Extracellular matrix-based adhesive composition

Abstract

The present invention relates to an extracellular matrix-based adhesive composition and, particularly, provides a bioadhesive, a preparation method therefor, a use thereof, etc., in which the bioadhesive comprises an extracellular matrix-containing hydrogel, methacryloyl-substituted gelatin, and a gelatin curing agent, and has excellent bioadhesion and elasticity.

Description

Extracellular matrix-based adhesive composition

[0001] The present invention relates to an extracellular matrix-based adhesive composition, and more particularly, to a bioadhesive based on an extracellular matrix and having excellent bioadhesiveness and elasticity, a method for producing the same, and uses thereof.

[0002] This patent was developed with the support of the Technology Development Project (S3318933) supported by the Ministry of SMEs and Startups (MSS, Republic of Korea).

[0003] Bioadhesives are a general term for adhesive materials used for bonding between living tissues such as skin, blood vessels, intestines, and bones, or for bonding artificial materials to living tissues. They primarily use substances that have adhesive properties for various biological samples such as cells and proteins. Bioadhesives can be applied in a variety of clinical fields, including tissue adhesives, hemostatic agents, tissue engineering scaffolds, drug delivery hydrogels, tissue fillers, and wound healing. Bioadhesives must have strong adhesive and cross-linking capabilities, maintain their function within the body for a long time, and, in addition, be able to bond instantly within the body in the presence of tissue fluids without generating heat or harmful substances or causing rejection reactions.

[0004] Currently commercialized or practical bioadhesives include cyanoacrylate instant adhesives, fibrin glues, gelatin glues, and polyurethane adhesives. However, synthetic polymer-based bioadhesives exhibit very weak adhesion in aqueous solutions within the body. Cyanoacrylate, gelatin, and polyurethane bioadhesives have been reported to be toxic in vivo, and their potential for adverse effects, including immune responses, is a significant limitation. Furthermore, fibrin-based bioadhesives currently used on patients, while exhibiting minimal side effects, have very low adhesive strength, limiting their utility. Furthermore, most bioadhesives currently commercialized or practical have low bioactivity, hindering self-healing of lesions and inhibiting tissue regeneration. To overcome these challenges, the development of an ideal bioadhesive that possesses strong adhesive and cross-linking capabilities, exhibits minimal side effects in vivo, and can aid tissue regeneration is essential.

[0005] Meanwhile, the cornea, a transparent, non-vascularized tissue on the anterior surface of the eye, serves as the outermost surface of the eye and protects it from the outside world. As the first layer of the eye through which light passes, it plays a crucial role in the refraction and transmission of light. However, because the cornea is constantly exposed to the external environment, it is easily injured and prone to various diseases. According to the World Health Organization, approximately 285 million people suffer from visual impairment, mostly due to corneal disease. Various corneal diseases and their associated complications exist, including dry eye, Sjogren's syndrome, and bacterial keratitis. Chronic corneal diseases cause corneal opacity, necessitating corneal transplantation, and more than 1.5 million new cases of corneal blindness are reported each year. Due to the shortage of donor tissue and the high cost of surgery, less than 5% of these patients receive transplantation. Corneal damage and infection can lead to corneal scarring and thinning of the corneal stroma, which can lead to vision loss.

[0006] Corneal transplantation is essential for corneal damage caused by severe corneal disease or trauma. However, worldwide, the number of corneal donors is significantly lower than the number of patients on the waiting list, resulting in an average waiting period of approximately eight years. While various approaches have been employed, including conservative treatments using tissue adhesives such as cyanoacrylate glue and fibrin glue, and amniotic membrane transplantation for wound protection and inflammation reduction, there are currently no treatments worldwide that promote healing and regeneration of the corneal epithelium or stroma in cases of fatal corneal damage. To overcome these limitations, there is a pressing need for the development of biomaterials for tissue repair that are highly transparent, biocompatible, easily engraft, and capable of regeneration and repair. In particular, biocomposites with high adhesiveness that adhere well to tissues even in humid environments such as the ocular surface are emerging as a new technology, as they can enhance the convenience of procedures in medical settings.

[0007] In this regard, the inventors of the present invention have developed an extracellular matrix-based bioadhesive in the form of a composition comprising an extracellular matrix-containing hydrogel and a gelatin curing agent, through Korean Patent Publication No. 10-2023-0050638 (Title: Extracellular matrix-based bioadhesive), wherein the extracellular matrix-containing hydrogel is gelatinized. However, the bioadhesive described above also has the disadvantage of having rather low adhesive strength and insufficient elasticity to compressive stimulation.

[0008] Accordingly, there is a constant need for the development of a bioadhesive that has excellent adhesive strength and elasticity to compressive stimuli similar to that of biological corneal tissue, making it suitable for use in corneal tissue repair.

[0009] [Prior Art Literature]

[0010] [Patent Document]

[0011] Republic of Korea Patent Publication No. 10-2023-0050638 (April 17, 2023)

[0012] The present invention is intended to solve the above-mentioned problems, and its purpose is to provide an adhesive composition having excellent viscosity and mechanical strength.

[0013] In addition, the present invention seeks to provide an adhesive composition having excellent bioadhesiveness as well as elasticity against compressive stimulation similar to that of biological corneal tissue.

[0014] In addition, the present invention provides a useful application as an adhesive biomaterial for healing and / or regenerating corneal defects and / or damages such as dry eye or corneal ulcers.

[0015] To achieve the above-described purpose, an extracellular matrix-based adhesive composition according to the present invention comprises an extracellular matrix-containing hydrogel, a gelatin curing agent, and methacryloyl-substituted gelatin.

[0016] Here, the methacryloyl-substituted gelatin can be included as GelMA (gelatin-methacryloyl) at a concentration of 5 to 15% (w / v).

[0017] In addition, the methacryloyl-substituted gelatin may be included as GelMA at a concentration of 5 to 10% (w / v).

[0018] Additionally, the methacryloyl-substituted gelatin can be included as GelMA at a concentration of 5% (w / v).

[0019] Additionally, the extracellular matrix-containing hydrogel may be a gelatinized decellularized extracellular matrix derived from the corneal stroma.

[0020] Additionally, the gelatin curing agent may be a photoinitiator comprising a combination of ruthenium and sulfuric acid.

[0021] In addition, the extracellular matrix-based adhesive composition according to the present invention can be used for corneal tissue repair.

[0022] Specific details of other embodiments are included in the detailed description and drawings.

[0023] The present invention is characterized by including a gelatin hardener and methacryloyl-substituted gelatin in an extracellular matrix-containing hydrogel, and thus has the effect of providing an adhesive composition having excellent viscosity and mechanical strength.

[0024] In addition, the present invention provides an adhesive composition that not only has excellent bioadhesiveness but also has elasticity to compression stimulus similar to that of living corneal tissue by including the methacryloyl-substituted gelatin in a specific ratio.

[0025] Since the present invention is suitable for use in corneal tissue repair, it can provide useful applications as an adhesive biomaterial for healing and / or regeneration of corneal defects and / or damages such as dry eye or corneal ulcers.

[0026] FIG. 1 is a schematic diagram showing a synthetic process of dityrosine activated by visible light according to an example of the present invention.

[0027] Figure 2 is a schematic diagram showing a process of synthesizing dopa from oxidized tyrosine according to an example of the present invention.

[0028] Figure 3 shows the results of protein amino acid analysis in a Co-dECM hydrogel according to an example of the present invention.

[0029] Figure 4 shows the results of measuring viscosity according to the GelMA addition ratio for an extracellular matrix-based adhesive composition according to one embodiment of the present invention.

[0030] Figure 5 shows the results of analyzing gelation kinetics according to the visible light irradiation time according to the addition ratio of GelMA in an extracellular matrix-based adhesive composition according to one embodiment of the present invention.

[0031] Figure 6 shows the results of analyzing the mechanical strength (storage modulus) of a gel in which photocrosslinking is induced according to the addition ratio of GelMA in an extracellular matrix-based adhesive composition according to one embodiment of the present invention.

[0032] Figure 7 shows the results of analyzing the adhesive strength (lap shear strength) according to the addition ratio of GelMA in an extracellular matrix-based adhesive composition according to one embodiment of the present invention.

[0033] Figure 8 shows the results of analyzing elasticity according to the addition ratio of GelMA in an extracellular matrix-based adhesive composition according to one embodiment of the present invention.

[0034] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0035] The terminology used in this application is only used to describe specific embodiments and is not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this application, it should be understood that the terms "comprise" or "have" indicate the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but do not exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0036] Terms such as first, second, etc. may be used to describe various components, but these components should not be limited by these terms. These terms are used solely to distinguish one component from another.

[0037] An extracellular matrix-based adhesive composition according to the present invention comprises an extracellular matrix-containing hydrogel, a gelatin curing agent, and methacryloyl-substituted gelatin.

[0038] The above-described extracellular matrix-containing hydrogel may be a hydrogel derived from or made based on an extracellular matrix. The term "extracellular matrix (ECM)" used in the present invention refers to the extracellular portion of animal tissue that typically provides structural support to animal cells and performs various other important functions. The extracellular matrix is ​​a characteristic that defines connective tissue in animals and is composed of various types of proteins, including collagen and glycosaminoglycans (GAG). This extracellular matrix may be the tissue of animals such as pigs or cows, and can be extracted from various organs.

[0039] In the present invention, the extracellular matrix is ​​preferably a decellularized extracellular matrix when considering bioapplication, etc. Decellularization is a technology for removing all xenogeneic cells that may induce an immune response in living tissue and separating the extracellular matrix. The present invention developed a biomaterial for adhesive tissue repair using decellularized extracellular matrix (dECM) as a main component. In particular, the decellularized extracellular matrix derived from corneal tissue contains a large number of proteins for maintaining the differentiation, activity, and homeostasis of corneal tissue cells, thereby promoting the regeneration of the corneal epithelium and stroma, and securing high transparency and mechanical properties, which are characteristics of the cornea. The decellularized extracellular matrix is ​​effective in minimizing the immune response during allograft or xenograft by removing cells that can act as antigens that induce an immune response. Since the type and number of cells and the physical properties of the tissue itself vary depending on the tissue, decellularization is performed using various chemicals such as acids, bases, storage solutions, hypertonic solutions, and detergents. In addition, the cellular matrix can be used while maintaining the structure of the tissue itself through only the decellularization process, but it can also be used by dissolving in an acidic solution after freeze-drying and pulverization, or by neutralizing it and making it into a hydrogel. In addition, considering the main application, it is more preferable that the cornea-derived decellularized extracellular matrix is ​​a cornea-derived decellularized extracellular matrix. The cornea-derived decellularized extracellular matrix is ​​derived from corneal stromal tissue, and in addition to the physical structure surrounding the cells, it contains proteins that help cell attachment or proteins that help cell growth and functional expression. The cornea-derived decellularized extracellular matrix preferably includes collagen fibers from which telopeptides have been removed.

[0040] In the present invention, the extracellular matrix-containing hydrogel may be gelatinized. The extracellular matrix-containing hydrogel may be gelatinized by heat denaturation of collagen, a component of the extracellular matrix. The term "gelatinized" used in the present invention refers to a state in which the extracellular matrix-containing hydrogel is denatured by heat and has rheological properties identical or similar to those of gelatin. Gelatin is a type of derivative protein obtained by treating collagen with hot water. It only swells in cold water, but dissolves in warm water, forming a sol and exhibiting flowability. Collagen, a major component of the extracellular matrix, denatures and dissolves when heated with water, and is eluted into a colloidal form to be converted to gelatin. Collagen, a major component of the extracellular matrix, exists in a coagulated state at the body temperature (approximately 37°C) at which the bioadhesive is applied. Therefore, it is difficult to evenly and easily apply a non-gelatinized extracellular matrix-containing hydrogel to the lesion site, resulting in inconvenience in use. In addition, non-gelatinized extracellular matrix-containing hydrogels do not harden easily even when a gelatin hardener is added, and the adhesive strength is greatly reduced when applied to a lesion.

[0041] In addition, the extracellular matrix-containing hydrogel may be a gelatinized decellularized extracellular matrix derived from corneal stroma. When the adhesive tissue repair biomaterial composition according to the present invention is based on the extracellular matrix derived from corneal tissue, it contains not only collagen constituting corneal tissue but also naturally derived ECMs constituting corneal tissue related to eye development, wound healing, tissue reconstruction, etc., and in particular, it also contains major ECMs related to maintaining corneal homeostasis, such as keratokan, lumican, and decorin, so that it can exhibit an excellent effect in regenerating damaged corneal tissue into clear and transparent original corneal tissue.

[0042] In addition, in the extracellular matrix-based adhesive composition according to the present invention, the content of the extracellular matrix-containing hydrogel is not particularly limited, and considering hydrogel forming ability, smooth thermal denaturation, uniform mixing with a gelatin curing agent, or ease of use or adhesive strength of the bioadhesive, it is possible to have 1 to 5% (w / v), preferably 2 to 4% (w / v), and more preferably 1.5 to 3% (w / v).

[0043] The adhesive composition according to the present invention, which includes such an extracellular matrix-containing hydrogel, not only has rheological properties identical to or similar to those of gelatin, but also has flowability at a temperature of 30°C or higher, can be evenly and easily applied to a damaged lesion area on the corneal surface, and can rapidly induce crosslinking using visible light.

[0044] The above gelatin curing agent is a substance that cures gelatin in the adhesive composition according to the present invention.

[0045] The term 'gelatin curing agent' used in the present invention refers to a substance or a combination of substances that crosslinks gelatin and converts it into a solid phase through simple addition, heat treatment, or light irradiation. Various inorganic compounds, such as salts containing polyvalent metal ions, or organic compounds, such as aldehydes and quinones, can be used as the gelatin curing agent. Generally, photoinitiators used as gelatin curing agents include lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), Irgacure 2959, etc. Specific examples of the gelatin curing agent in the present invention include a combination of ruthenium (Ru) and sodium persulfate (SPS), which induces curing of gelatin by irradiation with visible light (particularly, blue light); riboflavin, which induces curing of gelatin by irradiation with ultraviolet light; Or, there are combinations of EDC / NHS [(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride / Nhydroxysuccinimide], grape seed extract, dialdehyde starch, glutaraldehyde, etc., which induce hardening of gelatin by simple addition or predetermined heat treatment.

[0046] In an example of an extracellular matrix-based bioadhesive according to the present invention, the gelatin curing agent may be a photoinitiator comprising a combination of ruthenium and sulfuric acid, considering biosafety, ease of use, adhesiveness, etc. For example, it may be selected from a combination of ruthenium and sodium persulfate or riboflavin, and more preferably, it may be selected from a combination of ruthenium and sodium persulfate. It is known that the ruthenium and sodium persulfate oxidize aromatic residues including tyrosine when irradiated with visible light (particularly blue light), and the oxidized aromatic residues are converted into free radicals and form covalent bonds, for example, di-tyrosine covalent bonds, to induce a crosslinking reaction.

[0047] Figure 1 is a schematic diagram showing the synthesis process of dityrosine activated by visible light according to an example of the present invention. After ruthenium and sodium persulfate as photoinitiators are included in a bio-derived extracellular matrix hydrogel, when visible light is irradiated, Ru is synthesized in the presence of SPS as an electron acceptor. 2+ is Ru 3+ It is photodecomposed into Ru 3+ The presence of in turn oxidizes aromatic residues, including tyrosine, which is abundant in the extracellular matrix of living organisms, and the oxidized tyrosine group is further converted to a tyrosyl free radical and stabilized by forming a covalent dityrosine bond with nearby tyrosine moieties.

[0048] FIG. 2 is a schematic diagram showing a process for synthesizing DOPA from oxidized tyrosine according to an example of the present invention. The Ru / SPS cross-linking system can induce cross-linking very quickly due to the high absorbance of Ru in visible light and the chemical stability in the excited state. Since the bio-derived extracellular matrix is ​​rich in tyrosine transport proteins, the Ru / SPS system can promote the cross-linking of the hydrogel more quickly in a cell-friendly manner. In particular, tyrosine oxidized by Ru reacts with hydroxide ions (HO-) to form L-3,4-dihydroxyphenylalanine (L-Dopa), and since this DOPA has natural adhesive properties, it can increase the adhesive capacity of the bio-derived extracellular matrix hydrogel.

[0049] In addition, when the gelatin curing agent is selected from a combination of ruthenium and sodium persulfate, considering biosafety, adhesiveness, etc., the concentration of ruthenium in the bioadhesive is preferably 0.1 to 2 mM and the concentration of sodium persulfate is 1 to 20 mM, and more preferably the concentration of ruthenium is 0.2 to 1.5 mM and the concentration of sodium persulfate is 2 to 15 mM. The present inventors prepared an extracellular matrix-based adhesive composition by adding ruthenium and sodium persulfate, which are among visible light-activated photoinitiators, to a final concentration of 0.5-1 mM and 5-10 mM, respectively, at a ratio of 1:10, and stirring. Through this, crosslinking could be effectively induced through ionization using visible light and methacrylate polymerization of methacryloyl-substituted gelatin (GelMA).

[0050] The above methacryloyl-substituted gelatin may be methacrylated gelatin.

[0051] That is, the methacryloyl-substituted gelatin may be a methacryloyl-based gelatin, a type of hydrogel. For example, it may be a substance in which a methacrylate functional group is chemically introduced into gelatin to enable cross-linking.

[0052] The present inventors have conducted repeated studies to increase the adhesive strength of a bioadhesive comprising an extracellular matrix-containing hydrogel and a gelatin curing agent, and as confirmed in the examples and experimental examples described below, by further including methacryloyl-substituted gelatin, not only can excellent viscosity and mechanical strength be obtained, but also adhesive strength can be increased, thereby completing the present invention.

[0053] The present invention is characterized by including a gelatin hardener and methacryloyl-substituted gelatin in an extracellular matrix-containing hydrogel, and thus has the effect of providing an adhesive composition having excellent viscosity and mechanical strength.

[0054] In addition, as an example of the present invention, the methacryloyl-substituted gelatin can be included as GelMA (gelatin-methacryloyl) at a concentration of 5 to 15% (w / v), may be included at a concentration of 5 to 10% (w / v), and is preferably included at a concentration of 5% (w / v). As can be confirmed in the examples and experimental examples described below, when the methacryloyl-substituted gelatin is included in the above range, not only is it excellent in viscosity and mechanical strength (see FIGS. 4, 5, and 6), but also the adhesive strength and elasticity are the best (see FIGS. 7 and 8). That is, when the content of the methacryloyl-substituted gelatin is less than the above range, there is a disadvantage in that the adhesive strength, viscosity, mechanical strength, adhesive strength, and elasticity are all insufficient, and when it exceeds the above range, there is a disadvantage in that the adhesive strength is rather low and the elasticity is too large.

[0055] The present invention provides an adhesive composition having excellent bioadhesiveness and elasticity to compression stimulation similar to that of living corneal tissue by including the methacryloyl-substituted gelatin in a specific ratio.

[0056] As described above, the present invention has similar elasticity compared to living corneal tissue, and is therefore suitable for use in corneal tissue repair, and can provide useful applications as an adhesive biomaterial for healing and / or regeneration of corneal defects and / or damages such as dry eye or corneal ulcers.

[0057] Accordingly, the extracellular matrix-based adhesive composition according to the present invention can be used for corneal tissue repair. The composition of the present invention is a tissue repair material having adhesive properties, and especially, when composed of an extracellular matrix derived from the corneal stroma, it is clear and transparent and exhibits a light transmittance of 90% or more. In addition, since it exhibits adhesive properties, it exhibits high adhesive strength even on a moist ocular surface, has high elasticity against shear stimuli such as eye rubbing or blinking, and can be composed of an elastic biopolymer having elasticity similar to that of living tissue against a vertical pressing stimulus.

[0058] Furthermore, the adhesive composition according to the present invention can be used as a corneal repair biomaterial useful for healing and regenerating tissues in corneal damage and deficiencies caused by various causes, such as Sjogren's syndrome, neurotrophic keratitis, keratoconus, and corneal ulcers. This corneal tissue repair composition is biocompatible with corneal tissue, is biodegradable, and remains in the corneal tissue for more than 30 days after transplantation, and can be structurally replaced by regenerated autologous corneal tissue.

[0059] A method of using an extracellular matrix-based adhesive composition according to an embodiment of the present invention may include a step of applying the adhesive composition to a lesion site and applying heat or irradiating light. For example, when the extracellular matrix-based adhesive composition includes a combination of ruthenium and sodium persulfate as a gelatin curing agent, applying the adhesive composition to a lesion site and irradiating the composition with visible light, particularly blue light having a wavelength of 350 to 500 nm (preferably 400 to 450 nm), for 20 to 120 seconds (preferably 30 to 100 seconds) can form a bioadhesive film having excellent adhesive strength and elasticity.

[0060] In fact, when the adhesive composition according to the present invention is treated on the surface of the eye and irradiated with blue light for 30 seconds to 2 minutes, the adhesive strength (adhesive strength), which is the strength to withstand shear stress, can be secured up to 36 to 42 N.

[0061] In addition, when the adhesive composition according to the present invention is subjected to a load that acts substantially perpendicularly to the surface of the eye and is compressed to a depth of 100 μm, the compressive stress, which is a resistance force that occurs corresponding to the size of the load, was found to be approximately 9,500 to 11,000 μN, and thus, it was possible to impart properties similar to those of living corneal tissue.

[0062] The present invention may be better understood by the following examples, which are provided for illustrative purposes only and are not intended to limit the scope of protection defined by the appended claims.

[0063] Manufacturing example: Preparation of corneal-derived decellularized extracellular matrix (Co-dECM) hydrogel

[0064] A corneal-derived decellularized extracellular matrix (Co-dECM) hydrogel was prepared as follows. First, whole corneas excised from porcine eyes were washed with a PBS buffer solution containing 100 units / mL penicillin and 0.1 mg / mL streptomycin. Then, the epithelium and endothelium were removed from the corneal tissue, and a pure corneal stromal layer was obtained. The stromal tissue was then placed in a 20 mM ammonium hydroxide solution (NH4OH; 4.98 N aqueous solution) containing 0.5% Triton X-100 and stirred for approximately 4 hours. Thereafter, the stromal tissue was washed with distilled water and treated with a Tris-HCl (hypotonic Tris hydrochloride; pH 7.4) buffer solution for approximately 24 hours. Afterwards, the stromal tissue was placed in a 10 mM Tris-HCl solution containing 1% (v / v) Triton X-100 and stirred at 37°C for about 24 hr to obtain corneal-derived decellularized extracellular matrix (Co-dECM) tissue. Afterwards, the corneal-derived decellularized extracellular matrix (Co-dECM) tissue was sterilized by treating it with a 1% peracetic acid solution in 50% ethanol for about 10 hr. After completing the decellularization process, the corneal-derived decellularized extracellular matrix (Co-dECM) was freeze-dried overnight and ground into a fine powder using liquid nitrogen and a grinding device. 0.2 g Co-dECM powder was added to 10 ml of acetic acid solution (0.5 M) supplemented with 0.02 g pepsin and stirred for 3 days to obtain a homogeneous corneal-derived decellularized extracellular matrix hydrogel with a concentration of 2% (w / v). The above 2% (w / v) Co-dECM hydrogel was filtered through a 100 μm mesh and stored at 4°C for use in subsequent experiments.

[0065] Example: Preparation of an extracellular matrix-based adhesive composition

[0066] In the above manufacturing example, 2% (w / v) Co-dECM hydrogel was prepared by adding 10N sodium hydroxide solution and stirring to neutralize the pH to 7.0-7.4. Thereafter, the neutralized Co-dECM hydrogel was heated to 50-56°C and maintained for 20-40 minutes to undergo heat denaturation and obtain a gelatinized Co-dECM hydrogel. Thereafter, the gelatinized Co-dECM hydrogel was slowly cooled at room temperature, and when it reached approximately 37°C, GelMA was added at 0, 1, 3, 5, 10, 15, and 20% (w / v), respectively, and stirred to prepare a biomaterial composition. Here, ruthenium and sodium persulfate, which are among the visible light-activated photoinitiators, were added at a final concentration of 0.5 mM and 5 mM, respectively, at a ratio of 1:10, and stirred to prepare an extracellular matrix-based adhesive composition. The solvent used for both the ruthenium solution and the sodium persulfate solution was DPBS (Dulbecco's phosphate-buffered saline).

[0067] Experimental Example 1: Analysis of the amino acid composition of corneal-derived decellularized extracellular matrix (Co-dECM) hydrogels.

[0068] The protein amino acid composition of the cornea-derived decellularized extracellular matrix (Co-dECM) hydrogel prepared in the above manufacturing example was analyzed using a proteomics analysis method.

[0069] The results are as shown in Fig. 3. Fig. 3 shows the results of protein amino acid analysis in Co-dECM hydrogel according to an example of the present invention. As shown therein, the major amino acids constituting the proteins in Co-dECM hydrogel were glycine (Gly, 36%), alanine (Ala, 11.3%), proline (Pro 13.5%), and glutamic acid (Glu 9.4%), and it was confirmed that these values ​​are similar to the composition of collagen existing in a living body.

[0070] However, it was confirmed that tyrosine (Tyr, 0.39%), a target amino acid component for cross-linking and adhesive induction within ECM components, was contained in a relatively small amount.

[0071] Experimental Example 2: Viscosity Analysis of Extracellular Matrix-Based Adhesive Compositions

[0072] To analyze the rheological properties of the extracellular matrix-based adhesive composition manufactured in the above example, the viscosity was measured using a rheometer.

[0073] The results are as shown in Fig. 4. Fig. 4 shows the results of measuring the viscosity according to the GelMA addition ratio for the extracellular matrix-based adhesive composition according to one embodiment of the present invention. The experimental results showed shear thinning fluidic properties in which the viscosity decreased as the rotational shear rate increased under all experimental conditions, and it was confirmed that the higher the concentration of GelMA mixed into the gelatinized Co-dECM hydrogel, the higher the viscosity.

[0074] Experimental Example 3: Analysis of Gelation Kinetics and Mechanical Strength (Storage Modulus) of Extracellular Matrix-Based Adhesive Compositions

[0075] For the adhesive composition based on the extracellular matrix prepared in the above examples, the gelation dynamics were analyzed by irradiating it with blue light (400-500 nm) using a rheometer. When the adhesive composition according to the present invention is irradiated with blue light having a wavelength of about 400-500 nm, the tyrosine residues present within the gelatinized Co-dECM hydrogel are oxidized and converted into tyrosine free radicals, and form di-tyrosine covalent bonds with nearby tyrosine residues, thereby promoting curing.

[0076] Figure 5 shows the results of analyzing the gelation kinetics according to the visible light irradiation time according to the addition ratio of GelMA in an extracellular matrix-based adhesive composition according to one embodiment of the present invention. As shown here, the mechanical strength increased rapidly immediately after irradiation with blue light in all experimental groups, confirming that crosslinking was induced simultaneously with irradiation with blue light. Although blue light was continuously irradiated for 10 minutes thereafter, the increase in mechanical strength did not show as rapid a change as that observed at the moment of initial blue light irradiation.

[0077] Figure 6 shows the results of analyzing the mechanical strength (storage modulus) of the photocrosslinked gel according to the addition ratio of GelMA in the extracellular matrix-based adhesive composition according to one embodiment of the present invention. As shown therein, the mechanical strength of each experimental group composition in which gelation was induced upon blue light irradiation significantly increased as the mixing ratio of GelMA increased. That is, the 2% (w / v) Co-dECM hydrogel showed 4.3 ± 6.62 Pa, the 2% (w / v) Co-dECM hydrogel mixed with 5% (w / v) GelMA showed 512.7 ± 95.96 Pa, and the 10% (w / v) and 20% (w / v) GelMA mixed compositions showed values ​​of 1184.8 ± 36.49 Pa and 2976.8 ± 332.2 Pa, respectively.

[0078] Experimental Example 4: Analysis of the adhesive strength (lap shear strength) of an extracellular matrix-based adhesive composition.

[0079] For the extracellular matrix-based adhesive composition manufactured in the above example, the adhesive strength was analyzed by measuring and quantifying the shear stress strength using an Instron system.

[0080] Figure 7 shows the results of analyzing the adhesive strength (lap shear strength) according to the addition ratio of GelMA in an extracellular matrix-based adhesive composition according to one embodiment of the present invention. As shown therein, when GelMA was mixed into the gelatinized Co-dECM hydrogel according to the present invention at concentrations of 1, 3, 5, and 10% (w / v), it was confirmed that the shear stress strength significantly increased with increasing GelMA concentration. However, when GelMA was mixed at a concentration of 15% (w / v), the shear stress strength value actually decreased, and it was confirmed that it decreased further at a concentration of 20% (w / v).

[0081] According to this, it was found that when GelMA was mixed in an amount of 5% to 15% into Co-dECM hydrogel, it had remarkably excellent adhesive strength (36 ± 7 newton), and when GelMA was mixed in an amount of about 10%, it had the best adhesive strength, and it was found that mixing GelMA in an amount of about 5% could economically and efficiently produce an adhesive with excellent adhesive strength.

[0082] Experimental Example 5: Elasticity Analysis of Extracellular Matrix-Based Adhesive Compositions

[0083] For the extracellular matrix-based adhesive composition manufactured in the above example, elasticity in response to compression stimulation was measured.

[0084] That is, the extracellular matrix-based adhesive composition manufactured in the above example was irradiated with blue light (400-500 nm) for 2 minutes, and the elasticity of each gelled composition to compression stimulus was measured by comparison with the actual rat eye tissue through a compression test using CellScale's MicroTester G2 equipment.

[0085] Figure 8 shows the results of analyzing elasticity according to the addition ratio of GelMA in an extracellular matrix-based adhesive composition according to one embodiment of the present invention. As shown therein, when GelMA was mixed at concentrations of 5 and 10% (w / v) into the gelatinized Co-dECM hydrogel according to the present invention, it exhibited elasticity that was the same as or similar to that of actual ocular tissue when subjected to a compressive stimulus of 100 μm in depth. That is, it was confirmed that the composition mixed with 5 and 10% (w / v) GelMA had elasticity that could withstand the same degree of intraocular pressure as actual ocular tissue. In comparison, the composition mixed with GelMA at a concentration of 20% (w / v) exhibited elasticity that was 6 times higher than that of corneal tissue.

[0086] Accordingly, as a result of measuring the elasticity of the adhesive composition with various mixing ratios of GelMA, it was confirmed once again that when applying the adhesive composition of the present invention to the cornea, mixing 5-10% (w / v) of GelMA showed the strongest adhesive strength while having an elasticity similar to that of actual ocular tissue.

[0087] Although the present invention has been illustrated and described above with respect to specific preferred embodiments, it will be apparent to those skilled in the art that the present invention may be variously modified and changed without departing from the technical features or scope of the present invention as defined by the following claims.

Claims

1. An extracellular matrix-based adhesive composition comprising an extracellular matrix-containing hydrogel, a gelatin curing agent, and methacryloyl-substituted gelatin.

2. In paragraph 1, An extracellular matrix-based adhesive composition, characterized in that the above methacryloyl-substituted gelatin is contained as GelMA (gelatin-methacryloyl) at a concentration of 5 to 15% (w / v).

3. In paragraph 2, An extracellular matrix-based adhesive composition, characterized in that the above methacryloyl-substituted gelatin is included as GelMA at a concentration of 5 to 10% (w / v).

4. In paragraph 3, An extracellular matrix-based adhesive composition, characterized in that the above methacryloyl-substituted gelatin is included as GelMA at a concentration of 5% (w / v).

5. In paragraph 3, The above extracellular matrix-containing hydrogel is an extracellular matrix-based adhesive composition characterized in that the decellularized extracellular matrix derived from the corneal stroma is gelatinized.

6. In paragraph 5, An extracellular matrix-based adhesive composition, characterized in that the gelatin curing agent is a photoinitiator comprising a combination of ruthenium and sulfuric acid.

7. In any one of paragraphs 1 to 6, An extracellular matrix-based adhesive composition characterized in that it is used for corneal tissue repair.

Images