JP2537091B2 - Antithrombotic medical material and method for producing artificial blood vessel - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to biocompatible materials, particularly antithrombotic properties used for artificial blood vessels, artificial organs, catheters and the like, and antithrombotic properties excellent in tissue compatibility. The present invention relates to a medical material and a method for producing an artificial blood vessel.

[Prior Art] With the remarkable progress of medical care in recent years, various medical instruments such as artificial blood vessels, blood vessel catheters, extracorporeal blood circuits, and artificial organs have come to be widely used in places where they come into direct contact with blood. These medical instruments used in direct contact with blood are not only good in physical properties such as durability, but also excellent in biocompatibility such as antithrombotic property and histocompatibility. Is required.

Conventionally, for example, in artificial blood vessels, as artificial blood vessels having a wide diameter of 8 mm or more, one formed of porous polyester and one formed of porous polytetrafluoroethylene are used. The artificial blood vessel made of polyester utilizes the natural antithrombotic property of the endothelial cells that cover the blood vessel substrate after the artificial blood vessel is transplanted. In other words, the tissue penetrability into the porous polyester forming the artificial blood vessel is utilized. Therefore, in this artificial blood vessel biological tissue is likely to adhere to the inner surface, the formation of neointima on the inner surface of the artificial blood vessel is rapid, therefore, the invasion of biological tissue into the graft of the porous polyester forming the artificial blood vessel, The artificial blood vessel is well organized and excellent in long-term tissue repair. Furthermore, since the formed neointima grows firmly on the inner surface of the artificial blood vessel, it is characterized by less exfoliation and long-term patency. Further, the artificial blood vessel using porous polytetrafluoroethylene utilizes the antithrombotic property of its material, and has an excellent effect on the initial antithrombotic property.

[Problems to be Solved by the Invention] However, in the case of the artificial blood vessel using the above-mentioned porous polyester, when the inner diameter is large, a thrombus adheres to the inner surface immediately after transplantation, and thereafter, the endothelial cells cover the surface. Covers to form a surface structure similar to natural blood vessels. Therefore, a natural antithrombotic property can be obtained. But small diameter, especially 5m
An artificial blood vessel of m or less has a problem that it cannot be used because the artificial blood vessel is easily blocked by the attached thrombus.

Further, in the artificial blood vessel using the above-mentioned porous polytetrafluoroethylene, since the material used is inactive to the living body, the tissue repair tends to be extremely delayed. That is, since it is difficult for the biological tissue to enter the porous inner wall, the neointima is less likely to be formed in the central portion of the graft, delays organizing, and is formed on the biological tissue formed on the outer surface side and the inner surface side of the artificial blood vessel. Insufficient bonding with living tissue becomes a problem, and the neointima that has continued to grow from both anastomotic sites of the transplanted artificial blood vessel becomes easy to peel off. This exfoliated substance causes thrombus formation. In addition, after the artificial blood vessel was transplanted, the neointima was partially raised (thickened) in the vicinity of the anastomotic site on the peripheral blood flow side, which sometimes resulted in long-term occlusion.

Therefore, as a small-diameter artificial blood vessel, it is necessary to have antithrombogenicity in the initial stage, prevent thick attachment of living tissue, and form neointimal formation at an early stage. However, at present, cell proliferative properties such as neointimal formation and growth and antithrombotic properties tend to contradict each other.Giving antithrombotic properties generally suppresses platelet aggregation as much as possible. Yes, platelets contain many elements necessary for the cells to proliferate, and in particular, those elements are contained abundantly in α granules in platelets. However, it is said that the α granules are not released unless platelets aggregate. Therefore, at present, none of them have both antithrombotic properties and cell proliferative properties such as neointimal formation and growth.

[Means for Solving Problems] An object of the present invention is to have an antithrombotic property at an initial stage, prevent thick attachment of living tissue and formation of a thrombus, and further perform neointimal formation at an early stage. The present invention provides an antithrombotic medical material capable of exhibiting an antithrombotic effect for a long period of time by the formed neointima without peeling, and a method for producing an artificial blood vessel using the same.

What achieves the above object is a method for producing an antithrombotic medical material, which comprises coating a surface of a base material made of a polymer material with a fibrin layer, and then treating at least the surface of the fibrin layer with plasmin.

Then, the step of coating the surface of the base material with a fibrin layer is to contact the base material with a thrombin solution and then contact the fibrinogen solution with the base material to form a fibrin layer on the base material surface. Preferably there is.

Moreover, what achieves the above-mentioned object is to coat a fibrin layer on the inner surface of a tubular body formed of a polymeric material,
After that, at least the surface of the fibrin layer is treated with plasmin, which is a method for producing an artificial blood vessel.

Then, the step of coating the inner surface of the tubular body with a fibrin layer comprises contacting the tubular body with a thrombin solution and then contacting the fibrinogen solution with the tubular body to form a fibrin layer on the inner surface of the tubular body. It is preferably one.

Therefore, the antithrombotic medical material of the present invention will be described with reference to the embodiments shown in the drawings.

An antithrombogenic medical material 1 produced by the method for producing an antithrombogenic medical material according to the present invention has a fibrin layer 3 at least the surface of which is hydrolyzed on a surface of a base material 2 formed of a polymer material. ing.

 Therefore, description will be made with reference to FIG.

FIG. 1 is a sectional view of an embodiment of the antithrombotic medical material of the present invention.

In this antithrombotic medical material 1, a fibrin layer 3 is provided on the surface of a base material 2 formed of a polymer material, and the surface of this fibrin 3 is a hydrolyzed portion 4.

Polymer material 1 used for forming a substrate in the present invention
Examples of the material include synthetic polymer materials having excellent mechanical performance such as nylon, polyester, polyethylene, polypropylene, polyurethane, silicone, and polytetrafluoroethylene, or tissues and organs derived from living bodies such as natural blood vessels and ureters. Etc. can be used. Further, when a synthetic polymer material is used, its porous material is preferable from the viewpoint of the adhesiveness to fibrin. As the porous material, those formed by a stretching method, a solid-liquid layer separation method, or the like are preferable.

A sectional view of an example using this porous substrate is shown in FIG. When such a porous base material is used, as shown in FIG. 2, since the fibrin enters the pores of the base material to form a fibrin layer, the adhesiveness between the base material and the fibrin layer is further improved. It will be good. Further, FIG. 3 shows a cross-sectional view when a tubular body 5, which can be suitably used for an artificial blood vessel, in particular, a porous tubular body 5 formed of a polymer material is used as a base material. Also in this tubular body, similar to that shown in FIG. 2, the fibrin enters the pores of the tubular body to form the fibrin layer 3, so that the adhesion between the base material and the fibrin layer is good.

The fibrin layer 3 is then purified fibrinogen,
At least one selected from plasma and cryoprecipitate is used as a starting material. The cryoprecipitate is obtained by repeatedly freezing plasma and cooling and centrifuging to increase the fibrinogen concentration. Then, as a method of forming the layer 4 in which at least the surface of the fibrin layer 3 is hydrolyzed, it is possible to treat the fibrin layer with a proteolytic enzyme. And plasmin can be used suitably as a protease.

Fibrin is produced by the limited decomposition of fibrinogen by the action of thrombin to release fibrinopeptides A and B. Specifically, fibrinogen is hydrolyzed by the enzyme thrombin, and among its constituent peptide chains, Aα chain to fibrinopeptide A (FPA) and Bβ chain to fibrinopeptide B.
Releases (FPB) and becomes fibrin monomer. The fibrin monomer undergoes coagulation polymerization to become an unstable fibrin polymer (soluble fibrin polymer). Furthermore, the water-decomposable by thrombin, by activated activated factor XIII by Ca ²⁺ ions, between γ chains between fibrin molecules crosslinked in the presence of Ca ²⁺ ions (between lysine residues and glutamine residues (Iso peptide bond) and becomes a stable and stable fibrin polymer (insoluble fibrin polymer). The fibrin layer 3 in the present invention is formed of this insoluble fibrin polymer.

The plasmin used to hydrolyze the fibrin layer 3 is a proteolytic enzyme, the substrates are fibrin and fibrinogen, and it is said that it cleaves between the 43rd lysine and the 44th tryptophan of fibrin. It is being appreciated. The plasmin degradation product of fibrin has an antithrombin action. At least the surface of the fibrin layer 3 is hydrolyzed by treating the fibrin layer 3 with the above-mentioned plasmin, and the state of the hydrolyzed portion is α, β, which constitutes fibrin.
It is considered that the γ chain is in a state in which the residue (hydrolyzed part, fragments X, Y, D, E) that has been digested by plasmin and the part that has not yet been digested (unhydrolyzed part) are mixed. It is considered that the above-mentioned hydrolysis is mainly present in the surface layer of the fibrin layer which is the contact surface with plasmin. The abundance ratio of hydrolyzed moieties and unhydrolyzed moieties, the abundance ratio of fragments, and the existing forms of hydrolyzed moieties and unhydrolyzed moieties are considered to change depending on the treatment time of plasmin and the enzymatic activity of plasmin.

The hydrolysis by plasmin is preferably performed on the entire surface layer of the fibrin layer, but it is not always necessary. Further, the fibrin layer may be hydrolyzed at least in the surface layer portion in contact with blood. Also, the fibrin layer treated with plasmin tends to swell compared to before treatment.

The antithrombotic medical material of the present invention has a high antithrombotic property and biocompatibility by having the fibrin layer and the fibrin layer portion in which at least the surface layer is hydrolyzed.

In particular, cell proliferation is good, and as a result of cell spreading,
It is able to produce collagen and form an overall homogeneous neointima.

That is, the formed neointima grows and progresses on the hydrolyzed fibrin layer, and by replacing the fibrin layer with the collagen layer produced by the intimal cells themselves, a film that does not easily peel off is formed. . Therefore, at the site covered by the neointima, the hydrolyzed fibrin layer originally present does not exist, but the collagen layer produced by neointima cells becomes present.

The provision of the fibrin layer and the hydrolyzed portion thereof in the present invention allows the present inventors to prepare a model animal for intimal injury of the blood vessel in order to find out the conditions that the artificial blood vessel should have, and perform tissue repair over time. A detailed search of the process revealed that a fibrin layer was temporarily formed on the exposed inner elastic lamina where the intima was injured and replaced with neointima cells in only one week without occlusion. In the meantime, we obtained the findings suggesting the modification of fibrin by inflammatory cells, and based on these findings, the enzyme-modified fibrin was brought into direct contact with blood, and the neointimal membrane actually appeared early. It was recollected by the knowledge.

And this antithrombotic medical material can be used for medical devices having various blood contact surfaces. For example, artificial blood vessels that are blood-contacting artificial organs, catheters that are left in the body for a long time, artificial dialysers, artificial organs such as artificial lungs, and implantable artificial organs can be considered.

The antithrombotic medical material 1 of the present invention can be manufactured as follows.

First, a base material is made of a polymer material. As the base material 2, it is preferable that the fibrin layer 3 can be formed to the inside of the base material, and the fibrin layer 3 to be formed is fixed. From this point, a porous substrate is preferable. Further, the fibrin layer 3 can be more preferably fixed if the base material has hydrophilicity in order to enhance the affinity with the fibrinogen solution or the thrombin solution, which is the starting material for forming the fibrin layer.

Then, a fibrinogen solution is prepared using purified fibrinogen, plasma, cryoprecipitate, or the like. Then, after contacting the above substrate with a thrombin solution, this fibrinogen solution is contacted with the above substrate, and if the substrate surface and a porous substrate are inside, hydrolyzing fibrinogen with thrombin, A fibrin layer is formed on the surface of the base material. Next, the substrate having the above-mentioned fibrin layer on the inner surface is brought into contact with the plasmin solution. The plasmin treatment time (contact time between the plasmin solution and the fibrin layer) varies depending on the plasmin concentration in the plasmin solution, but is 1 second to 24 seconds.
The time is preferably 5 to 15 minutes. For example, if the plasmin solution is 0.05 u / ml, about 15 minutes is suitable.

Then, a soybean trypsin inhibitor or an antifibrinolytic agent was added to the plasmin solution, the enzymatic reaction was stopped, and the surface was hydrolyzed sufficiently to wash the surface of the antithrombotic medical agent of the present invention having a hydrolyzed fibrin layer. The material is created.

[Examples] Examples of the present invention will be specifically described below.

(Example 1) <Preparation of fibrinogen solution> A human fibrinogen preparation (grade L, manufactured by KabiVitrum) was dissolved in 50 ml of distilled water, and a gelatin-cellulophane column (manufactured by Seikagaku Corporation), and then a lysine-sepharose 4B column. (Made by Pharmacia) to remove fibronectin and plasminogen (plasmin),
It was sterilized by filtration through a sterilized 0.20 μm filter. The concentration of the fibrinogen solution after filter sterilization was measured by a coagulation time measuring method (Data-Fi, manufactured by Dade) and then freeze-dried.
Lyophilized fibrinogen was added to 20 mM CaCl ₂ , 2 mM MgCl ₂ , 1
A human fibrinogen solution was prepared by dissolving it in 5 mM Hepes buffer (PH7.5) containing 50 mM NaCl so that the fibrinogen concentration was 30 mg / ml.

<Formation of fibrin layer on substrate> For e-PTFE graft tube (expanded porous tube, trade name Gore-Tex, Gore Co.) with an inner diameter of 3 mm and a length of 30 mm
5 unit of 50 unit / ml thrombin (Mochida Pharmaceutical Co., Ltd.) was press-fitted, then 2.5 ml of the above fibrinogen solution was press-fitted, thoroughly washed with physiological saline, and then incubated at 37 ° C. for 24 hours, and the fibrin layer (thickness 5 μm ) Was prepared on the inner surface of the coated tubular body. Moreover, fibrin had penetrated into the pores of the e-PTFE graft tube.

<Hydrolysis treatment of fibrin layer> A tubular body having the above-mentioned fibrin layer on its inner surface is in 50 mM phosphate buffer, and a 0.05 μnit / ml plasmin solution (Boehringer Mannheim Yamanouchi Co., Ltd.) is prepared in this solution.
It was immersed at 37 ° C. for 15 minutes, and the surface of the fibrin layer was brought into contact with the plasmin solution. Then, soybean trypsin inhibitor (Boehringer Mannheim Yamanouchi) was added to a final concentration of 20.
It was added to 0 μg / ml and thoroughly washed with physiological saline to form a plasmin-treated hydrolyzed portion on the surface of the fibrin layer of the tubular body to obtain an artificial blood vessel (Example 1) of the present invention. .

The fibrin layer treated with plasmin was relatively firmly fixed to the inner surface of the tube and was not peeled off in a normal state. Further, the thickness of the fibrin layer treated with plasmin was 20 μm, and it was found that the fibrin layer was swollen before the treatment.

A tubular body having a fibrin layer whose surface was hydrolyzed as described above (Example 1) and a tubular body having a fibrin layer not subjected to plasmin treatment (Comparative Example 1) were each stained by a hematoxylin-eosin staining method. When observed with an optical microscope and an electron microscope, Comparative Example 1
In the tubular body of 1), one fibrin layer was recognized on the surface of the lumen of e-PTFE. Also, in the tubular body of Example 1, it was also observed as a layer, but as compared with the fibrin layer of the tubular body of Comparative Example 1, a layer that slightly swells and is poor in dyeability as observed by an optical microscope. Was recognized.

(Example 2) The same procedure as in Example 1 was performed except that a porous polyester tube (inner diameter: 3 mm, length: 30 mm) formed by plain weaving using polyester fibers (75 denier, 36 filaments) was used as a base material. An artificial blood vessel of the present invention (Example 2) was obtained.

(Example 3) As a base material, a Goraski artificial blood vessel (manufactured by Toki Trading Co., Ltd.), which is a polyester-made porous artificial blood vessel (inner diameter 3 m
m, length 30 mm) was performed in the same manner as in Example 1 to obtain an artificial blood vessel of the present invention (Example 3). Example 2
Also in the artificial blood vessel of Example 3, the fibrin layer treated with plasmin was relatively firmly fixed to the inner surface of the tube and was not peeled off in a normal state. Then, in the same manner as in Example 1, Example 2 and Example 3 were stained by the hematoxylin-eosin staining method and observed with an optical microscope and an electron microscope, but a fibrin layer was confirmed on the inner surface of the tubular body, As compared with the fibrin layer of the tubular body of Comparative Example 1, a layer that slightly swelled and was poor in dyeability was observed by observation with an optical microscope.

(Comparative Example 3) The porous polyester tube used in Example 2 was immersed in 5 ml of whole blood in a sterilized container and allowed to stand for 5 minutes. After blood was coagulated, anticoagulation treatment was performed with 1000u of heparin. 5 ml of whole blood was injected into the tube lumen to eliminate blood clots. Then, the lumen was further washed with physiological saline to obtain an artificial blood vessel of Comparative Example 3.

Comparative Example 4 The polyester porous artificial blood vessel used in Example 3 was treated in the same manner as Comparative Example 3 to obtain the artificial blood vessel of Comparative Example 4.

(Experiment 1) Rabbit abdominal aorta (3 mm) with Example 1, Comparative Example 1 and untreated e-PTFE graft tube (Comparative Example 2)
φ) was transplanted by end-to-end anastomosis, and the change over time was observed. During and after surgery, do not use any anticoagulant such as heparin,
Seven examples were carried out for Comparative Example 2, five cases were carried out for Comparative Example 1, and nine cases were carried out for Example 1.

As a result, in the case of Comparative Example 2, the entire graft was macroscopically red even one month after the transplantation, and as a result of the histological search, a migration (migration) image of cells from both anastomotic sites was observed. The blood contact surface other than the above is mainly composed of blood cell components and no other cells are observed, and even after 6 months after transplantation, the neointimal membrane was scarcely formed and there was a tendency for organizing to be delayed. Was given. On the other hand, in Comparative Example 1 and Example 1, the presence of cells forming the neointima in the central part of the graft was already observed 15 days after transplantation, but Example 1 is better in organic structure. Was being made.

Furthermore, when the state of organizing in the graft wall was observed, in Comparative Example 2, the cell invasion in the wall tended to be delayed, whereas in Comparative Example 1 and Example 1, the infiltration into the wall was early. Cell entry was observed and Example 1 tended to be superior.

Further, in Example 1, after 15 days, it was covered with a substantially homogeneous collagen layer which is considered to be produced by the cells constituting the tubular inner surface of the inner cavity surface. No thrombus was attached to the central part of the tubular body or the anastomosis. Furthermore, in the case of Example 1, the cells were completely replaced on the surface after one month.

From the results shown in Experiment 1, in the artificial blood vessel of the Example, cells proliferate favorably from both anastomotic parts of the tubular body, and as a result of spreading, collagen is produced and a neointima extending over the entire inner surface of the tubular body is formed. I understood that it was done.

(Experiment 2) The artificial blood vessels of Example 2, Example 3, Comparative Example 3 and Comparative Example 4 were transplanted to the abdominal aorta (3 mmφ) of the rabbit by end-to-end anastomosis, and when the base material of the artificial blood vessel was changed. Observed for changes. During and after surgery, no anticoagulant such as heparin was used. Further, upon transplantation, bleeding from the graft other than the anastomosis was not observed in any of the artificial blood vessels of Example 2, Example 3, Comparative Example 3 and Comparative Example 4. The anastomotic property and sutureability were better in Example 1 using the polyester knitted fabric than in Example 1 using e-PTFE as the base material. Also, the transplant is
Two examples were carried out for Example 2, three cases for Example 3, three cases for Comparative Example 3, and two cases for Comparative Example 4.

As a result, in the artificial blood vessels of Comparative Examples 3 and 4, there were 2 cases in Comparative Example 3 that were occluded 1 month after transplantation and 1 case in Comparative Example 4. Although he was patent for 1 month, red thrombus was prominently observed, and as a result of histological examination, formation of neointima was slightly observed, and most of the artificial blood vessel was occupied by thrombus. Was there.

In the artificial blood vessels of Examples 2 and 3, almost no adhesion of thrombus was observed one month after transplantation, and at this time point, it was confirmed that the cells forming the neointima were present on the entire inner surface of the artificial blood vessel. Furthermore, an almost homogeneous collagen layer that was considered to be produced by the cells constituting the neointima was confirmed. Therefore, in the artificial blood vessels of Examples 2 and 3, cells proliferated favorably from both anastomotic parts of the tubular body and spread, and as a result, collagen was produced and a neointima extending over the entire inner surface of the tubular body was formed. all right. As a result of this experiment, it was found that the effect of the present invention can be obtained without being influenced by the substrate used.

[Effects of the Invention] In the method for producing an antithrombotic medical material of the present invention, the surface of the polymer material is coated with a fibrin layer, and then at least the surface of the fibrin layer is treated with plasmin. Has an antithrombotic property, prevents thick attachment of living tissue and formation of thrombus, and further, because the blood contact part is replaced with living cells at an early stage, neointimal formation is good, and further detachment Without using the formed neointima, a medical material having a long-term antithrombotic effect can be easily produced.

[Brief description of drawings]

FIG. 1 is a schematic sectional view of an embodiment of the antithrombotic medical material of the present invention, FIG. 2 is a schematic sectional view of another embodiment of the antithrombotic medical material of the present invention, and FIG. It is a cross-sectional schematic diagram of one Example of the artificial blood vessel of this invention. 1 ... Antithrombogenic medical material, 2 ... Polymer base material, 3 ... Fibrin layer, 4 ... Hydrolyzed portion, 5 ... Porous tubular body

Claims (4)

(57) [Claims] 1. A method for producing an antithrombotic medical material, which comprises coating a surface of a base material made of a polymer material with a fibrin layer, and then treating at least the surface of the fibrin layer with plasmin. 2. The step of coating the surface of the base material with a fibrin layer comprises: contacting the base material with a thrombin solution;
The method for producing an antithrombotic medical material according to claim 1, wherein a fibrinogen solution is brought into contact with the substrate to form a fibrin layer on the substrate surface. 3. A method for producing an artificial blood vessel, which comprises coating the inner surface of a tubular body made of a polymeric material with a fibrin layer, and then treating at least the surface of the fibrin layer with plasmin. 4. The step of coating the inner surface of the tubular body with a fibrin layer comprises contacting the tubular body with a thrombin solution, and then contacting the fibrinogen solution with the tubular body to form a fibrin layer on the inner surface of the tubular body. The method for producing an artificial blood vessel according to claim 3, wherein the artificial blood vessel is formed.