 ~m in a thickness of 250 p.m from the inner surface of the tube and a fibril length of wm in another part of the tube, was treated with methyllithium and then methacrylic acid was graft polymerized on the EPTFE tube in the same manner as in Example 33. The grafted amount of methacrylic acid was about 231 pg per 1 cm of the tube.
25 The methacrylic acid-grafted EPTFE tube was treated with N-hydroxysuccinimide and then fibronectin in the same manner as in Example 33. The immobilized amount of fibronectin was 211 pg/cm.
_ _ 51 _ Next, the fibronectin-immobilized EPTFE tube was treated with polyallylamine and then heparin in the same manner as in Example 33. The immobilized amount of hanarin vemo , ~
UNIT/cm, and heparin was immobilized only on the nodes which appeared on the inner surface of the tube.
Example 36 The methacrylic acid-grafted EPTFE tube produced in the same manner as in Example 35 was treated with N-hydroxy-succinimide in the same manner as in Example 33, and then dipped in a solution of fibronectin (15 mg) and transferrin (15 mg) in phosphate buffer (pH = 11 ) (100 ml). The immobilized amounts of fibronectin and transferrin were 121 pg/cm and 118 p.g/cm, respectively.
Next, the fibronectin/transferrin-immobilized EPTFE
tube was treated with polyallylamine and then heparin in the same manners as in Example 33. The immobilized amount of heparin was 16 UNIT/cm, and heparin was immobilized only on the nodes which appeared on the inner surface of the tube.
Example 37 The methacrylic acid-grafted EPTFE tube produced in the same manner as in Example 35 was dipped in a solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (30 mg), fibronectin (15 mg) and transferrin (15 mg) in distilled water (100 ml), pH of which was adjusted to 5 with 1 N hydrochloric acid, for 24 hours. The immobilized amounts of fibronectin and transferrin were 131 ug/cm and 120 p.g/cm, respectively.
Next, the fibronectin/transferrin-immobilized EPTFE
tube was treated with polyallylamine and then heparin in the same I
= - 52 -manner as in Example 33. The immobilized amount of heparin was 16 UNIT/cm, and heparin was immobilized only on the nodes which appeared on the inner surface of the tube.
Comparative Example 12 An EPTFE tube having an inner diameter of 4 mm, an outer diameter of 5 mm and a fibril length of 30 p.m was used as an artificial blood vessel.
Example 38 The methacrylic acid-grafted EPTFE tube prepared in the 1 0 same manner as in Example 33 was treated with N-hydroxysuccinimide and then fibronectin in the same manner as in Example 33. The immobilized amount of fibronectin was 192 p.g/cm.
Next, the fibronectin-immobilized EPTFE tube in the uncompressed state was dipped in a 5 % solution of polyallylamine in phosphate buffer (pH = 11) for 12 hours, and Ipolyallylamine was immobilized on the inner and outer surfaces and also the pore surfaces of the tube. Then, the tube was dipped in a 10 % aqueous solution of heparin (available from SANOFI) for 1 hour and washed with water, followed by freeze drying. The immobilized amount of heparin was 85 UNIT/cm, and heparin was immobilized on all the surfaces of the tube, that is, the inner and outer surfaces and pore surfaces of the tube.
Example 39 The methacrylic acid-grafted EPTFE tube prepared in the same manner as in Example 33 was treated with N-hydroxy-succinimide in the same manner as in Example 33, and then dipped in a solution of fibronectin (15 mg) and transferrin (15 mg) in S3 ' 2 ~ ~ 7 ~ 7 _ phosphate buffer (pH = 11 ) (100 ml). The immobilized amounts of fibronectin and transferrin were 118 pg/cm and 109 pg/cm, respectively.
Next, the fibronectin/transferrin-immobilized EPTFE
tube in the uncompressed state was dipped in a 5 % solution of polyallylamine in phosphate buffer (pH = 11 ) for 12 hours, and polyallylamine was immobilized on the inner and outer surfaces and also the pore surfaces of the tube. Then, the tube was dipped in a % aqueous solution of heparin (available from SANOFI) for 1 hour 10 and washed with water followed by freeze drying. The immobilized amount of heparin was 83 UNIT/cm, and heparin was immobilized on all the surfaces of the tube, that is, the inner and outer surfaces and pore surfaces of the tube.
Exam Ip a 40 1 5 An EPTFE tube having an inner diameter of 4 mm, an outer diameter of 5 mm and a fibril length of 30 pm was treated with methyllithium and then methacrylic acid was graft polymerized on the EPTFE tube in the same manners as in Example 33 except that the amount of methacrylic acid was changed from 5 g 2 0 to 3 g. The grafted amount of methacrylic acid was about 220 p.g per 1 cm of the tube when calculated from the weight change.
The methacrylic acid-grafted EPTFE tube was treated with N-hydroxysuccinimide and then fibronectin in the same manner as in Example 33. The immobilized amount of fibronectin 2 5 was 192 ug/cm.
Comparative Example 13 _ An EPTFE tube, which had an~ inner diameter of 4 mm, an outer diameter of 5 mm and a fibril length of 60 pm in a.thickness of 250 ~m from the inner wall surface of the tube and a fibril length of 30 ~.m in other part of the tube, was used as an artificial blood vessel.
Ten artificial blood vessels {each having an inner diameter of 4 mm and a length of 4 cm) produced in each of Examples 33 to 40 and Comparative Examples 12 and 13 were implanted in carotid arteries of ten dogs. The patency ratio after one week and one month was checked, and the ratio of the surface area coverea witn endothelial cells was evaluated. The results are 1 0 shown in Table 4.
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Claims (10)
1. An artificial blood vessel comprising:
a tube formed from expanded polytetrafluoroethylene comprising fibrils and nodes connecting fibrils, wherein said tube has an inner surface and an outer surface, in which the inner surface of said tube and the surfaces of fibrils and nodes comprise a hydrophilic layer extending to a depth of between 5% and 96% of the wall thickness of said tube in a radial direction from the inner surface toward the outer surface of said tube, and a tissue-inducing substance which is immobilized on said inner surface and said surfaces of fibrils and nodes of the hydrophilic layer.
a tube formed from expanded polytetrafluoroethylene comprising fibrils and nodes connecting fibrils, wherein said tube has an inner surface and an outer surface, in which the inner surface of said tube and the surfaces of fibrils and nodes comprise a hydrophilic layer extending to a depth of between 5% and 96% of the wall thickness of said tube in a radial direction from the inner surface toward the outer surface of said tube, and a tissue-inducing substance which is immobilized on said inner surface and said surfaces of fibrils and nodes of the hydrophilic layer.
2. An artificial blood vessel according to claim 1, which further comprises an anti-thrombotic substance immobilized on at least a part of a surface of said tube which will be in contact with blood in use.
3. An artificial blood vessel according to claim 1, wherein said tissue-inducing substance is at least one material selected from the group consisting of a cell-adhesion material and a cell-growth material.
4. An artificial blood vessel according to claim 3, wherein said cell-adhesion material is fibronectin.
5. An artificial blood vessel according to claim 3, wherein said cell-growth material is at least one material selected from the group consisting of TGF-.alpha., transferrin, insulin and fibroblast growth factor.
6. An artificial blood vessel according to claim 2, wherein said anti-thrombotic substance is ionically immobilized to a polyamine or its salt which is covalently bonded through a polymer which has been graft polymerized on said surface of said tube which will be in contact with blood in use.
7. An artificial blood vessel according to claim 6, wherein said anti-thrombotic substance is heparin.
8. An artificial blood vessel according to claim 7, wherein said polyamine is at least one polyamine selected from the group consisting of polyallylamine, polyvinylamine and polyethylene imine.
9. An artificial blood vessel according to claim 7, wherein said polyamine has a molecular weight of between 1000 and 100,000, and 0 to 90 mole % of the amino groups of said polyamine are acetylated.
10. An artificial blood vessel according to claim 1, wherein said tissue-inducing substance is immobilized through covalent bond on said inner surface and said surfaces of pores by reacting said tissue-inducing substance with an ester of a carboxylic acid or a sulfonate ester of a hydroxyl group which has been formed on said inner surface and said surfaces of pores by graft polymerization.
Applications Claiming Priority (16)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP8027829A JPH09215746A (en) | 1996-02-15 | 1996-02-15 | Artificial blood vessel |
JP027824/1996 | 1996-02-15 | ||
JP2782596 | 1996-02-15 | ||
JP2782396 | 1996-02-15 | ||
JP027825/1996 | 1996-02-15 | ||
JP027829/1996 | 1996-02-15 | ||
JP2782496 | 1996-02-15 | ||
JP027823/1996 | 1996-02-15 | ||
JP076293/1996 | 1996-03-29 | ||
JP7629396A JP2986728B2 (en) | 1996-03-29 | 1996-03-29 | Artificial blood vessel |
JP153883/1996 | 1996-06-14 | ||
JP153880/1996 | 1996-06-14 | ||
JP153868/1996 | 1996-06-14 | ||
JP15386896A JP3014324B2 (en) | 1996-02-15 | 1996-06-14 | Artificial blood vessel |
JP15388096A JP3014325B2 (en) | 1996-02-15 | 1996-06-14 | Artificial blood vessel and method for producing the same |
JP15388396A JP2854284B2 (en) | 1996-02-15 | 1996-06-14 | Antithrombotic vascular prosthesis |
Publications (2)
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CA2197375A1 CA2197375A1 (en) | 1997-08-16 |
CA2197375C true CA2197375C (en) | 2003-05-06 |
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Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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CA002197375A Expired - Fee Related CA2197375C (en) | 1996-02-15 | 1997-02-12 | Artificial blood vessel |
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US (1) | US6053939A (en) |
EP (1) | EP0790042B1 (en) |
AU (1) | AU709305B2 (en) |
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DE (1) | DE69725198T2 (en) |
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-
1997
- 1997-02-12 CA CA002197375A patent/CA2197375C/en not_active Expired - Fee Related
- 1997-02-14 US US08/800,685 patent/US6053939A/en not_active Expired - Fee Related
- 1997-02-14 AU AU12682/97A patent/AU709305B2/en not_active Ceased
- 1997-02-14 EP EP97102455A patent/EP0790042B1/en not_active Revoked
- 1997-02-14 DE DE69725198T patent/DE69725198T2/en not_active Revoked
Also Published As
Publication number | Publication date |
---|---|
CA2197375A1 (en) | 1997-08-16 |
EP0790042A3 (en) | 1997-12-29 |
AU1268297A (en) | 1997-08-21 |
EP0790042B1 (en) | 2003-10-01 |
EP0790042A2 (en) | 1997-08-20 |
US6053939A (en) | 2000-04-25 |
DE69725198T2 (en) | 2004-06-03 |
DE69725198D1 (en) | 2003-11-06 |
AU709305B2 (en) | 1999-08-26 |
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