Extracellular matrix-based hybrid ink for 3D printing and its manufacturing method

Abstract

One embodiment of the present invention provides an extracellular matrix-based hybrid ink for 3D printing, which comprises an extracellular matrix having an amine group and a modified hyaluronic acid having an ethylenically unsaturated bond functional group introduced therein, characterized in that the extracellular matrix and the modified hyaluronic acid are in a crosslinked state due to a chemical bond formed between at least a portion of the amine groups present in the extracellular matrix and the ethylenically unsaturated bond functional group present in the modified hyaluronic acid. The hybrid ink according to one embodiment of the present invention has mechanical properties suitable for 3D printing due to the chemical crosslinking between the components, the extracellular matrix and the modified hyaluronic acid, and can control the drug release rate by adjusting the chemical crosslinking density.

Description

[Technical Field]

[0001] The present invention relates to an extracellular matrix-based hybrid ink, and more particularly to an extracellular matrix-based hybrid ink for 3D printing, which has mechanical properties suitable for 3D printing and can control drug release rate by adjusting crosslink density through chemical bonding between internal components, and a method for producing the same. [Background technology]

[0002] Cerebral ischemia occurs when insufficient blood flow to the brain leads to cerebral hypoxia and brain tissue death. Due to its severity, cerebral ischemia is known to be one of the most frequent causes of death worldwide. Treating ischemic brain tissue requires a continuous supply of angiogenic growth factors to maintain the patency of newly formed blood vessels and induce angiogenesis, in which capillaries extend from existing blood vessels to generate new blood vessels. The angiogenic process is a complex, multistep process involving the timely delivery of various angiogenic growth factors, which plays a role in providing a continuous supply of oxygen and nutrients necessary for cell permeability and metabolic maintenance. Therapeutic approaches that deliver angiogenic factors to promote angiogenesis have emerged as a promising strategy for the treatment of severe ischemic diseases.

[0003] Angiogenic factors that promote the early and mature stages of therapeutic angiogenesis are well known. Furthermore, the combined administration of angiogenic factors that act at each stage is also recognized as a beneficial method for promoting vascular regeneration. In particular, vascular endothelial growth factor (VEGF) is one of the key factors promoting the early stage of angiogenesis, inducing endothelial cell proliferation and the generation of immature blood vessels. However, concerns about the clinical use of VEGF have been raised due to its side effects, such as pro-inflammatory responses, including increased vascular permeability. Hepatocyte growth factor (HGF), another important angiogenic factor that promotes the mature stage of angiogenesis, exhibits synergistic effects when administered with VEGF, reducing the side effects of VEGF and promoting the growth of vascular endothelial cells. Therefore, the sequential release of these two angiogenic factors induces a more potent angiogenic response and prevents vascular regression and blood leakage. Therefore, the sustained administration of multiple angiogenic factors that mimic the angiogenic process is essential for the angiogenesis process in ischemic areas.

[0004] However, traditional growth factor administration methods, such as intravenous injection, often require high doses or repeated delivery to stimulate a therapeutic effect, resulting in reduced efficacy and serious side effects. To address this issue, these factors must be administered at physiologically appropriate times (time-controlled) and at appropriate sites (region-specific targeting). Therefore, the development of a soft and flexible hydrogel patch-type drug delivery system capable of tunable release of angiogenic factors and localized application to sensitive brain regions is necessary. 3D printing technology can be used to fabricate such patches. Using 3D printing technology and various types of biomaterials, controllable and user-definable patch-type delivery systems can be freely fabricated. Tuning the release of angiogenic factors can be achieved by adjusting 3D printing variables (e.g., system dimensions and design) or material properties (e.g., synthetic and naturally derived biomaterials, crosslinking density and concentration). Regarding biomaterials, extracellular matrix (ECM)-based hydrogels, which possess tissue properties and printability, have been proposed as a viable option.

[0005] Recently, research has reported the potential use of vascular tissue-derived decellularized extracellular matrix (VdECM) as a material for drug delivery. However, VdECM alone has the disadvantage of being difficult to use as a printable biomaterial ink due to its poor printability (G. Gao, JY Park, B.S. Kim, J.Jang, D.W. Cho, Advanced healthcare materials 2018, 7, 1801102).

[0006] Regarding extracellular matrix-based bioinks for 3D printing, Korean Patent Publication No. 10-1954953 describes a method for producing a thermosensitive hydrogel containing cell culture medium, hyaluronic acid, gelatin, fibrinogen, and glycerol, and a method for producing a decellularized extracellular matrix by decellularizing tissue extracted from the body and pulverizing it to a particle size of 0.05 to 100 μm. (c) solizing the temperature-sensitive hydrogel obtained in step (a) at 30-40°C; (d) mixing the decellularized extracellular matrix powder obtained in step (b) with the temperature-sensitive hydrogel in the sol state in step (c) by stirring to prepare a decellularized extracellular matrix-based bioink; and (e) mixing the decellularized extracellular matrix-based bioink prepared in step (d) with cells derived from a target tissue and pouring the mixture into a 3D bioprinter cartridge. (f) maintaining the 3D bioprinter cartridge of step (e) at 3 to 6°C for 8 to 15 minutes to induce temperature-induced gelation of the cells and decellularized extracellular matrix-based bioink in the cartridge; (g) 3D bioprinting a desired artificial tissue using the 3D bioprinter cartridge containing the cells and decellularized extracellular matrix-based bioink whose gelation has been induced in step (f); and (h) treating the desired artificial tissue 3D bioprinted in step (g) with a thrombin solution and maintaining the tissue at 20 to 25°C for 25 to 35 minutes to induce gelation by an enzymatic polymerization reaction of thrombin and fibrinogen. In addition, Korean Patent Publication No. 10-2302770 discloses a bioink composition for 3D printing that contains finely divided human tissue-derived components and biocompatible polymers, and the finely divided human tissue-derived components have a particle size of 10 to 1000 μm. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention was developed against the background of the prior art, and aims to provide an extracellular matrix-based hybrid ink for 3D printing that has mechanical properties suitable for 3D printing and can control the drug release rate, as well as a method for producing the same.

[0008] It is also an object of the present invention to provide a biomaterial-based drug delivery patch and a method for making the same.

[0009] It is also an object of the present invention to provide a biomaterial-based patch that can be used to promote angiogenesis or treat diseases associated with angiogenesis. [Means for solving the problem]

[0010] The present inventors developed an extracellular matrix-based hybrid ink that has mechanical properties suitable for 3D printing and can control the drug release rate by chemically bonding the amine groups of the extracellular matrix with the methacrylate groups of methacrylated hyaluronic acid through an aza-Michael addition reaction. The present inventors also added angiogenic growth factors that can promote cerebral angiogenesis to the extracellular matrix-based hybrid ink and then 3D printed it to fabricate a hydrogel patch in which the angiogenic growth factors are spatially compartmentalized and sequentially released. The present inventors implanted a patch containing two spatiotemporally compartmentalized angiogenic growth factors into the brain and monitored it using a photoacoustic microscope, confirming that it effectively promoted angiogenesis.

[0011] To achieve the above-mentioned object, one example of the present invention provides an extracellular matrix-based hybrid ink for 3D printing, which comprises an extracellular matrix having amine groups and modified hyaluronic acid into which ethylenically unsaturated bond functional groups have been introduced, characterized in that the extracellular matrix and the modified hyaluronic acid are in a crosslinked state due to chemical bonds formed between at least a portion of the amine groups present in the extracellular matrix and the ethylenically unsaturated bond functional groups present in the modified hyaluronic acid.

[0012] To achieve the above object, one example of the present invention provides a method for producing an extracellular matrix-based hybrid ink for 3D printing, comprising the steps of: preparing an extracellular matrix solution having a pH of 6.5 to 7.5; adding modified hyaluronic acid having an ethylenically unsaturated bond functional group introduced therein to the extracellular matrix solution and mixing them uniformly; and then conducting an aza-Michael addition reaction to obtain an ink composition in which the extracellular matrix and modified hyaluronic acid are crosslinked.

[0013] To achieve the above object, one example of the present invention provides a biomaterial-based drug delivery patch, characterized in that it is a multilayered patch including an inner core layer and an outer layer surrounding the inner core layer, wherein the inner core layer and the outer layer are made of an extracellular matrix-based hybrid ink, and the hybrid ink contains an extracellular matrix having an amine group, a modified biopolymer having an ethylenically unsaturated bond functional group introduced therein, and a growth factor, the extracellular matrix and the modified biopolymer constituting the hybrid ink being in a chemically crosslinked state, and the extracellular matrix-based hybrid ink constituting the inner core layer and the extracellular matrix-based hybrid ink constituting the outer layer have different chemical crosslink densities and contain different growth factors.

[0014] To achieve the above object, one example of the present invention provides a method for manufacturing a biomaterial-based drug delivery patch, comprising the steps of: preparing a first hybrid ink comprising a mixture of an extracellular matrix having amine groups, a modified biopolymer having an ethylenically unsaturated bond functional group introduced therein, and a first growth factor, wherein the extracellular matrix and the modified biopolymer are in a chemically crosslinked state; preparing a second hybrid ink comprising a mixture of an extracellular matrix having amine groups, a modified biopolymer having an ethylenically unsaturated bond functional group introduced therein, and a second growth factor different from the first growth factor, wherein the extracellular matrix and the modified biopolymer are in a chemically crosslinked state, and wherein the second hybrid ink has a different chemical crosslinking density from that of the first hybrid ink; 3D printing the second hybrid ink to form an inner core layer; and 3D printing the first hybrid ink to form an outer layer surrounding the inner core layer, thereby obtaining a multilayer structure.

[0015] To achieve the above object, one example of the present invention provides a biomaterial-based angiogenesis-promoting patch having a multilayer structure including an inner core layer and an outer layer surrounding the inner core layer, wherein the outer layer is made of a first extracellular matrix-based hybrid ink, and the inner core layer is made of a second extracellular matrix-based hybrid ink, wherein the first hybrid ink contains an extracellular matrix having amine groups, a modified biopolymer having an ethylenically unsaturated bond functional group introduced therein, and a first growth factor that promotes the early stage of angiogenesis, and the second hybrid ink contains an extracellular matrix having amine groups, a modified biopolymer having an ethylenically unsaturated bond functional group introduced therein, and a second growth factor that promotes the mature stage of angiogenesis, wherein the extracellular matrix and the modified biopolymer constituting the first and second hybrid inks are present in a chemically crosslinked state, and the second hybrid ink has a greater chemical crosslink density than the first hybrid ink.

[0016] To achieve the above object, one example of the present invention provides a method for manufacturing a biomaterial-based angiogenesis-promoting patch, the method comprising the steps of: preparing a first hybrid ink comprising a mixture of an extracellular matrix having amine groups, a modified biopolymer having an ethylenically unsaturated bond functional group introduced therein, and a first growth factor that promotes the early stage of angiogenesis, wherein the extracellular matrix and the modified biopolymer are present in a chemically crosslinked state; preparing a second hybrid ink comprising a mixture of an extracellular matrix having amine groups, a modified biopolymer having an ethylenically unsaturated bond functional group introduced therein, and a second growth factor that promotes the mature stage of angiogenesis, wherein the extracellular matrix and the modified biopolymer are present in a chemically crosslinked state, and the second hybrid ink has a higher chemical crosslink density than the first hybrid ink; 3D printing the second hybrid ink to form an inner core layer; and 3D printing the first hybrid ink to form an outer layer surrounding the inner core layer, thereby obtaining a multilayer structure.

[0017] The biomaterial-based pro-angiogenic patch can also be used to treat diseases associated with angiogenesis. [Effects of the Invention]

[0018] The hybrid ink according to one embodiment of the present invention has mechanical properties suitable for 3D printing due to chemical crosslinking between the components, extracellular matrix and modified hyaluronic acid, and the drug release rate can be controlled by adjusting the chemical crosslinking density. A multilayered patch in which different growth factors are spatially and temporally compartmentalized, fabricated by 3D printing the hybrid ink according to one embodiment of the present invention, can be used as a drug delivery system, to promote angiogenesis, or to treat diseases related to angiogenesis, as the different growth factors are released sequentially and sustainedly. [Brief explanation of the drawings]

[0019] [Figure 1]This study outlines the process for fabricating hybrid ink (HAVEM ink) from vascular tissue-derived decellularized extracellular matrix (VdECM) and methacrylated hyaluronic acid (HAMA) using dual crosslinking mechanisms, including chemical and thermal crosslinking. [Figure 2] In this study, VEGF and HGF, which are cerebrovascularization-inducing growth factors, were added to hybrid inks with different cross-linking densities, and a spatiotemporal compartmentalized cerebral angiogenesis-inducing (SCAI) patch was fabricated using a 3D printing process. The schematic process of the sequential release of VEGF and HGF from the SCAI patch was also shown. [Figure 3] This is a schematic diagram of the process for preparing HAVEM ink from vascular tissue-derived decellularized extracellular matrix (VdECM) and methacrylated hyaluronic acid (HAMA) through an aza-Michael addition reaction. [Figure 4] Nuclear magnetic resonance (NMR) spectra were measured for VdECM ink (4V0H), HAMA, and HAVEM ink (4V0.5H) to demonstrate the occurrence of cross-linking between vascular tissue-derived decellularized extracellular matrix (VdECM) and methacrylated hyaluronic acid (HAMA). [Figure 5] The above photograph shows the sol state of VdECM ink (4V0H), HAVEM ink (4V0.5H), and HAVEM ink (4V1H) due to differences in chemical cross-linking density. The graph below Figure 5 shows the viscosity of VdECM ink (4V0H), HAVEM ink (4V0.5H), and HAVEM ink (4V1H) measured at various shear rates and printing conditions at 4°C. [Figure 6]This shows the change in elastic modulus of VdECM ink (4V0H), HAVEM ink (4V0.5H), and HAVEM ink (4V1H) due to a temperature change from 4°C to 37°C. [Figure 7] This shows the storage modulus and loss modulus of VdECM ink (4V0H), HAVEM ink (4V0.5H), and HAVEM ink (4V1H) as a function of frequency change at a temperature of 37°C. [Figure 8] The release profiles of HGF encapsulated in HAVEM ink (4V0.5H) and HAVEM ink (4V1H) were measured over 7 days. [Figure 9] This is a schematic diagram of the process of fabricating the SCAI patch using 3D printing technology in this study. [Figure 10] The results show that the angiogenic effects of various angiogenic factors were measured by a tube formation assay. [Figure 11] The cross-sectional structure of the SCAI patch produced in this study and images of each cross section taken with a confocal microscope (scale bars: 200 μm). [Figure 12] The design structure of SCAI patches fabricated with various combinations of HAVEM inks and images of each patch at the printing, gelling, and vitrification steps. [Figure 13] The results of tensile strength measurement and swelling ratio of the SCAI patch that underwent the vitrification process produced in this study are shown below. [Figure 14] These are SEM images of various vitrified patches fabricated in this study (scale bars: 1 μm). In Figure 14, "4VOH" indicates a vitrified patch made of VdECM ink (4VOH), "4V0.5H" indicates a vitrified patch made of HAVEM ink (4V0.5H), and "4V1H" indicates a vitrified patch made of HAVEM ink (4V1H). [Figure 15]Schematic representation of the structure of the multilayered SCAI patch designed in this study for sequential sustained drug release of VEGF and HGF. [Figure 16] The cumulative release profiles of VEGF and HGF were evaluated over a one-month period for each of the two multilayer SCAI patches designed in this study for the sequential sustained drug release of VEGF and HGF. [Figure 17] This shows the results of an in vitro experiment in which an SCAI patch consisting of an outer layer formed using HAVEM Ink (4V0.5H) containing VEGF at a concentration of 0.05 μg / μl as Ink A and an inner core layer formed using HAVEM Ink (4V1H) containing HGF at a concentration of 0.05 μg / μl as Ink B was applied to human brain microvascular endothelial cells (HBMECs) and cultured. [Figure 18] The SCAI patch, which consists of an outer layer formed using HAVEM Ink (4V0.5H) containing VEGF at a concentration of 0.05 μg / μl as Ink A and an inner core layer formed using HAVEM Ink (4V1H) containing HGF at a concentration of 0.05 μg / μl as Ink B, was applied to human brain microvascular endothelial cells (HBMECs) and cultured. The cell viability was analyzed using a live / dead analysis kit (scale bars: 50 μm). [Figure 19] The results show that angiogenesis-related gene expression levels were analyzed when SCAI patches consisting of an outer layer formed using HAVEM Ink (4V0.5H) containing VEGF at a concentration of 0.05 μg / μl as Ink A and an inner core layer formed using HAVEM Ink (4V1H) containing HGF at a concentration of 0.05 μg / μl as Ink B were applied to human brain microvascular endothelial cells (HBMECs) and cultured. [Figure 20] This is a schematic diagram of an in vivo experiment in which the spatiotemporally compartmentalized SCAI patch fabricated in this study containing VEGF and HGF was implanted into the cerebral cortex of SD mice, followed by monitoring of the implanted area using the OR-PAM system. [Figure 21]This is a photoacoustic (PA) B-mode image (scale bars: 200 μm) of a cross section of the site where the patch was implanted to track changes in the SCAI patch over time in an in vivo experiment to evaluate cerebral angiogenesis behavior after SCAI patch implantation in this study. [Figure 22] This is an in vivo experiment conducted in this study to evaluate the behavior of cerebral angiogenesis after SCAI patch implantation. The changes in cerebral vascular structure in SD mice with and without SCAI patch treatment were shown using photoacoustic (PA) maximum amplitude projection (MAP) images. [Figure 23] Figure 22 shows cross-sectional PA B-mode images of the areas C1 and C2 indicated by dotted lines in the image of a mouse with an SCAI patch (scale bars: 400 μm). In the enlarged image of Figure 23 (scale bars: 1 mm), large blood vessels are highlighted by elliptical curves consisting of the following points: [Figure 24] This is the result of an in vivo experiment conducted in this study to evaluate cerebral angiogenesis behavior after SCAI patch implantation, in which optical resolution photoacoustic microscopy (OR-PAM) images were used to quantitatively analyze the blood vessel density in SD mice with and without SCAI patch treatment. [Figure 25] In this study, an in vivo experiment was conducted to evaluate the behavior of cerebral angiogenesis after SCAI patch implantation. The histological analysis results for the untreated and SCAI patch-implanted groups 14 days after SCAI patch implantation are shown in H&E staining images and immunohistochemistry (IHC) images (CD31) (scale bars: 50 μm). [Figure 26] This study was conducted to evaluate the behavior of cerebral angiogenesis after SCAI patch implantation in an in vivo experiment. The results are from a quantitative analysis of the number of blood vessels per unit area in the untreated and SCAI patch-implanted groups using immunohistochemistry (IHC) images 14 days after SCAI patch implantation. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention will be specifically described below.

[0021] The term "bioink" as used herein is defined as a cell-compatible material that can be 3-D printed. Bioink can be extruded through a needle at 0-37°C and subsequently gelled or solidified. Bioink can be formulated to be suitable for inkjet, laser-assisted, or microvalve 3-D printing equipment.

[0022] The term "extracellular matrix (ECM)" used in the present invention refers to the extracellular portion of animal tissues, which generally provides structural support to animal cells while also performing a variety of other important functions. The extracellular matrix is ​​a defining characteristic of connective tissue in animals and is composed of various forms of proteins, including collagen and glycosaminoglycans (GAGs). Such extracellular matrix may be tissue from animals such as pigs and cows, and can be extracted from a variety of sources.

[0023] The term "biopolymer" used in this invention is a general term for polymeric compounds synthesized within the bodies of living organisms, and representative examples include proteins, nucleic acids, and polysaccharides, and specific substances include hyaluronic acid and collagen.

[0024] The term "crosslinking density" used in the present invention is defined as the ratio of structural units (crosslinking points) that are crosslinked in water to the total structural units of a crosslinked polymer. Depending on the crosslinking density, the physical properties of the crosslinked polymer gel, such as swelling degree and elasticity, are significantly affected.

[0025] The term "growth factor" as used in the present invention refers to a protein involved in regulating the growth and differentiation of cells or organisms. It acts as a signal transmitter that transmits signals to the interior of cells by binding to specific receptors present on the cell surface, thereby regulating various cellular physiological phenomena such as cell differentiation, regeneration, and healing. Specific examples of growth factors include vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), epithermal growth factor (EGF), insulin-like growth factor (IGF), erythropoietin (EPO), fibroblast growth factor (FGF), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), keratinocyte growth factor (KGF), interleukin (IL), colony-stimulating factor (CSF), angiopoietin (ANG), platelet-derived growth factor (PDGF), placental growth factor (PGF), and transforming growth factor-α (transforming growth factor-α). These include TGF-α, transforming growth factor-β (TGF-β), matrix metalloproteinases (MMPs), and bone morphogenetic proteins (BMPs).

[0026] One aspect of the present invention relates to an extracellular matrix-based hybrid ink for 3D printing, which has mechanical properties suitable for 3D printing and can control drug release rate, and a method for producing the same.

[0027] The hybrid ink according to one embodiment of the present invention can be classified as a bioink or biomaterial ink because it is based on a cell-compatible biomaterial. The hybrid ink according to one embodiment of the present invention is a composition in which the components are uniformly mixed, and exists in a sol state or an intermediate state between sol and hydrogel to have the appropriate fluidity required for 3D printing.

[0028] An extracellular matrix-based hybrid ink for 3D printing according to one example of the present invention comprises an extracellular matrix having amine groups and modified hyaluronic acid with introduced ethylenically unsaturated bond functional groups.

[0029] The extracellular matrix may be derived from various organ tissues depending on the intended use of the hybrid ink. For purposes such as the manufacture of angiogenesis-promoting patches, described below, it is preferably derived from vascular tissue. Furthermore, for applications such as biotransplantation, the extracellular matrix is ​​preferably decellularized. The decellularization process effectively minimizes immune responses during allograft or xenograft transplantation by removing cells that can act as antigens inducing immune responses. Because the type and number of cells and the physical properties of the tissue vary depending on the tissue, decellularization can be performed using various chemicals, such as acids, bases, hypotonic solutions, hypertonic solutions, and detergents. The extracellular matrix may be used after only the decellularization process, maintaining the tissue structure. Alternatively, it may be dissolved in an acidic solution after freeze-drying and pulverization, or further neutralized to form a sol.

[0030] The ethylenically unsaturated bond functional group introduced into the modified hyaluronic acid is not particularly limited as long as it can undergo an aza-Michael addition reaction with an amine group present in the extracellular matrix, and can be selected from, for example, vinyl, allyl, acrylic, and methacrylic groups. In the extracellular matrix-based hybrid ink for 3D printing according to one embodiment of the present invention, the modified hyaluronic acid can be selected from methacrylated hyaluronic acid or acrylated hyaluronic acid, taking into consideration biocompatibility and reactivity with amine groups. The methacrylated hyaluronic acid has a chemical structure represented by the following Chemical Formula 1:

[0031] [Chemical formula 1] TIFF0007778423000001.tif4860

[0032] In an extracellular matrix-based hybrid ink for 3D printing according to one embodiment of the present invention, chemical bonds are formed between at least a portion of the amine groups present in the extracellular matrix and the ethylenically unsaturated functional groups present in the modified hyaluronic acid, thereby crosslinking the extracellular matrix and the modified hyaluronic acid, which are components of the hybrid ink. The chemical bonds between the amine groups present in the extracellular matrix and the ethylenically unsaturated functional groups present in the modified hyaluronic acid are formed via an aza-Michael addition reaction. The aza-Michael addition reaction is a nucleophilic conjugate addition reaction involving an N-nucleophile and an electrophile, such as an activated alkene. In the present invention, the amine groups present in the extracellular matrix act as the N-nucleophile, and the ethylenically unsaturated functional groups present in the modified hyaluronic acid act as the electrophile.

[0033] In an example of the present invention, the weight ratio of extracellular matrix to denatured hyaluronic acid in an extracellular matrix-based hybrid ink for 3D printing is preferably 1:0.01 to 1:0.8, more preferably 1:0.05 to 1:0.5, taking into account the chemical crosslinking density, which is a factor related to the mechanical properties, printability, or drug release control of the hybrid ink. Furthermore, the concentration of extracellular matrix in the hybrid ink is preferably 1 to 10% (w / v), more preferably 2 to 8% (w / v), taking into account the mechanical properties, printability, or drug release control of the hybrid ink. Furthermore, the concentration of denatured hyaluronic acid in the hybrid ink is preferably 0.01 to 8% (w / v), more preferably 0.1 to 4% (w / v), taking into account the mechanical properties, printability, or drug release control of the hybrid ink.

[0034] In addition, the extracellular matrix-based hybrid ink for 3D printing according to one embodiment of the present invention may further preferably contain growth factors for the production of drug delivery patches, angiogenesis promotion patches, etc., as described below. The growth factor may be selected from a variety of known growth factors depending on the specific application of the drug delivery patch manufactured by 3D printing the hybrid ink. Examples of the growth factor include vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), epithermal growth factor (EGF), insulin-like growth factor (IGF), erythropoietin (EPO), fibroblast growth factor (FGF), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), keratinocyte growth factor (KGF), interleukin (IL), colony-stimulating factor (CSF), angiopoietin (ANG), platelet-derived growth factor (PGF), and the like. The fibroblast growth factor may be one or more selected from the group consisting of PDGF, placental growth factor (PGF), transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β), matrix metalloproteinase (MMP), and bone morphogenetic protein (BMP).The concentration of growth factors in the hybrid ink may be selected from various ranges depending on the specific application, such as a drug delivery patch manufactured by 3D printing the hybrid ink. For example, it may be 0.001 to 1 g / L, and considering the drug delivery effect, it is preferably 0.01 to 0.5 g / L.

[0035] A method for preparing an extracellular matrix-based hybrid ink for 3D printing according to one embodiment of the present invention includes the steps of preparing an extracellular matrix solution having a pH of 6.5 to 7.5, adding modified hyaluronic acid having an ethylenically unsaturated bond functional group to the extracellular matrix solution, mixing the mixture uniformly, and then conducting an aza-Michael addition reaction to obtain an ink in which the extracellular matrix and modified hyaluronic acid are crosslinked. Furthermore, the method for preparing an extracellular matrix-based hybrid ink for 3D printing according to one embodiment of the present invention may further include the step of adding and mixing a growth factor to the ink so that a patch formed using the ink can be used as a drug delivery system or a disease treatment.

[0036] In the method for manufacturing an extracellular matrix-based hybrid ink for 3D printing according to one embodiment of the present invention, the technical features such as the extracellular matrix having an amine group, the modified hyaluronic acid having an ethylenically unsaturated bond functional group introduced therein, the aza-Michael addition reaction, and the growth factor are described above, and detailed description thereof will be omitted.

[0037] In a method for preparing an extracellular matrix-based hybrid ink for 3D printing according to one embodiment of the present invention, the step of preparing an extracellular matrix solution having a pH of 6.5 to 7.5 can be performed by dispersing an extracellular matrix having amine groups in water and then adjusting the pH, or by dissolving an extracellular matrix having amine groups in an acid solution and neutralizing it with a base. The type of acid solution used to dissolve the extracellular matrix is ​​not particularly limited. However, considering applications such as biotransplantation, a weak acid solution is preferred. The weak acid may be selected from acetic acid, citric acid, butyric acid, palmitic acid, oxalic acid, tartaric acid, malic acid, succinic acid, and the like. Furthermore, the pH of the extracellular matrix solution is preferably close to neutral, e.g., 6.8 to 7.4, in order to minimize adverse effects on the ink components and facilitate the aza-Michael addition reaction.

[0038] In a method for preparing an extracellular matrix-based hybrid ink for 3D printing according to one embodiment of the present invention, the extracellular matrix and modified hyaluronic acid are crosslinked after the aza-Michael addition reaction, which forms chemical bonds between at least some of the amine groups present in the extracellular matrix and the ethylenically unsaturated functional groups present in the modified hyaluronic acid. The aza-Michael addition reaction is carried out at a temperature of 1-10°C, preferably 2-8°C, for 2-24 hours, preferably 6-18 hours, to minimize negative effects on the extracellular matrix and modified hyaluronic acid and ensure smooth aza-Michael addition reaction.

[0039] In a method for preparing an extracellular matrix-based hybrid ink for 3D printing according to one embodiment of the present invention, the amount of modified hyaluronic acid added to the extracellular matrix solution is not significantly limited. However, taking into consideration the mechanical properties, printability, and drug release control of the hybrid ink, an amount of 1 to 80 parts by weight per 100 parts by weight of extracellular matrix is ​​preferred, and an amount of 5 to 50 parts by weight per 100 parts by weight of extracellular matrix is ​​even more preferred. Furthermore, taking into consideration the mechanical properties, printability, and drug release control of the hybrid ink, the concentration of the extracellular matrix in the extracellular matrix solution is preferably 1 to 10% (w / v), and even more preferably 2 to 8% (w / v). Furthermore, the modified hyaluronic acid is added to the extracellular matrix solution at a concentration of preferably 0.01 to 8% (w / v), and even more preferably 0.1 to 4% (w / v).

[0040] In a method for preparing an extracellular matrix-based hybrid ink for 3D printing according to an embodiment of the present invention, the amount of growth factor added to the ink can be selected from various ranges depending on the specific application, such as a drug delivery patch, prepared by 3D printing the hybrid ink. For example, the growth factor can be added to the ink to a concentration of 0.001 to 1 g / L, or, taking into consideration the drug delivery effect, can be added to a concentration of 0.01 to 0.5 g / L.

[0041] One aspect of the present invention relates to a biomaterial-based drug delivery patch with a multilayer structure in which drugs are spatially and temporally compartmentalized for the sequential sustained release of different drugs, and a method for manufacturing the same. In another example of the biomaterial-based drug delivery patch and method for manufacturing the same, technical features such as an extracellular matrix having amine groups, modified hyaluronic acid having ethylenically unsaturated bond functional groups introduced therein, aza-Michael addition reaction, and growth factors are described above, and detailed description thereof will be omitted.

[0042] A biomaterial-based drug delivery patch according to one example of the present invention can be fabricated by 3D printing the above-described extracellular matrix-based hybrid ink into a predetermined design structure.

[0043] A biomaterial-based drug delivery patch according to one embodiment of the present invention is a multi-layered patch including an inner core layer and an outer layer surrounding the inner core layer, the inner core layer and the outer layer being made of an extracellular matrix-based hybrid ink, and exhibits rheological properties corresponding to a hydrogel state or a vitrified gel state.

[0044] In one embodiment of the present invention, the hybrid ink of a biomaterial-based drug delivery patch comprises an extracellular matrix having an amine group, a modified biopolymer having an ethylenically unsaturated bond functional group introduced therein, and a growth factor. The modified biopolymer may be selected from the group consisting of a modified hyaluronic acid having an ethylenically unsaturated bond functional group introduced therein and a modified collagen having an ethylenically unsaturated bond functional group introduced therein. The ethylenically unsaturated bond functional group introduced into the modified biopolymer is not particularly limited as long as it is capable of undergoing an aza-Michael addition reaction with the amine group present in the extracellular matrix, and may be selected from the group consisting of vinyl, allyl, acrylic, and methacrylic groups. The modified hyaluronic acid may be selected from methacrylated hyaluronic acid or acrylated hyaluronic acid, taking into consideration biocompatibility and reactivity with amine groups. In addition, the denatured collagen may be selected from methacrylated collagen or acrylated collagen, taking into consideration biocompatibility and reactivity with amine groups. Methacrylated collagen is a denatured collagen in which a methacryl group is linked to an amine group present in collagen via a peptide bond, while acrylated collagen is a denatured collagen in which an acrylic group is linked to an amine group present in collagen via a peptide bond. The structure and manufacturing method of the methacrylated collagen or acrylated collagen are disclosed in various publicly known documents (e.g., U.S. Patent Publication No. 8,658,711; He Liang et al., Journal of Materials Chemistry B, 2018, 6, 3703-3715, etc.).The extracellular matrix and modified biopolymer that make up the hybrid ink are chemically crosslinked, starting with chemical bonds formed between at least some of the amine groups present in the extracellular matrix and the ethylenically unsaturated functional groups present in the modified hyaluronic acid via an aza-Michael addition reaction.

[0045] Furthermore, the extracellular matrix-based hybrid ink constituting the inner core layer and the extracellular matrix-based hybrid ink constituting the outer layer preferably have different chemical crosslink densities. Considering sequential sustained drug release, the chemical crosslink density of the extracellular matrix-based hybrid ink constituting the inner core layer is preferably greater than that of the extracellular matrix-based hybrid ink constituting the outer layer. The chemical crosslink densities of the extracellular matrix-based hybrid ink constituting the inner core layer and the extracellular matrix-based hybrid ink constituting the outer layer can be adjusted by adjusting the weight ratio of the extracellular matrix having an amine group to the modified biopolymer containing an ethylenically unsaturated bond functional group, which are components of the extracellular matrix-based hybrid ink. The primary component of the extracellular matrix-based hybrid ink is the extracellular matrix. The greater the amount of the modified biopolymer relative to the extracellular matrix in the hybrid ink, the greater the chemical bond density due to the formation of more chemical bonds through the aza-Michael addition reaction. For example, the weight ratio of the extracellular matrix to the modified biopolymer in the extracellular matrix-based hybrid ink constituting the inner core layer is preferably 1:0.2 to 1:0.5. Furthermore, the weight ratio of extracellular matrix to modified biopolymer in the extracellular matrix-based hybrid ink constituting the outer core layer is preferably 1:0.05 to 1:0.15. Furthermore, the concentration of extracellular matrix in the extracellular matrix-based hybrid ink constituting the inner core layer and the extracellular matrix-based hybrid ink constituting the outer layer is preferably 2 to 8% (w / v). Furthermore, the concentration of modified biopolymer in the extracellular matrix-based hybrid ink constituting the inner core layer is preferably 0.4 to 4% (w / v). Furthermore, the concentration of modified biopolymer in the extracellular matrix-based hybrid ink constituting the outer layer is preferably 0.1 to 1.2% (w / v).

[0046] In addition, the extracellular matrix-based hybrid ink constituting the inner core layer and the extracellular matrix-based hybrid ink constituting the outer layer contain different growth factors. Therefore, in a biomaterial-based drug delivery patch according to one embodiment of the present invention, the inner core layer and the outer layer contain different growth factors. The growth factors may be selected from a variety of known growth factors depending on the specific application of the drug delivery patch. For example, the growth factor may be selected from growth factors that induce angiogenesis, such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), epithermal growth factor (EGF), fibroblast growth factor (FGF), angiopoietin (ANG), platelet-derived growth factor (PDGF), placental growth factor (PGF), transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β), matrix metalloproteinase (MMP), and bone morphogenetic protein (BMP).

[0047] In a biomaterial-based drug delivery patch according to one embodiment of the present invention, if the extracellular matrix-based hybrid ink constituting the inner core layer has a higher chemical crosslink density than the extracellular matrix-based hybrid ink constituting the outer layer, the growth factors contained in the inner core layer are not only spatially compartmentalized relative to the growth factors contained in the outer layer based on their positional relationship, but also temporally compartmentalized based on the chemical crosslink density of the extracellular matrix-based hybrid ink, resulting in the growth factors contained in the inner core layer being released more slowly in vivo than the growth factors contained in the outer layer. Considering the drug delivery effect of the multilayered patch, the concentration of growth factors in the extracellular matrix-based hybrid ink constituting the inner core layer and the extracellular matrix-based hybrid ink constituting the outer layer is preferably 0.01 to 0.5 g / L.

[0048] A preferred embodiment of the present invention provides a biomaterial-based drug delivery patch having a multilayer structure in which a first layer, a second layer, and a third layer are sequentially laminated. The second layer comprises a second internal layer and two second external layers on both sides. The first layer, the two second external layers, and the third layer are composed of a first hybrid ink, and the second internal layer is composed of a second hybrid ink. The first and second hybrid inks contain an extracellular matrix having amine groups, a modified hyaluronic acid having an ethylenically unsaturated bond functional group, and a growth factor. The extracellular matrix and the modified hyaluronic acid constituting the first and second hybrid inks are present in a chemically crosslinked state. The first and second hybrid inks have different chemical crosslink densities. Considering sequential sustained drug release, it is preferable that the chemical crosslink density of the second hybrid ink be greater than that of the first hybrid ink. The growth factors constituting the first and second hybrid inks are selected from different growth factors.

[0049] A method for manufacturing a biomaterial-based drug delivery patch according to one embodiment of the present invention includes the steps of: preparing a first hybrid ink comprising a mixture of an extracellular matrix having amine groups, a modified biopolymer having an ethylenically unsaturated bond functional group introduced therein, and a first growth factor, wherein the extracellular matrix and the modified biopolymer are in a chemically crosslinked state; preparing a second hybrid ink comprising a mixture of an extracellular matrix having amine groups, a modified biopolymer having an ethylenically unsaturated bond functional group introduced therein, and a second growth factor different from the first growth factor, wherein the extracellular matrix and the modified biopolymer are in a chemically crosslinked state, and wherein the second hybrid ink has a different chemical crosslinking density from that of the first hybrid ink; 3D printing the second hybrid ink to form an inner core layer; and 3D printing the first hybrid ink to form an outer layer surrounding the inner core layer, thereby obtaining a multilayer structure. In a method for manufacturing a biomaterial-based drug delivery patch according to one embodiment of the present invention, a multilayer structure formed by 3D printing a first hybrid ink and a second hybrid ink, comprising an inner core layer and an outer layer surrounding the inner core layer, exists in an intermediate state between a sol and a hydrogel. The method for manufacturing a biomaterial-based drug delivery patch according to one embodiment of the present invention may further include a step of converting the multilayer structure into a hydrogel state by heat-treating the multilayer structure at 25-45°C, preferably 30-40°C, for 0.5-3 hours, preferably 0.6-1.5 hours, to obtain a multilayer structure in a hydrogel state. The method for manufacturing a biomaterial-based drug delivery patch according to one embodiment of the present invention may further include a step of drying the multilayer structure in a hydrogel state under ventilated conditions for 6-24 hours, preferably 8-18 hours, to obtain a vitrified multilayer structure.

[0050] In a method for manufacturing a biomaterial-based drug delivery patch according to one embodiment of the present invention, the second hybrid ink preferably has a higher crosslinking density than the first hybrid ink. In a method for manufacturing a biomaterial-based drug delivery patch according to one embodiment of the present invention, the first and second hybrid inks are extracellular matrix-based hybrid inks whose main component is extracellular matrix. The weight ratio of extracellular matrix to modified biopolymer in the first hybrid ink is preferably 1:0.05 to 1:0.15. Furthermore, the concentration of extracellular matrix in the first hybrid ink is preferably 2 to 8% (w / v), and the concentration of modified biopolymer is preferably 0.1 to 1.2% (w / v). The weight ratio of extracellular matrix to modified biopolymer in the second hybrid ink is preferably 1:0.2 to 1:0.5. Furthermore, the concentration of extracellular matrix in the second hybrid ink is preferably 2 to 8% (w / v), and the concentration of modified biopolymer is preferably 0.4 to 4% (w / v).

[0051] A method for manufacturing a biomaterial-based drug delivery patch according to a preferred embodiment of the present invention includes the steps of: preparing a first hybrid ink comprising a mixture containing an extracellular matrix having an amine group, a modified hyaluronic acid having an ethylenically unsaturated bond functional group introduced therein, and a first growth factor, wherein the extracellular matrix and the modified hyaluronic acid are present in a chemically crosslinked state; and preparing a second hybrid ink comprising a mixture containing an extracellular matrix having an amine group, a modified hyaluronic acid having an ethylenically unsaturated bond functional group introduced therein, and a second growth factor different from the first growth factor, wherein the extracellular matrix and the modified hyaluronic acid are present in a chemically crosslinked state. The method for manufacturing a biomaterial-based drug delivery patch according to a preferred embodiment of the present invention further includes the steps of: preparing a second hybrid ink, the second hybrid ink being in a chemically crosslinked state and having a different chemical crosslinking density from the first hybrid ink; 3D printing the first hybrid ink to form a first layer; 3D printing the second hybrid ink on the inner surface of the first layer and the first hybrid ink on both outer surfaces of the first layer to form a second layer comprising a second inner layer and two second outer layers; and 3D printing the first hybrid ink on the surface of the second layer to form a third layer, thereby obtaining a multilayer structure. A preferred embodiment of the present invention may further include the step of heat-treating the multilayer structure including the first, second, and third layers at 25-45°C, preferably 30-40°C, for 0.5-3 hours, preferably 0.6-1.5 hours, to obtain a multilayer structure in a hydrogel state. Furthermore, a preferred embodiment of the present invention may further include a step of drying the hydrogel-state multilayer structure under ventilated conditions for 6 to 24 hours, preferably 8 to 18 hours, to obtain a vitrified multilayer structure. The first and second hybrid inks have different chemical crosslinking densities, and considering sequential sustained drug release, the chemical crosslinking density of the second hybrid ink is preferably greater than that of the first hybrid ink. The first and second growth factors constituting the first and second hybrid inks are selected from different growth factors.

[0052] One aspect of the present invention relates to a multi-layered biomaterial-based angiogenesis promoting patch in which drugs are spatially and temporally compartmentalized for the sequential sustained release of different angiogenesis promoting factors, and a method for manufacturing the same. Compared to the biomaterial-based drug delivery patch and method for manufacturing the same described above, the biomaterial-based angiogenesis promoting patch and method for manufacturing the same according to the present invention have the same technical features as the biomaterial-based drug delivery patch and method for manufacturing the same, except that the growth factors contained in each layer are embodied as growth factors that promote the early stage of angiogenesis and growth factors that promote the mature stage of angiogenesis.

[0053] A biomaterial-based angiogenesis-promoting patch according to one embodiment of the present invention is a multilayered patch including an inner core layer and an outer layer surrounding the inner core layer. The outer layer is made of a first extracellular matrix-based hybrid ink, and the inner core layer is made of a second extracellular matrix-based hybrid ink. The first hybrid ink contains an extracellular matrix having amine groups, a modified biopolymer having ethylenically unsaturated bond functional groups, and a first growth factor promoting the initial stage of angiogenesis. The second hybrid ink contains an extracellular matrix having amine groups, a modified biopolymer having ethylenically unsaturated bond functional groups, and a second growth factor promoting the mature stage of angiogenesis. The extracellular matrix and the modified biopolymer constituting the first and second hybrid inks are chemically crosslinked, and the second hybrid ink has a higher chemical crosslink density than the first hybrid ink. In a preferred embodiment of the biomaterial-based angiogenesis-promoting patch according to the present invention, the modified biopolymer having ethylenically unsaturated bond functional groups is modified hyaluronic acid having ethylenically unsaturated bond functional groups. Furthermore, in the biomaterial-based angiogenesis-promoting patch according to the present invention, the first growth factor that promotes the initial step of angiogenesis may be one or more selected from the group consisting of vascular endothelial growth factor (VEGF), angiopoietin (ANG), platelet-derived growth factor (PDGF), placental growth factor (PGF), epithermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β), and matrix metalloproteinase (MMP).In addition, the second growth factor that promotes the maturation stage of angiogenesis may be one or more selected from the group consisting of hepatocyte growth factor (HGF), bone morphogenetic protein (BMP), and transforming growth factor-β (TGF-β).

[0054] A preferred embodiment of the present invention provides a biomaterial-based angiogenesis-promoting patch with a multilayer structure in which a first layer, a second layer, and a third layer are laminated in order. The second layer comprises a second internal layer and two external layers on both sides. The first layer, the two external layers, and the third layer are made of a first hybrid ink, and the second internal layer is made of a second hybrid ink. The first hybrid ink contains an extracellular matrix having amine groups, modified hyaluronic acid with ethylenically unsaturated bond functional groups, and a first growth factor that promotes the initial stage of angiogenesis. The second hybrid ink contains an extracellular matrix, modified hyaluronic acid with ethylenically unsaturated bond functional groups, and a second growth factor that promotes the mature stage of angiogenesis. The extracellular matrix and modified hyaluronic acid that comprise the first and second hybrid inks are chemically crosslinked. Furthermore, the first hybrid ink and the second hybrid ink have different chemical cross-linking densities, and considering the sequential sustained release of the drug, it is preferable that the chemical cross-linking density of the second hybrid ink is greater than the chemical cross-linking density of the first hybrid ink.

[0055] A method for manufacturing a biomaterial-based angiogenesis-promoting patch according to one embodiment of the present invention includes the steps of: preparing a first hybrid ink comprising a mixture of an extracellular matrix having amine groups, a modified biopolymer having an ethylenically unsaturated bond functional group introduced therein, and a first growth factor that promotes the early stage of angiogenesis, wherein the extracellular matrix and the modified biopolymer are present in a chemically crosslinked state; preparing a second hybrid ink comprising a mixture of an extracellular matrix having amine groups, a modified biopolymer having an ethylenically unsaturated bond functional group introduced therein, and a second growth factor that promotes the mature stage of angiogenesis, wherein the extracellular matrix and the modified biopolymer are present in a chemically crosslinked state, and wherein the second hybrid ink has a higher chemical crosslink density than the first hybrid ink; 3D printing the second hybrid ink to form an inner core layer; and 3D printing the first hybrid ink to form an outer layer surrounding the inner core layer, thereby obtaining a multilayer structure. Furthermore, the method for manufacturing a biomaterial-based angiogenesis-promoting patch according to one embodiment of the present invention may further include a step of heat-treating the multilayer structure including the inner core layer and the outer layer at 25-45°C, preferably 30-40°C, for 0.5-3 hours, preferably 0.6-1.5 hours, to convert the multilayer structure into a hydrogel state. Furthermore, the method for manufacturing a biomaterial-based angiogenesis-promoting patch according to one embodiment of the present invention may further include a step of drying the multilayer structure in a hydrogel state under ventilated conditions for 6-24 hours, preferably 8-18 hours, to obtain a vitrified multilayer structure.

[0056] A method for manufacturing a biomaterial-based angiogenesis-promoting patch according to a preferred embodiment of the present invention includes the steps of: preparing a first hybrid ink comprising a mixture containing an extracellular matrix having an amine group, a modified hyaluronic acid having an ethylenically unsaturated bond functional group introduced therein, and a first growth factor that promotes the initial stage of angiogenesis, wherein the extracellular matrix and the modified hyaluronic acid are present in a chemically crosslinked state; and preparing a first hybrid ink comprising a mixture containing an extracellular matrix having an amine group, a modified hyaluronic acid having an ethylenically unsaturated bond functional group introduced therein, and a second growth factor that promotes the maturation stage of angiogenesis, wherein the extracellular matrix and the modified hyaluronic acid are present in a chemically crosslinked state. The method includes the steps of preparing a second hybrid ink, in which the hyaluronic acid is present in a chemically cross-linked state and has a higher chemical cross-linking density than the first hybrid ink; 3D printing the first hybrid ink to form a first layer; 3D printing the second hybrid ink on the inside of the surface of the first layer and 3D printing the first hybrid ink on the outside of both sides of the surface of the first layer to form a second layer consisting of a second internal layer and two second external layers; and 3D printing the first hybrid ink on the surface of the second layer to form a third layer, thereby obtaining a multilayer structure.

[0057] The biomaterial-based angiogenesis-promoting patch according to the present invention can be used to prevent or treat angiogenesis-dependent diseases. Thus, the present invention provides a biomaterial-based patch for preventing or treating angiogenesis-dependent diseases.

[0058] The "angiogenesis-dependent disease" refers to a disease accompanied by symptoms of insufficient blood supply or insufficient angiogenesis. In the present invention, the angiogenesis-dependent disease is not particularly limited as long as it can be prevented or treated by promoting angiogenesis. For example, the angiogenesis-dependent disease may be one or more selected from the group consisting of ischemic disease, wound, burn, psoriasis, chronic ulcer, myocardial infarction, angina pectoris, pressure ulcer, alopecia, diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, glaucoma, diabetic foot ulcer, post-hypertension, and cerebrovascular dementia. The ischemic disease may be one or more selected from the group consisting of cerebral ischemia, cardiac ischemia, diabetic vascular heart disease, heart failure, myocardial hypertrophy, retinal ischemia, ischemic colitis, ischemic acute renal failure, stroke, cerebral trauma, and neonatal hypoxia.

[0059] The present invention will be described in more detail with reference to the following examples, which are provided to clearly illustrate the technical features of the present invention and are not intended to limit the scope of the present invention.

[0060] 1. Experimental Method (1) Production of vascular tissue-derived decellularized extracellular matrix (VdECM) and ink containing it Fresh porcine aortas purchased from a nearby slaughterhouse were minced into approximately 2 mm cubes and washed in deionized water for 6 hours to remove any remaining blood. The sliced ​​aortic tissue was then placed in a 0.3% (w / w) sodium dodecyl sulfate solution and stirred for 24 hours, followed by a 3% (w / w) Triton X-100 solution and stirred for 24 hours. The aortic tissue was then rinsed in phosphate-buffered saline (PBS) for 24 hours to remove the chemical detergent, and then decellularized by treating it with a nuclease solution containing 75 U / ml DNase and 50 mM magnesium chloride (MgCl2) in PBS at 37°C for 24 hours. The decellularized tissue was then sterilized by treating it with a 0.1% peracetic acid solution in 4% ethanol for approximately 4 hours and rinsed multiple times with deionized water and PBS to obtain vascular tissue-derived decellularized extracellular matrix (VdECM). The vascular tissue-derived decellularized extracellular matrix (VdECM) was then rapidly frozen at -80°C and lyophilized for approximately 48 hours to obtain powdered vascular tissue-derived decellularized extracellular matrix (VdECM).

[0061] 100 mg of lyophilized VdECM was dissolved in 7.5 ml of 0.5 M acetic acid solution with 15 mg of pepsin by stirring at approximately 120 rpm, and the solution was filtered through a 40 μm pore size mesh filter to remove undissolved particles, yielding a VdECM solution. Subsequently, 10 N sodium hydroxide solution was added to the VdECM solution, and the solution was neutralized to a pH of 7.4 by stirring, producing a VdECM ink in a sol state.

[0062] (2) Hybrid ink manufacturing VdECM and methacrylated hyaluronic acid (HAMA) were mixed in a neutral solution (pH 7) and then subjected to an aza-Michael addition reaction at 4°C for approximately 10-12 hours to synthesize a new hybrid bioink in a sol state, named "HAVEM ink." The acrylate functional group of HAMA acts as a Michael acceptor, and the amine functional group present in the VdECM component acts as a Michael donor, allowing the aza-Michael addition reaction to proceed. The aza-Michael addition reaction is a nucleophilic conjugate addition reaction involving an N-nucleophile and an electrophile, such as an activated alkene. The aza-Michael addition reaction is known to be the simplest and most efficient method for coupling amines to α,β-unsaturated carbonyl compounds without the need for a separate catalyst. Two types of HAVEM inks were prepared by adding different amounts of VdECM and methacrylated hyaluronic acid (HAMA) to a neutral solution: HAVEM ink (4V0.5H) and HAVEM ink (4V1H). The compositions of the two HAVEM inks are summarized in Table 1 below. As a control, VdECM ink (4VOH) was prepared by adding only VdECM to a neutral (pH 7) solution at a concentration of 4% (w / v).

[0063] [Table 1] TIFF0007778423000002.tif28128

[0064] In addition, to fabricate spatiotemporal compartmentalized cerebral angiogenesis inducing (SCAI) patches, a total of four types of bioinks were prepared by mixing vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) at concentrations of 0.05 μg / μl each with HAVEM ink (4V0.5H) or HAVEM ink (4V1H).

[0065] (3) Production of SCAI patches To develop a spatiotemporally separated dual growth factor release platform, three-layer circular patches were fabricated using a 3D printing system. The first, second outer, and third layers were printed using HAVEM ink mixed with VEGF (recombinant human VEGF 165; PeproTech, USA). The second inner layer was printed using HAVEM ink mixed with HGF (recombinant human HGF; PeproTech, USA). Each of the two types of HAVEM ink was loaded into a 3 ml syringe equipped with a 24G plastic nozzle (Musashi Engineering Company, Japan) and then loaded into a multi-head 3D printing system (T&R Biofab, Korea). All three layers were printed in a spiral pattern using a programmed G-code at a controlled temperature of 4°C. After printing each layer, a 20-second pause was allowed to prevent intermixing between the stacked layers. The printed three-layer circular SCAI patches were then placed in an incubator at 5% carbon dioxide and 37°C for 1 hour for gelation. The SCAI hydrogel patch was then dried in a well-ventilated place for approximately 10 to 12 hours for vitrification, and the vitrified SCAI patch was then used for subsequent cell and animal experiments.

[0066] (4) Physicochemical properties of 3D printing ink and SCAI patch Rheological analysis of the 3D printing biomaterial inks prepared above was performed using a 20 mm cone-and-plate Discovery Hybrid Rheometer-2 (TA Instruments, USA) system. Three neutralized inks (pH 7) were analyzed: 4% VdECM ink (4V0H); 4% VdECM ink containing 0.5% HAMA [HAVEM ink (4V0.5H)]; and 4% VdECM ink containing 1% HAMA [HAVEM ink (4V1H)]. Shear viscosity values ​​were measured at 4°C and 0.01 s to evaluate the flow behavior of each biomaterial ink. -1 From the 1000s -1 The gelation kinetics of each ink was determined under conditions of increasing shear rate from 4 to 37°C under a heating rate of 5°C / min. To confirm the storage modulus and loss modulus of the biomaterial ink according to frequency, a dynamic frequency change test was performed on the ink gelled at 37°C under a 2% strain condition in the range of 0.1 to 100 rad / s. Furthermore, to confirm that the ink components VdECM and HAMA were chemically crosslinked via the aza-Michael addition reaction mechanism, the freeze-dried ink was dissolved in DO. 1 H NMR (400 MHz) spectra were obtained using a Bruker Advance III HD 400 MHz NMR spectrometer. The patches prepared using the ink were then vitrified, and the tensile properties of the vitrified patches were confirmed using an MTS extensometer (AVX54, MTS). To prevent the vitrified patches from sliding during tensile property testing, rectangular vitrified patch samples (25 mm x 8 mm x 400 μm) were prepared and fixed using screw grips attached to the MTS extensometer.

[0067] (5) Growth factor release test Recombinant human VEGF 165 (100-20; PeproTech, USA) and recombinant human HGF (100-39H; PeproTech, USA) were mixed into each ink to a final concentration of 0.05 μg / μl. Multilayered patches were then laminated using 3D printing, followed by heat treatment and drying to fabricate vitrified SCAI patches. The vitrified SCAI patches underwent dual crosslinking, consisting of chemical and thermal crosslinking. Vitrified circular disk-shaped SCAI patches (8 mm diameter, 150 μm height) were placed in 1.1 ml of PBS solution at 37°C. Subsequently, 1 ml of the solution was removed at desired time points and stored at -80°C, and the amount of PBS solution removed was replaced accordingly. The amount of growth factors VEGF (DY293B; R&D Systems, USA) and HGF (DY294; R&D Systems, USA) released from each patch was quantified using an ELISA kit.

[0068] (6)Cell culture Human brain microvascular endothelial cells (HBMECs; Innoprot, Korea) were seeded in endothelial cell medium (ECM; Innoprot, Korea) supplemented with 5% (v / v) fetal bovine serum, 1% (v / v) endothelial cell growth supplement, and 1% (v / v) penicillin / streptomycin. The medium was changed every 2–3 days and cultured at 5% carbon dioxide and 37°C. When the cells reached 90% confluence, they were washed with Dulbecco's phosphate-buffered saline (DPBS) and treated with 0.25% trypsin-EDTA (Gibco, USA) solution at 5% carbon dioxide and 37°C for 4 minutes. The cells were then detached and used.

[0069] (7) rt-PCR Using a 12-well plate, place 1.5 x 10 HBMEC cells per well in 1.5 ml of medium. 4The cells were cultured at a density of 1000 μg / ml. In an experimental group, a SCAI patch was placed on a transwell inserted into the well to examine the effect of growth factors released by the patch on HBMEC growth. On days 3, 7, and 14 of culture, mRNA from the cells in the wells was extracted using RNAiso Plus (Takara Bio, Japan). The extracted RNA concentration and purity were measured using a Nanodrop Lite spectrometer (Thermo Fisher Scientific, USA). The extracted mRNA was then reverse-transcribed using the SuperScript IV First-Strand Synthesis System (Invitrogen Life Technologies, USA). Real-time PCR was performed using a StepOne Plus Real-Time Cycler (Applied Biosystems, USA) with SYBR-green I reagent (Takara, Japan). Data were analyzed using StepOne software v2.3, and the expression levels of each gene normalized to GAPDH expression were calculated as 2. -ΔΔCT The primer sequences used for real-time PCR were designed based on gene sequences published in NCBI and PubMed, and are summarized in Table 2 below.

[0070] [Table 2] TIFF0007778423000003.tif114157

[0071] (8) Evaluation of the effect of SCAI patches on cell experiments To evaluate in vitro cytotoxicity, a released SCAI patch was placed on a transwell inserted into each well, and 2 × 10 HBMECs were placed on the bottom of each well. 4Cells were added at a density of 1000 cells / ml. The cells were then cultured for 7 days at 37°C under 5% carbon dioxide conditions and then stained using a live / dead cell viability assay kit (LIVE / DEAD Cell Viability Assay, Thermo Fisher Scientific, USA). Live and dead cells were labeled with green and red fluorescent dyes, respectively, and observed under a fluorescence microscope (Nikon Instruments, Japan). Cell proliferation was quantified using a Cell Counting Kit-8 Assay Kit (Dojindo Molecular Technologies, Japan). After culturing the cells for the specified time in each of the patched and patchless control groups, they were incubated with a CCK-8 solution diluted 1:10 in fresh medium for 3 hours at 37°C. The treated solution was then transferred to a clear 96-well plate, and the absorbance at 450 nm was measured using a Multiskan™ GO Microplate Spectrophotometer (Thermo Fisher Scientific, USA).

[0072] (9) Evaluation of the effects of SCAI patches on animal experiments All animal studies were conducted in accordance with national guidelines after approval by the local Institutional Animal Care and Use Committee (POSTECH-2021-0022). Eight-week-old male Sprague-Dawley (SD) mice weighing 200–300 g were used for in vivo experiments. Mice were initially anesthetized using an induction chamber containing 4.0% isoflurane, and then maintained at 1.5% isoflurane throughout surgery. The head was fixed to a stereotaxic frame with ear bars, and body temperature was maintained with a heating pad. After shaving the head, a hemispheric skin incision was made. Remaining epidermis and remnants were removed with saline solution. A craniotomy was then carefully performed using a dental drill. The exposed brain tissue was completely covered with a 6 mm x 11 mm, 150–180 μm thick PDMS (Polydimethylsiloxane) membrane, sterilized, and then immersed in saline solution. The border of the PDMS membrane was attached to the skull using cyanoacrylate adhesive (Loctite, USA). Dental resin (OA2; Denkist Inc., Korea) was then applied along the edges of the PDMS membrane and exposed to UV light for 15 seconds to permanently bond it to the skull. Finally, the mice were given Baytril (5 mg / kg; Bayer, Germany) via subcutaneous injection and returned to their cages. Tylenol (0.5 mg / ml; Janssen, Korea) and Marboxyl (0.06 mg / ml; Vetoquinol, Korea) were then continuously provided in the drinking water to alleviate inflammation. Two weeks after surgery, brain tissue was harvested and analyzed using hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC). The functional angiogenic efficacy of the SCAI patch was evaluated based on the results.

[0073] (10) Photoacoustic monitoring of cerebral angiogenesis induced by SCAI patch To monitor cerebral angiogenesis, we used an optical resolution photoacoustic microscope (OR-PAM) system (OptchoM, Optcho, Korea). OR-PAM is a bioimaging tool for noninvasive exploration of volumetric microvessels. It generates ultrasound waves in blood vessels using a 532 nm nanopulse laser system (VPFL-G-10, Spectra-Physics, USA). The ultrasound waves generated by the system are measured by a customized ultrasound transducer with a center frequency of 20 MHz and a bandwidth of 60%. A wide field-of-view (FOV) image can be reconstructed by combining the segmented images. The segmented images were acquired using a galvanometer scanner (GVS001; Thorlabs, USA) and two linear motorized stages (L-509.10SD00; Physik Instrumente, Germany). The galvanometer scanner adjusts the laser pulses with a scanning field length of 1.6 mm (400 pixels) and a scanning rate of 50 Hz. A linear motorized stage with a maximum scanning range of 26 mm (step size of 5 μm) moves the scanning part of the OR-PAM system perpendicular to the scanning direction of the galvanometer scanner. The scanning range of the stage is adjusted depending on the size of the ROI. Another linear stage moves the scanning part in the same scanning direction as the galvanometer scanner to provide segmented images for different regions. The acquired segmented images were reconstructed with a wide-angle FOV image using a structural similarity (SSIM)-based volumetric image registration algorithm with pyramid blending. To calculate the density of newly generated blood vessels with a diameter of less than 50 μm, a mask was first created to remove the area outside the ROI, and then blood vessels with a diameter greater than approximately 150 μm were removed from the PA maximum amplitude projection (MAP) image of the mouse brain. The area of ​​the ROI was calculated as the area of ​​the mask. A Hessian-based vascular filter was then applied to extract blood vessels with a diameter greater than 50 μm from the filtered image, and a large vascular map was generated based on the extracted blood vessels. Finally, a map of small vessels less than 50 μm in diameter was reconstructed from the vascular filtering image, excluding the map of large vessels, and the area of ​​the small vessels was calculated based on the map of small vessels.

[0074] (11)Histological analysis The brains were removed, tissue samples were fixed in 4% paraformaldehyde solution, and the surgical site where the patch was attached was dissected. The tissue samples were then fixed in paraffin. For H&E staining experiments, the paraffin-containing tissue samples were sectioned transversely at a thickness of less than 5 μm. Images of the stained tissue were captured using a microscope system (Leica DM750; Leica).

[0075] (12) Immunohistochemistry Tissue samples fixed in 4% paraformaldehyde solution were hardened in paraffin and sectioned at 4 μm thickness for immunofluorescence staining. Subsequently, deparaffinization and rehydration steps were performed using buffer. The sections were then incubated in 10% hydrogen peroxide solution at room temperature for 10 minutes and then stained with a primary antibody against CD31 (1:1000; Abcam, UK). The stained sections were further incubated with a peroxidase-conjugated secondary antibody (Envision+Rabbit; DAKO, USA) for 30 minutes, followed by counterstaining with Mayer's hematoxylin. The resulting slides were imaged using a microscope system (Leica DM750; Leica).

[0076] (13) Statistical analysis All statistical analyses were performed using the GraphPad Prism 8.2.1 program (GraphPad Software, USA). The significance of differences between data was assessed using two-way ANOVA or t-tests. Statistical significance is expressed as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0077] 2. Experimental Results Figure 1 shows the schematic process for fabricating hybrid ink (HAVEM ink) from vascular tissue-derived decellularized extracellular matrix (VdECM) and methacrylated hyaluronic acid (HAMA) using dual crosslinking mechanisms, including chemical and thermal crosslinking. Figure 2 shows the schematic process for fabricating spatiotemporal compartmentalized cerebral angiogenesis-inducing (SCAI) patches by adding VEGF and HGF, which are cerebrovascular growth factors, to hybrid inks with different crosslinking densities and using a 3D printing process. The schematic process also shows the sequential release of VEGF and HGF from the SCAI patch.

[0078] As shown in Figures 1 and 2, the present inventors fabricated an elaborate SCAI hydrogel patch that compartmentalizes and sequentially releases two angiogenic growth factors using an extracellular matrix (ECM)-based hybrid ink and 3D printing technology. The hybrid ink was formed by combining vascular tissue-derived decellularized extracellular matrix (VdECM) with methacrylated hyaluronic acid (HAMA) using a chemical crosslinking method known as the aza-Michael addition reaction. The aza-Michael addition reaction is an ideal mechanism for fabricating SCAI patches because it does not require an exogenous catalyst and allows for the easy adjustment of angiogenic factor release patterns and printability by simply changing the chemical bond density. The present inventors spatially separated different growth factors inside and outside the SCAI patch to ensure sequential release of growth factors after patch implantation. To load the different growth factors, they used two types of hybrid inks (HAVEM inks) with different chemical crosslinking densities. The two types of hybrid inks (HAVEM inks) loaded with different growth factors were 3D printed to fabricate SCAI patches, which were then vitrified to create soft, flexible, and easy-to-manipulate patches for brain implantation. The therapeutic effect of the fabricated patch, which promotes angiogenesis, was demonstrated in vitro and in vivo. In particular, the present inventors used a label-free photoacoustic microscope to observe the angiogenesis pattern of cerebral microvessels over time 14 days after patch implantation. Quantitative results showed that the SCAI patch exhibited significant angiogenesis in the brain region.

[0079] (1) Physicochemical properties of hybrid ink (HAVEM ink) To load and release angiogenic factors at the desired amount and speed in a patch, we fabricated a new biomaterial-based dual-crosslinked hybrid ink from HAMA and VdECM, which we named "HAVEM ink." Figure 3 shows a schematic diagram of the process for fabricating HAVEM ink from vascular tissue-derived decellularized extracellular matrix (VdECM) and methacrylated hyaluronic acid (HAMA) through an aza-Michael addition reaction. First, during the chemical crosslinking process of HAVEM ink, the methacrylate groups of HAMA bond with the amine groups of VdECM through an aza-Michael addition reaction. The chemical crosslinking density could be controlled by adjusting the ratio of VdECM to HAMA.

[0080] The chemical bonds formed through the aza-Michael addition reaction were confirmed using NMR spectroscopy. Figure 4 shows the nuclear magnetic resonance (NMR) spectra of VdECM ink (4V0H), HAMA, and HAVEM ink (4V0.5H) to demonstrate the crosslinking between VdECM and methacrylated hyaluronic acid (HAMA). The characteristic NMR peaks associated with the methacrylate functional groups are highlighted at 1.9, 5.7, and 6.1 ppm.

[0081] The NMR spectrum of HAMA showed peaks at 1.9, 5.7, and 6.1 ppm, which are characteristic NMR peaks of the methacrylate groups contained in HAMA. However, these peaks were not observed at the same positions in the spectrum of the ink containing only VdECM (4V0H), indicating the absence of methacrylate groups. Furthermore, the NMR characteristic peaks corresponding to methacrylate groups could not be observed in the HAVEM ink groups (4V0.5H and 4V1H). This means that all of the methacrylate groups in HAMA successfully reacted with the amine groups in VdECM to form chemical crosslinks.

[0082] Next, we investigated the change in printability of HAVEM ink depending on the HAMA content. The top photograph in Figure 5 shows the sol state images of VdECM ink (4V0H), HAVEM ink (4V0.5H), and HAVEM ink (4V1H) with different chemical crosslink densities. The bottom graph in Figure 5 shows the viscosity of VdECM ink (4V0H), HAVEM ink (4V0.5H), and HAVEM ink (4V1H) measured at various shear rates and printing conditions of 4°C. As can be seen from Figure 5, the HAVEM inks (4V0.5H and 4V1H) with different chemical crosslink densities all had slightly higher viscosities than the ink containing only VdECM (4V0H), and exhibited shear thinning properties in their rheology. More specifically, as the chemical crosslink density increased, the ink's fluidity decreased and its viscosity increased. However, the viscosity of each HAVEM ink was barely affected by time.

[0083] To confirm the effect of controlled crosslink density on the mechanical properties of HAVEM ink, we monitored the change in complex modulus of the prepared ink by raising the temperature of the HAVEM ink from 4°C to 37°C. Figure 6 shows the change in the elastic modulus of the VdECM ink (4V0H), HAVEM ink (4V0.5H), and HAVEM ink (4V1H) as the temperature increased from 4°C to 37°C. The change in elastic modulus in Figure 6 allows us to understand the gelation kinetics of the ink. As can be seen from Figure 6, the HAVEM ink (4V1H) containing a higher HAMA concentration showed a tendency for the complex modulus to increase more significantly as thermal crosslinking progressed compared to the HAVEM ink (4V0.5H) containing a lower HAMA concentration and the VdECM ink (4VOH) containing VdECM alone. The time required to reach equilibrium was observed to be relatively longer for the HAVEM ink (4V1H), which has a higher chemical crosslink density than the other inks (4V0.5H, 4V0H). This is presumably because the increased proportion of HAMA in the ink leads to more chemical crosslinks, resulting in a larger complex modulus. At equilibrium, the complex modulus of the HAVEM ink (4V1H) was observed to be 1.91 and 3,045 times larger than those of the HAVEM ink (4V0.5H) and VdECM ink (4VOH).

[0084] Next, we examined the complex modulus of the ink in its gel state after chemical and thermal crosslinking. HAVEM (4V1H) ink exhibited a higher complex modulus than VdECM (4V0H) and HAVEM (4V0.5H) inks. Figure 7 shows the storage and loss moduli of VdECM ink (4V0H), HAVEM ink (4V0.5H), and HAVEM ink (4V1H) at 37°C. As can be seen from Figure 7, the hybrid ink exhibited higher structural fidelity with a higher crosslinking rate during the printing process. The chemical crosslink density of HAVEM ink could be altered to induce appropriate mechanical properties, which had a direct impact on regulating the release profile of angiogenic factors. Figure 8 shows the release profile of HGF encapsulated in HAVEM ink (4V0.5H) and HAVEM ink (4V1H) over a 7-day period. As can be seen from FIG. 8, it was confirmed that the HGF release from HGF-containing HAVEM ink (4V1H) was much slower than the HGF release from HGF-containing HAVEM ink (4V0.5H).

[0085] (2) Design and fabrication of SCAI patches using 3D printing technology Two types of HAVEM ink were used to fabricate the SCAI patch: HAVEM ink (4V0.5H) and HAVEM ink (4V1H). Figure 9 shows a schematic diagram of the process used to fabricate the SCAI patch using 3D printing technology. As shown in Figure 9, the patch structure was designed and 3D printed in a circular shape with three layers: an outer layer and an inner layer. The outer layer was loaded with VEGF (an angiogenesis factor involved in the early stage of angiogenesis) for early release, while the inner layer was loaded with HGF (an angiogenesis factor involved in the later stage of angiogenesis) for delayed release. Figure 10 shows the results of measuring the angiogenic effects of various angiogenic factors using a tube formation assay. Figure 10A shows a schematic diagram of the tube formation assay, and Figure 10B shows the total tube length measured after 48 hours for the untreated control, VEGF, HGF, and VEGF / HGF combination-treated samples. As shown in Figure 10, the combination of growth factors effectively induced rapid and extended tube formation in human brain microvascular endothelial cells (HBMECs), resulting in the highest total tube length after 48 hours compared to the group treated with only one growth factor. Based on these results, we fabricated 8 mm diameter circular SCAI patches using HAVEM ink containing VEGF (4V0.5H; Ink A) and HAVEM ink containing HGF (4V1H; Ink B). First, Ink A was printed along an 8 mm diameter spiral to form the first layer. To physically block HGF from the outside, Ink B was printed along a 6 mm diameter spiral to form the second inner layer at the inner position of the first layer. Subsequently, Ink A was printed under the same printing conditions to form the second outer and third layers. After printing each layer, a 20 second pause was performed, and the Z axis was raised 200 μm before printing the next layer.As a result, HGF was not only spatially compartmentalized during the printing process, but also temporally compartmentalized using HGF-containing HAVEM ink (4V1H), thereby preventing its release from the patch during the early stages of angiogenesis. In this study, the inner core layer was printed with a high-crosslink density HAVEM ink (4V1H; Ink B) containing HGF, and the outer layer completely surrounding the core layer was printed with a low-crosslink density HAVEM ink (4V0.5H; Ink A) containing VEGF. Because the first, second, and third layers of the patch were all made of HAVEM ink (4V0.5H; Ink A) containing VEGF, VEGF was released immediately after the patch was implanted, amplifying its effects on the early angiogenesis process.

[0086] Figure 11 shows the cross-sectional structure of the SCAI patch fabricated in this study and images of each cross-section taken with a confocal microscope (scale bars: 200 μm). Figure 12 shows the design structures of SCAI patches fabricated using various combinations of HAVEM inks, along with images of each patch during the printing, gelation, and vitrification steps. In Figure 12, the patch with design structure 1 (far left column) was fabricated by printing a circle using VdECM ink (4VOH). The patch with design structure 2 (middle column) was fabricated by printing the inner core layer with HAVEM ink (4V1H) and the outer layer surrounding the inner core layer with HAVEM ink (4V0.5H). The patch with design structure 3 (far right column) was fabricated by printing a lattice pattern using HAVEM ink (4V0.5H) (scale bars: 10 mm). As seen in Figures 11 and 12, the compartmentalized structure of the patch was well maintained even after the patch was printed and then subjected to a thermal crosslinking process. This demonstrates that HAVEM ink has excellent printability and can easily print desired structures even using a multi-material printing process. In fact, VdECM ink (4V0H) was less accurate than HAVEM ink (4V0.5H), making it difficult to print the desired SCAI patch shape. In contrast, HAVEM ink (4V0.5H) was confirmed to be capable of printing not only compartmentalized SCAI patch structures but also lattice-type hydrogel structures with a double-layer microporous structure. Therefore, the HAVEM ink developed in this study can be used to fabricate patches with a variety of structures.

[0087] The inner core layer was printed with HAVEM ink (4V1H), and the outer layer surrounding the inner core layer was printed with HAVEM ink (4V0.5H) to form an SCAI patch. This was then thermally crosslinked through a specified heat treatment and subsequently vitrified to facilitate implantation during surgery, handling, and storage. The tensile stress-strain characteristics were investigated to evaluate the mechanical properties of the vitrified SCAI patch. Figure 13 shows the tensile strength and swelling ratio of the vitrified SCAI patch fabricated in this study. In Figure 13, "Control" represents the SCAI patch without vitrification, and "SCAI patch" represents the SCAI patch with vitrification. As can be seen from Figure 13, the vitrified SCAI patch exhibited a tensile strength of 18.62 MPa, 12.46 times higher than the control sample without vitrification. Furthermore, when immersed in saline, the vitrified patch rapidly reabsorbed water and expanded up to 151%. The microstructure of the vitrified SCAI patch was imaged using scanning electron microscopy (SEM), revealing interconnected and porous structures that varied depending on the crosslink density. Figure 14 shows SEM images of the various vitrified patches fabricated in this study (scale bars: 1 μm). In Figure 14, "4VOH" indicates a vitrified patch made from VdECM ink (4VOH), "4V0.5H" indicates a vitrified patch made from HAVEM ink (4V0.5H), and "4V1H" indicates a vitrified patch made from HAVEM ink (4V1H). As can be seen from Figure 14, the density of the fiber morphology increases with increasing crosslink density. It was also confirmed that the density of the fiber morphology in the SEM images also increases with increasing crosslink density in each structure, from 4V0H to 4V0.5H to 4V1H.

[0088] (3) In vitro experiments to evaluate the sustained and sequential release profile and cellular behavior of SCAI patches After establishing the properties of the SCAI patch, the inventors of the present invention confirmed the release pattern of growth factors from the fabricated patch. To ensure the sequential sustained release of VEGF and HGF, they utilized two strategies. They adjusted the chemical crosslinking density of the HAVEM ink containing each growth factor and used 3D printing technology to fabricate a hydrogel patch that allowed the two angiogenic factors to be spatially and temporally compartmentalized.

[0089] Hydrogels with low chemical crosslinking density contain more water and therefore degrade faster than hydrogels with high chemical crosslinking density. Based on this principle, the higher the chemical crosslinking density of a hydrogel, the slower the release rate of growth factors encapsulated in the hydrogel. Because the SCAI patch of the present invention is a chemically crosslinked hydrophilic matrix, the degradation rate can be controlled by adjusting the density of chemical bonds, which in turn allows for the control of the release pattern of growth factors encapsulated in the hydrogel.

[0090] To verify this strategy for the sequential drug release of VEGF and HGF, two types of multilayered SCAI patches were fabricated, and the growth factor release profiles were confirmed for two experimental groups. Figure 15 shows a schematic diagram of the structure of the multilayered SCAI patch designed in this study for the sequential drug release of VEGF and HGF. As can be seen from Figure 15, the multilayered SCAI patch designed in this study consists of an inner core layer made of an HGF-containing hybrid ink (Ink B) and an outer layer surrounded by a VEGF-containing hybrid ink (Ink A). Figure 16 shows the cumulative release profiles of VEGF and HGF for each of the two multilayered SCAI patches designed in this study for the sequential drug release of VEGF and HGF over a one-month period. The left experimental group in Figure 16 shows the results of observing sustained and sequential drug release patterns for a multilayer SCAI patch with only spatially compartmentalized drug, consisting of an outer layer formed using HAVEM Ink (4V0.5H) containing VEGF at 0.05 μg / μL as Ink A and an inner core layer formed using HAVEM Ink (4V0.5H) containing HGF at 0.05 μg / μL as Ink B. The right experimental group in Figure 16 shows the results of observing sustained and sequential drug release patterns for a multilayer SCAI patch with spatiotemporal compartmentalization, consisting of an outer layer formed using HAVEM Ink (4V0.5H) containing VEGF at 0.05 μg / μL as Ink A and an inner core layer formed using HAVEM Ink (4V1H) containing HGF at 0.05 μg / μL as Ink B. As shown in Figure 16, VEGF loaded in the outer layer of the SCAI patch was released within one day (24 hours) at 50.07% ± 10.63% in both groups, with no significant difference between the two groups. On the other hand, HGF loaded in the inner core layer of the SCAI patch was released more slowly than VEGF in both groups because it was located in the inner layer, which was relatively protected from water erosion.Furthermore, as seen in the experimental group on the right, when the chemical crosslink density of the HAVEM ink was adjusted to create a difference between the external and internal conditions in terms of materials, sustained and sequential release patterns were more pronounced than when spatial separation alone was achieved. In the experimental group on the right, in which HGF was loaded into a different HAVEM ink (4V1H) than the outer layer by adjusting the chemical crosslink density of the outer and inner layers, it took 30 days to release approximately 50% of the initial HGF loading. In the experimental group on the left, in which HGF was loaded into the same HAVEM ink (4V0.5H) as the outer layer without adjusting the chemical crosslink density of the outer and inner layers, it took approximately 4 days to release a similar amount of HGF to that of the experimental group on the right.

[0091] To evaluate the therapeutic potential of the SCAI patch through angiogenesis promotion, its biocompatibility and in vivo function were verified through in vitro experiments. Figure 17 shows the results of an in vitro experiment in which human brain microvascular endothelial cells (HBMECs) were cultured with an SCAI patch composed of an outer layer formed using HAVEM Ink (4V0.5H) containing VEGF at a concentration of 0.05 μg / μL as Ink A and an inner core layer formed using HAVEM Ink (4V1H) containing HGF at a concentration of 0.05 μg / μL as Ink B. In the in vitro experiment shown in Figure 17, HBMEC cells were cultured with an SCAI patch placed on a transwell. As can be seen from Figure 17, when HBMECs were cultured with an SCAI patch for 7 days, no adverse effects were observed on cell viability and the proliferation rate of HBMECs was enhanced compared to the untreated group. Figure 18 shows the results of analyzing cell viability using a live / dead assay kit when SCAI patches were applied to human brain microvascular endothelial cells (HBMECs) and cultured. The patches consisted of an outer layer formed using HAVEM Ink (4V0.5H) containing VEGF at a concentration of 0.05 μg / μL as Ink A and an inner core layer formed using HAVEM Ink (4V1H) containing HGF at a concentration of 0.05 μg / μL as Ink B. The live / dead staining analysis results in Figure 18 cross-validated the cell proliferation results in Figure 17. Figure 19 shows the results of analyzing the expression levels of angiogenesis-related genes when SCAI patches composed of an outer layer formed using HAVEM Ink (4V0.5H) containing VEGF at a concentration of 0.05 μg / μL as Ink A and an inner core layer formed using HAVEM Ink (4V1H) containing HGF at a concentration of 0.05 μg / μL as Ink B were applied to human brain microvascular endothelial cells (HBMECs) and cultured. As can be seen from Figure 19, in the early stages of angiogenesis, the group that received the SCAI patch showed higher expression of angiogenesis-related genes than the untreated group, but the difference was not significant.Meanwhile, the expression of all angiogenesis-related gene markers, including FLT1, FLK1, ANGPT1, and PECAM, increased dramatically 14 days after application of the SCAI patch. These results indicate that the SCAI patch has a strong effect in promoting angiogenesis. VEGF and HGF released by the SCAI patch affect HBMEC cells, and the delayed release of HGF works synergistically with VEGF to effectively generate blood vessels.

[0092] (4) In vivo experiments to evaluate cerebrovascularization behavior after SCAI patch implantation To evaluate the in vivo therapeutic potential of the SCAI patch fabricated in this study, which consisted of an outer layer formed using HAVEM ink (4V0.5H) containing VEGF at 0.05 μg / μL as ink A and an inner core layer formed using HAVEM ink (4V1H) containing HGF at 0.05 μg / μL as ink B, we used the OR-PAM system to visualize the angiogenesis behavior of cerebral blood vessels induced by the SCAI patch over time. Figure 20 shows a schematic diagram of an in vivo experiment in which the SCAI patch fabricated in this study, containing spatiotemporally compartmentalized VEGF and HGF, was implanted into the cerebral cortex of SD mice and the implantation site was monitored using the OR-PAM system. The inventors anesthetized mice and performed craniotomy, removed the skull, and implanted the SCAI patch into the cerebral region. A thin PDMS membrane was then placed over the implantation site to allow penetration of laser and photoacoustic (PA) ultrasound for monitoring. Thereafter, photoacoustic imaging was performed on the area of ​​interest of the mouse for 14 days to monitor changes in blood circulation and cortical microvascular structure in the mouse brain.

[0093] Figure 21 shows photoacoustic (PA) B-mode images (scale bars: 200 μm) of a cross-section of the SCAI patch implanted site to track changes in the SCAI patch over time in an in vivo experiment to evaluate cerebrovascularization behavior after SCAI patch implantation. In Figure 21, the PA signal was weak inside the translucent SCAI patch due to low absorption of the laser beam, and the patch boundary was determined based on this and highlighted with a yellow dotted line. As can be seen in Figure 21, the patch thickness on day 0 after implantation was 140 μm, similar to the thickness of a vitrified SCAI patch. One day after implantation, the patch expanded with cerebrospinal fluid, increasing its thickness to 240 μm. Subsequently, the patch degraded, decreasing to approximately 70 μm by day 7, making it virtually indistinguishable in PA B-mode images. Furthermore, by day 14 after implantation, the patch had completely degraded, and the patch was no longer visible in PA B-mode images (result images not shown).

[0094] Figure 22 shows photoacoustic (PA) maximum amplitude projection (MAP) images of cerebral vascular structure changes in SD mice treated with and without SCAI patch in an in vivo experiment to evaluate cerebral angiogenesis after SCAI patch implantation. Figure 23 shows cross-sectional PA B-mode images of areas C1 and C2 indicated by dotted lines in the image of a mouse treated with an SCAI patch (scale bars: 400 μm). In the magnified image of Figure 23 (scale bars: 1 mm), large blood vessels are highlighted by elliptical curves consisting of dots. As can be seen from Figures 22 and 23, the implanted SCAI patch induced angiogenesis, and changes in cerebral microvasculature were observed over time. 14 days after implantation, numerous new microvasculature structures were observed growing at the SCAI patch implantation site, and many newly formed blood vessels with diameters of less than 50 μm were observed due to the synergistic effect of released VEGF and HGF. In contrast, no significant changes in microvasculature were observed in the untreated group. The original blood vessels partially covered by the newly generated blood vessels can be seen in the magnified image in Figure 22. The hidden original blood vessels are highlighted by dotted lines in the C1 and C2 regions in the MAP image in Figure 22, and can be clearly seen in Figure 23, which is a PA B-mode image of the C1 and C2 regions shown in Figure 22. In Figure 23, the contours of the original blood vessels and the newly generated blood vessels are displayed as an elliptical curve consisting of the points below and a curve consisting of the points above, respectively.

[0095] Quantitative analysis of the density of newly formed blood vessels was also performed. Figure 24 shows the results of quantitative analysis of blood vessel density in SD mice treated with and without SCAI patch treatment using optical resolution photoacoustic microscopy (OR-PAM) images from an in vivo experiment to evaluate cerebral angiogenesis behavior after SCAI patch implantation. Blood vessel density was determined as the ratio of the area of ​​blood vessels less than 50 μm in diameter to the implanted surface area. As can be seen from Figure 24, comparing the density obtained before and 14 days after SCAI patch implantation, the blood vessel density after patch implantation was 0.0609 ± 0.0052 mm on the 14th day after implantation. 2 / mm 2to 0.1999±0.0239mm 2 / mm 2 These results suggest that the SCAI patch fabricated in this study has therapeutic efficacy in promoting cerebral angiogenesis.

[0096] The inventors of the present invention also performed histological evaluation to verify the angiogenic efficacy of the SCAI patch, which showed results similar to those observed in the PAM images. Figure 25 shows the histological analysis results of the untreated and SCAI patch-implanted groups 14 days after SCAI patch implantation in an in vivo experiment conducted in this study to evaluate the behavior of cerebral angiogenesis after SCAI patch implantation. The results are shown in H&E staining and immunohistochemistry (IHC) images (CD31) (scale bars: 50 μm). The scale bars for the magnified images in Figure 25 are 20 μm. As can be seen from Figure 25, in the SCAI patch-implanted group, large blood vessels capable of supplying blood to the tissue surrounding the patch implantation site remained 14 days after patch implantation, and numerous capillaries of various diameters and lengths were also observed (indicated by black arrows). Some of the blood vessels appeared to extend from the original large vessels, providing clear evidence of angiogenesis (indicated by gray arrows). On the other hand, in the SCAI patch untreated group, no vascular features extending from the large vessels were observed. The inventors of the present invention also cross-validated the angiogenic effect of the implanted patch using immunohistochemistry (IHC) against mouse-specific vascular antibodies, and observed a similar trend to the H&E staining results (indicated by black arrows). Furthermore, the inventors of the present invention also performed a vascular angiogenesis test using a single unit area (1 mm 2 The degree of angiogenesis was quantified by counting the number of blood vessels per unit area. Figure 26 shows the results of a quantitative analysis of the number of blood vessels per unit area in the untreated and SCAI patch-implanted groups using immunohistochemistry (IHC) images 14 days after SCAI patch implantation in an in vivo experiment conducted in this study to evaluate the behavior of cerebral angiogenesis after SCAI patch implantation. As can be seen from Figure 26, the number of blood vessels per unit area in the SCAI patch-implanted group was 167 ± 13, while the number of blood vessels in the untreated group was 29 ± 5.2 Approximately 5.76 times more blood vessels were generated per unit area. Taken together, it can be concluded that the SCAI patch promotes angiogenesis and neovascularization, maintaining therapeutic efficacy for at least 14 days in vivo.

[0097] 3. Discussion and Conclusion The inventors of the present invention have fabricated a flexible patch-type drug delivery system that can release two angiogenic factors in the brain region during the angiogenesis process to promote cerebral angiogenesis. The main features of the fabricated SCAI patch are as follows:

[0098] (1) The HAVEM ink used to fabricate the patch can variably adjust the release profile by varying the bond density between the methacrylate functional group and the amine functional group.

[0099] (2) 3D printing technology can be used to spatially and temporally compartmentalize and sequentially release angiogenic growth factors.

[0100] Existing biofabrication strategies (e.g., mold casting, gas foaming, and freeze-drying) have been widely applied to produce biomaterial-based drug delivery systems. However, these methods inevitably involve numerous complex steps. To address this issue, the present inventors applied 3D printing technology to fabricate patches with spatially separated inner and outer layers in a single step, without multiple physical steps. This reduction in process steps reduces the overall fabrication time and helps maintain high biological activity of growth factors. Furthermore, traditional approaches often require additional processing under harsh environmental conditions (e.g., pH, temperature), which can also reduce the stability of protein-based drugs. However, the HAVEM ink developed by the present inventors does not form other chemically active substances, such as radicals, as intermediates during synthesis, and has mild crosslinking conditions without the need for a catalyst, which may also contribute to maintaining the biological activity of growth factors.

[0101] A patch containing spatiotemporally compartmentalized dual angiogenic factors has a significant effect on inducing cerebral angiogenesis. The present inventors investigated the efficacy of in vivo treatment using the OR-PAM system to monitor time-dependent changes in mouse cerebral cortical microvessels for 14 days. This imaging method does not require the use of labeling substances because it obtains vascular images by measuring PA waves generated by red blood cells present in the vasculature. The OR-PAM system can identify functional, integrated, newly formed blood vessels in a more effective and non-invasive manner than existing vascular imaging techniques.

[0102] The SCAI patch developed by the inventors of the present invention can serve as a reliable guide for further investigation into the possibility of neovascularization in cerebral ischemic areas. Furthermore, the sustained-release angiogenic growth factor-releasing patch system developed by the inventors of the present invention can also be applied to targeted treatment of intractable chronic cerebral ischemic diseases such as moyamoya disease. Moyamoya disease is a progressive obliterative arteriosclerosis characterized by occlusion of internal arteries and their branches. Treatment for moyamoya disease patients is primarily performed via surgical procedures (direct anastomosis and indirect anastomosis). However, in pediatric patients, the superficial temporal artery (STA) is too narrow for direct anastomosis to the middle cerebral artery. Therefore, indirect revascularization strategies, such as endovascular dural artery anastomosis (EDAS), are widely accepted as treatment strategies for pediatric moyamoya disease. The effectiveness of this surgical procedure is generally known to vary depending on the growth factor levels and angiogenic potential of the cerebrospinal fluid (CSF). The SCAI patch developed by the inventors of the present invention can show beneficial effects in promoting angiogenesis between the superficial temporal artery (STA) and the tunica medialis after indirect surgery simply by being placed near the site.

[0103] These results highlight the potential of spatiotemporally compartmentalized cerebral angiogenesis-inducing patches fabricated using HAVEM ink. HAVEM ink is a 3D printing ink with tunable crosslink density via aza-Michael addition reaction. In this study, we synthesized a decellularized extracellular matrix (dECM)-based hybrid ink with tunable crosslink density via aza-Michael addition reaction. Based on the improved printability of the hybrid ink, we fabricated complex patch-shaped structures using 3D printing technology. Hybrid inks with higher crosslink density have smaller pore sizes due to denser connections, effectively reducing the release rate of growth factors. Furthermore, 3D printing technology can be used to fabricate physically compartmentalized structures that can control the release of growth factors loaded within the hybrid ink. Therefore, the SCAI patch fabricated in this study effectively induces the sustained release of two angiogenic growth factors, inducing angiogenesis necessary for the treatment of ischemic disease. The SCAI patch-type drug delivery platform developed in this study overcomes the problems associated with conventional long-term drug delivery approaches. The patch, formed from HAVEM ink containing vascular tissue-derived decellularized extracellular matrix (VdECM) and methacrylated hyaluronic acid (HAMA), is a tissue-derived material with soft, flexible properties and versatile fabrication. The SCAI patch developed in this study can effectively induce angiogenesis when applied to patients with various acute or chronic cerebral ischemic diseases, making it a promising alternative therapeutic agent.Furthermore, if various other types of drugs are applied to the spatiotemporally compartmentalized patch or an additional layer capable of carrying additional drugs is formed, the spatiotemporally compartmentalized patch can effectively function as a drug delivery platform that must deliver drugs locally at a controlled dose, and can be used in a variety of applications such as the treatment of ischemic heart disease, diabetes, and the like, and coronavirus vaccines.

[0104] Although the present invention has been described through the above-mentioned embodiments, it is to be understood that the present invention is not limited thereto and various modifications can be made without departing from the scope and spirit of the present invention. Therefore, the scope of protection of the present invention should be interpreted as including all embodiments falling within the scope of the appended claims.

Claims

1. A hybrid ink comprising an extracellular matrix having an amine group and a modified hyaluronic acid having an ethylenically unsaturated bond functional group introduced therein, An extracellular matrix-based hybrid ink for 3D printing, characterized in that the extracellular matrix and the modified hyaluronic acid are in a crosslinked state due to chemical bonds formed between at least a portion of the amine groups present in the extracellular matrix and the ethylenically unsaturated bond functional groups present in the modified hyaluronic acid.

2. The extracellular matrix-based hybrid ink for 3D printing according to claim 1 , wherein the extracellular matrix is ​​a vascular tissue-derived decellularized extracellular matrix.

3. The extracellular matrix-based hybrid ink for 3D printing according to claim 1 , wherein the ethylenically unsaturated bond functional group is selected from a vinyl group, an acrylic group, or a methacrylic group.

4. 2. The extracellular matrix-based hybrid ink for 3D printing according to claim 1, wherein the modified hyaluronic acid is selected from methacrylated hyaluronic acid or acrylated hyaluronic acid.

5. The extracellular matrix-based hybrid ink for 3D printing according to claim 1, wherein the weight ratio of the extracellular matrix to the modified hyaluronic acid is 1:0.01 to 1:0.

8.

6. The extracellular matrix-based hybrid ink for 3D printing according to claim 1, characterized in that the concentration of the extracellular matrix in the hybrid ink is 1-10% (w / v) and the concentration of the denatured hyaluronic acid is 0.01-8% (w / v).

7. The extracellular matrix-based hybrid ink for 3D printing according to claim 1 , further comprising a growth factor.

8. The growth factors include vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), epithermal growth factor (EGF), insulin-like growth factor (IGF), erythropoietin (EPO), fibroblast growth factor (FGF), brain-derived neurotrophic factor (BDNF), and nerve growth factor (NERV). Growth factor (NGF), keratinocyte growth factor (KGF), interleukin (IL), colony-stimulating factor (CSF), angiopoietin (ANG), platelet-derived growth factor (PDGF), placental growth factor (PGF), transforming growth factor-α (TGF-α), and transforming growth factor-β (TGF-β).

8. The extracellular matrix-based hybrid ink for 3D printing according to claim 7, characterized in that it is composed of one or more proteins selected from the group consisting of transforming growth factor-β, transforming growth factor-β, transforming growth factor-β (TGF-β), matrix metalloproteinase (MMP), and bone morphogenetic protein (BMP).

9. The extracellular matrix-based hybrid ink for 3D printing according to claim 7, characterized in that the concentration of growth factors in the hybrid ink is 0.001 to 1 g / l.

10. providing an extracellular matrix solution having a pH of 6.5 to 7.5; and adding a modified hyaluronic acid having an ethylenically unsaturated bond functional group introduced therein to the extracellular matrix solution, mixing the mixture uniformly, and then carrying out an aza-Michael addition reaction to obtain an ink in which the extracellular matrix and the modified hyaluronic acid are crosslinked.

11. The method for producing an extracellular matrix-based hybrid ink for 3D printing according to claim 10, wherein the extracellular matrix is ​​a decellularized extracellular matrix derived from vascular tissue.

12. The method for producing an extracellular matrix-based hybrid ink for 3D printing according to claim 10, wherein the ethylenically unsaturated bond functional group is selected from a vinyl group, an acrylic group, or a methacrylic group.

13. 11. The method for producing an extracellular matrix-based hybrid ink for 3D printing according to claim 10, wherein the modified hyaluronic acid is selected from methacrylated hyaluronic acid or acrylated hyaluronic acid.

14. 11. The method for preparing an extracellular matrix-based hybrid ink for 3D printing according to claim 10, wherein chemical bonds are formed between at least a portion of the amine groups present in the extracellular matrix and the ethylenically unsaturated bond functional groups present in the modified hyaluronic acid by the aza-Michael addition reaction.

15. 11. The method for preparing an extracellular matrix-based hybrid ink for 3D printing according to claim 10, wherein the modified hyaluronic acid is added to the extracellular matrix solution in an amount of 1 to 80 parts by weight relative to 100 parts by weight of the extracellular matrix.

16. 11. The method for producing an extracellular matrix-based hybrid ink for 3D printing according to claim 10, wherein the concentration of the extracellular matrix in the extracellular matrix solution is 1 to 10% (w / v), and the denatured hyaluronic acid is added to the extracellular matrix solution to a concentration of 0.01 to 8% (w / v).

17. 11. The method for preparing an extracellular matrix-based hybrid ink for 3D printing according to claim 10, wherein the aza-Michael addition reaction is carried out at a temperature of 1 to 10° C. for 2 to 24 hours.

18. The method for preparing an extracellular matrix-based hybrid ink for 3D printing according to claim 10, further comprising the step of adding a growth factor to the ink and mixing the ink.

19. 20. The method for preparing an extracellular matrix-based hybrid ink for 3D printing according to claim 18, wherein the growth factor is added to the ink to a concentration of 0.001 to 1 g / L.

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