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KR101875264B1 - Bio-ink for fast gelation based on functional hydrogels and manufacturing method thereof - Google Patents

Bio-ink for fast gelation based on functional hydrogels and manufacturing method thereof Download PDF

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KR101875264B1
KR101875264B1 KR1020160011488A KR20160011488A KR101875264B1 KR 101875264 B1 KR101875264 B1 KR 101875264B1 KR 1020160011488 A KR1020160011488 A KR 1020160011488A KR 20160011488 A KR20160011488 A KR 20160011488A KR 101875264 B1 KR101875264 B1 KR 101875264B1
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hydrogel
hyaluronic acid
solution
acrylated
present
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KR1020160011488A
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Korean (ko)
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KR20170090755A (en
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박용두
이재연
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고려대학교 산학협력단
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    • CCHEMISTRY; METALLURGY
    • C09DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09DCOATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
    • C09D11/00Inks
    • C09D11/02Printing inks
    • CCHEMISTRY; METALLURGY
    • C09DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09DCOATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
    • C09D11/00Inks
    • C09D11/02Printing inks
    • C09D11/10Printing inks based on artificial resins
    • CCHEMISTRY; METALLURGY
    • C09DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09DCOATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
    • C09D4/00Coating compositions, e.g. paints, varnishes or lacquers, based on organic non-macromolecular compounds having at least one polymerisable carbon-to-carbon unsaturated bond ; Coating compositions, based on monomers of macromolecular compounds of groups C09D183/00 - C09D183/16

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  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Materials Engineering (AREA)
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  • Organic Chemistry (AREA)
  • Materials For Medical Uses (AREA)

Abstract

The present invention relates to a bio ink composition comprising a first hydrogel containing an acrylic group and a second hydrogel comprising a phenol group, and more particularly, to a bio ink composition containing a hydrogel, A bio-ink composition capable of rapidly forming a three-dimensional structure, and a method of producing the same.

Description

TECHNICAL FIELD [0001] The present invention relates to a high-speed curing bio-ink based on a functional hydration gel, and a method for manufacturing the same. BACKGROUND OF THE INVENTION < RTI ID = 0.0 >

More particularly, the present invention relates to a bio ink composition comprising a first hydrogel containing an acrylic group and a second hydrogel containing a phenol group, and a method for producing the same. And a manufacturing method thereof.

As 3D printing technology develops, 3D printing technology is being used in the medical field to manufacture products that require customization, such as hearing aids and dental prostheses. In particular, the laminate molding method, which is the main method of 3D printing, is widely used in the manufacture of small-sized medical products such as customized hearing aids, dental implants, and surgical implants that need to be customized for individual patients. Such lamination molding technology has a possibility to construct a cell culture without inserting a biocompatible substance or skeleton for tissue fixation, which can not be found in a natural cell tissue, and thus is highly likely to be used. 3D printing techniques designed for abiotic applications typically use organic solvents or crosslinking agents that are incompatible with living cells and biomaterials. In 3D bioprinting technology, however, one of the major issues is finding materials that are well compatible with biological materials and that can provide tissue structures with desired mechanical and functional properties.

At present, naturally derived polymers (alginate, gelatin, collagen, chitosan, fibrin and hyaluronic acid) and synthetic molecules (polyethylene glycol) obtained from animal or human tissues are widely used in the field of regenerative medicine. For example, in the case of artificial blood vessel materials, Polytetrafluoroethylene (PTFE) material which is stable to heat, easy to disinfect and has excellent hydrophobicity is used in consideration of the physical and chemical properties of the inner wall of vessels and mass transfer system. In addition, when replacing damaged bone or hard tissue, stainless steel or titanium material may be used. In the case of skin regeneration, 3D printing may be performed using biomaterial such as chitosan or collagen. Particularly, biocompatible chitosan is used for coating which enhances the bonding force between bones and implants by utilizing the property of stimulating bone cell activity. Hydrophilic hydroxyapatite, which is a calcium phosphate ceramic material, promotes bone growth, Good conformability and bone conduction are used as bone substitutes. In addition, hydrophilic and hygroscopic synthetic hydrogels can be easily controlled in their physical properties during the synthesis process, and thus they are being utilized in 3D bio-printing regenerative medicine fields. However, most of these hydrogels are hard to withstand mechanical loads, and have a disadvantage in that it is difficult to form a specific structure quickly due to a slow curing time.

In the field of tissue engineering and regenerative medicine, in order to restore damaged tissue and regeneration through proper delivery of cells and growth factors, which are therapeutic substances, various methods using supports in order to maximize the engraftment and differentiation regulation ability of stem cells in vivo Are being studied. For effective tissue regeneration, various growth factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF) Studies on techniques for promoting angiogenesis are needed. For example, a technique for smart release scaffold in which the degree of release of growth factors is controlled according to in vivo activity is useful in the field of regenerative medicine. In particular, when such a support is applied to the bio-ink of 3D printing technology, it can be utilized for research on organ structure reconstruction capable of maintaining growth factors and cell activity.

It is an object of the present invention to include a first hydrogel containing an acrylic group and a second hydrogel containing a phenol group so that the growth factor and cell activity can be maintained while the curing rate can be shortened to rapidly form a three- And a method for producing the same.

However, the problems to be solved by the present invention are not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

A first aspect of the present invention provides a bio ink composition comprising a first hydrogel comprising an acrylic group and a second hydrogel comprising a phenol group.

According to one embodiment, the first hydrogel may be contained in an amount of 2 to 15% by weight, and the second hydrogel may be included in an amount of 1 to 10% by weight.

According to one embodiment, the mixing ratio of the first hydrogel and the second hydrogel may be 1: 1 to 7: 1.

According to one embodiment, the bio-ink composition may further comprise a peptide comprising an amino acid sequence cleavable by MMP and a cysteine at the terminal, hydrogen peroxide, and HRP (Horseradish peroxidase).

According to one embodiment, the concentration of the hydrogen peroxide may be 0.05 to 0.1 mM.

According to one embodiment, the first hydrogel may be acrylated-hyaluronic acid (HA-ac), and the second hydrogel may be a tyraminized-hyaluronic acid (HA-Tyr).

According to one embodiment, the molecular weight of the acrylated-hyaluronic acid (HA-ac) may be from 10 kDa to 800 kDa.

According to one embodiment, the hardness of the bio-ink composition after curing may be 1 x 10 4 Pa to 1 x 10 6 Pa.

According to a second aspect of the present invention, there is provided a method of preparing a hydrogel, comprising: preparing a first hydrogel solution containing an acryl group; Preparing a second hydrogel solution comprising a phenol group; Mixing the first hydrogel solution and the second hydrogel solution; And adding a peptide, hydrogen peroxide, and HRP (Horseradish peroxidase) containing an amino acid sequence cleavable by MMP and cysteine to the mixture, to the mixture.

According to one embodiment, preparing the first hydrogel solution comprises dissolving a first hydrogel containing an acrylic group in a buffer solution at 2 to 15 wt%, and the second hydrogel solution is prepared by dissolving The preparing step may include the step of dissolving the second hydrogel containing the phenol group in the buffer solution in an amount of 1 to 10% by weight.

According to one embodiment, mixing the first hydrogel solution and the second hydrogel solution may include mixing the first hydrogel solution and the second hydrogel solution in a ratio of 1: 1 to 7: 1 have.

According to one embodiment, the concentration of hydrogen peroxide may be 0.05 to 1 mM.

According to one embodiment, the first hydrogel may be acrylated-hyaluronic acid (HA-ac), and the second hydrogel may be thylamin-hyaluronic acid (HA-Tyr).

The present invention relates to a bio ink composition comprising a first hydrogel containing an acrylic group and a second hydrogel containing a phenol group and a method for producing the same, wherein the growth factor and the activity of the cell are maintained while the curing rate is shortened Since it can form a three-dimensional structure quickly, it can be applied to bioprinting to form a structure by stacking it in layers, and it is possible to control the intensity so as to support the structure even after printing.

1 is a schematic view showing a method of manufacturing a bio ink composition according to an embodiment of the present invention.
2 is a schematic diagram of a peptide and a hydrogel containing the same according to an embodiment of the present invention.
3 is a photograph showing a cytotoxicity test on hydrogen peroxide concentration according to an embodiment of the present invention.
FIG. 4 is a graph illustrating gelation time according to concentration and ratio of each component according to an embodiment of the present invention.
5 is a rheological graph of a bio ink according to an embodiment of the present invention.

In the following, embodiments will be described in detail with reference to the accompanying drawings. Like reference symbols in the drawings denote like elements.

Various modifications may be made to the embodiments described below. It is to be understood that the embodiments described below are not intended to limit the embodiments, but include all modifications, equivalents, and alternatives to them.

The terms used in the examples are used only to illustrate specific embodiments and are not intended to limit the embodiments. The singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, the terms "comprises" or "having" and the like refer to the presence of stated features, integers, steps, operations, elements, components, or combinations thereof, But do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, or combinations thereof.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this embodiment belongs. Terms such as those defined in commonly used dictionaries are to be interpreted as having a meaning consistent with the contextual meaning of the related art and are to be interpreted as either ideal or overly formal in the sense of the present application Do not.

In the following description of the present invention with reference to the accompanying drawings, the same components are denoted by the same reference numerals regardless of the reference numerals, and redundant explanations thereof will be omitted. In the following description of the embodiments, a detailed description of related arts will be omitted if it is determined that the gist of the embodiments may be unnecessarily blurred.

A first aspect of the present invention provides a bio ink composition comprising a first hydrogel comprising an acrylic group and a second hydrogel comprising a phenol group.

As used herein, the term "hydrogel" means a water-swellable polymer that can be used as a medical material such as a drug delivery system or a biostructural synthetic material. Such a water-swellable polymer is a hydrophilic polymer that absorbs water but does not dissolve in water, that is, a hydrophilic polymer having a sufficient amount of water. The hydrophilic polymer includes natural polymers such as hyaluronic acid (HA), collagen, and polyvinyl alcohol (vinyl alcohol), PVA), polyhydroxyethyl methacrylate (PHEMA), and the like.

The hydrogel that can be used in the present invention is preferably a hyaluronic acid-based hydrogel. The hyaluronic acid is a biodegradable and biocompatible natural polymer having a broad molecular weight range of about 1,000 to 10,000,000 g / mole. This hyaluronic acid is a major component of the extracellular matrix (ECM) of the connective tissue and has been studied as a useful material for arthritis treatment, ophthalmic surgery, drug delivery and tissue engineering applications due to its unique viscoelastic properties and biological functions. It regulates hydration and forms a hydrated network with collagen fibers in the ECM and is also known to play an important role in wound healing and cell fluidity and differentiation in the process.

The bio ink composition of the present invention comprises a first hydrogel comprising an acrylic group. The acrylic group of the first hydrogel may bind to a polypeptide having a cysteine amino acid at the C 'terminus, more specifically, an SH group of cysteine may form an amide bond with an acryl group. (1-ethylbenzotriazole hydrate), EDC (1-Ethyl-3- (3-dimethylaminopropyl) -carbodimide), ADH (adipic acid dihydrazide) and NAS (NAcryloxysuccinimide) in order to introduce an acrylic group into the hydrogel. At least one selected may be used, but is not limited thereto. For example, it is possible to react Hyaluronic Acid with HOBT, EDC or ADH (adipic acid dihydrazide), dialyze and react with NAS (NAcryloxysuccinimide) to introduce acrylic into the hydrogel. The molecular weight of the first hydrogel into which the acrylic group is introduced may be from 10 kDa to 800 kDa, and preferably from 50 kDa to 300 kDa.

The bio-ink composition of the present invention comprises a second hydrogel comprising a phenol group. The second hydrogel containing phenolic groups can be cured by reacting with hydrogen peroxide and HRP (horseradish peroxidase). In order to introduce a phenol group into the hydrogel, at least one selected from the group consisting of trymine, hydroxyphenylacetic acid, hydroxypropionic acid and derivatives thereof may be used, but is not limited thereto. For example, when tyramine having a phenol group is introduced into hyaluronic acid, EDC. The carboxyl group of hyaluronic acid and the amine group of tyramine can be induced to bind by using HCl (1-Ethyl-3- (3-dimethylaminopropyl) -carbodiimide. Hydrochloride) and NHS (N-hydroxysuccinimide). The molecular weight of the second hydrogel into which the phenol group is introduced may be 50 kDa to 300 kDa, and preferably 90 kDa to 200 kDa.

According to an embodiment of the present invention, the first hydrogel may be acrylated-hyaluronic acid (HA-ac) and the second hydrogel may be a tyraminized-hyaluronic acid (HA-Tyr) Do not.

According to an embodiment of the present invention, the first hydrogel may be contained in an amount of 2 to 15 wt%, and the second hydrogel may be contained in an amount of 1 to 10 wt%. The content of the first hydrogel may vary depending on the molecular weight. For example, in the case of 50 kDa, 8-12 wt%, 230 kDa, 3-5 wt%, or 800 kDa, % ≪ / RTI > by weight. In addition, the second hydrogel may be contained in an amount of 1 to 10% by weight.

According to an embodiment of the present invention, the mixing ratio of the first hydrogel and the second hydrogel may be 1: 1 to 7: 1. The curing time may vary depending on the mixing ratio of the first hydrogel and the second hydrogel. For example, when the mixing ratio of the first hydrogel and the second hydrogel is 1: 1, 120 seconds, and may be 3 to 10 seconds when the mixing ratio of the first hydrogel and the second hydrogel is 7: 1.

According to an embodiment of the present invention, the bio ink composition may further comprise a peptide containing an amino acid sequence cleavable by MMP and a cysteine at the terminal, hydrogen peroxide, and HRP (Horseradish peroxidase).

As a peptide comprising an amino acid sequence cleavable by the MMP and a cysteine at the terminal, which can be contained in the bio ink composition of the present invention, this is a synthetic peptide in which cysteine is added to the functional peptide C terminal. The number of amino acids constituting the peptide is not limited, but preferably 10 to 300 amino acids are preferred. Peptides that can be used in the present invention include, for example, amino acid sequences of thymosin beta-4 derived Ac-SDKP peptide, (AcSDKPDGPQGIWQC), Substance P (RPKPQQFFGLMC), BMP-7 peptide (GQGFSPYKAVFSTQC), and osteopontin (PSKSNESHDHMDDMDDC) Or a peptide comprising an amino acid sequence capable of being delivered by plasmin

As used herein, the term "MMP" is an enzyme that dissolves the extracellular matrix and is one of the four major proteolytic enzymes (seminethreonine, aspartic-, cysteine- and metalloproteinase) The term "plasmin" is a proteolytic enzyme present in blood, for example, a proteolytic enzyme that dissolves fibrin involved in blood coagulation. The term "Thymosin beta-4" as used in the present invention is a very small-sized protein existing in vivo composed of 43 amino acids of 4.9 kDa in size. The thymosin beta-4 protein Actin binds to a single, separate form of G-actin to prevent the formation of F-actin, a form of G-actin binding. These G-actin and F-actin Is a protein that modulates the structure of the actin cytoskeleton and regulates cell migration by regulating the transcription of the actin cytoskeleton.

The peptides that can be used in the present invention are those in which a D (Aspartic acid, Asp) amino acid for peptidase cleavage is added between a thymosin beta-4 derived Ac-SDKP peptide and a peptide cleavable by MMP or plasmin Which can be cleaved by a prolyl endopeptidase or Asp-N-like protease in the body to release the Ac-SDKP peptide. Also, the peptides cleavable by MMP or plasmin can be any peptide sequence known in the art on MMP or plasmin cuts, for example, GPQGIWGQ for MMP and LIKMKPM for plasmin. In particular, when an MMP cleavage site is present, it can be cleaved by MMP and effectively release Ac-SDKP peptide to the target site. The thymosin beta-4-derived Ac-SDKP peptide is preferably acetylated.

The Ac-SDKP peptide (Ac-SDKP-D-GPQG-IWGQ-C, MW 1529.6) derived from thymosin beta-4 according to one aspect of the present invention is bound to a first hydrogel containing an acrylic group And hydrogen peroxide and HRP can bind with a second hydrogel containing a phenol group, so that they act as a cross-linking agent for the first hydrogel and the second hydrogel.

According to an embodiment of the present invention, the concentration of the hydrogen peroxide may be 0.05 to 0.1 mM. Hydrogen peroxide is a cytotoxic substance. When the concentration is higher than 0.1 mM, the cell viability may be lowered. When the concentration is lower than 0.05 mM, the gel may not be cured properly.

In addition, according to one embodiment of the present invention, the HRP is not a substance having a large cytotoxicity but may affect the curing time of the gel depending on the concentration, and it is preferable that the HRP is included in 1 to 10 units.

According to an embodiment of the present invention, the bio ink composition may further include PEG (Poly Ethylene Glycol) or a thiolated polyethylene glycol PEG-SH 4 as a crosslinking agent.

According to one embodiment of the present invention, the hardness measured after curing of the bio ink composition may be 1 x 10 4 Pa to 1 x 10 6 Pa. The hardness of the bio ink composition according to an embodiment of the present invention can be controlled by using the concentration of the hydrogel, the mixing ratio, the concentration of hydrogen peroxide, etc., and it is also possible to control the hardness so as to have a strong strength It is possible.

According to a second aspect of the present invention, there is provided a method of preparing a hydrogel, comprising: preparing a first hydrogel solution containing an acryl group; Preparing a second hydrogel solution comprising a phenol group; Mixing the first hydrogel solution and the second hydrogel solution; And adding a peptide, hydrogen peroxide, and HRP (Horseradish peroxidase) containing an amino acid sequence cleavable by MMP and cysteine to the mixture, to the mixture.

1 is a schematic view showing a method of manufacturing a bio ink composition according to an embodiment of the present invention.

The method for providing a bio ink composition of the present invention comprises preparing a first hydrogel solution containing an acrylic group and a second hydrogel solution containing a phenol group, respectively. According to an embodiment of the present invention, the step of preparing the first hydrogel comprises dissolving a first hydrogel containing an acrylic group in a buffer solution at 2 to 15 wt%, and the second hydrogel May comprise the step of dissolving the second hydrogel comprising phenolic groups in a buffer solution at 1 to 10% by weight. The content of the first hydrogel or the second hydrogel dissolved in the buffer solution may vary depending on the molecular weight of the hydrogel. For example, the hydrogel may be contained in an amount of 8 to 12 wt% in the case of 50 kDa, 3 to 5 wt% in the case of 230 kDa, or 2 to 3 wt% in the case of 800 kDa.

According to an embodiment of the present invention, mixing the first hydrogel solution and the second hydrogel solution comprises mixing the first hydrogel solution and the second hydrogel solution at a ratio of 1: 1 to 7: 1 . The curing time may vary depending on the mixing ratio of the first hydrogel and the second hydrogel. For example, when the mixing ratio of the first hydrogel and the second hydrogel is 1: 1, 120 seconds, and may be 3 to 10 seconds when the mixing ratio of the first hydrogel and the second hydrogel is 7: 1.

According to an embodiment of the present invention, the concentration of the hydrogen peroxide may be 0.05 to 1 mM. Hydrogen peroxide is a cytotoxic substance. When the concentration is higher than 0.1 mM, the cell viability may be lowered. When the concentration is lower than 0.05 mM, the gel may not be cured properly.

In addition, according to one embodiment of the present invention, the HRP is not a material having a high cytotoxicity but may affect the curing time of the gel depending on the concentration, and it is preferable that HRP is included in 1 to 10 units.

According to an embodiment of the present invention, the first hydrogel may be acrylated-hyaluronic acid (HA-ac), and the second hydrogel may be thylamin-hyaluronic acid (HA-Tyr).

≪ Preparation Example 1: Preparation of acrylated hydrogel >

(1-Ethyl-3- (3-dimethylaminopropyl) -carbodimide) (Sigma-Aldrich) was prepared by dissolving hyaluronic acid (Lifecore Biomedical Co., Chaska, MN, USA) in 40 ml of distilled water to make a 0.25 mmole hyaluronic acid solution. In the presence of 0.14 g (1.25 mmole) of 1-hydroxybenzotriazole hydrate (Fluka Chemical Co., Buchs, Switzerland) (Aldrich, Inc., St. Louis, Mo., USA) And reacted with adipic acid dihydrazide (Fluka Chemical Co., Buchs, Switzerland) (2.2 g, 12.5 mmole) at room temperature for 8 hours. Subsequently, the reactant prepared above is dialyzed in a 100 mM NaCl solution for 2.5 days using a dialysis membrane (MWCO 14,000, SpectraPor; Rancho Dominguez, CA, USA), and then dialyzed again in distilled water for 1 day. NAcryloxysuccinimide (Polyscience, Inc., Warrington, Pa., USA) (0.5 g, 3 mmole) was added to the dialyzed reactant and reacted for 12 hours. The prepared reaction product is dialyzed in 100 mM NaCl solution for 2.5 days, and then dialyzed again in distilled water for 1 day. Then, it is lyophilized for 3 days to obtain powdered acrylated hyaluronic acid. Thereafter, the acrylated hyaluronic acid was dissolved in a triethanolamine-buffer solution (TEA; 0.3 M, pH 8) for the preparation of the hydrogel solution.

To gel the hydrogel solution, a cross-linking agent, thiolated polyethylene glycol PEG-SH4 (MW 10,000) is added so that the molar ratio of acrylic group to thiol group is 1: 1. Subsequently, the peptide solution dissolved in physiological saline solution for immobilization of the peptide (Ac-SDKP-D-GPQG-IWGQC, MW 1529.6) can be mixed with the reaction mixture to prepare a gel in solution.

2 is a schematic diagram of a peptide and a hydrogel containing the same according to an embodiment of the present invention.

<Example 1: Cytotoxicity test of ultrafast curing bio-ink based on functional hydrated gel>

Hyaluronic acid HA-ac of 50 kDa synthesized through the above Preparation Example was dissolved in TEA buffer at 10 wt% and HA-tyr was dissolved in PBS at 3 wt%. The two solutions a HA-ac: 1 ratio were added to give mixed with 1x10 6 / 100ul a fibroblast with the preparation: HA-tyr = 7. In order to make 10 wt% 100ul hydrogel with HA-ac with 17% acrylic group through synthesis, a solution of crosslinking agent (HA-ac) was prepared by adding MMP-senstive peptide in consideration of the acrylic group bonded to the hyaluronic acid HRMP was fixed at 1.25 units, and the concentration of H2O was 1.2 mM, 0.6 mM, 0.1 mM, and 0.5 mM, respectively. 0.06 mM, respectively, and cured. The cured bio-ink hydrogel was cultured in DMEM (FBS10%, antibiotice 1%) medium for 3 days and then subjected to cytotoxicity test using Live and Dead kit.

3 is a photograph showing a cytotoxicity test on hydrogen peroxide concentration according to an embodiment of the present invention.

3, when the concentration of hydrogen peroxide is 1.2 mM or 0.06 mM, the survival rate of the cells is lowered.

Example 2: Curing speed test of ultrahigh-speed curing bio-ink based on functional hydrated gel [

Hyaluronic acid having a molecular weight of 230 kDa or 50 kDa was prepared and the gelation time according to the concentration, ratio, hydrogen peroxide concentration and HRP concentration of the first hydrogel and the second hydrogel was tested. HA-ac having a molecular weight of 230 kDa or 50 kDa was dissolved in 90 ul of 2.2 mg of TEA buffer, and MMP-sensitive petide as a crosslinking agent was dissolved in 10 ul of 0.8 mg of TEA buffer. 3 mg of HA-try was prepared and dissolved in 100 ul of PBS. Hydrogen peroxide was prepared to be 1 mM, 5 mM and 10 mM in the final gel solution of 200 ul, and HRP was dissolved in PBS at the concentration of 5 units and 10 units / 200ul gel. The prepared solutions were mixed according to respective ratios. HA-ac 90ul + MMPs 10ul + HA-tyr 80ul + H 2 O 2 10ul + HRP 10ul were mixed when mixed at a ratio of 1: 1, and HA-ac 144ul + MMPs 16ul + HA-tyr 32ul + H 2 O 2 4ul + HRP 4ul).

FIG. 4 is a graph illustrating gelation time according to concentration and ratio of each component according to an embodiment of the present invention.

Referring to FIG. 4, the gelation time was 30 seconds when HA-ac 3 wt% of 3, 3% HA-tyr was mixed with 1: 1 of 230 kDa and 5 units of HRP was mixed with 5 mM of hydrogen peroxide. In order to shorten the gelation time, the concentration of hydrogen peroxide and HRP was decreased to less than 5 seconds in 10mM and 10 units, respectively. On the contrary, when the concentration of hydrogen peroxide and HRP was 1mM and 5 units, 30 seconds. When 50 kD of HA-ac was used, the gelation time was longer than 2 min when 6 wt% of hydrogel solution was mixed with 3% HA-tyr 1: 1, 5 mM of hydrogen peroxide was mixed with 5 unit of HRP, A very soft gel was formed. On the other hand, when 5 g of HRP was mixed with 5 mM of hydrogen peroxide, the hydrogel solution containing 12 wt% of HA-ac was prepared, and the gelation time was shortened to 5 seconds or less. When the mixture ratio of HA-ac and HA-tyr was adjusted from 1: 1 to 3: 1 and the concentration of hydrogen peroxide was reduced to 1 mM in order to minimize cytotoxicity, while when mixed with 10 units of HRP, the gelation time Was within 10 seconds and the intensity was also measured as strong.

< Example  3: Functionality Hydrating Gel  Based curing speed test of ultrafast curing bio ink>

A rheometer was used to measure the physical properties of the bio ink according to one embodiment of the present invention. The first hydrogel and the second hydrogel were prepared as 10% HA-ac and 3% HA-tyr, respectively, and mixed at a ratio of 7: 1. Hydrogen peroxide was prepared by mixing 0.1 mM and 0.6 mM, and HRP concentration was fixed to 1.25 unit / 200ul. The prepared solutions were mixed immediately before the rheometer measurement and the hydrated gel was formed at 37 degrees using a rotatable rheometer and the values were monitored.

5 is a rheological graph of a bio ink according to an embodiment of the present invention.

Referring to FIG. 5, the physical properties of each hydrogel solution were analyzed. As a result, the hydrogel gel was rapidly formed as soon as the measurement was started, and a hard gel having a hardness of 1 x 10 4 Pa or more was formed.

Therefore, the bio-ink composition according to an embodiment of the present invention can be stacked with printing due to fast gelling time even when applied to bioprinting, and can be adjusted to have a strength to support the structure even after printing .

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. For example, if the techniques described are performed in a different order than the described methods, and / or if the described components are combined or combined in other ways than the described methods, or are replaced or substituted by other components or equivalents Appropriate results can be achieved.

Therefore, other implementations, other embodiments, and equivalents to the claims are also within the scope of the following claims.

Claims (13)

Acrylate-hyaluronic acid (HA-ac) and tyraminization-hyaluronic acid (HA-Tyr).
The method according to claim 1,
Wherein the acrylated-hyaluronic acid is contained in an amount of 2 to 15 wt%, and the tyraminized-hyaluronic acid is contained in an amount of 1 to 10 wt%.
The method according to claim 1,
Wherein the acrylated-hyaluronic acid and the tyraminized-hyaluronic acid are mixed in a weight ratio of 1: 1 to 7: 1.
The method according to claim 1,
A peptide comprising an amino acid sequence cleavable by MMP (matrix metalloproteinase) and a cysteine at the terminal; Hydrogen peroxide; And HRP (Horseradish peroxidase).
5. The method of claim 4,
Wherein the concentration of the hydrogen peroxide is 0.05 to 0.1 mM.
delete The method according to claim 1,
Wherein the molecular weight of the acrylated-hyaluronic acid is from 10 kDa to 800 kDa.
The method according to claim 1,
Wherein the hardness of the bio ink composition after curing is 1 x 10 4 Pa to 1 x 10 6 Pa.
Preparing an acrylated-hyaluronic acid (HA-ac) solution;
Preparing a solution of tyramine-hyaluronic acid (HA-Tyr);
Mixing the acrylated-hyaluronic acid solution and the tyramine-hyaluronic acid solution; And
Adding to said mixture a peptide comprising amino acid sequence cleavable by MMP (matrix metalloproteinase) and a cysteine at the end, hydrogen peroxide, and HRP (Horseradish peroxidase).
10. The method of claim 9,
Preparing the acrylated-hyaluronic acid solution comprises dissolving acrylated-hyaluronic acid in a buffer solution at 2 to 15 wt%
Wherein preparing the thylamine-hyaluronic acid solution comprises dissolving tyramine-hyaluronic acid in a buffer solution at 1 to 10 wt%.
10. The method of claim 9,
The step of mixing the acrylated-hyaluronic acid solution and the tyramine-hyaluronic acid solution comprises:
Wherein the acrylated-hyaluronic acid solution and the tyramine-hyaluronic acid solution are mixed at a weight ratio of 1: 1 to 7: 1.
10. The method of claim 9,
Wherein the concentration of the hydrogen peroxide is 0.05 to 1 mM.
delete
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