WO2018186611A2

WO2018186611A2 - Bioink and preparation method therefor

Info

Publication number: WO2018186611A2
Application number: PCT/KR2018/003353
Authority: WO
Inventors: 박찬흠; 김순희; 이정민; 연응규; 이영진; 서예빈
Original assignee: 한림대학교 산학협력단
Priority date: 2017-04-04
Filing date: 2018-03-22
Publication date: 2018-10-11
Also published as: WO2018186611A3; KR101983741B1; KR20180112436A

Abstract

The present invention relates to a bioink which is prepared from silk fibroin, a natural protein fiber, and from which biological tissues having improved cytocompatibility and outstanding mechanical properties can be prepared by means of a 3D printer, and a preparation method therefor. By 3D printing, the present invention can prepare a biological tissue having mechanical properties such as moisture absorbency, volume expansion rate, compressive strength, tensile strength, etc. In addition, the preparation of a bioink including silk fibroin as a base skeleton allows for the construction of a biostructure which causes almost no immune response in the body and thus is superb in biocompatibility.

Description

【Specification】

[Name of invention]

Bio ink and preparation method thereof

Technical Field

The present invention uses silk fibroin, one of the natural protein fibers.

As a bio ink, the present invention relates to a bio ink capable of producing a biological tissue having improved cell compatibility and excellent physical properties using a 3D printer, and a method of manufacturing the same.

Background Art

3D printing technology is to realize a three-dimensional shape, that is, a three-dimensional shape, based on a three-dimensional drawing, depending on the ink material used for printing, a curing method, and the driving principle of a 3D printer. Among them, they can be classified according to the classification of the American ASTM (Amer i can Society for Testing and Materials) F2792-12a.

First, the photopolymerization method, SLACstereo li thography (DCR) method and DLP (digital processing process) method, which uses a photocurable polymer that is cured in response to light as a base material, creates a three-dimensional shape by irradiating and curing light. ; A fused deposi- tion ion modeling (FDM) method of a material extrusion method, which is a method of continuously pushing a heated material at high pressure through a nozzle to produce a three-dimensional shape; Adhesive spraying method for producing a three-dimensional shape by combining the base material by discharging the adhesive of the liquid form on the base material of the powder form; A material injection method for discharging the material in solution form by jet ting and curing with ultraviolet light; High-energy direct irradiation method for melting three-dimensional sibling phase by melting raw materials with high energy sources such as laser and electron beam; Powder-laminated melting method for producing a three-dimensional shape by combining the high-energy beam and irradiating it on the powder-based base material; A sheet lamination method of laminating a thin film-like material with heat or an adhesive and laminating it in a three-dimensional shape; Etc.

In particular, as 3D printing technology gradually develops, it is possible to manufacture more precise and detailed 3D shapes, and it is applied to the medical or bio fields, and fine and large tissue structures that almost mimic medical device parts or actual human tissues are intact. It is used to manufacture human body model, skin tissue and body organ regeneration.

In the early stages, artificial supports for tissue engineering were manufactured by applying thermoplastic biocompatible polymers to FDM technology, which melted solid filaments and laminated them further to 3D printing, but recently, in addition to tissue engineering supports, surgical simulations, surgical implants, and personalized implants were manufactured. , a research and development is going to be applied in a variety of artificial blood vessels, artificial organs, such as medical biotechnology ^i.

In particular, the support for tissue engineering is very important for the selection of constituent materials and structural control technology. In other words, the supporter plays a role as a bridge connecting the tissue to regenerate the tissue lost through the self-recovery function, it should be excellent in cell affinity so that tissue regeneration is smoothly performed. Also, cells grow in three dimensions In order to be able to exchange nutrients and excreta well, it is desirable to have a pore structure that is three-dimensionally connected in a certain size region, while the biodegradation and biodegradation that is decomposed to the tissue regeneration rate during It must have mechanical strength to maintain its shape, and it must have excellent biostability. In particular, in regenerating hard tissues such as bones and teeth, it is important to secure mechanical properties according to regeneration sites.

Specifically, the support for tissue engineering must first be physically stable at the implant site, secondly, exhibit physiological activity that can regulate regenerative efficacy, and thirdly, form new tissue and then degrade in vivo. It must not have this toxicity.

Therefore, bioinks, which are materials for producing biostructures such as surgical simulations and surgical implants, personalized implants, artificial blood vessels, and artificial organs, have shown many limitations.

The characteristics of bio-inks required for 3D bioprinting include, firstly, excellent biocompatibility, and when applied to a 3D printer printed through a nozzle, a fine diameter dispensing nozzle (di spensing nozz le) is smoothly applied. It must have physical properties that can be passed through and printed in the desired pattern. In addition, it should be possible to maintain a mechanical support role while providing cell-specific signals after 3D printing. Recently, natural or synthetic hydrogel bio inks have been developed and used in the field of 3D bio printing (Republic of Korea Patent Publication No. 10-2017-0001444: 2017.01.04.) There are significant limitations in physical and biological aspects such as suitability, printing suitability, geometrical precision and precision.

[Detailed Description of the Invention]

[Technical problem]

The present invention provides a bio-ink that can be applied to a 3D printer and a method of manufacturing the same, by producing a bio-ink based on silk fibroin, which is a natural protein polymer as a main skeleton, to maintain excellent mechanical strength and cellular suitability of silk fibroin. I would like to.

Technical Solution

One embodiment of the present invention for achieving the above object, relates to a bio ink that can be used for 3D printing, preferably silk fibroin (Si lk Fibroin) and methacrylate (Methacryl ate) compound Polymerized high polymers; And a photoinitiator.

It is preferable to use the polymer formed by copolymerizing at least one methacrylate compound with the amino acid residue of silk fibroin.

The photoinitiator, lithium thienyl phenyl-2,4,6-trimethylbenzoylphosphinate (li thium phenyl- (2,4,6-trimethylbenzoyl) phosphinate, LAP), benzyl dimethyl ketal, acetophenone Acetophenone ， Benzoin methyl ether ether), diethoxyacetophenone, benzoyl phosphine oxide and benzoyl phosphine oxide, and may include one or more selected from the group consisting of 1-hydroxycyclohexyl phenyl ketone.

On the other hand, another embodiment of the present invention, a dissolution step of dissolving silk fibroin (Si lk Fibroin) in a solvent; Adding a methacrylate compound to a solution in which silk fibroin is dissolved, followed by stirring to prepare a polymer polymer; A drying step of lyophilizing and powdering the solution containing the polymer prepared by the polymerization step; And a mixing step of mixing a powder containing a high molecular polymer with water and a photoinitiator in water.

After the polymerization step, the solution containing the prepared polymer polymer in the dialysis tube, the dialysis step of removing impurities by immersing in water; The dialysis step, the dialysis step, the solution containing the prepared polymer polymer It is desirable to remove the impurities by immersing in 12 14 kDa cutof f dialysis tube, immersed in water for 3-5 days.

In the dissolving step, after dissolving the silk fibroin at a concentration of 0.05-0.35 g / ml in a solvent, it can be heated to a temperature of 50 ~ 70 ° C for 40-80 minutes.

In the polymerization step, methacrylate-based compound is added to the solution in which the silk fibroin is dissolved at a concentration of 141 to 705 mM, and after the methacrylate-based compound is added to the solution in which the silk fibroin is dissolved, the temperature is 50 to 70 ° C. It is preferable to stir at 200 to 400 rpm rotational speed for 2 to 4 hours at.

In the mixing step, 20 to 30 ^% of the powder containing the polymer polymer in water and 0.1 to 0.3% of the photoinitiator are mixed, but it is preferable that the sum of the water, the polymer and the photoinitiator does not exceed 100 wt%. .

The photoinitiator used in the mixing step, lithium thiphenyl phenyl-2,4,6-trimethylbenzoylphosphinate (li thium phenyl- (2,4,6-tr imethyl benzoydopinyll) phosphinate, LAP), benzyldimethyl Benzyl dimethyl ketal, acetophenone, benzoin methyl ether, diethoxyacetophenone, benzoyl phosphine oxide and 1-hydroxycyclonucleic phenyl ketone 1-hydroxycyclohexyl phenyl ketone) may be used that includes at least one selected from the group consisting of.

In the drying step, the solution containing the polymer polymer is frozen at -90 to -70 ° C for 10 to 14 hours, and then lyophilized for 40 to 60 hours at the same temperature as the freezing temperature.

On the other hand, another embodiment of the present invention is the aforementioned bio ink; Or a bio structure manufactured by 3D printing using the bio ink prepared by the aforementioned manufacturing method.

【Effects of the Invention】

The present invention is based on the silk fibroin, one of the natural protein fibers in a liquid state that can cause a gelation or curing reaction upon exposure to light through polymerization reaction. By preparing the bio ink composition, biological tissues having mechanical properties such as moisture absorption, volume expansion ratio, compressive strength, and tensile strength can be prepared.

In addition, by producing a bio ink based on silk fibroin as a basic skeleton, an immune reaction hardly occurs in the body, and thus a biostructure having excellent biocompatibility can be manufactured through 3D printing.

[Brief Description of Drawings]

1 is a schematic diagram schematically showing a hydrogel state of a bioink which is an embodiment of the present invention.

Figure 2 is a schematic diagram showing the manufacturing process of the bio ink of the present invention. Figure 3 is an embodiment of the present invention, a schematic chemical formula showing the polymerization reaction of silk fibroin and GMA.

FIG. 4 is a schematic view showing an embodiment of the present invention in which gel fibroin and GMA polymerized polymer (SGMA) are gelled.

Figure 5 is an embodiment of the present invention, FT-IR spectrum of the SGMA according to the concentration of GMA injected into the silk fibroin.

6 is an embodiment of the present invention, which is -NMR spectrum of SGMA according to the concentration of GMA injected into silk fibroin.

Figure 7 is a schematic diagram showing a 3D printing process in a DLP method using a bio ink prepared according to an embodiment of the present invention. .

Figure 8 is a graph showing the results of measuring the water absorption of the hydrogel printed by 3D printing using a bio ink prepared in another embodiment of the present invention. 9 is a graph showing the results of measuring the volume expansion rate of the hydrogel printed by 3D printing using a bio ink prepared according to another embodiment of the present invention. 10 to 16 are photographs or graphs showing an experimental procedure or test results of measuring the mechanical strength of a hydrogel printed by 3D printing using a bio ink prepared according to another embodiment of the present invention.

FIG. 17 is a graph showing storage modulus (G ′) and loss modulus (G ′ ¹ ) according to the shear strain of a hydrogel printed by 3D printing using a bio ink prepared according to another embodiment of the present invention.

FIG. 18 is a graph showing storage modulus (G ′) and loss modulus (G ′ ′) of a hydrogel printed by 3D printing using a bio ink prepared according to another embodiment of the present invention.

FIG. 19 is a graph showing the stiffness of hydrogels printed by 3D printing using bio inks containing different photoinitiator contents according to another embodiment of the present invention.

20 is a graph showing the stiffness of the hydrogel according to the curing time exposed to UV under 3D printing using different SGMA-3 content of different bio inks prepared by another embodiment of the present invention.

21 is a fluorescence micrograph showing the results of cytotoxicity test of the bio ink prepared according to another embodiment of the present invention.

22 is a cell proliferation experiment of a bio ink prepared by another embodiment of the present invention A graph showing the results.

[Form for implementation of invention]

Before describing in detail through the preferred embodiments of the present invention, the terms or words used in the present specification and claims should not be construed as being limited to the conventional or dictionary meanings, meanings corresponding to the technical spirit of the present invention To be interpreted as

Throughout this specification, when a part "includes" a certain component, it means that it can further include other components, without excluding other components unless specifically stated otherwise.

In each step, the identification code is used for convenience of explanation, and the identification code does not describe the order of each step, and each step may be performed differently from the stated order unless the context clearly indicates a specific order. have. That is, each step may be performed in the same order as specified, may be performed substantially simultaneously, or may be performed in the reverse order. Hereinafter will be described in more detail with respect to the bio ink of the present invention and its preparation method.

First, the bio ink of the present invention is a biomaterial capable of printing biostructures that can be utilized in medical and bio fields such as tissue engineering supports, surgical implants, personalized implants, artificial blood vessels, artificial organs, etc., via 3D printers. The biostructure consisting of a hydrogel formed as shown in FIG. 1 can be printed by exposing a liquid bio ink obtained by dissolving a polymer polymerized in a silk fibroin, which is a protein polymer, in water together with a photoinitiator to light (for example, UV). have.

In particular, the bio ink of the present invention may be applied to various types of 3D printers, but may be preferably applied to 3D printers based on digital processing (DLP).

In general, DLP (digi- tal process processing) (DLP) type 3D printer is a device for manufacturing a structure by mask projection image hardening. It is a shape to be molded by using a beam projector in a tank containing a liquid photocurable resin. It is a 3D printing method of manufacturing a structure by projecting light with light, and curing and laminating resin in the projected shape. This 3D printing technology of the DLP method projects the image from the beam projector in mask units (2D), which enables output without additional materials such as a support, and has excellent surface roughness and no noise during the printing process. There is this.

In particular, in the case of DLP type 3D printing, the structure is molded through the surface unit molding method using the resin in the liquid state as the ink, and thus the work speed can be uniformly and rapidly formed, which reduces the damage to the cells, thereby improving the cell compatibility. In addition, it is possible to print very fine surface roughness and precise structure, it is possible to produce a personalized implant with excellent fit when applied by the 3D bio printing method.

Specifically, the bio ink of the present invention can be used as a biomaterial Biocompatible materials (e.g. agarose, fibrinogen, fibroin, methacrylated hyaluronic acid (HAMA), thiolated hyaluronic acid, gelatin, gelatin methacrylate ( GelMA), Silicated Gelatin, Collagen, Alginate, Methyl Cellulose, Chitosan, Chitin, Synthetic Peptide, Polyethylene glycol based hydrogel, Polyglycolic acid (PGA), Poly-lact ic-co-glycolic acid ), Polylactic acid (PLA), poly (L-lactic acid) (PLLA), Polycaprolactone (PCL), PH 1 (Po 1 yhyd roxybutyrate), Polyhydroxyvalerate (PHV), PDO (Po 1 yd i ox anone), PTMC Polytrimethylene carbonate) and the like, and among them, fibroin having excellent biocompatibility and physical properties can be used as a base.

Fibroin can maintain rapid cross-link formation and mechanical stiffness, have excellent mechanical properties in terms of stability when formed into a hydrogel, and can create a microenvironment suitable for cell attachment and differentiation after the cells are printed. There is an advantage.

Thus, the bio ink of the present invention is fibroin. In addition, it contains a high molecular polymer prepared from silk fibroin obtained from silkworm cocoon, showing excellent cell compatibility, excellent compressive strength after printing, tensile strength and storage modulus. In addition, it can be applied to DLP type 3D printer.

More specifically, the bio ink according to the present invention includes a polymer polymer in which silk fibroin and methacrylate compound are polymerized; And a photoinitiator, wherein the polymer polymer and photoinitiator are dissolved in water to fill a liquid bio ink in a 3D printer, and the bio ink is exposed to light, preferably UV lltraviolet ray ultraviolet rays. The structure may be formed by hydrogel.

Silk fibroin, which forms the main skeleton of the bio ink of the present invention, is a natural protein polymer obtained by removing sericin from silkworm cocoon, and a polymer polymer can be prepared by polymerizing a single amino acid residue and a methacrylate compound in the silk fibroin. Preferably, at least one methacrylate compound is copolymerized to the lysine residue in the amino acid group in the silk fibroin to prepare a polymer polymer in which a methacrylate vinyl group is formed in the amine group of the silk fibroin.

For example, the polymer is a methacrylate-based compound as shown in FIG. 3, and polymerized with the silk fibroin by using glycidyl methacrylate (GMA) to meta-amine the amine in the silk fibroin molecule. The acrylate group (metacrylate groLip) may be a polymerized polymer (hereinafter, SGMA).

The photoinitiator may cause radical reaction by attacking the polymer when exposed to light, but may be used without particular limitation as long as it is a chemically stable compound that is not toxic to the human body.

For example, the photoinitiator included in the bio ink of the present invention is a free radical type photoinitiator, lithium phenyl— 2,4,6-trimethylbenzoylphosphinate (lithium phenyl- (2,4,6-trimethyl benzoyl) phosphinate, LAP), Benzyldimethyl ketal (benzyl dimethyl ketal, acetophenone, benzoin methyl ether, diethethacetophenone, benzoyl phosphine oxide and 1-hydroxycyclonuclear phenyl ketone (l- hydroxycyc lohexyl phenyl ketone) may be used that includes at least one selected from the group consisting of, but is preferably low cytotoxic lithium phenyl-2,4,6-trimethylbenzoylphosphinate (li thium phenyl- (2, 4, 6-tr imethyl benzoyl) phosphinate, LAP) can be used.

Specifically, when the bio ink is irradiated with light, the photoinitiator attacks the vinyl monomer of the polymer polymer to generate radical reaction, and the free radicals are cured through the polymerization reaction to form a structure.

Preferably, the bio ink according to the present invention may be a cell, growth factor, according to the use or characteristic of the biostructure to be prepared through 3D bioprinting in addition to the polymer and photoinitiator polymerized with the silk fibroin and methacrylate-based compounds mentioned above. Biodegradable polymers and the like may be further included as appropriate.

The content and structure of the specific components related to the bio ink will be described in more detail through the preparation method of the bio ink. On the other hand, another embodiment of the present invention relates to a method for producing a bio ink as shown in Figure 2 dissolution step of dissolving silk fibroin (Si lk Fibroin) in a solvent (Fig. 2 (a)); After adding methacrylate to a solution in which silk fibroin is dissolved, a polymerization step of preparing a polymer by stirring it (Fig. 2 (b)); Drying step of lyophilizing and drying the solution containing the polymer prepared by the polymerization step (Fig. 2 (d) and (e); and mixing step of mixing the powder and photoinitiator containing the polymer polymer in water) (FIG. 2 (f)) may be used to produce a bio ink.

The silk fibroin is a natural protein polymer, rejection reaction and immune reaction does not occur in the body, there is an advantage that the biocompatibility is excellent due to less inflammatory reaction. Silk fibroin is a silkworm cocoon (Bombyx mor i) silkworm cocoon raw silk as it is removed by sericin and impurities through the refining process, the method of refining silkworm cocoon silk is usually boiled over 10 hours with boiling water, diluted alkaline There is a method of treatment with a solution, and the technique for obtaining silk fibroin refined by removing sericin and impurities from silkworm cocoon can be used if it is a widely known method, so a detailed description thereof will be omitted.

Silk fibroin refined through the refining process is insoluble in dilute aqueous solution such as water, dilute acid or dilute base, and in order to dissolve silk fibroin in the solvent in the dissolution step, 50 ~ 70 ° C in lithium bromide solution It can be dissolved by heating to a temperature of 40-80 minutes. At this time, as the solvent used in the dissolution step, since the silk fibroin is not sufficiently dissolved in the dilute solution, preferably a lithium bromide (LiBr) solution or calcium chloride (CaCl ₂ ) solution of 8.0 to 10.0 M may be used. For example, a lithium bromide solution of 8.7 to 9.8 M may be used.

After adding a methacrylate compound to a solution in which the silk fibroin is prepared through the dissolution step, to prepare a polymer by stirring In the polymerization step, a polymer may be prepared by polymerizing a methacrylate group to an amino acid residue of silk fibroin, thereby forming a structure made of a hydrogel having improved mechanical properties such as compressive strength, tensile strength, and storage elasticity when exposed to light. have.

In this case, the amino acid residue refers to a structural unit from which H and 0H are separated from the molecular structure of various amino acids included in silk fibroin, and broadly means each amino acid constituting a peptide or protein.

Specifically, the polymerization step may be prepared by adding a methacrylate compound to a solvent in which the silk fibroin is dissolved, and then stirred at a speed of 200 to 400 rpm for 2 to 4 hours at a temperature of 50 ~ 70 ° C. .

The polymer included in the bio ink of the present invention is a polymer polymer prepared by copolymerizing silk fibroin (Sl ik Fibtoin, SF) and a methacrylate-based compound, and is a polymer polymer that is a basic skeleton of a biostructure manufactured during 3D printing. One or more methacrylate-based compounds may be copolymerized to the residues of amino acids included in the silk fibroin, thereby forming a polymer by forming a copolymer having a methacrylate group bonded to the residues of the amino acids. . Preferably, two methacrylate-based compounds may be polymerized to the residue of the amino acid contained in the silk fibroin to prepare a polymer, which may be cured upon exposure to light with a photoinitiator to form a hydrogel. Can be formed. In this case, the wavelength of light may be used to irradiate a light wavelength region in which the photoinitiator may initiate a radical reaction, thereby initiating gelation and curing reaction of the bio ink. In one example, the polymer copolymer is a combination of silk fibroin (SF) and glycidyl methacrylate (glyc idyl met hacry late, GMA) as shown in Figure 3, the amine in the silk fibroin molecule, preferably As the epoxy ring of glycidyl methacrylate is boiled, a polymer polymer (hereinafter, SGMA) in which a methacrylate group (metacrylate group) is polymerized to an amine of a lysine group in a silk fibroin molecule may be prepared. A polymer in which a methacrylate group is bonded to the amine through a nucleophilic semiungol while the epoxy ring of the methacrylate group is broken to the amine (# ₂ ) of the α-helix of the silk fibroin (SF) or the lysine group of the β-sheet. The polymer SGMA can be prepared.

Therefore, in order to prepare a preferred high polymer, the mixing ratio of the silk fibroin and the methacrylate-based compound mixed in the polymerization step is most important. In the dissolving step, it is preferable to add a methacrylate-based compound at a concentration of 141 to 705 mM in a solution in which silk fibroin is dissolved in a solvent at a concentration of 0.05 to 0.35 g / ml. When the ratio of the rate-based compound is out of the above range, the economic efficiency may be reduced due to the increase in the amount of unfinished silk fibroin or methacrylate-based compound, or the mechanical strength of the manufactured bio ink printed structure may be lowered. .

A solution containing the polymer prepared in order to remove impurities, which are ions contained in the solution containing the polymer polymer prepared through the polymerization step, that is, ions contained in the solvent used in the dissolution step, is placed in the dialysis stream. It is preferable to further include a dialysis step (FIG. 2 (c)) to be immersed in water. More preferably, before the dialysis step, the solution containing the polymer polymer may be filtered using a filter, and then a dialysis step may be performed to remove ions contained in the solution.

The dialysis step may use a dialysis tube that passes the ionic component but does not pass the polymer polymer, preferably in the ₁₂ ~ 14 kDa cutof f dialysis tube containing a solution containing the polymer prepared in water It is preferable to immerse for 3-5 days. When the dialysis time is less than 3 days, the removal of the ionic components contained in the solution containing the prepared polymer may not be sufficient, resulting in a decrease in biocompatibility or a decrease in mechanical strength of the structure manufactured by printing. If the dialysis time exceeds 5 days, there is no benefit over time, which may lower the economic efficiency.

The drying step of lyophilizing and drying the solution containing the polymer polymer passed through the dialysis step, the solution containing the polymer polymer in order to improve the circulation and storage of the liquid bio ink and to impart chemical stability of the bio ink All lyophilization and powdering are preferred.

Specifically, in the drying step, the dialysis proceeds sufficiently to completely freeze the solution containing the polymer polymer from which the ionic component is removed first at -90 to -70 ° C for 10 to 14 hours, and then for 40 to 60 hours. Freeze drying is preferably performed at the same temperature as the freezing temperature. The solution containing the freeze-dried polymer polymerizer is preferably powdered to have an appropriate size particle size through processing such as crushing and grinding.

Through the drying step, the powder containing the polymer may be prepared in the bio ink of the present invention through a mixing step of mixing with a photoinitiator in water. Preferably, in the mixing step, it is preferable to mix 20-30 wt% of the powder containing the polymer polymer in water and 0.01-1.3 wt% of the photoinitiator, wherein the sum of water, the polymer polymer and the photoinitiator is 100 \. It is desirable not to exceed ^%.

Since a detailed description of the photoinitiator has been mentioned above, it will be omitted here.

In this way, the bio ink of the present invention manufactured as described above may be gelled or cured by exposure to light having a wavelength at which the photoinitiator may initiate photopolymerization reaction as shown in FIG. 4 to form a hydrogel.

In detail, the bioinks of the present invention prepared by adding LAP, a photoinitiator, to a solution containing SGMA formed by polymerization of silk fibroin and glycidyl methacrylate (GMA) are used as light (ultraviolet, Exposure to UV) causes the photoinitiator LAP to attack the vinyl group of SGMA to generate free radicals, thereby binding within the chain of the double bond portion of the methacryl group of SGMA, between Not only covalent bonds are induced, but also the physical entanglement between the long chain of the polymer can be produced a structure of the hydrogel.

Therefore, the bio-structure in the hydrogel state may be manufactured by 3D printing, preferably DLP-type 3D printing using the aforementioned bio ink or the bio ink manufactured by the aforementioned manufacturing method. Hereinafter, an embodiment of the present invention will be described. However, the scope of the present invention is not limited to the following preferred embodiments, and those skilled in the art can implement various modified forms of the contents described herein within the scope of the present invention.

[Production Example 1]

Silkworm cocoon from Rural Crimson Korea, Korea. Cut »or /) into 4 pieces. 40 g of the cocoon was immersed in 1 L of 0.05 M sodium carbonate solution, heated to 100 ⁰ C for 30 minutes, and washed several times with distilled water. Then, it was dried at room temperature to obtain 31.1 g (about 80% yield) of silkworm cocoon, ie, silk fibroin, from which sericin was removed.

20 g of the obtained silk fibroin was added to 100 ml of an aqueous 9.3 M lithium bromide solution, which was then heated to 60 ^° C. for 1 hour to completely dissolve the silk fibroin in the aqueous lithium bromide solution (see FIG. 2A). .

GMG solution (Sigma-Aldrich, St. Louis, Missouri, USA) was added 2, 4, 6, 10 ml, respectively, and stirred for 3 hours at 300 rpm at a temperature of 60 ° C so that the silk fibroin and GMA could be fully polymerized. (b)). After filtration using a miracloth (Calbiochem, SanDiego, CA) filter, a 13 kDa cut-off dialysis tube contains a solution containing silk fibroin and SGMA, which is a combination of GMA, and then the dilution water is completely immersed in distilled water. After leaving for 4 days, the ions and impurities contained in the solution were dialyzed and removed (see FIG. 2C). The dialysis solution was frozen for 12 hours at an average temperature of -80 ⁰ C, then lyophilized for 48 hours (see FIG. 2 (d)), and then powdered to give SGMA powder (in order of GMA dosage, SGMA-l , SGMA-2, SGMA-3, SGMA-4) were prepared (see FIG. 2 (e)).

Experimental Example 1

In order to confirm the chemical structure of the SGMA powder prepared in Preparation Example 1, an FT-IR spectrum was measured using an infrared spectrophotometer (Frontier, PerkinElmer, Rodau, UK), and to confirm the degree of methacrylation of SGMA. ¾ NMR spectra were measured using a Buker DPX FT-NM instrument (Bruker Analytik GmbH, Karlsruhe, Germany).

Specifically, 0.001 g of SF, SGMA-1, SGMA-2, SGMA-3, and SGMA-4 powder prepared by adding different ratios of GMA in Preparation Example 1, together with 0.5 g of KBKPotassium bromide, FT-IR grade) Grinded evenly in a bowl. 5 shows the results of spectrum measurement using FT-IR (Froniter, PerkinElmer, Rodgau, UK).

In addition, 0.5 ing of SF, SGMA-1, SGMA-2, SGMA-3, SGMA-4 powder prepared in Preparation Example 1 to determine the degree of methacrylated SGMA 700 μΐ in a deuterium solvent (0 ， Sigma-Aklrich ), And filtered with a filter of 0.45 mm 3 and then measured the ^L H 匿 spectrum using a Buker DPX FT-NMR apparatus (Bruker Analytik GmbH, Karlsruhe, Germany) is shown in Figure 6 and Table 2 It was. Looking at the results of Figure 5, the FT-IR spectrum, SF, SGMA-1, SGMA— 2, SGMA-3,

All of SGMA-4 showed peaks at amide 1 (1639 cm ^"1 ), amide Π (1512 cm ^{" 1} ), and amide ΠΙ (1234 cm ^_1 ), representing 1105 halves of about 5000 amino acids contained in silk fibroin. Male amino acid. In addition, the CH-0H peak, which is weakly measured at 1238 ατΓ ¹ , represents an alcohol group formed by breaking the epoxy ring of GMA, and the peaks of 1165 CITT ¹ and 951 cnf ^{1 show} the resonance of CH ₂ of the methacrylate vinyl group of GMA. It was expected that the formed by the peak, it was confirmed that the intensity of the peak also increased as the amount of GMA contained.

Table 11

Referring to the result of FIG. 6, which is the ¾ NMR spectrum of NMR of SF, SGMA-1, SGMA-2, SGMA-3, and SGMA-4 powders prepared in Preparation Example 1, first, at 6.2 to 6δ = 5.8 to 5.6 ρρι. The peak observed is due to the methacrylate vinyl group, and the peak observed at δ = 1 · 8 ppm is believed to be due to the resonance of the G methyl group (-CH ₃ ), which gradually increases the intensity of the peak as the GMA content increases. It can be seen that increases.

In addition, it can be seen that the methylene signal of lysine (Lysin) gradually decreases at 5 = 2.9 ppm as the GMA content increases, which is determined to be modified by the polymerization of silk fibroin molecules and GMA. Through the degree of these peaks, the degree of denaturation of the methylene group of lysine of silk fibroin was estimated to be about 22-42% according to the change of GMA content.

Table 1 is a measurement of the width of each peak (vinyl group, lysine, methyl group of methacrylate) observed in FIG. 6 which is an NMR graph, and the degree of methacrylateization is ¾ NMR of FIG. 6. After measuring the graph area of the lysine group in the graph, it was calculated from the following equation (1). This is actually the case of polymerization reaction, methacrylate may occur in all secondary amine groups having semi-ungwoong in silk fibroin, but since the representative amino acid of the secondary amine group having semi-ungwoong in silk fibroin is lysine group The degree of methacrylated in silk fibroin was confirmed based on the freshness.

1- (Lysine group I of sgMA lysine group to pure silk fibro) ^χ 100 ... Equation (1) Therefore, looking at the results of FIG. 6 and Table 1, it was confirmed that the silk fibroin and GMA are polymerized by the above-mentioned manufacturing method to produce SGMA.

[Production Example 2]

SGMA-3 powder and lithium phenyl-2,4,6-trimethylbenzoylphosphinate powder (hereinafter referred to as LAP powder; Tokyo chemical industry, Tokyo, Japan) prepared in Preparation Example 1 were added to 1 L of water, respectively. After adding together, the SGMA '3 powder and the LAP powder were completely dissolved to prepare a bio ink.

[Table 2]

Experimental Example 2

In order to confirm the mechanical properties of the bio-ink prepared in Preparation Example 2, using the bio-ink prepared in Preparation Example 2 as shown in Figure 7 through a DLPC Digital light processing projector (365ι 皿, 3.5mW / cm ² ) A structure (10 × 10 × ² × ³ hexahedral specimens) was prepared. The specimens were designed in Solid works 2016 (Dassault systenmes, Waltham, USA), and the pieces were projected by the projector to produce 3D structures (samples) (print thickness; 50, number of base layers; 3, base layer curing time). 4 sec, number of buffer layers; 1, buffer layer curing time; 3 sec). In order to measure the water absorption capacity and the volume expansion rate of the manufactured 3D structure (sample), the specimens printed with 10 10 X 2mm ³ hexahedron were 0.3 0.5, 1, respectively in 37 ⁰ C PBS (phosphate reduced saline, ρΗ7 · 4). After soaking for 2, 3, 4, 5 hours, the weight was measured (W _swollen ), which was lyophilized to measure the weight (W _dry ). The weight of the lyophilized SGMA powder was measured and the weight of the water absorbed state was measured based on this (100%), and the water absorption capacity (Q) was derived through the following Equation (2). Table 3 shows.

Q = (W _swol ien-W _dry ) / W _dry X 100 (%) ... Equation (2) In addition, the volumetric expansion rate by water is equal to the X and γ axes of the 10x10x2 瞧³ cube immediately after the specimen is printed. after measuring the length, the expansion of the 10 10 ^χ ^χ 2ι丽³ expanded six-sided expansion in accordance with the X-axis and the elapsed time after the immersion relative to the γ-axis length (100%) X It was measured through the change in the length of the axis and expansion Y axis, the results are shown in Figure 9 and Table 3 below.

Referring to the results of FIGS. 8 and 9, the prepared specimen was 10% SGMA-3.

In the case of 1, it showed a high water absorption and high volume expansion rate, and as the content of SGMA-3 increased, the water absorption and volume expansion rate were lowered. In addition, compressive stress, compressive strain, elastic modulus, tensile stress, elongation and Young's modulus were measured to confirm the mechanical strength of the manufactured 3D structure, and the rheological properties (rheologi cal property) were measured to check the physical properties as ink. The measurement was performed, and the results are shown in Table 3 and Table 4 and FIGS. 10 to 20.

First, cylindrical specimens (diameter; 6 mm, height; 12 mm) by 3D printing using bio inks of Examples 1 to 3 ((10% SGMA-3 to 30% SGMA-3) to measure mechanical strength. It was manufactured and pressured at a displacement rate of 5 隱 / min using a universal test ing device (QM100S, QMESYS, South Korea) equipped with a lO kgf load shell (FIG. 10) compressive stress, modulus of elasticity (el ast ic modul us) was measured, and the tensile stress (tensi le stress) was measured by setting the elongation rate ¾! im / min at room temperature, and the Young's modulus was calculated as the slope of 50% strain deformation. The results of the experiment are shown in Table 3 and FIGS. 10 to 16.

[Table 3]

Referring to Table 3 and the results of FIGS. 10 to 16, the measured values also increase as the content ratio of SGMA-3 increases in all measurement results such as compressive stress, compressive strain, elastic modulus, tensile stress, elongation, and Young's modulus. In particular, in the case of Example 3 containing 30% SGMA-3, it can be seen that the compression force is increased by about 2 times compared to Example 2. Although the texture of Example 1, which is 10% SGMA-3, was too soft, The result was not obtained, but as the SGMA-3 content increased, it was confirmed that the measured values of tensile stress, elongation and Young's modulus increased.

In particular, Figure 16 is to confirm the strength or elasticity of the hydrogel, when a 7kg kettle bell on top of Example 3 which is 30% SGMA-3, Example 3 is a hydrogel weight of the In addition to supporting, after removing Kerbell after a certain period of time, it returned to the same shape as before the measurement. On the other hand, in order to confirm the optical cross-link reaction of Examples 1 to 3, the rheological properties were measured by using 0.1% strain and Anton Paar MCR 302 (Anton Paar, Zofingen, Swi tzer land) at 1 Hz frequency. 4 and FIGS. 17 and 18.

Table 4

Referring to Table 4 and the results of FIGS. 17 and 18, when the shear strain is 1% or less, the storage modulus (G ′) and the loss modulus (G ′ ′) show almost constant values. It was found that the elasticity and viscosity of the hydrogel of 3 were not affected by the shear force. In detail, as the content of SGMA-3 increased, the measured values of storage modulus and loss modulus gradually increased, and Example 3 containing 30% SGMA-3 showed the highest measured value.

In particular, in Examples 1 to 3, the storage modulus (G ') is about 6 times greater than the loss modulus (G' '), so the hydrogels of Examples 1 to 3 can be expected to behave in an elastomeric form. .

On the other hand, the phase change angle represents the state of the material to 6.4 ~ 9.1 ° (behavior like a solid '0 ⁰ ', behave like a liquid '90 ^ο ') Example 1 (10% SGMA-3) to 3 It can be seen that the hydrogel prepared through 3D printing using (30% SGMA-3) bio ink has a solid state. Therefore, when combining the results of Experimental Example 2 (results of FIGS. 8 to 18 and Tables 3 to 4), the physical properties of the bio inks of Examples l (10% SGMA-3) to 3 (30% SGMA-3) Looking at the properties, it can be seen that as the content of SGMA in the bio ink increases, the water absorption of the hydrogel gradually decreases, while the expansion rate decreases as the content increases. In addition, as a result of measuring the change in the increase of the specimen, the material contribution of SGMA to the 3D printing gradually decreased as the SGMA content was increased, which can be inferred from the increase of the transparency of the solution and the increase of the scattering of ultraviolet rays. there was.

In addition, as a result of measuring all the physical properties such as compressive strain, compressive strain, elastic modulus, tensile strain, elongation, and Young's modulus according to the increase of SGMA content in the bio ink, the measured value gradually increases with increasing SGMA content. It could be predicted that the silk fibroin polymer and GMA were polymerized by light irradiation and had excellent physical properties due to the entanglement between molecules and molecules. As a result, the reduction of the expansion rate according to the increase of the SGMA content in the bio ink could be expected to increase the degree of crystallization by increasing the chemical bond ½ degree in the molecule and increasing the rigidity and elasticity of the hydrogel.

In addition, as the content of SGMA in the bio ink increases, the rheological properties of phase change angle, storage modulus (G ′) and loss modulus (G ″) also increase significantly. G ') and the results of the loss modulus (G "), it can be expected that Example 3, in particular SGMA-3 of 30> has the highest form stability.

On the other hand, to monitor the SGMA rheological properties during UV curing at 0.1% strain and 1 Hz frequency, the storage modulus was measured according to the change of SGMA-3 and LAP content of photoinitiator. 19 and FIG. 20.

19 shows a tendency of stiffness of the hydrogel according to the change in the content of LAP, a photoinitiator, and the curing time of SGMA-3 (UV exposure for 4 seconds) of Examples 1 to 3 and 30, respectively, through FIG. 20. As it increased, it was confirmed that the storage modulus (G ′) of the hydrogel was increased. This could be expected to stabilize the hydrophobi c domain in which silk fibroin (SF) methacrylated by GMA with increasing exposure to UV light. In addition, in the case of 30% SGMA-3 (UV exposure for 4 seconds) of FIG. 20, when UV was blocked after 4 seconds, the storage modulus (G ′) rapidly decreased. On the other hand, the gelation point of SGMA according to the change of the content of SGMA, a polymer polymer in bio ink, and LAP, a photoinitiator, is defined as a gelation point by defining the intersection point of storage modulus (G ') and loss elasticity (G ") Measured.

Table 5

(Warning: ^' sec) Looking at the results of Table 5, the gel point is shortened from 41 seconds to 1 second as the photoinitiator LAP content was increased, and increased from 74 to 135 seconds as the content of SGMA increases Could. This gelation point was confirmed to depend on the photoinitiator LAP content, SGMA content, UV (light) exposure time in the bio ink.

Experimental Example 3

In order to confirm the cell suitability of the bio ink prepared in Preparation Example 2, Cytotoxicity (survival) and cell proliferation experiments were conducted.

Cells used for cytotoxicity and cell proliferation experiments were HeLa cells and NIH / 3T3 cells, which were purchased from ATCC (Manassas, Virginia). Cells DMEM (dulbecco 's modified Eagle medium )' in the medium 10v / v FBS (fetal bovine serum ), lv / v% penicillin 37 using a culture medium which streptomycin is added ⁰ C wetting a C0 _₂ (5% C0 ₂₎ Were incubated and the medium was changed every 3 days.

1 x 10 ⁶ of the cultured cells were mixed with the bio ink prepared in Preparation Example 2, and the mixed cell suspension 50, was dispensed into 96 well-plates, and then UV light (3.5 mW / ciTi ² ) was applied for 7 seconds. After irradiation, cell viability was observed for 14 days.

Cytotoxicity experiments were conducted using a LIVE / DEAD analysis kit (Life Technologies, USA), and observed with a fluorescence microscope to identify the cells with green fluorescence (calcein), that is, the living cells as shown in FIG. In addition, the cell proliferation experiment was carried out through CC-8 analysis (Do jindo molecular technologt, Rockville, USA) and the results are shown in FIG. As compared to GelMA (Comparative Example 1) and 10% SGMA-3 Example 1) measured with HeLa cells, as shown in FIG. 21, most cells showed green fluorescence on Day 1, indicating that living cells could be identified. have. However, as the experimental period gradually passed, the green fluorescence of GelMA (Comparative Example 1), that is, the distribution of living cells gradually decreased, whereas in the case of 10% SGMA-3 (Example 1), up to 7 days After 14 days of maintaining a certain number of cells it was confirmed that the cells proliferated.

Similar results were observed in experiments using NIH / 3T3 cells. Figure 22 is a graph showing the rate of cell proliferation, based on the number of cells of GelMA (Comparative Example 1) and 10% SGMA-3C Example 1) per day GelMA (Comparative Example 1) cells gradually over time While the number of cells decreased, 10% SGMA-3 Example 1) showed a slight increase in the number of cells until about 7 days, but after 14 days, the number of cells increased significantly, indicating that the cells proliferated. It was confirmed in all 3T3 cells. . Therefore, summarizing the results of Experimental Example 2 and Experimental Example 3, the polymer polymer formed by polymerizing silk fibroin into a basic skeleton can cross-link upon exposure to light together with a photoinitiator. 3D printing can be used to produce 3D biostructures with excellent cell suitability as well as excellent physical properties.

Industrial Applicability

The present invention is a polymer polymer in which the silk fibroin (Si lk Fibroin) and the methacrylate-based compound, which is a natural protein polymer, are polymerized; And photoinitiator]; and suggests a bio ink composition and a method of manufacturing the same, which can be applied to a 3D printer while maintaining excellent mechanical strength and cellular suitability of silk fibroin at an equivalent or higher. Absorption, volume expansion rate, compressive strength, Since almost no immune reaction occurs in a living tissue having mechanical properties such as tensile strength or in the body, an excellent biocompatibility can be manufactured through 3D printing, and thus there is industrial applicability.

Claims

[Range of request]

[Claim 11

Polymer polymers in which silk fibroin and methacrylate compounds are polymerized; And a photoinitiator.

[Claim 2]

The method of claim 1,

The high polymer,

Bio-inks, characterized in that formed by copolymerizing one or more methacrylate-based compounds in the amino acid residues of silk fibroin.

[Claim 3]

The method of claim 1,

The photoinitiator,

Lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate (l i thium

phenyl- (2,4,6-tr imethyl benzoyl) phosphinate, LAP, benzyl dimethyl ketal, acetophenone, benzoin methyl ether, diethoxyacetophenone , Benzoyl phosphine

Benzoyl phosphine oxide and 1-hydroxycyclonuclear phenyl

Bio ink, characterized in that it comprises at least one or more selected from the group consisting of ketone (1-hydroxycyclohexyl phenyl ketone).

[Claim 4]

A dissolution step of dissolving silk fibroin in a solvent;

Adding a methacrylate compound to a solution in which silk fibroin is dissolved, followed by stirring to prepare a polymer;

A drying step of lyophilizing and powdering the solution containing the polymer prepared by the polymerization step; And

A mixing step of mixing a powder and a photoinitiator containing a polymer polymer in water;

[Claim 5]

The method of claim 4,

After the polymerization step,

And a dialysis step of removing the impurities by placing the prepared solution containing the polymer polymer in the dialysis rib, and immersing in water.

[Claim 6]

The method of claim 5,

The dialysis step,

Put the solution containing the prepared polymer into 12 ~ 14 kDa cutof f dialysis flow

Method of producing a bio-ink, characterized in that impurities are removed by immersing in water for 3-5 days.

[Claim 7]

The method of claim 4, The dissolution step is,

After dissolving silk fibroin in a solvent at a concentration of 0.05-0.35 g / ml,

The method of manufacturing a bio ink, characterized in that for heating 40 to 80 minutes at a temperature of 50 ~ 70 ° C.

[Claim 8]

According to claim 14,

The polymerization step,

Method for producing a bio-ink, characterized in that the methacrylate-based compound in a concentration of 141 ~ 705 mM in a solution in which silk fibroin is dissolved.

[Claim 9]

According to claim 14,

The polymerization step,

After adding the methacrylate compound to the solution in which the silk fibroin is dissolved, the method for producing a bio ¾, characterized in that the stirring at 200 ~ 400 rpm rotation speed for 2 to 4 hours at 50 ~ 70 ° C temperature .

[Claim 10]

The method of claim 4,

The mixing step,

20 to 30 wt% of a powder containing a polymer polymer in water and 0.1 to 0.3% of a photoinitiator are mixed,

The sum of the water, the polymer and the photoinitiator is not more than 100 wt%, bio-ink manufacturing method.

[Claim 11]

The method of claim 4,

The photoinitiator,

Lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate (l i thium

phenyl- (2, 4, 6-tr imethyl benzoyl) phosphinate, LAP, benzyl dimethyl ketal, acetopherwne, benzoin methyl ether, diethoxyacetophenone Benzoyl phosphine

Benzoyl phosphine oxide and 1-hydroxycyclonuclear phenyl

Ketone (l-hydroxycyclohexyl phenyl ketone) comprising at least one selected from the group consisting of, bio-ink manufacturing method.

[Claim 12]

The method of claim 4,

The drying step,

After freezing the solution containing the polymer polymer for 10 to 14 hours at a temperature of -90 ~ -70 ° C,

A freeze-drying process for 40 to 60 hours at the same temperature as the freezing temperature, bio-ink manufacturing method.

[Claim 13]

The bio ink according to any one of claims 1 to 3; or A bio structure manufactured by 3D printing using the bio ink prepared by the method of any one of claims 4 to 12.