WO2003034945A1

WO2003034945A1 - Artificial vessel and process for producing the same

Info

Publication number: WO2003034945A1
Application number: PCT/JP2002/010986
Authority: WO
Inventors: Katsuko Sakai; Takashi Ushida; Tetsuya Tateishi
Original assignee: Katsuko Sakai; Takashi Ushida; Tetsuya Tateishi
Priority date: 2001-10-24
Filing date: 2002-10-23
Publication date: 2003-05-01
Also published as: JP2003126125A

Abstract

It is intended to provide an artificial vessel, which is excellent in biocompatibility, mechanical strength, antithrombotic properties, etc. and free from blood leakage and can be easily produced. An artificial vessel being free from blood leakage and excellent in antithrombotic properties, having almost smooth inner surface and showing favorable mechanical properties can be obtained within a short period of time by immersing a biologically active substance in a solution capable of setting to gel to give a solution containing the biologically active substance, incorporating this solution containing the biologically active substance into pores of a tube-shaped porous base material for artificial vessel, and then allowing the solution containing the biologically active substance, which has been incorporated into the pores of the porous base material for artificial vessel, to set to gel.

Description

Artificial blood vessel and its manufacturing method

The present invention relates to a vascular prosthesis for use in the treatment of various vascular diseases of the human body, including heart and brain diseases, and peripheral blood vessels of the extremities, and more particularly, to a porous vascular prosthesis substrate and gel. The present invention relates to an artificial blood vessel in which a living body and a bioactive substance are combined. Background art

Artificial blood vessels with large diameters (inner diameters of about 5 mm to about 30 mm) are used in the treatment of vascular diseases such as congenital diseases and arteriosclerosis. As such artificial blood vessels, human blood vessels made of materials such as polyester fibers and extensible fluorine tetrachloride compounds are used clinically. Large-diameter artificial blood vessels have achieved sufficient clinical results because they have a sufficient blood flow velocity even if made of such materials.

Artificial blood vessels of small diameter (with an inner diameter of about 0.5 mm to about 5 mm) are used for the treatment of vascular diseases such as arteriosclerosis, vasospasm, damage to peripheral blood vessels, aneurysm formation, and tumor invasion. Demand is very high. However, in the case of a small-diameter artificial blood vessel, because of the small diameter, it was not possible to obtain an artificial blood vessel having excellent antithrombotic properties capable of securing a sufficient blood flow velocity with the above-described materials. Further, the above-mentioned materials are non-biodegradable materials, have poor biocompatibility, and have a problem that foreign matter remains in the body even after forceful implantation.

Considering biocompatibility and antithrombotic properties, artificial cylindrical substances containing smooth muscle and having a smooth luminal surface capable of forming an endothelial cell layer on the luminal surface The formed hybrid artificial blood vessel model has been proposed.

As such a model, specifically, a model in which gel-like collagen mixed with smooth muscle is molded into a tubular structure has been proposed (Weinberg et al. Science, 1986, 231, p397-400). Hirai et al. ASAIO journal, 1994, p383-388). this According to the model, an artificial blood vessel having a smooth lumen surface can be formed in a short time. However, since such a tubular structure is fragile, it cannot be lifted with tweezers immediately after fabrication, and is extremely difficult to handle.In addition, it cannot withstand the mechanical environment such as blood pressure that exists in a living body. There was a problem that it was not possible.

In addition, a culture solution containing smooth muscle cells is directly inoculated into a biodegradable or non-biodegradable tubular structure, whereby the endothelial cells are cultured until the lumen surface is smoothed. A model for seeding has also been proposed (Niklason et al. Science, 1999, 284, p489-493, JP-A-2001-78750). According to these models, they have excellent mechanical strength and can withstand arterial vascular prostheses. However, it is known that a biodegradable or non-biodegradable tubular structure has a strong hydrophobicity and remarkably poor cell adhesion and proliferation. For this reason, there has been a problem that a long culture time of several months is required for culturing smooth muscle cells in a tubular structure until a smooth lumen surface is formed. This was not practical given the situation of patients who need artificial blood vessels. In addition, there is also a problem that the tubular structure has a low rate of capturing smooth muscle cells, and blood may leak from gaps in the tubular structure. In addition, according to the model, since the mechanical strength is high, there is a problem that friction occurs between the blood vessel and the own blood vessel after transplantation, a cut occurs at a joint, and blood may leak. . Ming disclosure

Therefore, an artificial blood vessel which is excellent in biocompatibility, mechanical strength, antithrombotic property, etc., does not leak blood, and is easy to manufacture is desired.

As a result of intensive studies, the present inventors have found that a cylindrical porous artificial blood vessel base material formed of a fiber-like or sponge-like material having an appropriate mechanical strength is converted into a cell-mixed solution. After impregnating under normal pressure or negative pressure conditions, allowing the cell-containing solution to be taken into the pores, and then gelling the solution, the cells can be densified in a short period of time to the fine details of the porous artificial blood vessel substrate. Have been found to be able to permeate and retain the present invention, and have completed the present invention. In addition, the present inventors have found that not only cells but also bioactive substances synthesized in vivo, genes encoding those bioactive substances, and pharmaceuticals synthesized or obtained in vitro can be obtained by the same method as described above. Pharmaceutical active ingredients such as E.C. compounds can be incorporated into porous artificial blood vessel substrates, and can be used as artificial blood vessels for various uses, including drug delivery systems and artificial organs other than artificial blood vessels. And found that the present invention was completed.

That is, in the present invention, there is provided an artificial blood vessel having a tubular porous artificial blood vessel base material, wherein the pores of the porous artificial blood vessel base material are impregnated with a gel solution containing a biologically active substance. .

Further, according to the present invention, in the method for producing an artificial blood vessel having a cylindrical porous artificial blood vessel base material, the method corresponds to any one of a temperature change, a pH change, a salt concentration change, and an enzyme reaction change. Preparing a biologically active substance-containing solution by impregnating the biologically active substance with the solution to be gelled; and preparing the biologically active substance-containing solution in the pores of the porous artificial vascular base under normal pressure or negative pressure conditions. And a step of gelling the bioactive substance-containing solution taken into the pores of the porous artificial blood vessel base material.

In the present invention, it is preferable that the porous artificial blood vessel substrate is composed of a biodegradable material or a non-biodegradable material. Moreover, the biodegradable material include polylactic acid, polyglycolic acid, copolymers of lactic acid and glycolic acid, polymalic acid, poly one ε- force caprolactone, _epsilon - copolymers of force caprolactone and lactic acid, ε-Co-prolatatatone and glycolic acid, _ε- Co-polymer of lactic prolactatone, lactic acid and dalicholic acid, poly-3-hydroxybutyrate, poly_4-hydroxybutyrate, and ぴ 3-hydroxybutyrate It is preferably at least one material selected from the group consisting of a copolymer of methacrylate and 4-hydroxybutylate. In addition, the non-biodegradable material includes polyethylene resin, polypropylene resin, polybutadiene resin, polystyrene resin, polychlorinated vinyl resin, polyacrylic resin, polymethacrylic resin, and polysulfone resin. And at least one material selected from the group consisting of polytetrafluoroethylene resins. In the present invention, the biologically active substance is preferably a cell and Z or a pharmaceutically active ingredient. The cells are preferably one or more cells selected from the group consisting of smooth muscle cells, fibroblasts, undifferentiated cells, hepatocytes, knee cells, and hematopoietic stem cells.

Further, in the artificial blood vessel according to the present invention, the bioactive substance-containing gel solution is at least one selected from the group consisting of a collagen gel solution, a fibrin gel solution, an acrylamide gel solution, an agarose gel solution, and an alginate gel solution. It is preferable that the gel solution contains a bioactive substance.

In the method for producing an artificial blood vessel according to the present invention, the solution to be gelled may be at least one solution selected from the group consisting of a collagen solution, a fibrin solution, an acrylamide solution, an agarose solution, and an alginic acid solution. preferable. Further, in the artificial blood vessel according to the present invention, an endothelial cell layer may be formed on a lumen surface of the porous artificial blood vessel base material.

Further, in the method for producing an artificial blood vessel according to the present invention, the porous artificial blood vessel base material may be formed by gelling the biologically active substance-containing solution taken into the pores of the porous artificial blood vessel base material. The method may further include the step of forming an endothelial cell layer. Further, in the present invention, the porous artificial blood vessel base material may be strengthened by a reinforcing member made of a biodegradable material or a non-biodegradable material. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a scanning electron microscope observation image of the inner cavity surface of the artificial blood vessel prepared in Example 1.

FIG. 2 is a scanning electron microscope observation image of the inner cavity surface of the artificial blood vessel prepared in Comparative Example 1.

FIG. 3 is a scanning electron microscope observation image of the lumen surface of the PLLA substrate prepared in Reference Example.

FIG. 4 is a cross-sectional image of the artificial blood vessel prepared in Example 1 by a fluorescence microscope. FIG. 5 is a cross-sectional image of the artificial blood vessel prepared in Comparative Example 1 by a fluorescence microscope. FIG. 6 is a cross-sectional image of the artificial blood vessel prepared in Example 2 observed by a transmission microscope. FIG. 7 is a fluorescent microscope cross-sectional image of the artificial blood vessel prepared in Example 2. BEST MODE FOR CARRYING OUT THE INVENTION

The artificial blood vessel according to the present invention has a cylindrical porous artificial blood vessel base material, and contains a bioactive substance-containing gel solution in its pores.

First, the porous artificial blood vessel base material used in the present invention will be described. The porous artificial blood vessel base material used in the present invention is porous and preferably has an open pore structure in which the pores are connected from the viewpoint of facilitating the incorporation of a bioactive substance into the pores. The percentage of pores is preferably from 70% to 99% of the base material, and more preferably from 80% to 95%.

Further, the porous artificial blood vessel substrate used in the present invention has a cylindrical shape.For example, when used as a small-diameter artificial blood vessel, the inner diameter is preferably 1 mm to 7 mm, and 1 mm to 5 mm. More preferably, it is still more preferably 1 mm to 4 mm.

Conventionally, in the treatment of arteriosclerosis, vasospasm, peripheral blood vessel damage, aneurysm formation, tumor invasion and other small-diameter vascular diseases, re-transplantation of autologous blood vessels at other sites or disease If the condition was systemic and autologous blood vessel transplantation was not appropriate, the limb was amputated and treated. When the artificial blood vessel according to the present invention is used as a small-diameter artificial blood vessel, even in the treatment of the above-mentioned vascular disease, it is possible to perform the treatment without performing the conventional treatment, thereby improving the urgency of surgery, It is preferable in consideration of the burden on the patient. When used as a large-diameter artificial blood vessel, the inner diameter is preferably from 5 mm to 70 mm, more preferably from 5 mm to 50 mm.

Examples of the material of the porous artificial blood vessel base material used in the present invention include a biodegradable material and a non-biodegradable material. A biodegradable material is preferable from the viewpoint of providing an artificial blood vessel in which components that are not derived from the living body are not dissolved after transplantation in consideration of foreign body reaction, immune reaction, and carcinogenicity in the living body. In addition, non-biodegradable materials are preferred from the viewpoint of maintaining mechanical strength because they do not dissolve after transplantation, and providing stable products at the current stage and at the current technological development level. . 02 10986

6 The biodegradable material is not particularly limited, but includes polydalicholic acid, polylactic acid, a copolymer of lactate and glycolic acid, polymalic acid, poly-coprolataton, and ε coprolataton and lactic acid. Polymers, copolymers of e-proprotatanone and glyco- / reic acid, copolymers of ε-proprotatanone, lactic acid and glycolic acid, poly-3-hydroxybutyrate, poly-1-hydroxybutyrate, Copolymers of 3-hydroxybutyrate and 4-hydroxybutyrate can be mentioned. From the viewpoint of the approval of the Ministry of Health, Labor and Welfare and the FDA (United States Drug Administration), polylactic acid, polyglycolic acid, and lactic acid And a copolymer of glycolic acid and glycolic acid.

The non-biodegradable material is not particularly limited, but is a polyethylene resin, a polypropylene resin, a polybutadiene resin, a polystyrene resin, a polyvinyl chloride resin, a polyacryl resin, a polymethacryl resin, a polysulfone resin. And polytetrafluoroethylene-based resin. From the viewpoint of approval by the Ministry of Health, Labor and Welfare and the FDA, polyethylene resin and polytetrafluoroethylene-based resin are preferable.

As a method for producing a tubular porous artificial blood vessel base material used in the present invention, for example, a foaming agent such as sodium chloride, ammonium hydrogencarbonate, or the like is mixed with a solution of the above-described material and molded into a tubular shape. After that, a method of foaming to make it porous can be mentioned. Such a method includes, for example, the method described in Nam et al. J. Biomed. Master Res. 2000, 53, pi-7.

Next, the bioactive substance-containing gel solution contained in the pores of the porous artificial blood vessel substrate will be described.

The gel solution containing a biologically active substance used in the present invention is a gel solution containing a biologically active substance, and it is preferable that the biologically active substance is uniformly mixed.

Examples of the biologically active substance contained in the gel solution include cells, pharmaceutically active ingredients, and the like, and one or more of these may be used. Such a biologically active substance can be appropriately determined according to the use of the artificial blood vessel.

As used herein, a pharmaceutically active ingredient refers to a component that exhibits one or more pharmacological activities in a living body, such as a physiologically active substance synthesized in a living body, and a physiologically active substance thereof. A gene encoding a sex substance, a pharmaceutical compound synthesized or obtained in vitro, and the like.

Cells used as biologically active substances include, for example, undivided cells such as stem cells and ES cells, smooth muscle cells, fibroblasts, hepatocytes, victory cells, and hematopoietic stem cells. it can.

Examples of the physiologically active substance used as a biologically active substance include proteins synthesized in vivo, such as insulin, anti-prion antibody, and anti-AIDS virus antibody. As an artificial blood vessel containing such a physiologically active substance as a biologically active substance, there is an application such as an artificial blood vessel that also treats diseases such as diabetes and infectious disease.

Examples of the gene used as a biologically active substance include a gene encoding the above-mentioned physiologically active substance, such as a gene encoding adenosine deaminase, a gene encoding an eighth blood coagulation factor, 9 Genes encoding blood coagulation factors, and the like. As an artificial blood vessel containing a gene as a bioactive substance, there is an application as an artificial blood vessel that also serves to treat diseases such as adenosine deaminase deficiency and hemophilia.

In addition, a pharmaceutical compound synthesized or obtained in vitro can be used as a biologically active substance. Examples of such pharmaceutical compounds include organic compounds, antibiotics produced by microorganisms existing in nature, and the like. Specific examples include penicillin, cyclophosphamide, actinomycin D, and 5-f / leuroushi / And pleomycin. An artificial blood vessel containing such a pharmaceutical compound as a biologically active substance has a use as an artificial blood vessel which also serves as an infectious disease, an anticancer treatment and the like.

When the porous artificial blood vessel base material is made of a biodegradable material, the gel solution contains at least smooth muscle cells and blast cells from the viewpoint of securing the mechanical strength after the artificial blood vessel transplantation. Preferably, undifferentiated cells are included.

When the bioactive substance is cells, the density of the bioactive substance to be mixed with the gel solution is preferably 1 × 10 ⁴ eell Zml to l × 10 ⁹ cel 1 Zm1, and 1 × 10 0 and still more preferably ⁶ a ce 1 1 / m 1~1 X 1 0 7 ce 1 1 Zm 1. The gel solution is a solution obtained by gelling a solution that gels due to changes in temperature, H, salt concentration, enzymatic reaction, etc., and is not particularly limited as long as it has no toxicity or carcinogenicity. it can. Examples of the gelling agent corresponding to the temperature change include collagen, acrylamide resin, and agarose. In addition, collagen that can be gelled in response to a pH change can be exemplified. Alginic acid or the like can be used as a gel that responds to changes in salt concentration. In addition, fibrin and the like can be mentioned as those that gel in response to the enzymatic reaction, and fibrinogen and the like can be mentioned as the enzyme to be gelled.

In the artificial blood vessel of the present invention, a gel solution containing a biologically active substance is taken into the pores of the porous artificial blood vessel base material. The bioactive substance-containing gel solution preferably occupies 50% or more of all pores of the porous artificial vascular base material, and 90%, from the viewpoint of preventing blood leakage and securing a scaffold for vascular endothelial cells. It is more preferable to account for the above.

In the artificial blood vessel according to the present invention, an endothelial cell layer may be formed on the lumen surface of the porous artificial blood vessel base material from the viewpoint of improving antithrombotic properties.

In the artificial blood vessel according to the present invention, in order to further improve the mechanical strength, the porous artificial blood vessel base material is strengthened by a strengthening member made of a biodegradable material or a non-biodegradable material. You may. The reinforcing member may be formed on the outer peripheral surface and / or the inner peripheral surface of the porous artificial blood vessel base material, or may be incorporated inside the porous artificial blood vessel base material. The reinforcing member formed on the outer peripheral surface of the porous artificial blood vessel base material has a substantially same inner diameter as the outer diameter of the porous artificial blood vessel base material, and has a cross section concentric with the base material. A coated tubular member can be mentioned. In addition, the capturing member formed on the inner peripheral surface of the porous artificial blood vessel base material has an outer diameter substantially equal to the inner diameter of the porous artificial blood vessel base material, and has a cross section concentric with the base material. And a cylindrical member covered with a base material. In any case, a material formed by weaving a fibrous biodegradable material or a non-biodegradable material into a mesh shape can be used. In addition, by incorporating such a mesh-shaped reinforcing member in advance at the time of molding the porous artificial blood vessel base material, the capturing member can also be incorporated into the porous artificial blood vessel base material. The biodegradation Examples of the porous material and the non-biodegradable material include those described above as the material for the porous artificial blood vessel base material.

Next, a method for producing an artificial blood vessel according to the present invention will be described.

First, a biologically active substance is mixed with a solution that gels in response to any of a temperature change, a pH change, a salt concentration change, and an enzyme reaction change to prepare a biologically active substance-containing solution. The biologically active substance to be mixed can be appropriately determined according to the use of the artificial blood vessel, and includes the substances described above. The density of the biological agent to be mixed in the solution to gel, if the biological agent is a cell is a ^{1 X 1 0 4 ce 1 l} Zm l~1 X 1 0 9 ce 1 1 Zm 1 It is more preferably 1 × 10 ⁶ ce 1 l / ml to 1 × 10 ⁷ ce 11 Zm 1.

Subsequently, the porous artificial vascular base material prepared in advance is impregnated with the solution containing the biologically active substance prepared as described above, and the biologically active substance is introduced into the pores of the substrate under normal pressure conditions, preferably under negative pressure conditions. Allow the containing solution to be incorporated.

The material and manufacturing method of the porous artificial blood vessel base material are as described above. Further, the porous artificial blood vessel base material may be captured by a capturing member. The reinforcing member and the reinforcing method are as described above.

The method for impregnating the porous artificial vascular base material into the biologically active substance-containing solution is as follows. There are several methods. In order to incorporate the biologically active substance into the substrate, the biologically active substance is impregnated until the biologically active substance is sufficiently incorporated into the pores of the porous artificial blood vessel substrate. For example, it is preferable that the bioactive substance-containing solution is impregnated with the porous artificial blood vessel substrate, and is left standing for 10 to 60 minutes, more preferably 30 to 60 minutes.

The pressure at the time of addition may be normal pressure, but from the viewpoint of efficiently incorporating the bioactive substance into the pores of the substrate, a pressure lower than the atmospheric pressure is preferable. This pressure is appropriately determined in consideration of the materials to be used, the pressure resistance of the equipment, the possibility of retaining the activity of the biologically active substance to be used, and the like.

Subsequently, the biologically active substance-containing solution taken into the pores of the porous artificial blood vessel substrate is gelled. The gelling conditions are set appropriately according to the solution to be used. Gelled In this case, from the viewpoint of forming a smooth lumen surface, it is preferable to further allow the mixture to stand for 10 minutes to 120 minutes under gelation conditions, and more preferably to allow the mixture to stand still for 30 minutes to 60 minutes. .

In the method for producing an artificial blood vessel according to the present invention, an endothelial cell layer can be formed on the luminal surface of the porous artificial blood vessel base material. The endothelial cell layer is easily formed by pouring a culture solution containing endothelial cells into the lumen of a porous artificial blood vessel substrate holding a solution containing a bioactive substance that has undergone genoleration in the pores and rotating the culture. be able to.

The number of rotations during the culture of endothelial cells is preferably from 0.5 rpm to 100 rpm, more preferably from 5 rpm to 10 rpm. The culture time is preferably from 10 minutes to 24 hours, and more preferably from 20 minutes to 60 minutes.

ADVANTAGE OF THE INVENTION According to this invention, the bioactive substance is contained at high density in the hole of a cylindrical porous artificial blood vessel base material, blood leakage does not occur, the lumen surface is almost smooth, and the porous artificial blood vessel Since it has a base material, an artificial blood vessel having excellent mechanical properties can be provided. Further, according to the present invention, a biologically active substance can be efficiently and rapidly incorporated into an artificial blood vessel in a short time. Example

Hereinafter, the present invention will be described specifically with reference to Examples.

Reference Example (Preparation of porous artificial blood vessel base material)

After 320 mg of polylactic acid having a weight-average molecular weight of about 300,000 was completely dissolved in 4 ml of black form and made liquid, 5 g of ammonium hydrogencarbonate crystals were mixed. After pouring the above-mentioned polymer into a mold with an inner diameter of 5 mm and an outer diameter of 7 mm and shaping it, remove the polymer from the mold and remove the polymer from the mold at 24 hours at 50 ° C to completely evaporate the form. Heated. Next, in order to produce a three-dimensional sponge structure, the cylindrical polymer was heated at 80 ° C for 10 minutes to elute ammonium carbonate. After thoroughly washing the cylindrical polymer with ultrapure water, freeze-dry it to obtain an inner diameter of about 5 mm and an outer diameter of about 7 A porous artificial blood vessel substrate (hereinafter, referred to as “PLLA substrate”) composed of polylactic acid with a length of about 2 mm and a length of about 2 Omm was prepared.

Example 1

A solution was prepared by mixing human normal aortic vascular smooth muscle cells at a concentration of 5 × 10 ⁶ cells / ml with a 0.39% collagen solution. Next, the PLLA substrate prepared in the reference example was placed in a glass tube and immersed in 1.4 ml of the above-mentioned collagen solution containing human normal aortic vascular smooth muscle cells, and this was pumped with a hydraulic pump (ULVAC, G The pressure was reduced according to -5), and the mixture was allowed to stand under negative pressure for 30 minutes. Subsequently, after inserting a tube with an outer diameter of 5 mm into the center of the PLLA substrate, the tube is moved to an incubator at 37 ° C and left standing for 60 minutes to gel the collagen solution in the PLLA substrate. Then, an artificial blood vessel was prepared.

Comparative Example 1

Human normal aortic vascular smooth muscle cells were mixed with a culture solution (Dulbecco's minimum essential medium (DMEM)) to prepare a cell-containing solution having a concentration of 5 × 10 ⁶ cells / ml. Next, the PLLA substrate prepared in Reference Example was placed in a glass tube, immersed in 1.4 ml of the above-described culture medium containing human normal aortic vascular smooth muscle cells, and this was immersed in a hydraulic pump ( , G-5) and allowed to stand under negative pressure for 30 minutes. Subsequently, after inserting a tube having an outer diameter of 5 mm into the center of the PLLA substrate, the tube was moved to an incubator at 37 ° C and left standing for 60 minutes to produce an artificial blood vessel.

(1) Comparison of the number of cells taken up

The numbers of human normal aortic vascular smooth muscle cells taken into the artificial blood vessels prepared in Example 1 and Comparative Example 1 were compared. For comparison, calculate the ratio (%) of the number of cells in the solution after immersing the PLLA substrate to the number of cells in the human normal aortic vascular smooth muscle cell solution before immersing the PLLA substrate. It was done by doing. The number of cells was determined by calculating the number of nuclei developed with 4,, 6-diamidino 2-phenylindole dihydrochloride (DAP I) using a fluorescence spectrophotometer. Since the immersion time is 30 minutes, smooth muscle cells do not proliferate during this time. Table 1 below shows the percentage of cells taken up. Example 1 Comparative Example 1 Percentage of cells taken up (%) 100% 18%

From Table 1, it can be seen that the artificial blood vessel prepared in Example 1 takes up cells extremely efficiently as compared to the artificial blood vessel prepared in Comparative Example 1.

(2) Comparison of the condition of the lumen surface of the artificial blood vessel

The state of the luminal surface of the artificial blood vessel prepared in Example 1 and Comparative Example 1 was examined. In order to stabilize the cells, each artificial blood vessel was further cultured for one day and used for observation. A scanning electron microscope (SEM) photograph (scale 100 m) of the luminal surface of the artificial blood vessel prepared in Example 1 is shown in FIG. Fig. 2 shows an EM photograph, and Fig. 3 shows an SEM photograph of the state of the lumen surface of the PLLA substrate prepared in Reference Example for reference. From FIGS. 1 and 2, the luminal surface of the artificial blood vessel of Comparative Example 1 is smooth without any holes, while the luminal surface of the artificial blood vessel of Comparative Example 1 is still large. I understand.

(3) Comparison of cell density on the luminal side of artificial blood vessel

The cell density on the luminal surface side of the artificial blood vessel prepared in Example 1 and Comparative Example 1 was examined. In order to stabilize the cells, each artificial blood vessel was further cultured for one day, and then a cross-sectional section (8 μππι thick) of each artificial blood vessel was prepared. The nucleus of human normal aortic vascular smooth muscle cells was Hoechst ( Hextone earth) (100 ηg / m1). FIG. 4 shows a fluorescence photograph of a cross section of the artificial blood vessel produced in Example 1, and FIG. 5 shows a fluorescence photograph of a cross section of the artificial blood vessel produced in Comparative Example 1. The dotted line in the figure indicates the lumen surface. 4 and 5 that the artificial blood vessel prepared in Example 1 'incorporates cells at a higher density than the artificial blood vessel prepared in Comparative Example 1. '

Example 2

A culture solution containing normal human umbilical vein vascular endothelial cells at a concentration of 5.0 × 10 ⁶ cells / ml was prepared in a volume of 1 ml, and poured into the lumen of the artificial blood vessel prepared in Example 1. Using a rotary culture device (Titech, RT-50), 5111 to 10 ^) An endothelial cell layer was formed by culturing for 60 minutes while rotating at a rotating speed.

(4) endothelial cell layer on lumen surface of artificial blood vessel

The state of the endothelial cells formed on the luminal surface of the artificial blood vessel prepared in Example 2 was examined. To stabilize the cell, after further 1 day culture artificial blood vessel, to produce the artificial blood vessel cross sections (thickness 8 _W m), and stained endothelial cells in Asechiru of low density lipoprotein (LDL). Fig. 6 shows a transmission micrograph of the cross section of the artificial blood vessel, and Fig. 7 shows a fluorescence photograph of the same cross section. The arrow in FIG. 7 indicates the endothelial cell layer. 6 and 7 that the endothelial cell layer is continuously formed on the luminal surface of the artificial blood vessel prepared in Example 2.

As described above, according to the present invention, it is possible to smooth the minimum cavity surface in direct contact with blood, which is indispensable for an artificial blood vessel, in a short period of time, and to efficiently and efficiently produce a bioactive substance in a short time. Incorporation into blood vessels is possible.

Further, according to the present invention, it is possible to provide an artificial blood vessel which does not cause blood leakage, has excellent antithrombotic properties, has a substantially smooth lumen surface, and has excellent mechanical properties.

Claims

The scope of the claims

1. In an artificial blood vessel having a cylindrical porous artificial blood vessel base material,

An artificial blood vessel obtained by impregnating the pores of the porous artificial blood vessel substrate with a gel solution containing a biologically active substance.

2. The artificial blood vessel according to claim 1, wherein the porous artificial blood vessel base material is made of a biodegradable material or a non-biodegradable material.

3. The biodegradable material is polylactic acid, polyglycolic acid, a copolymer of lactic acid and glycolic acid, polymalic acid, poly- ε-force prolactone, a copolymer of ε-force prolatataton and lactic acid, ε -Copolymer of prolactatone and glycolic acid, ε-Copolymer of prolactone and lactic acid and glycolic acid, poly-3-hydroxybutyrate, poly-4-hydroxybutyrate, and 3. The artificial blood vessel according to claim 2, wherein the artificial blood vessel is at least one material selected from the group consisting of a copolymer of 3-hydroxybutyrate and 4-hydroxybutyrate.

4. The non-biodegradable material is a polyethylene-based resin, a polypropylene-based resin, a polybutadiene-based resin, a polystyrene-based resin, a polychlorinated vinyl-based resin, a polyataryl-based resin, a polymethacryl-based resin, a polysulfone-based resin, and 3. The artificial blood vessel according to claim 2, wherein the artificial blood vessel is at least one material selected from the group consisting of polytetrafluoroethylene resins.

5. The artificial blood vessel according to any one of claims 1 to 4, wherein the biologically active substance is a cell and / or a pharmaceutically active ingredient.

6. The cell according to claim 5, wherein the cell is at least one cell selected from the group consisting of a smooth muscle cell, a fibroblast, an undifferentiated cell, a hepatocyte, a knee cell, and a hematopoietic stem cell. An artificial blood vessel as described.

7. The bioactive substance-containing gel solution is one or more gel solutions selected from the group consisting of a collagen gel solution, a fibrin gel solution, an acrylamide gel solution, an agarose gel solution, and an alginate gel solution, The artificial blood vessel according to any one of claims 1 to 6, wherein the artificial blood vessel is contained.

8. The artificial blood vessel according to any one of claims 1 to 7, wherein an endothelial cell layer is formed on a luminal surface of the porous artificial blood vessel base material.

9. The porous artificial blood vessel base material is strengthened by a reinforcing member made of a biodegradable material or a non-biodegradable material. The artificial blood vessel according to the above.

10. In the method for producing an artificial blood vessel having a cylindrical porous artificial blood vessel substrate, a solution that gels in response to any of temperature change, pH change, salt concentration change, and enzyme reaction change is used. Preparing a biologically active substance-containing solution by impregnating the biologically active substance;

Incorporating the biologically active substance-containing solution into the pores of the porous artificial blood vessel substrate under normal pressure or negative pressure conditions,

Gelling the biologically active substance-containing solution taken into the pores of the porous artificial blood vessel substrate;

A method for producing an artificial blood vessel, comprising:

11. The method for producing an artificial blood vessel according to claim 10, wherein the porous artificial blood vessel base material is formed from a biodegradable material or a non-biodegradable material.

12. The biodegradable material is polylactic acid, polyglycolic acid, a copolymer of lactic acid and glycolic acid, polymalic acid, poly-ε-force prolactone, or a copolymer of ε-force prolactone and lactic acid. A copolymer of ε-force prolactone and glycolic acid, a copolymer of ε-force prolactone, lactic acid and glycolenoic acid, poly-3-hydroxybutyrate, poly-4-hydroxybutyrate, The method according to claim 11, wherein the method is at least one material selected from the group consisting of a copolymer of 3-hydroxybutyrate and 4-hydroxybutyrate. .

13. The non-biodegradable material is a polyethylene resin, a polypropylene resin, a polybutadiene resin, a polystyrene resin, a polychlorinated vinyl resin, a polyacrylic resin, a polymethacrylic resin, a polysulfone resin, and a polyethylene resin. 12. The method for producing an artificial blood vessel according to claim 11, wherein the method is at least one material selected from the group consisting of trifluoroethylene-based resins.

14. The method for producing an artificial blood vessel according to any one of claims 10 to 13, wherein the biologically active substance is a cell and / or a pharmaceutically active ingredient.

15. The cell, wherein the cell is at least one cell selected from the group consisting of a smooth muscle cell, a fibroblast, an undifferentiated cell, a hepatocyte, a knee cell, and a hematopoietic stem cell. 14. The method for producing an artificial blood vessel according to item 14.

16. The solution according to claim 10, wherein the solution to be gelled is at least one solution selected from the group consisting of a collagen solution, a fibrin solution, an acrylamide solution, an agarose solution, and an alginic acid solution. 15. The method for producing an artificial blood vessel according to any one of 15.

17. The method for producing an artificial blood vessel according to any one of claims 10 to 16, wherein the bioactive substance-containing solution gelled in the pores of the porous artificial blood vessel base material is gelated. A method for producing an artificial blood vessel, further comprising a step of forming an endothelial cell layer on a lumen surface of a porous artificial blood vessel base material.

18. The method for producing an artificial blood vessel according to any one of claims 10 to 17, wherein the porous artificial blood vessel base material is a reinforcing member made of a biodegradable material or a non-biodegradable material. A method for producing an artificial blood vessel, wherein the artificial blood vessel is reinforced.