KR20090041271A - Method of controlling pore structure of nanofirous scaffold for tissue engineering and scaffold using the same - Google Patents

Abstract

A method for controlling the porous structure and the tissue engineering nanofiber supporter manufactured by using the same are provided, which enables the porosity which is similar to the extracellular matrix of the nature. A method for controlling the porous structure of the tissue engineering nanofiber supporter comprises: a step(S100) for preparing a mixture including the particle for the void formation and the nanofiber spreader solution; a step(S110) for preparing the nanofiber foam including the particle for the void formation using the prepared mixture; and a step(S120) for forming the nanofiber supporter in which the air gap is formed by removing the particle for the void formation of the nanofiber form.

Description

Method for controlling pore structure of nanofiber scaffold for tissue engineering and scaffold for tissue engineering manufactured using the same {METHOD OF CONTROLLING PORE STRUCTURE OF NANOFIROUS SCAFFOLD FOR TISSUE ENGINEERING AND SCAFFOLD USING THE SAME}

The present invention relates to the field of tissue engineering, and more particularly, to a method for controlling pore structure of a nanofiber support for tissue engineering and a support for tissue engineering prepared using the same.

Generally, tissue engineering refers to a science of artificially culturing or biological control of human or animal tissues for the purpose of treatment or industry, and using tissue engineering technology to culture cells in a scaffold. Preparing a support complex and using it to regenerate living tissue and organs.

The basic principle of this tissue engineering technique is to collect the necessary tissue from the patient's body, separate the cells from the tissue, and then culture the separated cells in a support to prepare a cell-support complex, and then, the prepared cell-support complex. It is implanted in the human body again.

In order to optimize the regeneration of biological tissues and organs in the tissue engineering technique, it is preferred to prepare a support similar to the biological tissues.

The basic requirement of the support is that the tissue cells should adhere to the support and form a framework capable of forming a tissue having a three-dimensional structure, and a non-toxic organism that does not cause blood coagulation or inflammatory reaction after implantation into a living body. Compatibility, etc.

For this reason, it is suitable for the support to be similar to an extracellular matrix that exists naturally in the body of an animal. Here, the extracellular matrix has a structure in which nano-sized fibers made of collagen have a network structure and other functional materials are present together.

However, the support according to the background art is generally manufactured in the form of a flat plate made of polystyrene. That is, since the support is prepared in two dimensions, there is a lack of space for cells to grow and divide.

Therefore, the problem to be solved by the present invention is to have a porosity similar to the natural extracellular matrix, the pore structure control method of the nanofiber support for tissue engineering and the tissue prepared using the same can adjust the porosity and pore size appropriately It is to provide an engineering support.

Other problems to be solved by the present invention are not limited to the above-mentioned problems, and other tasks not mentioned above may be clearly understood by those skilled in the art from the following description. There will be.

Method for controlling the pore structure of the nanofiber support for tissue engineering according to the present invention for achieving the problem to be solved is to prepare a mixture comprising a nanofiber dispersion solution and particles for forming pores insoluble in the nanofiber dispersion solution step; Preparing a nanofiber foam including the pore-forming particles by using the prepared mixture; And forming a nanofiber scaffold in which pores are formed by removing the particles for forming pores of the nanofiber foam.

On the other hand, the support for tissue engineering according to the present invention for achieving the above object is made of nanofibers having a three-dimensional structure and porosity of 90% or more.

Specific details of other embodiments are included in the detailed description and the drawings. Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments described below in detail with the accompanying drawings.

According to the present invention, when preparing a support for tissue engineering, the pore-forming particles are added to the nanofiber dispersion solution, and then the nanofiber foam is prepared using the same, and then the natural particles are removed by removing the pore-forming particles of the nanofiber foam. A support having a three-dimensional structure similar to the extracellular matrix of can be prepared. In addition, the desired pore size and porosity can be secured by adjusting the size and the amount of the particles for forming the pore.

Hereinafter, with reference to the accompanying drawings will be described in detail a method for preparing a support for tissue engineering and a support for tissue engineering prepared using the same.

1 is a process flowchart for schematically illustrating a method for preparing a support according to the present invention.

1, the support according to the present invention prepares a mixture including a nanofiber dispersion solution and particles for forming pores (S100), and then prepares a nanofiber foam including particles for forming pores ( S110), and then may be prepared by forming a nanofiber support (scaffold) in which the pores are formed (S120).

Hereinafter, a method for preparing a support according to the present invention will be described in more detail.

(1) Preparation process of the mixture

In order to prepare the mixture, first, an electrospinning spinning solution is prepared.

Specifically, the spinning solution may be appropriately selected from the type of spinning solution applied according to the use of the support. For example, the spinning solution may be prepared using a biodegradable natural polymer. Here, the biodegradable natural polymer resin may be one selected from the group consisting of collagen, fibrin, chitosan, hyaluronic acid, cellulose, polyamino acid, fibroin, keratin, elastin or derivatives thereof. Hereinafter, a case in which fibroin is used as the biodegradable natural polymer resin will be described as an example.

The fibroin can be obtained, for example, from a gamma cocoon having a structure in which two strands of fibroin are wrapped in a sericin envelope. Since fibroin has excellent biocompatibility and does not affect any surrounding tissues, the fibroin may be prepared in various forms such as powder, gel, and aqueous solution, and may be used in various foods and medicines.

When preparing the spinning stock solution using the gaze cocoon, sericin can be removed to maximize biocompatibility.

Subsequently, an aqueous solution of fibroin is prepared by drying, dissolving, and dialysis of the refined gamma cocoon, and then the fibroin in sponge form may be prepared by freeze-drying the aqueous solution of fibroin.

Thereafter, after dissolving the fibroin sponge in a predetermined solvent, the spinning stock solution may be prepared by removing impurities and bubbles.

Next, the prepared spinning stock solution is electrospun. At this time, an electrospinning apparatus including a radiating unit, a DC power supply unit, and a dispersion unit can be used.

The spinning unit receives the spinning stock solution, serves to spin the received spinning stock solution, and may use a syringe pump to maintain a constant spinning speed, but is not limited thereto.

The DC power supply unit is electrically connected to the radiation unit, and supplies a constant DC power to the radiation unit during nanofiber spinning.

The said dispersion part consists of a metal container grounded by a predetermined electric wire, for example. In this case, the dispersion portion may be filled with a coagulation solution, that is, a first solvent capable of dispersing and solidifying the spun nanofibers. In this case, the first solvent may vary depending on the type of nanofibers, and is not particularly limited, but may be selected from the group consisting of water, methanol, ethanol, hexane, heptane, ether, acetone, propanol, or a mixture thereof. Can be used.

When using the electrospinning apparatus having the above-described configuration, the electrospun nanofibers can be dispersed in the first solvent filled in the dispersion.

Next, the nanofiber dispersion solution may be prepared by mixing a second solvent for substituting the first solvent with the first solvent. Here, the reason for replacing the first solvent with the second solvent will be described in the nanofiber foam forming process described later.

Next, by adding the particles for forming pores to the nanofiber dispersion solution, a mixture including the nanofiber dispersion solution and the particles for forming pores can be prepared.

The pore-forming particles are insoluble in the nanofiber dispersion solution, and may be removed by a process described below, and the type thereof is not particularly limited, but is selected from the group consisting of sodium chloride, potassium chloride, calcium chloride, sucrose, and aluminum chloride. At least one particle can be used.

Particle diameter of the pore-forming particles may be 10 to 1000㎛, the pore-forming particles may be used 0.1 to 0.2g per 1ml nanofiber dispersion solution, but is not limited thereto.

(2) nano fiber foam preparation process

After preparing a mixture including the nanofiber dispersion solution and the particles for forming pores insoluble in the nanofiber dispersion solution, the mixture may be used to prepare a nanofiber foam including the particles for forming pores. It can be achieved by freeze drying the mixture.

At this time, since the first solvent may be unsuitable for the freeze-drying, as described above, the first solvent is replaced with the second solvent. Here, as the second solvent, for example, one selected from the group consisting of dioxane, carbon tetrachloride, trichloroethane, benzene, isopropanol, cyclohexane, or a mixed solvent thereof may be used. Among them, the dioxane is an organic solvent suitable for lyophilization because it is frozen at 10 ° C. or lower and sublimation occurs at a vacuum condition of 10 mTorr at room temperature. In addition, the sodium chloride particles, which are fibroin and pore forming particles, are not dissolved, and thus are suitable for shaping and freeze-drying them while maintaining the dispersed nanofiber form.

(3) nanofiber support forming process

In order to form the nanofiber support, first, the nanofiber foam may be vapor cross-linked using a predetermined vapor. The use of the steam crosslinking has the advantage of crosslinking the material susceptible to moisture because it does not have to wet the material to be crosslinked in the aqueous solution. Here, the vapor is not particularly limited since it may vary depending on the nanofiber foam to be crosslinked, but is not limited to glutaaldehyde, carbodiimide, diphenylphosphoryl azide, ethyldimethylaminopropylcarbodiimide, toylene diisocyanate and hexamethylene One selected from the group consisting of diisocyanates can be used.

Subsequently, the vapor crosslinked nanofiber foam may be immersed in a solution in which the pore forming particles are soluble. At this time, it is preferable that the residual toxicity of the steam used in the steam crosslinking is removed.

Subsequently, the immersed nanofiber foam may be washed with a predetermined solvent and then lyophilized to prepare a nanofiber support according to the present invention.

The nanofiber support according to the present invention has a three-dimensional sponge-like structure and may have a porosity of 90% or more. In addition, the pore size between the nanofibers forming the support may be 50 to 1000 μm depending on the particle diameter of the pore forming particle. Here, the porosity and the pore size may be adjusted by the amount and particle size of the pore forming particles.

Hereinafter, the support prepared according to the present invention through Examples and Comparative Examples will be described in detail. However, the present invention is not limited to the examples described below.

EXAMPLE

1.Comparison of Support Structure

In order to compare the structural characteristics of the support, a support of each of Example 1 and Comparative Example 1 according to the present invention was prepared as follows.

(1) Example 1

1) Preparation of Electrospinning Radiation Solution

In Example 1 of the present invention, the spinning stock solution was prepared using silk fibroin. Specifically, first, the gourd cocoon was added to an aqueous solution of 0.3% and 0.2% of Marseubi soap and sodium carbonate, respectively, in a ratio of 1:25 and treated at 100 ° C. for 1 hour, followed by repeated washing with distilled water to remove sericin. . The dried cocoons were completely dried, dissolved completely at 85 ° C. in a bath ratio of 1:20 using a calcium chloride / water / ethanol (molar ratio 1: 8: 2) mixed solvent, and dialyzed in distilled water for 3 days using a cellulose dialysis membrane to obtain salt and Ethanol was removed. The prepared silk fibroin aqueous solution was lyophilized to obtain a sponge.

The nanofiber stock solution was prepared by completely dissolving the prepared silk fibroin sponge in a concentration of 12% in 98% formic acid and removing impurities and bubbles.

2) Nanofiber Foam Preparation

The prepared spinning stock solution was molded into nanofibers using an electrospinning apparatus and dispersed in a dispersion part. Here, methanol was used as the coagulating solution in the dispersion, that is, the first solvent. Subsequently, the methanol was substituted with dioxane as a second solvent. Specifically, the process of adding the same volume of dioxane and methanol to the nanofiber dispersion solution and left for 12 hours for complete mixing after stirring was repeated three times.

Subsequently, sodium chloride particles having an average particle diameter of 400 μm were added to disperse to mix well with the nanofibers, filled into a glass container having an inner diameter of 11 mm, and then freeze-dried to prepare a nanofiber foam.

3) Nanofiber Support Formation

The nanofiber foam was placed in a desiccator saturated with glutaaldehyde vapor and treated for 24 hours to cause silk fibroin crosslinking. After crosslinking, the mixture was immersed in 0.1M aqueous solution of glycine and exchanged for 8 hours at 8 hours. Upon stirring, the sodium chloride particles melted to form the desired voids in the foam.

Thereafter, washing with PBS (phosphate buffer saline) three times, immersing, stirring for 24 hours, and washing three times with lyophilization to obtain a silk fibroin nanofiber support having a porous structure, and the photographs observed therefrom are shown in FIG. 2. Shown in

(2) Comparative Example 1

The nanofiber stock solution was prepared in the same manner as in Example 1, and the nanofibrous stock solution was electrospun and immediately freeze-dried to obtain a nanofiber support having a two-dimensional structure. Indicated. On the other hand, in Comparative Example 1, sodium chloride particles for forming voids were not added.

(3) Evaluation of support structure characteristic

Figure 2 is a photograph of the support prepared according to an embodiment of the present invention. 3 is a photograph of a support prepared according to a comparative example. Here, (a) of Figure 2 is a photograph showing the appearance of the support prepared according to an embodiment of the present invention, Figure 2 (b) and (c) of the support taken at different magnifications of the scanning electron microscope It is a photograph. In addition, Figure 3 (a) is a photograph showing the appearance of the support prepared according to the comparative example, Figure 3 (b) and (c) is a photograph of the support taken at different magnification of the scanning electron microscope.

The support according to an embodiment of the present invention, as shown in (a) of FIG. 2, has a three-dimensional structure, but the support according to the comparative example, as shown in (a) of FIG. It was shown to have a two-dimensional structure.

As such, since the support according to the embodiment of the present invention is manufactured in the form of a sponge having a three-dimensional structure, as shown in FIGS. 2B and 2C, a large amount of pores of a large size are formed in the support as a whole. I could see the inclusion. For this reason, the support according to an embodiment of the present invention was not only ensures a sufficient space for the cells to proliferate, it can be seen that it is advantageous for cell attachment. In addition, the support according to an embodiment of the present invention was found to be very suitable for culturing three-dimensional tissue.

On the other hand, since the support according to the comparative example is manufactured in the form of a non-woven fabric having a two-dimensional structure, as shown in (b) and (c) of FIG. I found it difficult.

2. Variation of porosity and elastic modulus of support

In order to determine the change in porosity and modulus of elasticity of the support according to the amount of sodium chloride particles added, supports of Examples 2 to 5 and Comparative Example 2 according to the present invention were prepared as follows.

(1) Example 2

Prepared in the same manner as in Example 1, but the support was prepared by adding 0.05 g of sodium chloride particles per 1 ml of nanofiber dispersion solution.

(2) Example 3

Prepared in the same manner as in Example 1, but a support was prepared by adding 0.10 g of sodium chloride particles per ml of nanofiber dispersion solution.

(3) Example 4

Prepared in the same manner as in Example 1, but a support was prepared by adding 0.15 g of sodium chloride particles per ml of nanofiber dispersion solution.

(4) Example 5

Prepared in the same manner as in Example 1, but a support was prepared by adding 0.20 g of sodium chloride particles per ml of nanofiber dispersion solution.

(5) Comparative Example 2

Prepared in the same manner as in Example 1, except that sodium chloride particles were added to prepare a support.

The porosity and modulus of elasticity of each of Examples 2 to 5 and Comparative Example 2 were measured, and the results are shown in FIGS. 4 and 5, respectively.

(6) Evaluation of porosity and modulus of elasticity

Figure 4 is a graph showing the porosity change of the support according to the amount of sodium chloride particles added when preparing the support according to an embodiment of the present invention.

Referring to Figure 4, when preparing the support according to an embodiment of the present invention, it can be seen that the porosity is affected by the amount of sodium chloride particles added. Specifically, when preparing a support according to an embodiment of the present invention, the porosity initially increases when the amount of sodium chloride particles is increased, but when the amount of sodium chloride particles added per 1 ml of the nanofiber dispersion solution is saturated, the porosity is saturated. It started to be. In this case, the porosity was found to be 90% or more.

5 is a graph showing a change in elastic modulus according to the amount of sodium chloride particles added when preparing a support according to an embodiment of the present invention.

Referring to Figure 5, when preparing the support according to an embodiment of the present invention, the more the amount of sodium chloride particles showed a tendency to lower the initial elastic modulus, when the amount of sodium chloride particles is more than 0.1 g / ml of relatively high strength Constant values were shown without degradation.

3. Change in pore size of support

In order to determine the change in pore size according to the sodium chloride particle size, the supports of Examples 6 to 8 and Comparative Example 3 according to the present invention were prepared by the following method.

(1) Example 6

Prepared in the same manner as in Example 5, but the support was prepared by adding sodium chloride particles having an average particle diameter of 150㎛.

(2) Example 7

Prepared in the same manner as in Example 5, but the support was prepared by adding sodium chloride particles having an average particle diameter of 200㎛.

(3) Example 8

Prepared in the same manner as in Example 5, but the support was prepared by adding sodium chloride particles having an average particle diameter of 400㎛.

(4) Comparative Example 3

Prepared in the same manner as in Example 5, except that sodium chloride particles were added to prepare a support.

The pore sizes of Examples 6 to 8 and Comparative Example 3 were measured, and the results are shown in FIG. 6.

6 is a graph showing a pore size change according to sodium chloride particle size when preparing a support according to an embodiment of the present invention.

Referring to Figure 6, when preparing the support according to an embodiment of the present invention, it can be seen that the pore size is affected by the sodium chloride particle size. Specifically, when preparing the support according to an embodiment of the present invention, the pore size was found to be approximately proportional to the particle size of sodium chloride.

4. Cell culture experiment using support

Figure 7 is a scanning electron micrograph showing the appearance of chondrocytes cultured in the support prepared according to an embodiment of the present invention. Here, (a) and (b) of Figure 7 is a scanning electron micrograph observing the appearance of chondrocytes with different magnification.

Referring to Figure 7 (a), it was found that the chondrocytes proliferated in three dimensions as a result of culturing the chondrocytes using the support prepared according to an embodiment of the present invention. In addition, it was confirmed that the chondrocytes were not only located in the periphery of the pores, but also the cells proliferated into the pores. In addition, as shown in Figure 7 (b), it was confirmed that the chondrocytes adhere well to the surface of the nanofibers.

Although the embodiments of the present invention have been described above with reference to the accompanying drawings, those skilled in the art to which the present invention pertains can realize that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. I can understand that.

Therefore, since the embodiments described above are provided to completely inform the scope of the invention to those skilled in the art, it should be understood that they are exemplary in all respects and not limited. The invention is only defined by the scope of the claims.

1 is a process flowchart for schematically illustrating a method for preparing a support according to the present invention.

Figure 2 is a photograph of the support prepared according to an embodiment of the present invention.

3 is a photograph of a support prepared according to a comparative example.

4 is a graph showing the porosity change of the support according to the amount of sodium chloride particles added when the support according to an embodiment of the present invention.

5 is a graph showing a change in elastic modulus according to the amount of sodium chloride particles added when preparing a support according to an embodiment of the present invention.

6 is a graph showing a pore size change according to sodium chloride particle size when preparing a support according to an embodiment of the present invention.

Figure 7 is a scanning electron micrograph showing the appearance of chondrocytes cultured in the support prepared according to an embodiment of the present invention.

Claims (16)

Preparing a mixture comprising a nanofiber dispersion solution and particles for forming pores insoluble in the nanofiber dispersion solution; Preparing a nanofiber foam including the pore-forming particles by using the prepared mixture; And Forming a nanofiber scaffold in which pores are formed by removing the particles for forming pores of the nanofiber foam; Method of controlling the pore structure of the nanofiber support for tissue engineering comprising a. The method of claim 1, wherein preparing the mixture comprises: Preparing a spinning stock solution; Electrospinning the prepared spinning stock solution; Dispersing the electrospun nanofibers in a coagulating solution; Preparing a nanofiber dispersion solution by mixing a solvent for replacing the coagulation solution with the coagulation solution; And Adding the pore forming particles to the prepared nanofiber dispersion solution Method of controlling the pore structure of the nanofiber support for tissue engineering comprising a. The method of claim 2, The coagulating solution is water, methanol, ethanol, hexane, heptane, ether, acetone, propanol, or a mixture of these solvents, characterized in that the pore structure control method of the nanofiber support for tissue engineering, characterized in that one. The method of claim 2, The solvent for replacing the coagulation solution is dioxane, carbon tetrachloride, trichloroethane, benzene, isopropanol, cyclohexane, or a mixture selected from the group consisting of a solvent mixture control method of the nanofiber support for tissue engineering, characterized in that . The method of claim 1, The nanofiber dispersion solution method of controlling the pore structure of the nanofiber support for tissue engineering, characterized in that it comprises a biodegradable natural polymer. The method of claim 5, The biodegradable natural polymer is collagen, fibrin, chitosan, hyaluronic acid, cellulose, polyamino acid, fibroin, keratin, elastin, or one selected from the group consisting of derivatives of tissue engineering nanofibrous support, characterized in that Way. The method of claim 1, The pore-forming particle is at least one particle selected from the group consisting of sodium chloride, potassium chloride, calcium chloride, sucrose and aluminum chloride method of adjusting the pore structure of the nanofiber support for tissue engineering. The method of claim 1, The pore structure control method of the nanofiber support for tissue engineering, characterized in that the particle diameter of the pore-forming particles is 10 to 1000㎛. The method of claim 1, Method for controlling the pore structure of the nanofiber support for tissue engineering, characterized in that using 0.1 to 0.2g of the pore-forming particles per ml of the nanofiber dispersion solution. The method of claim 1, wherein preparing the nanofiber foam comprises Freeze drying the mixture Method of controlling the pore structure of the nanofiber support for tissue engineering comprising a. The method of claim 1, wherein preparing the nanofiber support comprises Vapor cross-linking the nanofiber foam with steam; Immersing the vapor crosslinked nanofiber foam in a solvent solubilizing the pore forming particles; And Freeze drying the immersed nanofiber foam Method of controlling the pore structure of the nanofiber support for tissue engineering comprising a. The method of claim 11, The steam is for tissue engineering, characterized in that one of the steam selected from the group consisting of glutaraldehyde, carbodiimide, diphenylphosphoryl azide, ethyldimethylaminopropyl carbodiimide, toylene diisocyanate and hexamethylene diisocyanate Method for controlling pore structure of nanofiber support. A tissue engineering support made of nanofibers having a three-dimensional structure and having a porosity of 90% or more. The method of claim 13, The nanofiber interpore size constituting the support is a tissue engineering support, characterized in that 50 to 1000㎛. The method of claim 13, The nanofiber is a support for tissue engineering, characterized in that it comprises a biodegradable natural polymer. The method of claim 15, The biodegradable natural polymer is a support for tissue engineering, characterized in that one selected from the group consisting of collagen, fibrin, chitosan, hyaluronic acid, cellulose, polyamino acid, fibroin, keratin, elastin, or derivatives thereof.

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