KR102090958B1 - Sea Anemone-derived Silk Protein-Based Bioink for 3D Bioprinting and Uses Thereof - Google Patents

Abstract

The present invention relates to an anemone-derived silk protein-based 3D bio printing bio ink and its use, and more specifically, anemone-derived silk protein or a variant thereof; And a method of mixing and crosslinking the polymer to produce a 3D bio printer bio ink composition; A bio ink composition prepared according to the above method; And it provides a support for tissue engineering comprising the bio-ink composition. According to the present invention, the bio-ink composition of the present invention is capable of extrusion-based printing based on shear reduction characteristics, and can easily manufacture various types of three-dimensional structures through photocuring. The three-dimensional structure of the present invention can maintain its shape based on excellent physical properties, provides a bio-like environment for various cells, and provides excellent biocompatibility and biodegradability during biotransplantation, and thus is damaged in the field of tissue engineering and regenerative medicine. It can be used as a new platform for tissue replacement or artificial organ production for organ transplantation.

Description

Sea Anemone-derived Silk Protein-Based Bioink for 3D Bioprinting and Uses Thereof}

The present invention relates to a bio-ink for 3D bio-printing based on silk proteins derived from sea anemones and uses thereof.

3D bio-printing is a technology that prints living cells together with bio-ink to make a 3D artificial structure or support that can be implanted in the human body. The bio-printing technology has a problem of lack of organs for transplantation through the advantage of being able to produce a desired shape and structure. It is expected to be an effective solution. In order for these bio-inks to be actually utilized, they must have sufficient properties to maintain their structure as well as be able to continuously function without dying. That is, the bio ink serves as a support to connect the tissue and the tissue in order to regenerate the lost tissue through the self-recovery function, and for this purpose, it must have excellent cell affinity for smooth tissue regeneration. In addition, the cells grow well in three dimensions and have a pore structure that is well connected in three dimensions in a certain sized area so that exchange of nutrients and feces can be achieved well. It must have mechanical strength to maintain its shape during regeneration and excellent biosafety. In particular, in regeneration of hard tissues such as bones and teeth, it is important to secure mechanical properties according to regeneration sites.

Specifically, the support for tissue regeneration must first be physically stable at the implant site, second, it must exhibit physiological activity capable of regulating regeneration efficacy, and third, after formation of new tissue, it must be degraded in vivo, and fourth, Degradation products should not be toxic.

Conventional techniques using a synthetic polymer as a support manufacturing method have a considerable difficulty in being utilized as a bio-ink because of not only lacking cell-recognition but also having a hydrophobic surface. In addition, synthetic polymers are easy to maintain morphology on the basis of sufficient physical properties, but have the disadvantage of poor biocompatibility and biodegradability after biotransplantation.

In addition, when natural polymers are used, they have excellent biocompatibility and biodegradability, but they have too low physical properties, requiring a synthetic polymer-based support during the manufacturing process, and are unable to maintain their shape when applying the completed 3D structure. Bio ink development is being delayed.

To overcome this problem, a protein-based support can be used as a support.

The mechanical properties required for the protein-based support to maintain morphology during the regeneration of a 3D space biological tissue are not only various factors such as protein concentration, crosslinking method, type of crosslinking agent, concentration of crosslinking agent, post-treatment method, but also protein It may depend on the choice of material.

For example, the mechanical properties of the structural protein silk or collagen-based hydrogel are relatively high. Nevertheless, various searches for gel materials to increase mechanical properties were relatively insufficient.

Collagen, a representative example of structural proteins, is the main component of the connective tissues that make up our body and is the basic skeleton of the extracellular matrix. Collagen can be obtained through extraction in nature, but there is a problem of high immunity from other animals and high price due to difficulty in protein extraction and separation. As an alternative, attempts have been made to produce recombinant collagen, but it is not easy to artificially simulate the unique triple helix structure and modified hydroxyproline of collagen.

Silk protein, another structural protein, can be obtained in large quantities from silkworms, but has lower physical strength than spider silk. In the case of spider silk, due to the nature of eating spiders, mass breeding is impossible, so there is a limit to obtaining large amounts naturally. The repetitive sequences of silkworms and spiders are large, making it difficult to produce recombinant proteins under the E. coli system. Therefore, many studies have attempted to express a part of a repeat sequence that affects properties, or there have been studies to improve properties through cross-linking methods after their expression.

Protein-based materials research has already been conducted in developed countries such as the United States, Australia, and Japan. In addition to the search for new proteins such as ant silk and resilin, the production of silk-elastin fusion proteins is promoting the development of existing protein materials. The discovery and application of new genetic sequences have significance in that they can not only broaden the field of application based on structural, mechanical, and physicochemical properties unknown to the basics, but also secure a source material to gain a research advantage as a leading group. have. First of all, the little information and application examples of living organisms in the ocean reveal the importance and high potential of discovering new materials derived from marine organisms.

Among these marine-derived organisms, sea anemone is a generic term for sea anemone, belonging to the coral reef of the arachnid, arachnid, and mainly lives in the sea and lives on the reef, thus producing and secreting adhesive proteins. By doing so, you can firmly attach yourself to a wet solid surface, such as a rock in the sea.

Therefore, the use of an anemone-derived protein-based structural protein can be a method of eliciting more improved mechanical properties in the production of protein-based supports that have limited physical properties, and uses anemone-derived protein-based materials. Bio-ink is a biomaterial for printing that satisfies the physical stability, bioactive factor regulation, biodegradability and biocompatibility, which are conditions of the support, and may be a solution capable of effectively healing biological tissue by applying it to bioprinting.

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KR 10-2015-0042412; Composition for 3D printing, method for manufacturing same, and method for manufacturing 3D structure using same

Mechanically Durable and Biologically Favorable Protein Hydrogel Based on Elastic Silklike Protein Derived from Sea Anemone; Biomacromolecules. 2015 Dec 14; 16 (12): 3819-26. doi: 10.1021 / acs.biomac.5b01130. Epub 2015 Nov 16.

The present inventors made great efforts to develop a novel 3D bio printing bio ink suitable for providing a biomaterial. As a result, a bio-ink was produced by processing an additional cross-linking mechanism on silk proteins and natural polymers derived from sea anemones having sequence and structural properties similar to collagen or silk. The bio ink of the present invention not only has superior strength and elasticity as compared to silk fibroin, but also exhibits excellent physical properties that can contain moisture, and can be produced in the shape of a desired design without the use of synthetic polymers, thereby improving cell suitability, cell growth and differentiation. The present invention was completed by confirming that it is possible to exhibit an effect of increasing tissue engineering healing power.

Accordingly, one object of the present invention is an anemone-derived silk protein or a variant thereof; And it is to provide a method for producing a bio-ink composition for a 3D bio printer comprising the step of crosslinking by mixing the polymer.

In addition, another object of the present invention is to provide a bio-ink composition for a 3D bio printer prepared according to the above method.

In addition, another object of the present invention is to provide a cartridge for a 3D bio printer comprising the bio ink composition for the 3D bio printer.

In addition, another object of the present invention is to provide a 3D biomaterial comprising the bioink composition for the 3D bio printer.

In addition, another object of the present invention is to provide a tissue engineering support comprising the 3D biomaterial.

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

At this time, unless otherwise defined in the technical terms and scientific terms used herein, it has a meaning that is commonly understood by those skilled in the art to which this invention belongs.

In addition, repeated description of the same technical configuration and operation as the prior art will be omitted.

According to an aspect of the present invention, the present invention is an anemone-derived silk protein or a variant thereof; And it provides a method for producing a bio-ink composition for a 3D bio printer comprising the step of crosslinking by mixing the polymer.

In the present invention, the anemone-derived silk protein is an anemone-derived silk protein, but is not limited thereto, and preferably all the anemone-derived silk proteins described in Korean Patent Application Nos. 10-2012-0002161 and 10-2016-0079563. Included, the patent document is included in the present invention as a reference as a whole.

According to a preferred embodiment of the present invention, the anemone-derived silk protein is a protein consisting of the amino acid sequence of SEQ ID NO: 1.

In addition, the anemone-derived silk protein may be a polypeptide in which the amino acid sequence of SEQ ID NO: 1 is linked one or more times, preferably a polypeptide linked between 1 and 200 times, more preferably a polypeptide linked between 1 and 120 times. In addition, it may be a polypeptide comprising other amino acid sequences between 1 to 200 times or 1 to 120 times.

In the present invention, mutants of anemone-derived silk protein preferably contain additional sequences at the carboxyl or amino terminus of the anemone-derived silk protein under the premise of maintaining the adhesion of the anemone-derived silk protein, or some amino acids have different amino acids. It may be substituted with. For example, a polypeptide composed of 3 to 25 amino acids containing RGD is linked to the carboxyl or amino terminal of the anemone-derived silk protein, or 1 to 100% of the total number of tyrosine residues constituting the anemone-derived silk protein, preferably 5 to 100% may be substituted with 3,4-dihydroxyphenyl-L-alanine (DOPA).

The present invention discloses a bio-ink developed for 3D printing based on the characteristics of the anemone-derived protein and histological application thereof. The bio-ink for three-dimensional printing of the present invention is easily printable, and decomposes at an appropriate speed and sufficient properties to maintain its shape during printing and tissue regeneration. In addition, since it enables cell growth and differentiation, biocompatibility is excellent.

That is, the bio-ink produced according to the method of the present invention is excellent in printability, so it can be produced in the shape of a desired design without a separate synthetic polymer-based support, and is an excellent bio-ink in that it is capable of cell compatibility, cell growth and differentiation. Can be utilized.

In the present invention, preferably, the polymer is hyaluronic acid (hyaluronic acid, HA), chitosan (chitosan), collagen (collagen), alginate (alginate), gelatin, albumin (albumin), carrageenan (carrageenan), It can be selected from the group consisting of matrigel (matrigel), hemoglobin (hemoglobin), heparin (heparin), fibrin-gel (fibrin-gel) and agarose (agarose), more preferably hyaluronic acid (hyaluronic acid (HA)) , Chitosan or alginate, most preferably hyaluronic acid.

The bio-ink composition for a 3D bio-printer of the present invention may further include one or more bio-active substances as long as a desired effect can be achieved. The bioactive material is a material that exhibits a certain pharmacological activity when administered to a body or applied to a skin surface, but is not limited thereto, and may be one or more selected from the group consisting of drugs, enzymes, cells, and food additives. Preferably, anti-cancer agents, antibiotics, anti-inflammatory agents, hormones, hormone antagonists, interleukins, interferons, growth factors, tumor necrosis factors, endotoxins, lymphopoxys, eurokinases, streptokinases, tissue plasminogen activators, protease inhibitors, alkylphosphocholines, radiation It may be at least one selected from the group consisting of isotopic labeling agents, surfactants, cardiovascular drugs, gastrointestinal drugs and nervous system drugs.

In addition, the present invention is not limited to this, but preferably, the anemone-derived silk protein and the polymer may be formed by mixing in a weight ratio of 1: 0.01 to 100, and more preferably formed by mixing in a weight ratio of 1: 0.01 to 10. It may be, and even more preferably 1: 0.01 to 1 may be formed by mixing in a weight ratio, most preferably to be mixed in a 1: 0.1 weight ratio.

The type of solvent, a suitable pH, and a suitable temperature for preparing a bio ink composition for a 3D bio printer of the present invention are the same as known conditions under which the bio ink can be effectively formed.

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

That is, the object of the present invention is to provide a bio-like environment in a desired form through a crosslinked gel formulation of an anemone-derived silk protein and a polymer, and to prepare a bio ink composition capable of maintaining the form until a desired purpose is achieved. will be.

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

The crosslinking agent may use one selected from the group consisting of tyramine, hydroxyphenylacetic acid, hydroxypropionic acid, dopamine, epinephrine, and hydroxyethylaniline, and provides a tyrosine residue so that the crosslinking agent can be photocrosslinked through a photoreaction. As far as possible, it is not limited to this.

The bio-ink according to the present invention can solve the cytotoxicity problem that occurs during cross-linking by using conventional cross-linking by using an additional cross-linking mechanism without using ultraviolet rays, and a 3D scaffold for implantation that can be used as a bio ink for 3D bio-printing Can be produced.

Therefore, the present invention can successfully print a three-dimensional structure successfully based on a bio-ink mixed with an anemone-derived silk-like protein and hyaluronic acid.

In addition, the bio-ink based on silk-like protein derived from anemone is capable of extruding-based printing based on shear thinning properties and can maintain the printed form well by rapidly crosslinking through tyrosine photocrosslinking.

In addition, the platform can produce a printing structure that has better tensile strength, elasticity, and compressive strength than silk fibroin, and prints by imitating biological tissues such as blood vessels, ears, and nose, as well as precise grids with a printing interval of 400 μm or less. can do.

In addition, various cells such as mesenchymal stem cells, muscle cells, osteoblasts, and fibroblasts can adhere to and survive on the structure produced through the platform, and can induce differentiation into bone and cartilage forming cells.

In addition, it can be seen that the structure produced through this platform is similar to silk fibroin in biocompatibility and has better biodegradability.

The bio-ink composition according to the method of the present invention can control the physical properties of the crosslinked gel by adjusting the molecular weight of the polymer, and through this, it is possible to create an environment suitable for the target organ and target cells.

It is preferable that the polymer and the chemical functional group are mixed at a molar ratio of 1: 400 to 600, and if the range is less than 1: 400, there is a problem that the mechanical strength is lowered, and when it exceeds 1: 600, the mechanical strength is excessively high and excessive Movement of various materials may be limited by the degree of crosslinking, which is not preferable, but is not limited thereto. In addition, the polymer concentration is preferably 1 to 30 wt%, but if it is outside the above range, it is not preferable, but is not limited to, because of the possibility of being used as a bio ink due to high viscosity.

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

In addition, the present bio ink composition may further include at least one selected from cells, anti-adhesion materials, dyes, and drugs.

In addition, according to another aspect of the present invention, the present invention provides a cartridge for a 3D bio printer comprising the bio ink composition for a 3D bio printer described above.

In addition, according to another aspect of the present invention, the present invention provides a 3D biomaterial comprising the bioink composition for a 3D bio printer described above.

In addition, according to 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 comprising the 3D biomaterial, or an anti-adhesion agent. The drug may be used generally used antibiotics, anticancer agents, anti-inflammatory drugs, antiviral agents, antibacterial agents, proteins and peptides, but is not limited thereto.

The novel bio-ink composition produced using the silk-like protein and hyaluronic acid derived from the sea anemone of the present invention is capable of extrusion-based printing based on shear reduction characteristics, and facilitates various types of 3D structures through photocuring. Can be produced. The three-dimensional structure of the present invention can maintain its shape based on excellent physical properties, provides a bio-like environment for various cells, and provides excellent biocompatibility and biodegradability during biotransplantation, and thus is damaged in the field of tissue engineering and regenerative medicine. It can be used as a new platform for tissue replacement or artificial organ production for organ transplantation.

1 is a view showing the rheological properties of a bio-ink formed by mixing 20 wt% of anemone-derived silk protein and 2 wt% of hyaluronic acid. The hyaluronic acid of the polymer was mixed to change the rheological properties from Newtonian fluids to fluids with shear reduction properties. In addition, it can be seen that even when repeated shearing force was applied, the initial viscosity was restored to about 85%.
2 is a view showing the physical properties of the three-dimensional structure produced by the present platform. It can be seen that the tensile strength is about 2.5 times better and the elastic force is about 4 times better than the control silk fibroin.
3 is a view showing the compressive strength of the three-dimensional structure produced by the present platform. It can be seen that the compressive strength is superior to that of the control silk fibroin.
4 can print ears, grids, blood vessels, noses, and the like based on a computer design using the platform. As a result of checking the lattice through a scanning electron microscope, it can be confirmed that it is a three-dimensional structure having porosity.
FIG. 5 is a diagram showing the attachment and time of various cells on the 3D structure for 3 days. Based on the Cell Counting Kit-8 solution, the absorbance at 450 nm was examined.
6 is a diagram confirming through a Live-dead cell assay that various cells are alive on the 3D grid surface. Living cells are stained green and dead cells are stained red. In addition, F-actin was stained with green fluorescence to examine the interaction between cells and matrix. As a result, it was confirmed that all cells interact well with the substrate. It can be seen that the nucleus is dyed blue.
7 is a diagram showing the comparison of bone differentiation in a 2D plane and a 3D structure. Calcium deposition was examined by Alizarin Red S staining. It can be seen that the portion where the orange dye was densely deposited is distributed in a three-dimensional grid as calcium is deposited.
8 is a view showing a comparison of cartilage differentiation in a 2D plane and a 3D structure. The aggrecan formed in connection with cartilage differentiation can be confirmed by alcian blue staining. The darker the blue, the more aggrecan is formed. In addition, the cartilage differentiation-related factor, sex determining region Y-related high-mobility group box 9 (Sox9), was fluorescently stained to confirm that cartilage was well differentiated in the 3D structure.
9 is a view showing the biocompatibility, biodegradability, and immune response by placing the platform under the mouse. Silk fibroin was used as a control. Compared to the control group, this platform has a much faster biodegradability, and it can be confirmed that the immune response is hardly shown, and through this, it was confirmed that the biocompatibility is excellent.

Hereinafter, the examples are only intended to describe the present invention in more detail, and according to the gist of the present invention, the scope of the present invention is not limited by these examples. Those skilled in the art to which the present invention pertains It will be obvious to you.

Example 1. Fabrication of anemone-derived silk protein-based biomaterial ink and three-dimensional structure

1-1. Preparation of anemone-derived recombinant silk protein

The anemone-derived silk protein (SEQ ID NO: 1) used in the present invention is expressed as a recombinant protein using E. coli. In order to induce expression, IPTG (isopropyl-β-D-thiogalactopyranoside, 1 mM), which is an inducer, was added when the absorbance (OD600) of the culture medium was about 0.6 to 1.0 at 600 nm. The cultured cells are centrifuged at 4000 rpm for 15 minutes to recover the cells, and then suspended in lysis solution (lysis buffer, pH 8.0, 50 mM NaH2PO4, 300 mM NaCl), followed by a sonicator or a high-pressure homogenizer. ). The remaining whole cell lysate was centrifuged at 9000 rpm for 10 minutes to recover the supernatant. Here, a thermal shock of about 60 ° C was again applied. In this process, an insoluble fraction was taken again, dissolved in formic acid, dialyzed with water and freeze-dried. The production amount of anemone-derived collagen-like recombinant protein obtained by acid extraction is around 50 mg per liter.

1-2. Fabrication of anemone-derived silk protein-based biomaterial ink

The present inventors prepared a biocompatible bio ink by mixing hyaluronic acid, a polymer material, with the silk protein derived from anemone produced in Example 1-1.

Specifically, a bio ink was prepared as follows: 20 wt% of the silk protein derived from the anemone produced above and 2 wt% of hyaluronic acid of 1000 kDa were completely dissolved in formic acid.

In addition, the rheological properties of the bio-ink were confirmed.

As a result, as shown in Fig. 1, by mixing the hyaluronic acid of the polymer, the rheological properties were changed from Newtonian fluids to fluids having shear reduction properties, and despite the repeated shear strain, about 85% of the initial It can be seen that the viscosity is restored.

1-3. Fabrication of silk protein-based three-dimensional structures derived from sea anemones

The present inventors produced a three-dimensional structure using the biocompatible bio ink produced in Example 1-2.

Specifically, a structure was prepared as follows: A desired structure can be easily produced based on information about a structure to be printed using a computer-aided design. The bio-ink produced above may be injected into a syringe connected with nozzles of various diameters for printing. It was printed at a speed of 1000 mm / min with a pneumatic pressure of about 150 kPa. A blue light source with a wavelength of 450 nm was irradiated to fix the structure at the same time as printing.

Example 2. Characterization of silk protein-based three-dimensional structure derived from sea anemone

The present inventors tried to confirm the properties of the anemone-derived silk protein-based three-dimensional structure produced in Example 1.

First, the physical properties of structures fabricated with this platform were measured.

As a result, as shown in FIG. 2, the structure of the present invention has a tensile strength (left panel in FIG. 2) of about 4 times, and an elastic force (FIG. 2) compared to a structure made of a bio ink in which the structure of the present invention is a control silk fibroin and hyaluronic acid. The right panel) confirmed that about 6 times better.

In addition, the compressive strength of the three-dimensional structure produced by this platform was measured.

As a result, as shown in Figure 3, it was confirmed that the compressive strength of the structure of the present invention is superior to the control group silk fibroin.

In addition, based on computer design using this platform, the ear (FIG. 4A), lattice (FIG. 4B), and nose (FIG. 4D) 3D printed and observed through a scanning electron microscope grid (FIG. 4C), It has been confirmed that it is a three-dimensional structure having porosity, and this platform suggests that a support body that provides an environment similar to a human body as well as a structure that repeats a certain pattern form can be produced.

Example 3. Confirmation of cell adhesion and cell viability of anemone-derived silk protein-based three-dimensional structure

The present inventors tried to confirm cell adhesion and cell viability of the anemone-derived silk protein-based three-dimensional construct prepared in Example 1 above.

First, various cells on a 3D structure (fibroblasts (ATCC® CRL-1658?), Mesenchymal stem cells (purchased through Sciencell), myoblasts (AC30005, purchased through the Korea Research Institute of Bioscience and Biotechnology Microbial Resource Center) , After attaching osteoblasts ((ATCC® CRL-2593?)) And observing for 3 days over time, absorbance at 450 nm was measured using a Cell Counting Kit-8 solution.

As a result, as shown in FIG. 5, it was confirmed that the cells survived by attaching well to the 3D structure, and proliferated over time to maintain cell activity.

In addition, the survival rate of various cells on a 3D structure was measured through a live-dead cell assay. At this time, living cells are dyed green, dead cells are dyed red, and nuclei are dyed blue. In addition, F-actin was stained with green fluorescence to examine the interaction between cells and matrix.

As a result, as shown in FIG. 6, it was confirmed that all cell lines survived by remarkably interacting with the construct of the present invention as a substrate.

On the other hand, the 3D structure using the silk fibroin protein as a control group was not easy to maintain the structure due to the uneven molecular weight of the silk protein and the slow formation of covalent bonds during light crosslinking. For this reason, it was not possible to perform the experiment after forming a three-dimensional structure with silk fibroin.

Example 4. Confirmation of bone differentiation on an anemone-derived silk protein-based three-dimensional structure

The present inventors tried to confirm bone differentiation on an anemone-derived silk protein-based three-dimensional structure prepared in Example 1.

First, bone differentiation was compared in a 2D plane and a 3D structure. Bone differentiation is incubated with osteoblasts until 72% confluency is achieved. In addition, the culture medium in which the final concentration of 50 μg / mL ascorbic acid and 10 mM monobasic soduium phosphate was dissolved was replaced once every 3 days to induce differentiation for 3 weeks. Alizarin Red S staining, which can discriminate bone differentiation, was washed with PBS and immobilized using a 4% formalin solution. And it was dyed at room temperature for 30 minutes using 1% alizarin-red S solution with ammonium hydroxidefh pH 4.2. And after washing three times with distilled water, the degree of calcium deposition was confirmed.

As a result, as shown in FIG. 7, when calcium deposition was confirmed through Alizarin Red S staining, the portion strongly stained with orange was calcium deposited, and the grid on the three-dimensional structure of the present invention than in the 2D plane You can see that it is distributed a lot in This induces bone differentiation using a lattice on a three-dimensional structure, and thus, because it mimics an actual three-dimensional tissue, it is possible to induce more interactions between cells, resulting in efficient bone formation.

In addition, cartilage differentiation was compared in 2D plane and 3D structure. The mesenchymal stem cells were used as the cartilage, and the cartilage differentiation medium (Cat. No. 7551) of Sciencell was used, and the differentiation inducer TGF-Beta3 was added at a final concentration of 10 ng / ml. First, mesenchymal stem cells are harvested at a concentration of 10 ⁶ cells / ml, and centrifuged at 500 g for 5 minutes to make cell pellets. Be careful not to damage the cells and replace the cartilage differentiation medium once every 3 days, which induced differentiation for 3 weeks.

As a result, as shown in FIG. 8, when aggrecan formed in connection with cartilage differentiation was confirmed through alcian blue staining, the darker the blue, the more aggrecan was formed, and confirmed that the aggrecan was strongly stained in the three-dimensional structure of the present invention. Did. In addition, when fluorescence staining was performed on Sox9 (Sex determining region Y-related high-mobility group box 9), a factor related to cartilage differentiation, it was strongly stained in the 3D structure of the present invention, which was of the present invention than in the 2D plane. It can be seen that cartilage is significantly differentiated in the three-dimensional structure.

On the other hand, the 3D structure using the silk fibroin protein as a control group was not easy to maintain the structure due to the uneven molecular weight of the silk protein and the slow formation of covalent bonds during light crosslinking. For this reason, it was not possible to perform the experiment after forming a three-dimensional structure with silk fibroin.

Example 5. Confirmation of biocompatibility of anemone-derived silk protein-based three-dimensional structure

The present inventors tried to confirm the biodegradability and immune response after placing the anemone-derived silk protein-based three-dimensional structure platform prepared in Example 1 under the skin of mice. Cylindrical hydrogels prepared on the basis of the same amount of protein were placed subcutaneously in rats, and changes were observed over 2 and 4 weeks.

As a result, as shown in FIG. 9, when compared with the control group silk fibroin, it was confirmed that the platform has much faster biodegradability and almost no immune response, and thus biocompatibility was excellent.

Thus, the anemone-derived silk protein-based three-dimensional structure of the present invention can provide a safe and biocompatible three-dimensional environment, which can be usefully used as a composite support for tissue engineering.

<110> POSTECH ACADEMY-INDUSTRY FOUNDATION <120> Sea Anemone-derived Silk Protein-Based Bioink for 3D Bioprinting          and Uses Thereof <130> POSTECH1.56p <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 327 <212> PRT <213> sea anemone <400> 1 Met Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr   1 5 10 15 Gly His Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Asp              20 25 30 Pro Gly Asn Thr Gly Tyr Pro Gly Gln Asp Pro Gly Asn Thr Gly Tyr          35 40 45 Pro Gly Gln Asp Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly      50 55 60 Asn Thr Gly Cys Pro Gly Gln Gly Pro Gly Asn Thr Gly Cys Pro Gly  65 70 75 80 Gln Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr                  85 90 95 Gly Tyr Pro Gly Gln Gly Pro Ser Asn Thr Gly Tyr Pro Trp Gln Gly             100 105 110 Pro Gly Asn Thr Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro         115 120 125 Gly Asn Thr Gly His Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr Pro     130 135 140 Gly Gln Asp Pro Gly Asn Thr Gly Tyr Pro Gly Gln Asp Pro Gly Asn 145 150 155 160 Thr Gly Cys Pro Gly Gln Gly Pro Gly Asn Thr Gly Cys Pro Gly Gln                 165 170 175 Gly Ser Gly Asn Thr Gly Cys Pro Gly Gln Gly Ser Gly Asn Thr Gly             180 185 190 Cys Pro Gly Gln Gly Pro Gly Gln Gly Pro Gly Asn Thr Gly Tyr Pro         195 200 205 Gly Gln Gly Pro Gly Asn Thr Gly His Pro Gly Gln Gly Pro Gly Asn     210 215 220 Thr Gly Tyr Pro Gly Gln Asp Pro Gly Asn Thr Gly Tyr Pro Gly Gln 225 230 235 240 Asp Pro Gly Asn Thr Gly Cys Pro Gly Gln Gly Pro Gly Asn Thr Gly                 245 250 255 Cys Pro Gly Gln Gly Ser Gly Asn Thr Gly Cys Pro Gly Gln Gly Ser             260 265 270 Gly Asn Thr Gly Cys Pro Gly Gln Gly Pro Gly Gln Gly Pro Gly Asn         275 280 285 Thr Gly Tyr Pro Gly Gln Gly Pro Ser Asn Thr Gly Tyr Pro Gly Gln     290 295 300 Gly Pro Gly Asn Thr Gly Tyr Pro Gly Gln Gly Pro Gly Asn Thr Leu 305 310 315 320 Glu His His His His His His                 325

Claims (10)

An anemone-derived silk protein consisting of the amino acid sequence of SEQ ID NO: 1 or a variant thereof; And mixing hyaluronic acid (HA) and photo-crosslinking to form a hydrogel.
According to claim 1,
The 3D bio printer bio ink composition is characterized in that to increase the adhesion, viability and differentiation ability of the target transplanted cells.
delete delete delete According to claim 1,
Method, characterized in that the anemone-derived silk protein or a variant thereof and hyaluronic acid are mixed in a weight ratio of 1: 0.01 to 100.
A bio ink composition for a 3D bio printer prepared according to the method of claim 1, 2, or 6.
A cartridge for a 3D bio printer comprising the bio ink composition for a 3D bio printer of claim 7.
A 3D biomaterial comprising the bioink composition for a 3D bioprinter of claim 7.
A histological support comprising the 3D biomaterial of claim 9.

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