KR102155828B1 - Polymer film used for adhering bioprostheses and method for manufacturing the same - Google Patents

Abstract

A polymer film for bonding a bio-implantation material and a method of manufacturing the same are disclosed. The present invention increases the adhesion and spread of cells at the insertion site in the living body during the procedure of a bioprosthesis, and adhesion with cells according to the behavior of cells formed on the first surface of a polymer film that adheres to cells to increase biocompatibility. By including a nanostructure having a nano-pattern of a predetermined shape to control sex, the bio-implant can induce a stable state with the cells of the insertion site in the body, thereby increasing the success rate of the implantation procedure in the body.

Description

A polymer film for bonding bio-implants and its manufacturing method {POLYMER FILM USED FOR ADHERING BIOPROSTHESES AND METHOD FOR MANUFACTURING THE SAME}

The present invention relates to a polymer film for bonding a bio-implantation material and a method of manufacturing the same. More specifically, it relates to a polymer film and a method of manufacturing the same, in which cells are initially adhered to the bio-implant and are grown so that cells can perform normal cell activity at the insertion site after inserting the bio-implant into a living body.

The content described in this section merely provides background information on the present embodiment and does not constitute the prior art.

In recent years, due to the aging of the population, not only implant procedures due to accidents and aging, but also dental procedures for health are increasing, and procedures for wearing in vivo devices such as pacemakers are also rapidly increasing.

If such a procedure fails, the procedure site is not healed, and the biological implant inserted in the living body may be exposed to the outside.Therefore, the test subject is at risk of infection, and if the procedure is reoperated, the procedure has already failed, so the rate of failure of the reoperation There is a problem that becomes higher.

Conventional methods of treating the surface of a bioprosthesis, such as coating and etching, have a problem that is difficult to apply by creating a specific structure, and there is also a problem in that the range in which functionality can be given to the surface of the bioprosthesis is limited.

Registered Patent Publication No. 10-1654457 (2016.09.05.) Registered Patent Publication No. 10-1107223 (2012.01.25.) Unexamined Patent Publication No. 10-2016-0029941 (2016.03.16.)

The present invention prevents the failure of the procedure when implanting a bioprosthesis into a living body and attaches cells through focal adhesion (FA) to a polymer film that imparts functionality such as improving biocompatibility to the surface of the bioprosthesis. And by forming a nanostructure capable of controlling spreadability, the arrangement of the intracellular skeleton and the adhesion and spread of cells that affect the intracellular signal transduction system are controlled, thereby inducing cell adhesion at the beginning of the procedure and for stable cell regeneration. It is to provide a polymer film for bonding a bio-implantation material that can control the spreadability of and a method of manufacturing the same.

In order to achieve the above object, the polymer film for adhering to a bio-implant inserted into a living body according to an embodiment of the present invention allows the bio-implant to adhere to cells located at the insertion site in the living body. It may include a nanostructure having a nano-pattern of a preset shape formed on the first surface to adhere to the cell to adjust the adhesion to the cell according to the behavior of the cell.

Preferably, the size of the nanostructure having the nano-pattern of the preset shape or the space between the nano-structures according to the nano-pattern is the cell so that the cell adheres to the nano-structure and fills the space between the nano-structures. The cell adheres to the nanostructure by adjusting the size of the focal adhesion (FA) that connects the inside and outside of the cell, transmits a signal from the outside of the cell to the inside of the cell, and maintains the adhesion of the cell. It can be adjusted to directly control the degree of spreading, Cell Spreading.

Preferably, the nanostructure having the nano-pattern of the preset shape according to the shape of the cell is a groove array, a ridge array, a hole array, a pillar array, and orthogonal It may be any one shape selected from a type of mesh type.

Preferably, the size of the nanostructure having the nano-pattern of the preset shape or the spacing between the nano-structures according to the nano-pattern is the average area of the cells and the local adhesion to control the cell spreadability from the size of the local adhesion ( FA) can be adjusted by taking into account the relationship in which the average area is inversely proportional.

Preferably, a polymer monomer is grown on a second surface to be attached to the bio-implant in addition to the first surface on which the nanostructure is formed so as to adhere to the surface of the bio-implant by adjusting the difference between the surface energy of the bio-prosthetic implant. Polymer chains can be formed.

Preferably, the polymeric monomer is lactide, lactic acid, glycolide, glycolic acid, caprolactone, caprolactic acid, trimethylene ( trimethylene) and carbonate (carbonate).

Preferably, the polymer chain may be DNA or RNA, which is a biopolymer that complementarily binds according to a nucleotide sequence extracted from a test subject into which the bioartificial insert is to be inserted.

Preferably, after the nanostructure adheres to the cells located at the insertion site in the living body, the polymer film is biodegraded in the living body, so that pill polyethylene glycol, poly-L-lactic acid (poly(L-lactic acid)) acid)), polylactic-co-glycolic acid (PLGA), polyglycolic acid (PGA), polylactide (PLA), polycaprolactone (PCL), chitosan ( chitosan), polytrimethylenecarbonate (PTMC), or a mixture of two or more selected from the polymer group may include a group of synthetic polymers having biocompatibility.

Preferably, before the polymer film is biodegraded in the living body, the nanostructure is fixed to the insertion site while the cells regenerate at the insertion site in the living body after the bio-implant is implanted in the living body. After adhering to the regenerating cells, the polymer film may be biodegraded in the living body.

Preferably, the bio-artificial implant may be selected from the group consisting of orthopedic implants such as joints, spines, valves, dental implants, vascular stents, non-vascular stents, implantable injection devices, pace makers, and artificial organs. have.

Preferably, the bioprosthetic implant may be selected from the group consisting of titanium, stainless steel, cobalt, gold, silver, platinum, magnesium, or alloys thereof.

In another embodiment of the present invention for achieving the above object, a method of manufacturing a polymer film for bonding a bio-implant to be attached to a bio-implant inserted into a living body is, wherein the bio-implant is located at the insertion site in the living body. Forming a nanostructure having a nano-pattern of a predetermined shape on the surface of a mold material for controlling adhesion with the cell according to the behavior of the cell so as to adhere to the cell; And transferring the nanostructure formed on the mold material to the polymer film to form the nanostructure on the first surface of the polymer film that adheres to the cells.

Preferably, the size of the nanostructure having the nano-pattern of the preset shape or the space between the nano-structures according to the nano-pattern is the cell so that the cell adheres to the nano-structure and fills the space between the nano-structures. The cell adheres to the nanostructure by adjusting the size of the focal adhesion (FA) that connects the inside and outside of the cell, transmits a signal from the outside of the cell to the inside of the cell, and maintains the adhesion of the cell. It can be adjusted to directly control the degree of spreading, Cell Spreading.

Preferably, the size of the nanostructure having the nano-pattern of the preset shape or the spacing between the nano-structures according to the nano-pattern is the average area of the cells and the local adhesion to control the cell spreadability from the size of the local adhesion ( FA) can be adjusted by taking into account the relationship in which the average area is inversely proportional.

Preferably, the forming of the nanostructure may include forming a nanopit in a polymer mold from the nanostructure formed in the mold material using a nanoimprint lithography process; Pouring a polymer precursor into the polymer mold in which the nanofit is formed, and curing by covering the detachable outer film; Removing the outer film covering the cured polymer precursor; And separating the polymer precursor from which the outer film has been removed from the polymer mold.

Preferably, the step of attaching the polymer film with the nanostructure formed on the first surface to the bio-implantation; Further comprising, the step of attaching the polymer film to the bio-prosthetic implant comprises: activating the surface of the polymer film and the surface of the bio-prosthetic implant using a physical method or a chemical method, respectively; Forming a polymer monomer on the surface of the activated bio-implantation material and the surface of the polymer film, respectively; Growing a polymer chain by growing a polymer monomer formed on the surface of the bio-implantation material and the surface of the polymer film; And combining the grown polymer chains on the surface of the bio-implantation material and the surface of the polymer film, respectively.

Preferably, the activating step is performed by immersing the bio-implant and the polymer film in a basic solution such as lithium hydroxide, sodium hydroxide, potassium hydroxide, or calcium hydroxide, respectively, and the surface of the bio-implant and the polymer using a chemical method. Each surface of the film can be activated.

Preferably, the activating step is performed using a physical method of treating with plasma or an ion beam using a gas consisting of any one selected from argon, oxygen, nitrogen, water vapor and ammonia, or a mixture thereof, and Each of the surfaces of the polymer films can be activated.

According to the embodiment of the present invention as described above, by controlling the local adhesion of cells by using the nanostructure formed on the polymer film, by controlling the adhesion and spreading of the cell, the bio-implantation induces a stable state at the insertion site in the living body, It can increase the success rate of the procedure.

In addition, according to an embodiment of the present invention, since the biodegradable polymer film on which the nanostructured surface is formed is manufactured using a mold, it can be directly applied to conventionally commercially available bio-insertable products without additional treatment, thereby reducing cost. There is an advantage.

In addition, according to an embodiment of the present invention, the biodegradable polymer film provides functionality on the surface of a living body implant, and then is gradually decomposed in the body at the same time as tissue regeneration, so that it can be stably removed in the body without any other procedure.

The effects of the present invention are not limited to the above-mentioned effects, and other effects not mentioned will be clearly understood by those skilled in the art from the following description.

1 is a view showing the structure of a polymer film for bonding a bio-implantation material according to an embodiment of the present invention.
FIG. 2 is a view for explaining a polymer film for bonding a bio-prosthesis attached to a bio-prosthesis according to an embodiment of the present invention.
3 is a view for explaining an example of a nano-pattern or a micro-pattern of a preset shape formed on the surface of a polymer film according to an embodiment of the present invention.
4 is a diagram illustrating a functional group coated on the surface of a polymer film for bonding a bioprosthesis according to an embodiment of the present invention.
5 is a view showing the structure of a polymer film for bonding a bio-implantation material according to another embodiment of the present invention.
6 is a view for explaining a method of activating the surface of the bio-prosthesis adhesive polymer film 10 and the bio-prosthesis according to an embodiment of the present invention.
FIG. 7 is a diagram illustrating an interaction between a nanopillar array formed on a polymer film for bonding a bio-implantation material and fat-derived mesenchymal stem cells (ASCs) according to an embodiment of the present invention.
8 is a view for explaining an effective contact area in which cells adhere to the surface of a nanostructure in adhesion with cells according to the formation of a local adhesion (FA) according to an embodiment of the present invention.
FIG. 9 is a diagram illustrating that a morphological change of adipose-derived mesenchymal stem cells (ASCs) is induced by a nanostructure according to an embodiment of the present invention.
10 is a diagram illustrating a relationship between an average area of a local adhesion (FA) and a size of a nanostructure according to an embodiment of the present invention.
11 is a view showing the relationship between the size of local adhesion (FA) and the spreadability of adipose-derived mesenchymal stem cells (ASCs) controlled by a nanopillar array according to an embodiment of the present invention.
12 is a view for explaining a method of manufacturing a polymer film for bonding a bio-implantation material according to an embodiment of the present invention.
13 is a flowchart illustrating a specific method of forming a nanostructure on the surface of a polymer film according to an embodiment of the present invention.
14 is a diagram illustrating a detailed method of manufacturing a polymer film for bonding a bio-implantation material according to another embodiment of the present invention.
15 is a flowchart illustrating a specific method of attaching a polymer film having a nanostructure formed thereon to a bio-implantation according to an embodiment of the present invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. Advantages and features of the present invention, and a method of achieving them will become apparent with reference to the embodiments described below in detail together with the accompanying drawings. However, the present invention is not limited to the embodiments to be posted below, but may be implemented in a variety of different forms, and only these embodiments make the posting of the present invention complete, and common knowledge in the technical field to which the present invention pertains. It is provided to completely inform the scope of the invention to those who have it, and the invention is only defined by the scope of the claims. The same reference numerals refer to the same components throughout the specification.

Unless otherwise defined, all terms (including technical and scientific terms) used in the present specification may be used as meanings that can be commonly understood by those of ordinary skill in the art to which the present invention belongs. In addition, terms defined in a commonly used dictionary are not interpreted ideally or excessively unless explicitly defined specifically.

In the present specification, terms such as "first" and "second" are used to distinguish one component from other components, and the scope of the rights is not limited by these terms. For example, a first component may be referred to as a second component, and similarly, a second component may be referred to as a first component.

In the present specification, the identification code (eg, a, b, c, etc.) is used for convenience of description, and the identification code does not describe the order of each step, and each step is clearly in context. It may occur differently from the specified order unless a specific order is specified. That is, each of the steps may occur in the same order as specified, may be performed substantially simultaneously, or may be performed in the reverse order.

In this specification, expressions such as “have”, “may have”, “include” or “may include” indicate the existence of a corresponding feature (eg, a number, function, operation, or component such as a part). Points, and does not exclude the presence of additional features.

1 is a view showing the structure of a polymer film for bonding a bio-implantation material according to an embodiment of the present invention.

FIG. 2 is a view for explaining a polymer film for bonding a bio-prosthesis attached to a bio-prosthesis according to an embodiment of the present invention.

Referring to FIG. 1, a nanostructure 110 having a nano-pattern of a preset shape may be formed on a surface of a polymer film 10 for bonding a bio-implantation material according to an embodiment of the present invention.

The polymer film 10 including a nanostructure having a nano-pattern of a preset shape formed on the first surface 100 according to an embodiment of the present invention is formed by placing a bio-artificial implant inserted into a living body at the insertion site. It may have a use for facilitating the binding of the bio-implantation to the cells positioned at the insertion site to improve the bio-stability of the insert.

Therefore, attaching the polymer film 10 including the nanostructure 110 having a nano-pattern of a preset shape formed on the first surface 100 so as to adhere to the cells located at the insertion site according to an embodiment of the present invention The bioassembled implant may be positioned at the insertion site until the insertion site in the living body recovers after the insertion procedure.

Specifically, the polymer film 10 for bonding a bioartificial implant according to an embodiment of the present invention includes a nanostructure 110 having a nano-pattern of a preset shape for controlling adhesion with a cell according to the behavior of the cell. It may be a polymer film 10 having a nanostructure formed, and by combining the above-described nanostructured polymer film 10 with a bioartificial implant, the bioartificial implant is inserted into a living body and combined with the bioartificial implant. The polymer film 10 may be used to attach the cells present at the insertion site and the bio-implantation material.

The thickness of the polymer film 10 for bonding a bioprosthesis according to an embodiment of the present invention may be several tens to several hundred micrometers (μm) depending on the application environment, but the above-described example describes an embodiment of the present invention. It is only an example for and is not limited thereto.

Referring to FIG. 2 together, the bio-implant 20 according to an embodiment of the present invention includes orthopedic implants such as joints, spines, and valves, dental implants, vascular stents, non-vascular stents, implantable injection devices, and pacemakers. It may be selected from the group consisting of (pace maker) and artificial organs, but the above-described example is only an example for explaining an embodiment of the present invention and is not limited thereto.

The bioartificial implant 20 according to an embodiment of the present invention may be selected from the group consisting of titanium, stainless steel, cobalt, gold, silver, platinum, magnesium, or alloys thereof, but the above-described example is one of the present invention. It is only an example for describing the embodiment, but is not limited thereto.

Therefore, the polymer film 10 for bonding a bioprosthetic implant having a nano/micro structure having a nano/micro pattern of a preset shape according to an embodiment of the present invention is a method of implanting it on the surface of the bio-implant 20, It can be used directly in the existing bio-artificial implant 20. Therefore, since there is no need to separately process the surface of the bio-implant 20 that is previously inserted into the body, a living body in which a nano/micro structure 110 having a nano/micro pattern of a preset shape according to an embodiment of the present invention is formed. In the case of using the polymer film 10 for adhering an artificial implant, there is an advantage of possible cost reduction.

A nanostructure 110 having a nano-pattern of a preset shape may be formed on the surface of the polymer film 10 for bonding a bio-implantation material according to an embodiment of the present invention to enable interaction by adhering to cells.

Specifically, among the surfaces of the polymer film 10 for bonding a bio-implantation material according to an embodiment of the present invention, cells and cells on the first surface 100, which is a surface other than the attachment surface, which is the surface to be attached to the bio-implant 20, is It is possible to form a nanostructure 110 having a nano-pattern of a preset shape that enables interaction by bonding. The above-described bio-implant adhesion polymer film 10 has a nano structure or a micro structure, so that the reactivity of cells can be controlled.

On the first surface 100 of the polymer film 10 for bonding a bioprosthesis according to an embodiment of the present invention, a nano-pattern in a preset shape to control adhesion and spreadability by controlling the local adhesion (FA) of cells. The nanostructure 110 having a may be formed.

Specifically, the first surface 100 of the adhesive polymer film 10 according to an embodiment of the present invention controls the initial engraftment of the cells by controlling the local adhesion (FA) of the cells, thereby controlling the cells located at the insertion site. It can be grown stably, and the above-described engraftment includes reactions such as adhesion and spreadability of cells.

The nanostructure 110 having a nano-pattern having a predetermined shape described above may be in the form of a nanopillar array, and the localization formed by cells by adjusting the size of the nanopillar and the spacing of the nanopillar array described above. By adjusting the size of the adhesion (FA), it is possible to directly control the spreading of cells, which is the degree to which cells adhere and spread.

The above-described local adhesion (FA) indicates that the cell is connected to the inside and outside of the cell, transmits signals from the outside of the cell to the inside of the cell, and maintains the adhesion of the cell. While adhering to 110), it may be adjusted so that adhesion is achieved by jumping over the gap at the surface of the nanostructures 110.

Controlling the size of the above-described local adhesion (FA) increases the size of the integrin-ECM binding complex, which serves as a signaling medium to cells so that cell growth, division, survival or differentiation is possible. Can be adjusted based on

The size of the above-described local adhesion (FA) increases while forming a bridge connecting the spaces between the nanostructures 110, and specifically, the size control of the above-described local adhesion (FA) is performed on the surface of the nanostructure 110. Can be adjusted by rain.

The size of the local adhesion (FA) controlled by the surface ratio of the nanostructure 110 described above may increase while forming a bridge without filling the space between the surfaces of the nanostructure 110.

The increase in the size of the above-described local adhesion (FA) is to secure an adhesion area of cells greater than a certain level, and can be seen in FIGS. 7(c), 8, and 10 described later.

As the cell adheres to the nanostructure, changes in the mechanochemical signal according to the adhesion site that adheres to the nanostructure can be recognized by the cytoskeleton within the cell. Changes in local adhesion (FA) lead to changes in the cytoskeleton within the cell, which can alter the signal transduction from the point of attachment of the cell to the nanostructure. As a result, changes in local adhesion (FA) affect protein expression by altering signal transduction in cells, and thus can affect cellular activities such as cellular activity.

The above-described local adhesion (FA) may be connected to the cell nucleus through F-actin (F-actin), which is a protein that forms the intracellular skeletal structure. Through the above-described process, information such as structure, hardness, and orientation of a substance, which can be felt in local adhesion (FA), is transferred to the nucleus by cell signaling molecules to change protein synthesis, and ultimately, cell behavior and function. Is done.

A detailed description of the change in the above-described local adhesion (FA) will be described later.

In general, since proteins present on the surface of cells are different depending on the type of cell, the first surface 100 of the polymer film 10 for bonding a bioartificial implant according to an embodiment of the present invention has A nanostructure 110 having a nano-pattern of a preset shape may be formed to be suitable for adhesion with a protein.

That is, the polymer film 10 on which the nanostructure 110 having a nano-pattern of a predetermined shape is formed is directly adhered to the cells, and the bio-artificial implant 20 attached to the polymer film 10 is eventually a polymer film. Since it is indirectly adhered to the cell by 10, the polymer film 10 is a medium for bonding between the bio-implant 20 and the cell so that the bio-artificial implant 20 can be fixed at the insertion position. Can play a role.

In addition, a nanostructure 110 having a ridge array may be formed on the surface of the polymer film 10 for bonding a bioprosthesis according to an embodiment of the present invention to fit the shape of the myocardium.

In addition, a microstructure having a micro-pattern in a predetermined shape may be formed on the surface of the polymer film 10 for bonding a bioprosthesis according to an embodiment of the present invention to control the directionality of nerve cells.

The nano-patterns in a preset shape of the above-described nano structure 110 will be described with reference to FIG. 3.

3 is a view for explaining an example of a nano-pattern or a micro-pattern of a preset shape formed on the surface of a polymer film according to an embodiment of the present invention.

Referring to FIG. 3(a), the nano-pattern or micro-pattern of a preset shape according to an embodiment of the present invention may have a shape such as an annular shape in the form of a hole array according to the type of tissue and cells to be used. have.

In addition, referring to FIG. 3(b), the nano-pattern or micro-pattern of a preset shape according to an embodiment of the present invention may have an annular shape such as a pillar array according to the shape of the tissue and cell to be used. have.

In addition, referring to FIG. 3(c), the nano-pattern or micro-pattern of a preset shape according to an embodiment of the present invention may be a linear shape such as a groove array according to the type of tissue and cells to be used. have.

In addition, the above-described example is only an example for explaining an embodiment of the present invention, and is not limited thereto, and the nano-pattern or micro-pattern of the preset shape described above is a linear shape in the form of a ridge array. I can.

In addition, referring to FIG. 3(d), the nano-pattern or micro-pattern of a preset shape according to an embodiment of the present invention may have a shape such as an orthogonal mesh shape according to the shape of the tissue and cell to be used. have.

However, the examples of the nano-patterns or micro-patterns of the preset form described above are only examples for explaining an embodiment of the present invention, and are not limited thereto, and may be in various forms according to the cell tissue and the shape of the cell.

Referring back to FIGS. 1 and 2 together, the polymer film 10 for bonding a bio-implantation according to an embodiment of the present invention has a nanostructure or a microstructure, thereby controlling the behavior of cells and containing nanoparticles. According to the functional or functional group attachment, it may have a functionality for drug delivery.

The above-described drug delivery minimizes side effects and selectively delivers the necessary amount of drug to the treatment site in order to maximize efficacy and effect, so that healthy tissues are not exposed to the drug and at the same time, excellent therapeutic effects can be achieved with only a small amount of drugs. Denotes efficient delivery.

Various types of nano-patterns formed on the first surface 100 among the surfaces of the polymer film 10 for bonding a bioprosthesis to impart functionality to the polymer film 10 for bonding a bioprosthesis according to an embodiment of the present invention Branches can attach bio-friendly functional groups to a part of the nanostructure in the form of an array, and accordingly, by using a nanostructure having various types of nanopatterns formed on the surface of the polymer film 10 for bonding a bioartificial implant. It can precisely control cell adhesion.

When the above-described functional groups are attached in the form of an array, the array of functional groups can be adjusted according to the type of cell. Specifically, it is possible to map the distribution of phospholipids in the cell membrane and whether or not to combine proteins, and adjust the spacing of bio-friendly functional groups and the array density according to the mapping result.

In addition, the biodegradable polymer according to an embodiment of the present invention has a functional group for secreting substances that may affect cells such as antibiotics or differentiation factors during decomposition in vivo, Can be embedded in.

The functional groups coated on the surface of the polymer film will be described with reference to FIG. 4.

4 is a diagram illustrating a functional group coated on the surface of a polymer film for bonding a bioprosthesis according to an embodiment of the present invention.

Referring to FIG. 4, the surface of the polymer film 10 for bonding a bio-implantation material according to an embodiment of the present invention may be coated with a bio-friendly functional group 300 for providing selectivity for cell adhesion. have.

In addition, the polymer film 10 for adhesion to a bioartificial implant according to an embodiment of the present invention can directly contact an integrin protein using the amino acid sequence RGD (Arg-Gly-Asp) of the extracellular matrix protein. have. The above-described RGD may have a function of recognizing other cells while being attached to the outside of cells through integrin.

The above-described integrin is a membrane protein constituting the semi-transferring body, and has a function of regulating the binding between the epidermis and the dermis by acting as a knot connecting the basal cells and the basement membrane, and a signaling medium from the extracellular matrix (ECM) to the cells. It plays a role and has a function that is involved in cell growth, division, survival, and differentiation.

In addition, the polymer film 10 for bonding a bioprosthesis according to an embodiment of the present invention is formed by coating a polymer chain capable of complementary binding such as RNA or DNA on the surface of the above-described polymer film to adhere to the bioprosthesis. can do. A method of coating the polymer chain described above will be described later.

Referring back to FIGS. 1 and 2 together, in the case of a cell type or stem cells, depending on the type of cells to be differentiated, the polymer film 10 for bonding a bio-implantation material according to an embodiment of the present invention The size of the nanostructure 110 having a nano-pattern of a predetermined shape constituting the nanostructure and the interval between the nanostructures 110 may be adjusted from several nanometers (nm) to several micrometers (μm), respectively.

The stem cells described above maintain an undifferentiated state and can proliferate indefinitely, but represent cells capable of differentiating to have a specific function and shape given a certain environment and conditions.

Specifically, the size of the nanostructure 110 having a nano-pattern of a preset shape formed on the polymer film 10 for bonding a bio-implantation according to an embodiment of the present invention is used to control the arrangement of tissues and cells to be used. It may be adjusted to several to several hundred nanometers (nm) depending on the condition of the hazardous cell and organelles.

Alternatively, the size of the microstructure having a micro-pattern of a preset shape formed on the polymer film 10 for bonding a bio-implantation according to an embodiment of the present invention may be several to tens of micrometers ( μm) or tens to hundreds of micrometers (μm).

In addition, the interval between the nanostructures 110 having a nano-pattern of a preset shape formed on the surface of the polymer film 10 for bonding a bio-implantation according to an embodiment of the present invention may be several to several hundred nanometers (nm). In addition, by controlling the local adhesion (FA) of the cell membrane, cell activities such as cell differentiation or cell engraftment can be regulated based on a Mechanotransduction mechanism in vivo. The aforementioned Mechanotransduction refers to a part of energy conversion that is converted into a biochemical signal when a mechanical stimulus is applied to a cell.

According to another embodiment of the present invention, the polymer film 10 for bonding a bioartificial implant having a nano/micro structure having a nano/micro pattern of a preset shape may have a biodegradable or absorbent property.

Specifically, the material of each of the bioprosthesis 20 and the bioprosthesis adhesive polymer film 10 according to an embodiment of the present invention is polyethylene glycol, poly-L-lactic acid (poly(L-lactic acid)). acid)), polylactic-co-glycolic acid (PLGA), polyglycolic acid (PGA), polylactide (PLA), polycaprolactone (PCL), chitosan ( chitosan), polytrimethylene carbonate (PTMC), or a mixture of two or more selected from the above-described polymer group may include a group of biocompatible synthetic polymers.

However, the material of the above-described bio-implant 20 and the polymer film 10 for bonding the bio-implant is only an example for explaining an embodiment of the present invention and is not limited thereto, and the bio-implant 20 and The material of the polymer film 10 for bonding a bio-implantation material may include all polymers having biodegradability.

Therefore, due to the biodegradability of the biodegradable property of the polymer film 10 for bonding a bioartificial implant having a nano/micro structure having a nano/micro pattern of a preset shape according to an embodiment of the present invention, the bioartificial implant 20 and the The attached polymer film 10 can be inserted into the living body and adhered to the cells at the insertion position, and biodegradation after a certain period of time after adhesion with the cells removes only the bio-implant 20 and the attached polymer film 10 Accordingly, the cells are spread on the polymer film 10 and adhere to the polymer film 10 until the cells are stably regenerated at the insertion position in the living body before the polymer film 10 is biodegraded. The prosthesis 20 may be positioned at the insertion site until the cells located at the insertion site grow stably. Therefore, the polymer film 10 is used for adhesion to adhere the cells and the bio-implant 20, and can be naturally biodegraded and removed in the living body, thereby increasing the success rate of bio-insertion of the bio-implant 20 have.

Specifically, the polymer film 10 for bonding a bio-implantation material having a nano/micro structure having a preset nano/micro pattern according to an embodiment of the present invention is formed at the insertion site where the bio-implant 20 is implanted in the living body. After the bio-implant 20 is implanted in the living body before biodegradation, the cells regenerate at the insertion site in the living body, and the position of the bio-implant 20 is not fixed during the cell regeneration. The above-described nano/microstructure can adhere to the cells regenerating at the insertion site so that the bio-artificial implant 20 is fixed to the insertion site while the cells regenerate, and the polymer film 10 adheres to the regenerating cells described above. After that, after the biodegradation is gradually completed and cell regeneration is stably completed, the bio-artificial implant 20 may be positioned at the insertion site.

When the polymer film 10 for bonding a bioprosthesis according to an embodiment of the present invention is attached to the bioprosthesis 20, the surface to attach the polymer film 10 according to the surface of the bioprosthesis 20 Energy control is required, and a microstructure may be formed on the surface of the polymer film 10 or coating the surface of the polymer film 10 in order to control the surface energy described above.

In addition, the surface of the bio-prosthetic insert 20 and the surface of the polymer film 10 are attached by attaching the bio-implant 20 and the polymer film 10 for bonding the bio-prosthetic insert according to another embodiment of the present invention. Patterning techniques such as microelectromechanical systems (MEMS) can be used to increase the surface area of this mating surface and control the surface energy.

In addition, as a method for attaching the bio-implant 20 and the polymer film 10 for bonding the bio-implant according to another embodiment of the present invention, among the surfaces of the polymer film 10, the It is possible to increase adhesion by controlling the surface energy by coating a functional group on the adhesion surface to be attached to the surface.

In addition, as a method for attaching the bio-implant 20 and the polymer film 10 for bonding the bio-implant according to another embodiment of the present invention, roughness is formed on the surface of the polymer film 10 or a microstructure is formed. The above-described polymer film 10 is formed on the surface of the bio-implant 20 by controlling the surface energy of the polymer film 10 by adjusting the difference in surface energy of the surface of the bio-implant 20 to increase adhesion. ) Can be attached.

In addition, by the method of controlling the surface energy according to another embodiment of the present invention, complementary bonding is possible according to the chain order to the attachment surface where the surface of the bioartificial implant 20 and the surface of the polymer film 10 contact. Surface energy can be controlled by polymerizing a polymer monomer.

A specific method of attaching the above-described bio-implant 20 and the polymer film 10 for bonding the bio-prosthesis will be described later in FIGS. 5 and 6.

5 is a view showing the structure of a polymer film for bonding a bio-implantation material according to another embodiment of the present invention.

5 (a) and 5 (b), a method for attaching a bio-implant and a polymer film 10 for bonding a bio-implant according to another embodiment of the present invention. And a method of using a polymer coupling by forming a polymer on both adhesion surfaces, which are the surfaces of the polymer film 10, respectively.

Specifically, a nanostructure 110 having a nano-pattern of a preset shape on the first surface 100 to be adhered to cells among the surfaces of the polymer film 10 for bonding a bio-implantation according to another embodiment of the present invention May be formed, and a polymer monomer for attaching to the bio-implants on the second surface 200 corresponding to the attachment surface 200 to be attached to the bio-implants among the surfaces of the polymer film 10 for bonding the bio-implants. A polymer chain 210 in which is grown may be formed.

In order to control the local adhesion (FA) to the surface of the polymer film 10 for bonding a bioartificial implant according to another embodiment of the present invention, other than the first surface, which is the surface on which the nanostructure 110 is formed, is a cell adhesion surface. A polymer chain 210 capable of complementary bonding such as RNA or DNA can be formed on a second surface corresponding to the attachment surface to be attached to the bio-implantation material, and the polymer film 10 is formed by the formed polymer chain 210 It can be attached to the bioprosthesis.

In an embodiment of the present invention, the polymer film 10 for bonding a bioprosthesis and a method for attaching the bioprosthesis according to an embodiment of the present invention activates the surface of each of the above-described polymer film 10 and the bioprosthesis using a physical or chemical method. , Polymeric monomers are formed on the surface of each activated bioprosthesis and the surface of the polymer film, and the polymer chains 210 are grown after growing the polymeric monomers formed on the surface of each bioprosthesis and the surface of the polymer film. By doing so, the polymer film 10 and the bio-artificial implant can be attached.

A detailed method of activating the above-described polymer film 10 for bonding the bioprosthesis and the surface of the bioprosthesis will be described with reference to FIG. 6.

6 is a view for explaining a method of activating the surface of the bio-prosthesis adhesive polymer film 10 and the bio-prosthesis according to an embodiment of the present invention.

Referring to FIG. 6(a), the surface of the polymer film 10 for bonding a bioprosthesis according to an embodiment of the present invention is selected from argon, oxygen, nitrogen, water vapor, and ammonia, or a mixture thereof. It can be activated using a physical method of treating with plasma or ion beam using (200a). Accordingly, the second surface of the polymer film 10 for bonding a bioprosthesis according to an embodiment of the present invention may be the surface 201 in an activated state by a physical method.

In the case of activation by treatment with the above-described plasma or ion beam, the voltage is 500 [V] to 100 [kV], preferably 1 [kV] to 100 [kV], the power is 50 [W] to 10 [kW], preferably Preferably, 50 [W] to 200 [W], the time may be 1 second to 1 hour, preferably 10 seconds to 10 minutes. However, the above-described example is only an example for explaining an embodiment of the present invention, and is not limited thereto.

In addition, the surface of the bioprosthesis according to an embodiment of the present invention may also be activated using the same method as the above-described physical method.

Referring to FIG. 6(b), according to another embodiment of the present invention, using a chemical method of immersing the polymer film in a basic solution 200b such as lithium hydroxide, sodium hydroxide, potassium hydroxide, or calcium hydroxide, a polymer film ( 10) can activate the surface. Accordingly, the second surface of the polymer film 10 for bonding a bioprosthesis according to an embodiment of the present invention may be the surface 202 in an activated state by a chemical method.

The pH of the above-described basic solution 200b may be 9 to 12, and the temperature may be room temperature to 150°C, preferably 60°C to 100°C. However, the above-described example is only an example for explaining an embodiment of the present invention, and is not limited thereto.

In addition, the surface of the bioprosthetic according to an embodiment of the present invention may also be activated using the same method as the above-described chemical method.

Referring to FIG. 5 again, the polymeric monomers formed on the surface of the activated bioprosthetic implant and the surface of the polymer film according to an embodiment of the present invention are lactide, lactic acid, and glycolide. ), glycolic acid, caprolactone, caprolactic acid, trimethylene, and carbonate, but the above-described examples are It is only an example for explaining an exemplary embodiment, and is not limited thereto.

A polymer capable of substituting a polymer or atom having properties for disulfide bonding of a part of the polymer chain formed on the surface of the bioartificial implant and the surface of the polymer film according to an embodiment of the present invention, or complementarily bonding according to a specific chain sequence Or it can be substituted with an atom.

In addition, a polymer monomer capable of complementary binding according to the above-described chain sequence according to an embodiment of the present invention may include a biomaterial such as a DNA or RNA sequence. The above-described biomaterial such as DNA or RNA may be DNA or RNA extracted from a test subject attempting to be inserted into a living body, but is not limited thereto.

Therefore, the polymer chains formed on the surface of the bio-implants and the polymers or atoms substituted in the polymer chains 210 formed on the second surface 200 of the polymer film 10 by the above-described method are combined, A film can be attached.

In addition, as an attachment method according to another embodiment of the present invention, the polymer film may be attached to the surface of the bio-implant by using the van der Waals attraction or by inducing the electrostatic attraction of the polymer chain. It is only an example for explaining an embodiment of, but is not limited thereto.

In order to check whether the actual cell adhesion and spreadability can be adjusted according to the nanostructure 110 having a nano-pattern of a preset shape formed on the polymer film 10 according to an embodiment of the present invention, first fat The process of culturing and seeding adipose-derived mesenchymal stem cells (ASC) will be described.

In this experiment, adipose-derived mesenchymal stem cells (ASC) were cultured in low-glucose DMEM basal media at 37°C with 5% CO2.

Human adipose-derived mesenchymal stem cells (ASCs) cultured under the above-described conditions were used at passage 6 for all experiments, and seeded on a 12-well plate on which a nanopillar substrate was placed. ). A cell suspension of 2×10 4 cells was pipetted and removed after a certain period of time to keep only cells attached to the substrate. After 24 hours of incubation, the cells were washed with a phosphate buffer solution (PBS), and the washed cells were washed with immunofluorescence staining and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 -Diphenyltetrazolium bromide) was used to evaluate cell proliferation assay.

The above-described MTT cell proliferation assay is generally used to evaluate the viability and toxicity of cells, but in this experiment, the adhesion rate of cells with viability on the substrate on which the nanopillars are formed is measured through a calibration curve using the viability of cells evaluated by MTT. Measured.

To plot the calibration curve, a suspension of 1 x 10 3 to 1 x 10 6 (cells/mL) was prepared, and the cells were incubated at 37° C. for 12 hours overnight. After 500 μl of MTT reagent was dispensed, the cells were incubated for an additional 3 hours.

When the purple precipitate was observed using an optical microscope, 200 (μl) detergent reagent was carefully added and transferred to an enzyme linked immunosorvent assay (ELISA) plate. The absorbance in each well was measured using a micro plate reader on a 570 (nm) wavelength filter. The absorbance of adipose-derived mesenchymal stem cells (ASCs) attached to the nanopillars formed on the polymer film 10 was obtained by the same protocol and compared with a calibration curve for calculating adherent cells.

In this experiment, after culturing the cells on a nanopillar substrate formed on a polymer film for 24 hours, adipose-derived mesenchymal stem cells (ASCs) were added to Karnovsky's fixative (2% paraformaldehyde, 2% glutaraldehyde). )), and the above-described samples were dehydrated using ethanol (ascending gradual series, 50-100%).

Samples containing nanostructures before and after cell culture were sputtered with a gold layer using a sputter in a cluster system, and then observed with a scanning electron microscopy (SEM).

A scanning electron microscope (SEM) is another form of an electron microscope, exemplarily, an electron beam is focused on a point and scanned on the surface of a specimen. The detector collects the backscattered reflected secondary electrons or electrons from the surface and converts the collected electrons into a signal that is used to create an actual three-dimensional image of the specimen. During the above-described scanning process, the detector may receive smaller electrons from the depression of the surface, so the sub-regions of the surface appear darker in the resulting image. Scanning electron microscopy can provide magnification up to approximately 200,000 times, and may be higher.

To observe the cross section of the samples, the samples were subsequently processed using a Focused Ion Beam (FIB).

A focused ion beam (FIB) is similar to a scanning electron microscope that uses an electron beam except that an ion beam is scanned into the sample. The ion beam described above may be emitted from a liquid metal ion source (eg, gallium) having a spot size of less than about 10 nm.

In this experiment, after washing with a phosphate buffer solution (Phosphate-Buffered Saline, PBS), fat-derived mesenchymal stem cells (ASCs) were fixed with 4% paraformaldehyde in a phosphate buffer solution (PBS) at room temperature for 20 minutes.

Before staining, the immobilized cells were permeabilized with a permeabilization agent (0.5% Triton X-100) for 5 minutes, and 5% Bovine serum (BSA) in a phosphate buffer solution (PBS) for 60 minutes at room temperature (RT). albumin).

Samples were incubated with primary antibody diluted 5% in BSA for 1 hour at 37°C. The samples were washed three times with a phosphate buffer solution (PBS) to remove excess, and then incubated with a fluorescently labeled secondary antibody diluted 5% in BSA for 30 minutes at room temperature (RT).

In the last step, the cell nucleus was stained with a solution containing 4',6'diamino-2-phenylindol (4',6-diamidino-2-phenylindol, DAPI). Fluorescence images were acquired using a confocal microscope.

In this experiment, the fluorescence images of adipose-derived mesenchymal stem cells (ASCs) were analyzed to calculate the shape and size of the morphological parameters of local adhesion (FA), and the area of adipose-derived mesenchymal stem cells (ASCs) as the degree of spreadability. Also measured. The results of analyzing the data of the fluorescence image are expressed as mean ± standard deviation (SD).

Hereinafter, according to the adipose-derived mesenchymal stem cells (ASCs) cultured by the above-described method and the nanostructure 110 having a nano-pattern of a preset shape formed on the polymer film 10 using the above-described analysis method, Whether the adhesiveness and spreadability can be adjusted will be described later in FIGS. 7 to 11.

7 is a view for explaining a state in which the nano-pillar array formed on the polymer film for bonding a bio-implantation material and fat-derived mesenchymal stem cells (ASCs) are adhered according to an embodiment of the present invention.

FIG. 7(a) shows a nanostructure having a nano-pattern of a preset shape formed on a first surface of a polymer film for bonding a bioprosthesis according to an embodiment of the present invention, in the form of a nano-pillar array. According to the diameter (nm), spacing (nm) of the column array, the upper surface area of the nanopillar (μm2), and the area fraction (%) of the nanopillar array, the first to the first polymer films for bonding bioartificial implants were prepared. It shows the classification into 3 samples.

Referring to FIG. 7(a), it is shown that the size of the local adhesion (FA) according to an embodiment of the present invention is larger than the upper surface area of the nanopillars in the first sample. The above-described results indicate that the local adhesion (FA) increases in size beyond the spacing between the nanostructures, and the actual contact area of the local adhesion (FA) may be smaller than the proposed observation. It can be seen that the effective contact area fraction of adipose-derived mesenchymal stem cells (ASCs) increases linearly along the upper surface area of one nanopillar.

7(b) shows an image obtained by scanning electron microscope (SEM) of adipose-derived mesenchymal stem cells (ASCs) attached to the first to third samples.

Specifically, Figure 7 (b) is a nano-pillar formed in the polymer film to provide an ECM ligand for cell adhesion to the polymer film in which the nano-pillar array according to an embodiment of the present invention is coated with an ECM protein of fibronectin to the cell membrane. It shows the result of combining with the located integrin.

Referring to Figure 7(b), adipose-derived mesenchymal stem cells (ASCs) 30 according to an embodiment of the present invention are adhered to the upper surface of the nanopillar array 110 formed on the polymer film after 24 hours incubation. , Whether or not engraftment of cells indicating the degree of adhesion and spreadability of cells according to the nanopillar array 110 can be confirmed.

The density of adipose-derived mesenchymal stem cells (ASCs) 30 after culture for 24 hours was 28 ± 3 (cells/mm2) in the first sample, 26 ± 1 (cells/mm2) in the second sample, and the third sample. It was measured at 32 ± 9 (cells/mm2). The above results are interpreted as being about 55% -60% of the adhesion between the nanopillar array 110 and the adipose-derived mesenchymal stem cells (ASCs) 30 because the density of the seeding cells is about 50 (cells/mm2). can do. Therefore, it can be confirmed that the influence of the various sizes of the discrete nanostructures is very small in the adhesion rate of stem cells.

Specifically, the diagram enlarged to 1 micrometer (μm) at the bottom in FIG. 7(b) shows the adipose-derived mesenchymal stem cells (ASCs) 30 attached using a focused ion beam (FIB). As a result of observing the cross section, a gap between the nanopillar array 110 was observed in all of the first to third samples, and accordingly, the adipose-derived mesenchymal stem cells (ASCs) 30 were converted to the nanopillar array 110. ), only the upper surface of the cell is adhered, that is, only the upper surface of the nanopillar array 110 interacts with the adipose-derived mesenchymal stem cells (ASCs) 30.

The above-described results were essential prerequisites for localization of adhesion sites showing integrin-ECM clustering, and that the nanopillar array 110 discretely provides adhesion sites to adipose-derived mesenchymal stem cells (ASCs) 30 I can confirm.

Therefore, the above-described result is that adipose-derived mesenchymal stem cells (ASCs) 30 adhere only to the surface of the nanopillar array 110 and do not enter the gap corresponding to the empty space between the nanopillar arrays 110. I can confirm.

7(c) is a view for explaining a state in which adipose-derived mesenchymal stem cells (ASCs) 30 are adhered to each of a first sample and a third sample according to an embodiment of the present invention in FIG. 7(b) As shown, the spreadability of adipose-derived mesenchymal stem cells (ASCs) 30 is changed according to the size of the local adhesion (FA).

In FIG. 7(b), the spreadability of the adipose-derived mesenchymal stem cells (ASCs) 30 adhered to the nanopillar array 110 corresponding to the first sample and the nanopillar array 110 corresponding to the third sample respectively Other things can be seen, and FIG. 7(c) shows a state in which adipose-derived mesenchymal stem cells (ASCs) 30 are adhered to each of the first and third samples described above, see FIG. 7(c). If the size of the local adhesion (FA) on the surface of each of the nanopillar arrays 110 corresponding to the above-described first and third samples is large, the spread of adipose-derived mesenchymal stem cells (ASCs) 30 is less. Conversely, if the size of the local adhesion (FA) is small, adipose-derived mesenchymal stem cells (ASCs) 30 may spread more.

In this case, the nanopillar array 110 can control the size of the local adhesion (FA) in order to form the number of integrin-ECM bonds similarly regardless of the size of the surface of the nanopillar array 110.

This is because the size of the protein complex forming the local adhesion (FA) increases while forming bridges in the empty spaces between the nanopillar arrays 110 while exceeding the spacing of the nanopillar array 110 structure.

Accordingly, adipose-derived mesenchymal stem cells (ASCs) 30 may be adhered to each of the first and third samples while the above-described local adhesion (FA) and the nano-pillar array 110 are integrin-ECM bonded. Local adhesion (FA) can be confirmed from F-actin and α-actin of adipose-derived mesenchymal stem cells (ASCs) (30).

8 is a view for explaining an effective contact area in which cells adhere to the surface of a nanostructure in adhesion with cells according to the formation of a local adhesion (FA) according to an embodiment of the present invention.

8(a) shows the effective contact area between adipose-derived mesenchymal stem cells (ASCs) and the nanopillar array 110 according to local adhesion (FA).

When adipose-derived mesenchymal stem cells (ASCs) are attached to the above-described nano-pillar array 110, the effective contact area is between the nano-pillar arrays 110 covered by local adhesion (FA) among the areas of the local adhesion (FA). It represents the area in contact with the actual area of the nanopillar array 110 excluding the empty space 120.

Referring to FIG. 8(a), using the effective contact area between the adipose-derived mesenchymal stem cells (ASCs) and the nanostructures, the area ratio of the nanopillar array of each of the first to third samples in Table 1 (% ) Can be calculated, and the effective contact area described above can be found in Fig. 7(a) and Fig. 11(a) to be described later.

8(b) shows the result of calculating the effective contact area of the local adhesion (FA).

Referring to FIG. 8(b), the difference in the average size of the local adhesion (FA) between the first to third samples was considerably reduced, and the effective contact area of the local adhesion (FA) was 0.77 ± 0.52 (μm2) in the first sample. , 0.63 ± 0.39 (μm2) in the second sample and 0.82 ± 0.47 (μm2) in the third sample. The above-described results indicate that the size of the local adhesion (FA) is related to the area fraction of the nanopillars.

Referring to FIG. 7(c), the above-described experimental results are based on the actual area of the integrin-ECM complex formed on the surface of the nanopillar array 110 and the effective contact area of the surface of the nanopillar array 110. It can be seen that the size of) is controlled, and thus it can be confirmed that the spreadability of the cells is controlled according to the formation of intracellular skeletons such as actin filaments that can generate traction.

FIG. 9 is a diagram illustrating that a morphological change of adipose-derived mesenchymal stem cells (ASCs) is induced by a nanostructure according to an embodiment of the present invention.

9(a) shows fluorescently stained adipose-derived mesenchymal stem cells (ASCs) to confirm the expression and distribution of F-actin and vinculin, which are proteins involved in cell morphology and cell adhesion.

The above-described F-actin plays a role in forming an intracellular skeletal structure and is connected to a local adhesion (FA) to transmit a signal from the local adhesion (FA) into the cell, and maintain the shape of the cell.

The above-described vinculin is one of the types of proteins that make up a protein complex called local adhesion (FA), and local adhesion (FA) connects the inside and outside of cells, transmits signals from outside the cells to the inside, and adheres cells. Can play a role in maintaining.

In this experiment, the area of the cells was measured through the distribution of the F-actin protein forming the intracellular skeletal structure.

9(b), the average area of adipose-derived mesenchymal stem cells (ASCs) measured from a fluorescence image according to an embodiment of the present invention is 1894 ± 602 (μm2) in the first sample, and in the second sample. It can be seen that 2457 ± 1146 (μm2) and 3001 ± 1237 (μm2) in the third sample. It can be seen that the spreadability of the cells increases from the first sample to the third sample.

9(c) and 9(d) are diagrams showing differences in F-actin and vinculin expression between a plurality of samples. The above-described protein expression was compared based on the average pixel intensity per cell area, in which case the maximum intensity of one pixel corresponds to 255.

9(c) and 9(d), it can be seen that protein expression for both F-actin and vinculin is highest on the surface of the first sample, and as the nanostructure increases, F-actin and bin It can be seen that the protein expression for all of the coulin is slightly reduced.

The remarkable difference in the morphological change between the first to third samples described above was the size of the local adhesion (FA) at the edge of the adipose-derived mesenchymal stem cells (ASCs).

Since vinculin, which is an element of the protein complex, which is a local adhesion (FA), made an association between integrin adhesion and the actin cytoskeleton, F-actin and vinculin were usually used to confirm the local adhesion (FA). The size of the local adhesion (FA) according to an embodiment of the present invention may be specified by the area of vinculin at the edge of adipose-derived mesenchymal stem cells (ASCs).

10 is a diagram illustrating a relationship between an average area of a local adhesion (FA) and a size of a nanostructure according to an embodiment of the present invention.

Referring to FIG. 10(a), since the above-described cell spreadability is related to the local adhesion (FA) complex located at the edge, adipose-derived mesenchymal stem cells (ASCs) are located at the edges of the cells appearing while adhering to the nanostructure. Local adhesion (FA) linked to F-actin, which is used to confirm intracellular skeletal structure, can be confirmed.

Referring to FIG. 10B, it can be seen that the average area of the local adhesion (FA) decreases as the size of the nanostructure increases. Specifically, as the size of the nanostructure in the first to third samples increased, the area distribution of local adhesion (FA) narrowed, and the average area of the local adhesion (FA) was 3.00 ± 2.03 (μm2) in the first sample. , 2.30 ± 1.42 (μm2) in the second sample and 1.73 ± 0.79 (μm2) in the third sample.

Therefore, the size change of the local adhesion (FA) was small in the nanopillars with a large surface area, and the small nanopillars formed on the polymer film induced the size of the local adhesion (FA). The results of the area distribution and the average size indicate that when the size of the nanopillars formed on the polymer film is large, the adhesion site to which adipose-derived mesenchymal stem cells (ASCs) can adhere more stably is provided.

11 is a view showing the relationship between the size of local adhesion (FA) and the spreadability of adipose-derived mesenchymal stem cells (ASCs) controlled by a nanopillar array according to an embodiment of the present invention.

FIG. 11(a) is a diagram showing the relationship between the effective contact area of the nanopillars and the upper surface area of the nanopillars. Referring to FIG. 11(a), the coefficient of determination r2 is 0.99 in linear regression analysis, and the effectiveness of the nanopillars As the contact area increases, the upper surface area of the nanopillars is proportional. In the case of the first sample, the effective contact area of the nanopillars and the upper surface area of the nanopillars are both the smallest. It can be seen that both the upper surface areas of the pillars are larger than the first sample and the second sample.

FIG. 11(b) is a diagram showing the relationship between the upper surface area of the nanopillars and the average area of the local adhesion (FA). Referring to FIG. 11(b), as the upper surface area of the nanopillars increases, the It can be seen that the average area is inversely proportional to the decrease, and accordingly, the first sample with the smallest upper surface area of the nanopillars has a larger average area of local adhesion (FA) than the second and third samples, and the upper surface area of the nanopillars It can be seen that the largest third sample has a smaller average area of the local adhesion (FA) than the first and second samples.

Referring to FIG. 11(c), in the linear regression analysis, the coefficient of determination r2 is 0.97, and it can be seen that the average area of adipose-derived mesenchymal stem cells (ASCs) is inversely proportional to the average area of local adhesion (FA). The above-described correlation enables prediction of cell spreadability through local adhesion (FA) size measurement, and through this, the behavior of cells can be controlled using nanostructures.

Therefore, according to the experimental results of FIGS. 7 to 11 described above, it can be confirmed that the nanopillar array provides a limited adhesion site for integrin binding, which is an initial step for local adhesion (FA) formation, and cell spreadability Since it is related to differentiation into various cell lines, the polymer film having the above-described nanopillar array has the functionality to control adhesion, viability, and differentiation of stem cells, so that the polymer film having the above-described nanopillar array is It can be attached to provide the above-described functionality to the bio-artificial implant.

When the nanostructure of the polymer film for bonding a bioartificial implant according to an embodiment of the present invention is applied to stem cells, the differentiation process of stem cells may be controlled using the nanostructure. For example, the differentiation ability of stem cells may vary according to the spacing of nano-scale nano-contacts formed on the surface of the polymer film for adhesion of bio-implants, and osteoblasts and adipocytes of stem cells according to the nano-gap of the RGD protein. The differentiation ability of the furnace may be different.

12 is a view for explaining a method of manufacturing a polymer film for bonding a bio-implantation material according to an embodiment of the present invention.

Referring to FIG. 12, a nanostructure having a nano-pattern of a preset shape that adjusts adhesion with cells according to the behavior of cells so that the bio-prosthesis adheres to cells located at the insertion site in the living body is a mold material. ) To form on the surface (S1210).

A specific method of forming the nanostructure having the above-described nano pattern on the surface of the mold material will be described later in FIG. 14.

The nanostructure formed on the mold material is transferred to the polymer film to form a nanostructure on the first surface of the polymer film that adheres to the cells (S1220).

Specifically, as a method of transferring the nanostructure formed on the surface of the mold material according to an embodiment of the present invention to a polymer film, soft lithography, capillary force lithography (CFL), or micro/nano The nanostructure formed on the surface of the mold material may be transferred to a polymer film using a manufacturing method that replaces a microelectromechanical system (MEMS) process such as micro/nano imprinting with a nano process.

The above-described soft lithography refers to a method of transferring a micropattern to a substrate by making a fine pattern with an elastomeric stamp or mold, which is a flexible organic material.

The polymer film including a nanostructure having a nano-pattern of a predetermined shape formed on the first surface by the above-described method includes cells and bio-implants positioned at the insertion site so that the bio-implant inserted into the living body is positioned at the insertion site. It may have a use to facilitate bonding.

The shapes of the nanostructures having the nano-patterns of the preset shape described above may have various shapes as described above in FIG. 3, and detailed descriptions will be omitted.

In the method of manufacturing a polymer film for adhesion to a bioartificial implant according to an embodiment of the present invention, a flexible mold having a nanostructure characteristic having a function of cell control is manufactured, and a polymer precursor is poured into the manufactured flexible mold and separated. After the film is covered and cured, the outer film is removed, the couplingable polymer is attached, and then the polymer film is removed from the mold to prepare the above-described polymer film for bonding the bio-implant.

A specific method of forming a nanostructure on the first surface of the polymer film will be described later in FIG. 13.

A polymer film having a nanostructure formed on the first surface is attached to the bioartificial implant (S1230).

The bioprosthesis according to an embodiment of the present invention is a group consisting of orthopedic implants such as joints, spines, valves, etc., dental implants, vascular stents, non-vascular stents, implantable injection devices, pace makers, and artificial organs. In addition, the bio-prosthetic implant may be selected from the group consisting of titanium, stainless steel, cobalt, gold, silver, platinum, magnesium, or alloys thereof, but the above-described example describes an embodiment of the present invention. It is only an example for the following and is not limited thereto.

A specific method of attaching the above-described polymer film to the bio-artificial implant will be described later in FIG. 15.

13 is a flowchart illustrating a specific method of forming a nanostructure on the surface of a polymer film according to an embodiment of the present invention.

Referring to FIG. 13, a nanopit is formed in a polymer mold from a nanostructure formed in a mold material using a nanoimprint lithography process (S1221).

A specific method of forming a nanofit in the above-described polymer mold will be described with reference to FIG. 14.

A polymer precursor is poured into the polymer mold in which the nanofit is formed, and the detachable outer film is covered and cured (S1222).

Several drops of a polyurethane acrylate (PUA) precursor may be added to the center of the polymer mold having a nanofit structure according to an embodiment of the present invention.

The solution dropped in the center of the polymer mold having a nano-fit structure according to an embodiment of the present invention is covered with an 18 mm diameter and slightly etched glass coverslip for better adhesion, and then the mold having a nano-fit structure by capillary force. It can be made so that there is no empty space between the and coverslips.

The sample according to an embodiment of the present invention produced by the above-described method may be cured under ultraviolet (UV) (300mJ/cm2).

However, the above-described example is only an example for explaining an embodiment of the present invention, and is not limited thereto.

The outer film covering the cured polymer precursor is removed (S1223).

According to an embodiment of the present invention, after very carefully peeling off the cured solution from the polymer mold having a nanofit structure using a sharp razor, the sample may be additionally cured for termination overnight.

The polymer precursor from which the outer film has been removed is separated from the polymer mold (S1224).

Specifically, according to an embodiment of the present invention, after sterilization with ultraviolet (UV) light, fibronectin is coated to provide an ECM ligand for cell adhesion, and then a completely cured sample is prepared with ethyl alcohol and deionized water ( By rinsing with deionized (DI) water), a polymer film in which the above-described nanopillar array is formed can be prepared.

14 is a diagram illustrating a detailed method of manufacturing a polymer film for bonding a bio-implantation material according to another embodiment of the present invention.

When a nanostructure having a nano-pattern of a preset shape formed on a polymer film for bonding a bioprosthesis according to an embodiment of the present invention is a nano-pillar array, a method of manufacturing a polymer film will be described as an example, It is not limited thereto.

14(a) to 14(c), according to an embodiment of the present invention, a nano/micro structure 51 is formed on the surface of a mold material 50 such as Si, quartz, and glass. can do.

As a method of forming the nano/micro structure 51 on the surface of the above-described mold material 50, such as photolithography, electron beam lithography, dry and wet etching process. A microelectromechanical system (MEMS) process or a nanoelectromechanical system (NEMS) process may be used, but the above-described example is only an example for explaining an embodiment of the present invention. It is not limited.

According to an embodiment of the present invention, a quartz nanopillar array 51 may be generated from a quartz glass 50 using polystyrene beads 40. In the quartz nanopillar array 51 described above, the size of the nanopillars and the spacing between the nanopillar array 51 may be adjusted by the size of the polystyrene beads 40.

Capillary force lithography may be repeated twice to form the nanopillar array 110 on the polymer film 10 described above.

Referring to FIG. 14(d), it is the first time to create a polymer mold 60 having a nano-pit structure using the quartz nano-pillar array 51 manufactured by the above-described method according to an embodiment of the present invention. A second lithographic process can be used.

Colloidal lithography can be used to manufacture the polymer mold 60 having the nano-fit structure described above. When the above-described colloid lithography is used, there is an advantage in that various pitches can be controlled for a nanopillar array using nanoparticles of various sizes.

Referring to FIG. 14(e), to form a nanopillar array 110 on the polymer film 10 from the polymer mold 60 having a nano-fit structure generated by the first lithography process according to an embodiment of the present invention. A second lithographic process can be used.

In the method of transferring the nano/micro structure 51 formed on the surface of the mold material 50 according to an embodiment of the present invention to the polymer film 10, the mold is formed by the structure formed on the Si wafer and the above-described method. Any structure formed on the transferred polymer film can be used.

15 is a flowchart illustrating a specific method of attaching a polymer film having a nanostructure formed thereon to a bio-implantation according to an embodiment of the present invention.

Referring to FIG. 15, the surface of the polymer film and the surface of the bio-implant are activated using a physical method or a chemical method, respectively (S1231).

The surface of the polymer film for bonding a bioprosthesis according to an embodiment of the present invention and the surface of the bioprosthesis are each selected from argon, oxygen, nitrogen, water vapor, and ammonia, or by using a gas consisting of a mixture thereof. It can be activated using a physical method of treatment with an ion beam.

In addition, according to another embodiment of the present invention, a bio-implantation material using a chemical method of immersing the bio-implant adhesion polymer film and the bio-implantation material in a basic solution such as lithium hydroxide, sodium hydroxide, potassium hydroxide, or calcium hydroxide. It is possible to activate the surface of the adhesive polymer film and the surface of the bioartificial implant, respectively.

Since the detailed method of activating the surface of the above-described polymer film and the surface of the bio-implantation material has been described above, a detailed description will be omitted.

A polymeric monomer is formed on the surface of the activated bioprosthetic implant and the surface of the polymer film, respectively (S1232). Since the above-described polymeric monomer has been described above, a detailed description will be omitted.

Polymer chains are grown by growing polymeric monomers respectively formed on the surface of the bioprosthetic implant and the surface of the polymer film (S1233). Each of the grown polymer chains is bonded to the surface of the bio-implantation material and the surface of the polymer film (S1234).

A polymer capable of substituting a polymer or atom having properties for disulfide bonding of a part of the polymer chain formed on the surface of the bioartificial implant and the surface of the polymer film according to an embodiment of the present invention, or complementarily bonding according to a specific chain sequence Or it can be substituted with an atom.

A specific method of bonding the above-described bio-prosthesis and the polymer film for bonding the bio-prosthesis has been described above, so a detailed description thereof will be omitted.

The polymer film for bonding a bioartificial implant having a nanostructure having a nano-pattern having a preset shape according to an embodiment of the present invention may be manufactured by applying a nanoimprinting method, and accordingly, a roll-to-roll process. It can be applied to mass production methods including -to-roll process, R2R process).

The above-described roll-to-roll process (R2R process) is one of continuous processes, and refers to a process in which a thin material such as a film is wound on a rotating roller and a specific material is applied or a predetermined part is removed to give a new function. Since the above-described roll-to-roll process is advantageous for mass production, manufacturing cost can be lowered.

The above description is merely illustrative of the technical idea of the present invention, and those of ordinary skill in the technical field to which the present invention belongs can make various modifications, changes, and substitutions within the scope not departing from the essential characteristics of the present invention. will be. Accordingly, the embodiments disclosed in the present invention and the accompanying drawings are not intended to limit the technical idea of the present invention, but are for illustration, and the scope of the technical idea of the present invention is not limited by these embodiments and the accompanying drawings. . The scope of protection of the present invention should be interpreted by the following claims, and all technical ideas within the scope equivalent thereto should be construed as being included in the scope of the present invention.

10: polymer film 40: polystyrene beads
20: bioartificial implant 50: mold material
30: fat-derived mesenchymal stem cells 60: polymer mold

Claims (18)

In the polymer film attached to the bio-implantation inserted into the living body,
Having a nano-pattern of a preset shape formed on the first surface that adheres to the cells that adjusts adhesion with the cells according to the behavior of the cells so that the bio-implantation material adheres to the cells located at the insertion site in the living body It includes a nano structure,
In addition to the first surface on which the nanostructure is formed, a polymer chain formed of a group of polymers is included on the second surface to be attached to the bioprosthesis by controlling a difference between the surface energy of the bioprosthesis. Polymer film. The method of claim 1,
The size of the nanostructure having the nanopattern of the preset shape or the spacing between the nanostructures according to the nanopattern is,
A local area that connects the inside and outside of the cell so that the cell adheres to the nanostructure and fills the space between the nanostructures, transmits a signal from the outside of the cell to the inside of the cell, and maintains the adhesion of the cell A polymer film for bonding a bioartificial implant, characterized in that the size of the adhesion (Focal Adhesion, FA) is adjusted to directly control cell spreading, which is a degree of spreading while the cells adhere to the nanostructure. The method of claim 2,
According to the shape of the cell, the nanostructures having the nano-patterns of the preset shape include a groove array, a ridge array, a hole array, a pillar array, and an orthogonal mesh. A polymer film for bonding a bio-implantation material, characterized in that it is in any one form selected from (mesh) forms. The method of claim 2,
The size of the nanostructure having the nano-pattern of the preset shape or the interval between the nano-structures according to the nano-pattern is,
A polymer film for bonding a bioartificial implant, characterized in that it is adjusted in consideration of a relationship in which the average area of the cells and the average area of the local adhesion (FA) are inversely proportional to control the cell spreadability from the size of the local adhesion. delete The method of claim 1,
Polymeric monomers included in the polymer group are lactide, lactic acid, glycolide, glycolic acid, caprolactone, caprolactone, and tri A polymer film for bonding a bioprosthetic implant, characterized in that it can be selected from the group consisting of methylene and carbonate. The method of claim 1,
The polymer chain is a bio-artificial insert adhesive polymer film, characterized in that it is a biopolymer DNA or RNA that binds complementarily according to the nucleotide sequence extracted from the test subject to insert the bio-implant. The method of claim 1,
After the nanostructure adheres to the cells located at the insertion site in the living body, pill polyethylene glycol, poly-L-lactic acid (poly(L-lactic acid)) so that the polymer film is biodegraded in the living body , Polylactic-co-glycolic acid (PLGA), polyglycolic acid (PGA), polylactide (PLA), polycaprolactone (PCL), chitosan, Polytrimethylene carbonate (PTMC) or a biocompatible synthetic polymer group comprising a mixture of two or more selected from the polymer group. The method of claim 8,
Before the polymer film is biodegraded in the living body, after the bio-implant is implanted into the living body, the cells are regenerated at the insertion site in the living body, and the nanostructure is regenerated so that the bio-implant is fixed to the insertion site. The polymer film for adhesion to a bio-artificial implant, characterized in that the polymer film is biodegraded in the living body after adhesion to the cells. The method of claim 1,
The bio-artificial implant may be selected from the group consisting of orthopedic implants such as joints, spines, valves, dental implants, vascular stents, non-vascular stents, implantable injection devices, pace makers, and artificial organs. A polymer film for bonding a bio-implantation material. The method of claim 1,
The bio-prosthesis is a polymer film for bonding a bio-prosthesis, characterized in that it can be selected from the group consisting of titanium, stainless steel, cobalt, gold, silver, platinum, magnesium, or alloys thereof. In the method for producing a polymer film attached to a bio-implantation inserted into a living body,
A nanostructure having a nano-pattern of a preset shape that adjusts adhesion with the cells according to the behavior of the cells so that the bio-artificial implant adheres to the cells located at the insertion site in the living body is formed on the surface of a mold material. Forming on;
Transferring the nanostructure formed on the mold material to the polymer film to form the nanostructure on the first surface of the polymer film that adheres to the cells; And
In addition to the first surface on which the nanostructure is formed, forming a polymer chain formed of a group of polymers on the second surface to be attached to the bioprosthesis by controlling a difference between the surface energy of the bioprosthesis Method for producing a polymer film for bonding a bioartificial implant. The method of claim 12,
The size of the nanostructure having the nanopattern of the preset shape or the spacing between the nanostructures according to the nanopattern is,
A local area that connects the inside and outside of the cell so that the cell adheres to the nanostructure and fills the space between the nanostructures, transmits a signal from the outside of the cell to the inside of the cell, and maintains the adhesion of the cell A method for producing a polymer film for bonding a bioartificial implant, characterized in that the size of the adhesion (Focal Adhesion, FA) is adjusted to directly control the cell spreading, which is the degree to which the cells adhere to the nanostructure and spread. . The method of claim 13,
The size of the nanostructure having the nano-pattern of the preset shape or the interval between the nano-structures according to the nano-pattern is,
A method for producing a polymer film for bonding a bioartificial implant, characterized in that it is adjusted in consideration of a relationship in which the average area of the cells and the average area of the local adhesion (FA) are inversely proportional to control the cell spreadability from the size of the local adhesion. The method of claim 12,
Forming the nanostructure,
Forming a nanopit in the polymer mold from the nanostructure formed in the mold material by using a nanoimprint lithography process;
Pouring a polymer precursor into the polymer mold in which the nanofit is formed, and curing by covering the detachable outer film;
Removing the outer film covering the cured polymer precursor; And
Separating the polymer precursor from which the outer film has been removed from the polymer mold; Method for producing a polymer film for bonding a bioartificial implant comprising a. The method of claim 12,
Attaching the polymer film having the nanostructure formed on the first surface to the bio-artificial implant; Including more,
The step of attaching the polymer film to the bio-artificial implant,
Activating the surface of the polymer film and the surface of the bioartificial implant using a physical method or a chemical method, respectively;
Forming a polymer monomer on the surface of the activated bio-implantation material and the surface of the polymer film, respectively;
Growing a polymer chain by growing a polymer monomer formed on the surface of the bio-implantation material and the surface of the polymer film; And
A method for producing a polymer film for bonding a bio-implant, comprising: coupling the grown polymer chains to the surface of the bio-implant and the surface of the polymer film. The method of claim 16,
The activating step,
By immersing the bio-implant and the polymer film in a basic solution such as lithium hydroxide, sodium hydroxide, potassium hydroxide, or calcium hydroxide, respectively, using a chemical method, the surface of the bio-implant and the surface of the polymer film are activated. Method for producing a polymer film for bonding a bio-implantation material, characterized in that. The method of claim 16,
The activating step,
Activate the surface of the bio-implantation material and the surface of the polymer film by using a physical method of treating with plasma or an ion beam using any one selected from argon, oxygen, nitrogen, water vapor, and ammonia, or a mixture thereof. A method for producing a polymer film for bonding a bioartificial implant, characterized in that.

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