Hybrid bioink having decellularized extracellular matrix-based hydrogel mixed with gelatinized decellularized extracellular matrix-based hydrogel

Abstract

The present invention relates to a hybrid bioink in which a decellularized extracellular matrix-based hydrogel (dECM) and a gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) are mixed. In particular, the present invention can provide a flexible yet durable extracellular matrix-based bioink wherein GeldECM having dECM gelatinized therein and thus exhibiting enhanced properties such as softness, elasticity, and toughness, is incorporated, together with dECM, whereby the hybrid bioink exhibits significantly improved complex modulus and enhanced mechanical properties such as viscoelasticity and is endowed with a flexible yet durable property.

Description

Hybrid bioink combining decellularized extracellular matrix-based hydrogel and gelatinized decellularized extracellular matrix-based hydrogel

[0001] The present invention relates to a bioink, and more particularly to a hydrogel based on decellularized extracellular matrix (dECM), and more particularly to an extracellular matrix-based bioink with enhanced mechanical properties and flexible yet tough characteristics.

[0002] Decellularized extracellular matrix (dECM) is emerging as a tissue-specific biomaterial that mimics the unique microenvironment of a tissue. Compared to existing single-type ECM materials, such as collagen and laminin, dECM facilitates cell and tissue formation due to its similar composition of diverse biochemical environments.

[0003] Since the decellularized extracellular matrix (EDM) was manufactured in hydrogel form, research on bioinks based on EDM derived from various tissues has advanced. Not only has the remarkable regenerative potential of various target tissues been highlighted in vitro, but the application of these EDM-based bioinks and the tissues produced from them has also shown promising potential for ultimate clinical transplantation.

[0004] However, the crosslinking of decellularized extracellular matrix-based hydrogels primarily relies on thermal crosslinking mechanisms, which is significantly limited by the heat transport rate within the gel environment, resulting in inadequate structural stability of printed structures with complex geometries (e.g., tubular, curved, spherical). These limitations hinder the fabrication of flexible tissue constructs by high-resolution printing using decellularized extracellular matrix-based hydrogels. Therefore, to date, most tissue engineering approaches rely on polymer scaffolds to ensure the tissue geometry and mechanical stability of complex organ structures using decellularized extracellular matrix-based bioinks.

[0005] Additionally, various methods have been attempted to enhance the mechanical strength of decellularized extracellular matrix-based hydrogels, such as adding exogenous substances or modifying their chemical structure. Examples of exogenous substances include the addition of chemical cross-linkers, ionic cross-linking polymers, and reinforcing nanomaterials, while chemical modification typically involves methacrylation. However, these methods also have limitations. Chemical modification methods affect the physical composition of the decellularized extracellular matrix, thereby compromising its inherent bioactivity and thus undermining the original purpose of using decellularized extracellular matrix-based bioinks. Furthermore, ionic cross-linking is difficult to control at a rapid rate, and ionic cross-linked hydrogels tend to swell and dissolve in physiological, aqueous environments. Furthermore, the addition of nanomaterials increases the stiffness and brittleness of the bioink, which does not match the soft yet tough properties of natural soft tissue.

[0006] Recent studies have improved the printability of decellularized extracellular matrix-based bioinks using a biocompatible visible-light photoinitiator system. However, like other photocrosslinking methods, this method has limitations: it strengthens the hydrogel matrix through faster and more robust crosslinking of the printing ink, rather than improving the properties of the printing ink itself.

[0007] Accordingly, there is a constant need for the development of a decellularized extracellular matrix-based bioink that can manufacture flexible and soft tissue structures using the decellularized extracellular matrix and increase their stability.

[0008] [Prior Art Literature]

[0009] [Patent Document]

[0010] Republic of Korea Patent Publication No. 10-2023-0050638 (published on April 17, 2023)

[0011] The present invention is intended to solve the above-mentioned problems, and its purpose is to provide a bioink having remarkably excellent complex elastic modulus.

[0012] In addition, the present invention provides an extracellular matrix-based hydrogel that has enhanced mechanical properties such as viscoelasticity and is flexible yet tough.

[0013] In addition, the present invention aims to provide a bioink that is gelled with remarkably high strength, so that it can form much longer ink strands without breaking during 3D bioprinting, and can form tissue structures more stably while also exhibiting excellent cell viability.

[0014] One embodiment of the present invention for achieving the above-described purpose is a hybrid bioink comprising a decellularized extracellular matrix-based hydrogel (dECM) and a gelatinized decellularized extracellular matrix-based hydrogel (GeldECM).

[0015] Here, the bioink according to the present invention may further include a photoinitiator.

[0016] In addition, the decellularized extracellular matrix-based hydrogel (dECM) may be obtained by dissolving decellularized extracellular matrix in an acid solution and then neutralizing it.

[0017] In addition, the above gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) can be made by dissolving the decellularized extracellular matrix in an acid solution and then heat-treating the neutralized hydrogel to gelatinize it.

[0018] Additionally, the decellularized extracellular matrix-based hydrogel (dECM) and the gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) may be included in a weight ratio of 80:20 to 50:50.

[0019] Additionally, the decellularized extracellular matrix-based hydrogel (dECM) and the gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) can be included in a weight ratio of 80:20 to 60:40.

[0020] Additionally, the decellularized extracellular matrix-based hydrogel (dECM) and the gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) may be included in a ratio of 70:30 wt%.

[0021] In addition, the mixture containing the decellularized extracellular matrix-based hydrogel (dECM), the gelatinized decellularized extracellular matrix-based hydrogel (GeldECM), and the photoinitiator can undergo primary physical crosslinking, secondary crosslinking by light irradiation, and tertiary crosslinking by heat treatment.

[0022] In addition, the physical primary cross-linking may be performed at 1 to 10°C, the secondary cross-linking by light irradiation may be performed at visible light, and the tertiary cross-linking by heat treatment may be performed at 30 to 70°C.

[0023] Additionally, the tertiary cross-linking by the above heat treatment can be performed for 2 to 4 hours.

[0024] Additionally, the bioink according to the present invention may be used to manufacture a tissue structure mimic.

[0025] Meanwhile, another embodiment of the present invention is a method for producing a hybrid bioink comprising a step of mixing a decellularized extracellular matrix-based hydrogel (dECM) and a gelatinized decellularized extracellular matrix-based hydrogel (GeldECM).

[0026] Here, the step of mixing the decellularized extracellular matrix-based hydrogel (dECM) and the gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) may include a step of mixing a photoinitiator.

[0027] In addition, the decellularized extracellular matrix-based hydrogel (dECM) and the gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) may be mixed in a weight ratio of 80:20 to 50:50.

[0028] In addition, it is possible to further include a first cross-linking step of lowering the temperature of the mixture that has gone through the mixing step to 1 to 10°C; a second cross-linking step of irradiating the mixture that has gone through the first cross-linking step with visible light; and a third cross-linking step of treating the mixture that has gone through the second cross-linking step at 30 to 70°C.

[0029] Additionally, the above third cross-linking step may be performed for 2 to 4 hours.

[0030] Specific details of other embodiments are included in the detailed description and drawings.

[0031] The bioink according to the present invention is characterized in that it includes a decellularized extracellular matrix-based hydrogel (dECM) and a gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) in which the dECM is gelatinized to enhance properties such as softness, elasticity, and toughness, thereby providing a bioink having remarkably excellent complex elastic modulus.

[0032] In addition, the bioink according to the present invention has the effect of providing an extracellular matrix-based hydrogel with enhanced mechanical properties such as viscoelasticity through triple cross-linking and with flexible yet tough characteristics.

[0033] In addition, the bioink according to the present invention is characterized by the dECM and GeldECM being mixed in an optimal ratio, so that it is gelled with remarkably high strength, and thus can form much longer ink strands without breaking during 3D bioprinting, and can form tissue structures more stably, while also having excellent cell viability.

[0034] FIG. 1 is a schematic diagram illustrating changes in the internal network structure of a mixture of a decellularized extracellular matrix-based hydrogel (dECM) and a gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) according to one embodiment of the present invention, when the first, second, and third cross-linking methods are sequentially applied to the mixture.

[0035] Figure 2 is an experimental result showing the rheological properties of a gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) according to one embodiment of the present invention.

[0036] Figure 3 is a result of analyzing the mechanical properties according to gelatinization time for using a gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) according to one embodiment of the present invention as a tissue-specific rheological modifier.

[0037] Figure 4 is a result of analyzing the change in tyrosine residues according to gelatinization time in order to use a gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) according to one embodiment of the present invention as a tissue-specific rheological modifier.

[0038] Figure 5 is an experimental result showing the rheological properties according to the mixing ratio of a decellularized extracellular matrix-based hydrogel (dECM) and a gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) according to one embodiment of the present invention.

[0039] FIG. 6 is a photograph showing the result of extruding each of a hybrid bioink (MeTTi-dERS) and a decellularized extracellular matrix-based hydrogel (dECM) from a nozzle of a 3D printing device according to one embodiment of the present invention.

[0040] Figure 7 is a photograph evaluating the stability of a structure printed using a hybrid bioink (MeTTi-dERS) and a decellularized extracellular matrix-based hydrogel (dECM) according to one embodiment of the present invention.

[0041] Figure 8 is a photograph showing the viability of cells after encapsulating them in each of a hybrid bioink (MeTTi-dERS) and a decellularized extracellular matrix-based hydrogel (dECM) according to one embodiment of the present invention and then printing them.

[0042] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0043] The terminology used in this application is only used to describe specific embodiments and is not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this application, it should be understood that the terms "comprise" or "have" indicate the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but do not exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0044] Terms such as first, second, etc. may be used to describe various components, but these components should not be limited by these terms. These terms are used solely to distinguish one component from another.

[0045] Throughout this specification, "%" used to indicate the concentration of a particular substance is (weight / weight) % for solid / solid, (weight / volume) % for solid / liquid, and (volume / volume) % for liquid / liquid, unless otherwise noted.

[0046] One embodiment of the present invention is a hybrid bioink comprising a decellularized extracellular matrix-based hydrogel (dECM) and a gelatinized decellularized extracellular matrix-based hydrogel (GeldECM).

[0047] The present inventors have found that when a decellularized extracellular matrix-based hydrogel (dECM) is gelatinized, a gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) with enhanced properties such as softness, elasticity, and toughness can be produced, and based on this, the inventors have found that the GeldECM can be used as a tissue-specific rheological modifier in the production of bioink, and have produced a bioink containing the dECM and GeldECM, and have confirmed that such a bioink has enhanced mechanical properties such as viscoelasticity and is flexible yet tough, thereby completing the present invention.

[0048] The term "extracellular matrix (ECM)" used in the present invention refers to the extracellular portion of animal tissues that typically provides structural support to animal cells while also performing various other important functions. The ECM is a defining characteristic of connective tissue in animals and is composed of various types of proteins, including collagen and glycosaminoglycans (GAGs). This ECM can be derived from animal tissues such as pigs and cows, and can be extracted from various organs.

[0049] In the bioink according to one embodiment of the present invention, the extracellular matrix is ​​preferably a decellularized extracellular matrix when considering bioapplications, etc. The decellularized extracellular matrix is ​​effective in minimizing the immune response during allograft or xenograft by removing cells that can act as antigens that induce an immune response. Since the type and number of cells and the physical characteristics of the tissue itself differ depending on the tissue, decellularization is performed using various chemicals such as acids, bases, storage solutions, hypertonic solutions, and detergents. In addition, the cellular matrix can be used while maintaining the structure of the tissue itself through only the decellularization process, but it can also be used by dissolving it in an acidic solution after a freeze-drying and pulverization process, or by making it into a hydrogel through a neutralization process.

[0050] In an example of an extracellular matrix-based bioink according to the present invention, the decellularized extracellular matrix-based hydrogel (dECM) may be the extracellular matrix itself or a hydrogel containing the extracellular matrix as a main component. For example, the decellularized extracellular matrix-based hydrogel (dECM) may be obtained by dissolving the decellularized extracellular matrix in an acid solution and then neutralizing it.

[0051] Additionally, the term "gelatinized" used in the present invention refers to a state in which an extracellular matrix-containing hydrogel is denatured by heat and has rheological properties identical or similar to those of gelatin. Gelatin is a type of derived protein obtained by treating collagen with hot water. It only swells in cold water, but dissolves in warm water to form a sol, resulting in flowability.

[0052] Accordingly, in the extracellular matrix-based bioink according to an example of the present invention, the gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) preferably means one in which all or part of collagen, a component of the extracellular matrix, is gelatinized by heat denaturation. For example, the gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) can be obtained by dissolving decellularized extracellular matrix in an acid solution, neutralizing the solution, and heat-treating the resulting hydrogel to gelatinize it. The collagen, a major component of the extracellular matrix, denatures and dissolves when heated with water, and is eluted into a colloidal phase to be converted into gelatin. Since collagen, a major component of the extracellular matrix, exists in a solidified state at room temperature to which the bioink is applied, a non-gelatinized extracellular matrix-containing hydrogel is stiff and brittle, making it difficult to print with the bioink and causing inconvenience in use. In addition, non-gelatinized extracellular matrix-containing hydrogels have the disadvantage of not being easily cured even when a photoinitiator is added and having significantly reduced adhesive strength during printing.

[0053] However, compared to the decellularized extracellular matrix-based hydrogel (dECM), the gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) not only exhibited significantly superior complex elastic modulus, but also exhibited superior rheological properties at low temperatures and photocrosslinking properties upon light irradiation, enhanced viscoelasticity, and flexible and tough mechanical properties with reduced stiffness.

[0054] The bioink according to the present invention is characterized in that it includes a decellularized extracellular matrix-based hydrogel (dECM) and a gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) in which the dECM is gelatinized to enhance properties such as softness, elasticity, and toughness, thereby providing a bioink having remarkably excellent complex elastic modulus.

[0055] In addition, the bioink according to the present invention is characterized by including GeldECM as a tissue-specific rheological modifier together with the dECM, so that the viscoelastic strength, flexibility, and ductility of the hydrogel itself can be adjusted to suit the characteristics of the in vitro biomimetic body, thereby making it possible to more effectively produce an in vitro biomimetic body similar to an actual tissue.

[0056] As an example of the present invention, the bioink according to the present invention may further include a photoinitiator. The photoinitiator may be one that initiates or promotes crosslinking by light. Among these, the photoinitiator may be one that initiates crosslinking by visible light. Considering biosafety, ease of use, adhesiveness, etc., the photoinitiator may preferably be selected from a combination of ruthenium and sodium persulfate or riboflavin, and more preferably from a combination of ruthenium and sodium persulfate. It is known that the ruthenium and sodium persulfate oxidize aromatic residues including tyrosine when irradiated with visible light (particularly blue light), and the oxidized aromatic residues are converted into free radicals and form covalent bonds, such as di-tyrosine covalent bonds, to induce a crosslinking reaction. In addition, when the photoinitiator is selected from a combination of ruthenium and sodium persulfate, considering biosafety, adhesiveness, etc., it is preferable that the concentration of ruthenium in the bioink is 0.1 to 2 mM and the concentration of sodium persulfate is 1 to 20 mM, and it is more preferable that the concentration of ruthenium is 0.2 to 1.5 mM and the concentration of sodium persulfate is 2 to 15 mM.

[0057] As an example of the present invention, the decellularized extracellular matrix-based hydrogel (dECM) and the gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) may be included in a ratio of 80:20 to 50:50 wt%, preferably in a ratio of 80:20 to 60:40 wt%, and more preferably in a ratio of 70:30 wt%.

[0058] As can be confirmed in the examples and experimental examples described below, the inventors of the present invention tested the rheological properties according to the mixing ratio of the dECM and GeldECM, and as a result, the hybrid hydrogels (d7G3 and d5G5) containing 30% or more of GeldECM showed a much higher complex elastic modulus than dECM (d10G0) (see A, B of FIG. 5), and among them, it was confirmed that the d7G3 (70 wt% dECM and 30 wt% GeldECM) hybrid hydrogel had the best final complex elastic modulus value (see C, D of FIG. 5).

[0059] That is, in the mixing ratio of the dECM and GeldECM, if the content of GeldECM is less than the above range (if the content of dECM is greater than the above range), there is a disadvantage that the complex elastic modulus is low because gelatin chain entanglement or crosslinking is not formed well, and if the content of GeldECM is greater than the above range (if the content of dECM is less than the above range), there is a disadvantage that the complex elastic modulus is rather low during triple crosslinking including thermal crosslinking.

[0060] The bioink according to the present invention is characterized by the dECM and GeldECM being mixed in an optimal ratio, and has the best complex elastic modulus, and is gelled with remarkably high strength, so that it can form much longer ink strands without breaking during 3D bioprinting, and can form tissue structures more stably, while also having excellent cell viability.

[0061] As an example of the present invention, a mixture including the decellularized extracellular matrix-based hydrogel (dECM), the gelatinized decellularized extracellular matrix-based hydrogel (GeldECM), and a photoinitiator can undergo primary physical crosslinking, secondary crosslinking by light irradiation, and tertiary crosslinking by heat treatment.

[0062] In addition, the physical primary cross-linking may be performed at 1 to 10°C, the secondary cross-linking by light irradiation may be performed at visible light, and the tertiary cross-linking by heat treatment may be performed at 30 to 70°C.

[0063] Additionally, the tertiary cross-linking by the above heat treatment can be performed for 2 to 4 hours.

[0064] FIG. 1 is a schematic diagram illustrating changes in the internal network structure of a mixture of a decellularized extracellular matrix-based hydrogel (dECM) and a gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) according to one embodiment of the present invention, when first, second, and third cross-linking methods are sequentially applied to the mixture.

[0065] The hybrid bioink according to the present invention can initially form internal cross-links through physical entanglement. That is, the gelatin chains of GeldECM present within the hybrid hydrogel according to the present invention can have an entangled gelatin structure at low temperatures, thereby forming physical entanglement and spontaneously improving the complex elastic modulus in the pre-printing step, thereby enabling the hydrogel to immediately and precisely form a designed shape after extrusion.

[0066] In addition, the hybrid bioink according to the present invention can form internal crosslinks by secondary photo-crosslinking. That is, when a photoinitiator (photocuring agent) composed of ruthenium and sodium persulfate is mixed with a hydrogel including dECM and GeldECM and then irradiated with visible light, the tyrosine residues present within the hydrogel are oxidized to form di-tyrosine covalent bonds with nearby tyrosine residues, thereby promoting curing. Subsequently, when the hydrogel is irradiated with light during the printing process, secondary crosslinking occurs by the biocompatible visible light photoinitiator.

[0067] Next, the hybrid bioink according to the present invention can form internal crosslinks through tertiary thermal crosslinking. That is, when heat is applied to the hybrid hydrogel according to the present invention, the collagen chains of the dECM contained therein can be thermally crosslinked at 37°C to form collagen fibrils.

[0068] The present inventors have developed a triple sequential cross-linking method, as described above, by further incorporating a photoinitiator into a hydrogel comprising dECM and GeldECM. Through this triple cross-linking method, the present inventors sought to maximize the viscoelastic strength of the hybrid hydrogel itself. Indeed, the bioink according to the present invention exhibited enhanced mechanical properties, such as viscoelasticity, through triple cross-linking, and possessed flexible yet tough characteristics.

[0069] Meanwhile, another embodiment of the present invention is a method for producing a hybrid bioink, comprising a step of mixing a decellularized extracellular matrix-based hydrogel (dECM) and a gelatinized decellularized extracellular matrix-based hydrogel (GeldECM). The method or conditions for mixing the dECM and GeldECM are not particularly limited and may include various methods known in the art.

[0070] Here, the mixing ratio of the decellularized extracellular matrix-based hydrogel (dECM) and the gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) is not particularly limited, but may be mixed in a ratio of 80:20 to 50:50 wt%.

[0071] In addition, the step of mixing the decellularized extracellular matrix-based hydrogel (dECM) and the gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) may include a step of mixing a photoinitiator. The photoinitiator is as described above. The method or conditions for mixing the photoinitiator are not particularly limited and may include various methods known in the art.

[0072] In addition, it is possible to further include a first cross-linking step of lowering the temperature of the mixture that has gone through the mixing step to 1 to 10°C; a second cross-linking step of irradiating the mixture that has gone through the first cross-linking step with visible light; and a third cross-linking step of treating the mixture that has gone through the second cross-linking step at 30 to 70°C.

[0073] In order to form internal crosslinks by physical entanglement in the mixture that has gone through the mixing step, when treated at a relatively low temperature (lower than room temperature) in the range of 1 to 10°C, it is preferable to transform the gelatin chains of GeldECM into an entangled gelatin structure. In addition, in order to form internal crosslinks by photocrosslinking in the mixture that has gone through the first crosslinking step, when irradiating visible light, it is preferable to form di-tyrosine covalent bonds within the hydrogel. In addition, in order to form internal crosslinks by thermal crosslinking in the mixture that has gone through the second crosslinking step, it is preferable to transform the collagen chains of dECM into collagen fibrils in the mixture that has gone through the second crosslinking step. In addition, the above-mentioned third cross-linking step may be processed for 2 to 4 hours, as can be confirmed in the experimental example described below, and among them, it is more preferable to process for about 3 hours.

[0074] The bioink according to the present invention, manufactured as described above, is characterized by a complex modulus of 10 to 20 kPa. Preferably, the complex modulus is 13 to 17 kPa. When triple crosslinking is performed according to the present invention, the complex modulus of the hydrogel is significantly increased by a minimum of 3 times and a maximum of 32 times compared to double crosslinking.

[0075] In another embodiment of the present invention, there is provided a method for manufacturing an artificial tissue using a hybrid bioink manufactured by the aforementioned manufacturing method. The method for manufacturing an artificial tissue according to the present invention may include a step of printing an artificial tissue using a hybrid bioink; and a solidification step of gelling or cross-linking the printed artificial tissue.

[0076] The step of printing the artificial tissue refers to a bio-3D printing step of printing an artificial tissue, which is a three-dimensional structure, using a mixed hybrid bioink. The artificial tissue may be, for example, an organoid, an organ-on-a-chip, liver tissue, heart tissue, bone tissue, etc., and may be performed using a 3D printing device. The step of printing the artificial tissue may be performed by printing while cells or proteins are encapsulated in the bioink to manufacture a three-dimensional structure, or may be performed by printing while cells are not encapsulated to manufacture a three-dimensional structure and then culturing cells on the surface thereof.

[0077] Next, a solidification step is performed to gel or cross-link the printed artificial tissue. The solidification step may include a photoinitiator, light irradiation, or heat application. In other words, the present invention also allows for secondary and tertiary cross-linking processes after printing.

[0078] As described above, the present invention relates to a hybrid bioink in which dECM and GeldECM are mixed, and is particularly characterized by including GeldECM in which the dECM is gelatinized to enhance properties such as softness, elasticity, and toughness, thereby providing an extracellular matrix-based bioink that not only has a remarkably excellent complex elastic modulus but also has enhanced mechanical properties such as viscoelasticity and is endowed with flexible and tough properties.

[0079] Therefore, the bioink according to the present invention can control the viscoelastic strength, flexibility, and ductility of the hydrogel itself to suit the characteristics of the mimic to be produced by changing the mixing ratio of the dECM and GeldECM, and can also be modified as desired by the user, thereby making it possible to produce an in vitro mimic similar to the desired actual tissue.

[0080] Additionally, the bioink according to the present invention may further include other components in the hybrid hydrogel composition. For example, the bioink composition of the present invention may further include one or more cells or a cell culture medium. Furthermore, it may further include factors capable of promoting cell proliferation, cell differentiation, or extracellular matrix secretion. Furthermore, it may further include a biocompatible polymer compound.

[0081] The bioink according to the present invention, as described above, can be utilized for various purposes. It can be used as an ink for 2D, 3D, or 4D printing to produce various structures, as a bioadhesive, and for producing various tissue structure replicas.

[0082] The above tissue structure replica is a structure created by printing bioink using 3D bioprinting. The tissue structure replica can reproduce specific tissue functions and can be spatially organized in a form similar to actual tissue, making it suitable for use in tissue and organ regeneration and in creating biomodels for new drug development.

[0083] In the present invention, the tissue may be, but is not limited to, intestinal tissue, skin tissue, liver tissue, heart tissue, cartilage tissue, bone tissue, adipose tissue, muscle tissue, mucosal epithelial tissue, amniotic membrane tissue, or corneal tissue.

[0084] The present invention may be better understood by the following examples, which are provided for illustrative purposes only and are not intended to limit the scope of protection defined by the appended claims.

[0085] Hereinafter, in an embodiment of the present invention, in order to produce an in vitro intestinal structure mimic, a porcine small intestine-derived decellularized extracellular matrix (Small Intestine Submucosa-derived dECM, hereinafter referred to as SIS dECM) and a porcine small intestine-derived decellularized extracellular matrix (Small Intestine Submucosa-derived Gelatinized dECM, hereinafter referred to as SIS GeldECM) obtained by heat-treating the dECM were used to optimize the bioink according to the present invention.

[0086] The embodiments described in this specification are merely an ideal example of the present invention, and it is obvious to a person having ordinary skill in the art that a decellularized extracellular matrix can be prepared using various other tissues depending on the purpose.

[0087] Example 1: Preparation of decellularized extracellular matrix-based hydrogel (dECM)

[0088] Small Intestine Submucosa-derived dECM (SIS dECM) derived from porcine small intestine was prepared as follows.

[0089] First, the submucosa was separated from the pig small intestine, cut into 3 cm x 3 cm pieces, and washed with 70% ethanol solution for 2 hours. Then, the tissue was stirred in water containing 1% penicillin / streptomycin for 30 minutes. Afterwards, 25 x 10 -3 1% sodium dodecyl sulfate solution containing M ethylenediaminetetraacetic acid (EDTA) for 6 hours, and 25 x 10 -3The cells were removed by treating with 1% Triton X-100 solution containing M ethylenediaminetetraacetic acid (EDTA) for 24 hours. After washing with water for 12 hours, the cells were treated with 100% isopropyl alcohol for 2 hours. Then, 2.5 x 10 -3 The tissue was treated with 10 U / mL deoxyribonuclease dissolved in M ​​magnesium chloride solution for 2 hours. Afterwards, the tissue was sterilized by treating with 0.1% peracetic acid solution diluted in 4% ethanol for 2 hours, rinsed three times with secondary water for 30 minutes each, and washed with PBS buffer solution for 30 minutes to obtain small intestine-derived decellularized extracellular matrix (SIS dECM) tissue.

[0090] After completing the decellularization process, the small intestine-derived decellularized extracellular matrix (SIS dECM) was freeze-dried for more than 48 hours, ground into a fine powder using a grinder, and stored at -80°C until use. The freeze-dried SIS dECM was added to a 0.5 M acetic acid solution containing pepsin and dissolved for 7–10 days to obtain a small intestine-derived decellularized extracellular matrix-based hydrogel. The hydrogel was filtered through a 100 μm mesh and stored at 4°C. The dECM hydrogel was neutralized with a 10 N sodium hydroxide solution before use in subsequent experiments.

[0091] Example 2: Preparation of a gelatinized decellularized extracellular matrix-based hydrogel (GeldECM)

[0092] Using the porcine small intestine-derived decellularized extracellular matrix-based hydrogel (SIS dECM) prepared according to Example 1 above, a gelatinized porcine small intestine-derived decellularized extracellular matrix-based hydrogel (Small Intestine Submucosa-derived Gelatinized dECM, hereinafter referred to as SIS GeldECM) was prepared as follows.

[0093] That is, the hydrogel (SIS dECM) prepared according to Example 1 was heated to 60°C and maintained for 3, 12, 24, 72, and 120 hours to undergo thermal denaturation, thereby obtaining a gelatinized SIS dECM hydrogel (SIS GeldECM). After gelatinization, the GeldECM was stored at 4°C.

[0094] Example 3: Preparation of hybrid bioink

[0095] The SIS GeldECM prepared according to Example 2 was added to the SIS dECM hydrogel prepared according to Example 1 at a weight ratio of 100:0, 90:10, 70:30, and 50:50, respectively, and then ruthenium and sodium persulfate, which are biocompatible visible light photocuring agents, were added to final concentrations of 1 mM and 10 mM, respectively, to prepare a hybrid hydrogel. The solvent of the ruthenium solution and sodium persulfate solution was both DPBS (Dulbecco's phosphate-buffered saline). Thereafter, the prepared hybrid hydrogel was filtered through a 100 μm mesh to obtain a uniformly mixed hydrogel.

[0096] Then, the gelatin chains of GeldECM inside the hydrogel form physical entanglement at 4 degrees and are physically crosslinked for the first time at 4 degrees. Thereafter, the hybrid hydrogel was irradiated with visible light having a wavelength of about 400 to 450 nm, and then the tyrosine residues inside the hybrid hydrogel were oxidized and converted into tyrosine free radicals, and then di-tyrosine covalent bonds were formed with nearby tyrosine residues to promote curing and secondary crosslinking. Finally, the hybrid hydrogel was heated to 37°C, and then the SIS dECM hydrogel, which is mainly composed of collagen, was thermally crosslinked and tertiary crosslinked, thereby producing a hybrid bioink according to the present invention.

[0097] Experimental Example 1: Confirmation of the characteristics of GeldECM

[0098] Figure 2 is an experimental result showing the rheological properties of a gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) according to one embodiment of the present invention.

[0099] In the hybrid bioink according to the present invention, the optimal concentration of GeldECM, which acts as a tissue-specific rheological modifier, was determined.

[0100] GeldECM hydrogel was used by heat-denaturing SIS dECM hydrogel by heating it to 60°C and maintaining it for 3 hours according to the method of Example 2.

[0101] Then, the change in transparency of the GeldECM hydrogel obtained as above according to heat treatment was measured using a microplate spectrophotometer. Specifically, the absorbance of the dECM hydrogel and GeldECM hydrogel was measured at a wavelength of 300 to 800 nm, and the transparency was calculated using the formula that converts it to transmittance (transmittance = antilog (2-absorbance)). It was observed that part of the triple helix structure of collagen was unraveled by the applied heat and gelatinized, and that it had high transparency after gelatinization.

[0102] Additionally, changes in the complex modulus of the GeldECM hydrogel due to heat and light were measured using a rheometer equipped with a 20 mm diameter plate. Specifically, a time sweep analysis was performed while applying a temperature profile that cooled the GeldECM hydrogel from 37°C to 4°C over time, and the complex modulus at 2% strain was measured. As the temperature decreased, the GeldECM showed an increase in the complex modulus due to physical entanglement between gelatin chains (see Fig. 2A).

[0103] In addition, the change in complex modulus due to photocrosslinking was measured by irradiating GeldECM hydrogel (GeldERS) with visible light to which biocompatible visible light photocuring agents, ruthenium and sodium persulfate, were added to the GeldECM hydrogel at final concentrations of 1 mM and 10 mM, respectively. Specifically, the change in complex modulus due to photocrosslinking was measured at 10 mW / cm at 37°C. 2 Time sweep analysis was performed by irradiating the specimen with visible light starting at 180 s and continuing until the end of the experiment, and the complex modulus at 2% strain was measured. It was observed that the complex modulus increased due to light crosslinking only at 3% or more strain based on GeldECM (see B in Fig. 2).

[0104] Through this, it was expected that by mixing 3% or more of SIS GeldECM into a decellularized extracellular matrix-based hydrogel (dECM), the rheological properties at low temperatures could be enhanced and the photocrosslinking properties by light irradiation could be maximized.

[0105] FIG. 3 is a result of analyzing the mechanical properties according to gelatinization time for use as a tissue-specific rheological modifier of a gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) according to one embodiment of the present invention.

[0106] Each GeldECM was obtained by thermally denaturing SIS dECM hydrogel by heating it to 60°C and maintaining it for 0, 3, 12, 24, 72, and 120 hours. Then, the photocuring agents ruthenium and sodium persulfate were added to final concentrations of 1 mM and 10 mM, respectively, to prepare GeldERS.

[0107] Specifically, GeldERS was fabricated into uniform cylindrical specimens for compression tests. Light was irradiated for 2 minutes to crosslink GeldERS, and the sample was then shielded from light to prevent further photocrosslinking until the experiment. The fabricated sample was placed on the anvil equipped with a microtester and compressed using a microbeam with a 6 mm x 6 mm stainless steel plate attached to the end. The compression test was performed in displacement control mode set to a strain rate of 4% per minute. In addition, GeldERS was fabricated into uniform rectangular specimens for tensile tests. Both ends of the specimen were attached using tension forks equipped with the microtester, and the specimen was stretched at a constant rate of 2 mm / min until failure.

[0108] At this time, GeldERS gelatinized for 72 and 120 hours due to excessive protein degradation caused by long-term heat treatment were excluded as photocrosslinking was impossible.

[0109] Compression tests revealed a decrease in compressive modulus and yield strength after gelatinization (see Figures 3A, B, and C). Tensile tests revealed a decrease in tensile modulus and an increase in toughness and flexibility (see Figures 3D, E, F, and G). As shown here, by gelatinizing the stiff and brittle dECM and transforming it into GeldECM, we confirmed enhanced ductility, elasticity, and toughness.

[0110] FIG. 4 is a result of analyzing the change in tyrosine residues according to gelatinization time in order to use a gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) according to one embodiment of the present invention as a tissue-specific rheological modifier.

[0111] Changes in tyrosine residues due to thermal denaturation of GeldECM prepared differently depending on the heat treatment time were confirmed through amino acid analysis.

[0112] That is, the tyrosine content of each GeldECM obtained by heat-denaturing the SIS dECM hydrogel by heating it to 60°C and maintaining it for 0, 3, 12, 72, and 120 hours was quantified using an amino acid auto-analyzer.

[0113] Specifically, each of the above samples was hydrolyzed and separated using an ion exchange column. After post-column derivatization using ninhydrin, the samples were analyzed at wavelengths of 570 and 440 nm.

[0114] As a result, we observed that the tyrosine residue, which is important for photocuring, remained unchanged even after heat treatment for up to 72 hours. Furthermore, we confirmed that the tyrosine concentration was highest when heat-denatured (gelatinized) for 3 hours. Accordingly, GeldECM heat-denatured for 3 hours was used in the subsequent production of hybrid bioink.

[0115] Experimental Example 2: Confirmation of Bioink Characteristics According to Mixing Ratio

[0116] Figure 5 is an experimental result showing the rheological properties according to the mixing ratio of a decellularized extracellular matrix-based hydrogel (dECM) and a gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) according to one embodiment of the present invention. In Figure 5, it is indicated as d10G0 (ratio of 10 wt% dECM and 0 wt% GeldECM), d9G1 (ratio of 9 wt% dECM and 1 wt% GeldECM), d7G3 (ratio of 7 wt% dECM and 3 wt% GeldECM), and d5G5 (ratio of 5 wt% dECM and 5 wt% GeldECM).

[0117] That is, when hybrid hydrogels with different mixing ratios were sequentially crosslinked according to the above Example 3, the change in complex elastic modulus was measured using a rheometer equipped with a 20 mm diameter plate.

[0118] First, the hybrid hydrogel was maintained at 4 degrees for 30 seconds and then exposed to visible light at 10 mW / cm 2 The effects of sequential double cross-linking (gelatin chain entanglement at 4°C and visible light photocross-linking) were measured by time sweep analysis and investigated by the century. Hybrid hydrogels (d7G3 and d5G5) containing more than 30% GeldECM exhibited significantly higher complex moduli than dECM (d10G0) (see Fig. 5A, B).

[0119] In addition, to investigate the final effect of triple crosslinking through thermal crosslinking, the initial temperature of the hybrid hydrogel was maintained at 4 degrees for 180 seconds while irradiating the hybrid hydrogel with visible light at 10 mW / cm. 2 After investigating the century, time sweep analysis was performed while applying a temperature profile heating from 4 to 37 degrees. As a result, it was observed that triple cross-linking significantly increased the complex elastic modulus of the hydrogel by a minimum of 3 times and a maximum of 32 times compared to double cross-linking (see Fig. 5 C and D).

[0120] Accordingly, the d7G3 (70 wt% dECM and 30 wt% GeldECM) hybrid hydrogel with the highest final complex modulus was selected under optimized mixing conditions and named as Mechanically Tuned Tissue-specific bioink (MeTTi-dERS).

[0121] Experimental Example 3: Confirming the Functionality of Bioink

[0122] FIG. 6 is a photograph showing the results of extruding each of a hybrid bioink (MeTTi-dERS) and a decellularized extracellular matrix-based hydrogel (dECM) from a nozzle of a 3D printing device according to one embodiment of the present invention.

[0123] Here, the dECM was used as dERS manufactured by adding ruthenium and sodium persulfate, which are photocuring agents, to the SIS dECM hydrogel to a final concentration of 1 mM and 10 mM, respectively.

[0124] That is, the hybrid bioink (MeTTi-dERS) and dERS according to the present invention were printed using a microextrusion-based 3D printer and nozzles of various sizes. A 19G nozzle was used to evaluate the gelation state of the ink and the uniformity of the ink strands, and the dERS and MeTTi-dERS ink strands were extruded at a pressure of 5 kPa until they broke.

[0125] As a result, compared to dERS, MeTTi-dERS was able to form much longer ink strands (see Fig. 6), which confirmed that the MeTTi-dERS according to the present invention was gelled more strongly and could withstand more of the weight of the printing ink strand.

[0126] FIG. 7 is a photograph evaluating the stability of a structure printed using a hybrid bioink (MeTTi-dERS) and a decellularized extracellular matrix-based hydrogel (dECM) according to one embodiment of the present invention.

[0127] Here, the dECM was used as dERS manufactured by adding ruthenium and sodium persulfate, which are photocuring agents, to the SIS dECM hydrogel to a final concentration of 1 mM and 10 mM, respectively.

[0128] Before thermal cross-linking, tissues printed with dERS and MeTTi-dERS having the same aspect ratio were stirred on an orbital shaker at 300 rpm. As a result, the dERS tissue collapsed after 5 seconds, whereas the MeTTi-dERS tissue withstood stirring for 15 seconds and recovered to its original shape (see Fig. 7A).

[0129] Additionally, during the thermal cross-linking process by incubating the tissue at 37 °C for 40 minutes, the structure using dERS was observed to collapse, but the tissue structure using MeTTi-dERS was observed to continuously maintain the printed structure (see B in Fig. 7).

[0130] Accordingly, it was confirmed that the hybrid bioink (MeTTi-dERS) according to the present invention can not only disperse energy received from the outside but also stably culture tissues by further including a tissue-specific rheological modifier (GeldECM).

[0131] FIG. 8 is a photograph showing the viability of cells encapsulated in a hybrid bioink (MeTTi-dERS) and a decellularized extracellular matrix-based hydrogel (dECM) according to one embodiment of the present invention, and then printed with nozzles of various diameters.

[0132] Here, the dECM was used as dERS manufactured by adding ruthenium and sodium persulfate, which are photocuring agents, to the SIS dECM hydrogel to a final concentration of 1 mM and 10 mM, respectively.

[0133] Specifically, cells constituting the intestinal wall were placed in DMEM (Dulbecco's Modified Eagle's Medium, with high glucose) containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin / streptomycin and cultured under humidified 5% carbon dioxide atmosphere and 37°C temperature conditions. Thereafter, cells were encapsulated in each of the SIS dECM hydrogels and hybrid hydrogels manufactured in Examples 1 and 3 and printed using nozzles of various diameters. When a 19-gauge nozzle was used, both dERS and MeTTi-dERS showed a high viability of over 80%, and it was confirmed that there was no difference between the groups. In addition, even when a 24-gauge nozzle was used, an excellent cell viability of over 70% was observed (see Fig. 8).

[0134] Although the present invention has been illustrated and described above with respect to specific preferred embodiments, it will be apparent to those skilled in the art that the present invention may be variously modified and changed without departing from the technical features or scope of the present invention as defined by the following claims.

Claims

1. Hybrid bioink containing decellularized extracellular matrix-based hydrogel (dECM) and gelatinized decellularized extracellular matrix-based hydrogel (GeldECM).

2. In paragraph 1, A hybrid bioink characterized by further comprising a photoinitiator.

3. In paragraph 2, The above decellularized extracellular matrix-based hydrogel (dECM) is a hybrid bioink characterized in that the decellularized extracellular matrix is ​​dissolved in an acid solution and then neutralized.

4. In paragraph 2, The above gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) is a hybrid bioink characterized in that the decellularized extracellular matrix is ​​dissolved in an acid solution, neutralized, and then heat-treated to gelatinize the hydrogel.

5. In paragraph 2, A hybrid bioink characterized in that the decellularized extracellular matrix-based hydrogel (dECM) and the gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) are included in a weight ratio of 80:20 to 50:

50.

6. In paragraph 5, A hybrid bioink characterized in that the decellularized extracellular matrix-based hydrogel (dECM) and the gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) are included in a weight ratio of 80:20 to 60:

40.

7. In paragraph 5, A hybrid bioink characterized in that the decellularized extracellular matrix-based hydrogel (dECM) and the gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) are contained in a ratio of 70:30 wt%.

8. In paragraph 2, A hybrid bioink characterized in that the mixture containing the above decellularized extracellular matrix-based hydrogel (dECM), the gelatinized decellularized extracellular matrix-based hydrogel (GeldECM), and a photoinitiator undergoes primary physical crosslinking, secondary crosslinking by light irradiation, and tertiary crosslinking by heat treatment.

9. In paragraph 8, The above physical primary cross-linking is performed at 1 to 10°C, The secondary crosslinking by the above light irradiation is processing visible light, A hybrid bioink characterized in that the tertiary cross-linking by the above heat treatment is performed at 30 to 70°C.

10. In paragraph 9, A hybrid bioink characterized in that the tertiary cross-linking by the above heat treatment is performed for 2 to 4 hours.

11. In paragraph 9, A hybrid bioink characterized by a complex modulus of 10 to 20 kPa.

12. A hybrid bioink according to any one of claims 1 to 11, characterized in that it is used for manufacturing a tissue structure mimic.

13. A method for producing a hybrid bioink, comprising a step of mixing a decellularized extracellular matrix-based hydrogel (dECM) and a gelatinized decellularized extracellular matrix-based hydrogel (GeldECM).

14. In paragraph 13, The step of mixing the above decellularized extracellular matrix-based hydrogel (dECM) and the gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) is as follows: A method for producing a hybrid bioink, characterized in that it comprises a step of mixing a photoinitiator.

15. In paragraph 14, A method for producing a hybrid bioink, characterized in that the decellularized extracellular matrix-based hydrogel (dECM) and the gelatinized decellularized extracellular matrix-based hydrogel (GeldECM) are mixed in a weight ratio of 80:20 to 50:

50.

16. In paragraph 15, A first cross-linking step of lowering the temperature of the mixture that has undergone the above mixing step to 1 to 10°C; A second cross-linking step of irradiating visible light to the mixture that has undergone the first cross-linking step; and A method for producing a hybrid bioink, characterized in that it further includes a third cross-linking step of treating the mixture that has undergone the second cross-linking step at 30 to 70°C.

17. In paragraph 16, A hybrid bioink characterized in that the above-mentioned third cross-linking step is processed for 2 to 4 hours.

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