KR20190118430A - Patterning and transferring of bioactive material for improving biological property of artificial vascular graft - Google Patents

Abstract

The present invention relates to a transferring technique and patterning of a biomaterial for enhancing biological properties of artificial blood vessels and a method of manufacturing the same. The artificial blood vessel according to the present invention includes a flexible polymer film manufactured by solvent casting with a polymer solution; and a thin film-patterned ceramic and/or metal-containing bioactive material, which allows a pattern profile to be embossed or engraved through a photolithography process so that the polymer film does not peel off when the polymer film is bent, and which is transferred to and embedded in the polymer membrane surface when the thin film-patterned ceramic and/or metal-containing bioactive material is solvent cast with a polymer solution.

Description

Patterning and transferring of bioactive material for improving biological property of artificial vascular graft through patterning and transcription of biomaterials

The present invention relates to a technique for enhancing the biological properties of artificial blood vessels through the patterning and transcription of the biomaterial and a method of manufacturing the same.

When cholesterol or fat accumulates in the inner wall of blood vessels such as arteries or veins in the body, blood vessels are narrowed and hardened, thereby preventing smooth blood circulation and causing various vascular diseases such as angina pectoris, myocardial infarction and coronary artery disease. Today, the incidence of vascular diseases is increasing rapidly due to increased diabetes and obesity due to biased eating habits and static lifestyles, especially in developed countries. The mortality rate is also high and reported to be a very fatal disease. Non-surgical treatment of vascular disease is a method of suppressing additional stenosis by taking antianginal drug, but such drug treatment does not fundamentally resolve the stenosis site, and pain from angina continues even with continuous medication . As a method of treating vascular diseases, surgical treatments such as vascular graft surgery and vascular bypass surgery, which remove a hardened blood vessel through surgery and connect artificial blood vessels to the site, have emerged.

In the surgical method, a donor's artery or vein was initially collected and transplanted, but the success rate was not high due to rejection or hardening. On the other hand, autograft, which takes the patient's own healthy blood vessels and replaces the damaged part, has a low immune response after transplantation, which is the most successful method, but has a limited number of vessels that can be obtained at a single time. Since additional surgery is required, there is a disadvantage that the burden on the patient is increased. Accordingly, in order to solve this problem, recently, an operation of replacing an existing damaged blood vessel by using an artificial vascular graft made of synthetic polymer has been performed. As such, the synthetic polymers that can be selected for application to artificial blood vessels should be harmless to the human body and have a high in vivo compatibility. In addition, the body is not rejected by the immune system at the time of transplantation, it is good to be degraded in the body as the vascular tissue repaired. Furthermore, in order to prevent stenosis and vascular hardening that may occur after transplantation, it should be such that precipitation of proteins or lipids or thrombus formation and inflammatory reactions do not occur.

In general, since artificial blood vessels are used to replace blood vessels damaged by diseases such as trauma or arteriosclerosis, synthetic polymer materials that are easily molded and have similar mechanical properties to conventional blood vessels are widely used. In addition, a biodegradable polymer material is preferable for the future growth of vascular cells and repair of vascular tissues.

However, in the case of artificial blood vessels made of a polymer, due to its unique low biocompatibility, the body may have a rejection reaction or an inflammatory reaction resulting from the clogging of the blood vessels. In order to solve this problem, researches are being made to make artificial blood vessels from biopolymer materials such as collagen present in the body, but the mechanical properties thereof are significantly lower than those of synthetic polymer materials, and they are not widely used at high prices. . Therefore, it is required to introduce a material capable of inducing cell proliferation due to excellent biological properties on the existing high strength synthetic polymer-based artificial blood vessel inner wall.

A typical method of introducing a bioactive material to the surface of the vascular inner wall of the three-dimensional form is surface coating. In the general coating technique, ceramics or metals can be peeled off from the surface and cause side effects such as inflammatory reactions or blockage of blood vessels.

The present invention seeks to enhance the biological properties of the artificial blood vessel inner wall through the patterning and transcription of a bioactive material having excellent biocompatibility. For example, ceramic or metal patterns having high bioactive properties are used to help cell proliferation in the inner wall of blood vessels, thereby preventing occlusion of artificial blood vessels and enhancing their expandability.

A first aspect of the invention is a flexible polymer film (flexible polymer film) prepared by solvent casting (solvent casting) with a polymer solution; And a pattern profile is engraved or embossed through a photolithography process so that the polymer membrane is not peeled off when the polymer membrane is bent, such that the thin film patterned ceramic and / or metal-containing bioactive material is solvent-cast into the polymer solution. It provides an artificial blood vessel comprising a bio-active material containing a thin film patterned ceramic and / or metal, which is transferred to the surface and embedded in the surface of the polymer membrane.

A second aspect of the present invention is a flexible polymer film (flexible polymer film); And a thin film patterned ceramic and / or metal-containing bioactive material embedded on the surface of the polymer membrane such that the polymer membrane is not peeled off when the polymer membrane is bent. A first step of transferring a thin film patterned ceramic and / or metal containing bioactive material during solvent casting to thin film patterning the ceramic and / or metal containing bioactive material to be embedded on a flexible polymer film surface ; And a second step of forming a conduit by rolling the thin film patterned polymer membrane with the bioactive material.

The third aspect of the present invention is a polymer structure having a flexibility to form a stretch or curved surface; And a pattern profile is engraved or embossed through a photolithography process so that the surface of the polymer structure is not exfoliated when it is stretched or warped, thereby forming a thin film patterned ceramic and / or metal-containing bioactive material of the polymer structure. A thin film patterned ceramic and / or metal-containing bioactive material transferred to the surface of the polymer structure during molding and embedded in the surface of the polymer structure; It provides an elastic or flexible insert (implant) comprising a.

Hereinafter, the present invention will be described.

The present invention through a new method of patterning a thin film of a material having excellent biological properties and transfer it to the surface of the polymer during polymer molding,

A polymer structure having flexibility to form stretchable or curved surfaces; And

In order to prevent peeling when the surface of the polymer structure is stretched or bent, a pattern profile is embossed or embossed through a photolithography process to form a thin film patterned ceramic and / or metal-containing bioactive material to form the polymer structure. A thin film patterned ceramic and / or metal-containing bioactive material transferred to the surface of the polymer structure and embedded in the surface of the polymer structure;

It provides an elastic or flexible insert (implant) comprising a.

The present invention is applied to artificial blood vessels,

Flexible polymer film (flexible polymer film) prepared by solvent casting into a polymer solution; And

In order to prevent peeling when the polymer film is bent, a pattern profile is embossed or embossed through a photolithography process so that the thin film patterned ceramic and / or metal-containing bioactive material is solvent-cast into the polymer solution. on Thin film patterned ceramic and / or metal containing bioactive materials, which are transferred and embedded on the surface of the polymer film;

It provides an artificial blood vessel comprising a.

In the present invention, the polymer structure including the polymer membrane is a support containing a thin film patterned ceramic and / or metal-containing bioactive material, and may be made of a biocompatible polymer material that does not cause an inflammatory reaction. Non-limiting examples of polymeric materials that can be used as the support include lactide, caprolactone, glycolide, dioxanone, propylene, ethylene, vinyl chloride (vinylchloride), butadiene, methyl methacrylate, methacrylate, 2-hydroxyethlymethacrylate, carbonate, polyethylene terephalate or These copolymers can be used individually or in combination. As another example, chitosan / glycerol phosphate; Polyphosphazene; Polycaprolactone; Polycarbonate; Polycyanoacrylate; Polyorthoester; Polyhydroxyethyl methacrylamide lactate (Poly (N- (2-hydroxyethyl) methacrylamide-lactate)); Polypropylene phosphate (Poly (phosphate)); Polylactic-glycolic acid (poly (lactic-co-glycolic acid), PLGA); Polyethylene glycol-polyester copolymers (poly (ethyleneglycol) / polyester); Polyethylene glycol-poly (propylene glycol) / poly (propylene glycol), PEG / PPG) copolymers; Polyethylene glycol-polycaprolactone copolymers; Methoxypolyethylene glycol-polycaprolactone copolymers; Polyethylene glycol- (polylactic-glycolic acid) -polyethylene glycol triple polymer; (Polylactic-glycolic acid) -polyethyleneglycol- (polylactic-glycolic acid) triple polymer; Polyethylene glycol-polycaprolactone-polyethylene glycol triple polymer; Polycaprolactone-polyethylene glycol-polycaprolactone triple polymer etc. can be used individually or in combination. As another example, it may be a polylactic-glycolic acid copolymer (PLGA).

On the other hand, when an insert including an artificial blood vessel is inserted into the body and needs to be easily removed after the role is removed, a polymer support (structure) including a polymer membrane may be one that exhibits biodegradability. For example, a biodegradable polymer material is preferred for future growth of vascular cells and repair of vascular tissue. Non-limiting examples of biodegradable polymers include poly-L-lactide, polylactide-glycolide copolymers, polycaflolactone and combinations thereof.

In the case of the thickness of the polymer membrane used as an artificial blood vessel, since similar to the actual blood vessel thickness is advantageous for mechanical properties and tissue regeneration, the thickness may be 100 to 300 μm, which is generally used.

The thickness of the pattern made of ceramic and / or metal is preferably a thin film of 1 to 2 μm that is not too thick so that the polymer film does not peel off when the polymer film is bent.

The artificial blood vessel of the present invention is in a form containing a bioactive ceramic or metal pattern, and has excellent biocompatibility.

Artificial blood vessels according to an embodiment of the present invention, through the thin film patterned ceramic and / or metal-containing bioactive material embedded in the surface of the polymer membrane, the expansion of artificial blood vessels in a manner that induces the proliferation of cells in the vessel wall It can promote sex.

In the present invention, bioactive means that when the implant according to the present invention is inserted into the body, a predetermined effect can be exerted by a thin film patterned ceramic and / or metal-containing material on the surface. Adhesion, platelet adhesion inhibition, cell culture enhancement, a variety of biochemical signals, signal detection sensor, can provide the electrical signal.

Non-limiting examples of ceramics or metals contained in the thin film patterned bioactive material include hydroxy apatite, titanium, tantalum, gold or combinations thereof.

Hydroxy apatite (Cy ₅ (PO ₄ ) ₃ (OH)) is a representative component of the bone tissue in the body, has been found through many studies excellent in excellent biological properties and induction of cell proliferation.

Titanium is a material having excellent biocompatibility, and it is known that not only affinity with bone tissue, which is widely used, but also affinity with endothelial cells existing inside vascular tissues.

Tantalum is a material having excellent biocompatibility, and it is known that not only affinity with bone tissue, which is widely used, but also affinity with endothelial cells present in vascular tissues.

Gold is an electrically conductive and biocompatible material and can be used in fields such as in vivo sensors. Therefore, through the advantages of the present invention that can be patterned having any structure, it is possible to manufacture an electrical circuit, it can be a great advantage to be able to make a gold pattern.

Thus, using the artificial blood vessel according to the present invention, the cells can be cultured on the thin film patterned ceramic and / or metal-containing bioactive material while maintaining the attachment and inherent shape of the cells, thereby repairing the tissue. In addition, platelet adhesion can be suppressed on thin film patterned ceramic and / or metal containing bioactive materials. Platelets (Thrombocytes) come from the tear of the cytoplasm of megakaryocytes in the bone marrow, and produce about 400-8000 megakaryocytes. Therefore, there is no nucleus, the size is 0.5-2.5μm and has about 150 ~ 370 × 10 ⁹ per liter of blood. In general, when a foreign body penetrates into the body, it precipitates around it to generate a blood clot, and the side wall of the artificial blood vessel is recognized as a foreign material to generate a blood clot and block the blood vessel. Therefore, by inhibiting platelet adhesion, it is possible to prevent occlusion of artificial blood vessels.

In the embodiment of the present invention it was confirmed that the hydroxyapatite thin film pattern embedded in the inner wall of the artificial blood vessels can be cultured while maintaining human navel venous endothelial cell adhesion and cell specific characteristics, and platelet adhesion was suppressed.

After the artificial blood vessels are placed, the cells travel in biocompatible materials to create a uniform membrane on the inner walls of the blood vessels. As a result, uniform neovascularization that is not occluded replaces the site of the biodegradable artificial blood vessel and repairs it.

On the other hand, according to the invention a flexible polymer film (flexible polymer film); And a thin film patterned ceramic and / or metal-containing bioactive material embedded on the surface of the polymer membrane so as not to peel off when the polymer membrane is bent,

The ceramic and / or metal-containing biomaterial is transferred to a thin film-patterned ceramic and / or metal-containing bioactive material during solvent casting with a polymer solution during the polymer film manufacturing process and embedded on the surface of the flexible polymer film. A first step of thin film patterning the active material; And

The bioactive material may comprise a second step of rolling the thin film patterned polymer film to form a conduit.

At this time, when the thin film patterned bioactive material contains a ceramic, the first step is

Step 1-1 to form a photosensitive liquid pattern on the surface of the support through a photoresist process;

A first step of forming a metal thin film pattern on the surface of the support by depositing a metal thin film pattern corresponding to an intaglio portion of the photosensitive liquid pattern through physical deposition and then removing the photosensitive liquid pattern;

Applying a ceramic precursor-containing solution to the groove corresponding to the intaglio portion of the metal thin film pattern, and then removing the metal thin film pattern by forming a ceramic thin film pattern corresponding to the intaglio of the metal thin film pattern on the support surface; And

The method may include the steps 1-4 of transferring the ceramic thin film pattern formed on the support surface to the flexible polymer film.

On the other hand, when the thin film patterned bioactive material contains a metal, steps 1 to 3 may be omitted.

Step 1-1 to form a photosensitive liquid pattern on the surface of the support through a photoresist process;

A first step of forming a metal thin film pattern on the surface of the support by depositing a metal thin film pattern corresponding to an intaglio portion of the photosensitive liquid pattern through physical deposition and then removing the photosensitive liquid pattern; And

The method may include the steps 1-4 of transferring the metal thin film pattern formed on the support surface to the flexible polymer film.

In the pattern production of ceramic and / or metal containing bioactive materials according to the invention, it is preferred that the pattern has at least one of the following characteristics:

(i) for linear patterns, the width and spacing of the pattern should be 10-100 μm;

(ii) for circular patterns, the diameter and spacing of the patterns should be 5-20 μm;

(iii) Before making each pattern, make photoresist pattern by photolithography process.

(iv) In order to produce a pattern, an electron beam evaporator or a sputter should be used.

Unlike the conventional coating process, the transfer process of the present invention has an advantage in that a separate pattern is embedded on the surface of the polymer support to form a stable coating layer. If a flexible three-dimensional structure such as an artificial blood vessel is to be implemented, the coating layer is difficult to remain stable, while the pattern embedded through the transfer process of the present invention maintains stability even on curved surfaces.

The present invention enables the implementation of a neat pattern of micro size through the photolithography process, and unlike the existing process, the pattern produced on the substrate can be transferred to the above-described polymer of a flexible material and applied to three-dimensional structures such as blood vessels. Do. In addition, ceramics, which are bioactive materials that break more than conventional coatings, As the metal is patterned, it does not peel off even in a flexible structure.

In the embodiment of the present invention, a micro-sized pattern made of ceramic hydroxyapatite or metal titanium, tantalum or gold having excellent biocompatibility was produced.

When the tissue is repaired in the artificial blood vessel, the thin film pattern made of ceramic and / or metal may be separated when the polymer membrane is biodegraded, so the thin film pattern having a size that does not cause an inflammatory reaction is preferable.

The first step of the artificial blood vessel manufacturing method according to the invention transfers a thin film patterned ceramic and / or metal-containing bioactive material during solvent casting into a polymer solution during the polymer membrane manufacturing process, a flexible polymer membrane (flexible) thin film patterning a ceramic and / or metal containing bioactive material to be embedded in a polymer film surface.

The present invention performs solvent casting to transfer the micro-sized pattern to the synthetic polymer.

The polymer membrane formed in the first step may have a thickness equal to the thickness of the artificial blood vessel to be finally provided, and the width and length may be flat specimens each having an inner diameter and a length greater than that of the artificial blood vessel to be provided. At this time, the thickness of the artificial blood vessel manufactured may be 0.1 to 0.3 mm, but is not limited thereto. More specifically, the thickness and / or diameter of the artificial blood vessel may be adjusted in consideration of the blood flow volume, blood pressure, and blood vessel of the blood vessel defect to be implanted.

Preferably, in solvent casting, a solution may be used in which the polymer has a 5 to 10 weight percent volume (w / v%) of solvent.

Preferably, the polymer may be polylactide or polylactide-glycolide copolymer or polycaflolactone in pellet form.

Preferably, the solvent may be dichloromethane.

The first step of thin-film patterning a ceramic and / or metal-containing bioactive material so as to be embedded on a flexible polymer film surface may include the first-first step of forming a photoresist pattern on a surface of a support through a photoresist process. Include.

The photoresist process is also called photolithography, which collectively refers to the semiconductor exposure process technology. Mass production of devices in a short time is achieved by precisely producing a single disc called a mask and repeatedly copying patterns of the same shape using light. This is possible. The photolithography process alone does not produce a three-dimensional structure, but it can be combined with other unit processes such as thin film deposition and etching, and the lithography process enables partial etching by forming a selective protective film.

The basic shape of the photolithography equipment exposed using light is determined by the optical system used, and there are two types of proximity exposure methods and projection exposure methods. In general, the core of the technology is that the mask and the wafer are uniformly adhered so that a certain amount of light reacts evenly with the photoresist. The photosensitizer is a material that can selectively remove the lighted part and the non-lighted part during the subsequent developing process by using the characteristic that the solubility in the developer is changed by receiving light of a specific wavelength. In general, a photoresist that is commonly used to remove a portion selectively changed by the light using a developer, the case where the light is well melted by the developer is called a positive resist, and vice versa.

The first step of thin film patterning the ceramic and / or metal-containing bioactive material to be embedded on the surface of a flexible polymer film is a metal thin film pattern corresponding to the negative portion of the photoresist pattern through physical deposition. After the deposition (Deposition) to remove the photoresist pattern to form a metal thin film pattern on the surface of the supporter comprises the first step.

The method of manufacturing the thin film of the present invention may be performed by physical deposition, and the deposition may include methods using resistive heat, arc discharge, electron beam, laser, and the like.

In the exemplary embodiment of the present invention, the metal thin film pattern corresponding to the intaglio portion of the photoresist pattern is deposited by using electron beam, evaporation, or sputter deposition.

Vacuum deposition using an electron beam is a method of evaporating a material to be deposited on a substrate by heat generated by colliding hot electrons emitted from a filament by applying a very high voltage, and using a water cooling crucible under high vacuum (10 ^-5 torr or less). Therefore, high melting point materials can be deposited because the contamination, which is a disadvantage of the resistance heating type, is relatively small and focuses the hot electrons with high energy, and the deposition rate is easily controlled.

When the thin film patterned bioactive material contains ceramic, the first step is applying a ceramic precursor-containing solution to the groove corresponding to the intaglio portion of the metal thin film pattern and then reacting the ceramic thin film pattern to the intaglio of the metal thin film pattern Forming a surface on the surface of the support to remove the metal thin film pattern; And transferring the ceramic thin film pattern formed on the support surface to the polymer film by solvent casting. In steps 1-4, after the solvent is evaporated in the solvent casting process, the ceramic thin film pattern may be transferred in the process of physically separating the dried polymer film from the substrate.

However, in the case where the thin film patterned bioactive material contains a metal, the first step may be omitted in steps 1-3, and the metal thin film pattern formed on the surface of the support prepared in steps 1-2 may be solvent cast. It may include a step 1-4 to transfer to the polymer membrane. In steps 1-4, after the solvent is evaporated in the solvent casting process, the metal thin film pattern may be transferred in the process of physically separating the dried polymer film from the substrate.

The second step of the method for producing an artificial blood vessel according to the present invention is a step of forming a conduit by rolling a polymer membrane having a thin film patterned bioactive material. At this time, the bioactive material may roll the thin film patterned polymer film itself to form a conduit. For example, the artificial blood vessel according to the present invention may be prepared by manufacturing a composite of a polymer having a high biodegradability and excellent mechanical strength and a ceramic or metal in a thin film form, and then rolling it in a tubular form.

The polymer membrane having the thin film patterned bioactive material may be bent in a blood vessel shape to be sealed or bonded. The process of bending the thin film-patterned polymer membrane of the bioactive material into a blood vessel shape may be performed using a medical device for blood vessels such as plastic cannula, but is not limited thereto. At this time, the plastic cannula may be selected in consideration of the diameter of the artificial blood vessel to be manufactured.

Vascularly bent substrates can seal or adhere to each other so that the sides can be provided in the form of a closed tube so that blood does not leak. The suture may be performed using any method and / or means capable of suturing artificial blood vessels such as suture using a suture. The suture used in the suture is ligation of the blood vessels to bring about a hemostatic effect or to support the tissue until the damaged tissue is healed, so it must be inert, not flexible, and flexible, and damage the tissue. It should not be given and should not have foreign body action. As the suture, one or more selected from the group consisting of silk, nylon, Vicryl, Prolin, and Darkon may be used. Since the silk does not slip well, the knot is rarely loosened by tying only 3 to 4 times at the time of suture, and the silk is inexpensive, and is used for skin suture and intestinal suture and extensive fascia suture. Nylon is a polyamide polymer, and there are two types of nylon: monofilament and multifilament. Most monofilament nylon is used. The larger the first number, the thinner the thickness. The suture may be nylon of 6-0 to 10-0 monofilament, but is not limited thereto. On the other hand, the adhesion can be carried out using any method and / or means capable of adhering the artificial blood vessels using the adhesive for biological tissue. Biomaterial adhesives include, without limitation, materials used for the purpose of fixing tissues, wound closure, hemostasis, and air leakage prevention in various medical fields such as micro surgery, vascular surgery, lung surgery and plastic surgery, orthopedics, dentistry, etc. Can be used. As it is used in vivo, it must be biodegradable and free of toxicity and risk. In addition, since it is intended to be inserted into the body as artificial blood vessels, it is difficult to obtain sufficient adhesive strength because it is exposed to a wet state by body fluid or blood after transplantation, and only limited materials may be used because it should have characteristics such as fast adhesive strength and biocompatibility. Can be. As the adhesive for biological tissues, one or more selected from the group consisting of fibrin glue, gelatin glue, polyurethane adhesive, and cyanoacrylate adhesive may be used.

The bioactive artificial blood vessel according to the present invention can be effectively applied to cardiovascular and general vascular surgery as a substitute for general synthetic polymer artificial blood vessels having excellent body stability and cell affinity, causing rejection or inflammatory reactions.

1 is a schematic diagram showing a process for preparing a hydroxyapatite pattern and the artificial blood vessel by transferring it to a polymer membrane.
FIG. 2 is a scanning electron microscope photograph and an energy dispersive X-ray spectroscopy (EDS) photograph of (a) Example 1.2 and (b) Example 2.2. FIG.
3 is a result of analyzing the components of the complex produced according to Example 1 by Fourier transform infrared spectroscopy. Each black line represents a magnesium pattern on a silicon wafer, a red line represents a hydroxy apatite pattern on a silicon wafer, and a blue line represents poly-L-lactide (PLLA) to which a hydroxy apatite pattern is transferred.
Figure 4 is a photograph analyzed by the scanning electron microscope the results of the tensile test and the human mimic circulation test performed in accordance with Experimental Example 3. Respectively, (a) hydroxyapatite coated on the front of the tensile test, (b) hydroxyapatite pattern transferred on the tensile test, (c) hydroxyapatite coated on the front for 4 weeks human mimic dynamic flow conditions circulation Experiment, (d) Photograph immediately after the circulating experiment of the human mimic dynamic flow condition for 4 weeks after the hydroxyapatite pattern is transferred.
FIG. 5 shows human navel veins on the surface of poly-L-lactide to which (a) the poly-L-lactide film and (b) the hydroxyapatite pattern were transferred, respectively, after 1 day of cell division according to Experimental Example 4 Confocal laser scanning micrographs comparing the difference of endothelial cell growth and (e) the results of experiments comparing the proliferation of cells after 2 and 5 days, respectively, using MTS reagent.
6 shows platelet growth on the surface of poly-L-lactide to which (a) the poly-L-lactide film and (b) the hydroxyapatite pattern were transferred, respectively, 1 hour after platelet was dispensed according to Experimental Example 5; Scanning electron micrographs comparing the differences and (c) the number of platelets were measured and compared.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited by these examples.

EXAMPLE  One: Hydroxy  Preparation of Polymer Artificial Vessels Containing Apatite Patterns

As shown in FIG. 1, a hydroxyapatite pattern was prepared through a photolithography process and transferred to a polymer membrane to prepare an artificial blood vessel.

1.1 Hydroxy  Preparation of Apatite Patterns

The positive photoresist was coated to a thickness of 1 to 1.5 micrometers on the surface of the silicon wafer through a spin coater, and then heat-treated at 110 ° C. for 70 seconds to evaporate the solvent. After adjusting the position of the ultraviolet rays irradiated onto the surface through the mask aligner and the pre-made mask, the portion irradiated with the developer was removed to obtain various photoresist patterns.

Magnesium was deposited to a thickness of 1 micrometer at a rate of 1 nanometer per second using an electron beam deposition apparatus on a silicon wafer on which a photoresist pattern was formed, and then a portion of the photoresist was dissolved using acetone to leave only a magnesium pattern.

A silicon wafer having a magnesium pattern was treated in a 0.05 mol calcium ethylenediaminetetraacetic acid and 0.05 mol potassium phosphate mixed aqueous solution at 90 ° C. and pH 8.9 for 2 hours. It is reacted with hydroxy-apatite, and the generation, magnesium, magnesium is reacted with water and reaction to produce hydroxyl ion (OH ^{- -)} is the calcium ion (CA ²⁺⁾ and phosphate ions in the precursor solution (HPO ₄ ²⁾ As the process proceeds, the magnesium pattern is converted into a hydroxy apatite pattern by using the principle of gradually becoming an ion and disappearing and replacing the hydroxy apatite.

1.2 Hydroxy  Apatite Pattern Warrior

The hydroxyapatite pattern on the silicon wafer prepared according to Example 1.1 was immersed in the polymer solution upon solvent casting with a polymer solution in which the poly-L-lactide (PLLA) polymer was dissolved in dichloromethane at a concentration of 10 percent by volume. After 24 hours, when the solvent evaporated, the polymer thin film having the hydroxyapatite pattern was separated from the silicon wafer surface. Thin film and tubular structures prepared for residual solvent removal were stored in a vacuum desiccator for 24 hours. The prepared thin film may be bent into a blood vessel shape and then sutured or glued to form a tubular structure.

Comparative example  One: Hydroxy  Preparation of Apatite Front Coating Polymer Thin Film

In order to compare the stability with the poly-L-lactide thin film in which hydroxy apatite was introduced in a pattern form, a poly-L-lactide thin film coated with hydroxy apatite on the entire surface was prepared as a comparative group.

Specifically, magnesium was deposited to a thickness of 1 micrometer on a silicon wafer by using an electron beam deposition apparatus at a speed of 1 nanometer per second. The prepared magnesium-deposited silicon wafer was treated in 0.05 mol calcium ethylenediaminetetraacetic acid and 0.05 mol potassium phosphate mixed aqueous solution at 90 ° C. and pH 8.9 for 2 hours to convert the magnesium layer into a hydroxyapatite layer. Can be.

In a polymer solution in which a poly-L-lactide (PLLA) polymer was dissolved in dichloromethane at a concentration of 10 percent by volume, the hydroxyapatite layer on the silicon wafer prepared above was infiltrated into the polymer solution during solvent casting. After 24 hours, when all of the solvent had evaporated, the polymer thin film with the hydroxyapatite layer was separated from the silicon wafer surface. Thin film and tubular structures prepared for residual solvent removal were stored in a vacuum desiccator for 24 hours.

EXAMPLE  2: Preparation of Polymeric Artificial Vessels Containing Metal Patterns

2.1 Fabrication of metal patterns

The positive photoresist was coated to a thickness of 1 to 1.5 micrometers on the surface of the silicon wafer through a spin coater, and then heat-treated at 110 ° C. for 70 seconds to evaporate the solvent. After adjusting the position of the ultraviolet rays irradiated onto the surface through the mask aligner and the pre-made mask, the portion irradiated with the developer was removed to obtain various photoresist patterns.

Titanium was deposited to a thickness of 1 micrometer at a rate of 1 nanometer per second using an electron beam deposition apparatus on a silicon wafer on which a photoresist pattern was manufactured, and then the photoresist part was dissolved using acetone to leave only the titanium pattern.

Tantalum was deposited to a thickness of 1 micrometer on a silicon wafer on which a photoresist pattern was manufactured by using a sputtering apparatus, and then a portion of the photoresist was dissolved using acetone to leave only a tantalum pattern.

After the deposition of gold to a thickness of 1 micrometer at a rate of 1 nanometer per second using an electron beam deposition apparatus on a silicon wafer prepared photoresist pattern, the photoresist part was dissolved using acetone to leave only the gold pattern.

2.2 Transfer of Metal Patterns

Various metal patterns on the silicon wafer prepared according to Example 2.1 were immersed in the polymer solution upon solvent casting with a polymer solution in which the poly-L-lactide (PLLA) polymer was dissolved in dichloromethane at a concentration of 10 percent by volume.

After 24 hours, when the solvent evaporates, the polymer thin film It was separated on the silicon wafer surface. Thin film and tubular structures prepared for residual solvent removal were stored in a vacuum desiccator for 24 hours.

Experimental Example  1: Scanning Electron Microscope Analysis

In order to observe the surface of the patterns prepared in Examples 1 and 2, and analyzed by scanning electron microscopy (Scanning electron microscopy) after Pt coating is shown in FIG.

Referring to Figure 2 (a) it can be seen that the hydroxyapatite pattern of 20 to 30 μm in width is well formed and the transfer to the polymer layer is also well. Referring to Figure 2 (b) it can be seen that the 20 μm wide titanium pattern is well formed and the transfer to the polymer layer is also well.

Experimental Example  2: Fourier transform Infrared spectroscopy  analysis

Fourier-transform Infrared Spectroscopy (FTIR) was used to analyze the components of the hydroxyapatite obtained in Example 1 above.

Referring to FIG. 3, poly-L-lactide (PLLA), on which a magnesium pattern on a silicon wafer shown in black, a hydroxy apatite pattern on a silicon wafer shown in red, and a hydroxy apatite pattern shown in blue, are transferred, Indicates.

The red line salpimyeon 1026 cm ^- can confirm the hydroxyapatite distinctive phosphate (PO ₄ ^3-) peak at a ^first wavelength for.

The Blue-ray salpimyeon ^{1044 cm -1, 1088 cm -1,} 1184 cm -1, 1755 cm - carbon are found in poly-lactide -L- from ^one wavelength to-peak characteristics of the double bond between the oxygen is observed. Existing phosphate peaks expected to be present are identified as overlapping with the 1044 wavelength band.

Experimental Example  3: complex stability analysis

The mechanical stability of the hydroxyapatite pattern and the polymer composite obtained in Example 1 was analyzed through a tensile test and a human mimic circulation experiment.

For comparison with the poly-L-lactide thin film in which hydroxy apatite was introduced in the pattern form as in Example 1.3, the poly-L-lactide thin film coated on the entire surface of Comparative Example 1 hydroxyapatite was prepared.

In order to analyze the stability of the hydroxyapatite pattern when mechanical deformation is applied, the poly-L-lactide and the front-coated poly-L-lactide in which the hydroxyapatite is introduced in the pattern form are respectively 5% strain rate. After tensioning, the analysis was performed by scanning electron microscopy.

Referring to (a) of FIG. 4, when the hydroxyapatite is completely coated, the phenomenon in which the brittle ceramic hydroxyapatite layer is broken as deformation is applied and returns to a circular shape can be confirmed in a low magnification and a high magnification electron micrograph. Such surface cracks, when applied to actual blood vessels, can cause surface peeling which can lead to inflammatory reactions. On the other hand, in the case of the hydroxyapatite pattern in FIG. This means that the stability of the bioactive ceramic layer at the time of implantation in the body helps the stable recovery of cells.

In order to compare and analyze the stability of the hydroxyapatite pattern under dynamic flow conditions when used as a real artificial blood vessel, a biomimetic experiment was conducted for 4 weeks using a perfusion pump.

Poly-L-lactide and full-coated poly-L-lactide with hydroxyapatite introduced in a pattern form were rolled into a silicone tube and placed inside a silicone tube, followed by Dulbecco's phosphate-buffered saline (DPBS). ) Was introduced at a rate of 100 ml per minute, similar to the rate of blood flow in the body, and physiological saline was changed once every three days.

After four weeks of experiments, the shape of the surface hydroxyapatite was observed using a scanning electron microscope.

Referring to (c) of FIG. 4, when the hydroxyapatite is completely coated, the phenomenon in which the brittle ceramic hydroxyapatite layer is broken and peeled off as it is exposed to dynamic flow for 4 weeks can be confirmed by electron micrographs. Such surface exfoliation negatively affects the repair of vascular lining cells due to the absence of bioactive ceramics and may further lead to inflammatory responses. On the other hand, in the case of the hydroxyapatite pattern in FIG. 4 (d), despite the brittle ceramic, no cracking or peeling of the surface due to the dynamic flow was observed. This means that the stability of the bioactive ceramic layer at the time of implantation in the body helps the stable recovery of cells.

Experimental Example  4: complex vessel Endothelial cells  Culture experiment

Human umbilical vein endothelial cells were cultured to examine the bioactivity of the hydroxyapatite pattern and the polymer complex prepared in Example 1, and the shape of the cells grown on the surface of the complex was observed.

Before dispensing the cells, the complexes were washed in 70% by volume ethanol for 1 hour and sterilized and then UV treated for 2 hours.

A 1 ml cell suspension (cell concentration 3 × 10 ⁴ cells / mL) was added to a sterile 10 × 10 mm pure poly-L-lactide thin film and a hydroxyapatite patterned poly-L-lactide thin film. Busy. After incubation in a 95% carbon dioxide atmosphere incubator for 1 day, the cells were treated to observe the cell morphology.

The cell shape grown on the surface of the complex was confocal laser scanning by staining the cytoplasm and nucleus with red Phalloidin and blue 4 ', 6'diamino-2-phenylindole. Microscope, CLSM), and the results obtained are shown in (a), (b), (c) and (d) of FIG. 5.

5 is a type of vascular endothelial cells using (a, b) pure poly-L-lactide thin film and poly-L-lactide thin film on which (c, d) hydroxy apatite pattern prepared in Example 1 was transferred Low and high magnification confocal laser scanning microscopic images of human umbilical vein endothelial cells cultured on the surface for 1 day.

Referring to (a) of FIG. 5, it can be seen that cells on the pure poly-L-lactide surface do not extend the limbs well and have a distorted shape of the cellular part stained in red. Referring to FIG. 5B, which is enlarged, this phenomenon can be observed more reliably. Referring to (c) of FIG. 5, in the poly-L-lactide surface to which the bioactive hydroxyapatite pattern is transferred, cells are well attached to the poly-L-lactide and maintain their inherent shape. It can be seen that compared to many. In particular, referring to FIG. 5 (d) in which this is enlarged, the cells appear to be aligned on the hydroxyapatite pattern, which may help rapid cell repair on the surface of artificial blood vessels. As a result, it can be seen that the biocompatibility of the composite surface is generally improved as the hydroxy apatide pattern is introduced.

Cell proliferation comparison experiments were conducted to compare the proliferation rates of cells on the surface. Human umbilical vein endothelial cells were dispensed on the pure poly-L-lactide thin film and the poly-L-lactide thin film on which the hydroxyapatite pattern prepared in Example 1 was transferred and after 2 and 5 days, respectively, by MTS assay. The degree of proliferation of the cells was compared.

Referring to (e) of FIG. 5, it can be seen that at 2 days, more cells proliferate in the poly-L-lactide to which hydroxyapatite is transcribed compared to the pure poly-L-lactide. Similar trends appear after 5 days. As a result, it can be seen that as the hydroxyapatite pattern is transcribed, the proliferation of cells is also enhanced.

Experimental Example  5: Complex Platelet Culture Experiment

In order to determine the blood stability of the hydroxyapatite pattern and the polymer complex prepared in Example 1, the platelets grown on the surface of the complexes were cultured by culturing human platelets, and the platelets were counted and analyzed quantitatively.

Sterilization of the specimen was performed in the same manner as in Experiment 4, and the platelet solution mixed with saline was dispensed on the surface of the poly-L-lactide thin film on which the pure poly-L-lactide and hydroxyapatite patterns were transferred, for 1 hour. Incubated. After incubation, platelets were fixed with glutaraldehyde solution, and dehydrated stepwise using an aqueous ethanol solution and observed by scanning electron microscopy.

Referring to Figure 6 (a), it can be seen that the platelets are stretched wider and attached to a larger number of pure poly-L-lactide surface. Referring to FIG. 6B, the platelet is hardly observed in the hydroxyapatite portion of the picture, and the shape of the platelet is distorted. This is prominent in (c) of FIG. 6, in which the number of platelets is measured, and it can be seen that a statistically significant number of platelets is observed on the surface of poly-L-lactide to which hydroxyapatite is transferred. As a result, as the hydroxyapatite pattern is transcribed, it can be seen that the number of platelets that play an early role in the thrombogenesis in the body is markedly reduced, and thus the hydroxyapatite pattern is transcribed in the blood of poly-L-lactide. You can expect better stability.

Claims (15)

Flexible polymer film (flexible polymer film) prepared by solvent casting into a polymer solution; And
In order to prevent peeling when the polymer film is bent, a pattern profile is embossed or embossed through a photolithography process so that the thin film patterned ceramic and / or metal-containing bioactive material is solvent-cast into the polymer solution. A thin film patterned ceramic and / or metal containing bioactive material transferred to and embedded in the polymer film surface;
Artificial blood vessels comprising a.
The artificial blood vessel of claim 1, wherein the ceramic or metal contained in the thin film patterned bioactive material is selected from the group consisting of hydroxyapatite, titanium, tantalum, and gold.
The artificial blood vessel of claim 1, wherein the thin film patterned bioactive material is linear in the shape of a pattern.
The artificial blood vessel according to claim 3, wherein the width of the pattern and the spacing between the patterns, which are implemented through the photolithography process, are independently 10 to 100 μm.
The artificial blood vessel according to claim 1, wherein the thin film patterned bioactive material is in the form of a dot in the shape of a pattern.
The artificial blood vessel according to claim 5, wherein the diameter of the pattern and the distance between the patterns are independently 5-20 μm through a photolithography process.
The artificial blood vessel according to claim 1, wherein the polymer membrane has a thickness of 100 to 300 µm.
The artificial blood vessel according to claim 1, wherein the material of the polymer is a biodegradable polymer selected from the group consisting of poly-L-lactide, polylactide-glycolide copolymers, polycaflolactone and combinations thereof.
The tissue of claim 1, wherein the cells are cultured on a thin-patterned ceramic and / or metal-containing bioactive material while maintaining adhesion and cell-specific shape to repair the tissue. Artificial blood vessels characterized by being.
The artificial blood vessel according to any one of claims 1 to 8, wherein platelet adhesion is suppressed on the thin film patterned ceramic and / or metal-containing bioactive material.
Flexible polymer film; And a thin film patterned ceramic and / or metal-containing bioactive material embedded on the surface of the polymer membrane such that the polymer membrane is not peeled off when the polymer membrane is bent.
The ceramic and / or metal-containing biomaterial is transferred to a thin film-patterned ceramic and / or metal-containing bioactive material during solvent casting with a polymer solution during the polymer film manufacturing process and embedded on the surface of the flexible polymer film. A first step of thin film patterning the active material; And
And a second step of forming a conduit by rolling the thin film patterned polymer membrane as the bioactive material.
The method of claim 11, wherein the first step is
Step 1-1 to form a photosensitive liquid pattern on the surface of the support through a photoresist process;
A first step of forming a metal thin film pattern on the surface of the support by depositing a metal thin film pattern corresponding to an intaglio portion of the photosensitive liquid pattern through physical deposition and then removing the photosensitive liquid pattern;
Applying a ceramic precursor-containing solution to the groove corresponding to the intaglio portion of the metal thin film pattern, and then removing the metal thin film pattern by forming a ceramic thin film pattern corresponding to the intaglio of the metal thin film pattern on the support surface; And
Steps 1-4 for transferring the ceramic thin film pattern formed on the support surface to the flexible polymer film
Method for producing artificial blood vessels, characterized in that it comprises a.
The method of claim 11, wherein the first step is
Step 1-1 to form a photosensitive liquid pattern on the surface of the support through a photoresist process;
A first step of forming a metal thin film pattern on the surface of the support by depositing a metal thin film pattern corresponding to an intaglio portion of the photosensitive liquid pattern through physical deposition and then removing the photosensitive liquid pattern; And
Steps 1-4 to transfer the metal thin film pattern formed on the surface of the support to a flexible polymer film
Method for producing artificial blood vessels, characterized in that it comprises a.
The method for producing an artificial blood vessel according to claim 11, wherein the artificial blood vessel according to any one of claims 1 to 8.
A polymer structure having flexibility to form stretchable or curved surfaces; And
In order to prevent peeling when the surface of the polymer structure is stretched or bent, a pattern profile is embossed or embossed through a photolithography process to form a thin film patterned ceramic and / or metal-containing bioactive material to form the polymer structure. A thin film patterned ceramic and / or metal-containing bioactive material transferred to the surface of the polymer structure and embedded in the surface of the polymer structure;
Elastic or flexible insert (implant) comprising a.

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