Photointiator, bioink including the same, and methode of manufacturing hydrogel

Abstract

To provide a photointiator, a bioink including the same, and a method of manufacturing a hydrogel.SOLUTION: The present invention relates to a photoinitiator, a bioink including the same, and a method of manufacturing a hydrogel. According to the present invention, the photoinitiator of the present invention may have water solubility, high molar extinction coefficient, and low cytotoxicity. In addition, the method of manufacturing a hydrogel of the present invention may have high cell viability by increasing 3D printing efficiency due to a fast photo-curing rate.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to a photoinitiator, a bioink containing the same, and a method for producing a hydrogel.

Background Art

[0002] 3D bioprinting is widely known as an alternative that can precisely replicate the structural complexity of the natural cornea. A patient-customized corneal structure can be manufactured through 3D bioprinting. The cornea has advantages in manufacturing the cornea by 3D bioprinting in that it is completely avascular compared to other tissues, has a very low metabolic requirement, and has a relatively uniform cell composition. The bioink used in 3D bioprinting must have suitable biocompatibility and optical properties. Hydrogel-based bioinks exhibit high cell compatibility with living cells, have excellent mechanical stability after printing, and have excellent printing resolution.

[0003] 3D printers have the advantages of being a simple process, having a low cost, having no restrictions on the manufacturing form, and being capable of multi-variety and small-batch production. However, in the application to 3D bioprinting in the medical field, there is a limitation in that the material of the bioink is greatly restricted.

[0004] Hyaluronic acid (HA) in bioink materials is a natural linear polysaccharide found in various connective tissues such as skin, umbilical cord, and vitreous body. HA-based hydrogels can utilize high cell compatibility, biodegradability, and various functional groups of HA, and are suitable as bioprinting materials. In order to apply HA-based hydrogels as bioprinting materials, they must be rapidly produced through photocrosslinking.

[0005] The photoinitiator is one of the important factors that affect the photocrosslinking rate of the hydrogel and the properties of the hydrogel. For the photoinitiator to be applied to bioprinting, such as a hydrogel based on HA, it must have good water solubility, undergo a photocrosslinking reaction under visible light, have a fast photoreactivity, and excellent biocompatibility.

Summary of the Invention

Problems to be Solved by the Invention

[0006] One object of the present invention is to provide a photoinitiator.

[0007] Another object of the present invention is to provide a bioink containing the photoinitiator.

[0008] Still another object of the present invention is to provide a method for producing a hydrogel.

Means for Solving the Problems

[0009] To achieve the above object, the present invention provides a photoinitiator having the chemical structure of the following Chemical Formula 1, which is water-soluble and is activated by visible light to form free radicals:

[0010] [Chemical Formula 1]

Chemical Formula

[0011] In Chemical Formula 1, R1 is H or F, and R2 is Li + or Mg 2+ and.

[0012] In addition, the present invention provides a bioink composition containing distilled water; a plurality of hyaluronic acid methacrylate (HAMA) compounds having different molecular weights dissolved in the distilled water; and a photoinitiator having the chemical structure of the following Chemical Formula 1 dissolved in the distilled water:

[0013] [Chemical Formula 1] [Chem.]

[0014] In Chemical Formula 1, R1 is H or F, and R2 is Li + or Mg 2+ .

[0015] Moreover, the present invention provides a method for producing a hydrogel, including: dissolving a hyaluronic acid methacrylate (HAMA) compound having a plurality of molecular weights and a photoinitiator having the chemical structure of the following Chemical Formula 1 in distilled water to produce a bioink composition; and printing the bioink composition while irradiating visible light to crosslink the hyaluronic acid methacrylate (HAMA) compound:

[0016] [Chemical Formula 1] [Chem.]

[0017] In Chemical Formula 1, R1 is H or F, and R2 is Li + or Mg 2+ . [Advantages of the Invention]

[0018] According to the present invention, the photoinitiator of the present invention can have water solubility, a high molar extinction coefficient, and low cytotoxicity. Moreover, the method for producing a hydrogel of the present invention can increase the efficiency of 3D printing due to a fast photocuring rate and can have a high cell viability. [Brief Description of the Drawings]

[0019]

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Figure 8

[0020]

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Mode for Carrying Out the Invention

[0021] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. Since the present invention can be subject to various modifications and can have various forms, specific embodiments are illustrated in the drawings and described in detail in this specification. However, this is not intended to limit the present invention to a specific disclosed form, and it should be understood to include all modifications, equivalents, or alternatives included in the spirit and technical scope of the present invention. Similar reference numerals are used for similar components while explaining each drawing. In the accompanying drawings, the dimensions of the structures are illustrated in an enlarged manner compared to the actual ones for the sake of clarity of the present invention.

[0022] Terms such as first and second are used in the description of various components, but the components are not limited by the terms. The terms are used only for the purpose of distinguishing one component from another. For example, without departing from the scope of the rights of the present invention, the first component may be named the second component, and similarly, the second component may also be named the first component.

[0023] The terms used in this specification are merely used to describe specific embodiments and are not intended to limit the present invention. Singular expressions include plural expressions unless explicitly stated otherwise in the context. In this specification, terms such as "including" or "having" are intended to specify the existence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, and should not be construed as precluding the existence or addition possibility of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0024] Unless otherwise defined, all terms used herein, including technical and scientific terms, shall have the same meaning as commonly understood by one of ordinary skill in the art. Terms defined in commonly used dictionaries shall be interpreted to have a meaning consistent with the context of the relevant art and shall not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0025]

[0026] The photoinitiator according to the present invention has the chemical structure of Chemical Formula 1 below, is water-soluble, and can be activated by visible light to form free radicals:

[0027] [Chemical Formula 1] [Chemical Structure Diagram]

[0028] In Chemical Formula 1, R1 is H or F, and R2 is Li + or Mg 2+ .

[0029] In one embodiment, the solubility of the photoinitiator in water at 25 °C is 160-200 mM, but is not limited thereto.

[0030] In one embodiment, the molar extinction coefficient of the photoinitiator is 40-80 M -1 cm-1, but is not limited thereto.

[0031]

[0032] The bioink composition according to the present invention may include distilled water; a hyaluronic acid methacrylate (HAMA) compound dissolved in the distilled water; and a photoinitiator having the chemical structure of Chemical Formula 1 below dissolved in the distilled water:

[0033] [Chemical Formula 1] [Chemical Structure Diagram]

[0034] In the chemical formula 1, R1 is H or F, and R2 is Li + or Mg 2+ is.

[0035] In one embodiment, the hyaluronic acid methacrylate compound may have a molecular weight of about 1 to 2000 kDa.

[0036] As one example, the hyaluronic acid methacrylate compound may include a compound of a single molecular weight within the range of about 1 to 2000 kDa in molecular weight.

[0037] As another example, the hyaluronic acid methacrylate compound may include a first hyaluronic acid methacrylate compound having a molecular weight of 1 to 20 kDa and a second hyaluronic acid methacrylate compound having a molecular weight of 50 to 2000 kDa. For example, the hyaluronic acid methacrylate compound may include a first hyaluronic acid methacrylate compound having a molecular weight of about 10 kDa and a second hyaluronic acid methacrylate compound having a molecular weight of about 100 kDa.

[0038] In one embodiment, the hyaluronic acid methacrylate compound may include the first hyaluronic acid methacrylate compound and the second hyaluronic acid methacrylate compound in a molar ratio of 2 to 7:8 to 3.

[0039] In one embodiment, the bioink composition may include about 5 to 15% by mass of the hyaluronic acid methacrylate compound and about 0.02 to 5% by mass of the photoinitiator.

[0040] In one embodiment, the viscosity of the bioink composition is about 30 to 70 mPa·s, but is not limited thereto.

[0041] In one embodiment, the bioink composition may further include a light absorber. As one example, tartrazine is used as the light absorber.

[0042] In one embodiment, the bioink composition may further include cells. As one example, corneal stromal cells are used as the cells.

[0043] In one embodiment, the bioink composition may further include collagen.

[0044] In one embodiment, the bioink composition is a composition for corneal formation.

[0045]

[0046] Figure 1 is a flowchart of a method for manufacturing a hydrogel according to the present invention.

[0047] Referring to Figure 1, a method for manufacturing a hydrogel according to the present invention may include: manufacturing a bioink composition by dissolving a methacrylated hyaluronic acid (HAMA) compound and a photoinitiator having the chemical structure of Chemical Formula 1 below in distilled water; printing the bioink composition; and irradiating the printed bioink composition with visible light to crosslink the methacrylated hyaluronic acid (HAMA) compound.

[0048] [Chemical Formula 1]

Chemical Structure

[0049] In Chemical Formula 1, R1 is H or F, and R2 is Li + or Mg 2+ is.

[0050] In one embodiment, the methacrylated hyaluronic acid (HAMA) compound may have a single or multiple molecular weights within the range of about 1 to 2000 kDa.

[0051] In one embodiment, the thickness of the hydrogel is about 300 to 700 μm. As one example, the thickness of the hydrogel is about 500 μm.

[0052] In one embodiment, the hydrogel has a transmittance of about 70 to 98% at a wavelength of about 450 to 600 nm.

[0053] In one embodiment, the tensile strength of the hydrogel is about 150 to 250 kPa, the elongation at break is about 20 to 55%, and the toughness is about 10 to 40 kJ / m 3 but is not limited thereto. to this.

[0054]

[0055] Hereinafter, in order to facilitate the understanding of the present invention, examples will be given for detailed description. However, the following examples are illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more fully explain the present invention to those skilled in the art.

[0056]

[0057] <Production Example 1> Synthesis of Methacrylated Hyaluronic Acid (HAMA)

[0058] Hyaluronic acid (HA, about 0.5 g, molecular weight: about 100 kDa) was dissolved in distilled water (5 mL), and the pH was adjusted to 8.0 using sodium hydroxide (about 1 N) at about 0 to 5 °C to prepare a mixture. Four equivalents of methacrylic anhydride were added dropwise to the mixture at one drop per about 30 minutes based on the disaccharide unit of HA. The mixture was precipitated in ethanol, and the obtained solid was filtered, washed with ethanol, frozen at about -30 °C, and then freeze-dried and stored at -20 °C until use.

[0059]

[0060] <Production Example 2> Synthesis of Methacrylated Hyaluronic Acid (HAMA)

[0061] In the above Production Example 1, except that the molecular weight of hyaluronic acid was about 10 kDa, HAMA was produced in the same manner as in Production Example 1.

[0062]

[0063] <Example 1> Production of Photoinitiator

[0064] Dimethylphenylphosphonite (about 187 mg, about 1.1 mmol) was added to benzoyl chloride (about 140 mg, about 1 mmol) and 2-butanone (about 10 ml), and the mixture was stirred at room temperature for about 24 hours under nitrogen to prepare a first mixture. Lithium bromide (about 95 mg, about 1.1 mmol) was added to a 2-butanone (about 10 ml) solution to prepare a second mixture. The first mixture and the second mixture were combined, heated at about 60 °C for about 20 minutes, and then cooled to room temperature to form a reaction product. The reaction product was washed successively with 2-butanone and diethyl ether and then dried under vacuum to obtain a photoinitiator for bioprinting, which was named LBP (about 237 mg, about 94% yield).

[0065] 1 H NMR (600 MHz, D2O): 8.09 (d, 2H), 7.61 - 7.65 (m, 2H), 7.58 (t, 1H), 7.49 (t, 1H), 7.46 - 7.42 (m, 4H). 13 C NMR (151 MHz, D2O): 212.39, 211.61, 135.72, 135.43, 134.53, 133.54, 132.66, 131.91, 131.89, 131.70, 131.64, 129.18, 129.16, 128.83, 128.46, 128.36. 31 P NMR (243 MHz, D2O): 18.37. HRMS (ESI MS) m / z: calculated: 252.13 found: 253.06 ([M + H]+ detected)

[0066]

[0067] <Example 2> Production of Photoinitiator

[0068] In Example 1, except that 4-fluorobenzoyl chloride (about 158 mg, about 1 mmol) was used instead of the benzoyl chloride, a photoinitiator for bioprinting was produced by the same method as in Example 1 and named LFBP.

[0069] 1 H NMR (600 MHz, D2O): 8.18 - 8.17 (m, 2H), 7.64 - 7.61 (m, 2H), 7.50 (t, 1H), 7.44 - 7.42 (m, 2H), 7.17 (t, 2H). 13 C NMR (151 MHz, D2O): 210.53, 209.74, 167.03, 165.34, 133.41, 132.53, 132.27, 132.21, 131.99, 131.97, 131.95, 131.93, 131.71, 131.65, 128.46, 128.39. 31 P NMR (243 MHz, D2O): 18.59. HRMS (ESI MS) m / z: calculated: 270.12 found: 271.05 ([M + H]+ detected)

[0070]

[0071] <Example 3> Production of Photoinitiator

[0072] In Example 1, except that magnesium bromide (about 203 mg, about 1.1 mmol) was used instead of the lithium bromide, a photoinitiator for bioprinting was produced by the same method as in Example 1 and named MBP.

[0073] 1 H NMR (600 MHz, D2O): 8.14 - 8.13 (d, 2H), 7.70 - 7.65 (m, 2H), 7.65 - 7.60 (t, 1H), 7.44 - 7.42 (t, 2H), 7.51 - 744 (m, 4H).

[0074]

[0075] <Example 4> Bioink

[0076] LBP (about 1.0 g), Production Example 1 (about 5.0 g), Production Example 2 (about 5.0 g), and tartrazine (about 3 mM) were dissolved in about 100 ml of distilled water to produce bioink.

[0077]

[0078] <Example 5> Bioink

[0079] LBP (about 1.0 g), Production Example 1 (about 5.0 g), and Production Example 2 (about 5.0 g) were dissolved in about 100 ml of PBS.

[0080]

[0081] <Example 6> Hydrogel

[0082] 1.0 g) was dissolved to produce bioink. The bioink was irradiated with light (about 405 nm, ~6.8 mW / cm 2 about 1 5 seconds) using a DLP printer (Perfactory (registered trademark) Micro Plus HD, EnvisionTEC, Germany) in the shape of a dog-bone with a thickness of about 1 mm in accordance with the ASTM D1708 test method to produce a hydrogel.

[0083]

[0084] <Example 7> Hydrogel

[0085] LBP (about 1.0 g), Production Example 1 (about 7.0 g), and Production Example 2 (about 3.0 g) were dissolved in about 100 ml of distilled water to produce bioink. The bioink was irradiated with light (about 405 nm, ~6.8 mW / cm 2, and a hydrogel was produced by (about 15 seconds).

[0086]

[0087] <Example 8> Hydrogel

[0088] LBP (about 1.0 g), Production Example 1 (about 5.0 g), and Production Example 2 (about 5.0 g) were dissolved in about 100 ml of distilled water to produce a bioink. The bioink was irradiated with light (about 405 nm, ~6.8 mW / cm 2 , about 15 seconds) using a DLP printer (Perfactory (registered trademark) Micro Plus HD, EnvisionTEC, Germany) to produce a hydrogel in a dog-bone shape with a thickness of about 1 mm according to the ASTM D1708 test method.

[0089]

[0090] <Example 9> Hydrogel

[0091] LBP (about 1.0 g), Production Example 1 (about 3.0 g), and Production Example 2 (about 7.0 g) were dissolved in about 100 ml of distilled water to produce a bioink. The bioink was irradiated with light (about 405 nm, ~6.8 mW / cm 2 , about 15 seconds) using a DLP printer (Perfactory (registered trademark) Micro Plus HD, EnvisionTEC, Germany) to produce a hydrogel in a dog-bone shape with a thickness of about 1 mm according to the ASTM D1708 test method.

[0092]

[0093] <Example 10> Hydrogel

[0094] Dissolve LBP (about 1.0 g), Production Example 1 (about 5.0 g), and Production Example 2 (about 5.0 g) in about 100 ml of distilled water to produce a bioink. Using a DLP printer (Perfactory (registered trademark) Micro Plus HD, EnvisionTEC, Germany), irradiate the bioink with light (about 405 nm, ~6.8 mW / cm 2 , for about 15 seconds) to produce a hydrogel (hereinafter, bHAMA).

[0095]

[0096] <Example 11> Corneal hydrogel

[0097] Figure 2 is a schematic diagram of an example of a method for producing a corneal hydrogel according to the present invention . Figure 3 shows the structure of the corneal hydrogel according to the present invention. Referring to Figures 2 and 3, dissolve LBP (about 1.0 g), Production Example 1 (about 5.0 g), Production Example 2 (about 5.0 g), tartrazine (about 3 mM), and rabbit corneal stromal cells (1Х10 6 cells / mL) in about 100 ml of distilled water to produce a bioink. Using a DLP printer (Perfactory (registered trademark) Micro Plus HD, EnvisionTEC, Germany), irradiate the bioink with light (about 405 nm, ~6.8 mW / cm 2 , for about 15 seconds) to produce a hydrogel in the form of Figure 3. Immerse the hydrogel in DMEM medium and change the medium every 20 minutes to remove the LBP and tartrazine remaining in the hydrogel.

[0098]

[0099] <Comparative Example 1> Photoinitiator for bioprinting

[0100] Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), a commonly used photoinitiator for bioprinting, was used (hereinafter, LAP).

[0101]

[0102] <Comparative Example 2> Bioink

[0103] LBP (about 1.0 g), Production Example 1 (about 10.0 g), and tartrazine (about 3 mM) were dissolved in about 100 ml of distilled water to produce a bioink.

[0104]

[0105] <Comparative Example 3> Bioink

[0106] LBP (about 1.0 g), Production Example 2 (about 10.0 g), and tartrazine (about 3 mM) were dissolved in about 100 ml of distilled water to produce a bioink.

[0107]

[0108] <Comparative Example 4>

[0109] LBP (about 1.0 g) and gelatin methacryloyl (GelMA, about 10 g) were dissolved in about 100 ml of PBS.

[0110]

[0111] <Comparative Example 5>

[0112] LBP (about 1.0 g) and poly(ethyleneglycol)diacrylate (PEGDA, about 10 g) were dissolved in about 100 ml of PBS.

[0113]

[0114] <Comparative Example 6> Bioink

[0115] LBP (about 1.0 g) and Production Example 1 (about 10.0 g) were dissolved in about 100 ml of distilled water to produce a bioink. The bioink was formed into a dog-bone shape with a thickness of about 1 mm in accordance with the ASTM D1708 test method using a DLP printer (Perfactory (registered trademark) Micro Plus HD, EnvisionTEC, Germany) for light irradiation (about 405 nm, ~6.8 mW / cm 2 , about 15 seconds) to produce a hydrogel.

[0116]

[0117] <Comparative Example 7> Bioink

[0118] LBP (about 1.0 g) and Production Example 2 (about 10.0 g) were dissolved in about 100 ml of distilled water to produce a bioink. The bioink was formed into a dog-bone shape with a thickness of about 1 mm in accordance with the ASTM D1708 test method using a DLP printer (Perfactory (registered trademark) Micro Plus HD, EnvisionTEC, Germany) for light irradiation (about 405 nm, ~6.8 mW / cm 2 , about 15 seconds) to produce a hydrogel.

[0119]

[0120] <Comparative Example 8>

[0121] Gelatin methacryloyl (GelMA, about 10 g) was dissolved in about 100 ml of distilled water to produce a bioink. The bioink was irradiated with light (about 405 nm, ~6.8 mW / cm 2 , about 15 seconds) using a DLP printer (Perfactory (registered trademark) Micro Plus HD, EnvisionTEC, Germany) to a mean thickness of about 500 μm to produce a hydrogel (hereinafter, GelMA).

[0122]

[0123] <Comparative Example 9>

[0124] Polyethylene glycol diacrylate (PEGDA, about 10 g) was dissolved in about 100 ml of distilled water to prepare a bioink. The bioink was irradiated with light (about 405 nm, ~6.8 mW / cm 2 , about 15 seconds) using a DLP printer (Perfactory (registered trademark) Micro Plus HD, EnvisionTEC, Germany) to produce a hydrogel (hereinafter referred to as PEGDA).

[0125]

[0126] <Experimental Example 1> Performance Evaluation of Photoinitiators

[0127] Figure 4 is a graph evaluating the solubility of the photoinitiator for bio-printing according to the present invention in water at about 25°C. LBP, LFBP, and MBP showed maximum solubilities of about 170.5 mM, about 184.7 mM, and 163.2 mM, respectively, which were higher than that of LAP (about 159.7 mM). Through this, it was confirmed that LBP, LFBP, and MBP were dissolved in water without additional solvent and heating.

[0128] Figure 5 is a graph of the molar extinction coefficient (ε)-wavelength of the examples according to the present invention. The ultraviolet-visible absorption spectra of LBP, FLBP, MBP, and LAP were recorded with a spectrophotometer (Shimadzu UV-1 800) in distilled water using quartz cuvettes of about 10 mm, and the molar extinction coefficient was measured in M -1 ·cm -1 . The molar extinction coefficient is an inherent property that affects the efficiency of the photoinitiator. LBP (about 60 M-1·cm -1 ), LFBP (about 56 M -1 ·cm -1 ), and MBP (about 88 M -1 ·cm -1 ) were higher than that of LAP (about 29 M -1 ·cm -1) showed a molar extinction coefficient about two times or more higher than that in comparison. It was understood that LBP and LFBP showed similar absorption modes and that the replacement of a hydrogen atom with a fluorine atom, which is electrically positive, had no apparent effect on the performance of the molar extinction coefficient. Also, Li + ions were replaced with highly biocompatible Mg 2+ ions, and MBP had the most excellent characteristics of the molar extinction coefficient.

[0129] Figure 6 is a graph of the storage coefficient and loss coefficient - time of an example according to the present invention. To grasp the curing speeds of LBP, LFBP, MBP, and LAP, rheological properties were measured. The intersection point of the storage modulus (G') and the loss modulus (G'') is the gel formation point, and the curing speeds of the LBP, LFBP, MBP, and LAP can be grasped at the intersection point. Using a solution prepared by dissolving about 10 g of Production Example 1 and about 1.0 g of LBP, LFBP, MBP, or LAP in about 100 ml of distilled water, the rheological properties of LBP, LFBP, MBP, and LAP were measured. The measurement of the rheological properties was performed using a stress-controlled rheometer (MCR 302, Anton Paar, Austria) equipped with a photocuring system (405 nm) and a temperature-controlled water bath. The time-dependent changes in the storage modulus (G') and loss modulus (G'') of LBP, LFBP, MBP, and LAP were measured under light irradiation at about 405 nm (about 6.8 mW / cm 2 ) and 1 rad -1 vibration. A time-sweep vibration test was performed with plate-plate geometry at about 25°C, a frequency of about 1 Hz, an interval of about 0.4 mm, and a strain rate of about 1%. The intersection point of G' and G'' is the gel formation point, and LBP (about 2.76 s), LFBP (about 3.0 s), and MBP (about 4.0 s) showed a faster curing speed than LAP (about 4.73 s). A photoinitiator having a faster curing speed enables faster printing while maintaining the shape fidelity and can shorten the exposure time of cells to the photoinitiator.

[0130]

[0131] <Example 2> Cytotoxicity of Photoinitiator

[0132] Cell culture

[0133] All animal experiment procedures were approved by the Animal Experiment Ethics Committee of Pusan National University Hospital, Republic of Korea (approval number PNUH-2020-162). To obtain rabbit corneal stromal cells, rabbit eyes (New Zealand white rabbit adults) were enucleated from euthanized animals. Rabbit corneal stromal cells were cultured in a cell culture dish of about 100 mm with DMEM supplemented with about 10% FBS and penicillin-streptomycin (about 100 UI / ml). The cultured cells were maintained at about 37 °C in a sterilized incubator (Labogene, Seoul, Republic of Korea) containing about 5% CO2 until they reached the confluence point.

[0134] Murine NIH-3T3 fibroblasts purchased from ATCC (Manassas, VA, USA) were cultured in a cell culture dish of about 100 mm with DMEM supplemented with about 10% FBS and penicillin-streptomycin (about 100 UI / ml). The cultured cells were maintained at about 37 °C in a sterilized incubator (Labogene, Seoul, Republic of Korea) containing about 5% CO2 until they reached the confluence point.

[0135] Experiment on cytotoxicity

[0136] The cytotoxicities of LBP, MBP, and LAP were evaluated by measuring cell viability after exposing them to various concentrations. To analyze cell viability, fibroblasts were seeded in 24-well plates (24-well-plates, 2×10 4 cells / well), cultured in a sterilized incubator at about 37 °C for about 12 hours, and then the medium was removed. The LBP, MBP, and LAP were diluted in media at various concentrations (0, 0.025, 0.05, 0.1, and 0.2% (w / v)), and the fibroblasts were cultured in a sterilized incubator at about 37 °C for about 24 hours and then washed with PBS (1X).

[0137] Figure 7 is a graph of the cytotoxicity results of the examples according to the present invention. The cytotoxicity of LAP, LBP, and MBP against fibroblasts was compared at various concentrations from 0.025 to 0.2%. At all test concentrations, LBP and MBP showed lower cytotoxicity than LAP. At a concentration of about 0.2% (w / v), the cell viability of LAP was about 46%, while that of LBP and MBP was about 69% and 83%, respectively. In particular, it was found that substituting with highly biocompatible Mg 2+ ions has a clear effect on reducing cytotoxicity.

[0138]

[0139] <Experimental Example 3> Evaluation of the printing performance of bioink

[0140] Figure 8 is an image explaining the wood stack structure. Referring to Figure 8, the bioinks of Example 4, Comparative Example 2, and Comparative Example 3 were produced by light irradiation (about 405 nm, ~6.8 mW / cm 2 , about 15 seconds) using a DLP printer (Perfactory (registered trademark) Micro Plus HD, EnvisionTEC, Germany). A hydrogel having the structure of Figure 8 was fabricated and shown in Figure 9. In Figure 9, in Example 4, a wood stack structure with a unique check pattern was fabricated, while in Comparative Example 2 and Comparative Example 3, the structure collapsed.

[0141] Figure 10 is a graph evaluating the viscosity of the examples according to the present invention, and Figure 11 is a graph evaluating the dispersibility of the examples according to the present invention. To evaluate the viscosity of the bioink and the dispersion stability of the cells, rabbit corneal stromal cells (1Х10 6cells / mL) were respectively dispersed in Example 5, Comparative Example 4, and Comparative Example 5 (10% (w / v) in PBS (1X)). The viscosity of the 10% (w / v) bioink dissolved in PBS (1X) was measured using a viscometer (μVISC, RheoSense, USA) at about 25°C. After filling the mold placed between two glass slides separated at intervals of about 500 μm with the cell-bioink solution, the setting was held vertically. After about 1 hour, the dispersion stability of the cells in the bioink was evaluated using a confocal microscope (K1-Fluo, Nanoscopesystems, Republic of Korea). In Figure 10, the viscosities of Example 5, Comparative Example 4, and Comparative Example 5 were confirmed to be about 51.9 mPa·s, about 27.1 mPa·s, and about 8.6 mPa·s, respectively. Example 5 showed an advantageous high viscosity for long-term dispersion stability. In Figure 11, the cells dispersed in Comparative Example 4 and Comparative Example 5 having a low viscosity were precipitated within 1 hour. On the other hand, consistent cell dispersion was observed in Example 5.

[0142]

[0143] <Experimental Example 4> Cell viability of bioink

[0144] The rabbit corneal stromal cells cultured in Experimental Example 2 were used. The rabbit corneal stromal cells were cultured with LBP (about 1% (w / v)) and washed with PBS (1X) at intervals of about 30, 60, 120, 240, and 360 minutes. To measure cell viability, about 10 μl of WST-1 reagent (EZ-Cytox) was supplemented to the cells, and after culturing for about 1 - 2 hours, (AMR-100, Allsheng Co., Ltd., China) was used to measure cell viability by colorimetric analysis at about 450 nm.

[0145] Figure 12 is a graph of cell viability - time for an example according to the present invention. Although the cell viability gradually decreased over time, even after about 1 hour, the cell viability was maintained at about 85% or more. Considering that a structure of several centimeters can be fabricated within about 1 hour at a high printing speed (about 100 μm / min or more) of a DLP printer, when applying Example 4 to industry, it shows a cell viability higher than expected. Through this, the bioink according to the present invention does not have a negative impact on cell viability and can be used for 3D bioprinting. By combining the high molar absorption coefficient and low cytotoxicity in the visible region with the high printing speed of LBP and DLP and minimizing the light exposure time, the cell viability can be increased.

[0146]

[0147] <Experimental Example 5> Mechanical Properties of Hydrogel

[0148] The mechanical properties of Examples 6 to 9, Comparative Example 6, and Comparative Example 7 were measured using a tensile tester (34SC - 1, Instron, USA) at a strain rate of about 1 mm / min in the tensile mode and are shown in Figure 13. The tensile strength and elongation were measured at the maximum points of stress and strain rate, respectively, on the stress - strain rate curve, and the toughness was calculated from the region under the stress - strain rate curve to rupture. The elastic modulus was evaluated by obtaining the slope of the initial about 5% on the strain rate - stress curve.

[0149] Figure 14 is a graph showing the tensile strength, elongation, and toughness of an example according to the present invention. Examples 7 and 8 had improved tensile strength, elongation, and toughness compared to Comparative Example 6 and Comparative Example 7. Example 8 recorded the highest tensile strength (about 200.46 kPa), elongation (about 35%), and toughness (about 32.35 kJ / m 3 ) value.

[0150]

[0151] <Experimental Example 6> Optical Properties of Hydrogel

[0152] Figure 15 is a photograph of the transparency of an example according to the present invention. The cornea is the transparent part in front of the eye and allows light to pass through. Therefore, the optical transparency of the printed bioink is an important factor in corneal transplantation. Hydrogels were fabricated according to the corneal thickness (about 500 μm) in Example 10, Comparative Example 8, and Comparative Example 9. When Example 10, Comparative Example 8, and Comparative Example 9 were placed on the photograph and visually observed, Example 10 showed excellent sharpness and transparency. However, Comparative Example 8 produced an image with low transparency and blurring, and Comparative Example 9 was shown in a yellowish color tone.

[0153] Figure 16 is a graph of the transmittance measurement of an example according to the present invention. The transmittance was measured in the visible light region (400 - 800 nm) using a UV-Vis spectrometer (Libra S70, Biochrom, UK). Before measurement, it was immersed in PBS (1X) for about 1 hour to remove residual LBP and tartrazine. Example 10 and Comparative Example 9 showed high transmittances of about 91 - 97% and about 72 - 91%, respectively. In the case of Comparative Example 8, it showed a low light transmittance of about 73 - 88%.

[0154]

[0155] <Experimental Example 7> Corneal hydrogel

[0156] Figure 17 shows the shape of the corneal hydrogel of an example according to the present invention. Referring to Figure 17 , it was confirmed that the corneal hydrogel with a thin thickness (about 500 μm) has sectional adhesion and retains its structure even after removing LBP and tartrazine.

[0157] Figure 18 shows the results of cell viability - time of Example 11 according to the present invention. The cell viability of the hydrogel of Example 11 was evaluated. The method for evaluating the cell viability was carried out in the same manner as in Experimental Example 4. During the 14-day culture in the medium, corneal stromal cells in the hydrogel suffered cell damage during the photocuring process. However, even after the initial cell death, it maintained a good viability (about 80% or more).

[0158] Figure 19 is an image showing the characteristics of rabbit corneal stromal cells of Example 11 according to the present invention. The characteristics of the rabbit corneal stromal cells of Example 11 were investigated using immunohistochemical staining. The rabbit corneal stromal cells of Example 11 expressed vimentin and lumican and showed the same characteristic immunoreactivity as rabbit corneal stromal cells cultured on a 2D surface. Through this, it was confirmed that the phenotypic attributes of rabbit corneal stromal cells were not changed even after the 3D bioprinting process of the DLP substrate.

[0159]

[0160] As described above with reference to the preferred embodiments of the present invention, those skilled in the art will understand that the present invention can be variously modified and changed within the scope not departing from the spirit and scope of the present invention described in the following claims.

Claims

1. A photoinitiator having the chemical structure of the following Chemical Formula 1, being water-soluble, and being activated by visible light to form free radicals: [Chemical Formula 1] 【Chemical 1】 In the chemical formula 1, R １ is H or F, and R ２ is Li ＋ or Mg ２＋ is.

2. The photoinitiator according to Claim 1, wherein the solubility of the photoinitiator in water at 25 °C is 160 to 200 mM.

3. The molar extinction coefficient of the photoinitiator is 40 to 80 M －１ cm －１ The photoinitiator according to claim 1, which is such.

4. Distilled water, A methacrylate hyaluronic acid (HAMA) compound dissolved in the distilled water, A photoinitiator dissolved in the distilled water and having the chemical structure of the following Chemical Formula 1, A bioink composition comprising: [Chemical Formula 1] 【Chemical Formula 2】 In the above Chemical Formula 1, R １ is H or F, and R ２ is Li ＋ or Mg ２＋ is.

5. The methacrylate hyaluronic acid compound, A first methacrylate hyaluronic acid compound having a molecular weight of 1 to 20 kDa, A second methacrylate hyaluronic acid compound having a molecular weight of 50 to 2000 kDa, The bioink composition according to Claim 4, comprising:

6. The bioink composition according to Claim 5, wherein the methacrylate hyaluronic acid compound comprises the first methacrylate hyaluronic acid compound and the second methacrylate hyaluronic acid compound in a molar ratio of 2 to 7:8 to 3.

7. The bioink composition according to Claim 4, wherein the bioink composition comprises 5 to 15% by mass of the methacrylate hyaluronic acid compound and 0.02 to 5% by mass of the photoinitiator.

8. The bioink composition according to Claim 4, wherein the viscosity of the bioink composition is 30 to 70 mPa·s.

9. The bioink composition according to Claim 4, further comprising a light absorber.

10. The bioink composition according to Claim 9, wherein the light absorber comprises tartrazine.

11. The bioink composition according to Claim 4, further comprising cells.

12. The bioink composition according to Claim 11, wherein the cells comprise corneal stromal cells.

13. The bioink composition according to Claim 4, further comprising collagen.

14. The bioink composition according to Claim 4, which is for corneal formation.

15. Manufacturing a bioink composition by dissolving a methacrylate hyaluronic acid (HAMA) compound and a photoinitiator having the chemical structure of the following Chemical Formula 1 in distilled water; Printing the bioink composition while irradiating visible light to crosslink the methacrylate hyaluronic acid (HAMA) compound. A method for manufacturing a hydrogel, comprising: [Chemical Formula 1] [Chemical Formula 3] In the above Chemical Formula 1, R １ is H or F, and R ２ is Li ＋ or Mg ２＋ is.

16. The method for manufacturing a hydrogel according to claim 15, wherein the hyaluronic acid methacrylate (HAMA) compound has a single or multiple molecular weights within the range of 1 to 2000 kDa.

17. The method for manufacturing a hydrogel according to claim 15, wherein the thickness of the hydrogel is 300 to 700 μm.

18. The method for manufacturing a hydrogel according to claim 17, wherein the hydrogel has a transmittance of 70 to 98% at a wavelength of 450 to 600 nm.

19. The method for manufacturing a hydrogel according to claim 15, wherein the tensile strength of the hydrogel is 150 to 250 kPa.

20. The method for manufacturing a hydrogel according to claim 15, wherein the elongation ratio of the hydrogel is 20 to 55%.

21. The toughness of the hydrogel is 10 to 40 kJ / m ３ The method for producing a hydrogel according to claim 15, which is such.

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