KR102198398B1 - Non-solvent induced phase separation (NIPS)-based 3D plotting for porous scaffolds with core-shell structure - Google Patents

Abstract

The present invention relates to a technology for manufacturing a porous support using a non-solvent separation method, and a three-dimensional porous support having a porous core-dense shell structure can be manufactured using a biodegradable polymer having excellent biocompatibility and mechanical strength.

Description

Non-solvent induced phase separation (NIPS)-based 3D plotting for porous scaffolds with core-shell structure}

The present invention relates to a technique for manufacturing a porous core-dense shell structure scaffold using a non-solvent separation method.

Scaffolds made using 3D printers have the advantage of being able to provide personalized shapes to patients. In addition, the three-dimensional macro pore structure of the scaffold provided through the 3D printer has the advantage of increasing the regeneration speed of the tissue. On the other hand, the three-dimensionally connected microscopic pore structure formed inside the scaffold is advantageous in terms of inducing/stimulating cell adhesion, proliferation, and differentiation, as well as the ability to regenerate via tissue, which is similar to that of a living body.

1. Korean Patent Publication No. 10-2018-0001191

An object of the present invention is to prepare a porous scaffold (scaffold) having three-dimensional macro-size pores using a biodegradable polymer having excellent biocompatibility and mechanical strength.

The present invention includes the step of extruding a biodegradable polymer solution into a precipitation solution using a 3D printer,

The precipitation solution provides a method of preparing a porous support that is a mixed solution of water and alcohol.

In addition, the present invention is manufactured by the method for manufacturing the porous support described above,

The filaments constituting the porous support provide a porous support having a structure of a core having a porous structure and a shell having a dense structure.

The porous support according to the present invention can provide stability in vivo by using a biodegradable polymer with excellent biocompatibility, and since the biodegradable polymer has excellent mechanical strength, it can provide excellent mechanical strength in vivo. have.

In the porous support according to the present invention, the filaments constituting the porous support have a structure of a core having a porous structure and a shell having a dense structure, and this structure has an internal structure similar to that of a living body, so that the filaments constituting the porous support have a structure similar to that of a living body. In this case, it can induce/stimulate adhesion, proliferation and differentiation of cells as well as excellent mechanical strength.

Unlike other conventional techniques, the porous support according to the present invention has a dense structure in the shell (outer) portion of the filament, and thus the outer portion of the support may also have a dense structure without pores.

In addition, in the present invention, it is possible to control the thickness of the shell portion of the filament by adjusting the composition and ratio of the precipitation solution, through which the strength of the support can be artificially controlled.

1 is a schematic diagram showing a method of manufacturing a porous support according to the present invention, after transferring a polycaprolactone solution to a piston, a scaffold is manufactured in a precipitation solution according to a preset design using a 3D printer.
2 is a schematic diagram showing a state when a polycaprolactone solution extruded from a piston enters a precipitation solution. When the polycaprolactone solution is exposed to the precipitation solution, it is laminated according to a preset design, and the surface of the filament is solidified and has a pore structure through phase separation.
3 is a schematic diagram showing a cross section of a polycaprolactone filament according to the composition of a precipitation solution.
4 is a schematic diagram showing a structure of a polycaprolactone filament, that is, a polycaprolactone support (scaffold) according to the composition of a precipitation solution.
5 is an image showing a structure of a polycaprolactone filament, that is, a polycaprolactone support (scaffold) according to the composition of a precipitation solution.
6 is an image of a polycaprolactone support (scaffold) prepared according to the ratio of water and ethanol in a precipitation solution.
7 is a photomicrograph showing a change in the pore structure of the prepared polycaprolactone filaments according to the ratio of water and ethanol in the precipitation solution.
8 is an enlarged micrograph showing the change in the pore structure of the prepared polycaprolactone filaments according to the ratio of water and ethanol in the precipitation solution.
9 is a micrograph showing the core pore structure of the prepared polycaprolactone filaments according to the ratio of water and ethanol in the precipitation solution.
10 is a graph showing the tensile strength of the prepared polycaprolactone scaffold (support) according to the ratio of water and ethanol in the precipitation solution.

The present invention includes the step of extruding a biodegradable polymer solution into a precipitation solution using a 3D printer,

The precipitation solution relates to a method of manufacturing a porous support that is a mixed solution of water and alcohol.

Hereinafter, the porous support according to the present invention will be described in more detail.

In the present invention, a biodegradable polymer solution extruded into a precipitation solution using a 3D printer may be expressed as a filament, and a stack (structure, laminate) of the filaments may be expressed as a support or scaffold.

The method for preparing a porous support according to the present invention includes extruding a biodegradable polymer solution into a precipitation solution using a 3D printer.

In one embodiment, the manufacturing method of the present invention comprises the steps of preparing a biodegradable polymer solution;

Extruding a biodegradable polymer solution in a 3D printer capable of a 3D lamination system and laminating it in a precipitation solution;

Placing the stacked filaments, that is, the porous support, in the precipitation solution; And

It may include; removing the residual solvent and the precipitation solution from the porous support.

First, a biodegradable polymer solution is prepared by dissolving a biodegradable polymer in a solvent.

The kind of the biodegradable polymer is not particularly limited, and polycaprolactone (PCL), polylactide (PLA), polylactide glycolide random copolymer (polylactide glycolide, PLGA), polyglycolide , PGA) or mixtures thereof.

The solvent of the biodegradable polymer solution is not particularly limited as long as the biodegradable polymer can be dissolved, for example, dichloroethane (DCE), tetrahydrofuran (THF), chloroform, and acetone ( acetone), methylethylketone (MEK), and hexafluoro-2-propanol (hexafluoro-2-propano, HFP).

The concentration of the biodegradable polymer in the biodegradable polymer solution may be 12 to 25 wt%. It may have an appropriate viscosity for laminating the filament using a 3D printer in the above range. If it is less than 12 wt%, it is difficult for the extruded filament to have a uniform shape and there is a concern that it may collapse in the vertical direction.If it exceeds 25 wt%, it is difficult for the biodegradable polymer to be completely dissolved in the solvent, and the viscosity increases rapidly, resulting in a solution. There is a fear that it is difficult to extrude from the nozzle tip of the syringe.

In the present invention, in order to increase the solubility of the biodegradable polymer, a step of stirring the biodegradable polymer and a solvent at 40 to 70°C or 50 to 60°C may be further included. Stirring may be performed using a magnetic stirring method, a mechanical stirring method, or a vortex method, which is a stirring method generally used in the art. After the stirring is completed, it can be cooled to room temperature and used in the next step.

In the next step, the biodegradable polymer solution is extruded in a 3D printer capable of a three-dimensional lamination system and laminated in a precipitation solution.

In the above step, by laminating the biodegradable polymer solution in the precipitation solution in three dimensions, a porous support having various sizes of pores and pore structures in the polymer skeleton constituting the porous structure can be prepared.

At this time, the precipitation solution is a non-solvent, specifically, a mixed solution of water and alcohol. As the alcohol, ethanol, methanol, butanol, propanol, or a mixture thereof may be used. The ratio of water and alcohol in the precipitation solution may be 100:10 to 500, 100:10 to 300, or 100:10 to 100. The shell portion of the filament manufactured in the above range may have a dense structure.

In the present invention, the phase is separated by phase exchange between the solvent of the biodegradable polymer solution and the non-solvent of the precipitation solution, so that the biodegradable polymer solution is coagulated and laminated, and a pore structure is formed in the filament (see FIG. 2). The pores may have a micro size.

In one embodiment, the extrusion and lamination of the biodegradable polymer solution is performed using a 3D printer. By using the 3D printer, the user can program or design the layer in a desired structure. A biodegradable polymer solution can be extruded into the precipitation solution along the coordinates of a pre-designed 3D printer, and a support having a complex structure can be easily prepared. During lamination, the speed of the printer can be controlled according to the extrusion speed. The 3D printer is installed in connection with an extrusion system (extruder) for extruding filaments, and the filaments extruded from the extrusion system can be laminated according to a programmed design. Specifically, the extruded filaments may be laminated according to a desired direction and may be laminated several times.

Such a lamination may be laminated in three dimensions, and in one embodiment, the filaments may be laminated to have unidirectionality. The unidirectionality means that the filaments are directed in one direction based on one layer in which the filaments are stacked at regular intervals in the filament support in which the filaments are formed in multiple layers. That is, when the filaments are formed in multiple layers, one layer is formed so that the filaments have a certain distance in one direction, and the other layer in contact with the one layer has a certain distance in the direction perpendicular to the one layer. Can be formed to At this time, the interval between the filaments may be 10 to 1000 ㎛.

In order to induce proper solidification of the biodegradable polymer solution during three-dimensional lamination, the extrusion speed is preferably 1 to 5 mm/s. The extrusion rate refers to the rate of lamination of the filaments and the rate of phase exchange between the solvent and the non-solvent. For example, if the extrusion speed is less than 1mm/s, there is a risk that the upper filament collapses in the gravitational direction between the already stacked filaments and the filaments during 3D lamination, or a problem with difficult shape control may occur.If it exceeds 5 mm/s, The lamination speed is faster than the rate at which the polymer solution solidifies, and there is a concern that the collapse of the pore structure in the vertical direction may occur.

During the lamination, compressed air can be treated with 10 to 35 kPa. Since the viscosity also increases as the concentration of the biodegradable polymer solution increases, it is good to adjust the compressed air in the range as suggested for accurate lamination.

In addition, the diameter of the nozzle during extrusion may be 1,000 μm, 800 μm or less, or 600 μm or less.

The next step is to settle the stacked filaments, that is, the porous support in the precipitation solution.

Through the above step, complete phase separation between the solvent of the biodegradable polymer solution and the non-solvent of the precipitation solution is possible.

In this step, the settling time may be 1 to 30 minutes.

In the present invention, a step of removing a solvent, a precipitation solution, and impurities from the additionally prepared porous support is performed.

As a method of removing the solvent, freeze drying, vacuum drying, natural drying, etc. may be used, and an additional washing step may be included.

In the case of freeze drying, it may be carried out at a temperature of -80 to -10°C, specifically -50 to -20°C, and in the above temperature range, the freezing medium can be easily removed without damaging the laminate of the molded body.

A porous support in which the filaments are aligned in one direction is formed by the manufacturing method according to the present invention. The unidirectionality means that the filaments are directed in one direction based on one layer in which the filaments are stacked at regular intervals in the porous support in which the filaments are formed in multiple layers.

In addition, the present invention relates to a porous support manufactured by the above-described manufacturing method.

In one embodiment, the porous support has a structure in which filaments having an average diameter (diameter) of 300 to 1000 μm, 400 to 900 μm, or 500 to 800 μm are formed in one direction per layer.

The porous support has macropores having an average diameter (diameter) of 10 to 1000 µm or 100 to 900 µm or 200 to 800 µm between the filaments.

Particularly, in the porous support, the filaments constituting the support have a structure of a porous core and a dense shell. Here, the core and the shell are based on a cross section in a direction perpendicular to the direction in which the parliament is extruded, and the inside where the pores are formed is expressed as the core and the portion surrounding the inside where the pores are formed is expressed as a shell. The percentage of shell in the filament may be 1 to 50%.

This is due to the composition of the precipitation solution in the manufacturing method. When alcohol is used as the precipitation solution, filaments having micro or nano pores are formed. However, in the case of using a mixed solution of water and alcohol as in the present invention, pores are formed in the core and a filament having a dense structure in the shell portion, that is, having two structures can be produced.

In the present invention, the core has pores of 10 to 500 nm, 20 to 100 nm, or 30 to 50 nm.

That is, the porous support according to the present invention has a double pore structure of micro pores and macro pores.

In addition, the present invention relates to a product comprising the porous support.

Since the porous scaffold according to the present invention has a dual pore structure of micro pores/macro pores and excellent physical properties, it can be used not only to regenerate hard tissues such as bones or teeth of the human body, but also to regenerate various soft tissues such as cartilage, skin, ligaments, and muscles. .

In the present invention, a non-solvent separation method is used to prepare a porous support, whereby a porous support having a three-dimensionally connected pore structure while having a high porosity through exchange of a solvent and a non-solvent was prepared. The porous scaffold prepared through a non-solvent separation method may have a pore structure having a structure similar to that of a living body bone.

In addition, the present invention has a distinctive feature from the prior art in that it is possible to artificially impart a densified shell portion without a pore structure by controlling the type and content ratio of the precipitation solution. Such a densified shell can easily control the thickness of the shell by changing the composition of the precipitation solution, and the mechanical strength of the support can be controlled through the control of the shell thickness.

Advantages and features of the present invention, and a method of achieving them will become apparent with reference to embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in a variety of different forms, only the present embodiments are intended to complete the disclosure of the present invention, and the general knowledge in the technical field to which the present invention pertains. It is provided to completely inform the scope of the invention to those who have it, and the invention is only defined by the scope of the claims.

Example

Example 1.Core-shell Scaffold Produce

(One) Scaffold Preparation of solution for fabrication

After adding 10 g polycaprolactone (Sigma-Aldrich, St. Louis, MO, US) and 57.4 ml acetone in a 100 ml sealable glass bottle, mix it with a stirrer at 50° C. for 2 hours to make the polycaprolactone in acetone. A completely dissolved polycaprolactone solution was prepared. The polycaprolactone solution was cooled at room temperature for 2 hours.

(2) Through the cost sales separation method Scaffold making

In order to prepare a preset pore shape, a precipitation solution was prepared by mixing a set amount of ethanol and water.

The polycaprolactone solution prepared in (1) was filled in a 15 ml extrusion piston having a nozzle diameter of 0.5 mm.

After connecting the polycaprolactone solution contained in the extrusion piston to a 3D printer (Ez-Robo5), a pre-set (designed) scaffold (11.6 mm x 11.6 mm x) by applying a pressure of 3 mm per second and 25 kPa at a lamination rate 1.3mm) was prepared in the precipitation solution (see Fig. 1).

After the polycaprolactone scaffold was completed in the precipitation solution, the scaffold was exposed in the precipitation solution for 5 minutes for complete phase separation.

(3) Scaffold Solvent and Non-sale remove

The scaffold, completely phase-separated by the step (2), was washed with 200 ml distilled water for 5 minutes, and lyophilized for 24 hours to completely remove ethanol and distilled water remaining on the spherical support.

Experimental example 1. Changes in the composition of the filament according to the composition of the precipitation solution

In the present invention, Figure 2 is a schematic diagram showing a state when the polycaprolactone solution extruded from the piston enters the precipitation solution. When the polycaprolactone solution is exposed to the precipitation solution, the filaments are stacked according to a preset design, and the surface of the filament is solidified and has a pore structure through phase separation.

In the present invention, in order to confirm the change in the composition of the filaments according to the composition of the precipitation solution, the filaments were extruded into the precipitation solution while changing the ratio of water and ethanol constituting the precipitation solution.

3 is a schematic diagram showing a cross section of a polycaprolactone filament according to the composition of a precipitation solution.

As shown in FIG. 3, in the precipitation solution of 100% ethanol, the filaments exhibit a dense structure without shells. As the content of water increased, filaments having a core having a porous structure and a shell having a dense structure were prepared.

That is, it can be seen that the shell portion thickens as the water content in the precipitation solution increases.

4 is a schematic diagram showing a structure of polycaprolactone filaments, that is, a scaffold according to the composition of the precipitation solution, and FIG. 5 is an image of the scaffold.

As shown in FIG. 4, it can be seen that in the precipitation solution of 100% ethanol, only a pore structure without a shell is generated, and in the precipitation solution of 100% water, a structure of a porous core and a densely structured shell is formed.

In addition, as shown in the image of FIG. 5, it can be seen that polycaprolactone filaments are stacked regardless of the precipitation solution to prepare a scaffold. The scaffold prepared according to the present invention is the same as in the schematic diagram of Fig. 4, in the precipitation solution of 100% ethanol, only a pore structure without a shell is generated, and in the precipitation solution of 100% water, the core of a porous structure and the shell of a dense structure are Shows the structure.

Experimental example 2. Changes in the composition of filaments according to the ratio of water and ethanol in the precipitation solution

A scaffold was prepared by changing the ratio of water and ethanol in the precipitation solution as shown in Table 1 below. The preparation of the scaffold was prepared by the method of the examples.

The ratio of water and ethanol in the precipitation solution A 0 (water): 100 (ethanol) B 25: 75 C 50: 50 D 75: 25 E 100: 0

6 is an image of a polycaprolactone scaffold prepared according to the ratio of water and ethanol in a precipitation solution.

The size of the scaffold is 11.6mm x 11.6mm x 1.3mm, and it can be seen that the scaffold can be manufactured regardless of the composition and ratio of the precipitation solution.

In addition, FIG. 7 is a photomicrograph showing a change in the pore structure of the prepared polycaprolactone according to the ratio of water and ethanol in the precipitation solution, and FIG. 8 is a microscopic photo further enlarged. In addition, the thickness of the shell having a dense structure is shown in Table 2 below.

The ratio of water and ethanol in the precipitation solution Shell thickness (μm) A 0 (water): 100 (ethanol) 0 B 25: 75 9.7 C 50: 50 15.3 D 75: 25 24.9 E 100: 0 34.0

As shown in Figs. 7 and 8, as the proportion of water in the precipitation solution increases, the shell portion of the dense structure increases, which can be confirmed from the increase in the thickness of the shell portion as shown in Table 2.

In addition, FIG. 9 is a micrograph showing the core pore structure of the prepared polycaprolactone according to the ratio of water and ethanol in the precipitation solution.

As shown in FIG. 9, it can be seen that the pore structure of the core portion has a constant structure regardless of the composition of the precipitation solution.

Experimental example 3. According to the ratio of water and ethanol in the precipitation solution Scaffold Tensile strength measurement

The tensile strength of the scaffold used in Experimental Example 2 was measured, and is shown in FIG. 10 and Table 3 below.

The ratio of water and ethanol in the precipitation solution Tensile strength (MPa) A 0 (water): 100 (ethanol) 0.83 B 25: 75 1.04 C 50: 50 1.17 D 75: 25 1.25 E 100: 0 1.39

As shown in Fig. 10 and Table 3, it can be seen that as the content of water in the precipitation solution increases, that is, as the thickness of the densely structured shell portion increases, the tensile strength increases in proportion.

Claims (8)

Extruding the biodegradable polymer solution into a precipitation solution using a 3D printer,
The precipitation solution is a mixed solution of water and alcohol, and the ratio of water and alcohol in the precipitation solution is 100:10 to 500
A method for producing a porous support comprising a filament having a porous core and a dense shell structure.
The method of claim 1,
Biodegradable polymers include polycaprolactone (PCL), polylactide (PLA), polylactide glycolide (PLGA), polyglycolide (PGA), or a mixture thereof. Method for producing a porous support.
The method of claim 1,
The solvent of the biodegradable polymer solution is dichloroethane (DCE), tetrahydrofuran (THF), chloroform, acetone, methylethylketone (MEK), and hexafluoro-2-propanol. (hexafluoro-2-propano, HFP) a method for producing a porous support that is at least one selected from the group consisting of.
The method of claim 1,
Alcohol in the precipitation solution is ethanol (Ethanol), methanol (Methanol), butanol, propane, or a method for producing a porous support of a mixture thereof.
delete The method of claim 1,
A method for producing a porous support, further comprising the step of allowing the support extruded to the precipitation solution to stand in the precipitation solution for 1 to 30 minutes.
It is manufactured by the manufacturing method according to claim 1,
The filament constituting the porous support is a porous support having a structure of a core having a porous structure and a shell having a dense structure.
The method of claim 7,
The proportion of the shell in the filament is 1 to 50% of the porous support.

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