Artificial blood vessel, method for producing artificial blood vessel, and method for producing porous tissue regeneration substrate

Abstract

The present invention aims to provide a method for producing a porous tissue regeneration substrate that allows a wide choice of solvents and easy adjustment of the bulk density and pore size of the porous substrate. The present invention also aims to provide a method for producing an artificial blood vessel and an artificial blood vessel. The present invention relates a porous, tubular artificial blood vessel containing a bioabsorbable material, the artificial blood vessel including: a skin layer having a relatively small pore size as an innermost layer; and a porous layer positioned around the skin layer and having a relatively large pore size.

Claims

1. A method for producing a porous, tubular artificial blood vessel containing a bioabsorbable material, the method comprising: a dissolving step of preparing a uniform solution containing a bioabsorbable polymer dissolved therein using a bioabsorbable polymer, a solvent 1 having a relatively low solvency for the bioabsorbable polymer, a solvent 2 having a relatively high solvency for the bioabsorbable polymer and being incompatible with the solvent 1, and a common solvent 3 compatible with the solvent 1 and the solvent 2; an applying step of applying the uniform solution to a surface of a rod-shaped body; a precipitating step of cooling the uniform solution on the surface of the rod-shaped body to precipitate a tubular porous body containing the bioabsorbable polymer around the rod-shaped body; and a freeze-drying step of freeze-drying the tubular porous body to give a tubular artificial blood vessel.

2. The method for producing an artificial blood vessel according to claim 1, wherein the rod-shaped body contains a metal.

3. The method for producing an artificial blood vessel according to claim 1, further comprising the step of discharging ultrafine fibers containing a bioabsorbable material on the surface of the tubular porous body by electrospinning to form an ultrafine fiber nonwoven fabric layer on the tubular porous body.

4. A method for producing a porous tissue regeneration substrate containing a bioabsorbable polymer, the method comprising: a dissolving step of preparing a uniform solution containing a bioabsorbable polymer dissolved therein using a bioabsorbable polymer, a solvent 1 having a relatively low solvency for the bioabsorbable polymer, a solvent 2 having a relatively high solvency for the bioabsorbable polymer and being incompatible with the solvent 1, and a common solvent 3 compatible with the solvent 1 and the solvent 2; a precipitating step of cooling the uniform solution to precipitate a porous body containing the bioabsorbable polymer; and a freeze-drying step of freeze-drying the porous body containing the bioabsorbable polymer to give a porous tissue regeneration substrate.

5. The method for producing a porous tissue regeneration substrate according to claim 4, wherein two or more common solvents 3 are used, and a pore size of the resulting porous body is controlled by adjusting a mixing ratio between the two or more common solvents 3.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows electron micrographs of a cross section of a tubular artificial blood vessel obtained in Experiment Example 4.

(2) FIG. 2 shows an image of a blood vessel regenerated using the tubular artificial blood vessel obtained in Experiment Example 4, stained with HE.

(3) FIG. 3 shows an image of a blood vessel regenerated using the tubular artificial blood vessel obtained in Experiment Example 4, stained with von Kossa.

(4) FIG. 4 shows an electron micrograph of a cross section of an artificial blood vessel in which a skin layer is on the inner side and the pore size of a porous layer around the skin layer increases outward.

DESCRIPTION OF EMBODIMENTS

(5) The embodiments of the present invention will be described in more detail with reference to examples. The present invention however is not limited to these examples.

EXPERIMENT EXAMPLE 1

(6) (1) Production of Porous Tissue Regeneration Substrate

(7) At a room temperature of 25 C., 0.25 g of a L-lactide--caprolactone copolymer (molar ratio: 50:50) was mixed with a mixed solution containing 0.3 mL of water as the solvent 1, 2.0 mL of methyl ethyl ketone as the solvent 2, 1.0 mL of a mixture of acetone (common solvent 3-1) and ethanol (common solvent 3-2) as the common solvent 3. Thus, a non-uniform solution not dissolving the L-lactide--caprolactone copolymer was obtained.

(8) Subsequently, the obtained non-uniform solution was put in a glass tube with a diameter of 3.3 mm and heated at 60 C. to give a uniform solution containing the L-lactide--caprolactone copolymer dissolved therein.

(9) The obtained uniform solution was then cooled to 4 C. or 24 C. in a freezer to precipitate a porous body containing the L-lactide--caprolactone copolymer.

(10) The obtained porous body was immersed in an ethanol bath (50 mL) at 4 C. or 24 C. for 12 hours, and then immersed in a water bath (50 mL) at 25 C. for 12 hours for washing.

(11) Thereafter, the porous body was freeze-dried at 40 C. to give a cylindrical porous tissue regeneration substrate with a diameter of 3.0 mm and a height of 15 mm.

(12) Porous tissue regeneration substrates were produced at two different ratios between the common solvent 3-1 and the common solvent 3-2, 0.8:0.2 and 0.5:0.5.

(13) (2) Measurement of Pore Size and Bulk Density of Porous Substrate

(14) The pore size and bulk density of the obtained porous substrates were measured by the following method.

(15) Table 1 shows the results.

(16) (2-1) Measurement of Pore Size

(17) The cylindrical porous tissue regeneration substrate was cut, and an electron micrograph in the vicinity of the middle of the obtained cross section was taken at 1,000- or 8,000-fold magnification. The pore diameter (major axis) was measured at random 10 sites in the obtained electron microscopic image and the average was taken as the average pore size.

(18) (2-2) Measurement of Bulk Density

(19) The volume and weight of the obtained blood vessel substrate was measured. The mass was divided by the volume to determine the bulk density. Three measurements were performed for each substrate, and the average was taken as the bulk density.

(20) TABLE-US-00001 TABLE 1 Common solvent 3-1:Common solvent 3-2 0.8:0.2 0.5:0.5 Cooling Average pore size (m) 16.5 10.5 temperature Bulk density (kg/m.sup.3) 170 180 4 C. Cooling Average pore size (m) 80.6 5.54 temperature Bulk density (kg/m.sup.3) 250 230 24 C.

EXPERIMENT EXAMPLE 2

(21) At a room temperature of 25 C., 0.5 g of polylactide was mixed with a mixed solution containing 0.15 mL of water as the solvent 1, 6.0 mL of chloroform as the solvent 2, and 1.0 mL of a mixture of tetrahydrofuran (common solvent 3-1) and ethanol (common solvent 3-2) as the common solvent 3 while heating at 60 C. Thus, a uniform solution containing the polylactide dissolved therein was obtained.

(22) Subsequently, the obtained uniform solution was cooled to 80 C. in a freezer to precipitate a porous body containing the polylactide.

(23) The obtained porous body was immersed in an ethanol bath (50 mL) at 70 C. for 12 hours, and then immersed in a water bath (50 mL) at 25 C. for 12 hours for washing.

(24) The porous body was then freeze-dried at 40 C. to give a porous tissue regeneration substrate.

(25) Porous tissue regeneration substrates were produced at two different ratios between the common solvent 3-1 and the common solvent 3-2, 0.9:0.1 and 0.1:0.9.

(26) The pore size and bulk density of the obtained porous substrates were measured in the same manner as in Experiment Example 1.

(27) Table 2 shows the results.

(28) TABLE-US-00002 TABLE 2 Common solvent 3-1:Common solvent 3-2 0.9:0.1 0.1:0.9 Average pore size (m) 0.86 1.80 Bulk density (kg/m.sup.3) 229 236

EXPERIMENT EXAMPLE 3

(29) While heating at 60 C., 0.5 g of polylactide was dissolved in chloroform as the solvent 2. Subsequently, while continuing heating, acetone (common solvent 3-1) and then ethanol (common solvent 3-2) as the common solvent 3 were added in a total amount of 2.8 mL. Further, 0.22 mL of water as the solvent 1 was added, thus a uniform solution was obtained. The obtained uniform solution was cooled to 80 C. in a freezer to precipitate a porous body containing the polylactide.

(30) The obtained porous body was immersed in an ethanol bath (50 mL) at 70 C. for 12 hours, and then immersed in a water bath (50 mL) at 25 C. for 12 hours for washing.

(31) Thereafter, the porous body was freeze-dried at 40 C. to give a porous tissue regeneration substrate.

(32) Porous tissue regeneration substrates were produced at two different ratios between the common solvent 3-1 and the common solvent 3-2, 1.8:1.0 and 1.0:1.8.

(33) The pore size and bulk density of the obtained porous substrates were measured in the same manner as in Experiment Example 1.

(34) Table 3 shows the results.

(35) TABLE-US-00003 TABLE 3 Common solvent 3-1:Common solvent 3-2 1.8:1.0 1.0:1.8 Average pore size (m) 9.8 65.2 Bulk density (kg/m.sup.3) 177 166

EXPERIMENT EXAMPLE 4

(36) (1) Production of Artificial Blood Vessel

(37) At a room temperature of 25 C., 0.25 g of an L-lactide--caprolactone copolymer (molar ratio: 50:50) was mixed with a mixed solution containing 0.2 mL of water as the solvent 1, 2.5 mL of methyl ethyl ketone as the solvent 2, and 0.8 mL of acetone and 0.2 mL of ethanol as the common solvent 3. Thus, a non-uniform solution not dissolving the L-lactide--caprolactone copolymer was obtained.

(38) Subsequently, the obtained non-uniform solution was heated at 60 C. to give a uniform solution containing the L-lactide--caprolactone copolymer dissolved therein.

(39) A fluorine-coated stainless steel rod-shaped body with a diameter of 0.6 mm was placed in a glass tube with an inner diameter of 1.1 mm. The uniform solution was poured into the gap between the rod-shaped body and the glass tube. The uniform solution in this state was cooled to 30 C. in a freezer to precipitate a porous body containing the L-lactide--caprolactone copolymer around the rod-shaped body. The obtained porous body was immersed in an ethanol bath (50 mL) at 30 C. for 12 hours, and then immersed in a water bath (50 mL) at 25 C. for 12 hours for washing.

(40) Thereafter, the porous body was freeze-dried at 40 C. to give a tubular porous body.

(41) Polyglycolide and polylactide were separately dissolved into hexafluoroisopropanol to prepare a hexafluoroisopropanol solution with a polyglycolide concentration of 10% by weight and a hexafluoroisopropanol solution with a polylactide concentration of 10% by weight.

(42) The rod-shaped body with the tubular porous body therearound was used as a collector electrode. The hexafluoroisopropanol solutions were discharged onto the surface of the rod-shaped body using an electrospinning device. At this time, the hexafluoroisopropanol solutions prepared above were charged into two different nozzles, and discharged while rotating the rod-shaped body and reciprocating the nozzles multiple times. Thus, an ultrafine fiber nonwoven fabric layer was formed.

(43) The electrospinning was performed under the conditions of a voltage of 40 kV and a nozzle size of 23 G.

(44) Finally, the rod-shaped body was pulled out to give a tubular artificial blood vessel with an external diameter of about 1,090 m and an inner diameter of about 610 m.

(45) FIG. 1 shows electron micrographs of a cross section of the obtained tubular artificial blood vessel.

(46) The tubular artificial blood vessel had a three-layer structure composed of a skin layer having a relatively small pore size (average pore size of 4.3 m as measured in the same manner as in Experiment Example 1) as the innermost layer, a porous layer positioned around the skin layer and having a relatively large pore size (average pore size of 23.2 m as measured in the same manner as the skin layer), and an ultrafine fiber nonwoven fabric layer on the porous layer.

(47) (2) Evaluation of Blood Vessel Tissue Regeneration Performance

(48) The artificial blood vessel obtained in Experiment Example 4 was evaluated by an animal test by the following method. Part of the abdominal aorta of mice was removed and replaced with the artificial blood vessel obtained in Experiment Example 4. Ten mice were tested. At the time of week 8 after the operation, all the ten mice were alive and no blood vessel blockage was observed at all.

(49) At week 8 after the operation, the mice were euthanized by intraperitoneal administration of excess pentobarbital, and the graft sites were harvested. FIG. 2 shows a micrograph image of one of the obtained specimens stained with hematoxylin-eosin (HE). FIG. 3 shows a micrograph image of one of the obtained specimens stained with von Kossa for calcification evaluation.

(50) FIGS. 2 and 3 show regeneration of quite normal blood vessels without hypertrophy and calcification.

INDUSTRIAL APPLICABILITY

(51) The present invention provides a method for producing a porous tissue regeneration substrate that allows a wide choice of solvents and easy adjustment of the bulk density and pore size of the porous substrate. The present invention also provides a method for producing an artificial blood vessel and an artificial blood vessel.