Method for preparing decellularized matrix with enhanced biocompatibility on basis of supercritical carbon dioxide process

Abstract

According to a method according to one aspect, in the process of enhancing the biocompatibility of a decellularized matrix through supercritical carbon dioxide treatment, the immunogenicity of the decellularized matrix can be reduced with excellent efficiency, and at the same time, the content and bioactivity of bioactive substances can be maintained. Therefore, the decellularized matrix prepared by the method according to one aspect, and a composition and a biomaterial, comprising same, have low biotoxicity and excellent tissue regeneration ability, and thus can be usefully used as cosmetic or medical materials.

Description

Method for manufacturing decellularized substrates with enhanced biocompatibility based on a supercritical carbon dioxide process

[0001] The present application relates to a method for manufacturing a decellularized substrate having improved biocompatibility and physiological activity.

[0002] The extracellular matrix (ECM) is a non-cellular structure of tissues or organs, composed of various polymers secreted by cells. The ECM can be used as a natural scaffolding material, structurally intact and suited to biological purposes. Its specific composition and function can vary depending on the specific tissue type from which it originates.

[0003] Decellularization is the process of chemically separating extracellular matrix components from living tissue. While decellularization removes immunogenic components, such as cellular components, from living tissue, it preserves bioactive substances that contribute to the tissue's environment and function, such as the extracellular matrix and certain growth factor proteins. Therefore, decellularized matrices (hereinafter referred to as "decellularized matrices") containing various preserved bioactive substances, such as collagen, glycosaminoglycans, and various cytokines, promote tissue regeneration and provide a more natural, biomimetic microenvironment for cell growth and differentiation. These decellularized matrices can be used alone or in combination with other substances for tissue regeneration and other applications.

[0004] Meanwhile, in the process of decellularization or the process of processing the decellularized extracellular matrix manufactured through the process to reduce molecules or remove immunogenicity, depending on the reaction conditions including solvents, decomposing enzymes, temperature conditions, pH conditions, etc., the physiologically active substances in the decellularized matrix may be removed or denatured, thereby reducing the physiological activity. In addition, methods using existing surfactants, etc. may cause toxicity when applied to a living body because the decellularized matrix contains residual surfactants. In addition, depending on the reaction conditions, the osmotic pressure of the composition including the decellularized matrix may increase, and as the osmotic pressure increases, the level of mixing with other substances may decrease, and the problem of reduced complexing efficiency with other cosmetic or medical materials or ingredients may occur.

[0005] In addition, the extracellular matrix or decellularized extracellular matrix contains collagen, and at both ends of the collagen molecule, there is a part consisting of 12 to 27 amino acids called telopeptide. Since this telopeptide is the main cause of immune response, a process to remove the telopeptide is necessary to apply the decellularized matrix in vivo. Meanwhile, the existing process removes this telopeptide sequence using an acidic solution, enzyme, or a combination thereof. However, this existing method has the disadvantage of not only increasing osmotic pressure due to chemical acid or base treatment, making it difficult to complex with other substances, but also causing harmful effects to the human body due to additional acid or alkaline solution treatment. In addition, this multi-step treatment process is complicated, and there is a problem that the efficacy is reduced when applied to the body, such as the removal of active ingredients or bioactive substances in the extracellular matrix during the treatment process.

[0006] Accordingly, in order to solve the above problems, the inventors of the present invention have developed a technology for manufacturing a decellularized substrate material that maintains the content and physiological activity of bioactive substances in the decellularized substrate while reducing or eliminating the content of telopeptides, thereby exhibiting a reduced immune response or not causing an immune response when applied in vivo.

[0007] One aspect provides a method for producing a decellularized substrate having enhanced biocompatibility, the method comprising the step of treating a solution containing the decellularized substrate with supercritical carbon dioxide, wherein the step of treating the supercritical carbon dioxide is performed at 60 to 150°C.

[0008] Another aspect is to provide a decellularized substrate manufactured by the above method, a composition comprising the same, and a biomaterial comprising the same.

[0009] Another aspect provides a method for enhancing the biocompatibility of a decellularized substrate, the method comprising the step of treating a solution containing the decellularized substrate with supercritical carbon dioxide, wherein the step of treating with supercritical carbon dioxide is performed at 60 to 150°C.

[0010]

[0011] Other purposes and advantages of this application will be further clarified by the detailed description below, taken in conjunction with the appended claims and drawings. Any details not described herein will be readily apparent and inferred by those skilled in the technical field of this application or similar technical fields, and therefore, their description will be omitted.

[0012] Throughout this specification, whenever a part is referred to as "comprising" a component, this does not exclude other components, unless otherwise specifically stated, but rather implies the inclusion of other components. Furthermore, unless the context clearly dictates a specific order, steps may be performed in a different order than stated. That is, steps may be performed in the same order as stated, may be performed substantially simultaneously, or may be performed in the opposite order.

[0013]

[0014] One aspect provides a method for producing a decellularized substrate having enhanced biocompatibility, the method comprising the step of treating a solution containing the decellularized substrate with supercritical carbon dioxide, wherein the step of treating the supercritical carbon dioxide is performed at 60 to 150°C.

[0015] As used herein, “biocompatibility” means that a substance introduced into a native cell, tissue, organ, or organism performs its desired function without inducing a significant inflammatory response, immunogenicity, or cytotoxicity in the cell, tissue, organ, or organism. For example, a biocompatible substance means a substance that performs its desired function when introduced into an organism without inducing a significant inflammatory response, immunogenicity, or cytotoxicity in the native cell, tissue, organ, or organism.

[0016] In one specific example, the enhancement of biocompatibility may mean that, when the step of treating the decellularized substrate with supercritical carbon dioxide according to one embodiment is performed, the decellularized substrate exhibits a reduced inflammatory response, immunogenicity, or cytotoxicity upon bioapplication compared to when the step of not performing the step. For example, the enhancement of biocompatibility may include a reduction in the telopeptide content within the decellularized substrate.

[0017] As used herein, the term “decellularized extracellular matrix (dECM)” can be used interchangeably with “decellularized tissue”, “decellularized extracellular matrix”, or “decellularized material”. The decellularized matrix refers to a tissue or organ of a human or an animal such as a pig or cow that has been decellularized to remove cellular components such as the nucleus, cell membrane, and nucleic acids except for the extracellular matrix (ECM). On the other hand, a “decellularized organ” refers to an organ that has been decellularized while maintaining the structure of the entire organ, such as the heart or kidney. Since the purpose is to re-transplant cells or use the organ while maintaining the physical structure of the entire organ, it is a concept clearly distinct from the “decellularized matrix” of the present specification in that it is impossible to formulate an aqueous formulation of the extracellular matrix.

[0018] The extracellular matrix refers to the complex aggregate of biopolymers that fills the intracellular or extracellular space of tissues. It consists of various molecules synthesized by cells, secreted, and accumulated extracellularly, including fibrous proteins, complex proteins such as proteoglycans, and cell-adhesive proteins such as fibronectin and laminin.

[0019] The method for manufacturing the decellularized substrate with enhanced biocompatibility may not include a step of treating with an acid or alkaline solution. According to one embodiment, the method for manufacturing the decellularized substrate with enhanced biocompatibility includes, unlike conventional methods of treating with an acid or alkaline solution, a supercritical carbon dioxide treatment process performed at a specific temperature, and as an effect resulting therefrom, does not include a subsequent step of treating with an acid or alkaline solution and a subsequent step of treating with a decomposing enzyme, thereby increasing the efficiency of the manufacturing process and eliminating the need for a separate purification process. Accordingly, the decellularized substrate manufactured by the method according to one embodiment maintains the content of a physiologically active substance and its physiological activity, has a low telopeptide content and low immunogenicity, and thus exhibits excellent biocompatibility.

[0020] In one specific example, the step of treating the supercritical carbon dioxide may be performed at a temperature of 60 to 150°C. The step of treating the supercritical carbon dioxide may be performed within a reactor, and the temperature within the reactor may be 60 to 150°C. For example, the step of treating the supercritical carbon dioxide may be performed at a temperature within the reactor of 60 to 150°C, 60 to 145°C, 60 to 140°C, 60 to 135°C, 60 to 130°C, 60 to 125°C, 60 to 120°C, 60 to 115°C, 60 to 110°C, 60 to 105°C, 60 to 100°C, 60 to 95°C, 60 to 90°C, 60 to 85°C, 60 to 80°C, 60 to 75°C, 60 to 70°C, 65 to 150°C, 65 to 145°C, 65 to 140°C, 65 to 135°C, 65 to 130°C, 65 to 125°C, 65 to 120℃, 65 to 115℃, 65 to 110℃, 65 to 105℃, 65 to 100℃, 65 to 95℃, 65 to 90℃, 65 to 85℃, 65 to 80℃, 65 to 75℃, 65 to 70℃, 70 to 150℃, 70 to 145℃, 70 to 140℃, 70 to 135℃, 70 to 130℃, 70 to 125℃, 70 to 120℃, 70 to 115℃, 70 to 110℃, 70 to 105℃, 70 to 100℃, 70 to 95℃, 70 to 90℃, 70 to 85℃, 70 to 80℃, It may be performed under conditions of 70 to 150°C, 70 to 145°C, 70 to 140°C, 70 to 135°C, 70 to 130°C, 70 to 125°C, 70 to 120°C, 70 to 115°C, 70 to 110°C, 70 to 105°C, 70 to 100°C, 70 to 95°C, 70 to 90°C, but is not limited thereto.If the temperature is outside the above range, there is a concern that the decellularized substrate may not be sufficiently depolymerized or hydrolyzed, and the telopeptide content may not be sufficiently reduced.

[0021] In one specific example, the step of treating the supercritical carbon dioxide is performed within a reactor, and may be performed by injecting carbon dioxide so that the pressure within the reactor becomes 70 to 400 bar. For example, the pressure inside the reactor is 80 to 400 bar, 90 to 400 bar, 100 to 400 bar, 110 to 400 bar, 120 to 400 bar, 130 to 400 bar, 140 to 400 bar, 150 to 400 bar, 160 to 400 bar, 170 to 400 bar, 180 to 400 bar, 190 to 400 bar, 150 to 390 bar, 150 to 380 bar, 150 to 370 bar, 150 to 360 bar, 150 to 350 bar, 160 to 350 bar, 170 to 350 bar, 180 to 350 bar, 190 to 350 bar, 200 to Carbon dioxide gas may be injected into the reactor so as to be 350 bar, 210 to 350 bar, 220 to 350 bar, 230 to 350 bar, 240 to 350 bar, 250 to 350 bar, 260 to 350 bar, 270 to 350 bar, 280 to 350 bar, 290 to 350 bar, or 300 to 350 bar, but is not limited thereto. If the pressure within the reactor is low beyond the above pressure range, there is a concern that the decellularized substrate may not be sufficiently degraded or hydrolyzed, or the telopeptide content may not be sufficiently reduced.

[0022] In one specific example, the step of treating with supercritical carbon dioxide may be performed for 1 to 48 hours, and the pressure within the reactor may be maintained for 1 to 48 hours. For example, the step of treating with supercritical carbon dioxide may be performed for 1 to 36 hours, 1 to 30 hours, 1 to 24 hours, 1 to 18 hours, 6 to 36 hours, 6 to 30 hours, 6 to 24 hours, 6 to 18 hours, 6 to 17 hours, 6 to 16 hours, or 6 to 15 hours, but is not limited thereto. If the step of treating with supercritical carbon dioxide is performed for a shorter time than the above time, there is a concern that the decellularized substrate may not be sufficiently degraded or hydrolyzed, and thus the telopeptide content may not be sufficiently reduced.

[0023] In one specific example, the solution containing the decellularized matrix may be a solution containing a decellularized matrix powder. The decellularized matrix powder may be a dried powder of an extracellular matrix that has undergone a decellularization process, which may be obtained by drying the decellularized extracellular matrix and then pulverizing it. For example, the decellularized extracellular matrix may be dried and then pulverized. The drying method for preparing the extracellular matrix in a dried state may use a method commonly used in the art and is not particularly limited. Non-limiting examples of the drying method include an air drying method, a natural drying method, a spray drying method, a freeze drying method, and a reduced pressure drying method. These methods may be used alone or in combination of at least two methods. In one specific example, the decellularized matrix dried powder may be a freeze-dried powder of a decellularized matrix. The step of adding the decellularized matrix powder and distilled water to the reactor and the step of injecting supercritical carbon dioxide may be performed simultaneously or sequentially.

[0024] As used herein, the term "solution" may refer to a state in which two substances are mixed, for example, a state in which a solute and a solvent are mixed. In one specific example, the solution containing the decellularized matrix powder may refer to a mixture, suspension, or aqueous solution in which the decellularized matrix powder is contained in distilled water. The term "solution" as used herein may be used interchangeably with mixture, suspension, hydrate, composition, melt, aqueous solution, and the like.

[0025] In one specific example, the solution comprising the decellularized substrate may comprise a decellularized substrate powder, and may comprise a powder of the decellularized substrate and distilled water. The solution comprising the decellularized substrate powder may comprise 0.1 to 5 wt% of the decellularized substrate powder. In one specific example, the powder of the decellularized substrate may be contained in distilled water at 0.01 to 5 wt%, for example, 0.05 to 5 wt%, 0.1 to 5 wt%, 0.2 to 5 wt%, 0.3 to 5 wt%, 0.4 to 5 wt%, 0.5 to 5 wt%, 0.6 to 5 wt%, 0.7 to 5 wt%, 0.8 to 5 wt%, 0.9 to 5 wt%, 1.0 to 5 wt%, 0.05 to 4 wt%, 0.1 to 4 wt%, 0.2 to 4 wt%, 0.3 to 4 wt%, 0.4 to 4 wt%, 0.5 to 4 wt%, 0.6 to 4 wt%, 0.7 to 4 wt%, 0.8 to 4 wt%, 0.9 to It may be, but is not limited to, 4 wt%, 1.0 to 4 wt%, 0.05 to 3 wt%, 0.1 to 3 wt%, 0.2 to 3 wt%, 0.3 to 3 wt%, 0.4 to 3 wt%, 0.5 to 3 wt%, 0.6 to 3 wt%, 0.7 to 3 wt%, 0.8 to 3 wt%, 0.9 to 3 wt%, or 1.0 to 3 wt%.

[0026] In one specific example, the decellularized substrate may have a reduced content of telopeptides within the decellularized substrate or may have telopeptides removed.

[0027] In the present specification, reduction in the content of telopeptide or removal of telopeptide means that the content of telopeptide contained in the decellularized substrate is reduced compared to before performing the step of treating with supercritical carbon dioxide according to one aspect. For example, by treating a solution containing the decellularized substrate with supercritical carbon dioxide under temperature conditions of 60 to 150°C, the content of telopeptide in the decellularized substrate may be reduced by 80% or more. For example, the content of telopeptide contained in the decellularized substrate may be reduced by 60% or more, 65% or more, 70% or more, 75% or more, or 80% or more by performing the step of treating with supercritical carbon dioxide according to one embodiment. The decellularized substrate may have improved biocompatibility by reducing the content of telopeptide in the decellularized substrate or removing telopeptide.

[0028] According to a method according to one embodiment, it was confirmed that the telopeptide content can be reduced with an efficiency similar to or higher than that of the existing method, compared to a method of treating an existing acidic solution, an alkaline solution, or an acidic solution and an enzyme.

[0029] In one specific example, the decellularized substrate may contain a high content of a bioactive substance or an effective ingredient. For example, the decellularized substrate may mean that, even after treatment with supercritical carbon dioxide at a temperature of 60 to 150°C, the content of the bioactive substance or effective ingredient in the decellularized substrate is maintained at 80% or more, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% or more, compared to before treatment. According to the method according to one embodiment, it was confirmed that the residual amount of the bioactive substance or effective ingredient in the decellularized substrate was significantly higher compared to the case of treating with a conventional acidic solution, alkaline solution, or acidic solution and enzyme. For example, it was confirmed that the residual amounts of collagen and elastin in the decellularized substrate were each 95% or more. Through this, it was confirmed that, according to a method according to one embodiment, it is possible to manufacture a decellularized substrate having excellent physiological activity containing a large amount of physiologically active substances.

[0030] Therefore, it can be seen that, according to a method according to one embodiment, not only can the immunogenicity of a decellularized substrate be reduced with high efficiency to enhance biocompatibility, but at the same time, a decellularized substrate having excellent physiological activity can be manufactured by maintaining a high content of an effective ingredient or a physiologically active substance in the decellularized substrate.

[0031] In one specific example, the substrate may be an extracellular matrix derived from any one selected from the group consisting of skin tissue, heart tissue, adipose tissue, corneal tissue, bone tissue, brain tissue, vascular tissue, or cartilage tissue of a human or an animal such as a pig or cow, but is not limited thereto.

[0032] Meanwhile, the above method can be applied without limitation to techniques known in the art related to methods for decellularizing extracellular matrix.

[0033] For example, as a method for decellularizing the extracellular matrix, the decellularized matrix may be decellularized by a decellularization method including a step of injecting supercritical carbon dioxide into a reactor containing living tissue and ethanol so that the pressure within the reactor becomes 70 to 400 bar, and the step of injecting supercritical carbon dioxide into the reactor containing living tissue and ethanol may be performed at 31 to 60°C.

[0034] The decellularization method is, for example, a method in which the pressure in the reactor is 80 to 400 bar, 90 to 400 bar, 100 to 400 bar, 110 to 400 bar, 120 to 400 bar, 130 to 400 bar, 140 to 400 bar, 150 to 400 bar, 160 to 400 bar, 170 to 400 bar, 180 to 400 bar, 190 to 400 bar, 150 to 390 bar, 150 to 380 bar, 150 to 370 bar, 150 to 360 bar, 150 to 350 bar, 160 to 350 bar, 170 to 350 bar, 180 to 350 bar, 190 to 350 bar, It may include, but is not limited to, a step of injecting carbon dioxide gas into the reactor so that the pressure becomes 200 to 350 bar, 210 to 350 bar, 220 to 350 bar, 230 to 350 bar, 240 to 350 bar, 250 to 350 bar, 260 to 350 bar, 270 to 350 bar, 280 to 350 bar, 290 to 350 bar, or 300 to 350 bar.

[0035] The decellularization method may include, but is not limited to, a step of treating the supercritical carbon dioxide under conditions in which the temperature within the reactor is, for example, 31 to 55°C, 31 to 50°C, 31 to 45°C, 31 to 40°C, 31 to 39°C, 31 to 38°C, 31 to 37°C, 32 to 40°C, 33 to 40°C, 34 to 40°C, 35 to 40°C, 32 to 39°C, 33 to 39°C, 34 to 39°C, 35 to 39°C, 32 to 38°C, 33 to 38°C, 34 to 38°C, 35 to 38°C, 32 to 37°C, 33 to 37°C, 34 to 37°C, or 35 to 37°C.

[0036] The decellularization method may be to treat the supercritical carbon dioxide for 2 to 24 hours, and for example, the pressure within the reactor may be maintained for 2 to 24 hours. For example, the pressure within the reactor may be maintained for 3 to 23 hours, 4 to 22 hours, 5 to 21 hours, 6 to 20 hours, 6 to 19 hours, 6 to 18 hours, 6 to 17 hours, 6 to 16 hours, 6 to 15 hours, 6 to 14 hours, 6 to 13 hours, or 6 to 12 hours, but is not limited thereto.

[0037] The above decellularization method may not include a step of treating with an additional acid, base, or enzyme. The above decellularization method may further include a pretreatment step prior to the step of contacting the biological tissue with ethanol in a reactor. Through the pretreatment step, contaminants, fat, etc. of the biological tissue may be removed. In addition, the above decellularization method may further include a step of treating a mixture that has gone through the step of injecting supercritical carbon dioxide into a reactor containing the biological tissue and ethanol with a DNA-degrading enzyme, which serves to degrade DNA remaining in the mixture and remove immunogenicity of the mixture. The above decellularization method may further include a washing step of treating the mixture that has gone through the step of treating with the DNA-degrading enzyme with distilled water to remove residual contaminants.

[0038]

[0039] Another aspect is to provide a decellularized substrate manufactured by the above method. In the decellularized substrate, the terms or elements mentioned above are identical to those already mentioned.

[0040]

[0041] Another aspect provides a composition comprising the above-mentioned decellularized substrate. In the composition, the same terms or elements as those already mentioned are as described above.

[0042] In one specific example, the composition may be a hydrogel composition comprising a decellularized matrix. For example, the hydrogel composition may be a hydrogel composition comprising a decellularized matrix and hyaluronic acid according to one embodiment, or a hydrogel composition comprising a decellularized matrix and chitosan according to one embodiment. The hydrogel composition may further comprise any material suitable for preparing a hydrogel composition.

[0043] The term "hydrogel" herein refers to a three-dimensional network structure formed by cross-linking hydrophilic polymers via covalent or non-covalent bonds. Due to the hydrophilicity of the constituent materials, hydrogels absorb large amounts of water and swell in aqueous solutions and aqueous environments, but do not dissolve due to their cross-linked structure. Therefore, hydrogels with various shapes and properties can be produced depending on the constituents and manufacturing method. They generally contain a large amount of water, and thus may possess properties intermediate between those of a liquid and a solid.

[0044] The term "chitosan" as used herein may refer to a linear polysaccharide composed of D-glucosamine and N-acetylglucosamine. The chitosan may include a chitosan derivative in addition to pure chitosan. For example, the chitosan derivative may include at least one of methylated chitosan, carboxylmethylated chitosan, phthalated chitosan, esterified chitosan, amidated chitosan, or formylated chitosan.

[0045] In one specific example, the composition may be a tissue repair composition, which may be a material used for tissue repair. For example, it may refer to a filler similar to soft tissue injected into wrinkled skin or an area requiring volume, an adhesion prevention material between a surgical site and normal tissue, a tissue adhesive material, a wound dressing material for artificial skin, etc. The hydrogel may be applied to body parts such as the glabella, forehead, under-eye circles, crow's feet, nasolabial folds, cheeks, wrinkles around the mouth, and chin. The hydrogel must be biocompatible as a material directly applied to the human body. For example, when the hydrogel is filled into an area requiring volume, it must have excellent gel shape retention / persistence to form volume for a long period of time after injection. When the hydrogel is used to prevent adhesion at a surgical site, it must have tissue compatibility with the wound site and have little or no cytotoxicity. When used as a wound dressing, excellent retention / persistence is required to maintain adhesion and application. Furthermore, depending on the conditions of the hydrogel treatment, the skin surface may become uneven or the treatment results may be undesirable. Therefore, the hydrogel formed in the body must be easily degradable or deformable.

[0046] The above composition may have enhanced biocompatibility and physiological activity by including a decellularized substrate according to one embodiment, and thus may be usefully used as a composition for tissue repair.

[0047]

[0048] Another aspect is to provide a biomaterial comprising the above-mentioned decellularized substrate. In the biomaterial, the terms or elements mentioned above are identical to those already mentioned.

[0049] In one specific example, the biomaterial may be a biomaterial comprising the hydrogel composition.

[0050] In one specific example, the biomaterial may be a biomaterial for tissue repair.

[0051] The above biomaterial may have enhanced biocompatibility and physiological activity by including a decellularized substrate according to one embodiment, and thus may be usefully used as a biomaterial for tissue repair.

[0052]

[0053] Another aspect provides a method for enhancing the biocompatibility of a decellularized substrate, the method comprising the step of treating a solution containing the decellularized substrate with supercritical carbon dioxide, wherein the step of treating the supercritical carbon dioxide is performed at a temperature of 60 to 150°C. In the method for enhancing the biocompatibility of the decellularized substrate, the terms or elements mentioned are the same as those already mentioned above.

[0054] According to the method according to the aspect, not only can the biocompatibility of the decellularized substrate be improved, but also the content and physiological activity of the physiologically active substance can be maintained.

[0055] Accordingly, the decellularized substrate manufactured by the method according to the aspect has low biotoxicity and can be applied in vivo due to high biocompatibility and physiological activity, and thus can be usefully used as a biomaterial for tissue repair.

[0056] Figure 1a shows the result of treating a decellularized substrate using a supercritical carbon dioxide process according to one embodiment.

[0057] Figure 1b shows the results of treating a decellularized substrate using an HCl solution according to one embodiment.

[0058] Figure 1c shows the result of treating a decellularized substrate using a NaOH solution according to one embodiment.

[0059] Figure 2a shows the result of treating a decellularized substrate using a supercritical carbon dioxide process according to one embodiment.

[0060] Figure 2b shows the results of treating a decellularized substrate using an HCl solution according to one embodiment.

[0061] Figure 2c shows the result of treating a decellularized substrate using a NaOH solution according to one embodiment.

[0062] Figure 3 shows the results of confirming and comparing the content of telopeptide in the decellularized substrate according to the processing method through absorbance measurement.

[0063] Figure 4 shows the results of confirming and comparing the content of residual active ingredients in the decellularized matrix according to the treatment method through the hydroxyproline assay and the elastin assay.

[0064] Figure 5 shows the results of confirming the degree of immune response after injecting a composition including a decellularized material according to one embodiment into the skin of a mouse.

[0065] The present invention will be described in more detail through the following examples. However, these examples are provided for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0066]

[0067] Manufacturing Example 1. Manufacturing of decellularized substrate using supercritical carbon dioxide process

[0068] Pig skin tissue was prepared, keratin was removed with a knife, and fat was removed by stirring with 70% ethanol at room temperature for 4 hours. After that, 20 g of the skin tissue and 100 ml of 100% ethanol were placed in a supercritical fluid equipment (Ilshin Autoclave, Korea) reactor, and the temperature was set to 35°C. After that, carbon dioxide was injected to adjust the pressure to 300 bar, and the reaction was performed for 6 hours under the above temperature and pressure conditions. After that, the tissue was washed with an ethanol solution for 2 hours, and then with PBS and distilled water for 4 hours. The washed tissue was freeze-dried and then powdered using a freeze-miller.

[0069]

[0070] Example 1. Preparation of a decellularized matrix material with enhanced biocompatibility based on a supercritical carbon dioxide process.

[0071] The dry powder of the decellularized substrate manufactured as in Manufacturing Example 1 was mixed with distilled water at 2.0 wt% and placed in a supercritical fluid equipment (Ilshin Autoclave, Korea) reactor, and the temperature was set to 60°C. Thereafter, carbon dioxide was injected so that the pressure inside the reactor became 300 bar, and the reaction was performed under these conditions for 12 hours. The results of visual inspection of the decellularized substrate material manufactured in this manner are shown in Figures 1a and 2a.

[0072]

[0073] Comparative Example 1. Preparation of decellularized matrix material using acid

[0074] The dry powder of the decellularized substrate manufactured as in Manufacturing Example 1 was mixed at 2.0 wt% in a 0.1 N HCl solution. Thereafter, the temperature of the solution was adjusted to 60°C and the reaction was performed for 1 hour, 4 hours, and 15 hours. The results of visual inspection of the decellularized substrate material manufactured in this manner are shown in Figures 1b and 2b.

[0075]

[0076] Comparative Example 2. Preparation of decellularized substrate material using base

[0077] The dry powder of the decellularized substrate manufactured as in Manufacturing Example 1 was mixed at 2.0 wt% in a 0.2 N NaOH solution. Thereafter, the temperature of the solution was adjusted to 60°C and reacted for 1 hour, 4 hours, and 15 hours. The results of visual inspection of the decellularized substrate material manufactured in this manner are shown in Figures 1c and 2c.

[0078]

[0079] Comparative Example 3. Preparation of decellularized substrate material using acid and enzyme

[0080] Pepsin was mixed in a 0.1N HCl solution at 10 wt% relative to the decellularized substrate, and then the dry powder of the decellularized substrate prepared as in Manufacturing Example 1 was mixed in at 2.0 wt%. Thereafter, the solution was reacted at 4°C for 24 hours, and then the solution was filtered through a 100 kDa filter.

[0081]

[0082] Experimental Example 1. Confirmation of low molecular weight and formulation of decellularized matrix material.

[0083] In order to confirm the low molecular weight and formulation of the decellularized substrate material according to the supercritical carbon dioxide process according to one embodiment, the degree of low molecular weight and formulation of the decellularized substrate material of Example 1, Comparative Example 1, and Comparative Example 2 over time was confirmed.

[0084] As shown in Fig. 2b, when the decellularized substrate was treated with an acidic solution, precipitation occurred. In contrast, when the supercritical carbon dioxide process or base according to one embodiment was used, it was confirmed that the decellularized substrate powder was uniformly dispersed and dissolved within the solution without precipitation (Figs. 2a and 2c). Through this, it was confirmed that by treating the decellularized substrate according to the method of one embodiment, low molecular weight decellularized substrate and stable formulation formation are possible without additional acid, base, or enzyme treatment.

[0085]

[0086] Experimental Example 2. Confirmation of the immunogenicity removal efficiency of the supercritical carbon dioxide process.

[0087] To confirm the immunogenicity removal efficiency of the decellularized substrate in the supercritical carbon dioxide process according to one embodiment, the degree of telopeptide removal was measured using the sandwich ELISA (enzyme-linked immunosorbent assay) as follows.

[0088] Specifically, the telopeptide standard solution sample, the decellularized matrix material sample according to Comparative Examples 1 to 3, the decellularized matrix material sample according to Example 1, and the decellularized matrix material sample that was subjected to a supercritical carbon dioxide process (300 bar, 80°C, 16-hour reaction) in a similar manner to Example 1 and then additionally treated with a NaOH solution (0.2 N) (80°C, 8-hour reaction) ('supercritical + base hydrolysis' in Fig. 3) were each dispensed into a 96-well plate coated with a capture antibody. Afterwards, the plates were reacted at 37°C for 90 minutes and washed using a wash buffer. After treatment with a biotin-labeled detection antibody, the plates were reacted at 37°C for 60 minutes and washed using a wash buffer. After treatment with a HRP-streptavidin (Horseradish peroxidase-Streptavidin) conjugated solution. After reacting for 30 minutes at 37°C, the mixture was washed. After treating with a 3,3',5,5'-Tetramethylbenzidine (TMB) substrate solution and reacting for 15 minutes at 37°C, the reaction was stopped by adding a stop solution. Afterwards, the absorbance value at a wavelength of 450 nm was measured, and the telopeptide content in the sample was calculated using a standard curve.

[0089]

[0090] As a result, as shown in Fig. 3, when the decellularized substrate was treated using a base, it was confirmed that the telopeptide sequence was detected at a high level, and in this case, the telopeptide removal efficiency was confirmed to be low. In contrast, when the supercritical carbon dioxide process according to one embodiment was used, it was confirmed that the telopeptide sequence was not detected or was detected at a very low level. Through this, it was confirmed that the supercritical carbon dioxide process according to one embodiment can efficiently remove telopeptide without additional acid, base, or enzyme treatment. This indicates that the supercritical carbon dioxide process according to one embodiment can effectively remove the immunogenicity of the decellularized substrate, thereby enhancing biocompatibility.

[0091]

[0092] Experimental Example 3. Confirmation of the Residual Effective Ingredient Content of Decellularized Materials According to Treatment Method

[0093] In order to confirm the content of active ingredients remaining after treating a decellularized substrate material using a supercritical carbon dioxide process according to one embodiment, a hydroxyproline assay and an elastin assay were performed as follows.

[0094]

[0095] (3-1). Hydroxyproline assay to measure residual collagen

[0096] Comparative Examples 1 to 3 and the decellularized matrix material samples according to Example 1 and 12N HCl were mixed in a 1:1 ratio and hydrolyzed at 100°C for 3 hours. The hydroxyproline standard solution and the hydrolyzed samples were dispensed into a 96-well plate, and the solvent was completely evaporated. Thereafter, Chloramine-T reagent was added to the wells into which the samples were dispensed, and the mixture was reacted at room temperature for 5 minutes. After the reaction was completed, DMAB reagent was added, and the mixture was reacted at 60°C for 90 minutes. After the reaction was completed, the absorbance was measured at a wavelength of 560 nm. A hydroxyproline standard curve was constructed to calculate the hydroxyproline content of the samples, and then the collagen content was converted and quantified.

[0097]

[0098] (3-2). Performing an elastin assay to measure the remaining elastin.

[0099] Comparative Examples 1 to 3 and the decellularized matrix material solution according to Example 1 and oxalic acid were mixed and hydrolyzed at 100°C. A supernatant containing elastin was obtained using a centrifuge. An elastin precipitation reagent was added to the supernatant sample obtained in this way and an elastin standard solution, and after incubation at 4°C, an elastin precipitate was obtained using a centrifuge. Afterwards, a dyeing reagent was added, and after treatment at 4°C, centrifugation was performed to obtain a dyed elastin pellet. After treating a sample containing the pellet with a dye dissociation reagent, the absorbance was measured at a wavelength of 513 nm. The elastin content was calculated using an elastin standard curve.

[0100]

[0101] As a result, as shown in Fig. 4, in Comparative Examples 1 and 2 using acid or base ('acid hydrolysis' and 'base hydrolysis' in Fig. 4, respectively), and Comparative Example 3 using acid and enzyme ('HCl + pepsin hydrolysis' in Fig. 4), it was confirmed that the amount of collagen and elastin remaining in the decellularized matrix material after hydrolysis decreased. In contrast, in the case of the decellularized matrix material according to Example 1, it was confirmed that the amount of collagen and elastin was maintained at 95% or more even after hydrolysis. Through this, it was confirmed that when the decellularized matrix material was treated through the supercritical carbon dioxide process according to one embodiment, the amount of effective ingredients remaining in the decellularized matrix was significantly greater than when treated with acid, base, or enzyme, and thus it was confirmed that a bioactive substance in the extracellular matrix can be delivered into the body with excellent efficiency. This indicates that the method according to one embodiment can maintain the content and bioactivity of the bioactive substance in the decellularized matrix.

[0102]

[0103] Experimental Example 4. Confirmation of biocompatibility of decellularized materials according to one embodiment.

[0104] To confirm the biocompatibility of the decellularized material manufactured through the supercritical carbon dioxide process according to one embodiment, the degree of immune response occurrence upon in vivo application of the decellularized substrate was measured as follows.

[0105]

[0106] (4-1). Sample preparation

[0107] First, a 2 w / v% solution (dECM) of the decellularized matrix material according to Example 1, a solution (LTG-S Dermal filler) containing 2 w / v% carboxymethyl chitosan mixed with 0.5 w / v% of the decellularized matrix material according to Example 1, and a 2 w / v% hyaluronic acid hydrogel sample (HA filler) were prepared.

[0108] Afterwards, 100 μl of the above sample composition (HA filler, LTG-S Dermal filler, dECM) was injected into the skin of each mouse, and tissues were collected from the mice 1 day and 7 days later, pathological staining was performed, and the following items were measured through a microscope.

[0109]

[0110] (4-2). Confirmation of biocompatibility of decellularized substrates

[0111] To verify the biocompatibility of the decellularized substrate processed by the method according to one embodiment, the number of inflammatory cells and multinucleated cells migrating into the sample injection site was analyzed. Furthermore, the amount of collagen deposited into the sample injection site was measured by area, the amount of neovascularization and adipocytes induced into the sample injection site were measured, and the degree of necrosis of muscle cells surrounding the sample injection site was measured.

[0112] The results are shown in Fig. 5, Table 1, and Table 2.

[0113]

[0114] Group Measurement Time Inflammatory Cell Infiltration Multinucleated Cell Count HA Filler 1d0.0 ± 0.00.0 ± 0.07d3.0 ± 1.40.0 ± 0.0LTG-SDermal Filler 1d3.0 ± 0.00.0 ± 0.07d2.0 ± 0.00.0 ± 0.0ECM 1d0.0 ± 0.00.0 ± 0.07d0.0 ± 0.00.0 ± 0.0

[0115]

[0116] As shown in Table 1, when the decellularized substrate or hydrogel composite comprising the same was processed using the method according to one embodiment, insignificant inflammatory cell infiltration occurred, and no multinucleated cells were detected. This confirmed that the decellularized substrate or composite did not elicit a significant immune response. Therefore, it can be seen that the decellularized substrate according to one embodiment exhibits excellent biocompatibility.

[0117]

[0118] GroupCollagen depositionAngiogenesisAdipocyte infiltrationMuscle cell necrosisHA filler1d1.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.07d3.0 ± 0.01.7 ± 0.61.3 ± 2.30.0 ± 0.0LTG-SDermal filler1d3.0 ± 0.01.0 ± 0.00.0 ± 0.00.0 ± 0.07d2.0 ± 0.00.5 ± 0.70.0 ± 0.00.0 ± 0.0dECM1d0.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.07d0.0 ± 0.00.0 ± 0.00.0 ± 0.00.0 ± 0.0

[0119]

[0120] As shown in Table 2, the decellularized substrate and hydrogel composite comprising the same prepared by the method according to one embodiment exhibited low levels of collagen deposition and neovascularization. Furthermore, no adipocyte infiltration or muscle cell necrosis was observed. This confirms that the decellularized substrate according to one embodiment did not exhibit a significant immune response, demonstrating excellent biocompatibility.

[0121]

[0122] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A method for manufacturing a decellularized substrate with enhanced biocompatibility, the method comprising a step of treating a solution containing the decellularized substrate with supercritical carbon dioxide, wherein the step of treating the supercritical carbon dioxide is performed at 60 to 150°C.

2. A method according to claim 1, wherein the step of treating the supercritical carbon dioxide is performed in a reactor, and carbon dioxide is injected so that the pressure in the reactor becomes 70 to 400 bar.

3. A method according to claim 1, wherein the step of treating the supercritical carbon dioxide is performed for 1 to 48 hours.

4. A method according to claim 1, wherein the solution contains 0.1 to 5 wt% of a decellularized matrix powder.

5. A method according to claim 1, wherein the decellularized substrate has a reduced or eliminated content of telopeptide within the decellularized substrate.

6. A method according to claim 1, wherein the substrate is an extracellular matrix derived from any one selected from the group consisting of skin tissue, heart tissue, adipose tissue, corneal tissue, bone tissue, brain tissue, vascular tissue, or cartilage tissue.

7. A method according to claim 1, wherein the decellularized substrate is produced by a decellularization method including a step of injecting supercritical carbon dioxide into a reactor containing biological tissue and ethanol so that the pressure within the reactor becomes 70 to 400 bar, and the step of injecting supercritical carbon dioxide into the reactor containing biological tissue and ethanol is performed at a temperature of 31 to 60°C.

8. A decellularized substrate manufactured by the method of claim 1.

9. In claim 8, the decellularized substrate is a decellularized substrate in which the content of telopeptide in the decellularized substrate is reduced or removed.

10. A composition comprising the decellularized substrate of claim 8.

11. A biomaterial comprising the decellularized substrate of claim 8.

12. A method for improving the biocompatibility of a decellularized substrate, the method comprising a step of treating a solution containing a decellularized substrate with supercritical carbon dioxide, wherein the step of treating the supercritical carbon dioxide is performed at 60 to 150°C.

13. A method according to claim 12, wherein the step of treating the supercritical carbon dioxide is performed in a reactor, and carbon dioxide is injected so that the pressure in the reactor becomes 70 to 400 bar.

14. A method according to claim 12, wherein the step of treating the supercritical carbon dioxide is performed for 1 to 48 hours.

15. A method according to claim 12, wherein the substrate is an extracellular matrix derived from any one selected from the group consisting of skin tissue, heart tissue, adipose tissue, corneal tissue, bone tissue, brain tissue, vascular tissue, and cartilage tissue.

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