Photoinitiator, bio-ink containing same, and method for manufacturing hydrogel

Abstract

The present disclosure relates to photoinitiators, bio-inks comprising the photoinitiators, and methods of making hydrogels. According to the present disclosure, the photoinitiators of the present disclosure may have water solubility, high molar extinction coefficient, and low cytotoxicity. In addition, the method for manufacturing the hydrogel according to the present disclosure can improve 3D printing efficiency through a rapid photocuring rate, thereby having high cell survival.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of Korean Patent Application No. 10 - 2024 - 0002005, filed on January 5, 2024, with the Korean Intellectual Property Office, the entire disclosure of which is incorporated herein by reference for all purposes. Technical field

[0003] The present invention relates to a photoinitiator, a bioink containing the photoinitiator, and a method for manufacturing a hydrogel. Background art

[0004] 3D bioprinting is widely regarded as an innovative method for precisely replicating the structural complexity of the natural cornea. It can produce patient - specific corneal structures. Compared with other tissues, the cornea is completely avascular, requires minimal metabolism, and has a relatively uniform cell composition, making it particularly suitable for 3D bioprinting. Bioinks for 3D bioprinting must have appropriate biocompatibility and optical properties. Hydrogel - based bioinks show high cytocompatibility with living cells, excellent mechanical stability after printing, and good printing resolution.

[0005] 3D printing involves simple processes, which ensures low cost and allows the production of various products in small quantities without being restricted by the manufacturing form. However, in the context of 3D bioprinting for medical applications, the selection of bioink materials still has significant limitations.

[0006] Hyaluronic acid (HA) is a natural linear polysaccharide present in various connective tissues, including skin, umbilical cord, and vitreous humor. HA - based hydrogels are very suitable as bioprinting materials due to their high cytocompatibility, biodegradability, and the presence of numerous functional groups in HA. To use HA - based hydrogels for bioprinting, they must be rapidly fabricated by photocrosslinking.

[0007] Photoinitiators are key factors affecting the photocrosslinking rate and properties of hydrogels. For bioprinting applications (such as those using HA - based hydrogels), photoinitiators must exhibit high water solubility, photocrosslink under visible light, show rapid photoreactivity, and ensure excellent cytocompatibility.

[0008] [Prior art documents]

[0009] [Patent documents]

[0010] Korean Patent No. 10 - 2612626 Summary of the invention

[0011] Technical problems to be solved by the invention

[0012] The object of the present disclosure is to provide a photoinitiator.

[0013] Another object of the present disclosure is to provide a bioink comprising the photoinitiator.

[0014] Yet another object of the present disclosure is to provide a method for manufacturing a hydrogel.

[0015] Technical means for solving technical problems

[0016] To achieve the above objects, an embodiment of the present disclosure provides a photoinitiator having a chemical structure of the following Chemical Formula 1, and the photoinitiator is water-soluble and is activated by visible light to form free radicals:

[0017] [Chemical Formula 1]

[0018]

[0019] Wherein, in Chemical Formula 1, R1 is H or F, and R2 is Li + or Mg 2+ .

[0020] In addition, an embodiment of the present disclosure provides a bioink composition comprising distilled water, a methacrylated hyaluronic acid (HAMA) compound dissolved in distilled water and having various molecular weights, and a photoinitiator dissolved in distilled water, the photoinitiator having a chemical structure of the following Chemical Formula 1:

[0021] [Chemical Formula 1]

[0022]

[0023] Wherein, in Chemical Formula 1, R1 is H or F, and R2 is Li + or Mg 2+ .

[0024] In addition, an embodiment of the present invention provides a method for manufacturing a hydrogel, the method comprising: dissolving a methacrylated hyaluronic acid (HAMA) compound having various molecular weights and a photoinitiator having a chemical structure of the following Chemical Formula 1 in distilled water to prepare a bioink composition; printing the bioink composition while irradiating visible light to crosslink the methacrylated hyaluronic acid (HAMA) compound:

[0025] [Chemical Formula 1]

[0026]

[0027] Wherein, in Chemical Formula 1, R1 is H or F, and R2 is Li + or Mg 2+ .

[0028] Beneficial effects

[0029] According to the present disclosure, the photoinitiator of the present disclosure can have water solubility, a high molar extinction coefficient, and low cytotoxicity. In addition, due to the fast photocuring rate, the method for manufacturing a hydrogel of the present disclosure can have a high cell viability by improving the 3D printing efficiency. Description of the drawings

[0030] Figure 1 A flowchart showing a method for manufacturing a hydrogel according to the present disclosure is shown.

[0031] Figure 2 A schematic diagram showing an example of a method for manufacturing a corneal hydrogel according to the present disclosure is shown.

[0032] Figure 3 The structure of a corneal hydrogel according to the present disclosure is shown.

[0033] Figure 4 A graph showing the solubility of a photoinitiator for bioprinting according to the present disclosure in water at about 25 °C is shown.

[0034] Figure 5 A graph showing the molar extinction coefficient (ε)-wavelength of an example according to the present disclosure is shown.

[0035] Figure 6 A graph showing the storage modulus and loss modulus-time of an example according to the present disclosure is shown.

[0036] Figure 7 A graph showing the cytotoxicity results of an example according to the present disclosure is shown.

[0037] Figure 8 An image depicting a wooden stack structure is shown.

[0038] Figure 9 An image showing the evaluation of the Figure 8 structural reproducibility in an example according to the present disclosure is shown.

[0039] Figure 10 A graph showing the evaluation of the viscosity of an example according to the present disclosure is shown.

[0040] Figure 11 A graph showing the evaluation of the dispersibility of an example according to the present disclosure is shown.

[0041] Figure 12 A graph showing the cell viability-time of an example according to the present disclosure is shown.

[0042] Figure 13A graph showing the stress-strain curve according to an example of the present disclosure.

[0043] Figure 14 A graph showing the tensile strength, elongation at break, and toughness according to an example of the present disclosure.

[0044] Figure 15 An image showing the transparency according to an example of the present disclosure.

[0045] Figure 16 A graph showing the transmittance measurement according to an example of the present disclosure.

[0046] Figure 17 The shape of a corneal hydrogel according to an example of the present disclosure is shown.

[0047] Figure 18 The results of cell viability-time according to an example of the present disclosure are shown.

[0048] Figure 19 An image showing the characteristics of rabbit corneal stromal cells according to an example of the present disclosure is shown. Detailed Description of the Invention

[0049] Hereinafter, embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. The present disclosure may have various modifications and forms, and thus, specific embodiments will be illustrated in the drawings and described in detail in the specification. However, this does not mean that the present disclosure is limited to the specific forms disclosed, but should be understood to include all modifications, equivalents, or alternatives included within the spirit and technical scope of the present disclosure. When describing each drawing, like reference numerals are used to refer to like components. In the drawings, enlarged dimensions are given in the constructs to ensure the clarity of the present disclosure.

[0050] Terms such as "first" and "second" may be used to describe various components, but these components should not be limited by such terms. These terms are only used to distinguish one component from another. For example, without departing from the scope of the present disclosure, the first component may be named the second component, and similarly, the second component may also be named the first component.

[0051] The terms used in this application are only used to describe specific embodiments, but do not intend to limit the present disclosure. Unless the context clearly indicates otherwise, singular expressions include plural expressions. When used herein, it should be understood that terms such as "comprising / including", "include", or "have" are intended to specify the presence of the features, numbers, steps, operations, components, parts, or combinations thereof described in the application documents, but do not preclude the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0052] On the other hand, unless otherwise defined, all terms (including technical or scientific terms) used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant art, and should not be interpreted in an idealized or overly formal sense, unless expressly defined otherwise in this application.

[0053] The photoinitiator according to the present disclosure has a chemical structure of Formula 1 below, and the photoinitiator is water-soluble and is activated by visible light to form free radicals:

[0054] [Formula 1]

[0055]

[0056] Wherein, in Formula 1, R1 is H or F, and R2 is Li + or Mg 2+ .

[0057] In one embodiment, the solubility of the photoinitiator in water at 25 °C can be 160 mM to 200 mM, but is not limited thereto.

[0058] In one embodiment, the molar extinction coefficient of the photoinitiator can be 40 M -1 cm -1 to 80 M -1 cm -1 , but is not limited thereto.

[0059] The bioink composition according to the present disclosure may include distilled water, a methacrylated hyaluronic acid (HAMA) compound dissolved in the distilled water, and a photoinitiator dissolved in the distilled water, the photoinitiator having a chemical structure of Formula 1 below:

[0060] [Formula 1]

[0061]

[0062] Wherein, in Formula 1, R1 is H or F, and R2 is Li + or Mg 2+ .

[0063] In one embodiment, the methacrylated hyaluronic acid compound may have a molecular weight of about 1 kDa to 2000 kDa.

[0064] As one embodiment, the methacrylated hyaluronic acid compound may include a compound having a single molecular weight in the range of about 1 kDa to 2000 kDa.

[0065] As another embodiment, the methacrylated hyaluronic acid compound may include a first methacrylated hyaluronic acid compound having a molecular weight of 1 kDa to 20 kDa and a second methacrylated hyaluronic acid compound having a molecular weight of 50 kDa to 2000 kDa. For example, the methacrylated hyaluronic acid compound may include a first methacrylated hyaluronic acid compound having a molecular weight of about 10 kDa and a second methacrylated hyaluronic acid compound having a molecular weight of about 100 kDa.

[0066] In one embodiment, the methacrylated hyaluronic acid compound may include a first methacrylated hyaluronic acid compound and a second methacrylated hyaluronic acid compound in a molar ratio of 2-7:8-3.

[0067] In one embodiment, the bioink composition may contain about 5 to 15% by mass of the methacrylated hyaluronic acid compound and about 0.02 to 5% by mass of a photoinitiator.

[0068] In one embodiment, the viscosity of the bioink composition may be about 30 mPa·s to 70 mPa·s, but is not limited thereto.

[0069] In one embodiment, the bioink composition may further contain a light absorber. As an embodiment, tartrazine may be used as the light absorber.

[0070] In one embodiment, the bioink composition may further contain cells. As an embodiment, corneal stromal cells may be used as the cells.

[0071] In one embodiment, the bioink component may further contain collagen.

[0072] In one embodiment, the bioink component may be a composition for corneal formation.

[0073] Figure 1 A flowchart showing a method for manufacturing a hydrogel according to the present disclosure is shown.

[0074] See Figure 1 , the method for manufacturing a hydrogel according to the present disclosure may include dissolving a methacrylated hyaluronic acid (HAMA) compound and a photoinitiator having the chemical structure of Chemical Formula 1 below in distilled water to prepare a bioink composition; printing the bioink composition while irradiating visible light to crosslink the methacrylated hyaluronic acid (HAMA) compound:

[0075] [Chemical Formula 1]

[0076]

[0077] Among them, in Chemical Formula 1, R1 is H or F, and R2 is Li + or Mg 2+ .

[0078] In one embodiment, the methacrylated hyaluronic acid (HAMA) compound may have a single molecular weight or multiple molecular weights in the range of about 1 kDa to 2000 kDa.

[0079] In one embodiment, the thickness of the hydrogel may be about 300 μm to 700 μm. As an embodiment, the thickness of the hydrogel may be about 500 μm.

[0080] In one embodiment, the hydrogel has a transmittance of about 70% to 98% under light with a wavelength of about 450 nm to 600 nm.

[0081] In one embodiment, the tensile strength of the hydrogel may be about 150 kPa to 250 kPa, the elongation at break may be about 20% to 55%, and the toughness may be about 10 kJ / m 3 to 40 kJ / m 3 , but is not limited thereto.

[0082] Examples

[0083] Hereinafter, the present disclosure will be described in more detail by examples to help understand the present disclosure. However, the following examples are only intended to illustrate the content of the present disclosure, and the scope of the present disclosure is not limited to the following examples. The examples of the present disclosure are provided to more comprehensively elaborate the present disclosure to those skilled in the art.

[0084] <Preparation Example 1> Synthesis of Methacrylated Hyaluronic Acid (HAMA)

[0085] 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 with sodium hydroxide (about 1 N) at about 0 °C to 5 °C to prepare a mixture. Based on the disaccharide unit of HA, 4 equivalents of anhydrous methacrylate were added dropwise to the mixture for about 30 minutes. The mixture was precipitated in ethanol, then filtered, and the resulting solid was washed with ethanol, frozen at about -30 °C, and then freeze-dried and stored at -20 °C until use.

[0086] <Preparation Example 2> Synthesis of Methacrylated Hyaluronic Acid (HAMA)

[0087] Except that the molecular weight of hyaluronic acid in Preparation Example 1 was about 10 kDa, HAMA was prepared using the same method as in Preparation Example 1.

[0088] <Example 1> Preparation of Photoinitiator

[0089] Dimethyl phenylphosphonate (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 stirred at room temperature for about 24 hours in the presence of nitrogen to prepare a first mixture. Lithium bromide (about 95 mg, about 1.1 mmol) was added to a 2-butanone (about 10 mmol) solution to prepare a second mixture. The first mixture and the second mixture were mixed, heated at about 60 °C for about 20 minutes, and then cooled to room temperature to generate a reactant. The reactant was washed successively with 2-butanone and ether, and then dried under vacuum to obtain a photoinitiator for bioprinting, named LBP (about 237 mg, about 94% yield).

[0090] 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 (ESIMS) m / z: calculated value: 252.13 measured value: 253.06 ([M + H]+ detected)

[0091] <Example 2> Preparation of Photoinitiator

[0092] A photoinitiator for bioprinting was prepared using the same method as in Example 1, except that 4-fluorobenzoyl chloride (about 158 mg, about 1 mmol) was used instead of benzoyl chloride in Example 1, and it was named LFBP.

[0093] 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). 1313C 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 31P NMR (243 MHz, D2O): 18.59. HRMS (ESIMS) m / z: calculated: 270.12, found: 271.05 ([M + H]+ detected)

[0094] <Example 3> Preparation of Photoinitiator

[0095] A photoinitiator for bioprinting, named MBP, was prepared using the same method as in Example 1, except that magnesium bromide (about 203 mg, about 1.1 mmol) was used instead of lithium bromide in Example 1.

[0096] 1 1H 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).

[0097] <Example 4> Bioink

[0098] A bioink was prepared by dissolving LBP (about 1.0 g), Preparation Example 1 (about 5.0 g), Preparation Example 2 (about 5.0 g), and tartrazine (about 3 mM) in about 100 mL of distilled water.

[0099] <Example 5> Bioink

[0100] LBP (about 1.0 g), Preparation Example 1 (about 5.0 g), and Preparation Example 2 (about 5.0 g) were dissolved in about 100 mL of PBS.

[0101] <Example 6> Hydrogel

[0102] A bioink was prepared by dissolving LBP (about 1.0 g), Preparation Example 1 (about 9.0 g), and Preparation Example 2 (about 1.0 g) in about 100 mL of distilled water. According to the ASTM D1708 test method, a dog-bone-shaped hydrogel with a thickness of about 1 mm was prepared from the bioink using a DLP printer (Perfactory Micro PlusHD, EnvisionTEC, Germany) by light irradiation (about 405 nm, ~6.8 mW / cm 2 , about 15 s).

[0103] <Example 7> Hydrogel

[0104] A bioink was prepared by dissolving LBP (about 1.0 g), Preparation Example 1 (about 7.0 g), and Preparation Example 2 (about 3.0 g) in about 100 mL of distilled water. According to the ASTM D1708 test method, a dog-bone-shaped hydrogel with a thickness of about 1 mm was prepared from the bioink using a DLP printer (Perfactory Micro PlusHD, EnvisionTEC, Germany) by light irradiation (about 405 nm, ~6.8 mW / cm 2 , about 15 seconds).

[0105] <Example 8> Hydrogel

[0106] A bioink was prepared by dissolving LBP (about 1.0 g), Preparation Example 1 (about 5.0 g), and Preparation Example 2 (about 5.0 g) in about 100 mL of distilled water. According to the ASTM D1708 test method, a dog-bone-shaped hydrogel with a thickness of about 1 mm was prepared from the bioink using a DLP printer (Perfactory Micro PlusHD, EnvisionTEC, Germany) by light irradiation (about 405 nm, ~6.8 mW / cm 2 , about 15 seconds).

[0107] <Example 9> Hydrogel

[0108] A bioink was prepared by dissolving LBP (about 1.0 g), Preparation Example 1 (about 3.0 g), and Preparation Example 2 (about 7.0 g) in about 100 mL of distilled water. According to the ASTM D1708 test method, a dog-bone-shaped hydrogel with a thickness of about 1 mm was prepared from the bioink using a DLP printer (Perfactory Micro PlusHD, EnvisionTEC, Germany) by light irradiation (about 405 nm, ~6.8 mW / cm 2 , about 15 seconds).

[0109] <Example 10> Hydrogel

[0110] A bioink was prepared by dissolving LBP (about 1.0 g), Preparation Example 1 (about 5.0 g), and Preparation Example 2 (about 5.0 g) in about 100 mL of distilled water. A hydrogel with an average thickness of about 500 μm (hereinafter referred to as bHAMA) was prepared from the bioink using a DLP printer (Perfactory Micro Plus HD, EnvisionTEC, Germany) by light irradiation (about 405 nm, ~6.8 mW / cm 2 , about 15 seconds).

[0111] <Example 11> Corneal-shaped hydrogel

[0112] Figure 2 The figure shows a schematic diagram of one embodiment of a method for manufacturing a corneal-shaped hydrogel according to the present disclosure. Figure 3 The figure shows the structure of the corneal-shaped hydrogel according to the present disclosure. Refer to Figure 2 and Figure 3 , a bioink was prepared by dissolving LBP (about 1.0 g), Preparation Example 1 (about 5.0 g), Preparation Example 2 (about 5.0 g), tartrazine (about 3 mM), and rabbit corneal stromal cells (1×10 6 cells / mL) in about 100 mL of distilled water. As Figure 3 shown, a DLP printer (Perfactory MicroPlus HD, EnvisionTEC, Germany) was used to prepare the hydrogel with the bioink by light irradiation (about 405 nm, ∼6.8 mW / cm 2 , about 15 seconds). The hydrogel was immersed in DMEM medium and then changed every 20 minutes to remove the residual LBP and tartrazine in the hydrogel.

[0113] <Comparative Example 1> Photoinitiator for bioprinting

[0114] Lithium phenyl-2,4,6-trimethylbenzoylphosphate (LAP), a commercial photoinitiator for bioprinting (hereinafter referred to as LAP), was used.

[0115] <Comparative Example 2> Bioink

[0116] A bioink was prepared by dissolving LBP (about 1.0 g), Preparation Example 1 (about 10.0 g), and tartrazine (about 3 mM) in about 100 mL of distilled water.

[0117] <Comparative Example 3> Bioink

[0118] A bioink was prepared by dissolving LBP (about 1.0 g), Preparation Example 2 (about 10.0 g), and tartrazine (about 3 mM) in about 100 mL of distilled water.

[0119] <Comparative Example 4>

[0120] LBP (about 1.0 g) and methacrylated gelatin (GelMA, about 10 g) were dissolved in about 100 mL of PBS.

[0121] <Comparative Example 5>

[0122] LBP (about 1.0 g) and poly(ethylene glycol) diacrylate (PEGDA, about 10 g) were dissolved in about 100 mL of PBS.

[0123] <Comparative Example 6> Bioink

[0124] The bioink was prepared by dissolving LBP (about 1.0 g) and Preparation Example 1 (about 10.0 g) in about 100 mL of distilled water. According to the ASTM D1708 test method, a dog-bone-shaped hydrogel with a thickness of about 1 mm was prepared using a DLP printer (Perfactory Micro Plus HD, EnvisionTEC, Germany) by light irradiation (about 405 nm, ~6.8 mW / cm 2 , about 15 s) with the bioink.

[0125] <Comparative Example 7> Bioink

[0126] The bioink was prepared by dissolving LBP (about 1.0 g) and Preparation Example 2 (about 10.0 g) in about 100 mL of distilled water. According to the ASTM D1708 test method, a dog-bone-shaped hydrogel with a thickness of about 1 mm was prepared using a DLP printer (Perfactory Micro Plus HD, EnvisionTEC, Germany) by light irradiation (about 405 nm, ~6.8 mW / cm 2 , about 15 s) with the bioink.

[0127] <Comparative Example 8>

[0128] The bioink was prepared by dissolving methacrylated gelatin (GelMA, about 10 g) in about 100 mL of distilled water. Using a DLP printer (Perfactory Micro Plus HD, EnvisionTEC, Germany), a hydrogel with an average thickness of about 500 μm (hereinafter referred to as GelMA) was prepared by light irradiation (about 405 nm, ~6.8 mW / cm 2 , about 15 s) with the bioink.

[0129] <Comparative Example 9>

[0130] The bioink was prepared by dissolving poly(ethylene glycol) diacrylate (PEGDA, about 10 g) in about 100 mL of distilled water. Using a DLP printer (Perfactory Micro Plus HD, EnvisionTEC, Germany), a hydrogel with an average thickness of about 500 μm (hereinafter referred to as PEGDA) was prepared by light irradiation (about 405 nm, ~6.8 mW / cm 2 , about 15 s) with the bioink.

[0131] <Experimental Example 1> Evaluation of the Performance of Photoinitiators

[0132] Figure 4A graph showing the solubility of photoinitiators for bioprinting according to the present disclosure in water at about 25 °C is shown. LBP, LFBP, and MBP exhibited maximum solubilities of about 170.5 mM, 184.7 mM, and 163.2 mM, respectively, which are higher than that of LAP (about 159.7 mM). Therefore, it was determined that LBP, LFBP, and MBP can be dissolved in water without additional solvents and heating.

[0133] Figure 5 A molar extinction coefficient (ε)-wavelength graph according to an example of the present disclosure is shown. The ultraviolet-visible absorption spectra of LBP, FLBP, MBP, and LAP were recorded using a spectrophotometer (Shimadzu UV-1800) in distilled water with a quartz cuvette 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 ) showed a molar extinction coefficient about 2 times higher than that of LAP (about 29 M -1 ·cm -1 ) at about 405 nm. LBP and LFBP showed similar absorption patterns, indicating that replacing hydrogen atoms with negatively charged fluorine atoms had no significant effect on the performance of the molar extinction coefficient. In addition, MBP in which Li + ions were replaced with highly biocompatible Mg 2+ ions had the best characteristics in terms of the molar extinction coefficient.

[0134] Figure 6 A graph of the storage modulus and loss modulus-time according to an example of the present disclosure is shown. Rheological properties were measured to determine the curing rates of LBP, LFBP, MBP, and LAP. The intersection of the storage modulus (G') and the loss modulus (G") is at the gelation point, and the curing rates of LBP, LFBP, MBP, and LAP can be determined by the intersection. The rheological properties of LBP, LFBP, MBP, and LAP were measured using a solution in which about 10 g of Preparation Example 1 and LBP, LFBP, MBP, or LAP (about 1.0 g) were dissolved in about 100 mL of distilled water. The rheological properties were measured using a light curing system (405 nm) and a stress-controlled rheometer (MCR 302, Anton Paar, Austria) equipped with a temperature-controlled water bath. Under light irradiation at about 405 nm (about 6.8 mW / cm 2 ) and 1 rad -1Under the oscillation, the changes in storage modulus (G') and loss modulus (G") of LBP, LFBP, MBP, and LAP over time were measured. At approximately 25 °C, a time sweep vibration test was performed using a plate-plate geometry at a frequency of 1 Hz, a spacing of 0.4 mm, and a strain of 1%. The intersection of G' and G" is the gelation point, and LBP (approximately 2.76 s), LFBP (approximately 3.0 s), and MBP (approximately 4.0 s) showed a faster curing rate than LAP (approximately 4.73 s). A photoinitiator with a faster curing rate allows for faster printing while maintaining shape consistency and shortening the exposure time of cells to the photoinitiator.

[0135] <Experimental Example 2> Cytotoxicity of Photoinitiators

[0136] Cell culture

[0137] All animal experimental procedures have been approved by the Institutional Animal Care and Use Committee of Pusan National University Hospital (approval number: PNUH-2020-162). Rabbit eyes (adult white New Zealand rabbits) were removed from euthanized animals to obtain rabbit corneal stromal cells. The rabbit corneal stromal cells were cultured in a cell culture dish with a size of approximately 100 mm together with DMEM supplemented with approximately 10% FBS and penicillin-streptomycin (approximately 100 UI / mL). The cultured cells were maintained at approximately 37 °C in a sterile incubator (Labogene, Seoul, Korea) containing approximately 5% CO2 until confluence was reached.

[0138] Mouse NIH-3T3 fibroblasts purchased from ATCC (Manassas, VA, USA) were cultured in a cell culture dish with a size of approximately 100 mm together with DMEM supplemented with approximately 10% FBS and penicillin-streptomycin (approximately 100 UI / mL). The cultured cells were maintained at approximately 37 °C in a sterile incubator (Labogene, Seoul, Korea) containing approximately 5% CO2 until confluence was reached.

[0139] Cytotoxicity experiment

[0140] The cytotoxicity of LBP, MBP, and LAP was evaluated by measuring cell viability after exposure to different concentrations. For cell viability analysis, fibroblasts were seeded into 24-well plates (2×10 4 cells / well) and cultured at approximately 37 °C in a sterile incubator for approximately 12 hours, and then the medium was removed. LBP, MBP, and LAP were diluted in media containing different concentrations (0, 0.025, 0.05, 0.1, and 0.2% (w / v)), and then the fibroblasts were cultured at approximately 37 °C in a sterile incubator for approximately 24 hours, followed by washing with PBS (1X).

[0141] Figure 7 A graph showing the cytotoxicity results according to an example of the present disclosure. At different concentrations ranging from 0.025% to 0.2%, the cytotoxicities of LAP, LBP, and MBP against fibroblasts were compared. At all tested concentrations, LBP and MBP showed lower cytotoxicity than LAP. At a concentration of about 0.2% (w / v), the cell viability of LAP was found to be about 46%, while the cell viabilities of LBP and MBP were about 69% and 83%, respectively. In particular, by replacing with highly biocompatible Mg 2+ ions, it was found that the effect on reducing cytotoxicity was significant.

[0142] <Experimental Example 3> Evaluation of Bioink Printability

[0143] Figure 8 An image depicting a wooden stacked structure is shown. Referring to Figure 8 , a DLP printer (Perfactory Micro Plus HD, EnvisionTEC, Germany) was used to prepare the bioinks of Example 4, Comparative Example 2, and Comparative Example 3 by light irradiation (about 405 nm, ~6.8 mW / cm 2 , about 15 seconds). Hydrogels having the Figure 8 shown structure were prepared and are shown in Figure 9 . As Figure 9 shown, in Example 4, a wooden stacked structure with a unique lattice pattern was fabricated, but the structures in Comparative Example 2 and Comparative Example 3 collapsed.

[0144] Figure 10 A graph showing the evaluation of the viscosity according to an example of the present disclosure, Figure 11 A graph showing the evaluation of the dispersibility according to an example of the present disclosure. To evaluate the viscosity of the bioink and the dispersion stability of the cells, rabbit corneal stromal cells (1×10 6 cells / mL) were separately 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 at about 25 °C using a viscometer (μVISC, RheoSense, USA). The cell-bioink solution was filled into a mold placed between two glass slides with a spacing of about 500 μm between the two glass slides and kept vertically set. After about 1 hour, a confocal microscope (K1-Fluo, nanoscopesystems, Korea) was used to evaluate the dispersion stability of the cells in the bioink. In Figure 10Among them, it was found that the viscosities of Example 5, Comparative Example 4, and Comparative Example 5 were approximately 51.9 mPa·s, approximately 27.1 mPa·s, and approximately 8.6 mPa·s, respectively. Example 5 exhibited a high viscosity that was beneficial for long-term dispersion stability. In Figure 11 Among them, the cells dispersed in the low-viscosity Comparative Example 4 and Comparative Example 5 precipitated within 1 hour. On the other hand, in Example 5, consistent cell dispersion was observed.

[0145] <Experimental Example 4> Cell viability of the bioink

[0146] The rabbit corneal stromal cells cultured in Experimental Example 2 were used. The rabbit corneal stromal cells were cultured in LBP (approximately 1% (w / v)) and washed with PBS (1X) at intervals of approximately 30, 60, 120, 240, and 360 minutes. To measure cell viability, approximately 10 μL of WST-1 reagent (EZ-Cytox) was added to the cells and cultured for approximately 1 to 2 hours, and then cell viability was measured by colorimetry at approximately 450 nm using a microplate reader (AMR-100, Allsheng Co., Ltd., China).

[0147] Figure 12 A cell viability-time graph according to an example of the present disclosure is shown. Over time, the cell viability gradually decreased, but remained higher than 85% after approximately 1 hour. Considering that constructs of several centimeters are produced at a rapid printing rate (approximately 100 μm / min or higher) of a DLP printer within approximately 1 hour, when Example 4 is applied industrially, cell viability beyond expectations will be obtained. Therefore, the bioink according to the present disclosure can be used for 3D bioprinting without adversely affecting cell viability. Combining the high molar extinction coefficient in the visible light region with the rapid printing rate of LBP and DLP with low cytotoxicity can improve cell viability by minimizing the exposure time.

[0148] <Experimental Example 5> Mechanical properties of the hydrogel

[0149] The mechanical properties of Examples 6 to 9, Comparative Example 6, and Comparative Example 7 were measured using a tensile testing machine (34SC-1, Instron, USA) in tensile mode at a strain of approximately 1 mm / min, as Figure 13 shown. Tensile strength and elongation were measured at the maximum stress and strain points of the stress-strain curve, respectively, and toughness was calculated based on the area of the stress-strain curve up to the fracture point. The elastic modulus was evaluated by obtaining the initial slope of approximately 5% on the strain-stress curve.

[0150] Figure 14A graph is shown that illustrates the tensile strength, elongation at break, and toughness of examples according to the present disclosure. In Example 7 and Example 8, compared with Comparative Example 6 and Comparative Example 7, the tensile strength, elongation at break, and toughness are all improved. Example 8 has the highest record in terms of tensile strength (about 200.46 kPa), elongation at break (about 35%), and toughness (about 32.35 kJ / m 3 )

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

[0152] Figure 15 An image showing the transparency of an example according to the present disclosure is presented. Since the cornea is the transparent part at the front of the eye that transmits light, the optical transparency of the printed bioink is a key factor in corneal transplantation. In Example 10, Comparative Example 8, and Comparative Example 9, hydrogels were fabricated according to the thickness of the cornea (about 500 μm). When Example 10, Comparative Example 8, and Comparative Example 9 were placed on an image and visually observed, Example 10 showed excellent clarity and transparency. However, due to low transparency, Comparative Example 8 produced a blurred image, while Comparative Example 9 appeared yellow

[0153] Figure 16 A graph showing the transmittance measurement of an example according to the present disclosure is presented. The transmittance in the visible light region (400 to 800 nm) was measured using a UV-Vis spectrometer (Libra S70, Biochrom, UK). Before measurement, the samples were 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% to 97% and about 72% to 91%, respectively. In the case of Comparative Example 8, a low transmittance of about 73% to 88% was shown

[0154] <Experimental Example 7> Corneal-Type Hydrogel

[0155] Figure 17 The shape of a corneal-type hydrogel of an example according to the present disclosure is shown. Referring Figure 17 , it was found that the corneal-type hydrogel with a smaller thickness (about 500 μm) has compartmental adhesiveness, and the corneal-type hydrogel still maintains its structure even after removing LBP and tartrazine

[0156] Figure 18Shows the results of cell viability - time according to Example 11 of the present disclosure. The hydrogel of Example 11 was evaluated for cell viability. The cell viability evaluation method was carried out in the same manner as in Experimental Example 4. During the 14 - day culture in the medium, the corneal stromal cells in the hydrogel had cell damage during the photocuring process. However, even after the initial cell death, a good viability (about 80% or higher) was maintained.

[0157] Figure 19 Shows images identifying the characteristics of rabbit corneal stromal cells according to Example 11 of the present disclosure. The characteristics of the rabbit corneal stromal cells of Example 11 were studied by immunohistochemical staining. The rabbit corneal stromal cells of Example 11 expressed vimentin and lumican and exhibited the same unique immunotype as the rabbit corneal stromal cells cultured on a 2D surface. This indicates that the phenotype of the rabbit corneal stromal cells did not change even after the DLP - based 3D bioprinting process.

[0158] Although the preferred embodiments of the present disclosure have been described above, those skilled in the art will understand that various modifications and variations can be made to the present disclosure within a certain range without departing from the spirit and scope of the present disclosure set forth in the appended claims.

Claims

1. A photoinitiator, which has the chemical structure of the following Chemical Formula 1, and the photoinitiator is water-soluble and is activated by visible light to form free radicals: [Chemical Formula 1] Among them, In Chemical Formula 1, R1 is H or F, and R2 is Li + or Mg 2+ .

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

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

4. A bioink composition, which comprises: Distilled water; A methacrylated hyaluronic acid (HAMA) compound, which is dissolved in the distilled water; and A photoinitiator, which is dissolved in the distilled water and has the chemical structure of the following Chemical Formula 1: [Chemical Formula 1] Among them, In Chemical Formula 1, R1 is H or F, and R2 is Li + or Mg 2+ .

5. The bioink composition according to claim 4, wherein, The methacrylated hyaluronic acid compound comprises: A first methacrylated hyaluronic acid compound with a molecular weight of 1 kDa to 20 kDa; and A second methacrylated hyaluronic acid compound with a molecular weight of 50 kDa to 2000 kDa.

6. The bioink composition according to claim 5, wherein, The methacrylated hyaluronic acid compound contains the first methacrylated hyaluronic acid compound and the second methacrylated hyaluronic acid compound in a molar ratio of (2-7):(8-3).

7. The bioink composition according to claim 4, wherein, The bioink composition contains 5% to 15% by mass of the methacrylated 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 mPa·s to 70 mPa·s.

9. The bioink composition according to claim 4, wherein, The bioink composition further comprises a light absorber.

10. The bioink composition according to claim 9, wherein, The light absorber includes tartrazine.

11. The bioink composition according to claim 4, wherein, The bioink composition further comprises cells.

12. The bioink composition according to claim 11, wherein, The cells include corneal stromal cells.

13. The bioink composition according to claim 4, wherein, The bioink composition further comprises collagen.

14. The bioink composition according to claim 4, wherein, The bioink composition is used for corneal formation.

15. A method for manufacturing a hydrogel, which comprises: Dissolving a methacrylated hyaluronic acid (HAMA) compound and a photoinitiator having the chemical structure of the following Chemical Formula 1 in distilled water to prepare a bioink composition; Printing the bioink composition while irradiating visible light to crosslink the methacrylated hyaluronic acid (HAMA) compound: [Chemical Formula 1] Among them, in Chemical Formula 1, R1 is H or F, and R2 is Li + or Mg 2+ .

16. The method according to claim 15, wherein, The methacrylated hyaluronic acid (HAMA) compound has a single or multiple molecular weights in the range of 1 kDa to 2000 kDa, and The toughness of the hydrogel is 10 kJ / m 3 to 40 kJ / m 3 .

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

18. The method according to claim 17, wherein The transmittance of the hydrogel under light with a wavelength of 450 nm to 600 nm is 70% to 98%.

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

20. The method according to claim 15, wherein The elongation rate of the hydrogel is 20% to 55%.

21. The method according to claim 15, wherein, The toughness of the hydrogel is 10 kJ / m 3 to 40 kJ / m 3 .

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