Preparation method of acellular matrix for enhancing biocompatibility based on supercritical carbon dioxide process

Abstract

By means of the method according to one aspect, in a process of enhancing biocompatibility of an acellular matrix by supercritical carbon dioxide treatment, immunogenicity of the acellular matrix can be reduced with effective efficiency while maintaining a content of a physiologically active substance and physiologically active ability. Accordingly, the acellular matrix prepared by the method according to one aspect, the composition including the same, and the biomaterial have low biotoxicity and excellent tissue regeneration energy, and thus can be usefully used in cosmetic or medical materials.

Description

Technical Field

[0001] This application relates to a method for preparing a decellularized matrix, wherein the decellularized matrix has improved biocompatibility and physiological activity. Background Technology

[0002] The extracellular matrix (ECM) is a non-cellular structure of tissues or organs, composed of various macromolecules secreted from cells. The ECM can serve as a natural scaffolding material, possessing structural integrity and usable for biological purposes. Its specific composition and function can manifest in various forms depending on the specific tissue from which it is derived.

[0003] Decellularization is a process of separating extracellular matrix components from biological tissues through chemical treatment. During decellularization, immunogenic components such as cellular elements are removed from the tissues, while physiologically active substances involved in the tissue environment and function, such as the extracellular matrix and some growth factor proteins, are preserved. Therefore, various physiologically active substances preserved in this way, such as collagen, glycosaminoglycans, and various cytokines, are utilized in the decellularized matrix (hereinafter, decellularized matrix) to promote tissue regeneration and provide a more natural, biomimetic microenvironment for cell growth and differentiation. This decellularized matrix can be used alone or in combination with other substances for tissue regeneration, etc.

[0004] On the other hand, during the decellularization process, or during the treatment of the decellularized extracellular matrix prepared through this process to depolymerize or remove immunogenicity, depending on the reaction conditions including solvents, degrading enzymes, temperature conditions, pH conditions, etc., physiologically active substances in the decellularized matrix may be removed or denatured, leading to a reduction in physiological activity. Furthermore, with existing methods using surfactants, the decellularized matrix may contain residual surfactants, which may exhibit toxicity when applied to living organisms. Additionally, the osmotic pressure of compositions including decellularized matrices may increase depending on the reaction conditions; with increased osmotic pressure, the mixing level with other substances may decrease, and the efficiency of compounding with other cosmetic or medical materials or ingredients may also decrease.

[0005] Furthermore, the extracellular matrix, or decellularized extracellular matrix, contains collagen, with telopeptides consisting of 12 to 27 amino acids at both ends of the collagen molecules. These telopeptides are a major cause of immune responses; therefore, a process to remove telopeptides is necessary for the application of decellularized matrix in vivo. Existing processes use acidic solutions, enzymes, or combinations thereof to remove these telopeptide sequences. However, this existing method, using acid or base chemical treatment, leads to an increase in osmotic pressure, making it difficult to complex with other substances and potentially causing adverse effects on the human body due to the additional treatment with acidic or alkaline solutions. Moreover, this multi-step process is complex and suffers from reduced efficacy when applied in vivo; for example, effective components or physiologically active substances within the extracellular matrix may be removed during the process.

[0006] Therefore, in order to solve the above problems, the inventors have developed a technique for preparing decellularized mechanism materials that reduces or removes telopeptide content while maintaining the content and physiological activity of physiologically active substances in the decellularized matrix so that it exhibits a reduced immune response or no immune response when applied in vivo. Summary of the Invention

[0007] Technical issues

[0008] One aspect provides a method for preparing a biocompatible decellularized matrix, comprising the step of treating a solution containing the decellularized matrix with supercritical carbon dioxide, wherein the step of treating with supercritical carbon dioxide is performed at 60 to 150°C.

[0009] On the other hand, decellularized matrices prepared by the method, compositions comprising the same, and biomaterials comprising the same are provided.

[0010] On the other hand, a method for improving the biocompatibility of decellularized matrix is ​​provided, comprising the step of treating a solution containing decellularized matrix with supercritical carbon dioxide, wherein the step of treating with supercritical carbon dioxide is performed at 60 to 150°C.

[0011] Other objects and advantages of this application become more apparent from the appended claims, drawings, and detailed description below. Content not described herein is sufficient to be readily apparent and inferred by one of ordinary skill in the art to which this application pertains or similar art, and therefore such description is omitted.

[0012] Technical solution

[0013] Throughout this specification, when a section describes a component as "including," unless otherwise stated, it means that other components may be further included, not excluded. Furthermore, unless a specific order is explicitly stated in the context, the steps may be performed in a different order than explicitly stated. In other words, the steps may be performed in the same order as explicitly stated, substantially simultaneously, or in reverse order.

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

[0015] In this specification, "biocompatibility" means that a substance introduced into existing cells, tissues, organs, or individuals does not induce a significant inflammatory response, immunogenicity, or cytotoxicity in the organ and performs the required function. For example, a biocompatible substance is a substance that does not induce a significant inflammatory response, immunogenicity, or cytotoxicity in existing cells, tissues, organs, or individuals and performs the required function when introduced into an organism.

[0016] In one embodiment, the improved biocompatibility may refer to a reduction in inflammatory response, immunogenicity, or cytotoxicity when applied in vivo compared to when the step of treating the decellularized matrix with supercritical carbon dioxide according to one embodiment is performed, compared to when this step is not performed. For example, the improved biocompatibility may include a reduction in the content of terminal peptides in the decellularized matrix.

[0017] The term "decellularized extracellular matrix (dECM)" as used in this specification may be used interchangeably with "decellularized tissue," "decellularized extracellular matrix," or "decellularized material." Decellularized matrix refers to the product obtained by decellularizing tissues or organs of humans, pigs, or cattle to remove cellular components other than the extracellular matrix (ECM), such as the nucleus, cell membrane, and nucleic acids. "Decellularized organ" refers to an organ such as the heart or kidney obtained by decellularization while preserving the entire structure of the organ. Furthermore, considering that the purpose is for cell reimplantation or use while preserving the entire physical structure of the organ, it is not possible to prepare the extracellular matrix into a water-soluble formulation; this is a concept clearly distinct from "decellularized matrix" as used in this specification.

[0018] The extracellular matrix refers to a complex aggregate of biological macromolecules that fills the space within or outside of tissues. It consists of various types of molecules synthesized by cells and secreted and accumulated outside the cells, such as fibrin proteins, proteoglycans, and other complex proteins, as well as cell adhesion proteins like fibrous proteins and laminins.

[0019] The method for preparing a decellularized matrix with enhanced biocompatibility may exclude the step of treatment with acidic or alkaline solutions. According to one embodiment, the method for preparing a decellularized matrix with enhanced biocompatibility, unlike prior art methods using acidic or alkaline solutions, includes a supercritical carbon dioxide treatment process performed at a specific temperature. The advantage derived from this method is that, since it excludes subsequent steps involving treatment with acidic or alkaline solutions and subsequent steps involving treatment with degrading enzymes, the preparation process is highly efficient and does not require additional purification processes. Therefore, the decellularized matrix prepared according to the method of one embodiment retains the content and physiological activity of physiologically active substances, and exhibits low peptide content and low immunogenicity, thus demonstrating excellent biocompatibility.

[0020] In one embodiment, the supercritical carbon dioxide treatment step can be performed at a temperature of 60 to 150°C. The supercritical carbon dioxide treatment step can be performed inside a reactor, where the temperature can be between 60 and 150°C. For example, the supercritical carbon dioxide treatment step can be performed at reactor temperatures 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°C, 65 to 115°C, 65 to 110°C, 65 to 105°C, 65 to 100°C, 65 to 95°C, 65 to 90°C. ℃, 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℃ The experiment is performed at temperatures ranging from 70 to 85°C, 70 to 80°C, 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, and 70 to 90°C, but is not limited to these conditions. Outside of these temperature ranges, sufficient depolymerization or hydrolysis of the decellularized matrix may not occur, and the telopeptide content may not be sufficiently reduced.

[0021] In one embodiment, the step of using supercritical carbon dioxide treatment can be performed inside a reactor, and carbon dioxide can be injected to bring the pressure inside the reactor to 70 to 400 bar. For example, carbon dioxide gas could be injected into the reactor to create pressures of 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 350 bar, 210 to 350 bar, 220 to 350 bar, 230 to 350 bar, 240 to 350 bar. The pressure ranges are 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 are not limited to these. At pressures outside these ranges within the reactor, depolymerization or hydrolysis of the decellularized matrix may not occur sufficiently, or the telopeptide content may not be adequately reduced.

[0022] In one embodiment, the supercritical carbon dioxide treatment step can be performed for 1 to 48 hours, or the reactor pressure can be maintained as described above for 1 to 48 hours. For example, the supercritical carbon dioxide treatment step can 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, 6 to 15 hours, but is not limited thereto. If the supercritical carbon dioxide treatment step is performed for a shorter time than described above, the telopeptide content may not be sufficiently reduced due to insufficient depolymerization or hydrolysis of the decellularized matrix.

[0023] In one embodiment, the solution comprising the decellularized matrix may be a solution comprising decellularized matrix powder. The decellularized matrix powder may be a dry powder of extracellular matrix after the decellularization process, and may be a product obtained by drying and pulverizing the decellularized extracellular matrix. For example, it may be a product obtained by drying and pulverizing the decellularized extracellular matrix. As a drying method for preparing the cellular matrix into a dry state, methods commonly used in the art can be used, and there are no particular limitations. Non-limiting examples of the drying method include air drying, natural drying, spray drying, freeze drying, and vacuum drying. These methods may be used individually or in combination of at least two methods. In one embodiment, the dried decellularized matrix powder may be a freeze-dried powder of the decellularized matrix. The steps 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] In this specification, the term "solution" can refer to a mixture of two substances, such as a solute and a solvent. In one embodiment, the solution comprising decellularized matrix powder can refer to a mixture, suspension, or aqueous solution in which the decellularized matrix powder is contained in distilled water. In this specification, the term "solution" is used interchangeably with mixture, suspension, hydrate, composition, dissolved substance, aqueous solution, etc.

[0025] In one embodiment, the solution comprising the decellularized matrix may include decellularized matrix powder, or may include decellularized matrix powder and distilled water. The solution comprising the decellularized matrix powder may include the decellularized matrix powder at a concentration of 0.1 to 5% by weight. In one embodiment, the decellularized matrix powder may be included in distilled water at a concentration of 0.01 to 5% by weight, for example, at 0.05 to 5% by weight, 0.1 to 5% by weight, 0.2 to 5% by weight, 0.3 to 5% by weight, 0.4 to 5% by weight, 0.5 to 5% by weight, 0.6 to 5% by weight, 0.7 to 5% by weight, 0.8 to 5% by weight, 0.9 to 5% by weight, 1.0 to 5% by weight, 0.05 to 4% by weight, 0.1 to 4% by weight, 0.2 to 4% by weight, 0.3 to 5% by weight. 4% by weight, 0.4 to 4% by weight, 0.5 to 4% by weight, 0.6 to 4% by weight, 0.7 to 4% by weight, 0.8 to 4% by weight, 0.9 to 4% by weight, 1.0 to 4% by weight, 0.05 to 3% by weight, 0.1 to 3% by weight, 0.2 to 3% by weight, 0.3 to 3% by weight, 0.4 to 3% by weight, 0.5 to 3% by weight, 0.6 to 3% by weight, 0.7 to 3% by weight, 0.8 to 3% by weight, 0.9 to 3% by weight, or 1.0 to 3% by weight, but not limited thereto.

[0026] In one embodiment, the decellularized matrix is ​​formed by reducing the content of telopeptides in the decellularized matrix or by removing telopeptides.

[0027] In this specification, "reduction or removal of telopeptides" means that the content of telopeptides in the decellularized matrix is ​​reduced compared to before the step of treatment with supercritical carbon dioxide according to one aspect. For example, treating a solution containing the decellularized matrix with supercritical carbon dioxide at a temperature of 60 to 150°C can reduce the content of telopeptides in the decellularized matrix by 80% or more. For example, by performing the step of treatment with supercritical carbon dioxide according to one aspect, the content of telopeptides in the decellularized matrix can be reduced by 60% or more, 65% or more, 70% or more, 75% or more, or 80% or more. In the decellularized matrix, due to the reduction or removal of telopeptides within the decellularized matrix, biocompatibility can be increased.

[0028] The method according to one embodiment has been confirmed to reduce telopeptide content to a similar degree or with greater efficiency than existing methods that use acidic solutions, alkaline solutions, or acidic solutions or enzyme treatments.

[0029] In one embodiment, the decellularized matrix may include a high content of physiologically active substances or effective components. For example, the decellularized matrix may refer to a matrix in which, after treatment with supercritical carbon dioxide at a temperature of 60 to 150°C, the content of physiologically active substances or effective components remains at 80% or more of the level before treatment, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% or more. It has been confirmed by the method according to one embodiment that, compared with conventional treatments using acidic solutions, alkaline solutions, or enzymes, the residual amount of physiologically active substances or effective components in the decellularized matrix is ​​significantly higher. For example, it has been confirmed that the residual amounts of collagen and elastin in the decellularized matrix are 95% or more, respectively. Thus, it has been confirmed that, by the method according to one embodiment, a decellularized matrix with excellent physiological activity containing a large amount of physiologically active substances can be prepared.

[0030] Therefore, by means of the method according to one embodiment, the biocompatibility can be improved by efficiently reducing the immunogenicity of the decellularized matrix, while at the same time, the effective components or physiologically active substances in the decellularized matrix can be maintained at a high content to prepare a decellularized matrix with excellent physiological activity.

[0031] In one embodiment, the matrix may be an extracellular matrix selected from, but not limited to, skin tissue, heart tissue, adipose tissue, corneal tissue, bone tissue, brain tissue, vascular tissue, or cartilage tissue of humans, pigs, or cattle.

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

[0033] For example, as a method for decellularizing the extracellular matrix, the decellularized matrix may be a product obtained by decellularization through a method including the step of injecting supercritical carbon dioxide into a reactor comprising biological tissue and ethanol to bring the pressure inside the reactor to 70 to 400 bar, wherein the step of injecting supercritical carbon dioxide into the reactor comprising biological tissue and ethanol may be performed at 31 to 60°C.

[0034] The decellularization method may, for example, include injecting carbon dioxide gas into a reactor to achieve pressures of 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, or 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 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 not limited to these.

[0035] The decellularization method may include, for example, the step of using the supercritical carbon dioxide treatment at temperatures of 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, but is not limited thereto.

[0036] In the decellularization method, supercritical carbon dioxide can be used for treatment for 2 to 24 hours, for example, the pressure inside the reactor can be maintained for 2 to 24 hours. For example, the pressure inside the reactor can 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 decellularization method may not include steps involving additional acid, base, or enzyme treatment. The decellularization method may further include a pretreatment step before contacting the biological tissue with ethanol in the reactor. This pretreatment process removes contaminants, fats, etc., from the biological tissue. Furthermore, the decellularization method may further include treating a mixture after injecting supercritical carbon dioxide into a reactor containing biological tissue and ethanol with a DNA-degrading enzyme, wherein the immunogenicity of the mixture is removed by decomposing residual DNA in the mixture. The decellularization method may further include a washing step, wherein the mixture after the DNA-degrading enzyme treatment step is treated with distilled water to remove residual contaminants.

[0038] On the other hand, a decellularized matrix prepared by the method is provided. Regarding the decellularized matrix, the same terms or elements mentioned herein are as described above.

[0039] On the other hand, a composition comprising the decellularized matrix is ​​provided. Regarding the composition, the same terms or elements mentioned herein are as described above.

[0040] In one embodiment, the composition may be a hydrogel composition comprising a decellularized matrix. For example, the hydrogel composition may be a hydrogel composition according to one embodiment comprising a decellularized matrix and hyaluronic acid, or a hydrogel composition according to one embodiment comprising a decellularized matrix and chitosan. Furthermore, the hydrogel may further comprise any material suitable for preparing the hydrogel.

[0041] In this specification, the term "hydrogel" can refer to a three-dimensional network structure formed by cross-linking hydrophilic polymers through covalent or non-covalent bonds. Due to the hydrophilicity of its constituent materials, it has the property of absorbing a large amount of water and swelling in aqueous solutions and aqueous environments, yet remaining insoluble due to its cross-linked structure. Therefore, hydrogels with various forms and properties can be formed depending on the constituent components and preparation methods. Because they typically contain a large amount of water, they may exhibit intermediate properties between liquids and solids.

[0042] In this specification, the term "chitosan" can refer to a linear polysaccharide composed of D-glucosamine and N-acetylglucosamine. In addition to pure chitosan, the chitosan may also include chitosan derivatives. For example, the chitosan derivatives may include at least one of methyl chitosan, carboxymethyl chitosan, phthalated chitosan, esterified chitosan, aminated chitosan, or formylated chitosan.

[0043] In one embodiment, the composition may be a tissue repair composition or a substance used for tissue repair. For example, it may refer to a soft tissue-like filler material injected into the skin where wrinkles are present or areas requiring volume, a material used to prevent adhesion between the surgical site and normal tissue, a tissue adhesive, or a wound dressing substance for artificial skin. The hydrogel may be applicable to body areas such as the glabella, forehead, under-eye area, crow's feet, nasolabial folds, cheeks, mouth wrinkles, and jawline. As a material directly applicable to the human body, the hydrogel should be biocompatible. For example, when the hydrogel is used to fill areas requiring volume, it needs excellent shape retention / persistence to create a long-lasting volume effect after injection. When the hydrogel is used at surgical sites to prevent adhesion, it should have tissue compatibility at the wound site and low or no cytotoxicity. When used as a wound dressing substance, it needs excellent durability / persistence to maintain adhesion and spreadability. Furthermore, depending on the conditions of hydrogel application, the skin surface may become uneven or the application results may be unsatisfactory. Therefore, hydrogels that can be easily degraded or deformed from those already formed in the body are required.

[0044] The composition may have enhanced biocompatibility and physiological activity due to the inclusion of a decellularized matrix according to one embodiment, and therefore can be usefully used as a tissue repair composition.

[0045] On the other hand, a biomaterial is provided that includes the decellularized matrix. Regarding the biomaterial, the same terms or elements mentioned herein are as described above.

[0046] In one embodiment, the biomaterial may be a biomaterial comprising the hydrogel composition.

[0047] In one embodiment, the biomaterial may be a biomaterial for tissue repair.

[0048] The biomaterial may have enhanced biocompatibility and physiological activity due to including a decellularized matrix according to one embodiment, and can therefore be usefully used as a biomaterial for tissue repair.

[0049] On the other hand, a method for improving the biocompatibility of decellularized matrix is ​​provided, comprising the step of treating a solution containing decellularized matrix with supercritical carbon dioxide, said supercritical carbon dioxide treatment step being performed at 60 to 150°C. Regarding the method for improving the biocompatibility of decellularized matrix, the same terms or elements mentioned herein are as described above.

[0050] Beneficial effects

[0051] By following one approach, not only can the biocompatibility of decellularized matrix be improved, but the content of bioactive substances and physiological activity can also be maintained.

[0052] Therefore, the decellularized matrix prepared according to one method can be usefully used as a biomaterial for tissue repair because it has low biotoxicity and high biocompatibility and physiological activity, and can be applied in vivo. Attached Figure Description

[0053] Figure 1a shows the results of treating a decellularized matrix according to one embodiment using a supercritical carbon dioxide process.

[0054] Figure 1b shows the results of treating the decellularized matrix according to one embodiment with HCl solution.

[0055] Figure 1c shows the results of treating the decellularized matrix according to one embodiment with NaOH solution.

[0056] Figure 2a shows the results of treating a decellularized matrix according to one embodiment using a supercritical carbon dioxide process.

[0057] Figure 2b shows the results of treating the decellularized matrix according to one embodiment with HCl solution.

[0058] Figure 2c shows the results of treating the decellularized matrix according to one embodiment with NaOH solution.

[0059] Figure 3 The results were used to confirm and compare the content of terminal peptides in the decellularized matrix according to the treatment method by means of absorbance measurement.

[0060] Figure 4 The results were used to confirm and compare the content of residual active ingredients in the decellularized matrix according to the treatment method by hydroxyproline assay and elastin assay.

[0061] Figure 5 The results were used to confirm the extent of the immune response after intradermal injection of a composition comprising decellularized material according to one embodiment into mice. Detailed Implementation

[0062] The following description, through examples, provides a more detailed account. However, these examples are for illustrative purposes only, and the scope of the invention is not limited to these examples.

[0063] Preparation Example 1: Preparation of decellularized matrix using supercritical carbon dioxide process

[0064] A pretreatment process was performed in which porcine skin tissue was prepared, keratinized with a knife, and 70% ethanol was added and stirred at room temperature for 4 hours to remove fat. Then, 20g of the skin tissue and 100ml of 100% ethanol were added to a supercritical fluid reactor (Ilshin Autoclave Co., Ltd., South Korea), and the temperature was set to 35°C. Carbon dioxide was then injected, and the pressure was adjusted to 300 bar, and the reaction was carried out for 6 hours under the stated temperature and pressure conditions. The tissue was then washed with an ethanol solution for 2 hours, followed by washing with PBS and distilled water for 4 hours. After lyophilization, the washed tissue was powdered using a freeze-mill.

[0065] Example 1: Preparation of biocompatibility-enhanced decellularized matrix materials based on supercritical carbon dioxide process

[0066] The decellularized matrix powder prepared as described in Preparation Example 1 was mixed with distilled water at 2.0% by weight and then fed into a supercritical fluid reactor (Ilshin Autoclave Co., Ltd., South Korea), where the temperature was set to 60°C. Carbon dioxide was then injected to bring the pressure inside the reactor to 300 bar, and the reaction was carried out under these conditions for 12 hours. Figures 1a and 2a show the results of visually confirming the decellularized matrix material thus prepared.

[0067] Comparative Example 1: Preparation of decellularized matrix materials using acid

[0068] The decellularized matrix powder prepared as described in Preparation 1 was mixed into a 0.1N HCl solution at a weight of 2.0%. The solution was then cooled to 60°C and allowed to react for 1 hour, 4 hours, and 15 hours. Figures 1b and 2b show the results of visually confirming the decellularized matrix material thus prepared.

[0069] Comparative Example 2: Preparation of decellularized matrix materials using bases

[0070] The decellularized matrix powder prepared as described in Preparation Example 1 was mixed into a 0.2N NaOH solution at a weight of 2.0%. The solution temperature was then adjusted to 60°C, and the reaction was allowed to proceed for 1 hour, 4 hours, and 15 hours. Figures 1c and 2c show the results of visual confirmation of the decellularized matrix material thus prepared.

[0071] Comparative Example 3: Preparation of decellularized matrix materials using acids and enzymes

[0072] After mixing pepsin at 10% by weight based on the decellularized matrix into a 0.1N HCl solution, the dry powder of the decellularized matrix prepared as described in Preparation Example 1 was mixed in at 2.0% by weight. The solution was then reacted at 4°C for 24 hours, and then filtered using a 100kDa filter.

[0073] Experiment 1: Confirmation of depolymerization and formulation of decellularized matrix materials

[0074] The decellularized matrix materials of Examples 1, 1 Comparative Example, and 2 were confirmed to depolymerize and be formulated over time, in order to confirm the depolymerization and formulation of the decellularized matrix materials according to the supercritical carbon dioxide process of one embodiment.

[0075] As shown in Figure 2b, precipitation was observed when the decellularized solution was treated with an acidic solution. In contrast, when using a supercritical carbon dioxide process or base according to one embodiment, no precipitation was observed, and the decellularized powder was uniformly dispersed and dissolved in the solution (Figures 2a and 2c). This confirms that by treating the decellularized matrix according to the method of one embodiment, the decellularized matrix can be depolymerized and a stable dosage form can be formed without the need for additional acid, base, or enzyme treatment.

[0076] Experiment Example 2: Confirming the immunogenicity removal efficiency of the supercritical carbon dioxide process.

[0077] To confirm the immunogenicity removal efficiency of the supercritical carbon dioxide process according to one embodiment for decellularized matrix, the degree of telopeptide removal was confirmed using the Sandwich enzyme-linked immunosorbent assay (ELISA).

[0078] Specifically, the decellularized matrix material samples were obtained by further treating the decellularized matrix material with NaOH solution (0.2N) (80°C, 8 hours) using a supercritical carbon dioxide process (300 bar, 80°C, 16 hours) similar to that in Example 1. These samples included samples of telopeptide standard solution, decellularized matrix material samples according to Comparative Examples 1 to 3, decellularized matrix material samples according to Example 1, and decellularized matrix material samples obtained by further processing with NaOH solution (0.2N) (80°C, 8 hours). Figure 3Supercritical + alkaline hydrolysis was used to separate isolates into 96-well plates coated with capture antibody. The plates were then incubated at 37°C for 90 minutes followed by washing with wash buffer. After treatment with biotin-labeled detection antibody, the plates were incubated at 37°C for 60 minutes, followed by washing with wash buffer. Treatment with horseradish peroxidase-streptavidin binder solution was followed by incubation at 37°C for 30 minutes, followed by washing. Treatment with 3,3',5,5'-tetramethylbenzidine (TMB) matrix solution was incubated at 37°C for 15 minutes, followed by termination with stop solution. The absorbance at 450 nm was then measured, and the telopeptide content within the samples was calculated using a standard curve.

[0079] The result is confirmed, such as Figure 3 As shown, when base treatment was used on the decellularized matrix, telopeptide sequences were detected at high levels, confirming a decrease in telopeptide removal efficiency. In contrast, when using the supercritical carbon dioxide process according to one embodiment, telopeptide sequences were either not detected or detected at extremely low levels. This confirms that the supercritical carbon dioxide process according to one embodiment can effectively remove telopeptides without the need for additional acid, base, or enzyme treatment. This result indicates that the supercritical carbon dioxide process according to one embodiment can effectively remove the immunogenicity of the decellularized matrix, thereby improving biocompatibility.

[0080] Experiment Example 3: Confirm the residual effective component content of decellularized materials based on the treatment method.

[0081] After treating the decellularized matrix material using the supercritical carbon dioxide process according to one embodiment, in order to confirm the content of residual active ingredients, the following hydroxyproline assay and elastin assay were performed.

[0082] (3-1) Perform the hydroxyproline test to determine the amount of residual collagen.

[0083] The decellularized matrix material samples from Comparative Examples 1 to 3 and Example 1 were mixed at a 1:1 ratio with 12N HCl and hydrolyzed at 100°C for 3 hours. Hydroxyproline standard solution and the hydrolyzed sample aliquots were then plated in 96-well plates to allow complete solvent evaporation. Chloramine-T reagent was then added to the wells of the aliquots, and the reaction was carried out at room temperature for 5 minutes. After the reaction, dimethylamine borane (DMAB) reagent was added, and the reaction was carried out at 60°C for 90 minutes. After the reaction, the absorbance was measured at 560 nm. A hydroxyproline standard curve was constructed to calculate the hydroxyproline content in the samples, and the collagen content was then converted and quantified.

[0084] (3-2) Perform an elastin test to measure the amount of residual elastin.

[0085] Hydrolysis was performed at 100°C after mixing the decellularized matrix material solutions according to Comparative Examples 1 to 3 and Example 1 with oxalic acid. A supernatant containing elastin was obtained using a centrifuge. Elastin precipitation reagent was added to the obtained supernatant sample and elastin standard solution, and after incubation at 4°C, elastin precipitate was obtained using a centrifuge. Then, staining reagent was added, and after treatment at 4°C, centrifugation was performed to obtain stained elastin pellets. The absorbance at 513 nm was measured after treating the sample containing the pellets with a staining dissociation reagent. The elastin content was calculated using an elastin standard curve.

[0086] As a result, it was confirmed that... Figure 4 As shown, for Comparative Examples 1 and 2 using acids or bases (respectively...), Figure 4 Comparative Example 3 (using acid hydrolysis and alkaline hydrolysis) and the use of acid and enzymes. Figure 4 Regarding the 'HCl + pepsin hydrolysis' method, the amount of residual collagen and elastin in the decellularized matrix material is reduced after hydrolysis. It was also confirmed that, in comparison, the amount of collagen and elastin in the decellularized matrix material according to Example 1 remains at 95% or more after hydrolysis. This confirms that when the decellularized matrix material is treated by the supercritical carbon dioxide process according to one embodiment, the residual amount of active ingredients in the decellularized matrix is ​​significantly greater compared to treatment using acids, bases, or enzymes, thus enabling the efficient delivery of physiologically active substances from the extracellular matrix to the living body. This indicates that the method according to one embodiment can maintain the content and physiological activity of physiologically active substances in the decellularized matrix.

[0087] Experimental Example 4: Confirmation of the biocompatibility of the decellularized material according to one embodiment.

[0088] To confirm the biocompatibility of the decellularized material prepared by the supercritical carbon dioxide process according to one embodiment, the degree of immune response when the decellularized matrix is ​​applied in vivo is measured as follows.

[0089] (4-1) Sample preparation

[0090] First, a 2 w / v% solution of decellularized matrix material (dECM) according to Example 1, a solution obtained by mixing the decellularized matrix material according to Example 1 at 0.5 w / v% into a hydrogel containing 2 w / v% carboxymethyl chitosan (LTG-S dermal filler), and a 2 w / v% hyaluronic acid hydrogel sample (HA filler) were prepared.

[0091] Then, 100 μl of the sample (HA filler, LTG-S dermal filler, dECM) composition was injected into the skin of mice. After 1 day and 7 days, the tissue was recovered from the mice for pathological staining and the following items were measured under a microscope.

[0092] (4-2) Confirm the biocompatibility of decellularized matrix

[0093] To confirm the biocompatibility of the decellularized matrix treated according to the method of one embodiment, the number of inflammatory cells and multinucleated cells that migrated to the sample injection site were analyzed. Furthermore, the amount of collagen deposited at the sample injection site was measured based on area, the amount of neovascularization induced at the sample injection site and the amount of adipocytes were determined, and the degree of necrosis of muscle cells near the sample injection site was measured.

[0094] The result is as follows Figure 5 As shown in Table 1 and Table 2.

[0095] [Table 1]

[0096] As shown in Table 1, when the decellularized matrix prepared according to the method of one embodiment or the hydrogel complex comprising it was processed, minimal inflammatory cell infiltration occurred, and no multinucleated cells were detected. This confirms that the decellularized matrix or complex does not produce a significant degree of immune response. Therefore, the decellularized matrix according to one embodiment exhibits excellent biocompatibility.

[0097] [Table 2]

[0098] As shown in Table 2, the decellularized matrix prepared according to the method of one embodiment and the hydrogel complex comprising it exhibited low levels of collagen deposition and angiogenesis. Furthermore, no adipocyte infiltration or muscle cell necrosis was observed. Therefore, it is confirmed that the decellularized matrix according to one embodiment did not exhibit a significant immune response, demonstrating excellent biocompatibility.

[0099] The description above is merely exemplary, and those skilled in the art should understand that the invention can be easily modified into other specific forms without changing the technical concept or essential features. Therefore, the embodiments described above should be understood as exemplary in all respects, and not restrictive.

Claims

1. A method of preparing a decellularized matrix having improved biocompatibility, comprising the step of treating a solution comprising the decellularized matrix with supercritical carbon dioxide, wherein, The step of using supercritical carbon dioxide for treatment is performed at 60 to 150°C.

2. The method according to claim 1, wherein, The step of using supercritical carbon dioxide for treatment is performed inside a reactor, where carbon dioxide is injected to bring the pressure inside the reactor to 70 to 400 bar.

3. The method according to claim 1, wherein, The supercritical carbon dioxide treatment process is performed for 1 to 48 hours.

4. The method according to claim 1, wherein, The solution comprises 0.1 to 5% by weight of decellularized matrix powder.

5. The method according to claim 1, wherein, In the decellularized matrix, the content of terminal peptides within the decellularized matrix is ​​reduced or removed.

6. The method according to claim 1, wherein, The matrix is ​​an extracellular matrix derived from any one of the following groups: skin tissue, heart tissue, adipose tissue, corneal tissue, bone tissue, brain tissue, vascular tissue, or cartilage tissue.

7. The method according to claim 1, wherein, The decellularized matrix is ​​prepared by a decellularization method comprising the step of injecting supercritical carbon dioxide into a reactor comprising biological tissue and ethanol to bring the pressure inside the reactor to 70 to 400 bar, wherein the step of injecting supercritical carbon dioxide into the reactor comprising biological tissue and ethanol is performed at 31 to 60°C.

8. A decellularized matrix prepared by the method according to claim 1.

9. The decellularized matrix according to claim 8, wherein, In the decellularized matrix, the content of terminal peptides within the decellularized matrix is ​​reduced or removed.

10. A composition comprising the decellularized matrix according to claim 8.

11. A biomaterial comprising the decellularized matrix according to claim 8.

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

13. The method according to claim 12, wherein, The step of using supercritical carbon dioxide for treatment is performed inside a reactor, where carbon dioxide is injected to bring the pressure inside the reactor to 70 to 400 bar.

14. The method according to claim 12, wherein, The supercritical carbon dioxide treatment process is performed for 1 to 48 hours.

15. The method according to claim 12, wherein, The matrix is ​​an extracellular matrix derived from any one of the following groups: skin tissue, heart tissue, adipose tissue, corneal tissue, bone tissue, brain tissue, vascular tissue, or cartilage tissue.

Images