KR101875587B1 - Conduit for guided tracheal tissue regeneration and method for thereof - Google Patents

Abstract

The present invention relates to a conduit for guided trachea regeneration having a composite membrane structure consisting of a structure having a net structure and a microporous membrane layer having an asymmetric structure, and a manufacturing method thereof. According to the present invention, the conduit for guided trachea regeneration has a mechanical property capable of withstanding an external load during trachea regeneration to prevent tracheal stenosis during in vivo implantation. The interior and exterior of the conduit for guided trachea regeneration have an asymmetric microporous structure to properly deliver oxygen and nutrients, assist in binding to surrounding tissue such as cartilage, and allow surrounding tissue such as the cilium epithelial layer to properly adhere using the delivered oxygen and nutrients to effectively contribute to trachea regeneration. Also, the conduit for guided trachea regeneration can load a growth factor and a protein. The loaded growth factor and protein effectively proliferate and are divided into cilium epithelial cells important in trachea regeneration while being discharged in a sustained release form.

Description

Technical Field [0001] The present invention relates to a regeneration induction tube for guided tracheal tissue regeneration,

The present invention relates to an engine regeneration induction pipe and a method of manufacturing the same, and more particularly, to an engine regeneration pipe having an adequate mechanical strength as an engine and capable of providing an environment effective for regeneration of the engine and a method of manufacturing the same.

Trachea is the airway between the larynx and the bronchi, which serves as a passage for air to enter and leave the respiratory tract. Many of the recent accidents, drug addiction, and increased surgical outcomes due to the development of medical care, and the continued use of respiratory aids in many patients during intensive care unit management have resulted in a wide variety of organ damage, including congenital causes, trauma, malignant tumors, The cause of damage to the engine is increasing due to the cause.

In general, the method of treating injured organs is determined by the size of the damaged area, especially the length. When the lesion is long, such as congenital tracheal stenosis, or if it is accompanied by subglottic stenosis, And transplantation. However, it is complicated due to complicated surgical procedure and various stages of surgery are needed. It is not established as a standard treatment method because of the disadvantages such as damage occurring during autologous tissue smear.

In addition, although a method of transplanting a donor tissue of a dead person is recently reported, it is difficult to obtain a donor tissue. Even if a donor tissue is sought, complicated pretreatment can not be performed until the organ is transplanted. It remains a problem.

In order to solve such a problem, recently, treatment studies using tissue engineering techniques have been actively carried out not only by using artificial materials but also by using living tissue.

In the early stage of tissue regeneration for organ regeneration, we tried to regenerate the organ by simply constructing the scaffold to make the support for maintaining the organ, but the granulocyte formed because the respiratory mucosa was not regenerated on the inner surface of the scaffold And the formation of a successful organs such as the generation of pneumonia due to the inability of the foreign substances in the engine to be discharged to the outside.

In order to manufacture such an engine regeneration induction tube, various supports are used to manufacture an engine regeneration tube having an empty interior. However, since the physical properties of the support are weak, There was a problem that the circular shape could not be maintained and the shape was distorted to restrict air movement during breathing.

 In order to compensate for this, there has been an effort to fabricate an engine regeneration tube by wrapping the surface of the supporter with a corrugated structure of a straw, but the corrugated structure also has limitations in maintaining its physical properties.

Therefore, it is urgent to develop an organ regeneration induction tube capable of satisfactorily regenerating various tissues while having mechanical properties such that the organ stenosis does not occur.

 Cull DL, Lally KP, Mair EA. Ann Thorac Surg. 1990; 50: 899-901  Kim J, Suh SW, Shin JY. J Thorac Cardiovasc Surg. 2004; 128: 127-129  Kim HS, Suh H, Lee JH. Tissue Eng Regen Med. 2011; 8: 439-445

It is an object of the present invention to provide an engine regeneration pipe having sufficient mechanical strength as an engine and capable of providing an environment effective for regeneration of the engine.

Another object of the present invention is to provide a method for manufacturing an engine regeneration pipe having the above-described features.

The organ regeneration induction tube according to the present invention is characterized by having a composite membrane structure composed of a biodegradable polymer structure having a net structure and an asymmetric microporous membrane layer.

It is preferable that the thickness of the mesh of the biodegradable polymer structure having the net structure is 100 to 500 탆 and the interval of the net is 500 to 1000 탆.

The structure having the net structure preferably has a flexural strength of 0.1 to 0.8 MPa.

According to an embodiment of the present invention, the organ regeneration induction tube may be a human bronchial epithelial cell (HBEC), a basic fibroblast growth factor (bFGF), a hepatocyte growth factor (HGF) ), At least one growth factor selected from Transforming growth factor-beta (TGF-beta), and Epidermal growth factor (EGF) And the like.

According to another embodiment of the present invention, the surface of the organ regeneration induction tube contains at least one protein selected from the group consisting of collagen type IV, fibronectin, and laminin, Is discharged in a sustained manner from the engine regeneration induction pipe.

The asymmetric microporous membrane layer is characterized by an inner surface having an average pore size of 10 to 100 nm and an outer surface having an average pore size of 50 to 200 m.

The structure having the net structure and the polymer forming the asymmetric microporous membrane may be selected from poly (lactic acid), poly (glycolic acid), poly (lactic acid) having a weight average molecular weight of 1,000 to 1,000,000 g / mol, Poly (lactic acid-co-glycolic acid)], polycaprolactone, poly (lactic acid-co-ε-caprolactone) At least one biodegradable polymer selected from the group consisting of polyhydroxybutyric acid-co-hydroxyvaleric acid, polydioxanone, and polyphosphoester, or a biodegradable polymer selected from the group consisting of polyhydroxybutyric acid- or

And 0.1 to 20 parts by weight of a hydrophilic polymer having a weight average molecular weight of 1,000 to 1,000,000 g / mol based on 100 parts by weight of the biodegradable polymer.

The hydrophilic polymer may be selected from the group consisting of polyethylene oxide-polypropylene oxide copolymer (PEO-PPO copolymer), polyethylene oxide-polylactic acid copolymer (PEO-PLA) (PEO-PLGA), polyethylene oxide-polycaprolactone copolymer (PEO-PCL), and polyethylene oxide-poly (lactic-co-glycolic acid) (PEO), polyvinyl alcohol (PVA), polyoxyethylene alkyl ethers (Brij Series), polyoxyethylene castor oil derivatives (Cremophores), polyoxyethylene sorbitan fatty acid esters Polyoxyethylene sorbitan fatty acid esters (Tween Series), and polyoxyethylene stearates (polyoxyethylene stearates). It may be at least one selected from the group.

It is preferable that the tube for organ regeneration of the composite membrane structure is manufactured by using a cylindrical hydrogel as a support.

The cylindrical hydrogel support is preferably prepared by physically crosslinking the hydrophilic polymer.

The hydrophilic polymer used as the cylindrical hydrogel support includes alginic acid, hyaluronic acid, carboxymethyl cellulose (CMC), dextran, hydroxypropylmethylcellulose Ust [hydroypropyl methyl celluolse (HPMC)] , polyhydroxy ethylene methacrylate [polyhydroethyl methacrylate (polyHEMA)], polyvinylpyrrolidone, [poly (N -vinyl pyrrolidone (PVP )), poly-isopropyl acrylamide [poly ( N- isopropyl acrylamide (PNIPAAm)], polyvinyl alcohol (PVA), polyethylene oxide (PEO), and polyethylene oxide-polypropylene oxide ≪ / RTI >

The present invention also provides a method for preparing an organ regeneration induction tube having a composite membrane structure comprising a biodegradable polymer structure having an asymmetric microporous membrane and a net structure according to the present invention comprises the steps of preparing a cylindrical hydrogel support, Covering the biodegradable polymer structure with a biodegradable polymer solution, impregnating the biodegradable polymer solution with a hydrogel support coated with the net structure to form an asymmetric microporous membrane layer on the structure, and removing the cylindrical hydrogel support Which is produced by the above-mentioned method.

INDUSTRIAL APPLICABILITY According to the present invention, an engine regeneration pipe having mechanical properties capable of withstanding an external load during regeneration of an engine was manufactured.

Therefore, the organ regeneration induction tube according to the present invention has an appropriate physical property so that the organ is not stenosed when implanted, and the inside of the organ regeneration induction tube has an asymmetric microporous structure having nanopore and an outer microporous structure The outer micropores serve to facilitate the binding of oxygen and nutrients to surrounding tissues such as cartilage, and the inner nanopores are formed by the surrounding oxygen such as ciliated epithelium, So that it can be effectively adhered to the organ regeneration.

In addition, in the present invention, an induction tube having physical properties advantageous for regeneration of the organ as described above can be manufactured, and a growth factor and a protein can be loaded thereon. The growth factor and the protein are released into the sustained release over 40 days Effect. The growth factor and the protein are released in a sustained release form, and thus they are effectively proliferated and differentiated into ciliated epithelial cells which are important for regeneration of the organ.

1 is a view of a structure having a net structure according to the present invention (a, the thickness of the net; b, the distance between the net)
2 is a structure of an engine regeneration pipe of a composite membrane structure according to the present invention,
FIG. 3 is a schematic view showing a process for manufacturing an engine regeneration tube of a composite membrane structure according to the present invention,
FIG. 4 is a SEM photograph of an asymmetric porous membrane / mesh structure (3D printed mesh)
FIG. 5 shows the tensile strength measurement result of the asymmetric porous membrane / mesh structure (3D printed mesh)
6 is a result of measuring the bending strength of the asymmetric porous membrane / mesh structure (3D printed mesh)
FIG. 7 shows the results of cell mobility of growth factors and proteins using human bronchial epithelial cells (HBEC)
FIG. 8 shows (A) the loading amount of HGF in the organ regeneration induction tube equipped with the growth factors HGF and HGF / Col IV, (B) shows the release behavior of the HGF with time,
FIG. 9 shows the loading amount of collagen type IV in the organ regeneration induction tube equipped with growth factor HGF and HGF / Col IV, (B) shows the release behavior of collagen type IV with time,
FIG. 10 shows the results of a comparison between the control group (control), HGF alone group (HGF), collagen type IV only group (Col IV), and HGF and collagen type IV without HGF and collagen type IV The results of DNA content of bronchial epithelial cells cultured in the organ regeneration tube of the group (HGF / Col IV)
FIG. 11 shows the results of a comparison between the control group (HGF) and the group (HGF) in which only the collagen type IV was introduced (Col IV), and the group in which the HGF and the collagen type IV were not introduced (HGF / Col Ⅳ) in the bronchial epithelium and the ciliated epithelial cells, MUC5AC, β-tubulin Ⅳ, E-cadherin and ZO-1 RT-PCR of the cells,
FIG. 12 shows the results of a comparison between the control group (HGF) and the group (HGF) in which only the collagen type IV was introduced (Col IV), and the group in which the HGF and the collagen type IV were not introduced Western blot analysis was performed to confirm the differentiation of ciliated epithelial cells into β-actin (β-actin) and β-actin (HGF / Col Ⅳ)
FIG. 13 shows the results of a comparison between the control group (HGF) and the group (HGF) in which only the collagen type IV was introduced (Col IV), and the group in which the HGF and the collagen type IV were not introduced (HGF / Col Ⅳ) in the bronchial epithelial cells were examined by immunofluorescent staining. The results were as follows:
FIG. 14 is a graph showing the results of a comparison between HGF and a group without collagen type IV introduced into the control (control) group, HGF alone group (HGF), collagen type IV only group (Col IV), and HGF and collagen type IV Group (HGF / Col IV) in vivo ,
FIG. 15 shows the results of a comparison between the control group (HGF) and the group (HGF) without collagen type IV, the group with collagen type IV only (Col IV), and the group with HGF and collagen type IV (HGF / Col < IV >),
16 and 17 are micro-CT photographs of the tissue regeneration induction tube according to the present invention and the tissue regeneration induction tube according to Comparative Example 2 (FIG. 16) (Fig. 17).

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

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a,""an," and "the" include singular forms unless the context clearly dictates otherwise. Also, " comprise "and / or" comprising "when used herein should be interpreted as specifying the presence of stated shapes, numbers, steps, operations, elements, elements, and / And does not preclude the presence or addition of one or more other features, integers, operations, elements, elements, and / or groups.

TECHNICAL FIELD The present invention relates to an engine regeneration induction pipe having a composite membrane structure having a mechanical property excellent and a structure effective for regeneration of the engine and a method for manufacturing the same.

The tube for organ regeneration of the composite membrane structure according to the present invention comprises a biodegradable polymer structure having a net structure and a biodegradable polymer membrane layer.

It is apparent to those skilled in the art that the tube for regeneration of an organ of the present invention has a tube-shaped structure which is literally hollow inside, and that the thickness and length of the induction tube can be variously selected depending on the application to be used.

In the present invention, in order to solve the problem of distorted or deformed shape of the induction tube in which the material used as the conventional support is manufactured due to weak physical properties in manufacturing the tube regeneration induction tube, the surface of the supporter And then the biodegradable polymer membrane layer was formed to prepare an organ regeneration tube of the final composite membrane structure.

The term " structure having a net structure " according to the present invention means that the structure is repeated horizontally and vertically at regular intervals to form a mesh (mesh) as shown in FIG.

It is preferable that the thickness (a) of the net of the structure having the net structure is 100 to 500 탆 and the interval (b, interval) of the net is 500 to 1000 탆. When the net thickness (a) of the structure having the net structure is less than 100 탆, the physical properties are weak. When the net thickness exceeds 500 탆, the thickness is too thick, which is not preferable.

In addition, when the net structure spacing (b) of the net structure is less than 500 탆, the interval is narrow, moisture can not escape from the hydrogel used as a cylindrical support, the polymer porous membrane layer can not be formed smoothly, There is a problem that the physical properties are weak, which is not preferable.

In addition, the structure having the net structure according to the present invention can withstand the external force when the organ regeneration induction tube according to the present invention is transplanted, as long as the flexural strength satisfies the range of 0.1 to 0.8 MPa.

In addition, it is preferable that the structure having the net structure uses the same material as the microporous membrane layer having an asymmetric structure formed thereon. (Lactic acid), poly (glycolic acid), poly (lactic acid-glycolic acid) copolymer having a weight average molecular weight of 1,000 to 1,000,000 g / mol, co-glycolic acid], polycaprolactone, poly (lactic acid-co-ε-caprolactone), polyhydroxybutyric acid-polyhydroxybutyric acid copolymer acid-co-hydroxyvaleric acid, polydioxanone, and polyphosphoester, or may be one or more biodegradable polymers selected from the group consisting of 100 weight parts of the biodegradable polymer, More preferably 0.1 to 20 parts by weight of a hydrophilic polymer having 1,000 to 1,000,000 g / mol.

Meanwhile, the polymer scaffold for preparing the organ regeneration induction tube according to the present invention is preferably a hydrogel hydrophilic polymer in the form of a cylinder, and examples thereof include alginic acid, hyaluronic acid, carboxy Poly (methyl methacrylate) (polyHEMA), poly (methyl methacrylate), poly (methyl methacrylate), poly (methyl methacrylate) vinylpyrrolidone [poly (N -vinyl pyrrolidone (PVP )), poly-isopropyl acrylamide [poly (N -isopropyl acrylamide) ( PNIPAAm)], polyvinyl alcohol [polyvinyl alcohol (PVA)], polyethylene oxide [poly ( ethylene oxide (PEO), and polyethylene oxide-polypropylene oxide (PAO) copolymers.

Preferably, the cylindrical hydrogel polymer scaffold is prepared by preparing an aqueous solution of a hydrophilic polymer and crosslinking the polymer in a cylindrical mold having a thickness and a length suitable for each application.

The concentration of the hydrophilic polymer aqueous solution is preferably 0.1 to 50% by weight in view of obtaining a stable structure of the engine regeneration matrix.

The cylindrical polymeric scaffold according to the present invention is preferably structured in a hydrogel form in that it has a hydrogel form, and the crosslinking of the material for forming the hydrogel can be carried out by using a suitable cross-linking agent, applying ultraviolet rays or heat, Any known method may be used without particular limitation as long as it is a method.

In addition, when the microporous membrane layer having an asymmetric structure is formed on the outside thereof, the hydrogel polymer scaffold is finally removed from the organ regeneration induction tube and is temporarily used for manufacturing a tube-shaped induction tube .

However, when only the hydrogel polymer scaffold is used, the morphology of the organ regeneration inducing pipe to be manufactured may be modified. In the present invention, it is most preferable to use the structure having a net structure in a wrapped state of the hydrogel polymer scaffold desirable.

The microporous membrane layer having an asymmetric structure formed at the outermost periphery of the engine regeneration pipe of the present invention is composed of an inner surface having an average pore size of 10 to 100 nm and an outer surface having an average pore size of 50 to 200 m It is preferable to have an asymmetric porous structure.

In other words, the inner surface (the surface in contact with the hydrogel) has a pore size of about 100 nm which allows the surrounding tissues to adhere well through oxygen and nutrients to regenerate the ciliated epithelium, and the outer surface transmits oxygen and nutrients, (About 150 ㎛) to allow the organ regeneration induction tube to be more stably present in the human body by binding to the surrounding tissues such as the tissue, and has an asymmetric porous structure capable of selective permeation, .

Such an asymmetric porous structure is formed by a solvent-nonsolvent exchange and a phase separation phenomenon between a solvent and a non-solvent. Specifically, a cylindrical hydrogel polymer scaffold surrounded by a net structure is immersed in the biodegradable polymer solution N, N-dimethylacetamide, which is a solvent used in the polymer solution by solvent-nonsolvent exchange, and the water contained in the non-solvent hydrogel polymer scaffold meet and exchange with each other And precipitation is formed due to the diffusion of water. When this is completely washed in distilled water to remove residual solvent, the hydrogel polymer scaffold is removed and a composite membrane engine regeneration tube composed of a net structure and an asymmetric microporous membrane layer is formed Can be manufactured.

Such an asymmetric microporous membrane layer comprises a poly (lactic acid), a poly (glycolic acid), a poly (lactic acid-glycolic acid) copolymer having a weight average molecular weight of 1,000 to 1,000,000 g / poly (lactic acid-co-glycolic acid)], polycaprolactone, poly (lactic acid-co-ε-caprolactone), polyhydroxybutyric acid- Wherein the biodegradable polymer is at least one biodegradable polymer selected from the group consisting of polyhydroxybutyric acid-co-hydroxyvaleric acid, polydioxanone, polyphosphoester, And 0.1 to 20 parts by weight of a hydrophilic polymer having a weight average molecular weight of 1,000 to 1,000,000 g / mol.

Among the above biodegradable polymers, polycaprolactone (PCL) is most preferable as a material for producing an organ regeneration induction tube because of its excellent physical properties, flexibility and biocompatibility and a long biodegradation period.

The hydrophilic polymer may be selected from the group consisting of polyethylene oxide-polypropylene oxide copolymer (PEO-PPO copolymer), polyethylene oxide-polylactic acid copolymer (PEO-PLA ), Polyethylene oxide-poly (lactic-co-glycolic acid) copolymer (PEO-PLGA), polyethylene oxide-polycaprolactone copolymer (PEO- , Polyethylene oxide (PEO), polyvinyl alcohol (PVA), polyoxyethylene alkyl ethers (Brij Series), polyoxyethylene castor oil derivatives (Cremophores), polyoxyethylene sorbitan Polyoxyethylene sorbitan fatty acid esters (Tween Series), and polyoxyethylene stearates. It may be at least one selected from the group of binary, of which the polyethylene oxide-polypropylene oxide copolymer is most preferably the chain Pluronic series.

FIG. 2 shows the structure of the tube for regenerating the engine of the composite membrane structure manufactured according to the present invention.

The method for manufacturing an organ tube for regeneration of an organ membrane according to the present invention comprises the steps of preparing a cylindrical hydrogel support as illustrated in FIG. 3, preparing a biodegradable polymer structure having a net structure on the cylindrical hydrogel support, A step of forming a biodegradable polymer membrane layer on the structure by impregnating the hydrogel support coated with the net structure on the biodegradable polymer solution and removing the cylindrical hydrogel support, An organ regeneration induction pipe made of a composite membrane of a structure / asymmetric porous membrane layer can be manufactured.

The cylindrical hydrogel polymer scaffold is prepared by dissolving the polymer in water, which is a solvent, to prepare an aqueous solution. To make the polymer hydrogel polymer scaffold of various thicknesses and lengths, the polymer aqueous solution is dispersed in various sizes (1 to 100 mm) mm) and then crosslinked. The concentration of the polymer aqueous solution is preferably 0.1 to 5% by weight in view of obtaining a stable structure of the engine regeneration induction pipe.

Then, the thus prepared cylindrical hydrogel polymer scaffold having various thicknesses and lengths is covered with a structure having a net structure.

The thickness of the net of the structure having the net structure is preferably 100 to 500 탆, and the interval between the net is preferably 500 to 1000 탆.

In addition, the structure having the net structure according to the present invention can withstand the external force when the organ regeneration induction tube according to the present invention is transplanted, as long as the flexural strength satisfies the range of 0.1 to 0.8 MPa.

In addition, the biodegradable polymer structure having the net structure is preferably made of the same material as the biodegradable polymer membrane layer formed thereon.

In addition, the step of forming a biodegradable polymer layer having an asymmetric microporous structure on the structure by impregnation precipitation method is applied to the biodegradable polymer solution covered with the net structure structure.

The concentration of the biodegradable polymer solution used is preferably in the range of 0.1 to 50% by weight in view of obtaining the structure of the microporous membrane having an asymmetric structure and maintaining the physical properties of the engine regeneration tube to be finally produced. The solvent used for preparing the biodegradable polymer solution may be selected from the group consisting of tetraglycol, 1-methyl-2-pyrrolidinone (NMP), triacetin, benzyl alcohol, and the like.

The asymmetrically structured microporous membrane is formed by a solvent-nonsolvent exchange in which water in the Tetraglycol, N, N-dimethylacetamide and non-solvent hydrogel polymer supports, which are used in the polymer solution, a precipitation is formed due to diffusion. When the solution is completely washed with distilled water to remove residual solvent, the hydrogel polymer scaffold is removed and a composite membrane consisting of a net structure and an asymmetric microporous membrane layer made of a biodegradable polymer An engine regeneration induction pipe can be manufactured.

In the present invention, the surface of the hydrogel polymer scaffold is wrapped with a net structure. When the biodegradable polymer solution is impregnated with the biodegradable polymer, the biodegradable polymer solutions pass through the net structure of the structure to the hydrogel polymer scaffold Is spread.

Also, in the present invention, various growth factors and proteins can be mounted on the surface of the organ regeneration induction tube of the composite membrane structure.

The growth factors include human bronchial epithelial cells (HBEC), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), transforming growth factor- beta, TGF-beta, and epidermal growth factor (EGF).

Examples of the protein include collagen type IV, fibronectin, and laminin, but are not limited thereto.

The growth factor is preferably loaded with heparin or the like, but the method is not particularly limited. In addition, the protein may also be loaded onto the prepared organ regeneration induction tube by physical adsorption, and may be carried out according to a conventionally known method.

When the growth factor and the protein are mounted on the organ regeneration induction tube as in the present invention, the growth factor and the protein are released into the sustained release over 40 days.

Usually, the inner wall of the organ is covered with the ciliated epithelium. These cilia play an important role in discharging the foreign substances inhaled when they breathe, and it is known that regeneration of the ciliated layer is most important in regenerating the organ.

Therefore, in the present invention, when an induction tube having physical properties favorable for regeneration of an organ is manufactured, and the growth factor and the protein are loaded thereon, the growth factor and the protein are released in a sustained release form and the proliferation and differentiation .

In addition, the inner surface of the organ regeneration tube according to the present invention is expected to be more effective for regeneration of the organs because the surrounding tissue is well adhered through oxygen and nourishment to have a pore size of about 100 nm, in which the ciliated epithelium can be regenerated .

Hereinafter, preferred embodiments of the present invention will be described in detail. The following examples are intended to illustrate the present invention, but the scope of the present invention should not be construed as being limited by these examples. In the following examples, specific compounds are exemplified. However, it is apparent to those skilled in the art that equivalents of these compounds can be used in similar amounts.

Example  1: Manufacture of an engine regeneration tube using a net structure structure

1) Asymmetric microporous composite membrane production

PCL (12 wt%) and Pluronic F127 (5 wt%, PCL base) were mixed and dissolved in a solvent mixture of Tetraglycol and N, N-dimethylacetamide in a ratio of 2: 1 at 90 ° C to prepare a polymer solution Respectively. In addition, a cylindrical hydrogel containing a large amount of water was prepared by freezing / thawing the 15 wt% PVA aqueous solution.

A 3D printed mesh (material, PCL; net thickness of strand having a net structure of 300 mu m, mesh spacing of 900 mu m, bending strength of 0.19 MPa) having an inner diameter equal to the outer diameter of the cylindrical PVA hydrogel prepared above was placed .

The PVA hydrogel covered with the structure having the net structure was impregnated with the PCL / Pluronic F127 mixed polymer solution. When the polymer solution comes into contact with the PVA hydrogel, the solvent Tetraglycol, N, N-dimethylacetamide and the non-solvent exchange with each other by solvent-nonsolvent exchange, .

When the PVA hydrogel was immersed in distilled water for 30 minutes, the PVA hydrogel was removed, and at the same time, completely washed with distilled water to remove remaining solvent, and vacuum dried to prepare an asymmetric microporous membrane / 3D printed mesh composite organ regeneration pipe.

2) Introduction of heparin

The prepared tissue regeneration tube was immersed in heparin aqueous solution [3 mg / ml (2 wt% NaCl solution)] for 3 hours at 4 ° C to bind heparin. After washing with excess distilled water to remove unbound heparin, it was lyophilized to prepare an organ regeneration induction tube into which heparin was introduced.

3) Introduction of hepatocyte growth factor

An organ regeneration induction tube containing heparin was immersed in an aqueous solution of hepatocyte growth factor [200 ng / ml (PBS)] and allowed to stand in a refrigerator at 4 ° C for 6 hours. Growth factors that were not introduced into the surface of the organ regeneration tube were washed with excess PBS for one hour and then removed in 1 ml of buffer solution (PBS containing 0.1 wt% BSA) to prevent agglutination of the released growth factors Impregnated.

Example  2

After proceeding to 2) of Example 1, the collagen protein was adsorbed through the following 3) step.

3) Introduction of collagen type IV

Collagen type Ⅳ was physically adsorbed on the surface of the organ regeneration tube and immersed in collagen type Ⅳ aqueous solution [0.1 mg / ml (PBS)] for 1 hour at 37 ° C. Was washed with excess PBS and then impregnated with 1 ml of buffer (PBS containing 0.1 wt% BSA). (1, 3, 5, 7, 14, 21, 28, and 35 days) in a thermostat rotating at 37 ° C and 50 rpm to confirm the release behavior of collagen type IV. The amount of collagen type Ⅳ eluted from the obtained buffer solution was analyzed using a collagen type Ⅳ ELISA kit.

Comparative Example  1: Cylindrical PVA Hydrogel  Manufacture of induction tube for regeneration using support

The cylindrical PVA hydrogel prepared in Example 1 was used as a support and impregnated with a PCL / Pluronic F127 mixed polymer solution to prepare an engine regeneration induction tube.

Comparative Example  2: Study using only 3D printed mesh

In Example 1, only the 3D printed mesh was used as a support to impregnate a PCL / Pluronic F127 mixed polymer solution to prepare an organ regeneration induction tube.

Experimental Example  1: Structure verification

To observe the morphology of the regenerated induction tube, a 200 Å thick platinum coating was performed using an ion sputter (SC 7680, Quorum Technologies, UK), followed by scanning electron microscopy (SEM; S-3000N, Hitachi, Japan) And the inside and outside of the organ regeneration tube were observed.

As shown in FIG. 4, the inner surface (the surface in contact with the PVA hydrogel) has a pore size of about 100 nm in which the surrounding tissues adhere well through oxygen and nourishment to regenerate the ciliary epithelium, It can be observed that the asymmetric porous structure capable of selective permeation having a comparatively large pore size (about 150 탆) which allows nourishment to communicate well with surrounding tissues such as cartilage tissue, there was.

Experimental Example  2 : The tensile strength  (Tensile strength) measurement

Tensile tests were carried out to evaluate the mechanical properties of the manufactured regeneration tubes. The specimens were prepared with a diameter of 7 mm and a length of 20 mm. The specimens were placed in a UTM tensile machine (AG-5000G, Shimadzu, Japan). At this time, the tensile strength of the regeneration induction tube was evaluated at a cross-head speed of 50 mm / min. The tensile strength was measured by comparing the rabbit of Example 1 (Hybrid tube), Comparative Example 1 (Membrane), Comparative Example 2 (3D printed mesh), and a control group.

5, the maximum tensile strength of the asymmetric porous membrane / 3D printed mesh composite engine regeneration induction tube is compared with that of Comparative Example 1 (Membrane), Comparative Example 2 (3D printed mesh), and rabbit Which is higher than the tensile strength.

Experimental Example  3: Bending strength  Flexural strength measurement

The bending strength test was carried out on a UTM tensile machine (AG-5000G, Shimadzu, Japan) with a diameter of 7 mm and a length of 20 mm. The cross-head speed was 2.8 mm / The bending strength of the engine regeneration tube was evaluated to see if it could withstand the pressure. The tensile strengths were compared by measuring the rabbits as the above-mentioned Example 1 (Hybrid tube), Comparative Example 1 (Membrane), Comparative Example 2 (3D printed mesh) and a control group.

Referring to FIG. 6, it was judged that the maximum bending strength of the asymmetric porous membrane / 3D printed mesh compound induction tube reaches 60% of the rabbit organs, which is suitable for regenerating the organs.

However, the bending strength of the tube for regeneration of the engine according to Comparative Example 1 (Membrane) and Comparative Example 2 (3D printed mesh) was remarkably decreased. In the case of Comparative Example 1 (Membrane) using only a biodegradable membrane, the bending strength was so low that it was difficult to withstand the external pressure at the time of transplantation. Since the mechanical properties were weak and the shape of the organ was not maintained, It was judged that reproduction was impossible.

Experimental Example  4: Cell migration experiment

To determine growth factors and proteins that are effective in promoting the regeneration of ciliated epithelial cells in the trachea, cell migration experiments using human bronchial epithelial cells (HBEC) were performed. Bronchial epithelial cells were treated with Mytomycin C for 100% confluence on the surface of the culture dish to inhibit cell proliferation. Cells were separated by a scratch method and cultured in 100 ng / 35 mm dish of basic fibroblast growth (HGF), transforming growth factor-beta (TGF-β), and epidermal growth factor (EGF) Respectively. The mobility of bronchial epithelial cells following various growth factor treatments was compared by comparing the rate at which cells migrate into the empty space over time.

Collagen type Ⅳ, fibronectin and laminin were coated at a concentration of 10 ng / 35 mm dish. Bronchial epithelial cells were treated with Mytomycin C to inhibit cell proliferation, and cells were incubated for a certain period of time after the cells were separated by scratch method.

Referring to FIG. 7, the mobility of human bronchial epithelial cells (HBEC) was most active in the experimental group treated with HGF and the experimental group coated with collagen type IV as a result of the cell mobility test.

Experimental Example  5: growth factor Release behavior  Confirmation experiment

(1, 3, 5, 7, 14, 21, 28, and 35 days) in a thermostat rotating at 37 ° C and 50 rpm to confirm the release behavior of growth factors. The whole buffer solution was carefully taken at every fixed interval and the new buffer solution was exchanged again to collect the sample from which the growth factor was released. The amount of the growth factor eluted from the buffer solution from which the obtained growth factor was released was analyzed using an HGF ELISA kit.

Referring to FIG. 8, when HGF alone was introduced, about 120 to 140 ng / cm ^{2 of} HGF was detected, and 110 to 130 ng / cm ² of HGF / collagen type IV was detected (Fig. 8 (A)).

As shown in FIG. 8 (B), it was confirmed that HGF / Collagen type IV was introduced into the organ regenerating induction tube and HGF / Collagen type IV introduced heparin and growth factor It was not released much in the early stage and gradually released until 42 days.

Experimental Example  6: Collagen Release behavior  Confirmation experiment

Collagen type Ⅳ can be physically adsorbed on the surface of the organ regeneration tube, immersed in collagen type Ⅳ aqueous solution [0.1 mg / ml (PBS)], allowed to stand for 1 hour at 37 ° C, After washing with excess PBS to remove type IV, it was impregnated with 1 ml of buffer (PBS containing 0.1 wt% BSA). (1, 3, 5, 7, 14, 21, 28, and 35 days) in a thermostat rotating at 37 ° C and 50 rpm to confirm the release behavior of collagen type IV. The amount of collagen type Ⅳ eluted from the obtained buffer solution was analyzed using a collagen type Ⅳ ELISA kit.

Referring to FIG. 9, 1.5 to 2.0 μg / cm ^{2 of the} collagen type IV only group was detected, and 2.0 to 2.5 μg / cm ^{2 of the} HGF / collagen type IV introduced group (Fig. 9 (A)). In addition, as shown in Fig. 9 (B), the release of collagen type IV was gradually released until 28 days in both groups.

Experimental Example  7: proliferation of bronchial epithelial cells and Differentiation behavior  analysis ( In vitro )

Human bronchial epithelial cells (HBEC) were used for cell proliferation and differentiation. Airway epithelial cell basal medium (ATCC) supplemented with bronchial epithelial cell growth kit (ATCC) was used. First, in a cell incubator (CO ₂ incubator, 3154, Forma Scientific, Inc., USA) maintained at 37 ° C and 5% CO ₂ , 150 cm ² Bronchial epithelial cells were cultured in monolayer on a cell culture dish and centrifuged at 1,200 rpm for 5 minutes using 0.25% trypsin-EDTA (Gibco, USA) The cells were diluted with culture medium supplemented with growth kit (ATCC) and counted using a hemacytometer (Reichert Co., USA). To an organ regeneration tube prepared for cell culture, 1 x 10 ⁴ cells / cm ² Bronchial epithelial cells were placed in 12-well non-tissue culture PS plates (SPL, USA). After 2 hours, all of the culture medium was removed and 1 ml of fresh culture medium was added. The culture and differentiation behavior were confirmed after incubation at 37 ° C and 5% CO ₂ in a cell incubator for a certain period (7, 14 days). The culture medium was replaced with a fresh culture medium every two days. The bronchial epithelial cells of the proliferated and differentiated bronchial epithelial cells were treated with DNA content (Fig. 10), RT-PCR (Fig. 11), Western blot Immunofluorescent staining (Fig. 13) confirmed the differentiation behavior into ciliobacters.

For comparison with the present invention, HGF and a group not introducing collagen type IV were used as a control.

Referring to FIG. 10, it was confirmed that the proliferation was actively observed on day 14 rather than day 0, and that the concentration of DNA was not different in the groups other than the control group in which HGF and collagen type IV were not introduced there was.

In addition, RT-PCR was performed to confirm the mRNA level of the cells, and the behavior of differentiation into ciliated epithelial cells was confirmed by inserting bronchial epithelial cells into the organ regeneration induction tube into which HGF and collagen type IV were introduced. The epithelial markers MUC5AC, β-tubulin Ⅳ, E-cadherin and ZO-1 showed the highest expression values in the group in which HGF and collagen type Ⅳ were dual introduced. It was concluded that HGF and collagen type Ⅳ affected the differentiation into ciliated epithelial cells.

12, which is a result of Western blotting known as protein quantitative analysis to analyze more specific and objective cell differentiation, β-tubulin Ⅳ, a ciliated epithelial cell specific marker, And it was confirmed that the bands were thicker and thicker than the other groups in the group introduced with the dual, and the cells were most actively differentiated into the squamous epithelium cells.

13, which is a visual analysis of the differentiation behavior of the somatic embryonic cells through immunofluorescence staining, the blue nuclei were stained with the nuclei of the cells, and the distribution and proliferation of the cells were confirmed. Is a β-tubulin Ⅳ, a marker specifically expressed in ciliated epithelial cells, and is differentiated into ciliated epithelial cells. In the group introduced with Dual, it was confirmed that most of the cells were expressed in green, and the number of differentiation was increased. These results suggest that the organ regenerating induction tube into which HGF and collagen type Ⅳ are introduced as dual is most effectively differentiated into ciliated epithelial cells.

Experimental Example  8: Regeneration of organ tissue Induction ability  evaluation ( In vivo )

New Zealand White Rabbit (body weight, ~ 3.5 kg) was used to transplant the organ regenerating induction tube and evaluate the organ regeneration effect of the organ regenerating induction tube with HGF and collagen type IV introduced into the organ regeneration. (HGF), collagen type IV alone (Col Ⅳ), HGF and collagen type Ⅳ (HGF / collagen type Ⅳ), HGF (collagen type Ⅳ) Col Ⅳ).

Rabbit anesthesia was performed by intraperitoneal injection of 50 mg / kg of zoletil (Virbac, Carros, France) and 4.5 mg / kg of xylazine (Rumpun, Bayer, Korea). The hair of the rabbit surgical site was removed and sterilized before implanting the regenerated induction tube. Normal organs were removed by careful resection of 2 cm. The reconstructed tracheal tube was covered with the resected trachea 1 mm in thickness, and the overlapping sections of the tracheal regeneration tube were stitched with a micro suture (4-0 Vicryl, Johnson & Johnson, New Brunswick, NJ) And sutured. H & E staining was performed to determine the degree of regeneration after transplantation.

Referring to FIG. 14, in the group without control of HGF and collagen type IV, the ciliated epithelium was not regenerated. In all groups except for the control group without HGF and collagen type IV, Red circles, and arrows) were reproduced.

In addition, the thickness of the ciliary epithelial layer was measured. Referring to FIG. 15, the thickness of the ciliary epithelial layer was the best in the group in which HGF and collagen type IV were introduced at the same time as the other groups. HGF and collagen type Ⅳ at the same time will provide a good environment for regeneration of the organ.

Experimental Example  9: Regeneration of organ tissue Induction ability  evaluation ( In vivo )

As in Experimental Example 8, the H & E staining of the organ regeneration induction tube according to the present invention and the organ regeneration induction tube according to Comparative Example 2 was carried out to compare the performance thereof (FIG. 16) (Fig. 17).

Referring to FIGS. 16 and 17, when the 3D printed mesh is used alone as in Comparative Example 2, tissue is inserted between the meshes and granuloma is formed to confirm that the airway is blocked.

Claims (11)

A structure having a net structure, and
A microporous membrane layer having an asymmetric structure composed of an inner surface having an average pore size of 10 to 100 nm and an outer surface having an average pore size of 50 to 200 mu m formed on the structure having the net structure, tube.
The method according to claim 1,
Wherein the net of the structure having the net structure has a thickness of 100 to 500 mu m and an interval of the net is 500 to 1000 mu m.
The method according to claim 1,
Wherein the structure having the net structure satisfies a bending strength of 0.1 to 0.8 MPa.
The method according to claim 1,
The organ regeneration induction tube can be used for the treatment of various diseases such as bronchial epithelial cells (HBEC), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), transforming growth factor factor-beta, TGF-beta, and epidermal growth factor (EGF)
Wherein the growth factor is releasably released from the organ regeneration induction tube.
The method according to claim 1,
Wherein the surface of the organ regeneration induction tube contains at least one protein selected from collagen type IV, fibronectin, and laminin,
Wherein the protein is released in a sustained release form from the organ regeneration induction tube.
delete The method according to claim 1,
The structure having the net structure and the polymer forming the microporous membrane layer having an asymmetric structure may be formed of poly (lactic acid), poly (glycolic acid), or poly (lactic acid) having a weight average molecular weight of 1,000 to 1,000,000 g / ], Poly (lactic acid-co-glycolic acid), polycaprolactone, poly (lactic acid-co-epsilon-caprolactone) caprolactone), polyhydroxybutyric acid-co-hydroxyvaleric acid copolymer, polydioxanone, and polyphosphoester. The polyhydroxybutyric acid- Biodegradable polymer, or
Wherein the biodegradable polymer further comprises 0.1 to 20 parts by weight of a hydrophilic polymer having a weight average molecular weight of 1,000 to 1,000,000 g / mol based on 100 parts by weight of the biodegradable polymer.
8. The method of claim 7,
The hydrophilic polymer may be selected from the group consisting of polyethylene oxide-polypropylene oxide copolymer, polyethylene oxide-polylactic acid copolymer, polyethylene oxide-polylactic glycolic acid copolymer, polyethylene oxide-polycaprolactone copolymer, polyethylene oxide, polyvinyl alcohol, Wherein at least one selected from the group consisting of ethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan phthesic esters, and polyoxyethylene stearates is used.
The method according to claim 1,
Wherein the engine regeneration induction pipe of the composite membrane structure is manufactured by using a cylindrical hydrogel polymer as a support.
10. The method of claim 9,
The polymer may be selected from the group consisting of alginic acid, hyaluronic acid, carboxymethyl cellulose (CMC), dextran, hydropropyl methylcellulose (HPMC) ], polyhydroxy ethylene methacrylate [polyhydroethyl methacrylate (polyHEMA)], polyvinylpyrrolidone, [poly (N -vinyl pyrrolidone (PVP )), poly-isopropyl acrylamide [poly (N -isopropyl acrylamide) ( PNIPAAm) , At least one member selected from the group consisting of polyvinyl alcohol (PVA), polyethylene oxide (PEO), and polyethylene oxide-polypropylene oxide An organ regeneration induction tube having a composite membrane structure which is a hydrophilic polymer.
Preparing a cylindrical hydrogel support,
Covering the cylindrical hydrogel support with a structure having a net structure,
Impregnating the hydrogel support coated with the net structure on the polymer solution to form a microporous membrane layer having an asymmetric structure on the structure by substituting the solvent used in the polymer solution and the non-solvent used in the hydrogel support Step, and
And removing the cylindrical hydrogel support. The method of claim 1, wherein the microporous membrane layer has a net structure including an asymmetric structure.

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