JP2011130989A - Antithrombogenic modifier, medical instrument and porous collagen - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antithrombogenic modifier, which immobilizes an antithrombogenic agent on a surface without using an organic solvent, and also to provide a medical instrument having a blood contacting section whose surface is coated with the antithrombogenic modifier. <P>SOLUTION: The antithrombogenic modifier immobilizes an antithrombogenic agent on a blood contacting section of a medical instrument containing collagen as a main component and confers antithrombogenic property to the blood contacting section. The antithrombogenic agent is anionic, and the antithrombogenic agent is argatroban, nafamostat mesylate, warfarin, aspirin or the like. The medical instrument contains collagen as a main component, and the anionic antithrombogenic agent is immobilized on the surface of the blood contacting section of the medical instrument. The anionic antithrombogenic agent is immobilized on the surface of the blood contacting section at a concentration of not less than 0.01 and not more than 20 mg/cm<SP>2</SP>. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention can maintain excellent anticoagulability (antithrombogenicity) over a long period of time, and can be applied to living tissue or as a constituent material of various medical devices such as artificial organs and artificial blood vessels. The present invention relates to an antithrombotic modifier, and a medical device and porous collagen into which the antithrombotic modifier is permeated.

  When blood comes into contact with foreign matter, it has a property of coagulating due to the action of various components in the blood. Therefore, constituent materials of medical devices used for parts that come into contact with blood, such as artificial hearts, artificial heart valves, artificial blood vessels, vascular catheters, cannulas, cardiopulmonary bypasses, vascular bypass tubes, aortic balloon pumping, blood transfusion devices and extracorporeal circulation circuits Is required to have high anticoagulability.

  However, in many of the constituent materials of conventional medical devices, blood coagulation is unavoidable when used for a long period of time, which is not sufficient in terms of anti-blood coagulation sustainability.

  Therefore, when the above-mentioned medical device is applied to a patient, an anticoagulant such as heparin is usually used in combination. However, for example, when heparin is administered systemically, there is a problem that the risk of numerous bleeding lesions increases, and when heparin is administered systemically over a long period of time, abnormal lipid metabolism, platelet There are problems such as reduction and allergic reaction.

  As techniques for solving such problems, various techniques for fixing heparin on the surface of a medical material or releasing it gradually have been proposed. For example, in Patent Document 1, heparin is brought into contact with the surface of a polymer material having a cationic residue, and heparin is supported on the polymer material in an ionic bond, with polyvinyl chloride and acrylic acid or methacrylic acid. And anticoagulant medical materials for coating, in which heparin is ion-bonded to a copolymer thereof. For example, Patent Document 2 describes that polyamine or a salt thereof is immobilized on the surface of an artificial blood vessel, and heparin is immobilized on the surface of the polyamine or salt thereof by ionic bonding. For example, Patent Document 3 describes an antithrombotic material in which heparin is ionically bound to phospholipids in a phospholipid-polymer composite thin film formed on the surface of a substrate.

  In addition to heparin, as a drug for imparting antithrombogenicity to medical devices, the use of a drug called argatroban has attracted attention because of its physical properties. Argatroban is (2R, 4R) -4-methyl-1- [N2-((RS) -3-methyl-1,2,3,4-tetrahydro-8-quinolinesulfonyl) -L-arginyl] -2 -A compound corresponding to piperidinecarboxylic acid monohydrate, which is classified as an antithrombin agent among anticoagulants, and has an action of suppressing the initiation of blood coagulation reaction and its progress.

  Argatroban, unlike antithrombotic substances such as heparin and urokinase, has the property of being dissolved in an aprotic polar organic solvent, so there are several methods for imparting antithrombogenicity to medical devices using organic solvents. Or has been proposed. For example, Patent Document 4 describes a method in which a medical device such as a catheter tube is immersed in an organic solvent solution of argatroban dissolved in DMSO, DMF or the like to form an antithrombogenic film made of argatroban on the surface of the catheter tube. Yes. For example, Patent Document 5 describes a method in which argatroban is blended with a thermoplastic polymer material and melt-molded to form a catheter tube molded body containing argatroban in the entire tube. For example, Patent Document 6 describes a method of forming an antithrombogenic film on the surface of a medical device by applying and drying an organic solvent solution dissolved in DMF or DMSO together with a polymer material.

  However, in these technologies described above, since organic solvent is used when immobilizing heparin or argatroban on a medical device, the surface of a living tissue extracted from a host or a hybrid tissue composed of cells and a synthetic material is used. However, there is a problem in that they are damaged by organic solvents. Moreover, the material itself may be weak to an organic solvent like nitrile cellulose. In addition, when a medical device coated with a drug, such as a drug-releasing stent, is used, the polymer layer carrying the drug with an organic solvent may be peeled off. Furthermore, in the method of applying and immersing using an organic solvent, when using an organic solvent with a high boiling point, it takes time to dry, and the problem of environmental pollution due to solvent vapor and the possibility of fire due to ignition increases. There is a point.

  On the other hand, to repair and treat cartilage, skin, ligaments, blood vessels, pancreas, liver and other living tissues / organs damaged or lost due to accidents or illness, treatment with artificial organs or organ transplants Is done. However, in the case of an artificial organ, there are problems such as wear, looseness, and damage due to the artificial material. In the case of tissue transplantation, in addition to the problem of lack of donors, rejection reactions based on immune responses when the donor is another person. There is also a problem.

  Therefore, in recent years, regenerative medicine treatment methods are considered to be ideal methods because they do not require a donor as compared with organ transplantation. In the regenerative medicine treatment method, living cells are proliferated ex vivo, seeded on a base material used as a scaffold for living cells and tissues, cultured ex vivo, formed into living tissues, and then transplanted in vivo. Alternatively, living cells are seeded on a base material, embedded in the living body, and regeneration of living tissue is induced in the living body. Therefore, scaffold materials that induce and promote the formation of biological tissue and maintain the morphology of the biological tissue play a very important role. This scaffold material is not only biocompatible as a property that does not affect the living body, but also porous so that cells can enter easily and oxygen and nutrients can be supplied to the entering cells. Required.

  As the scaffold material, collagen has a particularly excellent affinity for cells, and therefore, a collagen sponge obtained by processing this into a sponge is widely used.

  For example, when a collagen sponge added with bFGF (basic fibroblast growth factor) is implanted subcutaneously, angiogenesis is increased and cell functions are satisfactorily expressed. Here, since blood has a property of coagulating by the action of various components in blood when it comes into contact with a foreign substance, the collagen sponge may be imparted with antithrombogenicity.

  Antithrombogenicity against a collagen sponge is imparted, for example, by performing a succinylation treatment on a collagen sponge as described in Patent Document 7. In the succinylation treatment, collagen is reacted with succinic anhydride or other anhydride, and as a result, a free amino group of the collagen molecule is acylated.

  However, since the succinylation reaction uses a large amount of succinic anhydride and the reaction time is not short, imparting antithrombogenicity by succinylation treatment is not a simple method. In addition, the succinylation reaction is to change the side chain amino group of collagen to a carboxyl group by succinylation, and as a result, the water absorption increases, so that sufficient mechanical strength is maintained for the period required for the regeneration of living tissue. There is a problem that it may not be possible to hold.

Japanese Patent No. 3341503 (2nd and 3rd pages) Japanese Patent No. 2854284 (2nd page) Japanese Patent No. 3372971 (page 3) JP-A-6-292718 (second page) JP-A-6-292717 (second page) JP 2000-60960 A (page 2) JP 08-336583 A (page 6)

  The present invention has been made in view of such problems, and an antithrombotic modifier capable of immobilizing an antithrombotic drug on the surface without using an organic solvent, and the antithrombotic modifier used as a blood contact portion. An object of the present invention is to provide a medical device fixed to the surface of the medical device. It is another object of the present invention to provide a porous collagen that is imparted with antithrombogenicity by a simple technique and that can maintain sufficient mechanical strength.

  The antithrombotic modifier according to the first aspect of the present invention is an antithrombotic modifier that immobilizes an antithrombotic agent on the blood contact portion of a medical device mainly composed of collagen and imparts antithrombogenicity. The antithrombotic drug is anionic.

  The antithrombotic agent is, for example, argatroban, nafamostat mesylate, warfarin, aspirin, and mixtures thereof.

  The concentration of the anionic antithrombotic drug is preferably 0.5 mg / ml or more and 200 mg / ml or less.

  A medical device according to a second aspect of the present invention is a medical device mainly composed of collagen, characterized in that an anionic antithrombotic drug is immobilized on the surface of the blood contact portion.

  The antithrombotic agent is, for example, argatroban, nafamostat mesylate, warfarin, aspirin, and mixtures thereof.

The anionic antithrombotic drug is preferably immobilized at 0.01 mg or more and 20 mg or less per cm ^{2 on} the surface of the blood contact portion.

  The porous collagen according to the third aspect of the present invention is characterized in that an antithrombotic drug containing at least one of heparin and low molecular weight heparin is infiltrated into the porous tissue.

  Since the antithrombogenic modifier of the present invention can be immobilized on the blood contact portion of a medical device mainly composed of collagen without using an organic solvent, there is little possibility of damaging a living tissue or the like extracted from the host. In addition, since the anionic antithrombotic drug is immobilized on the surface of the blood contact portion without using an organic solvent, the medical device of the present invention is less likely to be damaged. In addition, the porous collagen of the present invention retains the sufficient mechanical strength inherent in collagen, and a simple antithrombotic drug containing at least one of heparin and low molecular weight heparin can be obtained without using an organic solvent. The porous tissue is infiltrated by the technique.

It is the photograph which shows the adsorptivity of the anionic pigment | dye with respect to the connective tissue which has collagen as a main component, (a) is immediately after washing | cleaning, (b) is 3 hours after washing | cleaning, (c) is 24 hours after washing | cleaning. (D) is one week after washing. It is a photograph which shows the adsorptivity of the cationic pigment | dye with respect to the connective tissue which has collagen as a main component, (a) is immediately after washing | cleaning, (b) is 3 hours after washing | cleaning, (c) is 24 hours after washing | cleaning. (D) is one week after washing. It is a photograph which shows the adsorptivity of heparin with respect to collagen sponge, (a) is 1 hour after washing | cleaning, (b) is 24 hours after washing | cleaning. It is the photograph which shows the adsorptivity of a heparin solution (5 mg / mL) with respect to a connective tissue, (a) is immediately after toluidine blue dyeing | staining, (b) is a photograph of the cross section of the connective tissue immediately after toluidine blue dyeing | staining. (C) is one hour after washing and (d) is 24 hours after washing. It is a photograph figure of artificial blood vessel implantation to a rat abdominal aorta, (a) is a photograph figure which implanted an artificial blood vessel with an inside diameter of 1.5 mm, and (b) is a MRA image 3 months after transplantation. It is a photograph figure of artificial blood vessel implantation to the rabbit common carotid artery, of which (a) is a photograph figure in which an artificial blood vessel having an inner diameter of 3.0 mm is implanted, and (b) is an angiogram three months after transplantation. is there. It is a photograph figure of artificial blood vessel implantation to the beagle common carotid artery, of which (a) is a photograph figure in which an artificial blood vessel having an inner diameter of 5.0 mm is implanted, and (b) is an angiogram three months after transplantation. is there. It is a photograph figure of the transplantation of the artificial blood vessel to a beagle iliac artery, Among them, (a) is a photograph figure which embedded the Y-shaped artificial blood vessel, (b) is an angiogram 3 months after transplant. . FIG. 2 is a histological photograph of a rat abdominal transplant aorta, in which (a) is a hematoxylin-eosin (HE) stained image, (b) is a factor 8 immunostained image, and (c) is MTC ( (D) is an EWG (Elastica-Wangieson) staining diagram, and (e) is an α-SMA immunostaining diagram. It is a microscope figure which shows a connective tissue cross section, (a) is a fluorescence microscope figure in 10 mg / ml, (b) is a phase contrast microscope figure of the same visual field, (c) is a fluorescence microscope figure in 5 mg / ml. (D) is a phase contrast micrograph of the same visual field, (e) is a fluorescence microscopic diagram at 2.5 mg / ml, (f) is a phase contrast microscopic diagram of the same visual field, (g) Is a fluorescence micrograph at 1.25 mg / ml, and (h) is a phase contrast micrograph of the same field of view. It is a figure explaining the columnar core base material for creating the artificial blood vessel with a valve. It is a photograph figure explaining the columnar core base material which passed the mandrel to the center part. It is a photograph figure explaining an artificial blood vessel with a valve. It is a photograph figure which shows the blood contact surface 3 months after beagle pulmonary valve replacement surgery.

(Anti-thrombogenic modifier and medical device)
Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. The present inventor has found that connective tissue containing collagen as a main component selectively adsorbs an anionic dye through intensive research, and has completed the present invention based on this new finding.

  FIG. 1 is a photograph showing the adsorptivity of an anionic dye to connective tissue containing collagen as a main component, in which (a) is immediately after washing, (b) is 3 hours after washing, and (c) is After 24 hours of washing, (d) is one week after washing.

  When a connective tissue is prepared and an anionic dye eosin 1% aqueous solution is applied to the right side of the connective tissue, the connective tissue is stained red. Immediately after washing with physiological saline, staining with eosin remains on the right side of the connective tissue, as shown in FIG.

  Next, even after washing with physiological saline for 3 hours, staining with eosin remains on the right side of the connective tissue, as shown in FIG. 1 (b).

  Even after washing with physiological saline for 24 hours, eosin staining remains on the right side of the connective tissue, as shown in FIG. 1 (c).

  Furthermore, even after washing with physiological saline for 1 week, as shown in FIG. 1D, staining with eosin remains on the right side of the connective tissue.

  FIG. 2 is a photograph showing the adsorptivity of a cationic dye to connective tissue containing collagen as a main component, in which (a) is immediately after washing, (b) is 3 hours after washing, and (c) is After 24 hours of washing, (d) is one week after washing.

  When a connective tissue is prepared and a 1% aqueous solution of toluidine blue, which is a cationic dye, is applied to the right side of the connective tissue, the connective tissue is stained blue. Immediately after washing with physiological saline, staining with toluidine blue remains on the right side of the connective tissue as shown in FIG.

  However, after washing with physiological saline for 3 hours, as shown in FIG. 2 (b), the right side of the connective tissue is washed away, and staining with toluidine blue does not remain.

  As shown in FIG. 2 (c), staining with toluidine blue does not remain even after washing with physiological saline for 24 hours, and as shown in FIG. 2 (d), one week with physiological saline. Even after washing, no staining with toluidine blue remains.

  From these facts, it is understood that the cationic dye is difficult to fix to the connective tissue, but the anionic dye is adsorbed and fixed to the connective tissue.

  Therefore, it is possible to make it water-soluble by anionizing the antithrombotic agent. When a biological tissue mainly composed of collagen is immersed in this aqueous solution, the anionic antithrombotic agent penetrates quickly and ions The surface of the connective tissue can be covered with an antithrombotic drug without adsorbing and using an organic solvent, and antithrombogenicity can be imparted.

  Antithrombotic drugs are not particularly limited as long as they are anionized, and include all drugs having antithrombotic activity such as platelet aggregation inhibitors, antithrombin agents, anticoagulants, thrombolytic agents, For example, argatroban, nafamostat mesylate, warfarin, aspirin, and mixtures thereof.

  When the antithrombotic agent is argatroban represented by Formula 1 or nafamostat mesylate represented by Formula 2, the anionizing agent can be anionized by extracting hydrogen from the amino group. For example, an anion reagent such as sodium hydride, lithium hydride, n-butyllithium, s-butyllithium, t-butyllithium, phenyllithium, lithium diisopropylamide and the like can be used. .

  The amount of the anionizing agent is appropriately determined in consideration of the active hydrogen number of the amino group, the solubility of the amide ion generated after anionization, and the like. It is possible to act 0.9 equivalent of an anionizing agent.

  The reaction temperature of the anionization is appropriately determined in consideration of the solubility and stability of the anionizing agent and the anionized antithrombotic drug, and is typically from −100 ° C. to 160 ° C., preferably from −70 ° C. Anionization can be performed at 120 ° C.

  When the antithrombotic agent is aspirin represented by formula 3 or warfarin represented by formula 4, the anionizing agent is not particularly limited, but examples thereof include acetonitrile, acetone and the like. Aprotic solvents can be used.

  Next, the production method of the antithrombotic modifier according to this embodiment will be described below, taking as an example the case where argatroban is used as the antithrombotic agent and sodium hydroxide is used as the anionizing agent.

  First, argatroban powder is dissolved in an aqueous solution of sodium hydroxide as an anionizing reagent to obtain an aqueous solution of argatroban in sodium hydroxide. The sodium hydroxide aqueous solution of argatroban is transferred into a dialysis tube and dialyzed under running water until the pH becomes near neutral, and excess sodium hydroxide is discharged out of the dialysis tube. Thereafter, powdered anionic argatroban is obtained by freeze drying or vacuum drying.

  When immobilized on a medical device, an anionic antithrombotic drug aqueous solution is prepared by dissolving a powdered anionic antithrombotic drug in water. Then, this anionic antithrombotic drug aqueous solution is applied to the blood contact portion of the medical device to immobilize the antithrombotic drug.

  The concentration of the anionic antithrombotic drug in the anionic antithrombotic drug aqueous solution is, for example, 0.5 to 200 mg / ml, preferably 1 to 100 mg / ml. This is because if the concentration of the anionic antithrombotic drug is lower than 0.5 mg / ml, the concentration of the antithrombotic drug is low and it is difficult to immobilize it on the blood contact portion of the medical device. This is because if the concentration of the sex drug is higher than 200 mg / ml, the excess antithrombotic drug may have an unexpected effect.

  Although it does not specifically limit as a medical device, For example, artificial blood vessel, artificial heart, artificial heart valve, artificial heart-lung circuit, vascular catheter, vascular stent, cannula, artificial heart-lung machine, vascular bypass tube, hemodialysis circuit, aorta These include balloon pumping, blood transfusion devices, extracorporeal circulation circuits, and the like, and an anionic antithrombotic drug is immobilized on the blood contact portion of these medical devices. Here, in the cardiopulmonary circuit, the artificial heart, and the hemodialysis circuit, examples of the blood contact part include inner surfaces of circuit tubes, various connectors, housings, pumps, heat exchangers, heat exchanger housings, blood reservoirs, and the like. . The medical device includes a tissue obtained by decellularizing a living tissue. The decellularization method is not particularly limited. For example, a method of adding a surfactant, a method of repeating freezing and thawing, an ultrasonic treatment method, a method of immersing in a hypotonic solution, or a combination thereof Methods etc. can be used.

  In addition, the artificial blood vessel as the medical device includes a tissue body that is formed so as to cover an artificial object when it is embedded in a living body. Since such a tissue body contains fiber, it is excellent in physical strength and excellent in tissue compatibility and blood compatibility. When an artificial blood vessel is produced with a tissue body formed so as to cover the periphery of an artificial object implanted in the living body, generally, an artificial object such as silicon resin, vinyl chloride resin, or low density polyethylene is placed in the living body. After embedding and holding for a certain period of time, the tissue body formed on the surface of the artifact is decellularized with alcohol or the like to obtain a porous structure. When this tissue is used as an artificial blood vessel, it becomes possible to improve the antithrombogenicity by engrafting endothelial cells on the inner wall, but after transplanting as an artificial blood vessel, the luminal surface is completely covered with endothelial cells. It takes time to be converted. However, by using the antithrombotic modifier according to the present embodiment, sufficient antithrombotic properties can be imparted. Further, in coronary artery bypass surgery or the like, in an operation for replacing a blood vessel occluded by embolism with its own blood vessel, some endothelial cells may be lost when a tissue body is collected from the living body. However, even if a part of the endothelial cells on the inner wall is missing and the collagen tissue is exposed, the antithrombogenicity can be sufficiently imparted by using the antithrombotic modifier according to this embodiment.

For example, about 0.01 to 20 mg of an anionic antithrombotic drug per cm ² is preferably attached to the surface of the blood contact portion of the medical device. If the adhesion amount of the anionic antithrombotic drug is less than 0.01 mg, the degree of antithrombogenicity may be insufficient, while the adhesion amount of the anionic antithrombotic drug is more than 20 mg. This is because surplus antithrombotic drugs may flow out into the living body and bleeding spots may be generated.

  The medical device is mainly composed of collagen. The existence of 19 types of collagen has been reported due to the difference in structure, and there are cases where several different molecular species exist in collagen classified into the same type. Among them, type I, II, III and IV collagens are mainly used as raw materials for biomaterials. Type I is present in most connective tissues and is the collagen type that is present in the largest amount in the living body. Type II is collagen that forms cartilage. Although type III is small, it is often present at the same site as type I. Type IV is collagen that forms the basement membrane. Types I, II and III exist in the body as collagen fibers and mainly play a role of maintaining the strength of tissues or organs. Collagen that is the main component of the medical device is not particularly limited as long as it has fibrosis ability, but from the viewpoint of industrial use, I, II, III, or two or more of them It is preferable that it is a mixture.

  The origin of collagen is not particularly limited, and not only collagen extracted from vertebrates (hereinafter referred to as “animal collagen”) but also plant-derived collagen-like protein (hereinafter referred to as “plant collagen”). Is also included. The origin is not limited as long as it can be extracted artificially.

  Examples of vertebrates that are sources of animal collagen include fish, mammals, and birds. In recent years, the BSE (bovine spongiform encephalopathy) problem has become apparent and collagen products using raw materials derived from livestock including cow skin Therefore, the risk of infecting humans with pathogens is potentially pointed out, and fish-derived collagen is preferred from the viewpoints of safety and the amount of resources.

  As for fishes, chinless upper class, cartilaginous fish, herring eyes, carp eyes, catfish eyes, eel eyes, caraea eyes, sardine eyes, salmon eyes, scallops eyes, stagnation eyes, sardines eyes, crocodiles eyes, killer whales, antlers , Matouidae, Amphiidae, Spiraea, Gimmeidae, Codida, Ashiro, Puffer, Scorpiona, Bora, Sardine, Duck, Tauna, Scarlet, Perch, etc. Among them, cartilage fish, eel eyes, salmon eyes, cod eyes, flounder eyes, scorpion eyes, perch eyes, herring eyes, etc., especially sharks, eels, white salmon, trout, cod, walleye pollock, hake, flounder, hockey, Halibut, tuna, herring and the like are preferred. In addition, examples of mammals include cattle, pigs, goats, horses, and sheep, and examples of birds include chickens and the like. Among these, white salmon or perch tilapia is particularly preferably used.

  In addition, plant collagen is a glycoprotein present in the cell wall of a plant called extensin and contains hydroxyproline (former name: L-oxyproline) contained in animal collagen in the amino acid composition. When used as, it is expected to function similarly to animal collagen. Examples of the extraction source of plant collagen include celery family, mallow family, legume family, etc. Among them, carrot genus, velvet mallow genus, and soybean family are preferable, and darcas carrot, soybean, marshmallow are particularly preferable.

  In addition, about 40% of these collagens are present in skin and the like and about 20% in bones and the like in animal collagen. From the viewpoint of collagen yield, the collagen extraction site is bone or skin. (In the case of fish, scales are also included), etc., and in plant collagen, leaves, roots, etc. are preferred.

  Next, when forming a tubular tissue body as an artificial blood vessel replacement in a living body and treating the tubular tissue body with the antithrombotic modifier of the present invention, it is carried out as follows. That is, first, a silicone round bar is embedded in a living body, and after several months, a tubular connective tissue film is formed around the silicone round bar. When the silicone round bar is extracted from the connective tissue membrane, a tubular tissue body is formed in the living body. The antithrombotic modifier of the present invention is poured from any one end of the tubular tissue body. Thereafter, if the end of the tubular tissue body is anastomosed with a living blood vessel, an artificial blood vessel replacement operation is performed in which the affected living blood vessel is replaced with a new blood vessel.

  According to this technique, artificial blood vessel replacement can be performed while suppressing invasion, and anti-thrombogenicity can be imparted to a tubular tissue body in a living body without using an organic solvent.

(Porous collagen)
The porous collagen according to this embodiment is constituted by infiltrating a porous tissue with an antithrombotic drug containing at least one of heparin and low molecular weight heparin.

  The present inventor has found that heparin or low molecular weight heparin penetrates and is fixed in the porous tissue of porous collagen through intensive studies, and has completed the present invention based on this new knowledge.

  FIG. 3 is a photograph showing the adsorptivity of heparin to the collagen sponge, of which (a) is one hour after washing and (b) is 24 hours after washing. As the collagen sponge, for example, NMP collagen sponge (derived from pig skin) manufactured by Nippon Ham can be used.

  When heparin is immersed in a collagen sponge and a cationic pigment, toluidine blue is allowed to act, the collagen sponge is stained blue. After washing with physiological saline for 1 hour, heparin still remains as shown on the left side of FIG. On the other hand, when toluidine blue is immersed in collagen sponge, it is stained blue. After washing with physiological saline for 1 hour, toluidine blue is almost washed as shown on the right side of FIG. .

  Further, after washing with physiological saline for 24 hours, heparin still remains as shown on the left side of FIG. On the other hand, after washing the collagen sponge stained with only toluidine blue with physiological saline for 24 hours, as shown on the right side of FIG. 3B, toluidine blue is almost washed.

  On the other hand, FIG. 4 is a photograph showing the adsorptivity of a heparin solution (5 mg / mL) to which toluidine blue is bound to connective tissue, of which (a) is immediately after staining and (b) is immediately after staining. (C) is one hour after washing, and (d) is 24 hours after washing.

  The connective tissue can be prepared by, for example, the following method. That is, the skin of a beagle dog under anesthesia is incised, a silicone round bar having a diameter of 5 mm and a length of 30 mm is implanted subcutaneously, and after 1 month, the skin is incised again and the implanted silicone round bar is removed. A bag-shaped neoplastic connective tissue film (thickness: about 200 μm) is formed around the extracted silicone round bar, so that a new connective tissue is prepared by taking out the silicone round bar from the inside. .

  And as shown to Fig.4 (a), when a heparin solution couple | bonded with toluidine blue is immersed in a connective tissue, it will dye | stain blue.

  Next, when the section of the connective tissue stained blue is viewed, only the surface of the connective tissue is stained as shown in FIG.

  After washing with physiological saline for 1 hour, heparin was slightly washed away from the connective tissue as shown in FIG. 4 (c), and after further washing with physiological saline for 24 hours, FIG. ), Heparin is almost washed away.

  From these facts, it is found that heparin is difficult to solidify to connective tissue, but is adsorbed and immobilized to collagen sponge. It is considered that low molecular weight heparin is also adsorbed and immobilized on the collagen sponge.

  Therefore, it is possible to easily impart antithrombogenicity by impregnating heparin or low molecular weight heparin into porous collagen. Furthermore, in the present invention, no chemical reaction is applied to collagen. The original mechanical strength of collagen can be maintained. Furthermore, since imparting antithrombogenicity to the porous collagen according to the present invention does not use an organic solvent, the porous collagen is not damaged by the organic solvent.

  The average pore diameter of the fine pores of the porous collagen is not particularly limited, and an optimum value can be selected depending on the tissue or organ to be regenerated, for example, 10 to 400 μm, preferably 30 to 300 μm. . If the thickness is less than 10 μm, cells may not enter the porous body and cell adhesion may be extremely poor, or the adhered cells may not be able to extend three-dimensionally. This is because the tissue or organ may not be regenerated.

  It should be noted that the porous collagen of the present embodiment can contain a cell growth factor. The cell growth factor is not particularly limited as long as it promotes angiogenesis and enhances cell activity. For example, a cell growth factor having an angiogenesis action, specifically, basic fibroblast growth factor (bFGF) ), Acidic fibroblast growth factor (aFGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), plasma version-derived growth factor (PDGF), angiopoietin, transforming growth factor (TGF), etc. Can be made.

  The manufacturing method of the porous collagen of this embodiment is as showing below. That is, an aqueous collagen solution is prepared, poured into an appropriate mold, and freeze-dried at −50 to −80 ° C. for about 1 to 2 hours to obtain a porous collagen structure. The porous collagen according to the present embodiment is obtained by infiltrating the porous collagen structure with a solution of heparin or low molecular weight heparin.

  The heparin concentration in the solution of heparin or low molecular weight heparin in which the porous collagen structure is immersed is, for example, 0.5 to 200 mg / ml. This is because if the heparin concentration is lower than 0.5 mg / ml, the concentration of the antithrombotic drug is low and it is difficult to immobilize the porous structure. On the other hand, if the heparin concentration is higher than 200 mg / ml, This is because excessive heparin may flow out into the living body after the porous collagen is applied to the living body and the hemorrhagic lesion may be generated after the porous collagen is applied to the living body.

<Anionic argatroban aqueous solution>
Argatroban powder was dissolved in a 1N aqueous sodium hydroxide solution to obtain an aqueous sodium hydroxide solution of argatroban. This sodium hydroxide aqueous solution of argatroban was placed in a dialysis tube having a molecular weight cut off of 100, and dialysis was performed overnight under running water until the pH became near neutral to remove by-products and generated salts. Thereafter, powdered anionic argatroban was obtained by lyophilization. This powdery anionic argatroban was dissolved in water to obtain an aqueous anionic argatroban solution having a concentration of 5 mg / mL.

<Treatment with anionic argatroban aqueous solution>
The epidermis of the back of a rabbit that was locally anesthetized and shaved by a normal procedure was incised about 30 mm immediately after disinfection with isodine, and a sterilized silicone round bar was embedded under the subcutaneous tissue and sutured. The round bar made of silicone had a circular cross section, and had a diameter of 5.0 mm and a length of 30 mm. The suture site was disinfected with isodine twice a day, water was freely supplied, and an appropriate amount of ORC4 manufactured by Oriental Yeast Co., Ltd. was fed as feed. During the implantation period, there was no evidence of infection in the sutured area, and no antibiotics were required. One month after implantation, a silicone round bar was extracted by the same procedure as that at the time of implantation. A bag-shaped neoplastic connective tissue film having a thickness of about 200 μm was formed around the extracted silicone round bar. A silicone round bar was taken out from the inside, and an artificial blood vessel having an inner diameter of 5.0 mm was created from the resulting connective tissue membrane. Similarly, artificial blood vessels having inner diameters of 1.5 mm and 2.0 mm were created. Each artificial blood vessel was treated with the anionic argatroban aqueous solution.

  Next, the epidermis of the back of a rabbit that was locally anesthetized and shaved by a normal procedure was incised about 30 mm immediately after disinfection with isodine, and a sterilized silicone Y-shaped rod was implanted under the subcutaneous tissue and sutured. The silicone Y-shaped rod was formed of a main trunk and two limbs branched from the main trunk, and the angle between the limbs was 30 degrees. After passing through the same manner as above, a silicone Y-shaped rod was extracted one month after the implantation. A pouch-shaped neoplastic connective tissue film having a thickness of about 200 μm was formed around the extracted silicone Y-shaped rod. A silicone Y-shaped rod was taken out from the inside, a Y-shaped artificial blood vessel was created from the resulting connective tissue membrane, and treated with the above-mentioned anionic argatroban aqueous solution.

<Presence or absence of thrombosis in artificial blood vessels>
Next, administer a drug that is a mixture of 3 mL of ketamine hydrochloride (Ketaral for animals: Sankyo Yale Yakuhin Co., Ltd.), 5 mL of azaperone (Stressnil for animals: Sankyo Yakuhin Co., Ltd.), and 1 mL of atropine sulfate (Fuso Yakuhin Kogyo Co., Ltd.). The rats were initially anesthetized. Then, general anesthesia was performed by inhaling the rat with an isoflurane inhalant (Escaine: Merck Whey). Under general anesthesia, the abdomen was incised to expose the abdominal aorta. Approximately 3.0 cm of the exposed abdominal aorta is excised, and each stump is sutured by continuous anastomosis with an artificial blood vessel having an inner diameter of 1.5 mm produced above at this location with a suture, as shown in FIG. Then, an artificial blood vessel having an inner diameter of 1.5 mm was transplanted into the rat abdominal aorta. In the MRA observation image 3 months after transplantation, as shown in FIG. 5 (b), no thrombus formation was observed, and the tumor was patented without becoming cancerous or stenotic.

  Rabbits were given initial and general anesthesia as described above. Under general anesthesia, the chest was incised to expose the common carotid artery. Approximately 3.0 cm of the exposed common carotid artery is excised, and each stump is sutured by continuous anastomosis with an artificial blood vessel having an inner diameter of 2.0 mm prepared above at this location with a suture, as shown in FIG. Thus, an artificial blood vessel having an inner diameter of 2.0 mm was transplanted into the rabbit common carotid artery. In angiography 3 months after transplantation, as shown in FIG. 6 (b), no thrombus formation was observed, and the tumor was patented without becoming cancerous or stenotic.

  Moreover, the initial anesthesia and the general anesthesia were performed to the beagle similarly to the above. Under general anesthesia, the chest was incised to expose the common carotid artery. Approximately 3.0 cm of the exposed common carotid artery is excised, and each stump is sutured by continuous anastomosis with an artificial blood vessel having an inner diameter of 5.0 mm produced above at this location, as shown in FIG. Thus, an artificial blood vessel having an inner diameter of 5.0 mm was transplanted into the beagle common carotid artery. In angiography 3 months after transplantation, as shown in FIG. 7 (b), no thrombus formation was observed, and the tumor was patented without becoming cancerous or stenotic.

  Moreover, the initial anesthesia and the general anesthesia were performed to the beagle similarly to the above. Under general anesthesia, the abdomen was incised to expose the iliac artery. The exposed iliac arteries are excised, and the stumps are sutured by continuous anastomosis of the Y-shaped artificial blood vessel prepared above with a suture thread at this location, as shown in FIG. Transplanted into bone artery. In angiography 3 months after transplantation, as shown in FIG. 8 (b), no thrombus formation was observed, and the tumor was patented without becoming cancerous or stenotic.

<Tissue observation of artificial blood vessel transplantation site>
Next, the tissue of the abdominal transplantation site of a rat embedded with an artificial blood vessel having an inner diameter of 1.5 mm was observed. FIG. 9 (a) is an HE / E (hematoxin / eosin) staining diagram, FIG. 9 (b) is an immunostaining diagram of anti-hemophilic factor (factor 8), and FIG. 9 (c) is MTC (Masson). FIG. 9 (d) is an EWG (Elastica-Wangeson) staining diagram, and FIG. 9 (e) is an α-SMA immunostaining diagram. As shown in FIG. 9A, the cell and tissue structure at the transplantation site were normal. Endothelial cells are infiltrated on the surface (FIG. 9 (b)), and smooth muscle cells are contained in the matrix layer composed of collagen (FIG. 9 (c)) and elastin fibers (FIG. 9 (d)) in the wall. (FIG. 9 (e)) was infiltrated, and a tissue similar to a biological blood vessel was constructed. From this, it is understood that anionic argatroban imparted antithrombogenicity to the artificial blood vessel, and good healing occurred in the tissue at the transplantation site.

<Concentration of anionic argatroban>
Next, the cross-section of the connective tissue was observed when the concentration of the anionic argatroban in the anionic argatroban aqueous solution was changed to act on the connective tissue. The concentration of anionic argatroban was 1.25 mg / ml, 2.5 mg / ml, 5 mg / ml, 10 mg / ml. The epidermis of the back of a rabbit that has been anesthetized by local anesthesia is dissected quickly by disinfection with isodine, and a sterilized silicone round bar is inserted under the subcutaneous tissue and sutured. One month after implantation, the silicone round The rod was removed to obtain a neoplastic connective tissue membrane having a thickness of about 10 μm. The connective tissue was immersed in an anionic argatroban aqueous solution for 30 minutes and observed with a phase contrast microscope and a fluorescence microscope. The excitation light of the fluorescence microscope was 330 nm, and the observation light was 400 nm. The exposure time was 2.08 ms and the observation magnification was 20 times.

  FIG. 10A is a fluorescence microscope diagram at a concentration of 10 mg / ml, and FIG. 10B is a phase contrast microscope diagram of the same visual field. At a concentration of 10 mg / ml, as shown in FIG. 10 (a), anionic argatroban was clearly immobilized at the position of the connective tissue (shown in FIG. 10 (b)).

  Next, FIG.10 (c) is a fluorescence microscope figure in the density | concentration of 5 mg / ml, and FIG.10 (d) is a phase-contrast microscope figure of the same visual field. Even at a concentration of 5 mg / ml, as shown in FIG. 10 (c), the anionic argatroban was clearly immobilized at the position of the connective tissue (shown in FIG. 10 (d)).

  Next, FIG.10 (e) is a fluorescence microscope figure in the density | concentration of 2.5 mg / ml, and FIG.10 (f) is a phase-contrast microscope figure of the same visual field. Even at a concentration of 2.5 mg / ml, as shown in FIG. 10 (e), anionic argatroban was clearly immobilized at the position of the connective tissue (shown in FIG. 10 (f)).

  Next, FIG. 10 (g) is a fluorescence microscope diagram at a concentration of 1.25 mg / ml, and FIG. 10 (h) is a phase contrast microscope diagram of the same field of view. Even at a concentration of 1.25 mg / ml, as shown in FIG. 10 (g), anionic argatroban was clearly immobilized at the position of connective tissue (shown in FIG. 10 (h)).

  From the above, it is understood that anionic argatroban can be clearly immobilized on connective tissue in the case of an aqueous 1.25 to 10 mg / ml anionic argatroban solution.

<Creation of artificial blood vessel with valve>
Next, an artificial blood vessel with a valve was prepared using a columnar core substrate, and an anionic antithrombotic drug aqueous solution was applied to the artificial blood vessel with a valve to examine antithrombogenicity.

  The columnar core base material is made of silicone, and has two columnar parts, a valve forming part, and an overhang part as shown in FIG. The diameter of the columnar part was 14 mm. The valve formation site is a fitting site between the concave end surface of one columnar site and the convex end surface of the other columnar site, and forms a trilobal shape. The overhanging part is on the downstream side in the blood flow direction in the vicinity of the valve forming part.

  A hole is formed in the central part of the two columnar parts in the longitudinal direction of the blood vessel. As shown in FIG. 12, a needle-shaped mandrel is passed through the hole, the plurality of columnar objects are separated slightly and the centers are centered. After being joined together, they were implanted subcutaneously in a beagle dog.

  In 2 to 4 weeks after implantation, the outer peripheral surface of the columnar core substrate and the gap between the projections and depressions were completely covered with a connective tissue mainly composed of collagen and fibroblasts.

  When the columnar core substrate was extracted from the covering, a trilobal thin film artificial valve with a valve integrated with the surroundings was formed. The trilobal valve was connected at a position corresponding to the protruding end of the convex surface, but could be easily divided into three valves by incision.

  As shown in FIG. 13, the artificial blood vessel created in this way is configured to have a blood vessel part, a leaflet part, and an overhang part, and is very similar to a biological Valsalva sinus (aortic sinus). It was an artificial blood vessel with a valve.

  This artificial blood vessel with a valve was treated with an anionic argatroban aqueous solution having a concentration of 5 mg / mL prepared in the above <Anionic argatroban aqueous solution>. The beagle was subjected to initial anesthesia and general anesthesia, and the chest was opened under general anesthesia to expose the pulmonary artery. The exposed pulmonary artery was incised, the internal pulmonary valve was excised, and each stump was sutured to the incised pulmonary artery by continuous anastomosis with a sutured artificial blood vessel treated with an anionic argatroban aqueous solution.

  As a result of observation after 3 months of transplantation, as shown in FIG. 14, it was found that the blood contact surface after 3 months of beagle pulmonary valve replacement surgery had no thrombus formation and had high antithrombotic properties. . As described above, since the artificial blood vessel with a valve according to the present example has an overhanging portion corresponding to the Valsalva sinus, not only has an advantage that the backflow of blood hardly occurs, but also has antithrombogenicity without using an organic solvent. Therefore, there is a great advantage that the tissue damage is extremely small.

Claims (7)

An antithrombotic modifying agent that immobilizes an antithrombotic drug on a blood contact portion of a medical device mainly composed of collagen and imparts antithrombogenicity, wherein the antithrombotic drug is anionic. Antithrombotic modifier. The antithrombotic modifying agent according to claim 1, wherein the antithrombotic agent comprises at least one of argatroban, nafamostat mesylate, warfarin, and aspirin. The antithrombotic modifier according to claim 1 or 2, wherein the concentration of the anionic antithrombotic agent is 0.5 mg / ml or more and 200 mg / ml or less. A medical device comprising collagen as a main component, wherein an anionic antithrombotic drug is immobilized on the surface of a blood contact portion. The medical device according to claim 4, wherein the antithrombotic agent contains at least one of argatroban, nafamostat mesylate, warfarin, and aspirin. The anionic anti-thrombotic drugs, medical device according to claim 4 or 5, wherein the being 20mg or less fixed 1 cm ² per 0.01mg over the surface of the blood contact portion. A porous collagen, wherein an antithrombotic drug containing at least one of heparin and low molecular weight heparin is infiltrated into a porous tissue.

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