KR102145594B1 - Bioink compositions for visible light curing for 3D printing and method of manufacturing the same - Google Patents

Abstract

The present invention relates to a bio-ink composition for curing visible light for high-resolution 3D printing of DLP type and a method of manufacturing the same. The bioink composition according to the present invention can be cured using visible light as a light source, and a three-dimensional structure can be made using a DLP 3D printer. In addition, since it is possible to control the transmittance of light, it is possible to manufacture a hollow structure that was difficult to implement in the past, and it is useful for use as a bio-ink containing cells due to its high cellular compatibility. The bioink composition of the present invention is used to develop functional biomaterials, manufacture artificial organs for replacement of pharmaceuticals and cosmetics in animal experiments, print objects requiring cell loading and culture, and manufacture biochips and scaffolds that require 3D high resolution. It is applicable to technical fields such as.

Description

Bioink compositions for visible light curing for 3D printing and method of manufacturing the same}

The present invention relates to a bioink composition for curing visible light for 3D printing and a method of manufacturing the same.

3D printing technology is a technology that makes desired products through additive manufacturing using data obtained through 3D modeling. A stereolithography (SLA) method in which layers formed by curing by irradiating UV light on the surface of a photocurable resin and a fused deposition modeling (FDM) method in which solid filaments are melted and stacked and printed in three dimensions Was devised.

Recently, base technology and application technology using 3D printing technology are being actively implemented in the United States, Europe, and Japan. As 3D printing technology has been studied and applied technology has increased, interest in the field of medical engineering has also begun.

3D bioprinting is one of the technologies of 3D printing, and is a technology that produces tissues or organs by arranging living cells in a desired structure and pattern. Recently, with the development of a DLP (Digital Light Processing) method of manufacturing a 3D structure by sculpting in a plane unit using visible light, research on bio-ink for DLP is actively progressing.

Since the lamination is performed in units of surfaces rather than points and lines, the molding speed is remarkably fast, and since curing is performed using visible light, the cell compatibility is also excellent, which is drawing attention in the recent bioprinting field.

However, most of the bioprinting inks for DLP show optically transparent properties because they are used by dissolving a photocurable polymer precursor in water. Due to these characteristics, light passes through all of the polymer precursor solutions in fabricating a three-dimensional structure. For this reason, it is difficult to fabricate a hollow structure, and there is a disadvantage that the resolution in the Z axis is remarkably deteriorated.

The present inventors have made intensive research efforts to develop a bioink composition having a high resolution along the Z axis. As a result, the present inventors developed an additive (silk fibroin-melanosome complex) to increase the resolution of the bio-ink, which is cured by visible light, and the Z-axis resolution can be freely controlled by controlling the transmittance of visible light. The composition was developed. It was confirmed that the bioink composition of the present invention exhibits a high cell viability even after cell loading.

Thus, an object of the present invention is to provide a bioink composition.

Another object of the present invention is to provide a method for preparing a bioink composition.

One aspect of the present invention relates to a bioink composition for curing visible light for 3D printing, comprising a silk fibroin-melanosome complex and a biocompatible polymer compound.

Existing photocurable bioinks are intended to be applied to the fused deposition modeling (FDM) method, and their application to DLP (Digital Light Processing) type printing was very limited. In addition, due to the high transparency of the conventional bioink, it was not possible to implement a resolution in the Z-axis direction, and as a result, it was impossible to implement a structure with a void structure or an empty space. In the prior art, it is difficult to find a case in which a composition of an ink capable of satisfying the characteristics of a bio-ink with biocompatibility as an essential requirement is proposed.

Accordingly, the present inventors have made intensive research efforts to develop a bioink composition having a high resolution along the Z axis. As a result, the present inventors have developed a bioink composition that is cured by visible light through the development of an additive to increase the resolution of bioink, and can freely control the Z-axis resolution by controlling the transmittance of visible light. The bioink composition of the present invention exhibits a high cell viability even after cell loading.

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

The bioink composition for 3D printing of DLP type according to the present invention is cured using visible light as a light source. By controlling the Z-axis resolution, a structure with desired voids and empty spaces can be made, and compared to existing bioinks, it can be usefully used to manufacture a biocompatible structure of a complex structure or a cell-supporting hydrogel structure of various types.

The bioink composition of the present invention includes a silk fibroin-melanosome complex and a biocompatible polymer compound that is a precursor of a hydrogel.

In the present specification, "melanosome" refers to small particles containing melanin, and more specifically, refers to melanin nanoparticles.

In the present specification, "silk fibroin-melanosome complex" refers to a complex in which silk fibroin and melanosomes are combined, and silk fibroin incorporated with melanin nanoparticles is combined with melanin particles (melanosomes). In more detail, the "silk fibroin-melanosome complex" is a form in which melanosome nanoparticles are adsorbed on the surface of a silk fibroin hydrogel.

The melanosomal nanoparticles may be adsorbed into pores on the surface of the silk fibroin hydrogel. The pores on the surface of the silk fibroin hydrogel may be nanopores.

In the silk fibroin-melanosome complex of the present invention, the melanosomal nanoparticles may have a diameter of 500 nm or less.

According to an embodiment of the present invention, in the silk fibroin-melanosome complex of the present invention, the melanosomal nanoparticles may have a diameter of 50 nm to 100 nm.

According to another embodiment of the present invention, the melanosomal nanoparticles have a diameter of 60 nm to 100 nm, 70 nm to 100 nm, or 80 nm to 100 nm.

According to another embodiment of the present invention, the melanosomal nanoparticles have a diameter of 50 nm to 90 nm, 50 nm to 80 nm, or 50 nm to 70 nm.

According to another embodiment of the present invention, the melanosomal nanoparticles have a diameter of 60 nm to 90 nm, or 70 nm to 90 nm.

According to a specific embodiment of the present invention, the size of the melanosomal nanoparticles is 80 nm.

The bio-ink composition of the present invention contains a solution of a biocompatible polymer compound as a hydrogel precursor solution capable of curing visible light. According to an embodiment of the present invention, the biocompatible polymer compound solution may use distilled water, PBS, or a cell culture medium as a solvent. According to a specific embodiment of the present invention, the biocompatible polymer compound solution uses distilled water as a solvent.

The biocompatible polymer compound is, for example, polyethylene glycol (PEG), neophenyl glycol diacrylate (NPGDA) polyethylene oxide (PEO), polyacrylamide (PAAm), polyhydroxyethyl methacrylate (PHEMA). , Hyaluronic acid methacrylate (HAMA, Hyaluronic acid methacrylate), polyacrylic acid (PAA), polyvinyl alcohol (PVA), poly(N-isopropylacrylamide) (PNIPAM), polyvinylpyrrolidone (PVP), Polylactic acid (PLA), polyglycolic acid (PGA) and polycaprolactone (PCL), gelatin, gelatin methacryloyl (GelMA, Gelatin methacryloyl), alginate, carrageenan, chitosan, hydroxyalkylcellulose, alkylcellulose, silicone, At least one polymer compound selected from the group consisting of rubber, agar, carboxyvinyl copolymer, polydioxolane, polyacrylic acetate, polyvinyl chloride, and maleic anhydride/vinyl ether, a copolymer thereof, or a mixture thereof.

According to one embodiment of the present invention, the biocompatible polymer compound is polyethylene glycol containing an acrylate, vinylsulfone or thiol group.

According to another embodiment of the present invention, the biocompatible polymer compound is 4-arm or 8-arm polyethylene glycol containing an acrylate, vinylsulfone or thiol group.

According to another embodiment of the present invention, the biocompatible polymer compound is a compound containing polyethylene glycol hydroxy (4-arm PEG-hydroxyl, 20 kDa, PEG-OH).

According to a specific embodiment of the present invention, the biocompatible polymer compound is polyethylene glycol tetraacrylate (poly(ethylene glycol)-tetraacrylate, 4-arm PEG-acrylate, PEG-4A), polyethylene glycol maleimide (4-arm PEG -maleimide, PEG-4MAL), or polyethylene glycol vinylsulfone (4-arm PEG-vinylsulfone, PEG-4VS).

On the other hand, the biocompatible polymer compound may be used at a concentration suitable for supporting cells, for example, it may be used at a concentration of 2-10 (weight/volume)%.

According to one embodiment of the present invention, in the present invention, a 4% polyethylene glycol tetraacrylate solution was used as a concentration suitable for supporting cells. If the concentration range of the polyethylene glycol tetraacrylate solution is 2% or more, photocuring is possible, but when the concentration is increased to 10%, cell compatibility decreases.

The silk fibroin-melanosome complex of the present invention improves the mechanical strength of a hydrogel prepared as a precursor of the biocompatible polymer compound and improves cellular compatibility.

The bioink composition of the present invention is an ink composition for 3D printing that is cured by visible light. Therefore, the bioink composition of the present invention may additionally include a photoinitiator, a shellfish initiator, a crosslinking agent, or a mixture composition thereof.

According to one embodiment of the present invention, the photoinitiator is a photoinitiator in a visible light region. The photoinitiator in the visible light region is activated by visible light to photopolymerize a hydrogel made of a biocompatible polymer compound as a precursor. That is, a photopolymerization reaction occurs using a biocompatible polymer compound as a “monomer”. Visible light that can be used in the present invention may be, for example, a laser light having a wavelength of 400-650 nm such as He-Ne (633 nm), Ar (515 nm, 488 nm), YAG (532 nm), He-Cd (442 nm).

The visible light initiator or shellfish initiator that can be usefully used in the present invention may be selected from those known in the art, and more particularly, may be selected from known electron donating photoinitiators. Specifically, eosin (Eosin Y or Eosin B), N-phenylglycine, N,N-dialkylaniline compound, tertiary amine compound, organoborate salts, N-vinylpyrrolidone ), etc. Specific examples of the N,N-dialkylaniline compound include 4-cyano-N,N-dimethylaniline, 4-bromo-N,N-dimethylaniline, 4-acetyl-N,N-dimethylaniline, and 4-methyl -N,N-dimethylaniline, 4-amino-N,N-dimethylaniline, 4-ethoxy-N,N-dimethylaniline, 3-hydroxy-N,N-dimethylaniline, N,N,N', N'-tetramethyl-1,4-dianiline, 2,6-diethyl-N,N-dimethylaniline, pt-butyl-N,N-dimethylaniline, etc. are mentioned. Specific examples of the tertiary amine compound include triethylamine, dimethylethanolamine (DMEA), triethanolamine (TEA), and triisopropanolamine.

In the present invention, the visible light initiator or the shell initiator may be used alone or in combination of two or more, respectively.

According to an embodiment of the present invention, the photoinitiator is Eosin-Y, and the shellfish initiator is triethanolamine, and/or N-vinylpyrrolidone.

Cross-linking agents that can be used in the present invention include calcium chloride (CaCl ₂ ), MgCl ₂ and AlCl ₃ .

According to one embodiment of the present invention, the silk fibroin-melanosome complex may be included in a concentration of 0.01 to 1 (weight/volume)% based on the total bioink composition. According to another embodiment of the present invention, the silk fibroin-melanosome complex may be included in a concentration of 0.25 to 1 (weight/volume)% based on the total bioink composition.

It was confirmed that when the silk fibroin-melanosome complex was contained in a concentration exceeding 1% with respect to the total bioink composition, the viscosity of the precursor solution increased, resulting in poor printing stability. Therefore, in the present invention, the silk fibroin-melanosome complex is included in a concentration of 1 (weight/volume)% or less with respect to the total bioink composition.

In the bioink composition of the present invention, the silk fibroin may be included in a concentration of 0.01-1.0 (weight/volume)% relative to the silk fibroin-melanosome complex composition. When the concentration of silk fibroin exceeds 1.0%, the viscosity of the bio-ink increases, resulting in poor printing stability.

According to one embodiment of the present invention, the silk fibroin may be contained in a concentration of 0.25-1.0 (weight/volume)% relative to the silk fibroin-melanosome complex composition.

According to another embodiment of the present invention, the silk fibroin may be included in a concentration of 1.0 (weight/volume)% or less with respect to the silk fibroin-melanosome complex composition.

According to a specific embodiment of the present invention, the silk fibroin may be included in a concentration of 1.0 (weight/volume)% with respect to the silk fibroin-melanosome complex composition.

In the bioink composition of the present invention, the melanosomal nanoparticles may be included in a concentration of 0.01 (weight/volume)% or more with respect to the silk fibroin-melanosome complex composition.

According to another embodiment of the present invention, the melanosomal nanoparticles are 0.05 (weight/volume)% or more, 0.1 (weight/volume)% or more, 0.15 (weight/volume)% or more with respect to the silk fibroin-melanosome complex composition. , Or may be contained in a concentration of 0.2 (weight/volume)% or more.

According to a specific embodiment of the present invention, the melanosomal nanoparticles may be included in a concentration of 0.2 (weight/volume)% or more with respect to the silk fibroin-melanosome complex composition.

According to another embodiment of the present invention, the melanosomal nanoparticles may be included in a concentration of 0.05-0.2 (weight/volume)% relative to the silk fibroin-melanosome complex composition.

The photocurable bioink composition of the present invention can be used for DLP-type 3D printing, improves the resolution in the Z-axis direction to realize a void structure or empty space, and has excellent biocompatibility.

Another aspect of the present invention relates to a method for preparing a bio-ink for curing visible light comprising the following steps:

(a) mixing silk fibroin and melanosomal nanoparticles, and reacting at high temperature and high pressure of 100-140° C. and 10-20 psi pressure;

(b) obtaining by separating the silk fibroin-melanosome complex from the reaction product of step (a); And

(c) dispersing the silk fibroin-melanosome complex in a biocompatible polymer compound solution to prepare a bio-ink.

The manufacturing method of the bio-ink of the present invention is a method of manufacturing the bio-ink composition for curing visible light for 3D printing of the present invention, and descriptions thereof are omitted in order to avoid excessive redundancy in the present specification.

Hereinafter, the method for producing the bio-ink of the present invention will be described in detail.

Step (a): Preparation of silk fibroin-melanosome complex

First, silk fibroin and melanosomal nanoparticles are mixed and reacted at high temperature and high pressure at 100-140°C and 10-20 psi pressure.

Silk fibroin is obtained through a refining process that removes sericin that surrounds fibroin from silk.

According to one embodiment of the present invention, when the silk is boiled for 1 hour at 100° C. with sodium oilate and sodium carbonate, most of sericin is dissolved and removed. The sylph fibroin fibers are dissolved in a 9.3 M aqueous lithium bromide solution, and insolubles are removed by centrifugation. Thereafter, a silk fibroin solution is obtained by repeating dialysis with ultrapure water.

In the present invention, after dissolving the silk fibroin in distilled water, a hydrogel was prepared through ultrasonic treatment and used to prepare a melanosomal complex.

A silk fibroin-melanosome complex may be prepared using the silk fibroin solution (or silk fibroin hydrogel) and melanosomal nanoparticles of various concentrations.

Melanosomes are found in certain fish species and contain pigments that control the color of fish scales. Molecular motors, upon receiving a signal, transport melanosomes containing pigments around the cell or concentrate it in the cell center. The motor protein dynein serves to focus melanosomes to the center of the cell, or to the "minus end" of the microtubules. In contrast, the protein kinesin serves to disperse melanosomes around the cell, and is a plus end-oriented motor. The positive end of the microtubule is directed around the cell, so when kinesin disperses the melanosomes around the cell, the cell looks darker. When melanosomes are concentrated toward the center, the cells appear brighter. In nature, the diameter of melanosomes is observed up to about 500 nm.

According to an embodiment of the present invention, the melanosomes may be extracted from squid ink.

According to a specific embodiment of the present invention, viscous squid ink (melanosome particles 70%, purified water 20%, sodium chloride 9%, carboxymethyl cellulose 1%) was diluted 10-fold in distilled water, and a cellulose acetate membrane (MWCO, 12- 14 kDa) to obtain melanosomal nanoparticles by dialysis in distilled water at 60° C. for 7 days.

In the silk fibroin-melanosome complex of the present invention, the melanosomal nanoparticles may have a diameter of 500 nm or less.

According to an embodiment of the present invention, in the silk fibroin-melanosome complex of the present invention, the melanosomal nanoparticles may have a diameter of 50 nm to 100 nm.

According to another embodiment of the present invention, the melanosomal nanoparticles have a diameter of 60 nm to 100 nm, 70 nm to 100 nm, or 80 nm to 100 nm.

According to another embodiment of the present invention, the melanosomal nanoparticles have a diameter of 50 nm to 90 nm, 50 nm to 80 nm, or 50 nm to 70 nm.

According to another embodiment of the present invention, the melanosomal nanoparticles have a diameter of 60 nm to 90 nm, or 70 nm to 90 nm.

According to a specific embodiment of the present invention, the size of the melanosomal nanoparticles is 80 nm.

In the present specification, the "silk fibroin-melanosome complex" is a form in which melanosome nanoparticles are adsorbed on the surface of a silk fibroin hydrogel.

In the present invention, in order to prepare a silk fibroin-melanosome complex, silk fibroin hydrogel was mixed with melanosomal nanoparticles of various concentrations. The melanosomal nanoparticles are adsorbed to the nanopore on the surface of silk fibroin under a specific temperature and pressure.

According to an embodiment of the present invention, in the manufacturing step of the silk fibroin-melanosome complex, the concentration ratio of silk fibroin and melanosomes in the total mixed composition can be appropriately adjusted, but the concentration of silk fibroin in the silk fibroin-melanosome complex composition is 1.0% or less, the concentration of the melanosomal nanoparticles is preferably prepared at a concentration of 0.2% or more.

On the other hand, when the silk fibroin solution and melanosomes are reacted at high temperature and pressure, melanosomal nanoparticles are adsorbed on the surface of silk fibroin.

The adsorption reaction may be carried out in a temperature range of 100°C-140°C.

According to an embodiment of the present invention, the adsorption reaction may be carried out in a temperature range of 110°C-130°C. According to another embodiment of the present invention, the adsorption reaction may be carried out in a temperature range of 120°C.

The adsorption reaction may be carried out in a pressure range of 10-20 psi.

The adsorption reaction may be carried out by reacting for 10 to 30 minutes in the temperature and pressure range.

According to one embodiment of the present invention, the adsorption reaction is carried out for 20 minutes under conditions of 120° C. and 15 psi of silk fibroin and melanosomes.

Step (b): Isolation of silk fibroin-melanosome complex

Then, the silk fibroin-melanosome complex is separated from the reaction product of step (a).

According to one embodiment of the present invention, the silk fibroin/melanosome complex can be separated using mechanical separation. According to a specific embodiment of the present invention, the silk fibroin/melanosome complex may be separated by centrifugation.

The separated silk fibroin/melanosome complex can be dispersed in distilled water and used for ink production.

Step (c): dispersing in a biocompatible polymer compound solution

Finally, the silk fibroin-melanosome complex is dispersed in a biocompatible polymer compound solution to prepare a bioink.

The types of the biocompatible polymer compound are as described above.

In step (c), the silk fibroin/melanosome complex dispersed in the distilled water of step (b) is dispersed in a biocompatible polymer compound solution to prepare a bioink.

According to an embodiment of the present invention, the silk fibroin-melanosome complex dispersed in distilled water is added at a concentration of 0.01 to 1 (weight/volume)% based on the total bio-ink composition to finally prepare a bio-ink.

The concentration of melanosomal nanoparticles in the finally prepared bioink is 0.2 (weight/volume)% or more. The concentration of silk fibroin in the finally prepared bioink is 1.0 (weight/volume)% or less.

According to an embodiment of the present invention, in step (c), a visible light initiator, a shell agent, a crosslinking agent, and the like may be additionally added.

According to a specific embodiment of the present invention, in order to induce curing using visible light, eosin-Y, a visible light initiator, triethanolamine, and N-vinylpyrrolidone ) Was added.

The bioink finally prepared by the above-described method is an ink composition for DLP type 3D bioprinting, and the x, y, z axis resolution and transparency of the hydrogel can be adjusted by adjusting the amount of silk fibroin-melanosome complex added. .

In addition, by adding the silk fibroin-melanosome complex, the mechanical strength of the hydrogel can be improved, thereby realizing a pore structure by 3D printing.

The features and advantages of the present invention are summarized as follows:

(a) The present invention relates to a bioink composition for curing visible light for high-resolution 3D printing of DLP type and a method of manufacturing the same.

(b) The bioink composition according to the present invention is cured using visible light as a light source, and a three-dimensional structure can be made using a DLP 3D printer.

(c) In addition, since it is possible to control the transmittance of light, it is possible to manufacture a hollow structure that was difficult to implement in the past, and it is useful for use as a bio-ink containing cells because of its high cellular compatibility.

(d) The bioink composition of the present invention includes the development of functional biomaterials, production of artificial organs for replacement of pharmaceuticals and cosmetics for animal testing, printing of objects requiring cell loading and cultivation, and biochips requiring 3D high resolution. It can be applied to technical fields such as scaffold manufacturing.

1A is a scanning electron micrograph of the surface of a silk fibroin hydrogel according to the present invention, a scanning electron micrograph of melanosomal nanoparticles, and a scanning electron of a silk fibroin/melanosome complex adsorbed with melanosomal nanoparticles through an autoclave process. Show micrograph. White bar: 500 nm.
FIG. 1B shows the change in transparency of the hydrogel precursor solution according to the amount of silk fibroin/melanosome complex added to the polyethylene glycol tetraacrylate solution capable of curing visible light.
2A shows the configuration of a DLP type 3D printer according to the present invention. This is a method of manufacturing a hydrogel structure through visible light curing by transferring an image to a hydrogel precursor solution by placing a DLP projector on the top.
2B is a photograph of the DLP printing process.
2c, c and f show the design of a hollow pipe structure and a staircase structure, respectively. Black bar: 500 μm.
D and g of FIG. 2C are optical micrographs of a cross section of a hydrogel produced after printing of a polyethylene glycol tetraacrylate bioink containing no additives.
2C, e and h are optical micrographs of a cross section of a hydrogel prepared after printing a bioink containing silk fibroin/melanosome additive.
Figure 3a is an optical micrograph of the size of the visible light irradiation and the size of the cured hydrogel according to the concentration of melanosomal particles of the bio-ink used for DLP printing. White bar: 200 μm.
3B shows changes in the size of visible light irradiation and the size of the cured hydrogel according to the amount of melanosomal particles added.
3C shows the change in printing resolution according to the concentration of silk fibroin and melanosomes. Black bar: 1 cm.
3D shows the change in Z-axis printing thickness according to the concentration of silk fibroin and the concentration of melanosomes.
Figure 4a shows the storage elastic modulus change with respect to the shear rate according to the content of the silk fibroin/melanosome complex (SFM). 4% PEG4A + SFM (1% SF + 0.2 w/v% melanosomes)
Figure 4b shows the change in the elastic modulus of loss with respect to the shear rate according to the content of the silk fibroin/melanosome complex (SFM). 4% PEG4A + SFM (1% SF + 0.2 w/v% melanosomes)
4C is a surface scanning electron micrograph of a silk fibroin/melanosome complex hydrogel. White bars: 5 μm.
4D is a scanning electron microscope photograph of the surface of polyethylene glycol tetraacrylate hydrogel. White bars: 5 μm.
4E is a surface scanning electron micrograph of a polyethylene glycol tetraacrylate hydrogel containing a silk fibroin/melanosome complex. White bars: 5 μm.
Figure 5a is a non-added polyethylene glycol tetraacrylate hydrogel, polyethylene glycol tetraacrylate hydrogel containing 1 w / v% silk fibroin, polyethylene glycol tetraacrylate hydrogel containing 1 w / v% silk fibroin / melanosomes complex The fibroblasts are loaded on the gel to show metabolic activity according to the culture day.
5B shows a confocal laser micrograph through a live and dead test after culturing cells in the hydrogel for 13 days. Green represents live cells and red represents dead cells.

Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.

Throughout this specification, the “%” used to indicate the concentration of a specific substance is (weight/weight)% for solids/solids, (weight/volume)% for solids/liquids, and Liquid/liquid is (vol/vol) %.

The present invention is a high-resolution bioink composition for curing visible light containing silk fibroin hydrogel, melanosome nanoparticles, and polyethylene glycol tetraacrylate (poly(ethylene glycol)-tetraacrylate, PEG-4A) It provides, and preferably provides a bioink composition for a DLP printer containing a complex in the form of adsorbed melanosomal nanoparticles on the surface of the hydrogel of silk fibroin as an additive.

Example 1. Preparation of silk fibroin/melanosome complex additive

Silk fiber (Bombyx mori) is a fiber spun from silkworm cocoons, and is a long fiber with a triangular cross-section in which sericin in the outer layer surrounds two strands of fibroin, a protein-based inner layer.

The silk fibroin of the present invention is obtained through a refining process of removing sericin surrounding fibroin from silk. When the silk is boiled for 1 hour at 100° C. with sodium oilate and sodium carbonate, most of sericin is dissolved and removed.

The silk fibroin solution was obtained by dissolving sylph fibroin fibers in a 9.3 M aqueous lithium bromide solution, removing insoluble matters by centrifugation, and repeating dialysis with ultrapure water.

The melanosomal nanoparticles were extracted from squid ink (melanosome particles 70%, purified water 20%, sodium chloride 9%, carboxymethyl cellulose 1%). When purchasing squid ink, sodium chloride/carboxymethylcellulose mixed together was removed through a dialysis process and used. That is, the viscous squid ink was diluted 10-fold in distilled water and dialyzed in distilled water at 60° C. for 7 days using a cellulose acetate membrane (MWCO, 12-14 kDa) to obtain melanosomal nanoparticles. The melanosomal nanoparticles were obtained in a dispersed state in distilled water, which was used for adsorption of silk fibroin.

In order to prepare a silk fibroin/melanosome complex, various concentrations of silk fibroin hydrogel were mixed with various concentrations of melanosomal nanoparticles (dispersed in distilled water). Thereafter, the melanosomal nanoparticles were induced to be adsorbed on the surface of the silk fibroin hydrogel using an autoclave at 120° C. for 20 minutes. After centrifugation at 3000 RPM for 5 minutes, the supernatant was removed.

Then, the concentration of the silk fibroin/melanosome complex was adjusted to 0.25, 0.5, 0.75, and 1.0 (weight/volume)% with distilled water. The concentration of melanosomal nanoparticles of the finally prepared solution is 0.05, 0.1, 0.15, 0.2 (weight/volume)%, respectively, and the concentration of silk fibroin is 0.25, 0.5, 0.75, 1.0 (weight/volume)%, respectively (Fig. 3c).

Example 2. Preparation of silk fibroin/melanosome complex bioink

To synthesize polyethylene glycol tetraacrylate (PEG-4A), which constitutes a hydrogel precursor solution capable of visible light curing, for-am polyethylene glycol (4-arm PEG) was dissolved in dichloromethane and 3 equivalents of acryloyl Chloride (acryloyl chloride) and 3 equivalents of triethylamine were added, stirred in ice water for 30 minutes, and reacted at room temperature for 24 hours. After precipitation in ethyl ether and dissolving in distilled water, polyethylene glycol tetraacrylate (PEG-4A) was prepared through a dialysis process for 3 days with a 3.5 kDa cellulose acetate dialysis membrane.

The silk fibroin/melanosome complex diluted in distilled water was dispersed in a 4% (w/v) polyethylene glycol tetraacrylate solution, and finally the silk fibroin/melanosome complex was 0.25, 0.5, 0.75 and 1 (weight/volume)% A bio-ink was prepared containing at a concentration. In order to induce hardening using visible light, the visible light initiator Eosin-Y was dissolved at a concentration of 0.1 mM, and triethanolamine 0.3% and N-vinylpyrrolidone were dissolved as a shell reagent. It was dissolved in 0.3% to prepare a bio-ink.

Experimental Example 1. Surface analysis and chromaticity analysis

In order to analyze the surface structure of the silk fibroin/melanosome complex prepared in Example 1 of the present invention, the following experiment was conducted.

1A is a photograph showing a process of preparing a silk fibroin/melanosome complex by adsorbing melanosomal nanoparticles on a silk fibroin hydrogel as an additive for high-resolution DLP printing.

Figure 1b shows the transparency of the bioink according to the amount of silk fibroin / melanosomes added in a photograph. It shows the change in turbidity through the letters that appear after placing the letter "SNU" under the printed paper.

Referring to FIG. 1A, the surface of the silk fibroin hydrogel has a structure in which the surface is not smooth and pores exist, and it can be observed that the hydrogel precipitates when centrifugation is performed at 3000 RPM.

On the other hand, in the case of melanosomal nanoparticles, uniform particles with a diameter of about 80 nm could be observed, and it was confirmed that no precipitate was formed even when centrifugation was performed at 3000 RPM.

When silk fibroin hydrogel and melanosomal nanoparticles were mixed and subjected to a high-pressure high-temperature sterilization process, it was confirmed that the melanosomal nanoparticles were adsorbed on the silk fibroin hydrogel surface. As a result of centrifuging the complex at 3000 RPM, the supernatant appeared clear and transparent, indicating that most of the melanosomal nanoparticles were adsorbed on the silk fibroin surface.

Referring to FIG. 1B, it is possible to easily adjust the transparency of the bioink according to the amount of the silk fibroin/melanosome complex additive added. In the case of the polyethylene glycol tetraacrylate bioink containing no additives, it can be seen that the letters “SNU” are clearly visible because they are clear and transparent, whereas the letters “SNU” gradually disappear as the concentration of the additives increases.

Therefore, the visible light-curable bioink according to the present invention can change the transmittance of visible light according to the use of the silk fibroin/melanosome complex additive, and thus the change in resolution can be induced using a DLP 3D printer.

Experimental Example 2. Printing stability analysis of transparent bio-ink and opaque bio-ink

In order to analyze the printing stability of a structure made using the bio-ink composition for curing visible light prepared in Example 2 according to the present invention, a structure that is difficult to manufacture was manufactured using the existing transparent bio-ink.

Specifically, the design for the measurement of printing stability was designed by designing a hollow pipe structure and a staircase structure, and printing was performed. One bottom layer, five outer wall layers, and one ceiling layer were printed using bioink containing no additives and bioink containing silk fibroin/melanosome additives, respectively. As a result, it was confirmed that the structure of the structure manufactured through the transparent bio-ink formed a structure filled with voids (d in FIG. 2c) when the cross-section was observed with an optical microscope. On the other hand, in the case of the structure made of the bio-ink containing the additive, it was confirmed that voids were formed (e in FIG. 2c).

This is a result of stably forming pores due to the limit thickness that can be cured by forming the transmission limit distance of visible light in the bioink whose transparency is lowered by the additives in the printing of the last ceiling layer.

Likewise in the stepped structure shown in Fig. 2c, in the case of the structure manufactured using transparent bio-ink, since visible light penetrated indefinitely into the bio-ink, even the initially formed layer was cured. On the other hand, in the case of the bioink containing the additive, it was confirmed that the hydrogel was cured in the same structure as the intended design.

Therefore, it can be seen that the bio-ink containing the silk fibroin/melanosome additive according to the present invention has superior printing stability compared to the existing transparent bio-ink.

Experimental Example 3. Analysis of printing resolution according to the amount of silk fibroin/melanosome added

In order to analyze the printing resolution according to the additive content in the composition of the silk fibroin/melanosome-containing bioink according to the present invention, the following experiment was performed.

Specifically, the composite bioink prepared in Example 2 [4% PEG4A + SFM (1% silk fibroin + 0.05, 0.1, 0.15, or 0.2% melanosomes)] X, Y according to the concentration of silk fibroin/melanosome The resolution was analyzed. The size of the cured hydrogel was measured with an optical microscope by adjusting the visible light irradiation area to 200, 400, 600, 800, 1000, and 1200 micrometers, respectively, and shown in FIG. 3B.

When the size of the area irradiated with visible light and the measured size match, ideally, it was set as a bio-ink having a high resolution. The width of the cured hydrogel was analyzed with an optical microscope by adjusting the concentration of the additive (melanosome) to 0-0.2%. Meanwhile, in this experiment, the concentration of silk fibroin was fixed at a concentration of 1 (weight/volume)% relative to the silk fibroin/melanosome complex composition.

As a result, as the amount of additives (melanosomes) increased, it could be seen that the size of the measured width gradually approaches the size of the irradiated visible light width (see FIGS. 3A-3B). Through this, it can be seen that the presence or absence of an additive (melanosome) has a positive effect on the resolution in the X and Y axes.

On the other hand, even when the concentration of the additive (melanosome) was 0.2% or more, a result close to the abnormal value (width of the site irradiated with visible light) was shown (data not attached).

Figure 3c shows a map of the change in X, Y resolution according to the concentration when complexing silk fibroin concentration and melanosomal nanoparticles. It is a composition of bioink that shows higher resolution along the X and Y axes as it is filled with darker colors. As the concentration of silk fibroin and melanosomes used in the silk fibroin/melanosome complexation increased, it was confirmed that the resolution in the X and Y axes was improved.

3D is a result of evaluating the thickness of the hydrogel cured with visible light according to the concentration of the silk fibroin hydrogel and the concentration of the melanosomal particles used in preparing the silk fibroin/melanosome complex. It can be seen that the concentration of the complexed melanosomes has a high influence on the thickness of the hydrogel along the Z axis. On the other hand, as the concentration of silk fibroin hydrogel increases, the thickness decreases slightly. Through this, it can be seen that the silk fibroin/melanosome complex additive has a positive effect on the resolution in the Z-axis as well as the resolution in the X and Y axes.

Experimental Example 4. Evaluation of hydrogel strength according to the amount of silk fibroin/melanosome added

The mechanical strength of the structure made using the bioink composition for curing visible light prepared in Example 2 according to the present invention was analyzed. The following experiment was performed using a digital rheometer.

Samples for measurement were prepared with a diameter of 8 mm and a thickness of 2 mm.

Specifically, each construct was prepared and prepared with a bioink containing silk fibroin/melanosomes of 0, 0.25, 0.5, 0.75 and 1 w/v%.

The prepared sample was placed on a parallel plate geometry and measured at a frequency of 1 Hz in a shear strain-sweep mode. At this time, the applied force was fixed at 0.2 N and the gap was maintained at 2 mm.

Figure 4a shows the storage elastic modulus results according to the shear rate of the prepared hydrogel according to the content of the silk fibroin/melanosome complex.

Figure 4b shows the result of the loss elastic modulus according to the shear rate of the prepared hydrogel according to the content of the silk fibroin/melanosome complex.

Figures 4c, 4d and 4e show pictures of the surface after freeze-drying of a silk fibroin/melanosome complex hydrogel, a polyethylene glycol tetraacrylate hydrogel, and a polyethylene glycol tetraacrylate hydrogel containing a silk fibroin/melanosome complex, respectively. This is the result taken through a microscope.

It can be seen that as the content of the silk fibroin/melanosome complex increases, the storage elastic storage of the polyethylene glycol tetraacrylate hydrogel increases, so that the additive improves the properties of the hydrogel.

Experimental Example 5. Evaluation of the metabolic activity of fibroblasts according to the addition of silk fibroin/melanosome

Cellular compatibility of the construct made using the bio-ink composition for curing visible light prepared in Example 2 Air according to the present invention was confirmed. Fibroblasts (NIH 3T3 cells) were added to the bioink at a concentration of 1.5 X 10 ⁶ cells/ml to induce hardening of visible light, and then the metabolic activity of fibroblasts in the hydrogel was measured through alamarBlue assay. It was measured according to the culture day.

Figure 5a is an additive-free polyethylene glycol tetraacrylate hydrogel, a polyethylene glycol tetraacrylate hydrogel containing only silk fibroin hydrogel, and a polyethylene glycol tetraacrylate hydrogel containing silk fibroin / melanosomes complex, respectively, fibroblasts It shows the results of metabolic activity according to the culture day.

Compared with the non-added polyethylene glycol tetraacrylate hydrogel, the polyethylene glycol tetraacrylate hydrogel containing silk fibroin and silk fibroin/melanosome complex showed about twice as high metabolic activity. Through this, it can be seen that the additive has a positive effect on cell proliferation.

In addition, it was confirmed that fibroblasts no longer proliferated in the non-added polyethylene glycol tetraacrylate hydrogel after 10 days of incubation, while the polyethylene glycol tetraacrylate hydrogel containing the additives showed continuous cell growth. .

5B is a result of taking a fluorescence photograph of fibroblasts cultured for 13 days through a live and dead test using a confocal laser microscope.

In the case of manufacturing a structure using the high-resolution bio-ink for curing visible light according to the present invention, it can be seen that it exhibits superior cell stability compared to a hydrogel without using an additive.

Claims (11)

A bioink composition for curing visible light for 3D printing, including a silk fibroin-melanosome complex and a biocompatible polymer compound, in which melanosome nanoparticles are adsorbed on the surface of a silk fibroin hydrogel. delete The bioink composition according to claim 1, wherein the melanosomal nanoparticles are adsorbed into pores on the surface of the silk fibroin hydrogel. The bioink composition of claim 1, wherein the melanosomal nanoparticles have a diameter of 50 nm to 100 nm. The method of claim 1, wherein the biocompatible polymer compound is polyethylene glycol (PEG), neophenyl glycol diacrylate (NPGDA) polyethylene oxide (PEO), polyacrylamide (PAAm), polyhydroxyethyl methacrylate ( PHEMA), Hyaluronic acid methacrylate (HAMA), polyacrylic acid (PAA), polyvinyl alcohol (PVA), poly(N-isopropylacrylamide) (PNIPAM), polyvinylpyrrolidone (PVP) ), polylactic acid (PLA), polyglycolic acid (PGA) and polycaprolactone (PCL), gelatin, gelatin methacryloyl (GelMA, Gelatin methacryloyl), alginate, carrageenan, chitosan, hydroxyalkylcellulose, alkylcellulose, One or more polymer compounds selected from the group consisting of silicone, rubber, agar, carboxyvinyl copolymer, polydioxolane, polyacrylacetate, polyvinyl chloride, and maleic anhydride/vinyl ether, a copolymer thereof, or a mixture thereof. Bioink composition. The bioink composition of claim 5, wherein the polyethylene glycol (PEG) is a 4-arm or 8-arm polyethylene glycol containing an acrylate, vinylsulfone, or thiol group. The bioink composition of claim 1, wherein the bioink composition further comprises a photoinitiator, a shellfish initiator, a crosslinking agent, or a mixed composition thereof. The bioink composition of claim 1, wherein the concentration of the silk fibroin-melanosome complex is 0.01 to 1% (weight/volume) based on the total bioink composition. Method for producing a bio-ink for curing visible light comprising the following steps:
(a) Silk fibroin and melanosome nanoparticles are mixed and reacted at 100-140°C and 10-20 psi pressure, so that melanosomal nanoparticles are adsorbed on the surface of silk fibroin hydrogel Preparing a phosphorus silk fibroin-melanosome complex;
(b) separating and obtaining a silk fibroin-melanosome complex in a form in which melanosomal nanoparticles are adsorbed on the surface of a silk fibroin hydrogel from the reaction product of step (a); And
(c) preparing a bio-ink by dispersing the silk fibroin-melanosome complex, in which the melanosomal nanoparticles are adsorbed on the surface of the silk fibroin hydrogel, in a biocompatible polymer compound solution. The method of claim 9, wherein the concentration of the melanosomal nanoparticles in the bioink is 0.2% (weight/volume) or more with respect to the total bioink composition. The method of claim 9, wherein the concentration of silk fibroin in the bioink is 1.0% (weight/volume) or less based on the total bioink composition.

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