KR20200060037A - Polymer film used for adhering bioprostheses and method for manufacturing the same - Google Patents

Abstract

Disclosed is a polymer film for adhesion of a bio-artificial insert and a method for manufacturing the same. The present invention increases the adhesiveness and spreadability of cells at the insertion site in a living body during the procedure for inserting a bio-artificial insert, and adhesion to cells according to the behavior of cells formed on the first surface of the polymer film that adheres to the cells to increase biocompatibility. By including a nano-structure having a nano-pattern of a predetermined shape to control sex, the artificial prosthesis can induce a stable state with cells at the insertion site in vivo, thereby increasing the success rate of the implantation procedure in the body.

Description

Polymer film for adhesion of bio-artificial inserts and method for manufacturing the same {POLYMER FILM USED FOR ADHERING BIOPROSTHESES AND METHOD FOR MANUFACTURING THE SAME}

The present invention relates to a polymer film for adhesion to a bio-artificial insert and a method for manufacturing the same. More specifically, the present invention relates to a polymer film and a method for manufacturing a cell that is initially grown by adhering a cell with a bio-artificial insert so that cells can perform normal cell activity at the insertion site after the bio-insert is inserted into the body.

The contents described in this section merely provide background information for the present embodiment, and do not constitute a prior art.

Recently, due to the aging of the population, dental procedures for health as well as implant procedures due to accidents and aging are increasing, and procedures for wearing bio-insertable devices such as pacemakers are also rapidly increasing.

If such a procedure fails, the treatment site does not heal, and a bio-artificial implant inserted in the living body may be exposed to the outside. Accordingly, the subject is at risk of infection, and if the procedure is performed again, the failure rate of the reoperation has already failed. There is a problem that becomes higher.

Conventional methods of treating the surface of a bio-artificial insert, such as coating and etching, have a problem in that it is difficult to apply a specific structure, and there is also a limited range in which functionality can be provided to the surface of the bio-artificial insert.

The present invention prevents the failure of the procedure when a bio-artificial implant is implanted in vivo, and attaches cells through focal adhesion (FA) of cells to a polymer film that provides functionality such as improving biocompatibility on the surface of the bio-insert. And by forming a nano-structure that can control the spreadability of cells by controlling the placement and spread of cells affecting the intracellular signal transduction system, by inducing adhesion of cells at the beginning of the procedure and cells for stable cell regeneration It is to provide a polymer film and a method for manufacturing a bio-artificial insert adhesion that can control the spreadability of.

In order to achieve the above object, the polymer film for adhesion of a bio-artificial insert to be attached to a bio-artificial insert inserted in a living body according to an embodiment of the present invention is such that the bio-artificial insert adheres to cells located at an insertion site in the body. It may include a nano-structure having a predetermined pattern of nano-patterns formed on the first surface that adheres to the cells that control the adhesion to the cells according to the behavior of the cells.

Preferably, the size of the nano-structure having the nano-pattern of the predetermined shape or the gap between the nano-structures according to the nano-pattern while the cells adhere to the nano-structure while filling the space between the nano-structures It connects the inside and outside of the cell, transmits a signal from the outside of the cell to the inside of the cell, and controls the size of a focal adhesion (FA) that maintains the adhesion of the cell, while the cell adheres to the nanostructure It can be controlled to directly control cell spreading, which is the degree of spreading.

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

Preferably, the size of the nano-structure having the nano-pattern of the predetermined 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 ( The average area of FA) can be adjusted in consideration of the inverse relationship.

Preferably, a polymer monomer is grown on the second surface to be attached to the bio-artificial insert in addition to the first surface on which the nanostructure is formed to adjust the difference from the surface energy of the bio-artificial insert to adhere to the surface of the bio-artificial insert. Polymer chains can be formed.

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

Preferably, the polymer chain may be DNA or RNA, which is a biopolymer that complementarily binds according to a base sequence extracted from a subject to insert the bio-insert.

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

Preferably, before the biofilm is biodegraded in vivo, the nanostructure is such that the bioprosthesis is fixed to the insertion site while the cells are regenerated at the insertion site in the body after the bioinsert is implanted in the body. The polymer film can be biodegraded in vivo after the adhesion to the regenerating cells.

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

Preferably, the bio-artificial insert can 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 adhesion of a bio-artificial insert to be attached to a bio-artificial insert inserted in a living body, wherein the bio-artificial insert is located at an insertion site in the body Forming a nanostructure on a surface of a mold material having a nano-pattern of a predetermined shape that controls adhesion to the cell according to the behavior of the cell to adhere to the cell; And transferring the nanostructure formed in 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 nano-structure having the nano-pattern of the predetermined shape or the gap between the nano-structures according to the nano-pattern while the cells adhere to the nano-structure while filling the space between the nano-structures It connects the inside and outside of the cell, transmits a signal from the outside of the cell to the inside of the cell, and controls the size of a focal adhesion (FA) that maintains the adhesion of the cell, while the cell adheres to the nanostructure It can be controlled to directly control cell spreading, which is the degree of spreading.

Preferably, the size of the nano-structure having the nano-pattern of the predetermined 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 ( The average area of FA) can be adjusted in consideration of the inverse relationship.

Preferably, the step of forming the nanostructure comprises forming a nanopit in a polymer mold from the nanostructure formed in the mold material using a nanoimprint lithography process; Pouring a polymer precursor (precursor) into the polymer mold on which the nano-pit is formed and covering the separable outer film to cure; Removing the outer film covering the cured polymer precursor; And separating the polymer precursor from which the outer film is removed from the polymer mold.

Preferably, attaching the polymer film having the nano-structure on the first surface to the bio-artificial insert; Further comprising, the step of attaching the polymer film to the bio-artificial insert comprises activating the surface of the polymer film and the surface of the bio-artificial insert using physical or chemical methods, respectively; Forming a polymer monomer on the surface of each activated bio-artificial insert and the surface of the polymer film; Growing a polymer chain formed on the surface of the bio-insert and the polymer film, respectively, thereby growing a polymer chain; And bonding the grown polymer chains to the surface of the bio-artificial insert and the surface of the polymer film, respectively.

Preferably, the activating step of dipping the bio-artificial insert and the polymer film in a basic solution such as lithium hydroxide, sodium hydroxide, potassium hydroxide or calcium hydroxide, respectively, using a chemical method to surface the bio-artificial insert and the polymer. Each surface of the film can be activated.

Preferably, the activating step is performed using a physical method of treating with a plasma or an ion beam using a gas consisting of any one or a mixture of argon, oxygen, nitrogen, water vapor and ammonia, and the surface of the bio-artificial implant. Each surface of the polymer film may be activated.

According to the embodiment of the present invention as described above, by controlling the local adhesion of cells by using the nano-structure formed on the polymer film, by controlling the adhesion and spread of the cell, the artificial insert 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 having a nano-structured surface is manufactured using a mold, it is possible to directly reduce the cost because it can be directly applied to commercially available bio-insertable products without separate treatment. There are advantages.

In addition, according to an embodiment of the present invention, the biodegradable polymer film provides functionality on the surface of a bio-insert, and then decomposes slowly in the body at the same time as tissue regeneration and can be stably removed in vivo without other procedures.

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 adhesion of a bio-artificial insert according to an embodiment of the present invention.
2 is a view for explaining a polymer film for adhesion of a bio-artificial insert attached to a bio-artificial insert according to an embodiment of the present invention.
3 is a view for explaining an example of a nano pattern or a micro pattern in a preset form formed on the surface of a polymer film according to an embodiment of the present invention.
4 is a view for explaining a functional group coated on the surface of a polymer film for adhesion of a bio-artificial insert according to an embodiment of the present invention.
5 is a view showing the structure of a polymer film for adhesion of a bio-artificial insert according to another embodiment of the present invention.
6 is a view for explaining a method of activating the surface of the bio-artificial insert and the polymer film 10 for adhesion of the bio-artificial insert according to an embodiment of the present invention.
7 is a view for explaining a state in which the nano-pillar array formed on the polymer film for adhesion of a bio-artificial insert according to an embodiment of the present invention interacts with adipose-derived mesenchymal stem cells (ASCs).
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 formation of a local adhesion (FA) according to an embodiment of the present invention.
9 is a view for explaining that the morphological change of adipose-derived mesenchymal stem cells (ASCs) is induced by the nano-structure according to an embodiment of the present invention.
10 is a view for explaining the relationship between the average area of the local adhesive (FA) and the size of the nanostructure according to an embodiment of the present invention.
FIG. 11 is a view showing the relationship between the size of local adhesion (FA) of fat-derived mesenchymal stem cells (ASCs) regulated by a nanopillar array and spreadability of cells according to an embodiment of the present invention.
12 is a view for explaining a method for manufacturing a polymer film for adhesion of a bio-artificial insert 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 view for explaining in detail a method of manufacturing a polymer film for adhesion of a bio-artificial insert according to another embodiment of the present invention.
15 is a flowchart illustrating a specific method of attaching a polymer film formed with a nanostructure to a bio-artificial insert 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 methods for achieving them will be clarified with reference to embodiments described below in detail together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms, and only the embodiments allow the publication of the present invention to be complete, and general knowledge in the technical field to which the present invention pertains. It is provided to fully inform the holder of the scope of the invention, 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 commonly understood by those skilled in the art to which the present invention pertains. In addition, terms defined in the commonly used dictionary are not ideally or excessively interpreted unless specifically defined.

In this specification, terms such as “first” and “second” are for distinguishing one component from other components, and the scope of rights should not be limited by these terms. For example, the first component may be referred to as the second component, and similarly, the second component may also be referred to as the first component.

In this specification, the identification numbers (for example, a, b, c, etc.) in each step are used for convenience of explanation, and the identification numbers do not describe the order of each step, and each step is clearly in context. Unless a specific order is specified, it may occur differently from the specified order. That is, each step 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”, “can have”, “includes” or “can contain” indicate the existence of a corresponding feature (eg, a component such as a numerical value, function, operation, or part). Indicates, does not exclude the presence of additional features.

1 is a view showing the structure of a polymer film for adhesion of a bio-artificial insert according to an embodiment of the present invention.

2 is a view for explaining a polymer film for adhesion of a bio-artificial insert attached to a bio-artificial insert according to an embodiment of the present invention.

Referring to FIG. 1, a nanostructure 110 having a nano pattern in a predetermined shape may be formed on a surface of the polymer film 10 for adhesion of a bio-artificial insert according to an embodiment of the present invention.

The polymer film 10 including a nano structure having a nano-pattern of a predetermined shape formed on a first surface 100 according to an embodiment of the present invention is a bio-artificial by inserting a bio-artificial insert inserted into a living body at an insertion site In order to improve the bio-stability of the insert, it may have a use for facilitating the binding of the bio-insert with a cell located at the insertion site.

Accordingly, the polymer film 10 including the nanostructure 110 having a nano-pattern of a predetermined shape formed on the first surface 100 is attached to the cell located at the insertion site according to an embodiment of the present invention. The inserted artificial prosthesis may be placed at the insertion site until the insertion site in the body is recovered after the insertion procedure.

Specifically, the polymer film 10 for attaching a bio-artificial insert according to an embodiment of the present invention has a nano-structure 110 having a nano-pattern in a preset form for controlling adhesion to the cell according to the behavior of the cell. It may be a polymer film 10 having a nano-structure formed, and the above-described nano-structured polymer film 10 is combined with a bio-artificial insert, thereby inserting a bio-artificial insert into a living body, and thus having the above-described nano-structure combined with a bio-artificial insert. The polymer film 10 can be used to attach a cell and a bio-artificial insert present at the insertion position.

The thickness of the polymer film 10 for bonding an artificial prosthesis according to an embodiment of the present invention may be tens to hundreds of micrometers (μm) depending on the application environment, but the above-described examples illustrate one embodiment of the present invention. This is only an example and is not limited thereto.

Referring to FIG. 2 together, the bio-artificial insert 20 according to an embodiment of the present invention includes an orthopedic implant such as a joint, a spine, and a valve, a dental implant, a vascular stent, a non-vascular stent, an implantable injection device, and a face maker. (pace maker) and may be selected from the group consisting of artificial organs, but the above-described examples are merely examples for explaining an embodiment of the present invention and are not limited thereto.

The artificial prosthesis 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. This is only an example for explaining the embodiment, and is not limited thereto.

Therefore, the polymer film 10 for attaching a bio-artificial insert having a nano / micro structure having a nano / micro pattern in a preset form according to an embodiment of the present invention is implanted on the surface of the bio-artificial insert 20, It can be used directly in the existing artificial prosthesis 20. Therefore, since there is no need to separately treat the surface of the bio-artificial insert 20 inserted in the body, the nano / micro structure 110 having a nano / micro pattern of a predetermined shape according to an embodiment of the present invention is formed. In the case of using the polymer film 10 for adhesion of an artificial insert, there is an advantage that cost reduction is possible.

On the surface of the polymer film 10 for adhesion of a bio-artificial insert according to an embodiment of the present invention, a nano-structure 110 having a nano-pattern of a predetermined shape may be formed to adhere to cells and allow interaction.

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

The nano-pattern in a preset form to control adhesion and spreadability by controlling the local adhesion (FA) of cells on the first surface 100 of the polymer film 10 for adhesion of a bio-artificial insert according to an embodiment of the present invention Nano-structure 110 having a can be formed.

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

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

The above-described topical adhesion (FA) indicates that cells connect to the inside and outside of the cell, transmit signals from the outside of the cell to the inside of the cell, and maintain adhesion of the cells. While adhering to 110), the adhesion may be adjusted so that the adhesion is made over the gap on the surface of the nanostructures 110.

Controlling the size of the above-described local adhesion (FA) is to increase the size of the integrin-ECM binding complex, which acts as a signal transduction medium to cells to enable cell growth, division, survival, or differentiation. Can be adjusted based.

The size of the above-described topical adhesive (FA) is increased while forming a bridge connecting the space between the nanostructures 110. Specifically, the size of the above-described topical adhesive (FA) is controlled by the surface of the nanostructure 110. Can be controlled by rain.

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

Increasing the size of the above-described topical adhesion (FA) is to secure an adhesion area of a certain number of cells or more, and can be confirmed in FIGS. 7 (c), 8, and 10 described later.

As cells adhere to the nanostructure, changes in the mechanochemical signal according to the adhesion sites that adhere to the nanostructure can be recognized in the cytoskeleton within the cell. A change in local adhesion (FA) results in a change in the cytoskeleton within the cell, which can change the signal transduction from the attachment point where the cell is attached to the nanostructure. As a result, changes in local adhesion (FA) affect protein expression by altering signal transduction within cells, and thus can affect cell activity, such as cell activity.

The above-described topical adhesion (FA) may be connected to the cell nucleus through F-actin (F-actin) corresponding to a protein forming a skeletal structure in the cell. Through the above-described process, information such as the hardness and directionality of structures and substances that can be felt in the local adhesion (FA) is transferred to the nucleus by cell signaling molecules to change protein synthesis, and ultimately, the behavior and function of cells are changed. Is done.

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

In general, the protein present on the surface of the cell is different depending on the type of cell, so the first surface 100 of the polymer film 10 for adhesion of a bio-artificial insert according to an embodiment of the present invention is present on the surface of the cell. The nano-structure 110 having a nano-pattern of a predetermined shape suitable for adhesion with the protein may be formed.

That is, the polymer film 10 on which the nano-structure 110 having the above-described nano-pattern 110 is formed is directly adhered to the cell, and the polymer film 10 and the attached bio-artificial insert 20 are eventually polymer films. Since it is indirectly adhered to the cell by (10), the polymer film 10 is a medium for adhering between the bio-artificial insert 20 and the cell so that the bio-artificial insert 20 can be fixed at the insertion position. Can play a role.

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

In addition, a microstructure having a micro-pattern of a predetermined shape may be formed on the surface of the polymer film 10 for adhesion of a bio-artificial insert according to an embodiment of the present invention.

The nano-pattern of a predetermined shape of the above-described nanostructure 110 will be described with reference to FIG. 3 together.

3 is a view for explaining an example of a nano pattern or a micro pattern in a preset form 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 the micro-pattern in a preset form according to an embodiment of the present invention may have a shape such as a ring array-like annular shape depending on the type of tissue and cells to be used. have.

Also, referring to FIG. 3 (b), the nano-pattern or the micro-pattern in a preset form according to an embodiment of the present invention may be an annular form, such as a pillar array form, depending on the type of tissue and cells to be used. have.

In addition, referring to FIG. 3 (c), the nano-pattern or the micro-pattern in a preset form according to an embodiment of the present invention may be a linear form such as a groove array form depending on 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 above-described nano-pattern or micro-pattern in the form of a ridge array (ridge array) in the form of a linear form Can be.

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

However, the above-described examples of the nano-patterns or micro-patterns in the preset form are merely examples for explaining an embodiment of the present invention and are not limited thereto, and may be in various forms depending on cell tissues and cell types.

Referring again to FIGS. 1 and 2, the polymer film 10 for adhesion of a bio-artificial insert according to an embodiment of the present invention has a nano-structure or a micro-structure, and is capable of controlling the behavior of cells, and contains nanoparticles. Depending on the functionality or functional group attachment, it may have the functionality for drug delivery with the purpose.

The above drug delivery selectively delivers the required amount of drug to the treatment site in order to minimize side effects and maximize efficacy and effectiveness, so that healthy tissue is not exposed to the drug and at the same time, an excellent therapeutic effect can be achieved with only a small amount of drug. Shows efficient delivery.

Various types of nano patterns formed on the first surface 100 of the surface of the polymer film 10 for attaching a bio-artificial insert to impart functionality to the polymer film 10 for attaching a bio-artificial insert according to an embodiment of the present invention The branch can attach a bio-friendly functional group to a portion of the nano-structure in an array form, thereby using a nano-structure having various types of nano-patterns formed on the surface of the polymer film 10 for adhesion of a bio-artificial insert. Cell adhesion can be precisely controlled.

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 cells. Specifically, the phospholipid distribution of the cell membrane and the combination of proteins can be mapped, and the spacing and array density of bio-friendly functional groups can be adjusted according to the mapped result.

In addition, the biodegradable polymer according to an embodiment of the present invention has a functional group for secreting substances that can 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 above-described polymer film will be described with reference to FIG. 4 together.

4 is a view for explaining a functional group coated on the surface of a polymer film for adhesion of a bio-artificial insert according to an embodiment of the present invention.

Referring to FIG. 4, a bio-friendly functional group 300 for giving selectivity of cell adhesion may be coated on the surface of the polymer film 10 for adhesion of a bio-artificial insert according to an embodiment of the present invention. have.

In addition, the polymer film 10 for adhesion of a bio-artificial insert 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 RGD described above may have a function of recognizing other cells while being attached to the outside of the cell through the integrin.

The above-described integrin is a membrane protein constituting an anti-crosslinked body, and has a function of regulating the bond between the epidermis and the dermis by acting as a knot connecting the basement cell and the basement membrane. It plays a role, and has functions involved in cell growth, division, survival, and differentiation.

In addition, the polymer film 10 for attaching a bio-artificial insert 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 polymer film described above to adhere to the bio-artificial insert. can do. The method of coating the above-described polymer chain will be described later.

Referring back to Figures 1 and 2 together, in the case of a cell form or stem cells (stem cells) according to the type of cells to differentiate according to one embodiment of the present invention, the biofilm insert adhesive polymer film 10 of The size of the nano-structure 110 having a nano-pattern of a predetermined shape constituting the nano-structure and the spacing between the nano-structures 110 may be adjusted to several nanometers (nm) to several micrometers (μm), respectively.

The above-described stem cells represent cells capable of differentiating to have a specific function and morphology while maintaining an undifferentiated state and capable of proliferation indefinitely, but given a certain environment and conditions.

Specifically, the size of the nano-structure 110 having a nano-pattern of a predetermined shape formed on the polymer film 10 for adhesion of a bio-artificial insert according to an embodiment of the present invention is to control the arrangement state of tissues and cells to be used. It can be adjusted to several hundreds to hundreds of nanometers (nm) depending on the condition of the cells and organelles.

Alternatively, the size of the microstructure having a micro pattern of a predetermined shape formed on the polymer film 10 for attaching a bio-artificial insert according to an embodiment of the present invention may be several to several tens of micrometers depending on the size of tissue and cells to be used ( μm) or tens to hundreds of micrometers (μm).

In addition, the spacing between nanostructures 110 having a nano-pattern of a predetermined shape formed on the surface of the polymer film 10 for adhesion of a bio-artificial insert according to an embodiment of the present invention may be several to several hundred nanometers (nm). In addition, it is possible to control cellular activity such as cell differentiation or cell engraftment based on the mechanism of mechanical transduction in vivo by regulating the local adhesion (FA) of the cell membrane. The above-mentioned in vivo biomechanical transformation (Mechanotransduction) represents a part of energy conversion that is converted into a biochemical signal when a mechanical stimulus is applied to a cell.

The polymer film 10 for attaching a bio-artificial insert having a nano / micro structure having a nano / micro pattern in a preset form according to another embodiment of the present invention may have biodegradable or absorbent properties.

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

However, the materials of the above-described bio-artificial insert 20 and the polymer film 10 for attaching the bio-artificial insert are merely examples for explaining an embodiment of the present invention and are not limited thereto, and the bio-artificial insert 20 and The material of the polymer film 10 for adhesion of a bio-artificial insert may include all polymers having biodegradability.

Therefore, due to the biodegradable nature of the biofilm insert adhesive polymer film 10 having a nano / micro structure having a nano / micro pattern in a preset form according to an embodiment of the present invention, the bioartificial insert 20 and The attached polymer film 10 can be inserted into the living body to adhere to the cells at the insertion position, and after a certain period of time after adhesion with the cells, the biodegradation insert 20 and the attached polymer film 10 are removed. As a result, the cells are adhered to the polymer film 10 in a spread state on 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 artificial insert 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 as an adhesive purpose for adhering the cell and the bio-artificial insert 20, and can be biodegraded and removed naturally in vivo, thereby increasing the bio-insert success rate of the bio-artificial insert 20. have.

Specifically, the polymer film 10 for attaching a bio-artificial insert having a nano / micro structure having a preset nano / micro pattern according to an embodiment of the present invention at an insertion site where the bio-artificial insert 20 is implanted in the body Fixes a problem in which the implantation procedure fails due to the regeneration of cells at the insertion site in the body after the bio-artificial insert 20 is implanted in vivo before biodegradation, and the position of the bio-injection 20 is not fixed during cell regeneration. The nano / micro structure described above can adhere to the cells regenerating at the insertion site so that the bio-artificial insert 20 is fixed to the insertion site while the cells are regenerated, and the polymer film 10 adheres to the regenerating cells described above. Thereafter, the biodegradation insert 20 may be located at the insertion site after the biodegradation is gradually completed and the cell regeneration is stably completed.

When the polymer film 10 for attaching a bio-artificial insert according to an embodiment of the present invention is attached to the bio-artificial insert 20, the surface for attaching the polymer film 10 according to the surface of the bio-artificial insert 20 Energy control is required, and the surface of the polymer film 10 may be coated or the microstructure may be formed on the surface of the polymer film 10 for the above-described surface energy control.

In addition, the surface of the bio-artificial insert 20 and the surface of the polymer film 10 by attaching the bio-artificial insert 20 and the polymer film 10 for attaching the bio-artificial 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 abutting surface and control surface energy.

In addition, as a method for attaching the bio-artificial insert 20 and the polymer film 10 for attaching the bio-artificial insert according to another embodiment of the present invention, of the bio-artificial insert 20 of the surface of the polymer film 10 By attaching a functional group to the attachment surface to be attached to the surface, it is possible to increase the adhesion by controlling the surface energy.

In addition, as a method for attaching the bio-artificial insert 20 and the polymer film 10 for attaching the bio-artificial insert according to another embodiment of the present invention, a roughness is formed on the surface of the polymer film 10 or a microstructure is formed. By forming and controlling the surface energy of the polymer film 10 to adjust the surface energy difference of the surface of the bio-artificial insert 20 to increase the adhesion, the polymer film 10 described above on the surface of the bio-artificial insert 20 ) Can be attached.

In addition, as a method of controlling the surface energy described above according to another embodiment of the present invention, complementary bonding is possible according to the chain order on the attachment surface where the surface of the bio-artificial insert 20 and the surface of the polymer film 10 abut. The surface energy can be controlled by polymerizing the polymer monomer.

The detailed method of attaching the above-described bio-artificial insert 20 and the polymer film 10 for attaching the bio-artificial insert will be described later in FIGS. 5 and 6.

5 is a view showing the structure of a polymer film for adhesion of a bio-artificial insert according to another embodiment of the present invention.

5 (a) and 5 (b), the surface of the bio-artificial insert as a method for attaching the polymer film 10 for attaching the bio-artificial insert and the bio-artificial insert according to another embodiment of the present invention It is possible to use a method of forming a polymer on both surfaces of the polymer film 10 and attaching the polymer, respectively.

Specifically, among the surfaces of the polymer film 10 for adhesion of a bio-artificial insert according to another embodiment of the present invention, a nanostructure 110 having a nano pattern in a preset form is attached to a first surface 100 to be adhered to cells. The polymer monomer for attaching the bio-artificial insert to the second surface 200, which may be formed, and which corresponds to the attachment surface 200 to be attached to the bio-artificial insert among the surfaces of the polymer film 10 for attaching the bio-artificial insert The polymer chain 210 from which is grown may be formed.

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

According to an embodiment of the present invention, the method of attaching the polymer film 10 for attaching a bio-artificial insert and the bio-artificial insert activates the surfaces of each of the polymer film 10 and the bio-artificial insert described above using physical or chemical methods. , Forming a polymer monomer on the surface of the activated bio-artificial insert and the polymer film, and growing the polymer monomer formed on the surface of each bio-artificial insert and the surface of the polymer film, and then bonding the grown polymer chain 210 By doing so, the polymer film 10 and the bio-artificial insert can be attached.

The detailed method of activating the surface of the bio-artificial insert for attaching the polymer film 10 and the bio-artificial insert will be described with reference to FIG. 6.

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

Referring to Figure 6 (a), the surface of the polymer film 10 for attaching a bio-artificial insert according to an embodiment of the present invention is a gas consisting of any one or a mixture thereof selected from argon, oxygen, nitrogen, water vapor and ammonia It can be activated by using a physical method of treating with plasma or ion beam using (200a). Accordingly, the second surface of the polymer film 10 for adhesion of a bio-artificial insert according to an embodiment of the present invention may be a surface 201 in an activated state by a physical method.

When activated by treatment with the above-described plasma or ion beam, the voltage is 500 [V] to 100 [kV], preferably 1 [kV] to 100 [kV], and power is 50 [W] to 10 [kW], preferably Preferably, 50 [W] to 200 [W], and 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, but is not limited thereto.

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

Referring to Figure 6 (b), according to another embodiment of the present invention, the polymer film using a chemical method of dipping the polymer film in a basic solution (200b) such as lithium hydroxide, sodium hydroxide, potassium hydroxide or calcium hydroxide ( 10) can activate the surface. Therefore, the second surface of the polymer film 10 for adhesion of a bio-artificial insert according to an embodiment of the present invention may be a surface 202 activated by a chemical method.

The pH of the basic solution 200b described above may be 9 to 12, wherein the temperature may be from 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, but is not limited thereto.

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

Referring back to FIG. 5, the polymer monomers formed on the surfaces of the activated bio-artificial insert and the polymer film according to an embodiment of the present invention are lactide, lactic acid, and glycolide. ), Glycolic acid, caprolactone (caprolactone), caprolactic acid (caprolactic acid), may be any one selected from trimethylene (trimethylene) and carbonate (carbonate), but the above example is This is only an example for explaining an embodiment, but is not limited thereto.

A polymer capable of disulfide-bonding a part of a polymer chain formed on the surface of a bio-artificial insert and a polymer film according to an embodiment of the present invention, or a polymer capable of complementarily bonding according to a specific chain order. Or it can be substituted with an atom.

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

Thus, the polymer chains or atoms substituted on the polymer chains formed on the second surface 200 of the polymer film 10 and the polymer chains formed on the surface of the bio-artificial insert by the above-described method are combined with the bio-artificial inserts and polymers. A film can be attached.

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

First, in order to check whether or not control of adhesion and spreadability of actual cells is possible according to the nanostructure 110 having a nano-pattern of a predetermined shape formed on the polymer film 10 according to one embodiment of the present invention described above. Describe the process of culturing and seeding the derived mesenchymal stem cells (ASC).

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

Human adipose-derived mesenchymal stem cells (ASCs) cultured under the above conditions were used in passage 6 for all experiments and seeded in a 12-well plate on which a nanopillar substrate was placed. ). A cell suspension of 2 x 10 4 cells was pipetted and removed after a period of time to keep only the 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 immunofluorescence staining and MTT (3- (4,5-dimethylthiazol-2-yl) -2,5 -Diphenyltetrazolium bromide) was used to evaluate the 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 viability of cells evaluated with MTT is used to determine the adhesion rate of cells having viability on a nanopillar formed substrate through a calibration curve. It was measured.

To plot the calibration curve, 1 x 103 to 1 x 106 (cells / mL) suspension was prepared and the cells were incubated overnight at 37 ° C. for 12 hours. Cells were incubated for an additional 3 hours after 500 μl of the MTT reagent was dispensed.

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

In this experiment, after culturing the cells on a nanopillar substrate formed on a polymer film for 24 hours, fat-derived mesenchymal stem cells (ASCs) were Karnovsky fixative (2% Paraformaldehyde), 2% glutaraldehyde (Glutaraldehyde). )), And the samples described above were dehydrated using ethanol (ascending gradual series, 50-100%).

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

Scanning electron microscopy (SEM) is another form of electron microscopy, in which an electron beam is focused on a point and scanned on the surface of a specimen. The detector collects back-scattered reflected secondary electrons or electrons from the surface and converts the collected electrons into a signal used to generate a real three-dimensional image of the specimen. During the scanning process described above, the detector can receive smaller electrons from the depression of the surface, so the sub-regions of the surface appear darker in the generated image. The scanning electron microscope 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).

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

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

Before staining, the immobilized cells are permeabilized with a permeabilization agent (0.5% Triton X-100) for 5 minutes and 5% BSA (Bovine serum) in 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. Samples were washed 3 times with phosphate buffer (PBS) to remove excess, and then incubated with secondary antibody labeled with fluorescence 5% diluted in BSA for 30 minutes at room temperature (RT).

In the last step, the cell nuclei were 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, fluorescence images of adipose-derived mesenchymal stem cells (ASCs) are analyzed to calculate the shape and size, which are morphological parameters of local adhesion (FA), and the area of adipose-derived mesenchymal stem cells (ASCs) as a degree of spreadability. It was also measured. The result of analyzing the data of the fluorescence image is expressed as the mean ± standard deviation (SD).

The actual cells according to the adipose-derived mesenchymal stem cells (ASCs) cultured by the above-described method and the nano-structure 110 having a nano-pattern of a predetermined shape formed on the polymer film 10 using the above-described analysis method It will be described later in FIGS. 7 to 11 whether the adhesion and spreadability can be adjusted.

7 is a view for explaining a state in which the nano-pillar array formed on the polymer film for adhesion of a bio-artificial insert according to an embodiment of the present invention adheres to adipose-derived mesenchymal stem cells (ASCs).

Figure 7 (a) is a nanostructure having a nano-pattern of a predetermined shape formed on the first surface of the polymer film for bio-artificial insert adhesion according to an embodiment of the present invention in the form of a nano-column array, the above-described nano Depending on the diameter (nm) of the columnar array (nm), spacing (nm), the upper surface area (μm2) of the nanopillar and the area fraction of the nanopillar array (%), the polymer film for adhesion of bio-artificial inserts is 1 to 1 It shows the classification by 3 samples.

Referring to Figure 7 (a), it shows that the size of the local adhesive (FA) according to an embodiment of the present invention is larger than the upper surface area of the nanopillar in the first sample. The above results indicate that the local adhesion (FA) increases in size over the space between the nanostructures, and the actual contact area of the local adhesion (FA) may be smaller than the proposed observation. The effective contact area fraction of adipose-derived mesenchymal stem cells (ASCs) can be confirmed to increase linearly along the upper surface area of one nanopillar.

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

Specifically, FIG. 7 (b) is coated with a fibronectin ECM protein on a nanopillar formed on a polymer film to provide an ECM ligand for cell adhesion to a polymer film formed with a nanopillar array according to an embodiment of the present invention. It shows the result of combining with integrin located.

Referring to FIG. 7 (b), the 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 culturing for 24 hours. , It can be confirmed whether or not cells are engrafted indicating the degree of adhesion and spreadability of the cells according to the nanopillar array 110.

The density of adipose-derived mesenchymal stem cells (ASCs) 30 after the above-described 24 hour culture is 28 ± 3 (cells / mm2) in the first sample, and 26 ± 1 (cells / mm2) and the third sample in the second sample. At 32 ± 9 (cells / mm2). The above results indicate that the seeding cell density is about 50 (cells / mm2), so the adhesion rate between the nanopillar array 110 and the adipose-derived mesenchymal stem cells (ASCs) 30 is about 55% -60%. can do. Therefore, it can be confirmed that the effect of various sizes of the discrete nanostructures is very small in the adhesion rate of stem cells.

Specifically, in FIG. 7 (b), an enlarged view of 1 micrometer (μm) at the bottom of the adipose-derived mesenchymal stem cells (ASCs) 30 attached using a focused ion beam (Focused Ion Beam, FIB) As a result of observing the cross section, as a result of observation, a gap between the nanopillar arrays 110 was observed in all of the first to third samples, and accordingly, the fat-derived mesenchymal stem cells (ASCs) 30 were nanopillar arrays 110 It can be seen that cells adhere only to the upper surface of the), that is, only the upper surface of the nanopillar array 110 interacts with the fat-derived mesenchymal stem cells (ASCs) 30.

The above results were essential prerequisites for localization of the adhesion sites showing integrin-ECM clustering, and that the nanopillar array 110 provides discrete adhesion sites to adipose-derived mesenchymal stem cells (ASCs) 30. Can be confirmed.

Therefore, the above results indicate that the adipose-derived mesenchymal stem cells (ASCs) 30 adhere only to the surface of the nanopillar array 110 and do not enter into the gap corresponding to the empty space between the nanopillar arrays 110. Can be confirmed.

7 (c) is a view for explaining a state in which adipocyte-derived mesenchymal stem cells (ASCs) 30 are attached to each of the first sample and the third sample according to an embodiment of the present invention in FIG. 7 (b). It is a diagram showing that the spreadability of adipose-derived mesenchymal stem cells (ASCs) 30 changes according to the size of a local adhesion (FA).

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

At this time, the nanopillar array 110 is capable of controlling the size of the local adhesion (FA) is to form the number of integrin-ECM bonds similarly regardless of the size of the nanopillar array 110 surface.

This is because the size of the protein complex forming the local adhesion (FA) increases while forming a bridge in an empty space between the nanopillar arrays 110 while exceeding the spacing of the nanopillar arrays 110 structures.

 Accordingly, while the above-described topical adhesion (FA) and the nanopillar array 110 are integrin-ECM-binding, adipose-derived mesenchymal stem cells (ASCs) 30 may be adhered to each of the first and third samples. 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 to cells according to formation of a local adhesion (FA) according to an embodiment of the present invention.

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

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

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

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

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

Referring to FIG. 7 (c), the above-described experimental results show local adhesion (FA) by the actual area of the integrin-ECM complex formed on the surface of the nanopillar array 110 and the effective contact area of the nanopillar array 110 surface. It can be confirmed that the size of the control, and accordingly it can be seen that the spreadability of the cell is adjusted according to the formation of an intracellular skeleton such as actin filaments that can generate traction.

9 is a view for explaining that the morphological change of adipose-derived mesenchymal stem cells (ASCs) is induced by the nano-structure according to an embodiment of the present invention.

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

The above-described F-actin serves to form a skeletal structure in the cell 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 constituting a protein complex called a local adhesion (FA), and the local adhesion (FA) connects the inside and outside of the cell, transmits a signal from outside the cell to the inside, and adheres to the cell. It can serve to maintain.

In this experiment, the area of the cells was measured through the distribution of F-actin proteins that form the intracellular skeletal structure.

Referring to FIG. 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 views showing differences in F-actin and vinculin expression among a plurality of samples. The above-described protein expression was compared based on the average pixel intensity per cell area, where 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 becomes larger, F-actin and bin It can be seen that protein expression for all of the coolins is slightly decreased.

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

F-actin and vinculin were usually used to confirm local adhesion (FA), as vinculin, a component of the protein complex that is a local adhesion (FA), made an association between integrin adhesion and actin cytoskeleton. 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 view for explaining the relationship between the average area of the local adhesive (FA) and the size of the nanostructure according to an embodiment of the present invention.

Referring to Figure 10 (a), since the above-mentioned cell spreadability is related to the local adhesion (FA) complex located at the edge, the fat-derived mesenchymal stem cells (ASCs) are located at the edge of the cell appearing while adhering to the nanostructure. Local adhesion (FA) associated with F-actin, which is used to identify intracellular skeletal structures, can be identified.

Referring to Figure 10 (b), it can be seen that the average area of the local adhesive (FA) decreases as the size of the nanostructure increases. Specifically, the area distribution of the local adhesive (FA) was narrowed as the size of the nanostructure increased in the first to third samples, and the average area of the local adhesive (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) in the nano-pillar having a large surface area was small, and the nano-pillar having a small size formed on the polymer film induced the size of the local adhesion (FA), so that the above-described local adhesion (FA) was The results of the area distribution and the average size indicate that, when the nanopillar size formed on the polymer film is large, a fat-derived mesenchymal stem cell (ASCs) is provided with an adhesion site capable of adhesion.

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

FIG. 11 (a) is a view showing the relationship between the effective contact area of the nanopillar and the upper surface area of the nanopillar. Referring to FIG. 11 (a), the coefficient of determination r2 in the linear regression analysis shows 0.99, and the effectiveness of the nanopillar As the contact area increases, the upper surface area of the nanopillar also increases, so the effective contact area of the nanopillar and the upper surface area of the nanopillar are the smallest for the first sample. 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 view showing the relationship between the upper surface area of the nanopillar and the average area of the local adhesive (FA). Referring to FIG. 11 (b), as the upper surface area of the nanopillar increases, the local adhesive (FA) It can be seen that the average area is inversely proportional to decrease, and accordingly, the first sample having the smallest upper surface area of the nanopillar has a larger average area of local adhesion (FA) than the second and third samples, and the upper surface area of the nanopillar It can be seen that the largest third sample has a smaller average area of local adhesion (FA) than the first and second samples.

Referring to FIG. 11 (c), in the linear regression analysis, the determination coefficient r2 represents 0.97, and it can be confirmed 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 can predict cell spreadability through measurement of local adhesion (FA) size, and through this, the behavior of cells can be controlled using a nanostructure.

Accordingly, it can be confirmed that the nanopillar array provides a limited adhesion site for integrin binding, which is an initial step for forming a local adhesion (FA), according to the experimental results of FIGS. Since it is related to differentiation into various cell lines, the polymer film having the nanopillar array described above has a function of controlling adhesion, viability, and differentiation of stem cells, and thus the polymer film formed with the nanopillar array described above is a bio-artificial insert. It can be attached to provide the above-described functionality to the bio-insert.

The nanostructure of the polymer film for adhesion of a bio-artificial insert according to an embodiment of the present invention can control the differentiation process of stem cells using a nanostructure when applied to stem cells. 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 a bio-artificial insert, and osteoblasts and adipocytes of stem cells according to the nano-gap of RGD protein Differentiation capacity of the furnace may vary.

12 is a view for explaining a method for manufacturing a polymer film for adhesion of a bio-artificial insert according to an embodiment of the present invention.

Referring to FIG. 12, a nanostructure having a nano-pattern of a predetermined shape that controls adhesion to a cell according to the behavior of the cell so that the bio-artificial insert adheres to the cell located at the insertion site in the living body is a mold material (mold material) ) Is formed on the surface (S1210).

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

The above-described soft lithography shows a method of making a fine pattern with an elastic polymer stamp or mold, which is a flexible organic material, and transferring it to a substrate.

The polymer film including the nano-structure having a nano-pattern of a predetermined shape formed on the first surface by the above-described method, the bio-insert of the cell and the bio-insert inserted in the insertion site so that the bio-insert inserted in the body is located in the insertion site It can have a use for facilitating bonding.

The shape of the nano-structure having the nano-pattern of a predetermined shape as described above may be various shapes as described above in FIG. 3, and a detailed description thereof will be omitted.

The method of manufacturing a polymer film for adhesion of a bio-artificial insert according to an embodiment of the present invention produces a flexible mold having a nanostructure characteristic with a function of cell control, and a polymer precursor (precursor) is poured into the flexible mold and is removable. After the film is covered and cured to remove the outer film, the polymer film for adhesion to the bio-artificial insert can be prepared by removing the polymer film from the mold after attaching the coupling polymer.

A detailed method of forming the nanostructure on the first surface of the above-described polymer film will be described later in FIG. 13.

A polymer film having a nanostructure formed on a first surface is attached to a bio-artificial insert (S1230).

The artificial prosthesis according to an embodiment of the present invention is a group consisting of an orthopedic implant such as a joint, a spine, and a valve, a dental implant, a vascular stent, a non-vascular stent, an implantable injection device, a face maker, and an artificial organ It may be selected from, and the bio-artificial insert can also be selected from the group consisting of titanium, stainless steel, cobalt, gold, silver, platinum, magnesium or alloys thereof, but the above-described examples describe one embodiment of the present invention. This is only an example for, but is not limited to.

A specific method of attaching the above-described polymer film to the bio-artificial insert 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 detailed method of forming the nano-fit on the above-described polymer mold will be described with reference to FIG. 14 together.

A polymer precursor is poured into the polymer mold on which the nano-pit is formed, and a separable outer film is covered and cured (S1222).

A few drops of a polyurethane acrylate (PUA) precursor may be dropped in the center of the polymer mold of the nano-pit structure according to an embodiment of the present invention.

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

The sample according to an embodiment of the present invention produced by the above-described method may be cured under ultraviolet light (UV) (300 mJ / cm 2).

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

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

According to one embodiment of the present invention, the cured solution can be very carefully peeled from the polymer mold of the nano-fit structure using a sharp razor, and then the sample can be further cured for overnight termination.

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

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

14 is a view for explaining in detail a method of manufacturing a polymer film for adhesion of a bio-artificial insert according to another embodiment of the present invention.

In the case where the nanostructure having a nano-pattern of a predetermined shape formed on a polymer film for adhesion of a bio-artificial insert according to an embodiment of the present invention is a nanopillar array, a method for manufacturing a polymer film will be described as an example, It is not limited to this.

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

As a method of forming the nano / micro structure 51 on the surface of the mold material 50 described above, 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 examples are merely examples for explaining an embodiment of the present invention. It is not limited.

According to an embodiment of the present invention, a quartz nanocolumn array 51 may be generated from quartz glass 50 using polystyrene beads 40. In the above-described quartz nanopillar array 51, the size of the nanopillar and the distance 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 Figure 14 (d), using the quartz (quartz) nano-column array 51 prepared by the above-described method according to an embodiment of the present invention is the first to create a polymer structure 60 of the nano-pit structure A second lithography process can be used.

The colloidal lithography may be used to manufacture the polymer mold 60 having the above-described nano-fit structure. When using the colloidal lithography described above, it is possible to control various pitches for a nanopillar array using nanoparticles of various sizes.

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

In the method of transferring the nano / micro structure 51 formed on the surface of the mold material 50 to the polymer film 10 according to an embodiment of the present invention, the mold is formed by a structure formed on a 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 formed with a nanostructure to a bio-artificial insert according to an embodiment of the present invention.

15, the surface of the polymer film and the surface of the bio-artificial insert are activated using a physical method or a chemical method, respectively (S1231).

Plasma or the surface of the polymer film for adhesion of the bio-artificial insert and the surface of the bio-artificial insert according to an embodiment of the present invention using a gas consisting of any one or a mixture of argon, oxygen, nitrogen, water vapor and ammonia, respectively, or 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-artificial insert using a chemical method of immersing a biofilm and a polymer film for adhesion of a bio-artificial insert in a basic solution such as lithium hydroxide, sodium hydroxide, potassium hydroxide or calcium hydroxide, respectively The surface of the adhesive polymer film and the surface of the bio-insert can be activated, respectively.

Since the specific method of activating the surface of the polymer film and the surface of the bio-artificial insert, respectively, has been described above, a detailed description thereof will be omitted.

Polymer monomers are formed on the surfaces of the activated bio-artificial inserts and the polymer film (S1232). Since the above-described polymer monomer has been described above, detailed description will be omitted.

The polymer chains are grown by growing polymer monomers formed on the surface of the bio-insert and the polymer film (S1233). Each grown polymer chain is bound to the surface of the bio-artificial insert and the surface of the polymer film (S1234).

A polymer capable of disulfide-bonding a part of a polymer chain formed on the surface of a bio-artificial insert and a polymer film according to an embodiment of the present invention, or a polymer capable of complementarily bonding according to a specific chain order. Or it can be substituted with an atom.

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

The polymer film for adhesion of a bio-artificial insert having a nano-structure having a nano-pattern of a predetermined shape according to an embodiment of the present invention can be prepared by applying a nanoimprinting method, and accordingly a roll-to-roll process (roll 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 to apply a specific material or remove a predetermined portion to have a new function. Since the roll-to-roll process described above is advantageous for mass production, it is possible to lower the manufacturing cost.

The above description is merely illustrative of the technical spirit of the present invention, and those of ordinary skill in the art to which the present invention pertains may make various modifications, changes, and substitutions without departing from the essential characteristics of the present invention. will be. Therefore, the embodiments disclosed in the present invention and the accompanying drawings are not intended to limit the technical spirit of the present invention, but to explain the scope of the technical spirit of the present invention. . The scope of protection of the present invention should be interpreted by the claims below, and all technical spirits within the scope equivalent thereto should be interpreted as being included in the scope of the present invention.

10: polymer film 40: polystyrene beads
20: bio-artificial insert 50: mold material
30: fat-derived mesenchymal stem cells 60: polymer mold

Claims (18)

In the biofilm inserted into the living body and the polymer film attached to,
The bio-artificial insert has a nano-pattern of a predetermined shape formed on a first surface that adheres to the cell that controls the adhesion to the cell according to the behavior of the cell to adhere to the cell located at the insertion site in the living body. A polymer film for adhesion of a bio-artificial insert comprising a nano-structure. According to claim 1,
The size of the nano-structure having the nano-pattern of the preset form or the spacing between the nano-structures according to the nano-pattern,
A topical area that connects the inside and outside of the cell to fill the space between the nanostructures while the cell adheres to the nanostructure, transmits a signal from the outside of the cell to the inside of the cell, and maintains adhesion of the cell A polymer film for adhesion of a bio-artificial insert, by adjusting the size of adhesion (Focal Adhesion, FA) to directly control cell spreading, which is the degree to which the cells spread while adhering to the nanostructure. According to claim 2,
The nano-structure having the nano-pattern of the predetermined shape according to the shape of the cell is a groove array, a ridge array, a hole array, a pillar array and an orthogonal mesh A polymer film for attaching a bio-artificial insert, characterized in that it is any one selected from (mesh) forms. According to claim 2,
The size of the nano-structure having the nano-pattern of the preset form or the spacing between the nano-structures according to the nano-pattern,
A polymer film for bioadhesive adhesion, 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. According to claim 1,
A polymer chain in which a polymer monomer is grown on a second surface to be attached to the bio-artificial insert in addition to the first surface on which the nano-structure is formed to adjust the difference from the surface energy of the bio-artificial insert to adhere to the surface of the bio-artificial insert. Polymer film for adhesion of a bio-artificial insert, characterized in that formed. The method of claim 5,
The polymer monomers are lactide, lactic acid, glycolide, glycolic acid, caprolactone, caprolactic acid, trimethylene and A polymer film for attaching a bio-artificial insert, which can be selected from the group consisting of carbonates. The method of claim 5,
The polymer chain is a polymer film for attaching a bio-artificial insert, characterized in that the bio-polymer is DNA or RNA that complementarily binds according to a base sequence extracted from a test subject to insert the bio-artificial insert. According to claim 1,
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 as to be biodegraded. , Polylactic-co-glycolic acid (PLGA), polyglycolic acid (PGA), polylactide (PLA), polycaprolactone (PCL), chitosan, Polytrimethylene carbonate (Polytrimethylenecarbonate, PTMC) or a polymer film for adhesion of a bio-artificial insert, characterized in that it comprises a synthetic polymer group having a biocompatibility of a mixture of two or more selected from the polymer group. The method of claim 8,
Before the biofilm is biodegraded in vivo, the nanostructure is regenerated so that the bioartificial insert is fixed to the insertion site while the cells regenerate at the insertion site in the living body after the bioinsert is implanted in the living body. A polymer film for bioadhesive adhesion, characterized in that the polymer film is biodegraded in vivo after adhesion to a cell. According to claim 1,
The bio-artificial insert can be selected from the group consisting of orthopedic implants such as joints, spine, and valves, dental implants, vascular stents, non-vascular stents, implantable injection devices, face makers, and artificial organs. A polymer film for adhesion to a bio-artificial insert. According to claim 1,
The bio-artificial insert can be selected from the group consisting of titanium, stainless steel, cobalt, gold, silver, platinum, magnesium or alloys thereof. In the manufacturing method of the polymer film to be attached to the bio-artificial insert to be inserted into the living body,
A surface of a mold material is a nanostructure having a nano-pattern of a predetermined shape that controls adhesion to the cell according to the behavior of the cell so that the bio-artificial insert adheres to a cell located at an insertion site in the living body. Forming on; And
A method of manufacturing a polymer film for adhesion of a bio-artificial insert comprising; transferring the nano-structure formed on the mold material to the polymer film to form the nano-structure on a first surface of the polymer film that adheres to the cells. The method of claim 12,
The size of the nano-structure having the nano-pattern of the preset form or the spacing between the nano-structures according to the nano-pattern,
A topical area that connects the inside and outside of the cell to fill the space between the nanostructures while the cell adheres to the nanostructure, transmits a signal from the outside of the cell to the inside of the cell, and maintains adhesion of the cell A method for manufacturing a polymer film for bioadhesive adhesion, by adjusting the size of adhesion (Focal Adhesion, FA) to directly control cell spreading, which is the degree to which the cells spread while adhering to the nanostructure. . The method of claim 13,
The size of the nano-structure having the nano-pattern of the preset form or the spacing between the nano-structures according to the nano-pattern,
A method for manufacturing a polymer film for bioadhesive adhesion, characterized in that the average area of the cells and the average area of the local adhesion (FA) are adjusted in consideration of an inversely proportional relationship to control the cell spreadability from the size of the local adhesion. The method of claim 12,
The step of forming the nanostructure,
Forming a nanopit in a polymer mold from a nanostructure formed in the mold material using a nanoimprint lithography process;
Pouring a polymer precursor (precursor) into the polymer mold on which the nano-pit is formed and covering the separable outer film to cure;
Removing the outer film covering the cured polymer precursor; And
And separating the polymer precursor from which the outer film is removed from the polymer mold. The method of claim 12,
Attaching a polymer film having the nano-structure formed on the first surface to the bio-insert; Including more,
The step of attaching the polymer film to the bio-artificial insert,
Activating the surface of the polymer film and the surface of the bio-prosthesis respectively using a physical method or a chemical method;
Forming a polymer monomer on the surface of each activated bio-artificial insert and the surface of the polymer film;
Growing a polymer chain formed on the surface of the bio-insert and the polymer film, respectively, thereby growing a polymer chain; And
A method of manufacturing a polymer film for attaching a bio-artificial insert, comprising: bonding the polymer chain grown to the surface of the bio-artificial insert and the surface of the polymer film, respectively. The method of claim 16,
The activating step,
Soaking the bio-prosthesis and the polymer film in a basic solution such as lithium hydroxide, sodium hydroxide, potassium hydroxide or calcium hydroxide, respectively, to activate the surfaces of the bio-prosthesis and the polymer film using chemical methods, respectively. A method of manufacturing a polymer film for adhesion to a bio-artificial insert. The method of claim 16,
The activating step,
Activate the surface of the bio-prosthesis and the surface of the polymer film using a physical method of treating with a plasma or an ion beam using a gas composed of any one of argon, oxygen, nitrogen, water vapor and ammonia or a mixture thereof. A method of manufacturing a polymer film for adhesion of a bio-artificial insert, characterized in that.

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