KR102293725B1 - Method for preparing bioink comprising collagen for 3d printing and method for 3d printing using bioink prepared the same - Google Patents

Abstract

The present invention comprises the steps of (a) mixing a collagen acid solution to prepare an acid collagen solution; (b) concentrating the collagen acid solution to prepare a concentrated collagen acid solution; And (c) by adding the acidic solution to the concentrated collagen acid solution, based on the acidic solution of 0.01 N concentration, the collagen acid solution containing collagen at a concentration of 1.5 to 3.0% (w / v) 3D printing bio-ink preparing a; It relates to a method of manufacturing a bio-ink for 3D printing, comprising: and a 3D printing method using the bio-ink for 3D printing prepared accordingly. The 3D pattern prepared by this can be usefully used in regenerative medicine, and since it has strong mechanical properties by itself, there is an advantage in that there is no need for an auxiliary agent for strengthening physical properties, chemical crosslinking treatment, a support, and the like.

Description

A method for manufacturing a bio-ink for 3D printing containing collagen and a 3D printing method using the bio-ink prepared accordingly

The present invention relates to a method for manufacturing a bio-ink for 3D printing containing collagen and a 3D printing method using the bio-ink prepared accordingly, and more particularly, to a 3D printing method containing collagen that can be used without chemical crosslinking or support. It relates to a method of manufacturing bio-ink and a 3D printing method using the bio-ink prepared accordingly.

The domestic and overseas 3D printing market is growing rapidly, and the global market is growing rapidly every year. The medical field also occupies a significant portion of the industrial application of the 3D printing market, and the proportion of 3D printing in the medical field is expected to gradually increase in the future as patient-tailored technology is possible.

3D bioprinting technology is divided into inkjet, extrusion, and a method using a laser, and the ejection method is frequently used. In ejection-type bioprinting, by controlling the x, y, and z-axis movement of the micro nozzle, the bio-ink is discharged in the form of continuous filaments at the correct position, and the shape of the three-dimensional structure is completed by sequential stacking (layer-by-layer). Bio-ink used for 3D bioprinting should not only be non-cytotoxic, but also provide an environment suitable for cell growth and differentiation and should have printability.

Collagen is a protein that occupies the largest portion of the extracellular matrix, and controls the surrounding environment of cells and the functions of cells, which are essential in living tissues. Therefore, it is essential to secure 3D printing technology using collagen, but collagen bioinks used for 3D printing known to date each have their own limitations. For example, if a chemical cross-linking agent is added to increase physical properties to cross-link collagen, which is a natural material, it is difficult to say that the in vivo environment is imitated. In addition, in previous studies, 3D printing using collagen had a problem in that collagen 3D printing was possible only in the presence of a supporting material or a supporting bath.

Korean Patent Publication No. 10-2010560

The collagen bio-ink of the present invention is used as an auxiliary for strengthening physical properties by simultaneously printing a synthetic polymer that serves as a support to maintain its shape like the conventional bio-ink with weak physical properties, or a natural material using chemical cross-linking or UV light cross-linking. To provide a collagen bio-ink that can 3D print a desired pattern with only the collagen bio-ink without the hassle of inducing chemical denaturation of phosphorus collagen and using wet 3D printing in a supporting bath or coagulation bath. have.

According to one aspect of the present invention,

(a) mixing the collagen acid solution to prepare a collagen acid solution;

(b) concentrating the collagen acid solution to prepare a concentrated collagen acid solution; and

(c) By adding an acidic solution to the concentrated collagen acidic solution, based on an acidic solution having a concentration of 0.02 N, a bio-ink for 3D printing that is a collagen acid solution containing collagen at a concentration of 1.5 to 3.0% (w/v) manufacturing; There is provided a method of manufacturing a bio-ink for 3D printing comprising a.

The collagen may be extracted from one or more selected from animal skin, cartilage, organs, blood vessel walls, muscles, and fish scales.

The acidic solution may be any one selected from acetic acid aqueous solution, phosphoric acid aqueous solution, dilute hydrochloric acid aqueous solution, boric acid aqueous solution, and formic acid aqueous solution.

The acidic solution may have a concentration of 0.001 to 0.05 N.

In step (b), the concentration may be performed by permeation membrane concentration.

The permeable membrane concentration may be performed by a 2 to 200 kDa limiting filter.

The permeable membrane concentration may be performed by centrifugation at 4000 to 8000 rpm.

The centrifugation may be performed for 10 to 30 minutes.

After step (c),

(d) including a physiologically active factor in the bio-ink for 3D printing; may be additionally performed.

Most preferably,

In steps (a) and (c), the acidic solution uses an aqueous acetic acid solution having a concentration of 0.001 to 0.05 N, and in step (b), the concentration is a permeation membrane concentration using a 2800 to 3200 Da limiting filter, and the permeation Membrane concentration can be performed by centrifugation at 4800 to 5200 rpm for 18 to 22 minutes.

The physiologically active factor may be any one selected from nerve regeneration factor (Nerve growth factor, NGF), vascular endothelial growth factor (VEGF), and skin regeneration factor (Epidermal growth factor, EGF).

According to another aspect of the present invention,

There is provided a bio-ink for 3D printing manufactured according to the manufacturing method of the bio-ink for 3D printing.

According to another aspect of the present invention,

(1) forming a 3D collagen pattern by discharging the bio-ink for 3D printing by driving a jetting nozzle using a 3D printer;

(2) exposing the 3D collagen pattern to a basic solution vapor to prepare a neutralized 3D collagen pattern; and

(3) gelation of the neutralized 3D collagen pattern to prepare a gelled 3D collagen pattern; a 3D printing method comprising the is provided.

After step (3),

(4) washing the gelled 3D collagen pattern with PBS buffer, and carrying it; may be additionally performed.

The discharging in step (1) may be performed in a speed range of 5 to 10 mm/s, and a piston extrusion amount of 10 to 500 kPa.

The basic solution of step (2) may be any one selected from an aqueous ammonia solution, an aqueous methylamine solution, an aqueous magnesium hydroxide solution, and an aqueous aluminum hydroxide solution.

The exposure of the basic solution vapor in step (2) may be performed for 2 to 20 minutes.

The gelation in step (c) may be performed at 30 to 40 ℃.

In step (c), the gelation may be performed for 2 to 10 minutes.

Most preferably,

In step (1), the discharge is performed in a speed range of 5 to 10 mm/s, and in a range of 200 to 300 kPa piston extrusion, and in step (2), the basic solution is an aqueous ammonia solution, and 8 to 12 in ammonia vapor min, and the gelation in step (3) may be performed at 34 to 37° C. for 3 to 7 minutes.

According to another aspect of the present invention,

According to the 3D printing method, a 3D printed 3D pattern is provided.

The 3D printing pattern 3D-printed using the collagen bio-ink of the present invention is made of collagen, which is an in vivo material, so it has excellent biocompatibility and can be usefully used in regenerative medicine by containing various physiologically active factors therein. As it has strong mechanical properties, it has the advantage that it does not require an auxiliary for strengthening physical properties, chemical crosslinking treatment, and a support.

1 is a flowchart sequentially illustrating a method of manufacturing a bio-ink for 3D printing of the present invention.
2 is a flowchart sequentially showing the 3D printing method of the present invention.
3 is a schematic diagram showing the process of Examples 1 to 3;
4 is a photograph taken of the state of 3D printing according to Example 3.
5 shows the appearance of 3D printed collagen according to Example 3.

Hereinafter, various aspects and various embodiments of the present invention will be described in more detail.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those of ordinary skill in the art to which the present invention pertains can easily carry out the present invention.

However, the following description is not intended to limit the present invention to specific embodiments, and when it is determined that a detailed description of a related known technology may obscure the gist of the present invention in describing the present invention, the detailed description thereof will be omitted. .

The terminology used herein is only used to describe specific embodiments, and is not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly dictates otherwise. In the present application, terms such as "comprise" or "have" are intended to designate that a feature, number, step, operation, element, or combination thereof described in the specification exists, but is one or more other features or It should be understood that the existence or addition of numbers, steps, acts, elements, or combinations thereof, is not precluded in advance.

1 is a flowchart sequentially illustrating a method of manufacturing a bio-ink for 3D printing of the present invention. Hereinafter, a method of manufacturing the bio-ink for 3D printing of the present invention will be described with reference to FIG. 1 .

First, the collagen acid solution is mixed to prepare the collagen acid solution (step a).

The collagen is preferably extracted from at least one selected from animal skin, cartilage, organs, blood vessel walls, muscles, and fish scales.

Collagen is a type of protein in the form of long and thin fibers and plays a role in connecting cells to cells in the body. Therefore, collagen is always present in a site where a large number of multi-cells are gathered. Accordingly, collagen is not present in unicellular animals, but is present in large amounts in skin, bones, cartilage, and blood vessel walls of multicellular animals. In addition, collagen is an important component that accounts for a whopping 1/3 of the proteins that make up our body, and nutrients absorbed from the small intestine are delivered to each cell through collagen as a medium. Wastes can also be transported to blood vessels through collagen and discharged out of the body, and the transparent lens of the eye is also composed of collagen, and a part of the dentin of teeth, gums and periodontal fascia also contains collagen.

As the acidic solution, an aqueous acetic acid solution, an aqueous phosphoric acid solution, a dilute aqueous hydrochloric acid solution, an aqueous boric acid solution, an aqueous formic acid solution, etc. may be used, and preferably an acetic acid aqueous solution harmless to the human body may be used.

The acidic solution may have a concentration of 0.001 to 0.05 N, more preferably 0.005 to 0.03 N, and even more preferably 0.01 to 0.02 N concentration. If the concentration is lower than 0.001 N, a part of the acidified collagen may be neutralized and gelation may occur, and if the concentration is higher than 0.05 N, the structure of the collagen itself may be modified by acid, which is not preferable.

Next, the collagen acid solution is concentrated to prepare a concentrated collagen acid solution (step b).

The concentration is preferably carried out by a permeation membrane concentration, and such a permeation membrane concentration is preferably carried out by a limiting filter of 2 to 200 kDa, more preferably 2.5 to 100 kDa, even more preferably 3 to This can be done by a limiting filter of 10 kDa. When filtering with a size smaller than 2 kDa, the tube may not be able to withstand the speed because the rpm of the centrifuge must be increased to condense collagen. may not be preferable.

Meanwhile, the permeable membrane concentration is preferably performed by centrifugation at 4000 to 8000 rpm, more preferably 4500 to 6000 rpm, even more preferably 4800 to 5200 rpm. In the case of centrifugation at a rotation speed lower than 4000 rpm, it is difficult to sufficiently concentrate, and in the case of centrifugation at a rotation speed higher than 8000 rpm, a problem may occur that the tube cannot withstand centrifugal force.

The centrifugation is preferably performed for 10 to 30 minutes, more preferably 15 to 25 minutes, even more preferably 18 to 22 minutes.

Then, by adding an acidic solution to the concentrated collagen acidic solution, 3D collagen acidic solution containing collagen at a concentration of 1.5 to 3.0% (w/v) based on an acidic solution having a concentration of 0.02 N for printing Prepare the bio-ink (step c).

The acidic solution may be the same as the acidic solution used in step (a).

The collagen concentration of the finally prepared bio-ink for 3D printing is preferably 1.5 to 3.0% (w/v), more preferably 1.8 to 2.7% (w/v), based on an acidic solution having a concentration of 0.02 N, Even more preferably, it may be 2.0 to 2.5% (w/v). If the final collagen concentration is lower than 1.5% (w/v), mechanical properties may be weak and it may be difficult to maintain the shape, and if it is higher than 3.0% (w/v), ejection from the printer may not be smooth and it may be difficult to form a detailed pattern.

If necessary, the 3D for printing in bio ink bioactive factors to include can (step d).

The physiologically active factor may be nerve regeneration factor (Nerve growth factor, NGF), vascular endothelial growth factor (VEGF), skin regeneration factor (Epidermal growth factor, EGF), and the like.

The physiologically active factors may control the behavior of cells, and may be included in one or a plurality of pieces.

In particular, although not explicitly described in the following examples, in the method for manufacturing the bio-ink for 3D printing according to the present invention, in steps (a) and (c), the type and normal concentration of the acidic solution, step (b) In, bio-ink for 3D printing was prepared while varying the concentration method, the filtering conditions of the filter in the permeable membrane concentration, the rotation speed and time conditions of centrifugation.

As a result of checking the quality of the bio-ink for 3D printing produced in this way, unlike other conditions, only when all of the following conditions are satisfied, the ejection is smooth and the pattern resolution is high when printing the bio-ink for 3D printing. The mechanical properties are also very excellent, and the addition of the physiologically active factor in step (d) can be easily and uniformly distributed, and the bio-ink prepared according to the 3D printing method of the present invention, which will be described later, produces a 3D pattern. When it was manufactured and applied to the human body, the release rate of the physiologically active factor was the most suitable, and when applied to the human body, the cell behavior effect according to the physiologically active factor activity was very high. The conditions for remarkably increasing this effect are as follows.

In steps (a) and (c), the acidic solution uses an aqueous acetic acid solution having a concentration of 0.01 to 0.02 N, and in step (b), the concentration is a permeation membrane concentration by a 2800 to 3200 Da limiting filter, and the permeation Membrane concentration is performed by centrifugation at 4800 to 5200 rpm for 18 to 22 minutes.

The present invention provides a bio-ink for 3D printing manufactured according to the manufacturing method of the bio-ink for 3D printing.

2 is a flowchart sequentially showing the 3D printing method of the present invention. The 3D printing method of the present invention will be described with reference to FIG. 2 .

First, the 3D for printing Bio-ink using a 3D printer to drive a spray nozzle by discharging Form a 3D collagen pattern (step 1).

The discharging is preferably performed in a speed range of 5 to 10 mm/s and a piston pressure of 10 to 500 kPa, more preferably 100 to 400 kPa, even more preferably 200 to 300 kPa piston pressure. have.

If it does not reach the lower limit of the speed range, productivity is lowered, and when the upper limit of the speed range is exceeded, the formed pattern may be distorted. In addition, when the upper limit of the pressure range is exceeded, the collagen cannot be smoothly discharged in a linear form and may be discharged in an unbalanced shape in the form of a lump. It may not be able to be discharged smoothly from the

Next, a neutralized 3D collagen pattern is prepared by exposing the 3D collagen pattern to a basic solution vapor (step 2).

The basic solution is preferably any one selected from an aqueous ammonia solution, an aqueous methylamine solution, an aqueous magnesium hydroxide solution, and an aqueous aluminum hydroxide solution, and more preferably, an aqueous ammonia solution may be used.

It is preferable to use a basic aqueous solution having a concentration of 25 to 30% (v/v).

The exposure of the basic solution vapor is preferably made for 2 to 20 minutes, more preferably for 5 to 15 minutes, even more preferably for 8 to 12 minutes. A time exposure shorter than 2 minutes may not sufficiently neutralize the 3D collagen pattern, and exposure for a time longer than 20 minutes may result in complete neutralization, which is not only an unnecessary process, but also may lead to deformation of collagen molecules.

Next, the neutralized 3D collagen pattern gelation ( gelation ) let gelled Prepare a 3D collagen pattern (step 3).

It is preferably carried out at 30 to 40° C. for 2 to 10 minutes, and more preferably, it may be carried out at 34 to 37° C. for 3 to 7 minutes.

Finally, the above gelled 3D Collagen Pattern PBS washed with buffer, carry (Step 4).

In particular, although not explicitly described in the following examples, in the 3D printing method according to the present invention, the discharge rate and pressure in step (1), the type and vapor exposure time of the basic solution in step (2), step (3) ), 3D printing was performed while varying the gelation temperature and time conditions.

As a result of checking the quality of the 3D collagen pattern formed in this way, unlike other conditions, only when all of the following conditions are satisfied, the mechanical properties of the 3D collagen pattern and the accuracy of the pattern, i.e., resolution, are formed with very high resolution and can be applied to the human body. It can maintain its shape stably within the city organization. In addition, there were almost no residual impurities after washing with PBS buffer, so there were no adverse effects on the human body due to impurity deposition. The manufacturing conditions exhibiting such an effect are as follows.

In step (1), the discharging is performed in a speed range of 5 to 10 mm/s, and a piston pressure range of 200 to 300 kPa, and in step (2), the basic solution is an aqueous ammonia solution, and ammonia vapor for 8 to 12 minutes and the gelation in step (3) is carried out at 34 to 37° C. for 3 to 7 minutes.

Hereinafter, examples according to the present invention will be described in detail.

[ Example ]

Example 1: Preparation of bio-ink

An acidic solution of collagen at a concentration of 8 mg/ml (Corning® Collagen I, High Concentration, Rat Tail, dissolved in 0.02 N acetic acid) was placed in a permeable membrane concentrator (Amicon) with a 3000 Dalton (Da) limiting filter membrane. After centrifugation at 5000 rpm for 20 minutes, a concentrated collagen solution was taken. The concentration of the concentrated collagen solution was adjusted with 0.01 N acetic acid solution to complete the bio-ink, which is a collagen solution with a concentration of 22 mg/ml.

Example 2: Preparation of bio-ink containing bioactive factors

A bio-ink was completed in the same manner as in Example 1, except that Neural Growth Factor (NGF) was included in the bio-ink of Example 1 at a concentration of 0.5 μg/ml. The nerve regeneration factor was dissolved in 0.02 N acetic acid, mixed with an acidic collagen solution, and concentrated using Amicon.

Example 3: 3D printing

The bio-ink prepared in Example 1 was ejected at a speed of 7 mm/s and a pressure of 250 kPa while driving the injection nozzle in the x, y, and z axes using a 3D printer. A 3D pattern made of collagen gel was realized through repeated lamination. In order to neutralize the printed collagen pattern, it was exposed to ammonia vapor for 10 minutes in a closed system containing a 25% (v/v) aqueous ammonia solution. After 10 minutes, the pattern was removed from the ammonia chamber and gelation was induced in an oven at 37° C. for 5 minutes. Finally, it was thoroughly washed using PBS buffer solution, and immersed in PBS buffer solution for 2 hours. This is to prevent the harmful ammonia vapor remaining in the pattern, as it may adversely affect the cells.

3 is a schematic diagram showing the process of Examples 1 to 3, FIG. 4 is a photograph taken of the 3D printing according to Example 3, and FIG. 5 is a 3D printed collagen according to Example 3. .

In the above, although embodiments of the present invention have been described, those of ordinary skill in the art can add, change, delete or add components within the scope that does not depart from the spirit of the present invention described in the claims. The present invention may be variously modified and changed by, etc., and this will also be included within the scope of the present invention.

Claims (21)

(a) mixing collagen and an acidic solution to prepare an acidic solution of collagen;
(b) concentrating the collagen acid solution to prepare a concentrated collagen acid solution; and
(c) By adding an acidic solution to the concentrated collagen acidic solution, based on an acidic solution having a concentration of 0.02 N, a bio-ink for 3D printing, which is a collagen acid solution containing collagen at a concentration of 1.5 to 3.0% (w/v) manufacturing; and
(d) including a physiologically active factor that controls the behavior of cells in the bio-ink for 3D printing;
The acidic solution has a concentration of 0.001 to 0.05 N,
The concentration is carried out by permeable membrane concentration so that collagen is concentrated in the permeable membrane,
The physiologically active factor is any one selected from nerve regeneration factor (Nerve growth factor, NGF), vascular endothelial growth factor (VEGF), and skin regeneration factor (Epidermal growth factor, EGF),
The bio-ink for 3D printing is a method of manufacturing a bio-ink for 3D printing, characterized in that it is for releasing the physiologically active factor. According to claim 1,
The collagen is a method of manufacturing a bio-ink for 3D printing, characterized in that it is extracted from at least one selected from animal skin, cartilage, organs, blood vessel walls, muscles, and fish scales. According to claim 1,
The acidic solution is an aqueous solution of acetic acid, an aqueous phosphoric acid solution, a dilute aqueous hydrochloric acid solution, an aqueous boric acid solution, and an aqueous formic acid solution. delete delete According to claim 1,
The permeable membrane concentration is a method for producing a bio-ink for 3D printing, characterized in that performed by a 2 to 200 kDa limiting filter. According to claim 1,
The permeable membrane concentration is a method for producing a bio-ink for 3D printing, characterized in that performed by centrifugation at 4000 to 8000 rpm. 8. The method of claim 7,
The centrifugation is a method for producing a bio-ink for 3D printing, characterized in that it is performed for 10 to 30 minutes. delete According to claim 1,
In steps (a) and (c), the acidic solution uses an aqueous acetic acid solution,
In step (b), the concentration is a permeation membrane concentration by a 2800 to 3200 Da limiting filter, and the permeation membrane concentration is performed by centrifugation at 4800 to 5200 rpm for 18 to 22 minutes for 3D printing bio A method for producing ink. delete Claims 1 to 3, claims 6 to 8, and claim 10, bio-ink for 3D printing manufactured according to any one selected from. (1) forming a 3D collagen pattern by discharging the bio-ink for 3D printing of claim 12 by driving a jetting nozzle using a 3D printer;
(2) exposing the 3D collagen pattern to a basic solution vapor to prepare a neutralized 3D collagen pattern; and
(3) gelation of the neutralized 3D collagen pattern to prepare a gelled 3D collagen pattern; 3D printing method comprising a. 14. The method of claim 13,
After step (3),
(4) Washing the gelled 3D collagen pattern with a PBS buffer solution, and supporting the 3D printing method, characterized in that performing additionally. 14. The method of claim 13,
3D printing method, characterized in that the ejection of step (1) is performed in a speed range of 5 to 10 mm/s, and a piston pressure range of 10 to 500 kPa. 14. The method of claim 13,
The basic solution of step (2) is a 3D printing method, characterized in that any one selected from ammonia aqueous solution, methylamine aqueous solution, magnesium hydroxide aqueous solution, and aluminum hydroxide aqueous solution. 14. The method of claim 13,
3D printing method, characterized in that the exposure of the basic solution vapor in step (2) is performed for 2 to 20 minutes. 14. The method of claim 13,
3D printing method, characterized in that the gelation in step (3) is carried out at 30 to 40 ℃. 19. The method of claim 18,
3D printing method, characterized in that the gelation in step (3) is performed for 2 to 10 minutes. 14. The method of claim 13,
The discharging in step (1) is performed in a speed range of 5 to 10 mm/s, and in a range of 200 to 300 kPa piston extrusion amount,
In step (2), the basic solution is an aqueous ammonia solution, exposed to ammonia vapor for 8 to 12 minutes,
3D printing method, characterized in that the gelation in step (3) is carried out at 34 to 37 ℃ for 3 to 7 minutes. A 3D pattern 3D printed according to the 3D printing method of claim 13 .

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