Hyaluronic acid–gelatin-based hydrogel composition

Abstract

The present invention relates to a hydrogel composition for 3D printing and, particularly, to: a hyaluronic acid–gelatin-based hydrogel composition including a hyaluronic acid derivative and a gelatin derivative, which exhibits excellent biocompatibility, structural stability, and mechanical properties; and an injectable gel, a hydrogel for 3D printing, and an artificial organ-mimicking hydrogel prepared therefrom, and an application thereof.

Description

Hyaluronic acid-gelatin-based hydrogel composition

[0001] The present invention relates to an injectable and 3D printing hydrogel composition, and more specifically to a hyaluronic acid-gelatin-based hydrogel composition having excellent biocompatibility, structural stability, and mechanical properties including a hyaluronic acid derivative and a gelatin derivative, a 3D printing hydrogel prepared therefrom, and applications.

[0002] Recently, in the fields of tissue engineering and regenerative medicine, hydrogels derived from biopolymers are emerging as key scaffolding materials due to their biocompatibility and biodegradability. These hydrogels, known for their hydrophilicity, can absorb and retain significant amounts of water and bodily fluids and can be composed of natural polymers of biological tissues by combining them with proteins such as collagen and gelatin. Hydrogels exhibit a soft and flexible texture and mechanical properties similar to biological tissues, and can be utilized for tissue regeneration by using proteins, polysaccharides, etc., to enhance cell and biocompatibility.

[0003] Due to these unique characteristics, they are establishing themselves as optimal scaffolds that promote cell adhesion, growth, and differentiation in a manner very similar to the natural environment. Among various biomolecules, polysaccharides have been particularly emphasized due to their structural similarity to the extracellular matrix and support the three-dimensional expansion and proliferation of embedded cells.

[0004] Hyaluronic acid (HA) is a representative example of a polysaccharide recognized for its essential role in the extracellular matrix of various tissues and its potential as a carrier for cell culture. Therefore, to enhance mechanical elasticity and maintain the structural integrity of hydrogels, these polysaccharides are often modified with reactive groups that enable crosslinking. This modification process not only strengthens the physical properties of hydrogels but also improves their natural compatibility with biological systems, thereby solidifying their role in creating scaffolds and bioinks that encapsulate cells within a multidimensional framework for tissue regeneration.

[0005] 3D bioprinting, one of the latest biotechnologies, is widely utilized in tissue engineering and regenerative medicine to create complex artificial organ and tissue structures that closely resemble natural organs and tissues. The limited self-regeneration capabilities of human tissues have sparked significant interest in tissue regeneration and accelerated the development of the field of regenerative medicine, which aims to replace or repair damaged organs or tissues. To address the increasing prevalence of injuries resulting from industrialization and modernization, innovative and successful repair technologies are required. Among cutting-edge methods, 3D bioprinting is recognized as an interesting and promising option, and printable hydrogels can also be used as injectable hydrogels.

[0006] Some examples of the applications of 3D printing and bioprinting in tissue engineering, regenerative medicine, new drug testing, and organoids include injectable hydrogels for bone and cartilage growth, the fabrication of bioprinted tissue engineering scaffolds and devices, skin tissue engineering, and hydrogel systems used in biomedicine. Injectable gels offer the advantage of being able to directly apply bioink to damaged defects and complexly shaped damaged areas, while bioprinting is advantageous for tissue engineering applications when using hand-written movable pens (in vivo tissue regeneration) and stationary bioprinters and bioreactors that utilize in vitro tissue regeneration processes.

[0007] However, the skin's limited intrinsic regenerative capacity and the need for large-area tissue regeneration present significant challenges in the context of injury and degeneration. Furthermore, to address issues such as the repair of cartilage damage involving complex structural shapes, the regeneration of elastic tissues in the heart and blood vessels, and the regeneration of conduit-like nerves, it is necessary to develop advanced injectable hydrogels capable of providing an environment for cell growth, differentiation, and tissue regeneration; biomaterials applicable to bioprinting pens for manual writing; and bioprinting tissue engineering used in fixed locations. Additionally, there is a need to develop bioinks capable of satisfying the physicochemical properties required for stem cell differentiation and the development of organoids for regenerative organs. Moreover, there is a need for the development of various materials, including self-bonding hydrogels that facilitate the easy mixing of cells and drugs, as well as materials for skin tissue regeneration and nerve regeneration in patients with diabetic foot ulcers. Furthermore, in the field of tissue engineering, researchers are exploring various methods to find innovative and effective biomaterials capable of mimicking the complex microenvironments and physicochemical and biological properties of natural tissues, such as biomaterials composed of proteins and polysaccharides, diabetic foot ulcers, nerves, and cartilage.

[0008] [Prior Art Literature]

[0009] [Patent Literature]

[0010] (Patent Document 1) Republic of Korea Published Patent No. 10-2018-0049712

[0011] The present invention aims to solve the above-mentioned problems by providing a hyaluronic acid-gelatin-based hydrogel composition having excellent biocompatibility, structural stability, and mechanical properties, comprising oxidized hyaluronic acid (OHA)-cystamined gelatin (CyGel).

[0012] In addition, as a means to minimize chemical modification of the components of the hydrogel, a hyaluronic acid-gelatin-based hydrogel composition with excellent biocompatibility is provided by simply mixing oxidized hyaluronic acid (OHA), gelatin, and cystamine.

[0013] In addition, the present invention aims to solve the above-mentioned problems by preparing a self-crosslinked hydrogel through a Schiff base mechanism comprising cystamined hyaluronic acid (HACys), oxidized carboxymethyl cellulose (OCMC), and adipic acid dehydrazide gelatin (GelADH), thereby providing a hyaluronic acid-gelatin-based hydrogel composition with excellent biocompatibility, structural stability, and mechanical properties.

[0014] In addition, the present invention provides a ternary polymer composition by adding carboxymethyl cellulose to the main chain of a hyaluronic acid-gelatin polymer to improve bioprinting properties.

[0015] In addition, the present invention can enhance the physical properties of a hydrogel by adding a low molecular weight erythritol substance to the above hydrogel (hyaluronic acid-gelatin, hyaluronic acid-carboxymethylcellulose) to increase the degree of cross-linking. Since the physical properties of the hydrogel with added erythritol can improve mechanical strength and control the sugar composition in the culture medium, it provides a tissue regeneration hydrogel suitable for treating diabetic patients.

[0016] In addition, the present invention can be utilized as a more efficient tissue regeneration material by controlling the degree of crosslinking to control drug delivery, control the physical properties of bioink tissue regeneration structures, stacking of bioprinted structures, skin regeneration over large areas, and inducing tissue regeneration by directly injecting into damaged sites in vivo (e.g., cartilage, blood vessels, heart, etc.) using a digital bioprinting pen on damaged tissues with complex shapes.

[0017] The above and other objects and advantages of the present invention will become apparent from the following description describing preferred embodiments.

[0018] The above objective can be achieved by a hyaluronic acid-gelatin-based hydrogel composition comprising oxidized hyaluronic acid (OHA) and cystamined gelatin (CyGel).

[0019] The above-mentioned oxidized hyaluronic acid (OHA) and cystamine-modified gelatin (CyGel) are mixed in a weight ratio of 1:0.5 to 1:2.

[0020] The above objective can be achieved by a method for preparing a hyaluronic acid-gelatin-based hydrogel composition comprising the steps of: preparing a hyaluronic acid (HA) solution; adding an oxidizing agent to the hyaluronic acid (HA) solution and performing an oxidation reaction; removing the oxidizing agent after the reaction is completed and dialyzing to prepare oxidized hyaluronic acid (OHA); preparing a gelatin solution; adding a cysteamine compound to the gelatin solution and performing an amide bonding reaction; dialyzing after the reaction is completed to obtain cystamine-containing gelatin (CyGel); and mixing and reacting the oxidized hyaluronic acid (OHA) and cystamine-containing gelatin (CyGel) to prepare a hydrogel.

[0021] The above oxidation reaction is characterized by including a step of stirring for at least 24 hours at room temperature of 21 to 24°C.

[0022] The above amide bonding reaction is characterized by including a step of stirring at 40 to 45°C for 24 hours or more.

[0023] The above-mentioned oxidized hyaluronic acid (OHA) and cystamine-modified gelatin (CyGel) are mixed in a weight ratio of 1:0.5 to 1:2.

[0024] The above objective can be achieved by a hyaluronic acid-gelatin-based hydrogel composition comprising oxidized hyaluronic acid (OHA), cystamine, and gelatin (Gel). If necessary, compounds such as EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) may be added.

[0025] The above objective can be achieved by a hyaluronic acid-gelatin-based hydrogel composition comprising the carboxymethyl cellulose oxide (OCMC), cystamined hyaluronic acid (HACys), and adipic acid dihydrazide gelatin (GelADH).

[0026] The above carboxymethyl cellulose (OCMC), cystamined hyaluronic acid (HACys), and adipic acid dihydrazide gelatin (GelADH) are characterized by being mixed in a weight ratio of 1:1 to 2:1 to 2.

[0027] The above objective comprises the steps of: preparing a carboxymethyl cellulose (CMC) solution; adding an oxidizing agent to the carboxymethyl cellulose (CMC) solution and performing an oxidation reaction; preparing oxidized carboxymethyl cellulose (OCMC) by dialysis after the oxidation reaction is completed; preparing a hyaluronic acid (HA) solution; performing an amide bonding reaction by adding a coupling agent to the hyaluronic acid (HA) solution, and preparing cystamine-modified hyaluronic acid (HACys) by adding a cystamine compound and reacting; preparing a gelatin solution; mixing adipic acid dihydrazide (ADH) into the gelatin solution and performing a reaction by adding a coupling agent; and obtaining adipic acid dihydrazide gelatin (GelADH) by dialysis after the reaction is completed. This can be achieved by a method for preparing a hyaluronic acid-gelatin-based hydrogel composition comprising the step of mixing and reacting the oxidized carboxymethyl cellulose (OCMC), cystamined hyaluronic acid (HACys), and adipic acid dihydrazide gelatin (GelADH) to prepare a hydrogel.

[0028] The above carboxymethyl cellulose (OCMC), cystamined hyaluronic acid (HACys), and adipic acid dihydrazide gelatin (GelADH) are characterized by being mixed in a weight ratio of 1:1 to 2:1 to 2.

[0029] The method is characterized by adding erythritol and reacting it together in the step of preparing the above hydrogel.

[0030] The above erythritol is characterized by being included in an amount of 1 to 5 weight percent based on the total weight of the hydrogel.

[0031] According to the present invention, oxidized hyaluronic acid (OHA)-cystamine modified gelatin (CyGel) are each prepared, and a hydrogel with a unique Schiff reaction-based self-crosslinking matrix can be prepared by utilizing the chemical interaction between them.

[0032] In addition, according to the present invention, biocompatibility can be improved by preparing an oxidized hyaluronic acid-gelatin hydrogel without using the crosslinking agent used in the process of preparing the above-mentioned cystamine-gelatin compound, including oxidized hyaluronic acid (OHA) and cystamine and gelatin (Gel).

[0033] In addition, according to the present invention, cystamine-modified hyaluronic acid (HACys), oxidized carboxymethyl cellulose (OCMC), and adipic acid dehydrazide gelatin (GelADH) are each prepared, and by grafting them, a cross-linked hydrogel can be prepared through a Schiff base mechanism formed between an amine modified group (HA and gelatin) and an aldehyde modified CMC.

[0034] These hydrogels can minimize cytotoxicity issues by maximizing the purity of the gel precursor, and have excellent biocompatibility and mechanical properties. In particular, they can produce various 3D structures (meniscus, pyramids, lattice structures) of up to 50 layers with high precision and excellent post-printing structural shape stability without the need for a scaffold or post-processing.

[0035] Furthermore, the hydrogel according to one embodiment of the present invention has excellent biocompatibility and can demonstrate excellent potential for 3D bioprinting of self-supporting structures in skin tissue engineering, bone tissue engineering, and regenerative medicine. This can be used in various ways in the field of tissue regeneration by tissue engineering.

[0036] Furthermore, according to the present invention, erythritol has the effect of significantly improving hydrogen bonding and gelation performance with polysaccharides and protein polymers. In particular, the balance of strength, flexibility, and structural stability was best maintained up to 5% (w / v), whereas as the concentration increased, viscous hydrogels were formed, limiting usability. In addition, HACys-OCMC-GelADH-based hydrogels containing erythritol demonstrated controllable mechanical properties, degradation rates, and biological activity in sugar-free media. By controlling the amount of erythritol, properties highly suitable for 3D bioprinting and tissue engineering applications for diabetic patients can be exhibited.

[0037] Thus, the erythritol-induced gelation mechanism supporting the development of low-calorie functional hydrogels and the hydrogel system utilizing it can provide the potential for tissue repair and next-generation biomaterials for specific immunologically damaged groups by leveraging the synergistic effects of the components as a promising strategy for regenerative medicine.

[0038] However, the effects of the present invention are not limited to those mentioned above, and other unmentioned effects will be clearly understood by those skilled in the art from the description below.

[0039] Figure 1 is a figure showing the Fourier transform infrared spectroscopy (FTIR) analysis results of hydrogels according to Examples 1 to 3 of the present invention.

[0040] Figure 2 is a figure showing the Fourier transform infrared spectroscopy (FTIR) analysis results of hydrogels according to Examples 4 to 7 of the present invention.

[0041] Figure 3 is a figure showing the scanning electron microscope (SEM) analysis results of hydrogels according to Examples 1 to 3 of the present invention.

[0042] Figure 4 is a figure showing the scanning electron microscope (SEM) analysis results of a hydrogel according to Example 4 of the present invention.

[0043] Figure 5 shows the results of measuring the periodic compression characteristics (5 cycles) of the hydrogels of Examples 1 to 3 in Experimental Example 5.

[0044] Figure 6 is a figure showing the results of measuring the hardness of the hydrogels of Examples 1 to 3 in Experimental Example 5.

[0045] Figure 7 is a figure showing the results of measuring the gumminess and chewiness of the hydrogels of Examples 1 to 3 in Experimental Example 5.

[0046] Figure 8 is a figure showing the results of measuring the cohesion of the hydrogels of Examples 1 to 3 in Experimental Example 5.

[0047] Figure 9 is a figure showing the results of measuring the resilience and springiness of the hydrogels of Examples 1 to 3 in Experimental Example 5.

[0048] Figure 10 is a figure showing the results of measuring the adhesiveness of the hydrogels of Examples 1 to 3 in Experimental Example 5.

[0049] Figure 11 shows the results of measuring the periodic compression characteristics (5 cycles) of the hydrogel of Example 4 in Experimental Example 5.

[0050] Figure 12 is a figure showing the results of measuring the hardness of the hydrogel of Example 4 in Experimental Example 5.

[0051] Figure 13 is a figure showing the results of measuring the adhesiveness of the hydrogel of Example 4 in Experimental Example 5.

[0052] Figure 14 is a figure showing the results of measuring the cohesion of the hydrogel of Example 4 in Experimental Example 5.

[0053] Figure 15 is a figure showing the results of measuring the resilience and springiness of the hydrogel of Example 4 in Experimental Example 5.

[0054] Figure 16 is a figure showing the results of measuring the gumminess and chewiness of the hydrogel of Example 4 in Experimental Example 5.

[0055] Figure 17 is a figure showing the rheological analysis (viscosity) results of the hydrogels of Examples 1 to 3 in Experimental Example 6.

[0056] Figure 18 is a figure showing the results of measuring the percentage of porosity (%) of the hydrogels of Examples 1 to 3 in Experimental Example 6.

[0057] Figure 19 is a figure showing the measurement results of the average swelling rate (%) of the hydrogels of Examples 1 to 3 in Experimental Example 6.

[0058] FIG. 20 is a figure showing the results of the performance degradation test (degradation) of the hydrogels of Examples 1 to 3 in Experimental Example 6.

[0059] Figure 21 is a figure showing the rheological analysis (viscosity) results of the hydrogel of Example 4 in Experimental Example 6.

[0060] Figure 22 is a figure showing the results of the performance degradation test (decomposition) of the hydrogel of Example 4 in Experimental Example 6.

[0061] Figure 23 is a figure showing the measurement results of the average swelling rate (%) of the hydrogel of Example 4 in Experimental Example 6.

[0062] Figure 24 is a figure showing the measurement results of the porosity percentage (%) of the hydrogel of Example 4 in Experimental Example 6.

[0063] FIGS. 25 to 30 are drawings showing the results of measuring mechanical properties in Experimental Example 7 of the hydrogels of Examples 1 to 3.

[0064] FIGS. 31 to 34 are drawings showing the results of measuring the mechanical properties of the hydrogel of Example 2 in Experimental Example 8.

[0065] FIG. 35 is SEM images for morphological analysis of various 3D printed structures using the hydrogel of Example 2 of Experimental Example 9.

[0066] Figure 36 is SEM image for morphological analysis of various 3D printed structures using the hydrogel of Example 4 of Experimental Example 9.

[0067] Figure 37 is a diagram showing the results of Experimental Example 10.

[0068] Figure 38 is a diagram showing the results of Experimental Example 11.

[0069] Figure 39 is a diagram showing the results of cell viability and the degree of damage according to the rotational speed (rpm) of the bioprinting pen of Experimental Example 12.

[0070] The present invention will be described in detail below with reference to the embodiments and drawings. These embodiments are presented merely as examples to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these embodiments.

[0071] Furthermore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains, and in the event of a conflict, the description in this specification including definitions shall prevail.

[0072] To clearly explain the proposed invention in the drawings, parts unrelated to the description have been omitted, and similar parts throughout the specification have been given similar reference numerals. Furthermore, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components. Additionally, the term "part" as described in the specification refers to a single unit or block that performs a specific function.

[0073] In each step, identification codes (1st, 2nd, etc.) are used for convenience of explanation and do not describe the order of the steps; the steps may be performed differently from the specified order unless a specific order is clearly indicated in the context. That is, the steps may be performed in the same order as specified, substantially simultaneously, or in the reverse order.

[0074] All technical terms used in this invention, unless otherwise defined, are used in the sense generally understood by those skilled in the art in the relevant field of this invention. Furthermore, while preferred methods or samples are described herein, similar or equivalents are also included within the scope of this invention. The contents of all publications cited as references in this specification are incorporated by reference in their entirety into this invention.

[0075]

[0076] Example 1: Hyaluronic acid oxidized (OHA)-cystamined gelatin (CyGel) hydrogel

[0077] One aspect of the present invention provides a hyaluronic acid-gelatin-based hydrogel.

[0078] Specifically, the above hydrogel may be formed by using hyaluronic acid oxidized (OHA) and cystamined gelatin (CyGel) to form a matrix through Schiff base bonding (hereinafter referred to as 'OHA-CyGel hydrogel').

[0079] The above OHA-CyGel hydrogel is a hydrogel that combines gelatin and hyaluronic acid (HA) through Schiff base bonding formed by the combination of the aldehyde group of OHA and the amine group of CyGel, and can improve biocompatibility, structural stability, and mechanical stability compared to conventional hyaluronic acid-gelatin-based hydrogels.

[0080] The above OHA-CyGel hydrogel is a hydrogel that combines gelatin and hyaluronic acid (HA) through Schiff base bonding formed by the combination of the aldehyde group of OHA and the amine group of cystamine and gelatin, and can improve biocompatibility compared to conventional hyaluronic acid-gelatin-based hydrogels.

[0081]

[0082] For example, it is preferable that the oxidized hyaluronic acid (OHA) and cystamined gelatin (CyGel) be mixed in a weight ratio of 1:0.5 to 1:2, specifically, a weight ratio of 1:1.

[0083]

[0084] Cystamine can be an oxidized form of cysteamine. Since the disulfide bonds of cystamine are found in cell surface proteins, this molecule can easily interact with cells. Cystamined gelatin can be conjugated with OHA to facilitate interaction with cells and reduce the degradation rate of gelatin-hyaluronic acid hydrogels.

[0085] In the above OHA-CyGel hydrogel, the formation of Schiff base bonds, which are dynamic covalent bonds between aldehyde groups and amine groups, plays a role in improving stability in the biological and hydrated states along with the formation of the hydrogel.

[0086] Incorporating cystamine, which has a double amine group, into gelatin, a natural polymer derived from collagen, can create special gelatin modifications. These modifications react with the aldehyde groups found in oxidized hyaluronic acid (OHA). These chemical interactions can facilitate the development of hydrogels distinguished by enhanced physical and biological properties, making them suitable for use in 3D printing applications.

[0087] These Schiff base reactions help replace the use of toxic chemical crosslinking agents, such as formaldehyde, glutaraldehyde, and carbodiimide, which are critical in tissue engineering applications. Furthermore, by fabricating hydrogels that fully utilize the properties of gelatin and hyaluronic acid biopolymers, they hold significance as materials for injectable and bioprinted tissue engineering and regenerative medicine.

[0088]

[0089] According to one embodiment of the present invention, a method for preparing the oxidized hyaluronic acid (OHA)-cystamined gelatin (CyGel) hydrogel comprises the step of preparing a hyaluronic acid (HA) solution;

[0090] The method may include the steps of: adding an oxidizing agent to the hyaluronic acid (HA) solution and performing an oxidation reaction; removing the oxidizing agent after the reaction is completed and dialyzing to produce oxidized hyaluronic acid (OHA); preparing a gelatin solution; adding a cysteamine compound to the gelatin solution and performing an amide bonding reaction; dialyzing after the reaction is completed to obtain cystamine-modified gelatin (CyGel); and mixing and reacting the oxidized hyaluronic acid (OHA) and cystamine-modified gelatin (CyGel) to produce a hydrogel.

[0091] In other words, by preparing oxidized hyaluronic acid (OHA) and cystamine-modified gelatin (CyGel) separately, then preparing each as a precursor solution and mixing them to self-react, a hyaluronic acid-gelatin-based hydrogel can be prepared, thereby overcoming the limitations of individual polymers and the toxicity issues of crosslinking agents to obtain biocompatible properties. Sodium iodate (NaIO4) oxidation of hyaluronic acid (HA) provides an aldehyde group, whereas cystamine functionalization of gelatin provides an amine group for the formation of an imine bond (C=N) using a Schiff base reaction.

[0092] In order to improve the shape, pore diameter, printability, cell viability, and physicochemical properties of the hyaluronic acid-gelatin hydrogel, the mixing ratio of the OHA and CyGel composition may be a weight ratio of 1:0.5 to 1:2, more specifically, a weight ratio of 1:0.8 to 1:1.2, preferably a weight ratio of 1:1.

[0093]

[0094] Example 2: Cystaminated hyaluronic acid (HACys)-carboxymethyl cellulose oxidized (OCMC)-adipoyl dehydrazide gelatin (GelADH) hydrogel

[0095] Another aspect of the present invention provides a hyaluronic acid-gelatin-based hydrogel.

[0096] Specifically, the above hydrogel may be formed by creating a ternary copolymer hydrogel matrix through self-bonding crosslinking using a combination of three types of polymers: cystamine-modified hyaluronic acid (HACys), carboxymethyl cellulose oxidized (OCMC), and adipic acid dihydrazide gelatin (GelADH) (hereinafter referred to as 'HACys-OCMC-GelADH hydrogel').

[0097] Specifically, in order to further improve structural and mechanical properties compared to conventional hyaluronic acid-gelatin-based hydrogels, hyaluronic acid (HACys) incorporated with cystamine is formed to improve the structural integrity and mechanical stability of the hydrogel under physiological conditions through disulfide crosslinking, and a HACys-OCMC-GelADH hydrogel can be prepared by incorporating carboxymethyl cellulose oxide (OCMC) and adipic acid dihydrazide (GelADH) to form a double crosslink through a Schiff base mechanism between an amine modified group (HA and gelatin) and an aldehyde modified CMC.

[0098]

[0099] Oxidized carboxymethyl cellulose (OCMC), a cellulose derivative, is a modified form of methyl cellulose (CMC) through oxidation, in which the hydroxyl groups of CMC are converted to dialdehyde at the C2 / C3 of the glucose unit, where two adjacent hydroxyl (-OH) bonds are located.

[0100] The above oxidized carboxymethyl cellulose (OCMC) has excellent biodegradability, biocompatibility, and the ability to form a stable hydrogel, provides shape stability when manufacturing structures by bioprinting, and has excellent mechanical properties.

[0101] Gelatin extracted from collagen contains physiologically active peptide sequences that support cell adhesion, proliferation, and differentiation. By modifying this gelatin with adipic acid dihydrazide to provide -NH2 functional groups, the amine-restricted gelatin can secure additional crosslinking sites, thereby improving the mechanical properties and stability of the hydrogel.

[0102] The above HACys-OCMC-GelADH hydrogel can not only be mechanically strong due to the synergy of the combination of its components but also enhance cell viability and function. Accordingly, a hydrogel composition containing the above hydrogel exhibits characteristics suitable for 3D bioprinting, a cutting-edge technology capable of precisely fabricating complex tissue structures. Since hydrogel-based scaffolds can be printed with high spatial resolution and the scaffold structure can be controlled, 3D bioprinting can be applied as an ideal platform for developing tissue engineering structures for the repair and regeneration of bone, cartilage, nerve, and myocardial tissues.

[0103] For example, the carboxymethyl cellulose oxide (OCMC), cystamined hyaluronic acid (HACys), and adipic acid dihydrazide gelatin (GelADH) are preferably mixed in a weight ratio of 1:1 to 2:1 to 2, specifically 1:1:1.

[0104] Meanwhile, the above HACys-OCMC-GelADH hydrogel contains erythritol, and by forming hydrogen bonds between erythritol and the polysaccharide-protein network to increase the organized structure, properties such as structural stability, mechanical properties, degradation rate, and bioactivity of the hydrogel can be further improved.

[0105] Sucrose, which is similar to erythritol, also helps with moisture attachment or retention by promoting intermolecular interactions, and the addition of sucrose can stabilize the hydrogel by increasing the overall hydrogen bond structure and preventing moisture separation. Additionally, polyols are reducing sugars with alcohol groups added instead of aldehydes, and can also contribute to improving gel properties.

[0106] However, the glycemic index and caloric value of erythritol are 0 and 0.2 kcal / g, respectively, whereas those of sucrose are 100 and 3.9 kcal / g, respectively. In other words, erythritol is not absorbed into the bloodstream and is excreted in the urine, or is rapidly and effectively absorbed in the small intestine (60–90%), which can prevent the laxative side effects that occur when other polyols are consumed in excess, and it has excellent potential as a sugar-free substitute for specific patient groups.

[0107] The erythritol may be included in an amount of 0.1 to 10 weight%, specifically 1 to 7 weight%, more specifically 1 to 5 weight%, preferably 5 weight%, based on the total weight of the hydrogel.

[0108]

[0109] A method for preparing a HACys-OCMC-GelADH hydrogel according to another embodiment of the present invention is as follows.

[0110] The method for preparing the above HACys-OCMC-GelADH hydrogel comprises the steps of: preparing a carboxymethyl cellulose (CMC) solution; adding an oxidizing agent to the carboxymethyl cellulose (CMC) solution and performing an oxidation reaction; preparing oxidized carboxymethyl cellulose (OCMC) by dialysis after the oxidation reaction is completed; preparing a hyaluronic acid (HA) solution; performing an amide bonding reaction by adding a coupling agent to the hyaluronic acid (HA) solution, and preparing cystamine-modified hyaluronic acid (HACys) by adding a cystamine compound and reacting; preparing a gelatin solution; mixing adipic acid dihydrazide (ADH) into the gelatin solution and performing a reaction by adding a coupling agent; and obtaining adipic acid dihydrazide gelatin (GelADH) by dialysis after the reaction is completed. and a step of preparing a hydrogel by mixing and reacting the oxidized carboxymethyl cellulose (OCMC), cystamined hyaluronic acid (HACys), and adipic acid dihydrazide gelatin (GelADH);

[0111] In the step of preparing the above hydrogel, erythritol can be added and reacted together.

[0112] The erythritol may be included in an amount of 0.1 to 10 weight%, specifically 1 to 7 weight%, more specifically 1 to 5 weight%, based on the total weight of the hydrogel.

[0113]

[0114] Bioink for 3D printing

[0115] The above hydrogel may be used as an ink composition for 3D printing (or bioprinting) itself, or may be included in an ink composition.

[0116] The above hydrogel may have a content range of, for example, 0.1 to 100 weight% with respect to the total weight of the ink composition.

[0117] When the above hydrogel is included in the above 3D printing (or bioprinting) ink composition, in addition to the above hydrogel, known additive components commonly used in the field may be included without limitation.

[0118] For example, the bio-ink composition may additionally contain one or more selected from the group consisting of cells, cell culture media, bioactive substances, and additives. The additives may be any biological or non-biological, including gels, nano or microparticles, biomolecules, polymers, crosslinking agents, and mixtures thereof.

[0119] The above cells may be one or more selected from the group consisting of, but not limited to, stem cells, embryonic stem cells, mesenchymal stem cells, adipose-derived stem cells, hematopoietic stem cells, osteoblasts, myoblasts, tenocytes, neuroblasts, fibroblasts, glioblasts, germ cells, hepatocytes, renal cells, Sertoli cells, chondrocytes, epithelial cells, cardiovascular cells, keratinocytes, smooth muscle cells, cardiomyocytes, glial cells, endothelial cells, hormone-secreting cells, immune cells, pancreatic islet cells, and neurons.

[0120] A bioink can be manufactured by incorporating the above-mentioned stem cells into a hydrogel. The bioink can be quantitatively separated and dispensed, and the differentiation of stem cells can be induced and applied to 3D cell culture or 2D cell culture to be used for the manufacture of organoids.

[0121] The cell culture medium described above may include, but is not limited to, any medium suitable for the target cell, and any medium suitable for cell lines is known in the industry, such as MEM medium, RPMI1640 medium, DMEM medium, IMDM medium, etc. Additionally, the cell culture medium may additionally include, but is not limited to, vitamins, growth factors, cytokines, antibiotics, erythritol, etc.

[0122] The above-mentioned bioactive substances, for the purpose of proliferating cells or promoting the secretion of extracellular matrix, include but are not limited to: transforming growth factor-beta (TGF-β), fibroblast growth factor (FGF), bone morphogenic protein (BMP), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), nerve growth factor (NGF), hepatocyte growth factor (HGF), placental growth factor (PIGF), granulocyte colony stimulating factor (G-CSF), and cell culture media such as ascorbate 2-phosphate. It may include one or more selected from the group consisting of additives.

[0123] However, the types of the above additives are not limited thereto.

[0124]

[0125] According to one embodiment of the present invention, a bioink composition comprising the hyaluronic acid-gelatin-based hydrogel can be provided as an injectable agent, cell therapy agent, 3D cell culture structure, drug delivery system, organoid material, etc. that can be directly injected into a small defect site.

[0126] According to one embodiment of the present invention, a 3D printing method may be provided comprising the steps of: including a bioink composition containing the hyaluronic acid-gelatin-based hydrogel in a syringe; filling a 3D printer; and performing 3D printing to form a three-dimensional structure.

[0127] The above three-dimensional structure may include, for example, tissue engineering structures, artificial organ structures (e.g., organoids), but is not limited thereto, and may further include various structures intended as final targets in the field of 3D bioprinting without limitation.

[0128]

[0129] Hereinafter, the structure of the present invention and the resulting effects are to be explained in more detail through specific embodiments and comparative examples. However, these embodiments are intended to explain the present invention more specifically, and the scope of the present invention is not limited to these embodiments.

[0130]

[0131] [Preparation Example 1: Synthesis of Hyaluronic Acid Oxide (OHA)]

[0132] 1 g of hyaluronic acid (HA) was added to 100 ml of deionized water and mixed using a magnetic stirrer for 2 hours at room temperature (21–24°C) to prepare a homogeneous solution. Then, 2.5 mmol (0.535 g) of sodium periodate was dissolved in 5 ml of deionized water and added to the solution to perform an oxidation reaction. The oxidation reaction was carried out in a dark chamber at room temperature (21–24°C) for 24 hours using a magnetic stirrer. Afterward, 2.5 mmol (0.155 g) of ethylene glycol was added and the reaction was continued for 1 hour to remove unreacted oxidizing agent. The solution was then dialyzed with water for 1 week. Subsequently, the dialyzed solution was freeze-dried at -80°C for 5 days. The resulting white solid material was stored at -4°C for further study. The mechanism of Preparation Example 1 is shown in Reaction Scheme 1 below.

[0133] <Reaction Equation 1>

[0134]

[0135] [Preparation Example 2: Synthesis of Cystaminated Gelatin (CyGel)]

[0136] 5g of gelatin was completely dissolved in DI water at 45°C, 4g of cystamine dihydrochloride was added to the reaction mixture, and NaOH equivalent to HCl was added to the reaction mixture to neutralize HCl molecules and stirred for 30 minutes. Then, EDC (0.9g) (optional) and HOBT (0.9g) were added to the reaction mixture under stirring. The reaction solution was continuously stirred at 40°C for 24 hours. The stirred product was dialyzed in NaCl solution (0.3 M) for 2 days, in ethanol (25% v / v) for 1 day, and then in distilled water for 2 days. The sample was then transferred and frozen. Finally, the sample was freeze-dried and stored at 4°C. The mechanism for the synthesis process of Preparation Example 2 is shown in Reaction Scheme 2 below.

[0137] <Reaction Equation 2>

[0138]

[0139]

[0140] [Preparation Example 3: Synthesis of Hyaluronic Acid Oxidized-Cystamine Gelatin Hydrogel]

[0141] Various HA solution concentrations (1:0.5, 1:1, 1:2) according to Preparation Example 1 were independently prepared in ultrapure distilled water using a vortex of 1000 rpm for 4 hours at room temperature (18–24°C). Then, CyGel solutions of various concentrations according to Preparation Example 2 were also prepared in ultrapure distilled water. Subsequently, Preparation Examples 1 and 2 were introduced into a Vortex device to form a uniform hydrogel. The mechanism of Preparation Example 3 is shown in Reaction Scheme 3 below, and information regarding gel formation and time is recorded in Table 1 below (the ratio of HA to gelatin hydrogel was based on the molar calculation and deformation ratio of individual samples).

[0142] <Reaction Equation 3>

[0143]

[0144] HA : Gelatin HA (mg / ml) Gelatin (mg / ml) Gel formation time (seconds) Example 11 : 0.5 100 50 20~30 Example 21 : 1 100 100 20~30 Example 31 : 2 100 200 20~30

[0145] [Preparation Example 3-1: Synthesis of Hyaluronic Acid-Cystamine-Gelatin Hydrogel]

[0146] Oxidized hyaluronic acid (OHA) and a gelatin solution (cystamine to gelatin ratio of 4:5) were added to ultrapure water and prepared using a vortex at 100 rpm for 4 hours at room temperature. Homogeneous hyaluronic acid-gelatin gels were prepared by mixing HA and gelatin at various concentrations (1:0.5, 1:1, 1:2) using a vortex. Hydrogel formation was confirmed by inverting the tube, and the gelation time was recorded. The hydrogel can be further stabilized using the crosslinking agent EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide). After dialyzing the hydrogel in water for one week, it was concentrated with polyethylene glycol (PEG) and extruded.

[0147]

[0148] [Preparation Example 4: Synthesis of Carboxymethyl Cellulose Oxide (OCMC)]

[0149] 2.0 g of sodium carboxymethylcellulose (CMC) (400-800 cps: medium viscosity, degree of substitution: 0.65-0.90) was dissolved in 200 mL of distilled water, then 24 mL of 6.7% (w / v) NaIO4 solution was added, and an oxidation reaction was carried out in a dark environment at room temperature (24°C) for 24 hours. Afterward, ethylene glycol was added to stop the reaction, the reaction mixture was dialyzed with distilled water, and then freeze-dried. The mechanism of Preparation Example 4 is shown in Reaction Scheme 4 below.

[0150] <Reaction Equation 4>

[0151]

[0152]

[0153] [Preparation Example 5: Synthesis of Cystaminated Hyaluronic Acid (HACys)]

[0154] 0.4 g of sodium hyaluronate was dissolved in 10 mL of MES buffer (0.1 M, pH 6.5). Then, 0.570 g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC.HCl; MW: 191.70 kDa) and 0.340 g of N-hydroxysuccinimide (NHS) were added, and the reaction solution was stirred at 37°C for 15 minutes. Subsequently, 0.340 g of cystamine dihydrochloride (MW: 225.20) (Cys) was added, and the reaction was carried out at 37°C for 24 hours. The reaction solution was dialyzed with NaCl solution (0.1 M) for 2 days and with distilled water for 2 days, and then freeze-dried to obtain HACys. The mechanism of Preparation Example 5 is shown in Reaction Scheme 5 below.

[0155] <Reaction Equation 5>

[0156]

[0157]

[0158] [Preparation Example 6: Synthesis of Adipic Acid Dehydrazide Gelatin (GelADH)]

[0159] 3.0 g of gelatin was added to 300 ml of distilled water and stirred overnight. Next, 2.2 g of adipic acid dihydrazide (ADH; MW: 174.20 kDa) was added directly to the gelatin solution and stirred for 30 minutes to prepare a mixture. Meanwhile, HOBt solution (0.45 g in 10 ml DMSO) and EDC·HCl solution (0.46 g in 10 ml distilled water) were added drop by drop to the mixture, and dilute HCl solution was added to adjust the pH of the solution to 5.0. The reaction was carried out overnight at room temperature (24°C) at 37°C. The reaction-completed mixture was dialyzed with NaCl solution (0.3 M) for 2 days, ethanol (25% v / v) for 1 day, and distilled water for 2 days, after which the sample was transferred and frozen. Subsequently, the sample was freeze-dried and stored at 4°C. The mechanism of Preparation Example 6 is shown in Reaction Scheme 6 below.

[0160]

[0161] <Reaction Equation 6>

[0162]

[0163]

[0164] [Preparation Example 7: Synthesis of HACys-OCMC-GelADH Hydrogel]

[0165] As shown in Table 2 below, hydrogels were prepared by setting different compositional ratios of HACys, OCMC, and GelADH (1:1:1, 2:1:1, 1:2:1, and 1:1:2), dissolving each component in distilled water, and gently stirring. Then, the HACys-OCMC-GelADH hydrogels of Examples 4 to 7 were prepared by individually adding erythritol at various concentrations (0%, 1%, 3%, 5%, 7%) and mixing manually. Gelation occurred rapidly through self-crosslinking and hydrogen bonding, and the gelation time was recorded and verified by inverting the vial. As a result of testing the stability of the prepared HACys, OCMC, and GelADH hydrogels, they remained stable for 50 days at physiological temperature (37°C).

[0166]

[0167] HACys(10%) OCMC(10%) GelADH(10%) Erythritol Example 4 1ml 1ml 0%, 1%(0.25mmol), 3%(0.74mmol), 5%(1.23mmol), 7%(1.72mmol) Example 5 2ml 1ml 0%, 1%(0.25mmol), 3%(0.74mmol), 5%(1.23mmol), 7%(1.72mmol) Example 6 1ml 2ml 1ml 0%, 1%(0.25mmol), 3%(0.74mmol), 5%(1.23mmol), 7%(1.72mmol) Example 7 1ml 1ml 2ml 0%, 1%(0.25mmol), 3%(0.74mmol), 5%(1.23 mmol), 7%(1.72 mmol)

[0168] It was confirmed that a gel was formed within 20 to 30 seconds when combined with HACys solution, OCMC solution, and GelADH solution with varying erythritol content (0%, 1%, 3%, 5%, 7%). In other words, rapid gelation occurs due to the Schiff base reaction between the present amine and aldehyde groups, and the gelation time can be maintained at a constant level. However, among them, the hydrogel of Example 4, in which each component was mixed in a 1:1:1 ratio and erythritol was added, showed the best results in improving texture characteristics by promoting hydrogen bonding. This can be confirmed through the experimental examples described below.

[0169]

[0170] <Experimental Example 1: Oxidation Analysis>

[0171] For the oxidized hyaluronic acid (OHA) prepared according to Preparation Example 1 and the oxidized carboxymethyl cellulose (OCMC) prepared according to Preparation Example 4, the degree of oxidation was confirmed by evaluating the aldehyde concentration using the hydroxylamine hydrochloride titration method.

[0172] For the experimental method, 25 mL of a hydroxylamine hydrochloride solution (0.25 mol / L) containing methyl orange reagent was mixed with 0.1 g each of freeze-dried OHA and OCMC, respectively, and left at room temperature for 24 hours. After the aldehyde was converted to an oxime, the released hydrochloric acid was titrated with a 0.1 mol / L sodium hydroxide solution until the endpoint was reached, changing the solution from red to yellow. The average of three experiments represents the oxidation degree (oxidation %) of OHA and OCMC.

[0173] As a result of measuring the actual aldehyde content of oxidized hyaluronic acid (OHA) using the hydroxylamine hydrochloride titration method according to Experimental Example 1 described above, an oxidation degree of 49.13% was found. As a result of measuring the actual aldehyde content of oxidized carboxymethyl cellulose (OCMC), it was confirmed that an oxidation degree of 49.13% was found.

[0174]

[0175] <Experimental Example 2: Measurement of Amine Content>

[0176] The amine content of cystamine-modified gelatin (CyGel) prepared according to Preparation Example 2, cystamine-modified hyaluronic acid (HACys) prepared according to Preparation Example 5, and adipic acid dihydrazide gelatin (GelADH) prepared according to Preparation Example 6 was measured, respectively.

[0177] Specifically, approximately 0.2–0.3 g of samples were dissolved in 50 mL of distilled water using bromophenol blue as an indicator. Then, the blue solution was titrated with a 0.2 N HCl solution until a distinct color change from blue to yellow was observed, indicating an endpoint. Parallel titrations were performed on gelatin used as a blank. Subsequently, based on these titration results, the total amine value was calculated and expressed as mg KOH / g of the sample.

[0178] As a result, the total amine content was confirmed to be 211.74 mg KOH / g for CyGel and 74.15 mg KOH / g for gelatin. The difference in total amine content between CyGel and gelatin was considered as the degree of modification and calculated as 64.98%. GelADH was confirmed to be 211.74 mg KOH / g and HACys as xyz mg KOH / g. The difference in total amine content between GelADH and gelatin was considered as the degree of modification and calculated as 64.98%.

[0179]

[0180] <Experimental Example 3: FTIR Analysis>

[0181] Each hyaluronic acid-gelatin hydrogel prepared according to Examples 1 to 7 was dried in a freeze dryer and subjected to Fourier Transform Infrared (FTIR) spectroscopy. An FTIR spectrometer (Agilent Cary 660, USA) was used to scan all samples, with a scan range of 400–4000 cm⁻¹. -1...was done. The results are shown in Figures 1 and 2 below.

[0182] FIG. 1 is a diagram showing the Fourier Transform Infrared Spectrometer (FTIR) analysis results of hydrogels according to Examples 1 to 3 of the present invention, and

[0183] Referring to FIG. 1, a hydrogel prepared according to one embodiment of the present invention shows a specific amide band typical of a protein. CyGel, a new gel derivative, was synthesized by carbodiimide chemistry.

[0184] a) In the HA-gelatin composition, the stronger band is at 1630 cm⁻¹ -1 It is located in the center, which is caused by a combination of C=O stretching vibrations from the unreacted aldehyde group and C=N stretching vibrations from the Schiff base. The Schiff base was formed between the amino group of CyGel and the aldehyde group of OHA as a result of imine bond formation.

[0185] b) 1745 cm -1 The band appears only at the HA-gelatin (1:0.5) ratio and not at other ratios, namely HA-gelatin (1:1) and HA-gelatin (1:2), which may be an indication of unreacted OHA, which may have carbonyl groups that do not participate in the formation of Schiff bases of the amine groups of CyGel.

[0186] c) is 1735cm next to the fingerprint area -1 It shows the clear presence of a shoulder peak (shoulder) proving aldehyde formation.

[0187] The cystamine variant of CyGel is 1236 cm in d). -1 (Amid III), 1525 cm -1 (Amide II), 1633 cm -1 (Amide I), 3283 cm -1 It is indicated by an increase in the peak at (amide A), and the disulfide bond (SS) is at 549 cm⁻¹ -1 It is observed in.

[0188] 3283 cm⁻¹ in the CyGel spectrum -1 The peak of (amide A) is stronger than any other peak in the same region of the gelatin spectrum and may represent -NH2 stretching vibrations from grafted cystamine. 549 cm⁻¹ -1 The peak is much stronger in the CyGel spectrum than in the gelatin A spectrum, suggesting the presence of SS binding of cystamine. 1525 cm⁻¹ -1 The amide II band located at is attributed to the NH bending and CN stretching vibrations, and at 1633 cm -1 The amide I band peak at is attributed to the C=O stretching vibration.

[0189]

[0190] Figure 2 is a diagram showing the Fourier transform infrared spectroscopy (FTIR) analysis results of a hydrogel according to Example 4 of the present invention.

[0191] Referring to Fig. 2, the 1540 cm⁻¹ of the FT-IR spectra of HA and HA-Cys in a) -1 The peak corresponds to the NH bending vibration representing the amino group, and b) shows CMC and OCMC, with the aldehyde group of OCMC (C=O stretching) at 1735 cm⁻¹. -1 It was confirmed that an oxidation reaction occurred.

[0192] c) In this case, GelADH is amide I (1633 cm⁻¹), indicating the presence of a gelatin backbone and a hydrazide group. -1 ), amide II (1525 cm -1 ) and NH stretching (3283 cm -1 Typical protein peaks such as ) were confirmed, and in the hydrogels of Example 4 prepared with various erythritol contents (0%–7%), significant changes and additional peaks related to erythritol could be identified in the FT-IR spectra. Specifically, at 1540 cm⁻¹ -1Judging by the nearby peak, the appearance confirmed the formation of C=N in the hydrogel of Example 4.

[0193] d) When examining the XRD patterns of the hydrogel and erythritol of Example 4, it was confirmed that erythritol exhibited characteristic diffraction peaks at 14.7°, 19.6°, 20.2°, 24.5°, 27.8°, 28.3°, 29.6°, and 31.2°. However, it was confirmed that the matrix of the hydrogel without erythritol (0%) exhibited an amorphous structure.

[0194] On the other hand, as the erythritol concentration increased from 1% to 7%, the XRD spectrum showed that the crystal peak corresponding to erythritol gradually appeared and enhanced. In particular, the peaks around 19.6° and 29.6° became clearer, which is interpreted as erythritol successfully penetrating and influencing the crystal structure of the hydrogel network.

[0195]

[0196] <Experimental Example 4: Morphological Analysis>

[0197] For the freeze-dried hyaluronic acid-gelatin hydrogels prepared according to Examples 1 to 7, the surface morphology was analyzed using a scanning electron microscope (SEM: TESCAN VEGA3, Korea). Pore diameter and wall thickness were examined in the SEM images using ImageJ software (NIH, USA). For all samples, three photographs of each sample were examined. The surface of the cross-sectional hydrogel samples was randomly selected at sampling locations (50 per sample).

[0198]

[0199] Figure 3 is a figure showing the scanning electron microscope (SEM) analysis results of hydrogels according to Examples 1 to 3 of the present invention.

[0200] Referring to FIG. 3, the porous surface and cross-sectional structure of each HA-gelatin hydrogel according to Example 1 (HA-gelatin 1:0.5), Example 2 (HA-gelatin 1:1), and Example 3 (HA-gelatin 1:2) could be confirmed in SEM images. All cross-linked gels with pores of different micron ranges exhibited a porous network topology. That is, it can be confirmed that the hydrogel prepared according to one embodiment of the present invention has highly connected pores, which can promote cell adhesion, proliferation, and nutrient transport.

[0201] More specifically, the pore size distribution histogram area of ​​the dry and cross-sectional HA-gelatin hydrogel [HA-gelatin (1:0.5) (A1, A2, A3), HA-Gelatin (1:1) (B1, B2, B3) and HA-Gelatin (1:2) (C1, C2, C3)] was measured (ImageJ software): 5761.598 μm (max: 17831.832 μm, minimum: 1108.981 μm), HA-Gelatin (1:1): 12731.12 μm (max: 56800.36 μm, minimum: 1704.667 μm) and HA-gelatin (1:2): 17523.05 μm (max: 111584.3 μm, minimum: 2912.785 μm).

[0202] Meanwhile, the pore size of the HA-gelatin gel varied depending on the components and composition, and the gel area increased as the concentration of CyGel increased. HA-gelatin (1:0.5) exhibited the smallest pore size among the samples used because the amount of CyGel used in the process was small. On the other hand, HA-gelatin (1:2) showed a larger pore size because the amount of CyGel in the hydrogel sample was twice as large. This may be because a larger amount of unreacted CyGel in the sample resulted in a larger pore size. HA-gelatin (1:1) shows a uniform pore size and high Schiff's base formation of OHA and CyGel because it provides equal amounts of amine and aldehyde groups compared to the other two concentrations to form imine bonds.

[0203]

[0204] Figure 4 is a figure showing the scanning electron microscope (SEM) analysis results of a hydrogel according to Example 4 of the present invention.

[0205] Referring to Fig. 4, for the hydrogel of Example 4, the microstructures of the hydrogel (a) not containing erythritol, the hydrogel (b) containing 1% erythritol, and the hydrogel (c) containing 5% erythritol were each analyzed using SEM.

[0206] Hydrogels without erythritol exhibited a relatively porous and loosely interconnected network, suggesting that the connections between polymer chains are weak and sparse, which may result in somewhat lower mechanical properties such as hardness and elasticity. In other words, the absence of erythritol lowers the density of the structure, limiting its ability to provide robust mechanical support.

[0207] In contrast, the hydrogel containing 5% erythritol exhibited a much denser and more organized high-density polymer network. The addition of erythritol strengthens intermolecular bonds, improving the structural integrity of the hydrogel and forming a denser and more stable network. This is attributed to intermolecular hydrogen bonding between the hydroxyl groups of erythritol and the functional groups of HACys, OCMC, and GelADH components. The microstructure of this hydrogel, characterized by high crosslinking density, demonstrates excellent mechanical properties such as high hardness, elasticity, and cohesion, as well as the ability to control the delivery of encapsulated drugs and cells, hydrogel degradation, and tissue regeneration rates.

[0208] In other words, the structural network of a hydrogel containing 5% erythritol can exhibit enhanced functionality in applications such as 3D bioprinting and tissue engineering, and the reinforced high-density hydrogel structure suggests that it can improve the delivery and retention of therapeutic agents for regenerative medicine by improving compatibility with bioactive molecules.

[0209]

[0210] <Experimental Example 5: Measurement of Mechanical Properties>

[0211] The mechanical texture characteristics of each hydrogel in Examples 1 to 4 were measured using a Stable Micro System (TA. XT plus texture analyzer, Surrey, UK). The mechanical properties of three different HA-gelatin hydrogel formulations in a cylindrical shape (10 mm diameter) under compression were evaluated. To compare mechanical properties, each form (three combinations) of HA-gelatin hydrogel was evaluated using TPA at a 30% strain. TPA parameters: Load cell 50 N; Test speed: 0.5 mm sec1; Post-test speed: 5 mm sec1; Interval: 5 sec; Target mode: Distance; Trigger type: Button. Results were calculated by selecting TPA settings and activating the TPA macro. Three different strains of 30% were applied for five cycles of periodic compression analysis. Similarly, 30% strain was used to test all three scaffold materials. The results are shown in Figures 5 to 16 below, respectively.

[0212]

[0213] Referring to FIGS. 5 to 10 together, it was confirmed that as the gelatin content of the gel sample increased, the compressive strength and other mechanical properties of the sample increased significantly. Compared to gels with similar amounts of Example 1 (HA-gelatin (1:0.5)) and Example 2 (HA-gelatin (1:1)), the strength at 30% compression increased threefold in the gel with twice the amount of HA-gelatin (1:2). In addition, as hardness increased, the amount of CyGel also increased.

[0214] Meanwhile, HA-gelatin (1:0.5): 5.2623g, HA-gelatin (1:1): 12.6516g, HA-gelatin (1:2): 16.9653g; similarly, as the CyGel level of the hydrogel increased, the chewiness and elasticity also increased. This is interpreted to be because the mass content of the hydrogel increases as the hydrogel concentration increases. Cohesion, elasticity, and resilience did not change significantly even when the amount of CyGel in the hydrogel varied, but adhesion decreased as the amount of CyGel in the hydrogel increased. It was confirmed that increasing the amount of CyGel in the hydrogel formulation is effective in enhancing stability and improving the physical, mechanical, and biological properties of the hydrogel. The strength of the gel is reflected in hardness, and the texture when the gel is crushed is reflected in chewiness and stickiness.

[0215] Referring together to FIGS. 11 to 16, the results of texture profile analysis (TPA) of hydrogels containing erythritol at various concentrations (0%, 1%, 3%, 5%, 7%) (hereinafter referred to as hydrogel without erythritol (HCG-E0) / hydrogel containing 1% erythritol (HCG-E1) / hydrogel containing 3% erythritol (HCG-E3) / hydrogel containing 5% erythritol (HCG-E5) / hydrogel containing 7% erythritol (HCG-E7)) show that, regarding the mechanical and adhesive properties of the hydrogels such as compression force, hardness, adhesion, cohesiveness, resilience, elasticity, viscosity, and chewiness, the hydrogel containing 5% erythritol achieves an optimal balance of strength, flexibility, and usability, whereas the hydrogel containing 7% erythritol It was confirmed that the hydrogel exhibited excessive viscosity, which reduced its applicability.

[0216] The force-compression curve in Fig. 11 shows that mechanical strength gradually increases as the erythritol concentration increases. HCG-E5 strikes a desirable balance, providing sufficient structural integrity without being excessively stiff, whereas HCG-E7 exhibited the highest force during compression, reflecting its high crosslinking density and viscosity. However, this stiffness raises concerns that it may limit adaptability to applications requiring flexibility, such as wound healing and tissue scaffolds.

[0217] Figure 12 shows the hardness of the hydrogels increasing with erythritol concentration. HCG-E5 exhibits high hardness but is easy to manage, making it suitable for biomedical applications such as drug delivery systems and scaffolds where durability is important. However, HCG-E7 has excessively high hardness, which may make it difficult to use in soft tissue environments, and its high viscosity makes it difficult to handle and process, raising concerns about reduced practicality.

[0218] Referring to Fig. 13, HCG-E5 can maintain appropriate adhesion and exhibit effective surface interaction without excessive stickiness. This characteristic can be particularly useful for wound dressings where balanced adhesion is essential for both patient comfort and ease of removal. On the other hand, HCG-E7 has high viscosity and exhibits excessive adhesion, which makes handling complex and raises concerns about poor overall usability.

[0219] Referring to Fig. 14, the cohesive strength values ​​of all formulations remain high, indicating the structural stability of the hydrogel. Among them, HCG-E5 exhibited excellent cohesive strength retention characteristics.

[0220] Referring to Fig. 15, elasticity and resilience are also affected by the erythritol concentration. Among them, HCG-E5 exhibits optimal elasticity and resilience, allowing it to recover its shape after deformation, which is a very important characteristic for applications requiring repetitive mechanical loading, such as tissue scaffolds. On the other hand, HCG-E7 exhibits high elasticity, but there is a concern that its adaptability may be poor due to extreme stiffness.

[0221] Referring to Figure 16, stickiness and chewiness are also affected by the erythritol concentration. HCG-E5 strikes a balance between mechanical rigidity and flexibility, whereas HCG-E7 exhibits excessively high values ​​reflecting viscosity and stiffness. Due to these characteristics, there is a concern that HCG-E7 may not be suitable for applications requiring dynamic adaptability.

[0222]

[0223] <Experimental Example 6: Measurement of Physical Properties>

[0224] To confirm the physical properties of each hydrogel in Examples 1 to 7, viscosity, porosity percentage, expansion ratio analysis, and degradation tests were performed. The specific experimental methods followed known techniques, and the measurement results are shown in FIGS. 17 to 24.

[0225] Figure 17 is a figure showing the rheological analysis (viscosity) results in Experimental Example 6.

[0226] The gelation time of each hydrogel in Examples 1 to 3 was measured.

[0227] Referring to Fig. 17, the gelation time was between 20 and 30 seconds after correction with viscosity data, and it was confirmed that the gelation time decreased significantly as the concentration of CyGel increased. Meanwhile, there was no significant difference in the gelation time of the hydrogel HA-gelatin (1:2), but it was confirmed that the viscosity gradually increased over time.

[0228]

[0229] Figure 18 shows the results of measuring the porosity percentage (%) in Experimental Example 6. For each hydrogel of Examples 1 to 3, the porosity percentage value of the HA-gelatin gel sample was measured by the liquid displacement method.

[0230] Each hydrogel sample was prepared, frozen at -75°C, and then freeze-dried as described above to perform the experiment. The porosity value of the liquid displacement experiment can represent the free space occupied by ice or water crystals in addition to the pore volume of the scaffold.

[0231] Referring to Fig. 18, the porosity of each hydrogel was found to be over 90%. The HA-gelatin (1:2) gel had the highest porosity, but the results were not significantly different from the other examples. Similarly, the average pore diameter of the cross-linked hydrogels varied depending on the test group, but there was no noticeable difference in the overall bulk porosity of the hydrogels. This is interpreted as being due to the fact that the total mass and water content used in the synthesis of the hydrogels were the same.

[0232]

[0233] Figure 19 is a figure showing the measurement results of the average swelling rate (%) in Experimental Example 6.

[0234] Referring to Fig. 19, the weight change determined by the expansion test verified the morphological change observed in the SEM of Experimental Example 2. Among the three different compositions of HA-gelatin gels, the HA-gelatin (1:2) gel containing the largest amount of CyGel showed superior performance in terms of expansion behavior compared to the other two HA-gelatin (1:0.5) and HA-gelatin (1:1) gels.

[0235] Meanwhile, the HA-gelatin (1:0.5) gel exhibited the least swelling. This aligns with the SEM images and pore size distribution area of ​​the HA-gelatin gel, where the pore size was lowest in the HA-gelatin (1:0.5) sample and largest in the HA-gelatin (1:2) sample. Since the HA-gelatin (1:1) gel has the highest level of crosslinking, its swelling ratio matches that of the other mixtures.

[0236] However, the HA-Gelatin (1:2) gel exhibited the highest swelling ratio because it contained twice the amount of CyGel in the hydrogel sample. This is interpreted as being due to the greater swelling caused by the inclusion of more unreacted CyGel in the sample, despite the high hydration rate of hyaluronic acid. When tested frequently between 1 and 12 hours, the gel reached equilibrium within 1 hour and exhibited similar behavior thereafter.

[0237]

[0238] Figure 20 is a figure showing the results of the performance degradation test (decomposition) in Experimental Example 6.

[0239] The in vitro degradation of the gel was measured in PBS (pH 7.4) at 37°C.

[0240] Referring to Fig. 20, a decrease in gel mass over time was observed in all gels for 49 days under normal physiological conditions. The degradation of the hydrogel depends primarily on two criteria: the degradable chemical structure and the physicochemical structure of the hydrogel network. In the present invention, the cross-linking of the two polymer components forms Schiff bonds mainly through aldehyde and amine groups. The breakdown of these bonds is generally attributed to cleavage by hydrolysis.

[0241] After 49 days, the weight loss rate (%) of the gel was found to be 85.55%, 85.61%, and 83.25% for the HA-gelatin (1:0.5), HA-gelatin (1:1), and HA-gelatin (1:2) gels, respectively. Among the three combinations, the HA-gelatin (1:2) gel showed less degradation after 49 days compared to the other two combinations.

[0242] As the CyGel content of the gel decreased, the degradation became higher and faster. However, in the HA-gelatin (1:2) gel, the greater the amount of CyGel, the better the stability was shown, which may be attributed to the high cross-linking density and hydrophobicity initiated by the cystamine group of gelatin.

[0243] Meanwhile, in the case of the HA-gelatin (1:0.5) gel, although the aldehyde group provided more crosslinking than the HA-gelatin (1:1) gel, the low CyGel content caused relatively faster degradation compared to the HA-gelatin (1:2) gel.

[0244]

[0245] Referring again to FIGS. 18 and 19, the HA-gelatin (1:2) sample was found to have a larger pore diameter and consequently expand faster due to greater water absorption. Consequently, this leads to faster hydrolysis of the bonds compared to the expansion ratio of other examples with relatively smaller pore diameters. Additionally, HA-gelatin (1:1) has a higher pore diameter and expansion ratio compared to the HA-gelatin (1:0.5) gel, which corresponds to faster degradation. However, the lower expansion ratio due to the high crosslinking density observed in the HA-gelatin (1:2) gel indicated a much lower degradation rate. The HA-gelatin (1:2) gel may have an additional advantage in forming polymer electrolyte complexes because the interaction groups of the polymer side chains are identical in this combination.

[0246]

[0247] Figure 21 is a figure showing the rheological analysis (viscosity) results of the hydrogel of Example 4 in Experimental Example 6.

[0248] Referring to Figure 21, all formulations exhibited shear thinning behavior in which viscosity decreased as the rotational speed (RPM) increased. When erythritol was added, viscosity increased with concentration. The hydrogel without erythritol (HCG-E0) showed the lowest viscosity, whereas the formulation containing 7% erythritol (HCG-E7) exhibited the highest viscosity due to enhanced cross-linking and hydrogen bonding interactions. This indicates that erythritol contributes to a denser network structure, allowing the hydrogel to form a more stable structure at higher erythritol concentrations. However, the high viscosity of HCG-E7 raises concerns that it may hinder applicability in bioprinting and other dynamic applications due to reduced fluidity.

[0249]

[0250] Figure 22 is a figure showing the results of the performance degradation test (decomposition) of the hydrogel of Example 4 in Experimental Example 6.

[0251] Referring to Figure 22, a gradual decrease in weight was observed in all formulations over 14 days. The degradation rate decreased as the erythritol concentration increased, with HCG-E7 exhibiting the slowest degradation. The enhanced stability of HCG-E7 is attributed to the high density of the cross-linking network formed by the interaction of erythritol hydroxyl groups with the hydrogel components. In contrast, HCG-E0 exhibited the fastest degradation rate, indicating a loose structure and weak intermolecular interactions. These results suggest that the degradation rate of the hydrogel can be controlled by adjusting the erythritol concentration.

[0252]

[0253] Figure 23 is a figure showing the measurement results of the average swelling rate (%) of the hydrogel of Example 4 in Experimental Example 6.

[0254] Referring to Fig. 23, the addition of erythritol slightly reduced the swelling ability of the hydrogels, with HCG-E0 exhibiting the highest swelling ratio and HCG-E7 exhibiting the lowest swelling ratio. This trend is interpreted as being due to the fact that as the erythritol concentration increases, the crosslinking density increases, thereby limiting water absorption. Although the reduced swelling of HCG-E7 improves mechanical stability, there is a concern that its use may be limited in applications requiring high water retention, such as drug delivery systems.

[0255]

[0256] Figure 24 is a figure showing the measurement results of the porosity percentage (%) of the hydrogel of Example 4 in Experimental Example 6.

[0257] Referring to Fig. 24, the porosity of the hydrogel remained consistently high in all formulations, and minimal differences were observed between various erythritol concentrations. This indicates that erythritol does not significantly change the overall porosity of the hydrogel network.

[0258]

[0259] <Experimental Example 7: Measurement of mechanical properties of microporous meniscus structure (1)>

[0260] Microporous meniscus structures were fabricated by 3D printing using each hydrogel according to Examples 1 to 3 (and the hardness, adhesion, elastic recovery, and mechanical properties were measured according to known evaluation methods).

[0261] The periodic expansion reaction for each thin film of Examples 1 to 3 was irradiated in expansion mode using a TPA machine expanded by 50%. The film thicknesses were set to 0.7 mm, 1.1 mm, and 1.5 mm for the HA-gelatin films of Example 1 (1:0.5), Example 2 (1:1), and Example 3 (1:2), respectively. The measurement results are shown in Figures 25 to 30, respectively.

[0262]

[0263] Referring to FIGS. 25 to 30 together, the hardness values ​​indicate the strength and structural stability of the hydrogel under specific compression. As can be seen in FIG. 27, the hardness value of the 1:2 meniscus sample was noticeably higher than that of the other two hydrogels (meniscus 1:0.5 and meniscus 1:1). Furthermore, all meniscus samples of Examples 1 to 3 not only had higher hardness values ​​than previously reported hydrogels but also had an elastic recovery rate that was more than 70% higher on average. The elastic recovery value is an indicator of the hydrogel's ability to rebound after compression. Ultimately, the resilience value of the hydrogel indicates how well the hydrogel can withstand elastic collapse without energy loss.

[0264] Furthermore, the resilience values ​​of the 1:1 meniscus hydrogel were noticeably higher than those of the other evaluated samples. One of the important hydrogel characteristics in tissue engineering is adhesion, which measures the gel's ability to adhere to various surfaces. In comparison, the adhesive properties of the meniscus samples decreased somewhat as hardness and stability increased. The chewiness and stickiness of the 1:2 meniscus showed higher values ​​than the other two meniscus samples.

[0265] In addition, the meniscus 1:2 microporous hydrogel exhibited superior performance compared to other compositions in terms of hardness and elasticity. However, the meniscus 1:1 was found to have better stability under compression.

[0266] In other words, the meniscus 1:1 hydrogel can be utilized as a recommended biomaterial in the field of tissue engineering due to its high elastic recovery, moderate hardness, and low adhesion properties.

[0267]

[0268] <Experimental Example 8: Measurement of mechanical properties of microporous meniscus structure (2)>

[0269] A microporous meniscus structure was fabricated by 3D printing using the hydrogel according to Example 2, and then the mechanical properties (restorative force, adhesion, hardness, and elastic recovery force) were measured under various compressions (30% and 60%). The results are shown in FIGS. 31 to 34.

[0270] Referring to FIGS. 31 to 34 together, the hardness of the meniscus sample according to Example 2 increased as the strain increased, and when various strains (e.g., 30% and 60%) were applied, the adhesive strength also increased as the strain increased. According to the statistics, the resilience value of the sample did not change significantly even when the strain increased, and rather, it was confirmed that the elastic recovery improved as the strain increased.

[0271]

[0272] <Experimental Example 9: Analysis of Morphological Characteristics of Various 3D Printed Structures>

[0273] 3D printing was performed using the hydrogels of Examples 2 and 4 to form a meniscus, a pyramid structure, a grid structure (2 cm Υ 2 cm), and a hollow ring structure, and then each SEM image was examined. The optimal settings for printing the given structures using a metal needle (27G) and a predetermined G-code were a pressure of 100 kPa - 150 kPa, 25°C, and a stage travel speed of 100 m / sec (S Video V1). The results of the morphological characteristic analysis are shown in Figures 35 and 36.

[0274] Referring to Fig. 35, it is shown that all complex printed structures have a porous surface topology. In particular, a pyramid-shaped (b) HA-gelatin-based 3D printed structure with about 100 layers and a height of 1.5 cm demonstrated excellent stability and shape integrity. This allowed us to confirm how high the surface adhesion of the hydrogel is, as the printed structure adhered very well to the substrate without additional adhesive.

[0275] A clear and stable porous strut structure could be confirmed on the top surface (A3, B3 and C3) of the structure using high-magnification SEM images.

[0276] The structure exhibited uniform pore sizes, and since the composition of the HA-gelatin hydrogel used for printing was identical, the pore sizes did not vary. The meniscus was printed with 50% fill, and the perimeter is indicated above. The perimeter of the object is the outer wall, which determines the object's external shape. The area inside the perimeter is called the infill. After running each generated G-code file through a G-code tool, the extrusion volume was compared with the output of the G-custom file. High-magnification SEM images of the meniscus showed that the average pore size on the surface was 759.048 ± 169.897 µm.

[0277] (c) shows the basic 3D structure of the grid structure, and (c1, c2, b3) show high-magnification SEM images. Here, the average pore size was confirmed to be 1265.42 ± 800.919 μm. For this sample, there was no noticeable shrinkage or change in shape during the freeze-drying process relative to the overall size of the printed structure. There was also little difference between the cross-section and the surface of the sample.

[0278] (b) shows the 3D-printed pyramidal hydrogel structure, and (b2, b3) show SEM images. The 3D-printed pyramidal structure exhibits excellent stability even in the absence of a support. This characteristic enables the fabrication of a stable bioink with excellent fidelity and stability even after printing. The aforementioned optimal setup was achieved through a ring-like continuous printing path. The measured pore size was consistent at 1251.95 ± 409.522 μm SEM.

[0279] After freeze-drying (b1), the wall width or thickness of the pyramid decreased by about 30% compared to the initial printed sample (b). These results show that the printed structure can be stored for a long time even though the wall thickness is reduced due to drying. These results indicate that the pores inside the microporous hydrogel are larger than the pores outside.

[0280] This high porosity is expected to aid in cell migration, growth, and differentiation by transporting metabolic substances and waste products. This demonstrates that it is possible to print diverse and complex structures with excellent stability and fidelity. This implies that microporous hydrogels can reversibly alter their chemical bonds in response to applied shear stress, thereby returning to their original physical configuration.

[0281] In addition, these hydrogels can maintain their shape without post-processing after printing. Furthermore, the resolution or size of the printed structure can be changed or adjusted for specific applications by modifying the synthesis settings and bioink composition. This method can also be extended to fabricating nerve conduits by manufacturing them into various biomaterials, scaffolds for bone tissue engineering, or stents. In other words, it has been successfully demonstrated that large and complex self-supporting structures can be printed quickly and reliably using the bioink gel according to the present invention.

[0282]

[0283] Referring to Fig. 36, in a), the meniscus structure was printed with 50% infill, showing the perimeter defining the infill area responsible for the outer wall and internal integrity. In the high-magnification SEM image, the average pore size was 85.32 ± 17.53 μm, confirming that it represents a well-distributed and uniform network suitable for the exchange of nutrients and metabolites in tissue engineering applications. Furthermore, the shape and dimensions of the structure remained intact even after freeze-drying, and no significant shrinkage or deformation was observed.

[0284] In b), the 2cm x 2cm lattice structure exhibited excellent structural integrity, as seen in the high-magnification SEM image. The average pore size was 75.78 ± 12.63 μm, indicating an open and interconnected porous structure. This structure demonstrated minimal structural changes during the freeze-drying process, proving the stability of the HCG-E (5%) hydrogel. This porous network is expected to enhance cell migration, growth, and differentiation, making it suitable for use as a scaffold in tissue engineering applications.

[0285] In C), the hollow ring structure exhibited excellent fidelity with a uniform wall thickness and a stable circular shape. SEM analysis revealed an average pore size of 54.66 ± 15.47 μm, confirming consistent porosity on both the inner and outer surfaces. The ring maintained its structural integrity and dimensions even after freeze-drying. This stability suggests potential for application in vascular stents or nerve conduits where long-term structural integrity is critical.

[0286]

[0287] <Experimental Example 10: Cell Viability Analysis>

[0288] A cytotoxicity evaluation (MTT) was performed on the hydrogels prepared according to Examples 1 to 3. The evaluation results are shown in Fig. 37.

[0289] Referring to Fig. 37, (a) shows the MTT results regarding the cytotoxicity levels of all samples in indirect contact with L929 (i.e., HA-gelatin (1:0.5), HA-gelatin (1:1), and HA-gelatin (1:2). Cytocompatibility was confirmed for all hydrogels after 3 days, and they exhibited cell viability similar to that of the control group. In other words, a general trend in cell viability was observed in all sample groups after 3 days of culture. Considering that cells require time to adapt to a new environment, this can be explained by the number of cells that successfully attached to the intended surface (the lag phase). Cell development and proliferation in the new microenvironment manifest as an increase in cell viability after 3 days, which is more pronounced in the samples.

[0290] (b) shows the total GAG content measured on days 1, 3, and 7 in cultures of L929 cells cultured on HA-gelatin hydrogels of various compositions (1:0.5, 1:1, 1:2). Consistent GAG levels over 7 days across all hydrogel ratios indicate that the cells are actively producing matrix components, which are a crucial role for fibroblasts in tissue repair and wound healing processes. As the cells proliferate and mature, and synthesize more extracellular matrix components, GAG synthesis is expected to increase over time.

[0291] High-magnification images of L&D, DAPI / actin (red rhodamine-phaloidin for F-actin), and GAG assays clearly show excellent intercellular contact and communication formed after hydrogel degradation. L929 fibroblast cultures appear to be well supported by HA-gelatin hydrogel scaffolds, which may influence the fabrication of tissue engineering structures. It appears that using fabricated HA-gelatin structures for 3D bioprinting of hydrogels encapsulated at high cell density will enable the loading of fully functional tissue regeneration samples.

[0292] The interaction between L929 cells and the HA-gelatin hydrogel was confirmed through H&E staining. The cytoplasm of the cells stained red showed a spreading pattern. After 7 days of cell growth in the hydrogel, high-density cells were observed in all samples, consistent with the DAPI staining results. After the ECM was completely degraded, intercellular connectivity and communication could be confirmed in the viability (L / D) results on day 7 (e).

[0293] After 7 days of culture, the cytoskeleton of the cells showed improved structural strength and diffusion pattern. Live / dead data on day 1 showed that cells in HA-gelatin (1:1) exhibited better proliferation and growth than the other two samples, namely HA-gelatin (1:0.5) and HA-gelatin (1:2), and on day 7, it showed that the living cells were evenly dispersed in the gel matrix.

[0294] Through 3 days of cell culture, further evidence was confirmed that these hydrogels promote cell development in HA-gelatin (1:0.5) and HA-gelatin (1:1). High cell viability was confirmed in the live / death analysis results at all time points (e), and live cell density increased significantly from day 1 to day 7. Consistent green fluorescence within the hydrogel matrix indicates not only the survival of L929 cells but also their metabolic activity and overall health status. To evaluate the effect of surface modification on the cellular performance of the prepared samples in terms of cell viability, MTT cell viability assays were performed on HA-gelatin scaffolds at various concentrations. The cells used were mouse fibroblasts (L929).

[0295] (f) shows DAPI(4',6-diamidino-2-phenylindole) stained images of blue nuclei on the surface of various HA-gelatin hydrogel compositions on days 1 and 7 using mouse fibroblast skin (L929) cells. On day 1, little cell attachment and proliferation were observed. However, on day 7, cell proliferation appears to be strong, as the volume of cell nuclei increased noticeably and the actin cytoskeleton was more widely distributed. The wide dispersion of fibroblast L929 cells suggests that the cells prefer low-density hydrogel networks.

[0296]

[0297] <Experimental Example 11: Cell Compatibility of Hydrogels for Bone Tissue Engineering>

[0298] Cell compatibility (MC3T3) experiments to confirm cell adhesion, growth, and proliferation patterns were performed on the hydrogels prepared according to Examples 1 to 3 for up to 7 days. The experimental results are shown in Fig. 38.

[0299] In Fig. 38, (a) shows high-magnification images of DAPI staining on day 1 and day 7, (b) shows Live / Dead analysis images of various HCG hydrogels using MC3T3 cells, (c) shows H&E and MT staining results, and (d) shows MTT analysis results.

[0300] Specifically, referring to Figures 38(a) and (b) together, uniform growth and proliferation of MC3T3 cells within the hydrogel matrix were confirmed through DAPI staining, which displays cell nuclei in blue. In particular, on day 7, DAPI / Phalloidin (red) staining indicated that the cells in the HCG-E5 hydrogel were well spread, suggesting that intercellular communication had been established. The presence of F-actin in the cytoplasm acts as an indicator of cell health, reflecting active growth and proliferation. In contrast, the HCG-E0 and HCG-E1 hydrogels showed fewer actin filaments than HCG-E5, suggesting that erythritol is incorporated into HCG-E5 to more strongly support cell growth.

[0301] These results suggest that erythritol-enhanced hydrogels can be promising candidates for bone regeneration by effectively promoting osteoblast activity and extracellular matrix production. Additionally, the injectable nature of these hydrogels enables minimally invasive surgical applications, which is interpreted as being useful for tissue regeneration treatment of small, irregular cartilage defects commonly seen in cartilage-related diseases.

[0302] The results of the viability / death analysis on Day 0 showed that living cells were uniformly distributed throughout the gel matrix. Observations over 7 days confirmed that the hydrogel effectively supported cell growth. The 3D bioprinted structures created using the roller head BioOpen demonstrated that cells proliferated evenly throughout the hydrogel matrix. By Day 7, the cells had expanded to the point where they covered almost the entire gel structure. In other words, the increase in cell density with higher erythritol concentrations indicated that erythritol significantly enhances cell growth and proliferation.

[0303] The H&E and MT staining results shown in Fig. 38(c) confirm that higher erythritol concentrations in the multi-component bioink promote extracellular matrix formation. The MT-stained samples show an increase in collagen (green) in the HCG-E5 hydrogel, suggesting that higher erythritol levels increase the likelihood of bone tissue formation and structural hardening. Overall, these results suggest that erythritol has a positive effect on the in vitro tissue regeneration of scaffolds printed with bioink.

[0304] Referring to Fig. 38(d), the MTT analysis results showed that all hydrogel samples were non-toxic and beneficial for cell growth. In particular, the HCG-E5 hydrogel exhibited the highest cell proliferation among all bioprinted samples, with a significant increase observed on day 5. This suggests that while higher concentrations of erythritol may affect the cell growth rate, the presence of erythritol can significantly enhance cell viability. Thus, it was proven that 3D printability and cell proliferation can be effectively optimized by controlling the erythritol content.

[0305]

[0306] <Experimental Example 12: Experiment on Cell Viability and Damage by Bioprinting Pen Rotation Speed ​​(rpm)>

[0307] Cell viability was evaluated when 500,000 cells were mixed in a 1 mL gel. Cell viability was compared using the method of Example 11 on the day of mixing with bioprinting pen rotational speeds (rpm) of 40, 70, and 100 rpm, in comparison to the results of mixing using a spatula (control group). In the control group, where cells were loaded into the gel with a spatula, the cells were not evenly distributed, whereas the greatest cell damage was observed when cells were loaded with a spatula.

[0308]

[0309] Referring to Figure 39, cell damage caused by bioprinting showed a tendency to increase as the rotation speed increased; however, while there was a slight increase between RPM 70 and 100, a significant difference of about twofold was observed between rotation speeds of 40 and 70. Among the samples in which cells were mixed inside the gel using Biopen, the most homogeneously mixed condition was the sample mixed at a rotation speed of 100. Although lower than the control group, cell damage was highest in the bioprinting pen experimental group. Therefore, it was observed that a cell concentration of 500,000 / mL and a rotation speed of approximately 70 rpm are appropriate for use in terms of balancing cell dispersion and cell viability.

[0310]

[0311] A hyaluronic acid-gelatin-based hydrogel according to one embodiment of the present invention can produce multilayer 3D printed gel structures without a support material and can exhibit effects such as advantageous self-crosslinking, high controllability, high cell viability, mechanical elasticity, biocompatibility, rapid gelation, adaptability to 3D bioprinting, interconnected microporous structure, shear exfoliation, slow degradation, and improved stability after printing.

[0312] Thus, using 3D bioprinting, it is possible to manufacture more complex biological structures similar to natural tissues and organs, with multiple layers and a uniform distribution of biofactors.

[0313]

[0314] A hyaluronic acid-gelatin-based hydrogel according to one embodiment of the present invention can be applied in various fields, such as 3D bioprinting of soft tissues, organ regeneration engineering, biomaterials for tissue engineering, and other biomedical applications.

[0315]

[0316] In this specification, only a few examples among the various embodiments performed by the inventors are described; however, the technical concept of the present invention is not limited or restricted thereto, and it is obvious that it can be modified and implemented in various ways by those skilled in the art.

Claims

1. A hyaluronic acid-gelatin-based hydrogel composition comprising oxidized hyaluronic acid (OHA) and cystamined gelatin (CyGel).

2. In Paragraph 1, A hyaluronic acid-gelatin-based hydrogel composition characterized by the above-mentioned oxidized hyaluronic acid (OHA) and cystamined gelatin (CyGel) being mixed in a weight ratio of 1:0.5 to 1:

2.

3. Step of preparing a hyaluronic acid (HA) solution; A step of adding an oxidizing agent to the above hyaluronic acid (HA) solution and then performing an oxidation reaction; A step of producing oxidized hyaluronic acid (OHA) by removing the oxidizing agent after the reaction is complete and then dialysis; Step of preparing a gelatin solution; A step of adding a cysteamine compound to the above gelatin solution and then performing an amide bonding reaction; A step of obtaining cystamine-modified gelatin (CyGel) by dialysis after the reaction is complete; and A method for preparing a hyaluronic acid-gelatin-based hydrogel composition comprising the step of mixing and reacting the above-mentioned oxidized hyaluronic acid (OHA) and cystamined gelatin (CyGel) to prepare a hydrogel.

4. In Paragraph 3, A method for preparing a hyaluronic acid-gelatin-based hydrogel composition, characterized in that the oxidation reaction comprises a step of stirring at room temperature of 21 to 24°C for at least 24 hours.

5. In Paragraph 3, A method for preparing a hyaluronic acid-gelatin-based hydrogel composition, characterized in that the above amide binding reaction includes a step of stirring at 40 to 45°C for 24 hours or more.

6. In Paragraph 3, A method for preparing a hyaluronic acid-gelatin-based hydrogel composition, characterized in that the oxidized hyaluronic acid (OHA) and cystamined gelatin (CyGel) are mixed in a weight ratio of 1:0.5 to 1:

2.

7. A hyaluronic acid-gelatin-based hydrogel composition comprising carboxymethyl cellulose oxide (OCMC), cystamined hyaluronic acid (HACys), and adipic acid dihydrazide gelatin (GelADH).

8. In Paragraph 7, A hyaluronic acid-gelatin-based hydrogel composition characterized by the above-mentioned carboxymethyl cellulose (OCMC), cystamined hyaluronic acid (HACys), and adipic acid dihydrazide gelatin (GelADH) being mixed in a weight ratio of 1:1 to 2:1 to 2.

9. Step of preparing a carboxymethyl cellulose (CMC) solution; A step of adding an oxidizing agent to the above carboxymethyl cellulose (CMC) solution and then performing an oxidation reaction; A step of producing oxidized carboxymethyl cellulose (OCMC) by dialysis after the oxidation reaction is completed; Step of preparing a hyaluronic acid (HA) solution; A step of preparing cystamine-modified hyaluronic acid (HACys) by adding a coupling agent to the above hyaluronic acid (HA) solution to perform an amide bonding reaction, and then adding a cystamine compound and reacting it; Step of preparing a gelatin solution; A step of mixing adipic acid dihydrazide (ADH) into the above gelatin solution and adding a coupling agent to perform a reaction; A step of obtaining adipic acid dehydrazide gelatin (GelADH) by dialysis after the reaction is complete; and A method for preparing a hyaluronic acid-gelatin-based hydrogel composition comprising the step of mixing and reacting the above-mentioned carboxymethyl cellulose (OCMC), cystamined hyaluronic acid (HACys), and adipic acid dihydrazide gelatin (GelADH) to prepare a hydrogel.

10. In Paragraph 9, A method for preparing a hyaluronic acid-gelatin-based hydrogel composition, characterized in that the carboxymethyl cellulose oxide (OCMC), cystamined hyaluronic acid (HACys), and adipic acid dihydrazide gelatin (GelADH) are mixed in a weight ratio of 1:1 to 2:1 to 2.

11. In Paragraph 9, A method for preparing a hyaluronic acid-gelatin-based hydrogel composition, characterized by adding and reacting erythritol together in the step of preparing the hydrogel.

12. In Paragraph 11, A method for preparing a hyaluronic acid-gelatin-based hydrogel composition, characterized in that the erythritol is included in an amount of 1 to 5 weight% based on the total weight of the hydrogel.

13. Method for preparing a hyaluronic acid-cystamine-gelatin hydrogel composition by mixing cystamine, a gelatin compound, and oxidized hyaluronic acid.

14. A bioink composition comprising a hyaluronic acid-gelatin-based hydrogel according to claim 1.

15. A bioink composition comprising a hyaluronic acid-gelatin-based hydrogel according to claim 7.

16. A bioink composition comprising a hyaluronic acid-gelatin-based hydrogel according to claim 13.

17. In any one of paragraphs 14 through 16, A step of filling the above bioink composition into a 3D printer; and A 3D bioprinting method comprising the step of forming a three-dimensional structure by performing 3D printing.

18. In Paragraph 17, A 3D bioprinting method characterized in that the above-described three-dimensional structure includes a tissue engineering structure and an artificial organ structure.

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