KR20230106823A - Manufacturing method for bioink composition for 3D bioprinting using chitosan derivatives of high degree of substitution - Google Patents

Abstract

The present invention relates to a method for manufacturing a bioink composition for three-dimensional (3D) bioprinting that can form a stable 3D structure immediately upon extrusion without a chemical additive by introducing a galyl group into chitosan, a natural polymer, to form self-crosslinking through multiple interactions. The method for manufacturing a bioink composition for 3D bioprinting of the present invention comprises the steps of: dissolving chitosan in a solvent, to prepare a chitosan solution; preparing chitosan into which a galloyl group with a degree of substitution of 30 % is introduced; performing dialysis to remove unreacted substances; freeze-drying to obtain chitosan into which a galloyl group is introduced, in powder form; preparing a chitosan solution into which a galloyl group is introduced; and preparing hydrogel.

Description

Manufacturing method for bioink composition for 3D bioprinting using chitosan derivatives with high degree of substitution {Manufacturing method for bioink composition for 3D bioprinting using chitosan derivatives of high degree of substitution}

The present invention relates to a bioink composition capable of 3D bioprinting without chemical additives or photoinitiators.

Recently, as we enter an aging society, the demand for organ transplantation increases every year, but there is a shortage of organ donors. It is getting attention. 3D bioprinting technology is a concept that combines 3D printing technology and biotechnology, and is a technology that produces artificial tissues and organs by stacking living cells in desired shapes. It can reproduce complex biological tissue based on medical image information such as CT or MRI, and create accurate structures through digital design such as CAD, so it has high reproducibility and precision, and it is possible to repeatedly manufacture.

The basic printing methods of 3D bioprinting are the inkjet method, which prints by spraying small-sized droplets, the extrusion method, which pushes materials with pneumatic pressure or a piston, and curing by light. There is a stereolithography method in which a light source is irradiated to a photosensitive resin. 3D bioprinting is similar to conventional 3D printing and printing methods, but the difference is that biocompatible polymers are used as bioinks to print living cells.

Bioink contains living cells or biomolecules, and must provide physical properties for 3D processing and a biological environment for cells to perform their intended functions. Accordingly, the bioink must have excellent cell affinity and be able to protect cells from physical stress generated during the printing process. In addition, physical properties required in the printing process, such as repeatability of 3D patterning, productivity, and nozzle clogging, are required. In order to develop excellent bioprinting technology, it is essential to develop excellent bioink technology.

Currently, various polymer materials are being researched and developed as bioinks, and since synthetic polymers are toxic, their applications are limited, so bioinks using natural polymers with biocompatibility are mainly developed. Among these natural polymers, chitosan is a natural polysaccharide obtained from crustaceans and exhibits excellent biocompatibility, biodegradability and antibacterial properties, and is widely applied in the field of medicine and pharmacy. However, due to its slow gelation rate and weak mechanical properties, commercial chitosan-based bioinks do not exist, and studies are being conducted to improve structural stability by improving the weak mechanical properties of these natural polymers. Currently commercially available GelMa (gelatin methacryloyl) and CollMA (collagen methacryloyl) bioinks use a photoinitiator to introduce crosslinking to increase structural stability, but the use of photoirradiation and photoinitiator may damage cells. In addition, although alginate bioink can form a hydrogel through ionic crosslinking in the presence of calcium ions, an additional secondary crosslinking process is required due to its low structural stability.

Accordingly, the development of a better bioink is required.

Korean Patent Publication No. 10-2021-0110120 Korean Patent Registration No. 10-2145594

In order to solve the above problems, an object of the present invention is to provide a chitosan-based bioink composition capable of self-crosslinking without chemical additives and a method for preparing the same, while having fast gelation characteristics by preparing a chitosan polyphenol derivative having a high substitution degree. to be

In order to achieve the above object, the present invention provides a method comprising: 1) preparing a chitosan solution by dissolving chitosan in a solvent; 2) Gallic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxy in the chitosan solution preparing chitosan having a gallol group having a degree of substitution of 30% by adding and reacting with succinimide (N-hydroxysuccinimide; NHS); 3) dialysis to remove unreacted substances; 4) Freeze-drying to obtain chitosan having a gallol group introduced therein in powder form; 5) adding the gallol group-introduced chitosan to distilled water or pH 4.5 2-(N-morpholino)ethanesulfonic acid buffer (MES buffer) and stirring to prepare a gallol group-introduced chitosan solution; and 6) preparing a hydrogel by immersing or extruding the chitosan solution into which the gallol group is introduced in a phosphate buffer solution having a pH of 7.4, wherein the molar ratio of the chitosan, gallic acid, EDC, and NHS is 1:1:1.1:1.1 It provides a method for producing a bioink composition for 3D bioprinting, characterized in that.

The chitosan is characterized in that it has a degree of deacetylation of 75 to 85% and a molecular weight of 50,000 to 190,000 Da.

In step 2), the reaction is characterized in that the reaction is stirred for 6 hours to 24 hours under nitrogen gas at pH 4.5 to 6.0.

In step 3), the dialysis is performed for 6 to 12 hours.

In the step 6), the immersion or extrusion is characterized in that it is performed for 1 hour to 12 hours.

The hydrogel is characterized in that it contains a chitosan solution into which 9 wt% of a gallol group is introduced.

In one aspect, the present invention provides a bioink composition for 3D bioprinting prepared by the above manufacturing method and a support for tissue engineering comprising the same.

The 3D bioprinting is characterized by using a needle diameter of 25G (0.25 mm).

In another aspect, the present invention comprises the steps of 1) preparing a chitosan solution by dissolving chitosan in a solvent; 2) Gallic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxy in the chitosan solution preparing chitosan having a gallol group having a degree of substitution of 30% by adding and reacting with succinimide (N-hydroxysuccinimide; NHS); 3) dialysis to remove unreacted substances; 4) Freeze-drying to obtain chitosan having a gallol group introduced therein in powder form; 5) adding the gallol group-introduced chitosan to distilled water or pH 4.5 2-(N-morpholino)ethanesulfonic acid buffer (MES buffer) and stirring to prepare a gallol group-introduced chitosan solution; and 6) preparing a hydrogel by immersing or extruding the chitosan solution into which the gallol group is introduced in a phosphate buffer solution having a pH of 7.4, wherein the molar ratio of the chitosan, gallic acid, EDC, and NHS is 1:1:1.1:1.1 It provides a 3D bioprinting method, characterized in that.

The 3D bioprinting conditions are characterized by a hydrogel containing a chitosan solution into which 9 wt% of gallol groups are introduced, a printing speed of 3 mm/s, and an extrusion amount of 0.5%.

The bioink composition according to the present invention introduces a gallol group into chitosan, a natural polymer, and forms self-crosslinking through multiple interactions, so that a stable three-dimensional structure can be formed immediately after extrusion without chemical additives and post-processing, and the printing process is simple. , excellent printability.

In addition, it is biocompatible by using natural polymers instead of synthetic polymers, and it is possible to mix cells suitable for target tissues, so that bioinks with various functions can be manufactured.

In addition, it has excellent structural stability and mechanical properties due to its fast gelation property, and exhibits excellent morphological stability for a long period of time, so it can be used for tissue regeneration scaffolds, customized scaffolds (eg prosthetic limbs, prosthetic arms, artificial noses, artificial jawbones, etc.), artificial tissues and organs, etc. There are benefits that can be widely applied across industries.

1 shows a method for preparing a chitosan derivative into which a gallol group is introduced.
Figure 2 shows the gelation mechanism and printing process of the hydrogel of chitosan into which gallol group is introduced.
3 shows the results of chemical structure analysis of chitosan derivatives into which a gallol group is introduced. (a) ¹ H-NMR spectra, (b) degree of substitution according to gallic acid content, (c) UV-vis spectra, (d) XRD spectra, (e) ATR-IR spectra.
4 shows the results of chemical structure analysis of chitosan derivatives and hydrogels into which a gallol group is introduced. (a) ATR-IR spectra, (b) UV-vis spectra.
5 shows the rheological behavior analysis results of the hydrogel of chitosan into which the Galol group was introduced. (a) shear thinning, (b) thixotropic behavior.
6 shows the results of analyzing the printability of the chitosan-based bioink according to the concentration. (a) 3D structure image and optical image, (b) change in filament diameter, (c) change in pore diameter.
7 shows the results of analyzing the printability of chitosan-based bioink according to the needle diameter. (a) Optical image of the 3D structure, (b) filament diameter change, (c) pore diameter change.
8 shows the mechanical strength analysis results according to the crosslinking time of the hydrogel of chitosan into which the gallol group was introduced.
9 shows the results of analysis of the gelation behavior and structural stability of the 3D structure according to the pH of the coagulation bath of the hydrogel of chitosan into which a Gallol group was introduced.

Hereinafter, it will be described in detail with reference to embodiments of the present invention. In describing the present invention, if it is determined that a detailed description of a related known configuration or function may obscure the gist of the present invention, the detailed description will be omitted.

Conventional natural polymer-based bioinks not only have weak mechanical properties, but also can cause toxicity by crosslinking by photocrosslinking, ionic crosslinking, or enzymatic crosslinking, and have disadvantages in that the process is complicated and expensive. Accordingly, the present invention synthesizes a chitosan-polyphenol derivative with a high degree of substitution in order to solve the problems caused by the weak physical properties and the use of chemical additives of conventional natural polymer-based bioinks, and forms a chitosan hydrogel by crosslinking them to obtain a new 3D biomaterial. A bioink composition for printing was prepared.

Chitosan is a natural polysaccharide obtained by deacetylating chitin produced in crustaceans, and has biocompatibility, biodegradability, non-toxicity and antibacterial properties. However, its use is limited due to its low water solubility and high stiffness and brittleness. In the present invention, in order to overcome these limitations, gallic acid, a phenolic compound, was introduced into chitosan to improve its physicochemical and biological properties. Gallic acid is a phenolic compound with three hydroxyl groups (-OH), which can be combined in various ways, and has excellent antioxidant performance, antibacterial properties, and cell compatibility. In particular, gallic acid can form a quinone structure when oxidized to form covalent bonds with chitosan such as Schiff base and Michael addition, in addition to hydrogen bonds and π-π interactions can form non-covalent bonds. Figure 2 shows the gelation mechanism and printing process of chitosan hydrogel in which gallol groups are introduced. Due to the presence of a large amount of gallol groups, gelation is performed immediately upon extrusion in a PBS buffer solution (pH 7.4), enabling the production of structurally stable 3D structures. do.

The chitosan-polyphenol derivative is obtained by introducing a gallol group into chitosan through an EDC/NHS coupling reaction between chitosan and gallic acid.

The chitosan-polyphenol derivative induces cross-linking covalent bonds by oxidation of gallol groups in a PBS solution (pH 7.4), which is a pH condition in vivo, to prepare a bioink composition for 3D bioprinting without chemical additives such as crosslinking agents or initiators. can

In addition, the prepared chitosan derivative bioink composition having a high degree of substitution according to the present invention is directly extruded into a PBS solution to induce gelation immediately after printing, so that a structurally stable 3D structure can be prepared without a post-treatment process, and the shape can be maintained for a long time in the body. can keep This provides a new 3D bioprinting method that solves the problems caused by the use of chemical additives.

A method for preparing a bioink composition for 3D bioprinting according to the present invention includes the steps of 1) preparing a chitosan solution by dissolving chitosan in a solvent; 2) Gallic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxy in the chitosan solution preparing chitosan having a gallol group having a degree of substitution of 30% by adding and reacting with succinimide (N-hydroxysuccinimide; NHS); 3) dialysis to remove unreacted substances; 4) Freeze-drying to obtain chitosan having a gallol group introduced therein in powder form; 5) adding the gallol group-introduced chitosan to distilled water or pH 4.5 2-(N-morpholino)ethanesulfonic acid buffer (MES buffer) and stirring to prepare a gallol group-introduced chitosan solution; and 6) preparing a hydrogel by immersing or extruding the chitosan solution into which the gallol group is introduced in a phosphate buffer solution having a pH of 7.4, wherein the molar ratio of the chitosan, gallic acid, EDC, and NHS is 1:1:1.1:1.1 It is characterized by being

For example, in step 1), the solvent for preparing the chitosan solution may be an aqueous acetic acid solution, but is not limited thereto.

Step 2) is a step of introducing a gallol group into chitosan through an EDC/NHS coupling reaction between chitosan and gallic acid, and the degree of substitution of the chitosan derivative can be controlled by adjusting the ratio between chitosan and gallic acid. The chitosan and gallic acid are preferably mixed in a molar ratio of 1:0.1 to 1:2, more preferably in a molar ratio of 1:1. In addition, it is preferable that the chitosan, EDC, and NHS are mixed in a molar ratio of 1:1.1:1.1, but is not limited thereto. For example, in the case of a chitosan derivative having a degree of substitution of about 30%, the molar ratio of chitosan, gallic acid, EDC, and NHS may be 1:1:1.1:1.1, but is not limited thereto.

In addition, in step 2), the pH of the reaction solution is preferably adjusted to 4.5 to 6.0 to prevent oxidation of the chitosan derivative during the reaction, more preferably pH 5.0, but is not limited thereto. At this time, the reaction time may be preferably 6 hours to 24 hours, more preferably 8 hours to 16 hours, but is not limited thereto.

Step 3) is a step of removing unreacted gallic acid, EDC, and NHS. It is preferable to adjust the pH of the dialysis solution to 4.5 to 6.0 to prevent oxidation of the chitosan derivative during the dialysis process, more preferably the pH. It is desirable to adjust to 5.0. The dialysis process is preferably performed for 6 to 12 hours, more preferably 7 to 10 hours, but is not limited thereto.

Step 5) is a step of preparing a chitosan derivative solution by dissolving the chitosan derivative in a solvent, distilled water or a 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution. (pH 4.5), but is not limited thereto.

Step 6) is a step of forming a hydrogel by inducing a cross-linked covalent bond by oxidation of a gallol group in the chitosan derivative solution at pH 7.4, which is an in vivo pH condition. At this time, no chemical additives are required and only pH change is used. Thus, a hydrogel is formed through self-crosslinking between gallols. In this case, the crosslinking time may be preferably 1 hour to 24 hours, more preferably 1 hour to 20 hours, and still more preferably 1 hour to 16 hours, but is not limited thereto.

In another aspect, the present invention includes a bioink composition for 3D bioprinting prepared by the above manufacturing method and a support for tissue engineering including the same.

The 3D bioprinting preferably uses a needle diameter of 25 G (0.25 mm), but is not limited thereto.

In another aspect, the present invention provides a method comprising: 1) preparing a chitosan solution by dissolving chitosan in a solvent; 2) Gallic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxy in the chitosan solution preparing chitosan having a gallol group having a degree of substitution of 30% by adding and reacting with succinimide (N-hydroxysuccinimide; NHS); 3) dialysis to remove unreacted substances; 4) Freeze-drying to obtain chitosan having a gallol group introduced therein in powder form; 5) adding the gallol group-introduced chitosan to distilled water or pH 4.5 2-(N-morpholino)ethanesulfonic acid buffer (MES buffer) and stirring to prepare a gallol group-introduced chitosan solution; and 6) preparing a hydrogel by immersing or extruding the chitosan solution into which the gallol group is introduced in a phosphate buffer solution having a pH of 7.4, wherein the molar ratio of the chitosan, gallic acid, EDC, and NHS is 1:1:1.1:1.1 It provides a 3D bioprinting method, characterized in that.

In step 6), the chitosan derivative solution is directly extruded into the PBS buffer solution to induce gelation immediately after printing, thereby manufacturing a structurally stable 3D structure without post-processing. The high substitution chitosan derivative according to the present invention has a high gelation rate and improved gel strength due to a large amount of covalent bonds caused by gallol oxidation. In addition, the printability and mechanical strength of the structure can be controlled through various process parameters.

The 3D bioprinting conditions are preferably, but not limited to, a hydrogel containing a 9 wt% gallol group-introduced chitosan solution, a printing speed of 3 mm/s, and an extrusion amount of 0.5%.

Hereinafter, examples of chitosan derivatives and hydrogels into which a gallol group is introduced according to the present invention will be described in detail, but the present invention is not limited to the following examples.

< Example 1> Galolgiga Preparation of introduced chitosan derivatives

Referring to FIG. 1 , a chitosan derivative having a gallol group introduced therein is prepared by forming an amide bond between an amine group (—NH ₂ ) of chitosan and a carboxyl group (—COOH) of gallic acid through an EDC/NHS reaction. Chitosan (CS) has a degree of deacetylation of 75 to 85% and has a molecular weight of 50,000 to 190,000 Da.

First, 1 g of chitosan was dissolved in 100 mL of an acetic acid aqueous solution (0.5%) to prepare a 1 wt% chitosan solution. Then, gallic acid (GA), EDC (1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide), and NHS (N-hydroxysuccinimide) were added to the chitosan solution in a molar ratio of 1:0.1 to 2. In the case of a chitosan derivative having a degree of substitution of about 30%, the molar ratio of chitosan to gallic acid is 1:1, and the molar ratio of chitosan to EDC and NHS is 1:1:1. The pH of the reaction solution was adjusted to 5.0 to prevent oxidation during the reaction, and the reaction was stirred for 12 hours at room temperature under nitrogen gas. In order to remove unreacted gallic acid, EDC and NHS, it was dialyzed in an aqueous solution of pH 5.0 for 8 hours. Lyophilization was performed for 7 days to prepare a chitosan derivative in powder form.

< Example 2> Galolgiga Introduced Chitosan Hydrating gel manufacturing

A chitosan derivative having a gallol group was added to distilled water or MES (2-(N-morpholino)ethanesulfonic acid) buffer solution (pH 4.5), stirred at room temperature for 2 hours, and then the prepared solution was mixed with PBS buffer solution (pH 7.4). A hydrogel was prepared by immersion or extrusion.

< Experimental example 1> Galolgiga Chemical structure analysis of introduced chitosan derivatives

Through ¹ H-NMR analysis, changes in the chemical structure and degree of substitution of the chitosan derivative into which the gallol group was introduced were calculated. Chitosan and chitosan-gallic acid derivatives were dissolved in CD ₃ COOD/D ₂ O (1%), and spectra of each solution were obtained using ¹ H-NMR spectroscopy. The degree of substitution was calculated through the peak area of aromatic protons present in gallic acid relative to acetyl group protons present in the chitosan structure. 3a and 3b, a gallol group was introduced into the chitosan polymer chain, and a peak due to benzene ring appeared at around 7 ppm, showing a high degree of substitution of up to about 30%. As a result of UV-vis analysis, a gallol group-induced peak appeared at 270 nm in the chitosan derivative having a gallol group introduced (Fig. 3c), and as a result of XRD analysis, it was confirmed that the crystallinity decreased due to the introduction of a gallol group (Fig. 3d). Referring to FIG. 3e, as a result of ATR-IR analysis, in the case of chitosan derivatives into which a gallol group was introduced, the peak intensity due to the hydroxyl group (-OH) group of the benzene ring increased at 3,383 cm ^-1 and ranged from 1,648 cm ^-1 to 1,635 cm The peak due to amide I was shifted to ^-1 and a more intense peak appeared. In addition, the peak expressed by the amine group of chitosan at 1,584 cm ^-1 was removed as the gallol group was introduced in the case of chitosan derivatives, and the peak by amide II shifted from 1,560 cm ^-1 to 1,531 cm ^-1 and a more intense peak appear. Through the above results, it was confirmed that the gallol group was successfully introduced into the chitosan polymer chain.

< Experimental example 2> Galolgiga introduced chitosan derivatives and of hydrogel chemical structure analysis

Through ATR-IR and UV-vis analysis, changes in the chemical structure of the chitosan derivative and the hydrogel in which the gallol group was introduced were confirmed. Referring to FIG. 4a, as a result of ATR-IR analysis, in the case of the chitosan hydrogel into which the gallol group was introduced, the gallol group was transformed into a quinone structure, and the peak due to the hydroxy group (-OH) was shifted from 3,214 cm ^-1 to 3,271 cm ^-1 . In addition, a peak at 1,694 cm ⁻¹ due to a C=N bond appeared, and a peak at 1,640 cm ⁻¹ due to biphenol having a modified benzene ring.

Referring to FIG. 4b, as a result of UV-vis analysis, as a hydrogel was formed, the gallol group was oxidized at 350 to 400 nm, and a peak caused by semiquinone intermediates and radicals appeared, and at around 500 nm, the gap between the gallol group and the amine group of chitosan A peak due to binding appeared. Through this, it was confirmed that a hydrogel was formed through covalent bonding by Schiff base and Michael addition reaction.

< Experimental example 3> Galolgiga Introduced Chitosan of hydrogel rheological behavioral analysis

In applying bioink to 3D printing, the rheological behavior of bioink acts as an important factor. The rheological behavior of the chitosan hydrogel into which gallol groups were introduced was measured using a rheometer having a plate shape (diameter: 20 mm, gap: 1 mm). At this time, the strain and frequency are 1% and 1 rad/s. Figure 5a shows the shear thinning behavior in which the viscosity decreases with increasing shear force. As a result, when extruding the hydrogel, the viscosity decreases due to shear thinning behavior, thereby facilitating extrusion. 5b shows the thixotropic behavior of the hydrogel, which means that after extrusion, the hydrogel recovers to its original shape by elastic restoring force and can stably maintain the shape of the printed 3D structure.

< Experimental example 4> Analysis of printability of chitosan-based bioink according to concentration

6 is data obtained by evaluating the printability according to the concentration of the hydrogel using the chitosan-based hydrogel having a high degree of substitution prepared in the above example. At this time, the optimal conditions were established through analysis of various variables (concentration, printing speed, extrusion amount, needle diameter, etc.) for printing conditions, and then printing in a PBS buffer solution (pH 7.4) after putting a chitosan solution into which gallol groups were introduced into a syringe. proceeded. When hydrogels with concentrations of 7 wt%, 9 wt%, and 11 wt% were prepared and extruded, a large amount of galol groups introduced into the chitosan polymer chain in the PBS buffer solution were oxidized to form covalent bonds, resulting in 3D gelation immediately after extrusion. structure was formed. The diameter and pores of the prepared 3D structure were measured through an optical microscope.

As a result, the hydrogel of 7 wt% concentration spread the solution due to its low viscosity, forming thick filaments and circular pores, and the hydrogel of 11 wt% concentration formed uneven filaments and crushed pores due to its high viscosity. did On the other hand, the hydrogel at the concentration of 9 wt% had an appropriate viscosity and formed pores with a uniform square filament surface. Therefore, it was confirmed that the use of a hydrogel having a concentration of 9 wt% made it possible to manufacture a 3D structure having excellent shape stability and easy extrusion.

< Experimental example 5> You guys in diameter Analysis of printability of chitosan-based bioink according to

7 is data obtained by evaluating printability according to needle diameter using the chitosan-based hydrogel having a high degree of substitution prepared in the above example. When needle diameters of 21G (0.514 mm) and 23G (0.337 mm) were used, thick filaments and circular pores were formed. When needle diameters of 28G (0.184 mm) were used, non-uniform filaments were formed and Aggregation occurred. On the other hand, when a needle diameter of 25 G (0.25 mm) was used, uniform filaments were continuously formed. Therefore, printing was performed by setting the final printing conditions to 9 wt% hydrogel, 3 mm/s printing speed, and 0.5% extrusion amount.

< Experimental example 6> Galolgiga Introduced Chitosan of hydrogel in bridge time Analysis of mechanical strength and structural stability according to

8 is data obtained by measuring the mechanical strength of the hydrogel of chitosan into which gallol groups were introduced according to the crosslinking time. The hydrogel was prepared in a cylindrical shape (diameter: 15 mm, height: 10 mm), stabilized in a PBS buffer solution for each time, and then the compressive strength of the hydrogel was measured using a texture analyzer (TA). . High compressive strength was exhibited even with a short crosslinking time, and as the crosslinking time increased, the compressive strength increased and showed excellent compressive strength of up to about 350 kPa.

9 is an evaluation of the structural stability of the hydrogel according to the pH of the coagulation bath. In weakly acidic (pH 4.5) or neutral (pH 6.0) conditions, crosslinking was not formed and the shape of the 3D structure was not maintained due to dissolution or decomposition. . On the other hand, under the same weakly basic (pH 7.4) condition as the in vivo condition, it gelled immediately after extrusion to form a 3D structure having structural stability. From these results, it can be seen that the pH condition has an important effect on the formation of gelation, and it is possible to prepare a 3D structure with excellent structural stability through rapid gelation formation by preparing a chitosan derivative with a high degree of substitution. The 3D structure prepared in this way not only has morphological stability and biocompatibility, but also can be maintained for a long time in the body, so it is considered that it can be applied as a substitute for tissues and organs.

As described above, it was confirmed through chemical structure analysis that the chitosan derivative having a large amount of gallol group introduced therein was successfully prepared, and through rheological analysis, it was confirmed that it had suitable properties as a bioink for 3D bioprinting, and it had excellent printability and printability. of mechanical strength was realized.

The above description is merely illustrative of the present invention, and those skilled in the art will be able to make various modifications without departing from the essential characteristics of the present invention. Accordingly, the embodiments disclosed in this specification are intended to explain, not limit, the present invention, and the spirit and scope of the present invention are not limited by these embodiments. The protection scope of the present invention should be construed by the following claims, and all technologies within the equivalent range should be construed as being included in the scope of the present invention.

Claims (11)

1) preparing a chitosan solution by dissolving chitosan in a solvent;
2) Gallic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxy in the chitosan solution preparing chitosan having a gallol group having a degree of substitution of 30% by adding and reacting with succinimide (N-hydroxysuccinimide; NHS);
3) dialysis to remove unreacted substances;
4) Freeze-drying to obtain chitosan having a gallol group introduced therein in powder form;
5) adding the gallol group-introduced chitosan to distilled water or pH 4.5 2-(N-morpholino)ethanesulfonic acid buffer (MES buffer) and stirring to prepare a gallol group-introduced chitosan solution; and
6) preparing a hydrogel by immersing or extruding the chitosan solution into which the gallol group is introduced in a phosphate buffer having a pH of 7.4;
Method for producing a bioink composition for 3D bioprinting, characterized in that the molar ratio of chitosan, gallic acid, EDC and NHS is 1: 1: 1.1: 1.1
The method of claim 1, wherein the chitosan has a degree of deacetylation of 75 to 85% and a molecular weight of 50,000 to 190,000 Da.
The method for preparing a bioink composition for 3D bioprinting according to claim 1, wherein in step 2), the reaction is stirred for 6 hours to 24 hours under nitrogen gas at a pH of 4.5 to 6.0.
The method of claim 1, wherein in step 3), the dialysis is performed for 6 to 12 hours.
The method of claim 1, wherein in step 6), the immersion or extrusion is performed for 1 hour to 12 hours.
The method of claim 1, wherein the hydrogel comprises a chitosan solution into which 9 wt% of a gallol group is introduced.
Bioink composition for 3D bioprinting prepared by the manufacturing method according to any one of claims 1 to 6
The bioink composition for 3D bioprinting according to claim 7, wherein the 3D bioprinting uses a needle diameter of 25G (0.25 mm).
Support for tissue engineering comprising the bioink composition according to claim 7
1) preparing a chitosan solution by dissolving chitosan in a solvent;
2) Gallic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxy in the chitosan solution preparing chitosan having a gallol group having a degree of substitution of 30% by adding and reacting with succinimide (N-hydroxysuccinimide; NHS);
3) dialysis to remove unreacted substances;
4) Freeze-drying to obtain chitosan having a gallol group introduced therein in powder form;
5) adding the gallol group-introduced chitosan to distilled water or pH 4.5 2-(N-morpholino)ethanesulfonic acid buffer (MES buffer) and stirring to prepare a gallol group-introduced chitosan solution; and
6) preparing a hydrogel by immersing or extruding the chitosan solution into which the gallol group is introduced in a phosphate buffer having a pH of 7.4;
3D bioprinting method, characterized in that the molar ratio of the chitosan, gallic acid, EDC and NHS is 1: 1: 1.1: 1.1
11. The 3D bioprinting method according to claim 10, wherein the 3D bioprinting conditions are a hydrogel containing a chitosan solution into which 9 wt% of gallol groups are introduced, a printing speed of 3 mm/s, and an extrusion amount of 0.5%.

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