KR102241492B1 - Artificial blood vessels of double layer structure formed by nanofibers with different arrangements and a preparation method thereof - Google Patents

Abstract

The present invention is an inner wall formed of nanofibers uniaxially arranged in parallel with the direction of blood flow; And an outer wall formed of randomly arranged nanofibers, wherein the nanofibers forming the inner wall include a biodegradable synthetic polymer and a natural polymer composite, and the nanofibers forming the outer wall are biodegradable. An outer wall composed of a synthetic polymer and a ceramic composite; It relates to an artificial blood vessel having a bilayer structure of nanofibers having different arrangements, and a method of manufacturing the same. Specifically, it was confirmed that the biocompatibility and regeneration rate of vascular endothelial cells were improved by introducing natural polymers and uniaxially arranging the nanofibers in the bloodstream direction in the inner wall. In addition, it was confirmed that ceramic particles were evenly dispersed and randomly arranged in the nanofibers forming the outer wall, thereby enhancing biocompatibility and reinforcing physical properties to resemble actual blood vessels.

Description

[Artificial blood vessels of double layer structure formed by nanofibers with different arrangements and a preparation method thereof]

The present invention is an inner wall formed of nanofibers uniaxially arranged in parallel with the direction of blood flow; And an outer wall formed of randomly arranged nanofibers, wherein the nanofibers forming the inner wall include a biodegradable synthetic polymer and a natural polymer composite, and the nanofibers forming the outer wall are biodegradable. An outer wall composed of a synthetic polymer and a ceramic composite; It relates to an artificial blood vessel having a bilayer structure of nanofibers having different arrangements, and a method of manufacturing the same.

Cardiovascular diseases such as blockage of coronary arteries or peripheral arteries due to blood clots due to heart disease or accumulation of cholesterol, etc. account for 31% of the world's mortality. The incidence of cardiovascular disease is gradually increasing due to the increase of the elderly population and unhealthy eating habits. In order to treat this, vascular bypass surgery and vascular graft surgery, which facilitate the supply of blood by taking anti-anginal drugs or replacing surgically damaged blood vessels, are required. In the case of surgical surgery, autologous or artificial blood vessels are mainly used.However, the use of autologous blood vessels has the advantage that the immune response is low and the success rate is high. There are limitations such as blood vessel conditions. Therefore, in recent years, there is an increasing demand for surgery to replace or bypass damaged blood vessels using artificial blood vessels.

Currently commercially available artificial blood vessels include expanded polytetrafluoroethylene (ePTFE) or polyethylene terephthalate (Dacron). blood vessels). In the case of small-diameter blood vessels with an inner diameter of less than 6 mm, biocompatibility is excellent because there is a high risk of artificial blood vessel occlusion after transplantation due to thrombus formation, intimal hyperplasia, and physical incompatibility. Material should be used. In addition, it must have physical properties similar to actual blood vessels, suitable to prevent expansion, rupture, aneurysm, etc. from occurring against long-term hemodynamic stress, and inflammatory reactions occur after transplantation or fibrous capsule formation. It shouldn't be. In the future, new vascular tissues are regenerated along artificial blood vessels, and as a result, the implanted artificial blood vessels are preferably decomposed in the body.

Biodegradable biomaterials generally used include synthetic polymers such as polylactic acid (PLA) and polycaprolactone (PCL), or natural polymers such as collagen and hyaluronic acid. Natural polymers have excellent biological properties, but they are expensive, have weak physical properties, and have a relatively high decomposition rate, so there is a risk that new vascular tissues may be decomposed before regeneration. In the case of synthetic polymers, physical properties and decomposition rates are suitable for use as artificial blood vessels, but their inherent biocompatibility is low, so rejection reactions in the body may occur. Therefore, it is advantageous to use a composite of synthetic polymers, natural polymers, and other bioactive materials.

In terms of structure, if a mat in the form of nanofibers is produced using electrospinning, an artificial blood vessel composed of nanofibers having a structure similar to that of an actual extracellular matrix and having nano-sized pores can be produced. However, in the case of small-diameter artificial blood vessels, long-term patency of the transplanted blood vessel can be secured only when an endothelium that can prevent muscle cell hyperproliferation and platelet adhesion and activity is rapidly formed. To this end, if the nanofibers forming the inner wall of the artificial blood vessel are arranged in the direction of blood flow, the growth direction of endothelial cells can be guided in the same direction, and cell activity can also be promoted. However, since the uniaxially arranged nanofiber mat has low structural resistance and suturability due to blood pressure acting in a direction other than the fiber arrangement direction, it is difficult to be used as such.

Patent Document 1: Korean Registered Publication No. 10-1633226

As a result of intensive research efforts to ensure that the polymer complex has a decomposition rate and biocompatibility suitable for the growth of new vascular tissues and physical properties similar to those of an actual blood vessel, the present inventors introduced a biocompatible polymer and a bioactive inorganic material, and the inner wall was the same As a result of combining the double-layered nanofiber structure to have a uniaxial arrangement and an arbitrary arrangement of the outer wall, the endothelial cell regeneration rate was improved, structural resistance and suturing firmness were confirmed, and the present invention was completed.

A first aspect of the present invention is a structure composed of an inner wall of a biodegradable synthetic polymer/natural polymer composite and an outer wall of a biodegradable synthetic polymer/ceramic composite in which the nanofibers constituting the artificial blood vessels; And a structure consisting of a double-layer tubular structure having a thickness of 200 to 300 nm, a total thickness of 450 to 500 μm, and an inner diameter of 2 to 10 mm. And the inner wall nanofibers provide an artificial blood vessel consisting of a structure including a uniaxial arrangement parallel to the blood flow direction, and the outer wall nanofibers an arbitrary arrangement.

A second aspect of the present invention provides a method of manufacturing the artificial blood vessel of the first aspect in which the thickness of the inner wall and the outer wall and the size of the inner diameter can be adjusted without layer separation.

Hereinafter, the present invention will be described in more detail.

The present invention is an inner wall formed of nanofibers uniaxially arranged in parallel with the direction of blood flow; And

As an artificial blood vessel of a double-layer structure comprising an outer wall formed of randomly arranged nanofibers, the nanofibers forming the inner wall include a biodegradable synthetic polymer and a natural polymer composite, and the nanofibers forming the outer wall are synthesized biodegradable. An outer wall composed of a polymer and ceramic composite; characterized in that it includes, an artificial blood vessel having a nanofiber double-layer structure having a different arrangement, and a method of manufacturing the same.

The biodegradable synthetic polymers include poly(L-lactic acid-co-ε-caprolactone) (Poly(L-lactide-co-ε-caprolactone)), polylactic acid (PLA), polyglycolic acid (PGA), poly( D,L-lactic acid-co-glycolic acid) (poly(D,L-lactide-coglycolide);PLGA), poly(caprolactone), diol/diacid aliphatic polyester, polyester-amide/polyester-urethane , Polyethylene oxide, poly(valerolactone), poly(hydroxybutyrate), and poly(hydroxyvalerate), but one or more synthetic polymers selected from the group consisting of, but are not limited thereto. However, polycaprolactone, one of the biodegradable synthetic polymers, has a relatively slow decomposition rate compared to other materials, providing sufficient time for new blood vessels to regenerate, and electrospun polycaprolactone has similar properties to actual blood vessels. Therefore, it is preferable as an artificial tissue support.

The biodegradable natural polymer includes any one or more of one or more natural polymers selected from the group consisting of collagen, gelatin, elastin, silk fibroin, chitosan, chitin, alginic acid, and hyaluronic acid, but is not limited thereto.

The "ceramic" may be a term collectively referring to an inorganic compound that is a non-metallic solid. The outer wall must be able to support the entire artificial blood vessel structurally and physically. In addition, if biocompatibility is improved by enhancing the hydrophobicity of the biodegradable polymer, it is advantageous for reducing the immune response and regenerating blood vessels. In order to provide artificial blood vessels that exhibit such effects, ceramics, which are inorganic materials, can be dispersed in nanofibers forming the outer wall. For example, silica is a bioactive material that enhances the biocompatibility of the material.

Silica has excellent bioactivity and biodegradability, so it is used in various medical fields. In addition, bioactive silica produced by using the sol-gel method is suitable for other bioactive silica production methods in terms of particle distribution of uniform size, possibility of controlling chemical components in the manufacturing process of the composite, and ease of manufacture. Compared to this, it is widely used. When such bioactive silica is used in an organic/inorganic composite based on a polymer, it is expected to increase the mechanical properties as well as the improvement of biological properties. Accordingly, in the present invention, silica nanoparticles are evenly dispersed using a sol-gel method to have a stronger strength than a pure biopolymer.

The nanofibers are actually composed of nanofibers having an extracellular matrix of 50 to 500 nm, and thus may have a thickness in this range. For example, the thickness is preferably 200 to 300 nm.

The thickness of the artificial blood vessel is similar to the actual blood vessel thickness. Specifically, it is 400 to 600 μm, but is not limited thereto. For example, it is preferably 450 to 500 μm.

The inner diameter of the artificial blood vessel is similar to the inner diameter of the actual blood vessel 10 mm to 2 mm, and more specifically includes a large or medium diameter vessel of 10 mm to 6 mm, more preferably a small diameter vessel of 6 mm to 2 mm It includes the inner diameter of.

In the present invention, for structural similarity to the actual extracellular matrix, the entire composition of the artificial blood vessel may be composed of a double layer. Specifically, there is a layer of endothelial cells arranged in parallel with the direction of blood flow on the inside, and a layer with high strength that can physically support the entire blood vessel on the outside.

In the nanofiber arrangement of the inner wall, platelets and endothelial cells can be competitively attached to the inner wall, and if platelet adhesion is predominant, there is a high risk of occurrence of blood clots or inflammatory reactions, so the compatibility between the endothelial cells and the inner wall is excellent. It is to do. For example, the inner wall may be configured such that the nanofibers are uniaxially arranged parallel to the blood flow direction, so that the endothelial cells can elongate and migrate in the same direction. This is to provide a rapid endothelial layer formation effect.

Like the extracellular matrix, it is composed of fibers having a thickness between 50-500 nm.

In order to provide an artificial blood vessel having such an effect, the inner wall is composed of a biodegradable synthetic polymer and a natural polymer. More preferably, it may be composed of polycaprolactone and collagen composite nanofibers.

The outer wall must be able to support the entire artificial blood vessel structurally and physically. In addition, if biocompatibility is improved by enhancing the hydrophobicity of the biodegradable polymer, it is advantageous for reducing the immune response and regenerating blood vessels. In order to provide artificial blood vessels that exhibit such effects, ceramics, which are inorganic materials, can be dispersed in nanofibers forming the outer wall.

"Ceramic" may be a generic term for an inorganic compound that is a non-metallic solid. For example, silica is a bioactive material that enhances the biocompatibility of the material.

Silica has excellent bioactivity and biodegradability, so it is used in various medical fields. In addition, bioactive silica produced by using the sol-gel method is suitable for other bioactive silica production methods in terms of particle distribution of uniform size, possibility of controlling chemical components in the manufacturing process of the composite, and ease of manufacture. Compared to this, it is widely used. When such bioactive silica is used in an organic/inorganic composite based on a polymer, it is expected to increase the mechanical properties as well as the improvement of biological properties. The "sol-gel process" is a sol by dispersing silica fine particles obtained from flame hydrolysis of a sol in which tens or hundreds of mm of colloidal particles obtained by hydrolysis or dehydration condensation are dispersed in a liquid. It is a reaction that causes the fluidity of sol to be lost due to agglomeration and condensation of colloidal particles, resulting in a gel of a porous body. Accordingly, in the present invention, silica nanoparticles are evenly dispersed using a sol-gel method to have a stronger strength than a pure biopolymer.

The manufacturing method of the present invention comprises the steps of (a) mixing a biodegradable synthetic polymer solution and a natural polymer solution and then electrospinning to produce a uniaxially arranged nanofiber inner wall mat;

(b) winding the inner wall mat produced in step (a) on a circular rod having a diameter of 10 mm to 2 mm;

(c) mixing the biodegradable synthetic polymer solution and the ceramic, and then electrospinning on the inner wall mat of step (b) to generate the randomly arranged outer nanofiber walls.

The "electrospinning" is a continuous electrospinning method, and it is possible to manufacture an artificial blood vessel having the above-mentioned structure without layer separation.

For example, the electrospinning of step (a) is preferably performed under the conditions of a voltage of 15 to 25 kV and a fluid velocity of 0.1 to 0.3 ml/h.

For example, the electrospinning of step (c) is preferably performed under the conditions of a voltage of 10 to 20 kV, a fluid velocity of 0.7 to 1.3 ml/h, and a spinning distance of 10 to 20 cm.

In the step (a), in order to obtain a uniaxial arrangement, it is preferable that the cylindrical substrate is rotated at a high speed at 2500 to 3500 laps per minute and electrospinning.

The "circular rod" of step (b) can adjust the diameter of the artificial blood vessel. For example, first, an inner wall mat is produced, and then a metal rod of a specific diameter coated with a water-soluble polymer is wound, and then the outer wall mat is fabricated on it, so that the diameter of the artificial blood vessel or the thickness of the inner/outer wall can be adjusted as necessary.

For example, the circular rod in step (b) is connected to a motor that rotates 100 per minute, and the distance between the motor and the substrate is 5 to 10 cm, and the radiation distance is preferably performed under conditions of 10 to 20 cm.

The step (c) includes being evenly dispersed in the biocompatible synthetic polymer by a ceramic sol-gel process.

Provides a small-diameter artificial blood vessel having biocompatibility, structural stability, and physical properties similar to actual blood vessels, manufactured by electrospinning in which the aperture size is adjustable and the double layer structure and arrangement method of the inner and outer walls are different. Compared with the improved blood vessel regeneration rate, the endothelium can be rapidly formed after transplantation, and has long-term patency, so that it can be usefully used in the field of vascular surgery.

1 is a diagram schematically showing a process of manufacturing a double-layer artificial blood vessel by a continuous electrospinning method. R and A corresponding to the inner wall mean an arbitrary arrangement and a uniaxial arrangement, and both consist of polycaprolactone/collagen. PCL and PCL/Si corresponding to the outer wall mean pure polycaprolactone and polycaprolactone/silica composite, respectively, and all of them are randomly arranged. Comparative Example 1 describes a method of preparing R-PCL, Comparative Example 2 A-PCL, Comparative Example 3 R-PCL/Si, and Example 1 A-PCL/Si.
2A is a diagram showing the results of observing the polycaprolactone/collagen inner wall arranged uniaxially, and (b) the polycaprolactone/silica composite outer wall arranged randomly with a scanning electron microscope.
3 is a diagram showing the results of analyzing components of specimens prepared in Example 1 and Comparative Examples 1 and 2 by Fourier transform infrared spectroscopy.
4 shows the tensile strength (a) and the degree of deformation at fracture (b), which are the results obtained through a tensile test, of the physical properties of A-PCL/Si obtained in Example 1, R-PCL, R-PCL/Si, A-PCL. It is a diagram compared with the value of.
5 is a diagram showing the suture resistance strength of A-PCL/Si obtained in Example 1 compared with R-PCL/Si of the same thickness.
6 is a diagram showing the experimental results by dispensing and culturing endothelial cells on the uniaxially arranged inner wall and the randomly arranged inner wall. 6-(a) is a confocal laser scanning micrograph comparing the patterns of cells attached to a specimen after culturing for one day. 6-(b) is a diagram schematically showing a graph in which a relative distance was measured by observing the distance the cells moved along the cell prohibition zone over time. 6-(c) is a diagram comparing the degree of cell proliferation after 3 days and 5 days through MTS assay.
Figure 7 is after dispensing fibroblasts on the outer wall prepared differently according to the presence or absence of silica, Figure 7-(a) compares the adhesion pattern after 1 day culture, and Figure 7-(b) is 3 days, 5 days later The degree of proliferation of is compared through the MTS assay.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.

Example 1. Preparation of artificial blood vessels in which the inner wall is uniaxially arranged in a polycaprolactone/collagen complex, and the outer wall is randomly arranged in a polycaprolactone/silica complex

1.1 Uniaxially arranged polycaprolactone/collagen complex inner wall

Polycaprolactone and collagen were dissolved in a hexafluoroisopropanol solvent at a ratio of 4:1 for 24 hours. For electrospinning, a syringe pump capable of discharging a solution at a constant rate, a DC high voltage generator, and a grounded substrate were prepared. In order to obtain a uniaxially arranged nanofiber mat, the substrate was cylindrical and rotated at high speed at 3000 revolutions per minute. 3 ml of the mixed solution was placed in a syringe and the solution was discharged at 0.2 ml per hour through a 20 G metal needle, while a high pressure of 20 kV was applied to the metal needle. The inner wall of the nanofiber mat uniaxially arranged on the substrate rotating at high speed was formed.

1.2 Arbitrarily arranged polycaprolactone/silica composite outer wall

The inner wall of the uniaxially arranged polycaprolactone/collagen mat prepared in Example 1 was wound on a circular rod coated with a water-soluble polymer having a diameter of 3 mm. The circular rod is connected to a low-speed motor that rotates 100 revolutions per minute, and the motor is positioned between a high-pressure metal injection needle and a ground-connected substrate. The distance between the injection needle and the substrate is 15 cm, and the distance between the motor and the substrate is 5 cm. Thereafter, the tetraethyl orthosilicate, purified water, and hydrochloric acid of 1 molar concentration are reacted at 5:1:0.02 to prepare a silica zero gel solution, and can be evenly mixed with a polycaprolactone solution dissolved in 20% in hexafluoroisopropane. Mix for 1 hour so that.

While 5 ml of the mixed solution was discharged to the substrate in a stationary state at 1 ml per hour, a voltage of 15 kV was applied to a metal injection needle to prepare an outer wall arbitrarily arranged on the inner wall. The finished double layer mat was dried under vacuum for 24-48 hours to avoid residual solvent.

1.3 Collagen crosslinking in the inner wall

To increase the stability of the collagen contained in the inner wall, double-layer artificial blood vessels were placed in a solution of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide in 70% ethanol for 24 hours. Collagen cross-linking was formed by immersion. Then, the water-soluble polymer was dissolved through a washing process with 70% ethanol and distilled water to separate the rod from the artificial blood vessel. As a result, a bilayer small-diameter artificial blood vessel having an inner wall of a polycaprolactone/collagen complex arranged uniaxially and an outer wall of a polycaprolactone/silica complex arranged randomly was prepared.

Comparative Example 1. Preparation of artificial blood vessels in which the inner wall is randomly arranged in a polycaprolactone/collagen complex, and the outer wall is randomly arranged in a polycaprolactone.

1.1 Randomly arranged polycaprolactone/collagen complex inner wall

The same solution as 1.1 in Example 1 was spun onto the substrate in a stationary state to prepare a randomly arranged polycaprolactone/collagen composite inner wall. At this time, other electrospinning conditions such as the release rate and voltage intensity of the solution were the same. However, in order to equalize the thickness of the formed mat, the volume of the spinning solution was varied by calculating the size ratio of the high-speed rotating substrate and the stationary substrate. After finishing the outer wall fabrication, drying under vacuum, chemical crosslinking, and washing were performed.

1.2 Randomly arranged polycaprolactone outer wall

The inner wall of the composite prepared in Comparative Example 1.1 is wound around a circular rod. The circular rod is connected to a low-speed motor that rotates 100 revolutions per minute, and the motor is positioned between a high-pressure metal injection needle and a ground-connected substrate. The distance between the injection needle and the substrate is 15 cm, and the distance between the motor and the substrate is 5 cm. A solution in which a polycaprolactone polymer was dissolved in hexafluoroisopropane at a concentration of 10 mass percent per volume was prepared. At the same time, 0.1 ml of the solution was discharged on the inner wall at an hourly rate, and a voltage of 13 kV was increased to prepare a randomly arranged outer polycaprolactone wall.

1.3 Collagen crosslinking in the inner wall

It was prepared in the same manner as 1.3 in Example 1.

Comparative Example 2. Preparation of artificial blood vessels in which the inner wall is uniaxially arranged with a polycaprolactone/collagen complex, and the outer wall is randomly arranged with polycaprolactone

2.1 Uniaxially arranged Polycaprolactone/collagen complex inner wall

It was prepared in the same manner as 1.1 in Example 1.

2.2 Randomly arranged polycaprolactone outer wall

The inner wall of the composite prepared in Comparative Example 2.1 was wound around a circular rod. The circular rod is connected to a low-speed motor that rotates 100 revolutions per minute, and the motor is positioned between a high-pressure metal injection needle and a ground-connected substrate. The distance between the injection needle and the substrate is 15 cm, and the distance between the motor and the substrate is 5 cm. A solution in which a polycaprolactone polymer was dissolved in hexafluoroisopropane at a concentration of 10 mass percent per volume was prepared. At the same time, 0.1 ml of the solution was discharged on the inner wall at an hourly rate, and a voltage of 13 kV was increased to prepare a randomly arranged outer polycaprolactone wall.

2.3 Collagen crosslinking in the inner wall

It was prepared in the same manner as 1.3 in Example 1.

Comparative Example 3. Preparation of artificial blood vessels in which the inner wall is randomly arranged in a polycaprolactone/collagen complex, and the outer wall is randomly arranged in a polycaprolactone/silica complex

3.1 Randomly arranged polycaprolactone/collagen complex inner wall

It was prepared in the same manner as 1.1 of Comparative Example 1.

3.2 Preparation of randomly arranged polycaprolactone/silica composite

It was prepared in the same manner as 1.2 in Example 1.

3.3 Collagen crosslinking in the inner wall

It was prepared in the same manner as 1.3 in Example 1.

Experimental Example 1. Scanning electron microscope analysis

In order to confirm the structure and thickness of the nanofibers constituting the double-layer mat prepared in Example 1, it was observed by scanning electron microscopy after Pt coating.

Figure 2 (a) is a uniaxially arranged polycaprolactone/collagen nanofibers with a thickness of 250 nm. 2(b) is a polycaprolactone/silica nanofiber arranged in random order, and the thickness of the nanofibers forming the inner wall and the outer wall is the same as 250 nm in thickness. This is advantageous in lowering the possibility of delamination by affecting the continuity between the two layers. In addition, breakage of nanofibers or formation of beads were not observed in both layers, and a double-layer mat made of nanofibers of uniform thickness and having nano-sized pores was well formed.

Experimental Example 2: Fourier Transform Infrared Spectroscopy Analysis

In order to analyze characteristic functional groups for each of the inner and outer walls obtained in Example 1, comparison was made using Fourier-transform Infrared Spectroscopy (FTIR).

In FIG. 3, black indicates polycaprolactone, blue indicates polycaprolactone and collagen complex constituting the inner wall of artificial blood vessels, and red indicates polycaprolactone and silica complex constituting the outer wall.

Following the blue line, characteristic peaks of amide binding of collagen were found in the ^{1650 cm -1} and 1536 cm ^{-1 wavelengths.} In addition, a broad peak appeared due to hydrogen bonds formed between the carboxyl group of polycaprolactone and the amide group of collagen in the ^{3304 cm -1 wavelength range.}

In the red line, the peak due to Si-O-Si bonding due to the inclusion of silica ^{appeared in the 1065 and 785 cm -1} wavelength band, and the peak due to Si-OH vibration was in the 935 cm ^-1 wavelength band.

Experimental Example 3: Measurement and comparison of artificial blood vessel properties

In order to be used surgically, having similar physical properties to actual blood vessels is essential for preventing blood vortex or swelling of blood vessels at the junction between surrounding blood vessels and artificial blood vessels after transplantation.

Tensile test and suture rigidity test were performed on the double-layer mat obtained in Example 1, and the ultimate tensile strength, fracture strain, and suture retention strength were compared with actual blood vessels. .

As a comparative group, an inner wall randomly arranged according to Comparative Examples 1 and 2, an outer wall made of a single polycaprolactone material, an inner wall arranged uniaxially, an outer wall made of a single polycaprolactone material, and an inner wall randomly arranged and an outer wall of a polycaprolactone/silica composite material Was set.

The double-layer mat was cut into a rectangular shape having a size of 10 mm (short axis) * 40 mm (long axis), and was sufficiently wetted in Phosphate-Buffered Saline (PBS) to prepare. However, the direction of the long axis of the rectangle and the direction of the nanofiber arrangement on the inner wall are the same.

Tensile test was performed by pulling the double-layer mat in the long axis direction until fracture occurred. As a result, as shown in Fig. 4, it was confirmed that the specimen having the polycaprolactone/collagen composite inner wall arranged uniaxially and the polycaprolactone/silica composite outer wall randomly arranged among the specimens having different combinations had the greatest tensile strength. This is in the range of 1.4 to 11.1 Mpa, which is an actual coronary artery tensile strength value of 3.2 MPa or more. It was confirmed that the outer wall of the polycaprolactone/silica composite had a significant effect on improving the overall structural properties when compared according to the presence or absence of silica in the outer wall having the same inner wall structure. In addition, the degree of deformation at fracture was 65.7%, which also showed similar values to the actual blood vessels.

Pass a single 4-0 polyglycolide suture 2 mm from the short end of the mat with a size of 10 mm (short)*50 mm (long axis)*450 μm (thickness) moistened in phosphate buffered saline, and then screw the loop. made. The resistance strength of the suture was measured by pulling the end of the mat and the suture in opposite directions to each other. Suture resistance strength is defined as the maximum force applied before the suture completely tears the mat.

As shown in FIG. 5, the specimen having the uniaxially arranged inner walls exhibited relatively low suture resistance strength compared to the specimen having the randomly arranged inner walls. However, thanks to the double-layer structure having an outer wall with improved physical properties due to the introduction of silica, the suture resistance strength of 2.5 N, which is 2 N or more, which is the minimum value required for clinical use, was confirmed.

Experimental Example 4: Cell culture experiment

The specimens prepared in Example 1 and Comparative Example 1 were prepared in a size of 10 mm * 10 mm, washed in 70 vol% ethanol for 3 hours, and sterilized under ultraviolet light for 1 hour, followed by cell experiments.

Endothelial cells were dispensed for the inner wall and fibroblasts were dispensed for the outer wall, and the pattern and proliferation of cells attached to the specimen were compared. In particular, for the inner wall, the cell migration rate, which is closely related to the rapid formation of the endothelial cell layer after artificial blood vessel transplantation, was also observed in clinical practice.

In the case of endothelial cells, to ^{5 * 10 4 cells / mL,} 10 to the mobile test ^{* 10 4 cells / mL, 1} * 10 4 cells / mL in the cell to a proliferation test suspension to the adhesion test to each specimen is immersed fully It was dispensed by 1 ml. The adhesion experiment was observed after culturing under 5% carbon dioxide for 1 day after cell dispensing. In the migration experiment, a screen was installed when dispensing cells, cultured for 1 day to form a cell inhibition zone, and if a sufficient amount of cells covered the surface of the specimen, the screen was removed and the medium was replaced, and then cultured again. Made it. The cell migration distance was observed after 0, 3, and 24 hours from the time of removal of the occlusion membrane. In the proliferation experiment, the degree of proliferation was compared through MTS assay on the 3rd and 5th days after cell dispensing.

For the outer wall, an experiment was conducted to compare the adhesion pattern and the degree of proliferation of fibroblasts. Adhesion experiment yen 5 * 10 ⁴ cells / mL, proliferation experiments yen 2 * 10 ⁴ cells / mL of each specimen immersed in the suspension having a cell concentration and incubated under the same conditions as described above. Cells were cultured for 1 day for adhesion and 3 and 5 days for proliferation and then analyzed.

After washing and fixing the cells attached to the specimen, the cytoplasm was stained with red phalloidin, and the cell nucleus was stained with blue 4',6' diamino-2-phenylindole (DAPI). It was observed with a confocal laser scanning microscope (CLSM).

As shown in (a) of FIG. 6, it was confirmed that the adhesion pattern of the cells was different according to the arrangement of the polycaprolactone/collagen nanofibers. In the case of random arrangement, the cells extend in all directions, but in the case of uniaxial arrangement, the cytoplasm part extends in the same direction. In both specimens, the cells were well stretched while maintaining the pattern, and the cell compatibility of the material was confirmed to be excellent. This has the advantage of minimizing the inflammatory response after transplantation, and preventing platelet adhesion and activity by making superior adhesion in the action of endothelial cells competitively with platelets. As a result, it is possible to solve problems such as blood clot formation and restenosis of blood vessels, and helps rapid endothelium formation along the inner wall of artificial blood vessels.

As shown in (b) of FIG. 6, comparing the moving distance of the endothelial cells according to the nanofiber arrangement, as a result of culturing the cells for 24 hours after removal of the shielding membrane, the cells in the uniaxially arranged polycaprolactone/collagen nanofiber mat All of the forbidden areas have been removed by newly migrated cells. On the other hand, in the case of random arrangement, the cell migration rate was relatively slow, so that the cell restricted zone remained. In addition to the previous test of adhesion, it was confirmed that the uniaxially arranged nanofiber mat has a positive effect on rapid vascular endothelial regeneration.

As shown in (c) of FIG. 6, it was confirmed that the degree of cell proliferation increased as the culture time increased regardless of the cell arrangement, and through this, it was confirmed that the polycaprolactone/collagen complex was a material having excellent endothelial cell affinity. I did. After culturing for a sufficient period of time, the uniaxially arranged endothelial cells generated structural signals with surrounding cells due to the rapid formation of the endothelial layer, thereby showing a high degree of proliferation. The degree of proliferation showed a significant difference from the randomly arranged nanofibers on the 5th day.

As shown in (a) of FIG. 7, with the introduction of silica, the size of the cytoplasm of the cells attached to the specimen and the number of attached cells per unit area increased. It was confirmed that by fabricating the silica composite, not only the physical properties of the material were improved, but also the cellular compatibility of the material was improved.

As shown in (b) of FIG. 7, significantly more cells proliferated in the polycaprolactone/silica complex compared to pure polycaprolactone on the 3rd day, and on the 5th day, both specimens showed excellent proliferation.

Claims (14)

An inner wall formed of nanofibers uniaxially arranged parallel to the direction of blood flow; And
As an artificial blood vessel of a double layer structure, comprising an outer wall formed of randomly arranged nanofibers,
The nanofibers forming the inner wall are composed of a composite of a biodegradable synthetic polymer and a natural polymer, wherein the biodegradable synthetic polymer is polycaprolactone (PCL), and the natural polymer is collagen,
The nanofibers forming the outer wall are composed of a composite of biodegradable synthetic polymer and ceramic, wherein the biodegradable synthetic polymer is polycaprolactone (PCL), and the ceramic is silica zero gel,
An artificial blood vessel with a nanofiber bilayer structure with different arrangements.
The method of claim 1,
The biodegradable natural polymer is a cross-linked, artificial blood vessel of a nanofiber bilayer structure with different arrangements.
delete delete The method of claim 1,
The nanofibers have a thickness of 200 to 300 nm, wherein the artificial blood vessels of the nanofiber bilayer structure with different arrangements.
The method of claim 1,
The total thickness of the artificial blood vessel is 450 to 500 μm and the inner diameter is 2 mm to 10 mm, the artificial blood vessel of the nanofiber bilayer structure with different arrangements.
(a) Mixing the biodegradable synthetic polymer solution and the natural polymer solution and then electrospinning to create a uniaxially aligned nanofiber inner wall mat, wherein the biodegradable synthetic polymer is polycaprolactone (PCL), and the natural polymer is collagen. Characterized in that;
(b) winding the inner wall mat produced in step (a) on a circular rod having a diameter of 2 mm to 10 mm;
(c) mixing the biodegradable synthetic polymer solution and ceramic, and then electrospinning it on the inner wall mat of step (b) to generate an arbitrarily arranged outer wall of nanofibers, wherein the biodegradable synthetic polymer is polycaprolactone (PCL). And, the ceramic is a silica-zero gel; containing,
A method for manufacturing artificial blood vessels of a nanofiber double layer structure with different arrangements.
The method of claim 7,
After immersing the nanofiber double-layer mat produced after step (c) in an ethanol solution containing 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, biodegradable natural Crosslinking the polymer; a method for producing an artificial blood vessel of a nanofiber bilayer structure in a different arrangement including.
delete delete The method of claim 7,
The electrospinning of step (a) is performed under the conditions of a voltage of 15 to 25 kV and a fluid velocity of 0.1 to 0.3 ml/h.
The method of claim 7,
The uniaxial arrangement of the step (a) is obtained by electrospinning a cylindrical substrate by rotating at a high speed of 2500 to 3500 laps per minute, a method of manufacturing an artificial blood vessel having a nanofiber double layer structure with different arrangements.
The method of claim 7,
The step (b) is performed by setting the distance between the motor and the substrate to 5 to 10 cm and the spinning distance to 10 to 20 cm while the circular rod is connected to a motor that rotates 100 per minute. A method for producing artificial blood vessels with a nanofiber double layer structure.
The method of claim 7,
The electrospinning of step (c) is performed under the conditions of a voltage of 10 to 20 kV and a fluid velocity of 0.7 to 1.3 ml/h.

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