KR102005737B1 - Novel Bioink for 3D Bioprinting and Uses Thereof - Google Patents

Abstract

The present invention relates to a novel bio-ink for 3D bio-printing and its use, and a method for producing a bio-ink composition comprising mixing a natural polymer with a mussel adhesive protein or a variant thereof and performing a cross-linking reaction; A bio ink composition prepared according to the above method; And a tissue engineering support comprising the bio-ink composition.

Description

Novel bio-ink for 3D bio-printing and its use [0002]

The present invention relates to novel bio-inks for 3D bio-printing and their use.

 3D bioprinting is a technology to produce 3D artificial structures or scaffolds that can be implanted in the human body by printing living cells with bio-inks. The bio-printing technology can produce desired shapes and structures, Is expected to be an effective solution. In order for these bio-inks to be practically used, it is essential that they not only have sufficient physical properties to maintain the structure, but also that living cells can function continuously without dying. In other words, bio-ink acts as a support to connect tissues and tissues in order to regenerate tissues lost through self-repairing function, and cell-affinity should be excellent so that tissue regeneration can be smoothly performed. In addition, the cells have three-dimensionally well-connected pore structures in a uniform size region so that the cells can grow well in three dimensions and exchange nutrients and excrements, and biodegradability It must have mechanical strength to maintain its shape during regeneration, and should be biocompatible. Especially, in the hard tissue regeneration such as bone and teeth, it is important to secure the mechanical properties according to the regeneration site.

Specifically, the support for tissue regeneration must first be physically stable at the site of transplantation, secondly, exhibit a physiological activity that can regulate regeneration efficacy, third, decompose in vivo after forming a new tissue, Decomposition products should not be toxic.

Techniques using a synthetic polymer, which is a conventional method for producing a support, are not only lacking in cell-recognition but also have a hydrophobic surface, which makes it difficult to utilize it as a bio-ink. In addition, when natural polymers are used, development of bio-ink is delayed due to the disadvantage that it is impossible to fabricate a three-dimensional scaffold with too low physical properties.

On the other hand, the coacervate is a kind of colloidal material formed when the anionic polyelectrolyte and the cationic polyelectrolyte are mixed under specific conditions. When the coacervate is formed, the absorbance of the solution increases, and the external solution It exists separately. During formation of the coacervate, the participating electrolyte separates from the solution and condenses and remains liquid. At this time, the surface tension decreases and the viscosity changes. Coacervate can also occur by mixing a protein with a polymer electrolyte of opposite nature (C.G. deKruif et al., 2004, Current Opinion in Colloid and Interface Science 9, 340-349). Techniques for immobilizing functional materials such as drugs, enzymes, cells, and food additives in microcapsules due to the low surface tension of coacervate have also been reported. (Schmitt C. et al., 1998, Critical Review in Food Science and Nutrition 8, 689-753).

The mussel, a marine creature, produces and secretes adhesive proteins that can be firmly attached to wet solid surfaces such as rocks in the oceans, and are not affected by wave shocks or seawater buoyancy effects. (JH Waite et al., 1983, Biological Review 58, 209-231; HJ Cha et al., 2008, Biotechnology Journal 3, 631-638). These mussel adhesive proteins are known as strong natural adhesives when compared to the currently known chemical synthetic adhesives, and they are flexible enough to bend while exhibiting high tensile strength of about twice as much as epoxy resin. In addition, mussel adhesive proteins have the ability to adhere to a variety of surfaces such as plastics, glass, metals, teflon and biomaterials, and can be attached to wet surfaces within minutes. These properties have not yet been an issue in the field of chemical adhesives. In addition, it is known that adhesive proteins do not attack human cells or cause immune reactions, and are thus likely to be applied to medical fields such as adhesion of biopsies or adhesion of broken teeth during surgery (J. Dove et al., 1986, Journal of American Dental Association 112, 879). Particularly, the mussel adhesive protein can also be used in the field of surface adhesion of cells. The surface adhesion technique of cells is one of very important technologies required for cell culture and tissue engineering. For example, for cell and tissue culture, (M. Tirrell et al., 2002, Surf. Sci., 500, 61-83), because it is a technique of efficiently sticking to the surface of a cell.

Therefore, bio-ink using cationic protein, in particular, coacervate based on mussel adhesive protein, is a biotic material for printing satisfying physical stability, regulation of physiologically active factor, biodegradability and biocompatibility, Which can be a solution to the healing of living tissue.

Application No. 10-2010-0075717

Accordingly, the present inventors have made an effort to develop a new bio-ink for bio-printing suitable for providing a bio-material. As a result, the inventors of the present invention produced a coacervate-based bio-ink by treating the cationic protein mussel adhesive protein (MAP) and the natural polymer with an additional crosslinking mechanism, and when using the same, exhibited excellent physical properties without using a synthetic polymer, The present invention has been completed.

Accordingly, an object of the present invention is to provide a method for producing a bio ink composition for a 3D bio-printer, which comprises cross-linking a coacervate formed by mixing a natural polymer with a mussel adhesive protein or a mutant thereof.

Another object of the present invention is to provide a bio ink composition for a 3D bio-printer produced by the above method.

It is still another object of the present invention to provide a cartridge for a 3D bio printer including the bio ink composition for the 3D printer.

It is still another object of the present invention to provide a 3D biomaterial including the bio ink composition for the 3D printer.

Still another object of the present invention is to provide a tissue engineering support comprising the 3D bio-material.

Hereinafter, the present invention will be described in more detail.

Here, unless otherwise defined in the technical terms and scientific terms used herein, unless otherwise defined, those skilled in the art will understand what is commonly understood by those skilled in the art.

Repeated descriptions of the same technical constitution and operation as those of the conventional art will be omitted.

According to one aspect of the present invention, there is provided a method for producing a bio ink composition for a 3D bio-printer, which comprises cross-linking a coacervate formed by mixing a natural polymer with a mussel adhesive protein or a mutant thereof.

In the present invention, the mussel adhesive protein is an adhesive protein derived from mussel, including, but not limited to, all the mussel adhesive proteins described in WO2006 / 107183 or WO2005 / 092920.

According to a preferred embodiment of the present invention, the mussel adhesive protein or a variant thereof comprises a protein consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; Or a fusion protein in which at least one amino acid sequence selected from the above group is linked, more preferably a protein consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, Most preferably a protein consisting of the amino acid sequence of SEQ ID NO: 1.

In the present invention, the mutants of the mussel adhesive protein preferably include additional sequences at the carboxyl terminal or amino terminal of the mussel adhesive protein under the condition that the adhesive property of the mussel adhesive protein is maintained, or some amino acids are substituted with other amino acids Lt; / RTI > More preferably, a polypeptide consisting of 3 to 25 amino acids including RGD is linked to the carboxyl terminal or amino terminal of the mussel adhesive protein, or a polypeptide comprising 1 to 100% of the total number of tyrosine residues constituting the mussel adhesive protein, And 5 to 100% thereof may be substituted with 3,4-dihydroxyphenyl-L-alanine (DOPA).

RGD (Arg Gly Asp Ser), RGDC (Arg Gly Asp Cys), RGDV (Arg Gly Asp Val), RGDSPASSKP (Arg Gly Asp Ser Pro), GRGDS (Gly Arg Gly Asp Ser), GRGDTP (Gly Arg Gly Asp Ser Pro), GRGDSP (Gly Arg Gly Asp Ser Pro), GRGDSPC Cys) and YRGDS (Tyr Arg Gly Asp Ser).

A mutant of a mussel adhesive protein to which a polypeptide consisting of 3 to 25 amino acids including RGD is linked at the carboxyl terminal or amino terminal of the mussel adhesive protein is not limited thereto but preferably a polypeptide consisting of the amino acid sequence of SEQ ID NO: Lt; / RTI >

The mussel adhesive protein of the present invention is not limited thereto, but it may be inserted into a conventional vector which is designed for expressing an external gene so that it can be mass-produced by a genetic engineering method. The vector may be suitably selected according to the type and characteristics of the host cell for producing the protein, or may be newly produced. A method of transforming the vector into a host cell and a method of producing a recombinant protein from the transformant can be easily carried out by a conventional method. Methods for selecting, producing, transforming, and expressing recombinant proteins described above can be easily carried out by those skilled in the art, and some modifications of the conventional methods are included in the present invention.

In the present invention, the natural polymer may be any natural polymer material capable of forming a coacervate by binding to the cationic mussel adhesive protein. Preferably, the natural polymer is a polymer having a pI (isoelectric point) lower than that of the cationic mussel adhesive protein, More preferably a polymer having a pI value of 2 to 6, and still more preferably a polymer having a pI value of 2 to 4. If the pI value is more than or less than the above-mentioned pI value, it is difficult to form coacervate, so that it is preferable to use an anionic polymer within the pI range.

In the present invention, preferably, the natural polymer is selected from the group consisting of hyaluronic acid (HA), chitosan, alginate, gelatin, albumin, carrageenan, hemoglobin, Heparin, and more preferably hyaluronic acid (HA), chitosan or alginate, and most preferably hyaluronic acid.

The average molecular weight of the anionic polymer may have a molecular weight selected from the group consisting of, but not limited to, 1 kDa to 300 kDa, more preferably 10 kDa to 100 kD, more preferably 17 kDa to 59 kDa, and most preferably 17 kDa, A molecular weight of 35 kDa or 59 kD. If the molecular weight is more than or less than the molecular weight, coacervate may not be formed.

The bio-ink composition for 3D bio-printers of the present invention may further include one or more bioactive substances as long as the desired effects can be achieved. The bioactive substance may be one or more selected from the group consisting of drugs, enzymes, cells, and food additives, although it is not limited thereto, and it may be a substance exhibiting a certain pharmacological activity when administered to a living body or applied to the skin surface. Preferably, the anticancer agent is selected from the group consisting of anticancer agents, antibiotics, anti-inflammatory agents, hormones, hormone antagonists, interleukins, interferon, growth factors, tumor necrosis factor, endotoxin, lymphotoxin, urokinase, streptokinase, tissue plasminogen activator, An isotope labeling substance, a surfactant, a cardiovascular drug, a gastrointestinal drug, and a nervous system drug.

Preferably, the mussel adhesive protein and the natural polymer may be mixed in a weight ratio of 1: 0.01 to 100, most preferably 7: 3.

The kind of the solvent, the optimum pH, and the optimum temperature for producing the bio ink composition for the 3D bio-printer of the present invention are the same as known conditions in which the coacervate can be effectively formed.

In the crosslinking, a tyrosine residue contained in the natural polymer may be photocrosslinked through a photoreaction by adding a crosslinking agent.

That is, an object of the present invention is to prepare a bio ink composition capable of capturing cells through a cross-linked gel formulation of a mussel adhesive protein and a natural polymer and maintaining its biological activity.

For example, the bio-ink composition according to the present invention can be manufactured with a hyaluronic acid hydrogel system in which tyramine is conjugated to hyaluronic acid, thereby enhancing physical properties through an additional crosslinking mechanism.

The crosslinking agent may be one selected from the group consisting of tyramine, hydroxyphenylacetic acid, hydroxypropionic acid, dopamine, epinephrine, and hydroxyethylaniline. The crosslinking agent may provide a tyrosine residue so that the crosslinking agent can photo- As long as it is possible.

The bio-ink according to the present invention can solve the problem of cytotoxicity that occurs during crosslinking because it does not use ultraviolet rays but uses the additional crosslinking mechanism to perform ordinary cross-linking. The bio-ink according to the present invention can be used as a bio-ink for 3D bio- .

The bio ink composition according to the method of the present invention can regulate the physical properties of the crosslinked gel through controlling the molecular weight of the natural polymer, thereby making it possible to create an environment suitable for the target organ or target cell.

It is preferable that the biopolymer and the chemical functional group are mixed at a molar ratio of 1: 400 to 600. If the ratio is less than 1: 400, the mechanical strength is lowered. If the ratio is more than 1: 600, the mechanical strength is excessively increased The migration of various substances may be restricted by excessive crosslinking, which is not preferable, but is not limited thereto. The concentration of the polymer is preferably 1 to 30 wt%. If the concentration is out of the above range, the possibility of not being used as a bio-ink due to a high viscosity is not preferable, but is not limited thereto.

In addition, the bio-ink composition may further include, but is not limited to, a solvent of acetic acid, distilled water, or a buffer solution. Specifically, the buffer solution is one selected from 2- (n-morpholino) ethanesulfonic acid, 4- (4,6-Dimethoxy-1,3,5-thiazin-2-yl) -4-methylmorpholinium chloride and Phosphate buffer saline Or more.

In addition, the bio-ink composition may further include at least one selected from the group consisting of a cell, a substance for preventing adhesion, a dye and a drug.

According to another aspect of the present invention, there is provided a cartridge for a 3D bio-printer including the above-described bio ink composition for a 3D printer.

According to still another aspect of the present invention, there is provided a 3D biomaterial including the above-described bio ink composition for a 3D printer.

According to still another aspect of the present invention, there is provided a tissue engineering support comprising the 3D biomaterial.

In addition, according to the present invention, it is possible to provide a drug delivery system containing the 3D bio-material, or an adhesion inhibitor. The drug may be used in general, but is not limited to, antibiotics, anticancer agents, anti-inflammatory agents, antiviral agents, antibacterial agents, proteins and peptides.

The bio-ink composition of the present invention provides a tissue engineering support as a novel platform for the production of artificial organs for organ transplantation using coacervate and an additional cross-linking mechanism, and has excellent biocompatibility using biopolymers without synthetic polymer, A bio-ink capable of retaining physical properties and a three-dimensional scaffold artificial structure using the same. Therefore, it is expected that it will be possible to regenerate the tissue by applying it to a biological tissue having a body defect.

Figure 1 shows the yield of binding confirmation of hyaluronic acid and tyramine according to various molecular weights. The highest absorbance at 275 nm for tyramine, and its concentration and absorbance are proportional to each other. The absorbance standard curve according to the concentration of tyramine was prepared, and then the absorbance of the diluted hyaluronic acid was measured and the yield was calculated.
FIG. 2 shows turbidity measured to determine coacervate production according to the mixing conditions of HA and MAP. The experiment was carried out in PBS (phosphate-buffered saline, pH 7.8). The optimal concentration ratio of MAP and HA was 7: 3 (wt: wt).
FIGS. 3A to 3C show the results before and after crosslinking of Alg-Tyr and MAP mixture (FIG. 3A), gelatin and MAP mixture (FIG. 3B), HA-Tyr and MAP (FIG.
Fig. 4 is a graph showing the viscosity measured according to the angular frequency before photocuring.
FIG. 5 is a graph showing changes in storage modulus and loss modulus (measured by a rheometer) of G 'and G''after cross-linking of a hydrogel prepared through an additional crosslinking mechanism of coacervate with mussel adhesive protein and hyaluronic acid ).
Figure 6 compares the compressive modulus of the hydrogel-mussel adhesive protein-based coacervate of the present invention with the GelMA series known in the art.
FIG. 7 shows a three-dimensional structure produced using the hydrogel-mussel adhesive protein-based coacervate of the present invention.
Fig. 8 shows the cytotoxicity results of the three-dimensional structure using the hydrogel-mussel adhesive protein-based coacervate.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined by the appended claims. It will be obvious to you.

Example

Example 1. Fabrication of a natural polymer substance with tyrosine group conjugated

In order to increase the physical properties of a bioabsorbable 3D bioinf ink, the present inventors have made a natural polymer material conjugated with a tyrosine group in a polymer substance.

First, in order to impart photo-crosslinking properties to chitosan, alginate and hyaluronic acid present in nature in the blue light, a tyrosine or tyrosine acetic acid is added to these tyrosine reactors, (Hydrophenylacetic acid) to produce Chi-Tyr, Alg-Tyr and HA-Tyr.

Specifically, in the case of HA-Tyr, HA was dissolved in DW at a concentration of 0.2 wt%. After the addition of 49.84 mM EDC and NHS, 74.76 mM of tyramine was added, and the pH was adjusted to 5.5, followed by overnight reaction. In the case of Alg-Tyr, Alginate was dissolved in DW at a concentration of 1.0 wt%. To this solution, 5 mM EDC, NHS and tyramine were added, and the pH was adjusted to 5.5, followed by overnight reaction. In case of Chi-Tyr, 1.0 wt% was dissolved in 1.0 wt% of Acetic acid solution. After the addition of 5 mM EDC, NHS and Hydrophenylacetic acid, the pH was adjusted to 5.5 and reacted overnight. Then, all of the above three substances were purified through dialysis for at least 2 days and then used in the following examples through lyophilization.

In addition, the binding confirmation yields of hyaluronic acid and tyramine according to various molecular weights (111, 500 and 1010 kDa) were confirmed. Tilamine, which has the highest absorbance at 275 nm, utilizes that its concentration and absorbance are proportional to each other.

As a result, as shown in Fig. 1, an absorbance standard curve corresponding to the concentration of tyramine was prepared, and then the absorbance of the diluted hyaluronic acid was measured to calculate the yield.

Example 2. Preparation of Recombinant Mussel Adhesion Protein (MAP)

2-1. Production of Recombinant Mussel Adhesion Protein fp-151

The mussel adhesive protein fp-151 (SEQ ID NO: 1) used in the present invention is a mussel adhesive protein fp-1 (SEQ ID NO: 1) which is used in the present invention, so that a decapeptide composed of 10 amino acids repeated 80 times in the natural mussel adhesive protein fp- (Genbank No. AAS00463 or AY521220) was inserted between two fp-1 mutants and then produced in Escherichia coli (DS Hwang et al., &Quot; Biomaterials 28, 3560-3568, 2007).

Specifically, the fp-1 mutant (hereinafter referred to as 6xAKPSYPPTYK) in which the peptide consisting of AKPSYPPTYK was repeated six times in the amino acid sequence of fp-1 (Genbank No. Q27409 or S23760) 6xAKPSYPPTYK was combined with 6xAKPSYPPTYK at the C-terminus of Mgfp-5 to prepare fp-151 of SEQ ID NO: 1. The specific preparation of the mussel adhesive protein is the same as described in WO2006 / 107183 or WO2005 / 092920, which is incorporated herein by reference in its entirety.

2-2. Production of Recombinant Mussel Adhesion Protein fp-151-RGD

Fg-151-RGD of SEQ ID NO: 2 was prepared by adding the GRGDSP sequence selected from the fibronectin RGD group to the C-terminus of fp-151 of Example 1-1.

2-3. Production of Recombinant Mussel Adhesion Protein fp-131

The mussel adhesive protein fp-131 was obtained by replacing the gene (Genbank No. BAB16314 or AB049579) of the mussel adhesive protein Mgfp-3A naturally present between two fp-1 mutants in the same manner as fp-151 of Example 1-1 And then produced by Escherichia coli.

Specifically, an fp-1 mutant (hereinafter referred to as 6xAKPSYPPTYK) in which the peptide consisting of AKPSYPPTYK is repeated six times in the amino acid sequence of fp-1 (Genbank No. Q27409 or S23760) 6xAKPSYPPTYK was combined with 6xAKPSYPPTYK at the C-terminus of Mgfp-3 to prepare fp-131 of SEQ ID NO: 3.

Example 3. Preparation of coacervate based on hydrogel-mussel adhesive protein and confirmation of the possibility of photocrosslinking

The present inventors have found that the recombinant mussel adhesive protein (MAP) fp-151 is mixed with the tyramine conjugated hydrogel (Chi-Tyr, Alg-Tyr, HA-Tyr) prepared in Example 1, The mussel adhesive protein-based coacervate was formed and treated with a light-treating agent to confirm the solidification state of the hydrogel-mussel adhesive protein-based coacervate.

Coacervate is a kind of colloid produced by mixing anionic electrolytic polymer and cationic electrolytic polymer at specific ratios at specific pH conditions. Since the absorbance of the solution increases when the coacervate is formed, the absorbance is mainly measured to confirm the formation of the coacervate (V. Ducel et al., Colloids and Surfaces a-Physicochemical and Engineering Aspects, 232, 239-247 , 2004).

First, a mixed solution of recombinant mussel adhesive protein fp-151 and Chi-Tyr, Alg-Tyr and HA-Tyr was prepared and absorbance was measured.

As a control, a mixed solution of gelatin and a recombinant mussel adhesive protein fp-151 was used.

As a result, it was confirmed that coacervate was formed when HA-Tyr and MAP were mixed.

Turbidity was also measured to determine the amount of coacervate produced according to the mixing conditions of HA and MAP. The experiment was carried out in PBS (phosphate-buffered saline, pH 7.8).

As a result, as shown in FIG. 2, it was confirmed that the highest amount of coacervate was formed when the optimum concentration ratio of mussel adhesive protein and HA-Tyr was 7: 3 wt%.

Then, sodium persulfate and Tris (bipyridine) ruthenium (II) chloride were added to give a concentration of 1 mM to add photo-crosslinking function. Thereafter, blue light of 452 nm was irradiated to confirm the hydrogel formation.

FIGS. 3A to 3C show cross-linking test results for Alg-Tyr and MAP mixture 3a, Gelatin and MAP mixture 3b, HA-Tyr and MAP 3c coacervate samples.

As a result, it was confirmed that the shape was not changed and solidified within a short time.

Example 4. Physical properties of the hydrogel-mussel adhesive protein-based coacervate formulation

Among the formulations prepared in Example 1, HA-Tyr was used to confirm the physical properties of the coacervate imparted with photo-crosslinking properties.

First, the viscosity according to the angular frequency before the photo-curing was measured.

As a result, as shown in FIG. 4, it was confirmed that the initial viscosity of the material itself is considerably high, but the shear-thinning property of decreasing viscosity with shear stress is applied.

In addition, storage modulus and loss modulus were measured. G 'and G "after cross-linking of the hydrogel prepared through the additional cross-linking mechanism of the coacervate of mussel adhesive protein and hyaluronic acid were measured via a rheometer. The crosslinking reaction was crosslinked using SPS (sodium persulfate) under a photoredox catalyst, rubpy (Tris (bipyridine) ruthenium (II) chloride).

As a result, as shown in FIG. 5, it was confirmed that the loss elastic modulus before the photo-crosslinking exists in a liquid state higher than that of the photo-crosslinking, and the gel has a higher storage elastic modulus after photo-crosslinking.

Compressive modulus of the hydrogel-mussel adhesive protein-based coacervate of the present invention and the GelMA series known in the art were also compared.

As a result, as shown in FIG. 6, the physical properties of the hydrogel-mussel adhesive protein-based coacervate according to the present invention are four times higher than those of the simple GelMA materials (15% GelMA to 30 kPa) Which is higher than that of the conventional method.

Example 5. Production of a three-dimensional structure by biomaterial ink using a hydrogel-mussel adhesive protein-based coacervate and a 3D printer

The capability of constructing a three-dimensional structure using the hydrogel-mussel adhesive protein-based coacervate of the present invention was confirmed. I used the INKREDIBLE + device sold by CELLINK. The solution coming out through the nozzle of the device was bridged using blue light. When one layer was completely accumulated, the same output was repeatedly produced.

As a result, as shown in FIG. 7, it was confirmed that a structure having a repeated pattern pattern can be fabricated.

Example 6. Cytotoxicity of a 3-dimensional structure using the hydrogel-mussel adhesive protein-based coacervate

To examine how the survival rate of HUVEC cells changed after 48 hours from the treatment of exudates, the cytotoxicity of the constructs prepared in Example 5 was examined.

As a result, as shown in Fig. 8, the survival rate of the cells was 98.9%, indicating no cytotoxicity.

Therefore, the hydrogel-mussel adhesive protein-based coacervate of the present invention can provide a safe and biocompatible three-dimensional structure, which can be usefully used as a composite scaffold for tissue engineering.

<110> POSTECH ACADEMY-INDUSTRY FOUNDATION <120> Novel Bioink for 3D Bioprinting and Uses Thereof <130> POSTECH1-31p-1 <150> 10-2016-0125014 <151> 2016-09-28 <160> 3 <170> KoPatentin 3.0 <210> 1 <211> 196 <212> PRT <213> Artificial Sequence <220> <223> FP-151 <400> 1 Met Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr   1 5 10 15 Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala              20 25 30 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro          35 40 45 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ser Ser Glu      50 55 60 Gly Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His Tyr His Ser  65 70 75 80 Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys                  85 90 95 Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser Gly             100 105 110 Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr         115 120 125 Lys Lys Tyr Tyr Gly Gly Ser Ser Ala Lys Pro Ser Tyr Pro Pro Thr     130 135 140 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 145 150 155 160 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys                 165 170 175 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro             180 185 190 Pro Thr Tyr Lys         195 <210> 2 <211> 202 <212> PRT <213> Artificial Sequence <220> <223> FP-151-RGD <400> 2 Met Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr   1 5 10 15 Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala              20 25 30 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro          35 40 45 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ser Ser Glu      50 55 60 Gly Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His Tyr His Ser  65 70 75 80 Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys                  85 90 95 Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser Gly             100 105 110 Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr         115 120 125 Lys Lys Tyr Tyr Gly Gly Ser Ser Ala Lys Pro Ser Tyr Pro Pro Thr     130 135 140 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 145 150 155 160 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys                 165 170 175 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro             180 185 190 Pro Thr Tyr Lys Gly Arg Gly Asp Ser Pro         195 200 <210> 3 <211> 172 <212> PRT <213> Artificial Sequence <220> <223> FP-131 <400> 3 Met Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr   1 5 10 15 Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala              20 25 30 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro          35 40 45 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Pro Trp Ala      50 55 60 Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly Gly  65 70 75 80 Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp Asn                  85 90 95 Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Gly Ser Ala             100 105 110 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro         115 120 125 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro     130 135 140 Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr 145 150 155 160 Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Leu                 165 170

Claims (10)

A method for preparing a bio-ink composition for a 3D bio-printer, comprising the steps of:
(a) preparing a hyaluronic acid hydrogel in which hyaluronic acid (HA) is conjugated with tyramine;
(b) mixing a mussel adhesive protein or a mutant thereof with the hyaluronic acid hydrogel in step (a) to form a coacervate,
Wherein said mussel adhesive protein or its variants comprises a protein consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; Or a fusion protein in which at least one amino acid sequence selected from the group is linked; And
(c) photo-crosslinking the coacervate of step (b).
delete delete delete delete The method according to claim 1, wherein in step (b), the mussel adhesive protein or a variant thereof and hyaluronic acid hydrogel are mixed at a weight ratio of 1: 0.01 to 100.
6. A bio-ink composition for a 3D bio-printer produced according to the method of claim 1 or 6.
A cartridge for a 3D bio-printer comprising the bio-ink composition for a 3D printer according to claim 7.
A 3D bio-material comprising the bio-ink composition for a 3D printer of claim 7.
A tissue engineering support comprising the 3D biomaterial of claim 9.

Images