Gelatin-coated acellular dermal tissue and use thereof

Abstract

The present invention relates to a gelatin-coated acellular dermal tissue and a use thereof. More specifically, the present invention relates to: an acellular dermal tissue in which gelatin derived from human skin tissue is crosslinked on the surface of the acellular dermal tissue; and a use thereof for treating skin damage. The gelatin-coated acellular dermal tissue according to the present invention has predetermined physical properties so as to provide ease of handling, and also eliminates toxicity and transplant rejection responses, and thus can be effectively used for treating patients with skin tissue damage.

Description

Gelatin-coated acellular dermal tissue and its use

[0001] The present invention relates to acellular dermal tissue coated with gelatin and its use. More specifically, it relates to acellular dermal tissue cross-linked with human skin tissue-derived gelatin on the surface of the acellular dermal tissue and its use in treating skin damage.

[0002] The human skin is composed of the epidermis, the outermost layer, the dermis, and the subcutaneous tissue. The epidermis is composed of epithelial cells, differentiated into multiple layers from the basement membrane (BM), which firmly connects the epidermis and dermis, as well as melanocytes and immune cells. Beneath the epidermis lies the dermis, which is primarily composed of fibroblasts and the extracellular matrix (ECM), which is composed of collagen and elastin. The skin maintains its shape through the interaction of these various cells and constituents, and performs various functions, including temperature regulation and acting as a barrier against the external environment.

[0003] Specifically, the basement membrane, which forms the boundary between the epidermis and dermis, acts as a barrier, allowing liquid substances to pass through but controlling the movement of inflammatory and tumor cells between the two layers. The basement membrane facilitates the attachment of epidermal cells, acting as a support, regulating epidermal formation, and participating in wound healing.

[0004] Meanwhile, as the need for substitute skin has increased in the fields of cosmetic and reconstructive plastic surgery for extensive burns, skin ulcers or skin damage caused by diabetes, and skin tissue depressions caused by accidents, trauma, aging, etc., the development of biomaterials and artificial skin that can replace skin has been progressing rapidly.

[0005] Currently applied skin substitutes are in the form of directly applying decellularized dermis (Korean Patent No. 10-1362402) with the donated basement membrane layer removed, biodegradable coverings such as collagen and small intestinal submucosa, and biocompatible synthetic polymers such as PLGA, or artificial skin cultured with skin-derived cells on collagen or polymer sponge (Korean Patent Publication No. 10-2023-0169028).

[0006] Collagen is a protein that makes up most of the dermal layer of the skin. It is commercially available in various forms, but the difficulty in the manufacturing process makes it expensive. In particular, when collagen is used as an artificial skin support, it has the disadvantage of not being able to replicate the dermal layer as is.

[0007] Furthermore, decellularized dermis, intended to eliminate the occurrence of immune rejection, suffers from the disadvantage of physicochemical decellularization methods, which damage the basement membrane, which serves as the support between the epidermis and dermis, making it difficult to preserve the structure and properties of the dermal layer. Consequently, this not only reduces the convenience of handling artificial skin grafts during surgery, but also ultimately negatively impacts the surgical outcome.

[0008] Accordingly, the inventors of the present invention conducted research to develop an acellular dermal graft material that overcomes the problems of the prior art, and as a result, established a process for manufacturing acellular dermal tissue capable of implementing properties similar to dermal tissue with a basement membrane. By cross-linking human skin tissue-derived gelatin on acellular dermal tissue to form a basement membrane-mimicking layer that functions as a support, the present invention was completed by manufacturing acellular dermal tissue that not only has the desired properties but also maintains a high content of extracellular matrix such as collagen.

[0009] To achieve the above purpose, one aspect of the present invention provides acellular dermal tissue coated with gelatin.

[0010] Another aspect of the present invention provides a method for producing acellular dermal tissue coated with the gelatin.

[0011] Another aspect of the present invention provides a skin damage treatment agent comprising the acellular dermal tissue coated with the gelatin or the acellular dermal tissue manufactured according to the manufacturing method.

[0012] The gelatin-coated acellular dermal tissue according to the present invention is cross-linked acellular dermal tissue obtained by uniformly applying human skin tissue-derived gelatin to one or both sides of acellular dermal tissue without a basement membrane layer, and has superior physical properties compared to acellular dermal tissue in which the basement membrane is not removed. The cross-linked human skin tissue-derived gelatin not only functions as a basement membrane-mimetic layer to provide physical properties similar to the basement membrane, but also preserves various minerals, trace elements, and other components contained in human skin tissue, thereby further enhancing the tissue regeneration effect of the final acellular dermal tissue graft material produced. In addition, since no other artificially synthesized or animal-derived components are used, the safety of the artificial skin graft material can be maximized. Accordingly, the gelatin-coated acellular dermal tissue according to the present invention not only has the physical properties and ease of handling, but also eliminates toxicity and graft rejection, and can thus be usefully utilized in the treatment of patients with damaged skin tissue.

[0013] Figure 1 schematically illustrates a process for manufacturing acellular dermal tissue coated with gelatin according to one embodiment of the present invention.

[0014] Figure 2 schematically illustrates a process for manufacturing gelatin derived from human skin tissue used in manufacturing acellular dermal tissue coated with gelatin according to one embodiment of the present invention. Specifically, the process schematically illustrates a) a raw material preparation step, b) an acid or alkali treatment step to induce skin tissue expansion, c) a heat treatment step to dissolve gelatin, d) a gelatin solidification step through drying, and e) a basement membrane-mimicking dermal product manufacturing step by coating human acellular dermal tissue with gelatin.

[0015] Figure 3 is a photograph of a sealed packaged sample according to one embodiment of the present invention. Specifically, the samples are acellular dermal tissue that has undergone decellularization but has not had its basement membrane layer removed (control group 1: ADM sample with BM), acellular dermal tissue with its basement membrane layer removed (control group 2: ADM sample without BM), and acellular dermal tissue coated with gelatin (test group: ADM without BM + gelatin cross-linked sample).

[0016] Figure 4 shows the results of measuring the thickness of a sample according to one embodiment of the present invention.

[0017] Figure 5 shows the results of visually measuring the elasticity of a sample according to one embodiment of the present invention.

[0018] Figure 6 shows the results of cytotoxicity measurement of a sample according to one embodiment of the present invention.

[0019] Figure 7a shows the results of measuring the viscoelasticity of a sample according to one embodiment of the present invention.

[0020] Figure 7b shows the results of quantitative analysis of storage modulus (G') and loss modulus (G") versus shear strain for a sample according to one embodiment of the present invention.

[0021] Figure 8 shows the results of measuring the collagen content of a sample according to one embodiment of the present invention.

[0022] Figure 9 shows the results of H&E staining (Hematoxylin & Eosin staining) of a sample according to one embodiment of the present invention.

[0023] Figure 10 shows the results of MT staining (Masson trichrome staining) of a sample according to one embodiment of the present invention.

[0024] Gelatin-coated acellular dermal tissue

[0025] One aspect of the present invention provides acellular dermal tissue coated with gelatin.

[0026] As used herein, the term "dermis" refers to a layer of skin composed primarily of collagen fibers, interwoven with elastic fibers made of elastin. The dermis comprises the majority of the skin, providing nutrients and support to the epidermis and protecting it from external damage. It also has the ability to store moisture, regulate body temperature, and act as a sensory receptor, as well as regenerate the skin by interacting with the epidermis.

[0027] As used herein, the term "acellular dermal matrix (ADM)" refers to a bio-derived skin substitute in the form of an extracellular matrix (ECM) composed of collagen and elastin, etc., which is a dermal layer matrix obtained from human or animal skin through acellularization technology. The acellular dermal matrix may be used interchangeably with decellularized dermal tissue.

[0028] The acellular dermis described above is a biomaterial obtained by removing cells that can induce an immune response from skin isolated from an organism, and can be used to restore skin by transplantation in patients with skin defects caused by burns, traffic accidents, ulcers, etc. In addition, the acellular dermis, which is a skin tissue for human transplantation, can be used not only for the reconstruction of full-thickness skin, nasal septum, and dural defects of the brain and spinal cord, but also for reconstructive plastic surgery and cosmetic surgery such as correction of sunken scars, correction of hemifacial atrophy, nipple reconstruction, and lip augmentation.

[0029] The above acellular dermis must selectively remove only cellular antigens that are targets of immune response while maintaining various structural proteins and components without damaging the three-dimensional structure of the dermal layer within the skin tissue.

[0030] Furthermore, acellular dermis must possess appropriate elasticity and critical load to maintain stability during application to damaged tissue or skin to restore the damaged area, even when the treatment site is moved. Acellular dermis must be a material that does not damage the surrounding normal tissue and must be easy to treat.

[0031] Gelatin derived from human skin tissue and its production

[0032] In the present specification, the acellular dermal tissue coated with gelatin may also be used interchangeably as a basement membrane-mimicking acellular dermis or acellular dermis including a basement membrane-mimicking layer.

[0033] The term "gelatin" as used herein refers to a denatured protein obtained by hydrolyzing a natural polymer, such as collagen present in skin tissue.

[0034] In one specific example, the gelatin may be obtained by hydrolyzing collagen components within skin tissue, including but not limited to, acid or alkaline treatment and heat treatment. In this case, the gelatin may be obtained by hydrolyzing collagen components within skin tissue, including the following steps:

[0035] i) A step of treating decellularized dermal tissue with acid or alkali by supercritical fluid extraction;

[0036] ii) a step of neutralizing acid or alkali-treated dermal tissue; and

[0037] iii) A step of heat-treating the neutralized dermal tissue.

[0038] Specifically, the human skin tissue-derived gelatin of the present invention can be produced by hydrolyzing collagen components within skin tissue, as described below. More specifically, the method may additionally include the following decellularized dermal tissue production processes: a) to c):

[0039] a) A step of separating skin tissue separated from an object into the epidermis layer and the dermis layer.

[0040] The separation of the epidermis and dermis can be performed using methods known in the art. Typically, the separation of the epidermis and dermis can be performed using various proteolytic enzymes, such as dispase, thermolysin, and trypsin.

[0041] Additionally, the epidermal and dermal layers can be separated by varying the ionic strength of the solution. Specifically, the epidermal and dermal layers can be separated by treating with a 1 M or higher sodium chloride (NaCl) solution or a 20 mM EDTA solution at 37°C for 14 to 32 hours.

[0042] In one specific example, the separation of the epidermal layer and the dermal layer can be performed by treating with 1 M NaCl for 24 hours under temperature conditions of 37°C, but is not limited thereto.

[0043] b) A step of extracting the separated dermal layer with a supercritical fluid.

[0044] In the present invention, a supercritical fluid can be used to produce decellularized dermal tissue by extracting lipid components, specifically, phospholipid components, which are the main components of cell membranes, from dermal tissue separated from an individual based on solubility and decellularizing them.

[0045] The above supercritical fluid may be selected from the group consisting of carbon dioxide gas, ammonia gas, nitrogen gas, nitric oxide (NO) gas, nitrogen dioxide (NO2) gas, nitrous oxide (N2O) gas, sulfur dioxide gas, hydrogen gas, water vapor, saturated hydrocarbons, unsaturated hydrocarbons, aromatic compounds, and mixed gases thereof. Specifically, it may be carbon dioxide gas, but the type is not limited thereto as long as it is a supercritical fluid that can efficiently manufacture dermal tissue by removing most of the cells of the dermal tissue while maintaining the mechanical properties as well as the structural form of the dermal tissue. When carbon dioxide gas is used as the above supercritical fluid, carbon dioxide has a low critical temperature (31°C) and critical pressure (73 bar), so it can be easily adjusted to supercritical conditions, and has the advantages of being widely present in nature, colorless, odorless, harmless to the human body, and chemically stable.

[0046] In the present invention, the supercritical extraction step may be performed under pressure conditions of 0 to 1000 bar, 30 to 900 bar, 60 to 800 bar, 90 to 700 bar, 120 to 600 bar, 150 to 500 bar, or 200 to 400 bar.

[0047] Specifically, the pressure of the supercritical extraction step may be, but is not limited to, 0 bar or more, 50 bar or more, 100 bar or more, 150 bar or more, 200 bar or more, 250 bar or more, 300 bar or more, 350 bar or more, 400 bar or more, 450 bar or more, 500 bar or more, 550 bar or more, 600 bar or more, 650 bar or more, 700 bar or more, 750 bar or more, 800 bar or more, 850 bar or more, 900 bar or more, or 950 bar or more.

[0048] Additionally, the pressure of the supercritical extraction step is not limited thereto, but may be 1000 bar or less, 950 bar or less, 900 bar or less, 850 bar or less, 800 bar or less, 750 bar or less, 700 bar or less, 650 bar or less, 600 bar or less, 550 bar or less, 500 bar or less, 450 bar or less, 400 bar or less, 350 bar or less, 300 bar or less, 250 bar or less, 200 bar or less, 150 bar or less, 100 bar or less, or 50 bar or less.

[0049] The pressure conditions of the above supercritical extraction step are not limited to the range of conditions that can efficiently manufacture dermal tissue by removing most of the cells of the dermal tissue while preserving the mechanical properties of the dermal tissue and the structural form of the tissue.

[0050] In the above supercritical extraction step, in addition to the supercritical fluid, a cosolvent may be further included. The cosolvent may be one or more solvents selected from the group consisting of ethanol, water, methanol, hexane, petroleum ether, acetonitrile, acetone, ethyl acetate, and methylene chloride. Preferably, ethanol may be further included as a cosolvent.

[0051] The above co-solvent is added for the purpose of increasing the extractability and improving the solubility of the supercritical fluid, extracting lipids in the separated dermal tissue, specifically phospholipids of the cell membrane, thereby removing most of the cells of the dermal tissue. However, as long as the mechanical properties of the dermal tissue and the structural form of the tissue are preserved, the type thereof is not particularly limited.

[0052] In the present invention, the supercritical extraction step may be performed under temperature conditions of, but not limited to, about 31°C to about 40°C, about 31°C to about 39°C, about 32°C to about 38°C, about 33°C to about 37°C, about 34°C to about 36°C, or about 35°C.

[0053] In the present invention, the supercritical extraction step may be performed for, but is not limited to, about 3 hours or less. Preferably, the supercritical extraction step may be performed for, but is not limited to, about 60 minutes to about 180 minutes, about 70 minutes to about 170 minutes, about 80 minutes to about 160 minutes, about 90 minutes to about 150 minutes, about 100 minutes to about 140 minutes, about 110 minutes to about 130 minutes, or about 120 minutes.

[0054] c) A step of washing the dermal layer extracted with supercritical fluid with a phosphate buffer solution.

[0055] Washing using the above phosphate buffer solution can wash away any remaining solution and impurities present in the dermal tissue after supercritical fluid extraction.

[0056] i) A step of treating acid or alkali to decellularized dermal tissue by supercritical fluid extraction.

[0057] The collagen component in the dermal tissue can be primarily hydrolyzed by acid or alkali treatment of the decellularized dermal tissue through supercritical fluid extraction.

[0058] The above acid treatment may be performed by treatment with any one acid solution selected from the group consisting of, but not limited to, hydrochloric acid (HCl), sulfuric acid (H2SO4), nitric acid (HNO3), acetic acid (CH3OOH), and formic acid (HCOOH).

[0059] The above alkali treatment may be performed by treatment with any one basic solution selected from the group consisting of, but not limited to, sodium hydroxide (NaOH), potassium (K), barium (Ba), sodium carbonate (Na2CO3), sodium acetate (C2H3NaO2), calcium oxide (CaO), and calcium hydroxide (Ca(OH)2).

[0060] In one specific embodiment of the present invention, the acid treatment may be performed using hydrochloric acid. Specifically, the hydrochloric acid may have a concentration of, but is not limited to, about 0.05 N to about 1.5 N, about 0.07 N to about 1.3 N, about 0.09 N to about 1.1 N, or about 1 N. At this time, the pH value may be at least 1.5 or less. In addition, the hydrochloric acid may be treated for at least 3 hours or more, or at least 7 days or less. Specifically, the hydrochloric acid may be treated for, but is not limited to, at least 3 hours or more, at least 4 hours or more, at least 5 hours or more, at least 6 hours or more, at least 7 hours or more, at least 8 hours or more, at least 9 hours or more, at least 10 hours or more, at least 11 hours or more, or at least 12 hours or more. Additionally, the hydrochloric acid may be treated for, but is not limited to, at least 7 days or less, at least 6 days or less, at least 5 days or less, at least 4 days or less, at least 3 days or less, at least 2 days or less, or at least 1 day or less.

[0061] In one specific example of the present invention, the alkali treatment may be performed using a saturated solution of calcium hydroxide (Ca(OH)2). Specifically, the calcium hydroxide may have a concentration of, but is not limited to, about 0.05 N to about 1.5 N, about 0.07 N to about 1.3 N, about 0.09 N to about 1.1 N, or about 1 N. At this time, the pH value may be at least 11 or more. In addition, the calcium hydroxide may be treated for at least 3 hours or more, or at least 7 days or less. Specifically, the calcium hydroxide may be treated for, but is not limited to, at least 3 hours or more, at least 4 hours or more, at least 5 hours or more, at least 6 hours or more, at least 7 hours or more, at least 8 hours or more, at least 9 hours or more, at least 10 hours or more, at least 11 hours or more, or at least 12 hours or more. Additionally, the calcium hydroxide may be treated for, but is not limited to, at least 7 days or less, at least 6 days or less, at least 5 days or less, at least 4 days or less, at least 3 days or less, at least 2 days or less, or at least 1 day or less.

[0062] The collagen component in the decellularized dermal tissue may be reduced to a molecular weight ranging from about 50,000 to about 100,000 Da by hydrolysis according to the above acid or alkali treatment, but is not limited thereto.

[0063] ii) A step for neutralizing acid or alkali-treated dermal tissue.

[0064] The above acid or alkali treated dermal tissue can be neutralized by alkali or acid treatment.

[0065] The above neutralization may be performed through alkaline solution treatment if the neutralization is performed after acid treatment, or through acidic solution treatment if the neutralization is performed after alkaline treatment. In this case, the neutralization may be adjusted to a pH range of 6.0 to 8.0, but is not limited thereto.

[0066] Any basic solution or acidic solution known in the art can be used as each of the above basic solution and acidic solution.

[0067] The neutralization treatment may be performed by treating with an acid or alkaline solution having a concentration ranging from about 0.05 N to about 1.5 N for at least 0.5 hours. Specifically, when neutralization treatment is performed after acid treatment, it may be performed by treating within the concentration range of calcium hydroxide described above for at least 0.5 hours. In addition, when neutralization treatment is performed after alkaline treatment, it may be performed by treating within the concentration range of hydrochloric acid described above for at least 0.5 hours.

[0068] iii) Step of heat-treating the neutralized dermal tissue

[0069] The collagen component in the dermal tissue can be secondarily hydrolyzed by heat treatment of the above-mentioned neutralized dermal tissue.

[0070] The above heat treatment conditions are not particularly limited as long as collagen, a major component in the dermal tissue, is hydrolyzed and gelatinized, thereby causing the collagen to have a predetermined molecular weight.

[0071] The collagen component in the decellularized dermal tissue may be reduced to a molecular weight ranging from about 1,500 to about 30,000 Da by hydrolysis according to the above heat treatment, but is not limited thereto.

[0072] In one specific example of the present invention, the heat treatment may be performed using heat generated by radiation sterilization using an E-beam (Electron beam) or gamma rays, etc. In this case, the radiation sterilization using an E-beam (Electron beam) or gamma rays, etc. is not limited thereto, but may be irradiated at about 10 kGy to about 25 kGy.

[0073] Additionally, in one specific example of the present invention, the heat treatment may be performed by a process of heating at a temperature of about 40°C to about 80°C.

[0074] The above heat treatment may be performed for, but is not limited to, about 5 minutes to about 15 minutes.

[0075] The gelatin finally obtained after hydrolysis of collagen within the dermal tissue by the above heat treatment may be solidified through drying, but is not limited thereto. The drying may be performed using, but is not limited to, vacuum drying, hot air drying, heat drying, freeze drying, or other methods.

[0076] Additionally, the solidified gelatin may be powdered through a crushing or grinding process. The powdering may be performed using, but is not limited to, a grinder such as a freeze grinder or ultrasonic grinder, a mixer, a hand blender, a plunger mill, a cutting mill, a hammer mill, a ball mill, a disk mill, etc.

[0077] In one specific example of the present invention, the powdered gelatin can be prepared in a hydrated state by dissolving it in a physiologically acceptable solvent.

[0078] The physiologically acceptable solvents include, but are not limited to, distilled water, purified water, saline, phosphate buffer solution (PBS), HBSS (Hank's balanced salt solution), TBS (Tris buffered saline), TAPS (N-Tris(hydroxy-methyl)methyl-3-aminopropanesulfonic acid) buffer solution, Bicine (N,N-Bis(2-hydroxyethyl) glycine) buffer solution, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer solution, TES (NTris(hydroxymethyl)methyl-2-aminoethanesulfonicd acid) buffer solution, PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid) buffer solution, cacodylate buffer solution, MES (2-(N-morpholino)ethanesulfonic acid) buffer solution, MEM (Minimum Essential Media), DMEM (Dulbecco's Modified Eagle Media), RPMI1640, IMDM (Iscove's Modified Dulbecco's Media), Defined Keratinocyte-SFM (without BPE (bovine pituitary extract)), Keratinocyte-SFM (with BPE), KnockOut D-MEM, AmnioMAX-II Complete Medium, AmnioMAX-C100 Complete Medium, and mixtures thereof.

[0079] In one specific embodiment of the present invention, the gelatin may be included in an amount of about 4 wt% to about 30 wt% based on the total weight of the physiologically acceptable solvent in which it is dissolved. Preferably, the gelatin may be included in an amount of about 6 wt% to about 29 wt%, about 8 wt% to about 28 wt%, about 10 wt% to about 27 wt%, about 12 wt% to about 26 wt%, about 14 wt% to about 25 wt%, about 16 wt% to about 24 wt%, about 18 wt% to about 23 wt%, about 19 wt% to about 22 wt%, or about 20 wt% to about 21 wt%, but is not limited thereto.

[0080] Gelatin coating on acellular dermal tissue

[0081] In the present specification, the acellular dermal tissue coated with gelatin can be manufactured by applying, i.e. coating, the gelatin derived from human skin tissue as described above to decellularized dermal tissue.

[0082] As used herein, the term "skin tissue" is composed of the epidermis, the outermost layer, the dermis layer beneath it, and subcutaneous tissue. The epidermis layer comprises epithelial cells differentiated into multiple layers from the basement membrane (BM), which firmly connects the epidermis and dermis, as well as melanocytes and immune cells. The dermis layer, beneath the epidermis, is primarily composed of fibroblasts and an extracellular matrix composed of collagen and elastin.

[0083] In the present invention, the skin tissue is not limited thereto, but refers to a dermal layer from which the epidermal layer including the basement membrane of the skin tissue has been removed, and the terms skin tissue and dermal layer can be used interchangeably. The skin tissue may be skin tissue of homologous or heterologous origin. The homologous species refers to a human, and the heterologous species may be a mammal including, but not limited to, an animal other than a human, such as a monkey, a chimpanzee, an orangutan, a horse, a cow, a pig, a dog, and a rabbit.

[0084] In one specific example, the skin tissue may be decellularized dermal tissue derived from skin tissue isolated from an individual.

[0085] In one specific example, the decellularized dermal tissue can be used as a substrate on which the gelatin is coated, and can be cut to a size suitable for the application site as an acellular dermal tissue graft. Furthermore, the decellularized dermal tissue can also be used as a raw material for producing gelatin derived from human skin tissue.

[0086] Typically, the dermis contains more than 90% collagen, but is not limited thereto. However, the decellularized dermal tissue according to the present invention can be hydrolyzed to obtain gelatin (MW of about 1,000 to about 100,000 Da) having a triple helix structure by any treatment, such as treatment with acid, alkali, collagenase, or heating, thereby destroying the triple helix and thus inducing collagen denaturation.

[0087] The term "decellularization," as used herein, refers to a novel method for producing artificial scaffolds by removing cells from an entire organ while maintaining the original structure of the desired transplanted tissue or organ. During the decellularization process, cellular components are removed from the tissue, but the extracellular matrix and some growth factor proteins are preserved. Therefore, the various extracellular matrix components, including collagen, fibronectin, and elastin, preserved within the decellularized tissue provide a three-dimensional microenvironment similar to that within the intact tissue, thereby enhancing the survival, proliferation, and differentiation of cultured cells.

[0088] In the present invention, decellularization can be performed by supercritical fluid extraction without surfactant treatment, but is not limited thereto.

[0089] The term "supercritical fluid extraction" or "supercritical extraction" as used herein refers to a method of separating substances using a supercritical fluid that has properties intermediate between those of a gas and a liquid that exist above its critical point, i.e., its critical temperature and pressure. The supercritical fluid extraction utilizes the principle of solvent extraction, in which soluble components contained in a raw material are dissolved in a supercritical fluid due to the difference in solubility between the raw material to be extracted and the supercritical fluid, and the principle of distillation, in which solute molecules contained in the raw material move from a high-density condensed phase to a low-density expanded phase, the supercritical fluid, as an evaporation phenomenon.

[0090] The term "supercritical fluid," as used herein, refers to a gaseous substance under normal conditions but a fluid at a critical temperature and pressure above the critical temperature. Suitable supercritical fluids for use in the present invention are not particularly limited, but include, for example, carbon dioxide, nitrogen, nitrous oxide, methane, ethylene, propane, and propylene. Preferably, carbon dioxide, which has a critical temperature of 31°C and a critical pressure of 72.8 atm, can be used.

[0091] In the present invention, decellularization may be performed by adding a "co-solvent" in addition to the supercritical fluid during supercritical fluid extraction. The co-solvent may be added for purposes such as increasing the extractability and improving the solubility of the supercritical fluid, and may include, but is not limited to, ethanol, methanol, petroleum ether, acetonitrile, hexane, and the like as the co-solvent. In this case, the co-solvent may preferably be ethanol.

[0092] The decellularized dermal tissue of the present invention, i.e., acellular dermal tissue, may have cells removed by 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%, compared to the original tissue isolated from the subject, i.e., compared to tissue that has not been decellularized.

[0093] In one specific example, gelatin-coated acellular dermal tissue can be produced by coating the decellularized dermal tissue with gelatin derived from human skin tissue as described above.

[0094] In the present invention, the gelatin coated on the acellular dermal tissue can function as an artificial basement membrane layer that mimics the basement membrane layer that functions as a support for human skin tissue.

[0095] As used herein, the term "basement membrane" refers to a thin membrane, approximately 50 to 100 nm thick, located between the epidermis and dermis, composed of extracellular matrix components such as laminin, type IV collagen, and proteoglycans. The basement membrane functions as a nutrient supply and protective barrier between the epidermis and dermis. Furthermore, it facilitates the attachment of epidermal cells, acting as a support, and not only regulates epidermal formation but also participates in the wound healing process.

[0096] The gelatin functioning as the artificial basement membrane layer may be coated on one or both sides of the acellular dermal tissue, but is not limited thereto.

[0097] The term "coating" as used herein refers to forming a new layer of a certain thickness by modifying a specific substance on a target surface. The target surface and the coating material may be modified through various chemical bonds, such as ionic bonds, covalent bonds, and hydrogen bonds. In the present invention, the target surface may refer to the surface of acellular dermal tissue, and the coating material may refer to gelatin derived from human skin tissue.

[0098] In one specific example of the present invention, the acellular dermal tissue can be coated with gelatin by cross-linking the acellular dermal tissue by uniformly applying gelatin to one or both sides of the acellular dermal tissue at least once, but is not limited thereto.

[0099] Specifically, the acellular dermal tissue can be coated by cross-linking gelatin by applying gelatin using, but not limited to, dip coating, spray coating, ultrasonic spray coating, aerosol printing, etc.

[0100] The process of applying gelatin to the above acellular dermal tissue and cross-linking treatment is not limited thereto, but can be performed repeatedly to obtain acellular dermal tissue of a desired thickness.

[0101] As used herein, the term "crosslinking" refers to a bonding reaction that links one polymer chain to the same or different chains, and a "crosslinker" refers to any compound capable of inducing the bonding reaction. In the bonding reaction, the linkage may take the form of a covalent bond or an ionic bond, and as the number of crosslinking bonds increases, solubility and thermoplasticity decrease, but the mechanical strength increases and the elasticity of the polymer compound increases.

[0102] In one specific example, the coating of gelatin on the acellular dermal tissue can be performed by treating with any one cross-linking agent selected from the group consisting of formaldehyde, glutaraldehyde, glyceraldehyde, dialdehyde cellulose (DAC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM), spermine, putrescine, spermidine, 1,4-butandiol diglycidyl ether (BDDE), and ethylene glycol diglycidyl ether (EGDGE). and is not limited to these.

[0103] basement membrane-mimicking acellular dermis

[0104] In the present specification, the acellular dermal tissue coated with gelatin, which functions as the artificial basement membrane layer, can be a skin substitute with improved mechanical properties while maintaining histological form, and can have the characteristics described below.

[0105] In one specific example of the present invention, the acellular dermal tissue may have a thickness of, but is not limited to, about 0.3 mm to about 5.5 mm. The thickness of the acellular dermal tissue is, for example, about 0.3 mm to 0.9 mm, about 0.4 mm to about 5.5 mm, about 0.5 mm to about 5.4 mm, about 0.6 mm to about 5.4 mm, about 0.7 mm to about 5.3 mm, about 0.8 mm to about 5.3 mm, about 0.9 mm to about 5.2 mm, about 1.0 mm to about 5.2 mm, about 1.0 mm to about 1.4 mm, about 1.5 mm to about 5.1 mm, about 1.5 mm to 1.9 mm, about 2.0 mm to about 5.1 mm, about 2.0 mm to about 2.4 mm, about 2.5 mm to about 5.0 mm, about 2.5 mm to about 2.9 mm, about 3.0 mm to about 5.0 mm, about 3.0 mm to about It can be 3.4 mm, about 3.5 mm to about 4.9 mm, about 3.5 mm to about 3.9 mm, about 3.7 mm to about 4.9 mm, about 3.8 mm to about 4.8 mm, about 3.9 mm to about 4.8 mm, about 4.0 mm to about 4.7 mm, about 4.0 mm to about 4.4 mm, about 4.1 mm to about 4.7 mm, about 4.2 mm to about 4.6 mm, about 4.3 mm to about 4.6 mm, about 4.4 mm to about 4.5 mm, or about 4.5 mm to about 4.9 mm.

[0106] In one specific example of the present invention, the acellular dermal tissue may have a viscosity of, but is not limited to, about 8,000 Pa·s to about 9,500 Pa·s. The viscosity of the acellular dermal tissue may be, for example, about 8,050 Pa·s to about 9,400 Pa·s, about 8,100 Pa·s to about 9,300 Pa·s, about 8,150 Pa·s to about 9,200 Pa·s, about 8,200 Pa·s to about 9,100 Pa·s, about 8,250 Pa·s to about 9,000 Pa·s, about 8,300 Pa·s to about 8,900 Pa·s, about 8,350 Pa·s to about 8,800 Pa·s, about 8,400 Pa·s to about 8,700 Pa·s, about 8,450 Pa·s to about 8,600 Pa·s, or about 8,500 Pa·s to about 8,550 Pa·s.

[0107] The acellular dermal tissue according to the present invention may contain a collagen content of about 350 μg / mg or more based on dry weight. Preferably, the acellular dermal tissue may contain about 350 μg / mg to about 500 μg / mg, about 360 μg / mg to about 490 μg / mg, about 370 μg / mg to about 480 μg / mg, about 380 μg / mg to about 470 μg / mg, about 390 μg / mg to about 460 μg / mg, about 400 μg / mg to about 450 μg / mg, about 410 μg / mg to about 440 μg / mg, or about 420 μg / mg to about 435 μg / mg, but is not limited thereto.

[0108] The acellular dermal tissue according to the present invention may contain a collagen content of about 70% or more compared to the original tissue. Preferably, it may contain about 70% to about 100%, about 75% to about 99%, about 80% to about 97%, about 82% to about 95%, about 84% to about 93%, about 85% to about 91%, about 86% to about 90%, or about 87% to about 89%, but is not limited thereto.

[0109] The gelatin-coated acellular dermal tissue according to the present invention is an acellular dermal tissue cross-linked by uniformly applying gelatin to the surface of the acellular dermal tissue without a basement membrane layer, and has superior mechanical properties compared to acellular dermal tissue from which the basement membrane has not been removed. Accordingly, the gelatin-coated acellular dermal tissue according to the present invention not only has superior mechanical properties and thus facilitates manipulation during transplantation surgery, but also eliminates toxicity and graft rejection through decellularization by supercritical fluid extraction without the use of a surfactant, and thus can be utilized as a graft material for treating patients with damaged skin tissue.

[0110] Method for producing acellular dermal tissue coated with gelatin

[0111] Another aspect of the present invention provides a method for producing gelatin-coated acellular dermal tissue. The method for producing gelatin-coated acellular dermal tissue may comprise the following steps:

[0112] a) A step of decellularizing dermal tissue with a supercritical fluid to obtain decellularized dermal tissue;

[0113] b) a step of hydrolyzing collagen components in decellularized dermal tissue to obtain gelatin; and

[0114] c) A step of coating the decellularized dermal tissue obtained in step a) with the gelatin obtained in step b).

[0115] The above “gelatin”, “coating”, “acellular dermis”, “supercritical fluid” and “decellularization” are as described above.

[0116] One specific example of manufacturing the gelatin-coated acellular dermal tissue of the present invention may be as follows:

[0117] a) A step of decellularizing dermal tissue with a supercritical fluid to obtain decellularized dermal tissue.

[0118] The cells of the dermal tissue can be decellularized through the above supercritical fluid extraction. The decellularization through the above supercritical fluid extraction is as described in step b) of the above 'Gelatin derived from human skin tissue and its preparation'.

[0119] b) A step of hydrolyzing collagen components in decellularized dermal tissue to obtain gelatin.

[0120] Gelatin can be obtained by inducing denaturation of the collagen component within the decellularized dermal tissue by hydrolyzing the collagen component through acid or alkali treatment and heat treatment. The gelatin obtaining process is as described throughout steps i) to iii) of the above 'Gelatin derived from human skin tissue and preparation thereof'.

[0121] c) A step of coating the decellularized dermal tissue obtained in step a) with the gelatin obtained in step b).

[0122] By coating the gelatin obtained in step b) on the surface of the decellularized dermal tissue, a basement membrane-mimicking layer that functions as a nutrient supply and protective barrier and as a skin support can be formed.

[0123] The coating may be cross-linked by applying gelatin to one or both sides of the dermal tissue. Specifically, the coating may be cross-linked by applying gelatin to one or both sides of the dermal tissue at least once by a method such as dip coating, spray coating, ultrasonic spray coating, or aerosol printing, but is not limited thereto.

[0124] The above cross-linking treatment can be performed using any one cross-linking agent selected from the group consisting of formaldehyde, glutaraldehyde, glyceraldehyde, dialdehyde cellulose (DAC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM), spermine, putrescine, spermidine, 1,4-butandiol diglycidyl ether (BDDE), and ethylene glycol diglycidyl ether (EGDGE).

[0125] After obtaining acellular dermal tissue of the desired thickness through repeated gelatin application and cross-linking processes, an additional washing process can be performed. The cross-linking agent used is not particularly limited as long as the toxicity of the cross-linking agent is removed through the washing process.

[0126] The above acellular dermal tissue can be sterilized after the gelatin coating step of step c).

[0127] As used herein, the term "sterilization" refers to the process of killing or eliminating all vegetative cells and spores of fungi, bacteria, viruses, and protozoa present on and within an object, thereby rendering it sterile. Sterilization methods include physical, chemical, and mechanical methods, and an appropriate sterilization method can be selected and implemented based on the characteristics of the object to be sterilized or its composition.

[0128] In one specific example, the gelatin-coated acellular dermal tissue, also known as basement membrane-mimicking acellular dermal tissue, can be sterilized using eBeam or gamma rays.

[0129] In one specific example, the sterilized basement membrane-mimicking acellular dermis may be further subjected to, but is not limited to, hermetically sealed packaging.

[0130] The basement membrane-mimicking acellular dermis manufactured through the above series of processes may have a thickness of, but not limited to, about 0.3 mm to about 5.5 mm, and a viscosity of, but not limited to, about 8,000 Pa·s to about 9,500 Pa·s.

[0131] In addition, the acellular dermal tissue may contain, but is not limited to, about 350 μg / mg or more of collagen based on dry weight, and may contain about 70% or more of collagen compared to the original tissue.

[0132] Uses of gelatin-coated acellular dermal tissue

[0133] Another aspect of the present invention provides a skin damage treatment agent comprising the acellular dermal tissue coated with the gelatin or the acellular dermal tissue manufactured according to the manufacturing method.

[0134] The above “gelatin”, “coating” and “acellular dermis” are as described above.

[0135] The above gelatin-coated acellular dermal tissue can be used as a skin substitute, i.e., a skin damage treatment agent, in the fields of cosmetic surgery and reconstructive surgery for skin ulcers or skin damage caused by extensive burns, diabetes, etc., and skin tissue depression caused by accidents, trauma, aging, etc.

[0136] Specifically, the acellular dermal tissue according to the present invention forms an artificial support layer simulating a basement membrane layer by coating gelatin derived from human skin tissue on dermal tissue without a basement membrane layer, thereby realizing excellent mechanical properties, while at the same time eliminating toxicity and graft rejection by decellularization through supercritical fluid extraction without using a surfactant, and can be used in the fields of dermatology, plastic surgery, gynecology, surgery, neurosurgery, urology, and otolaryngology as a skin restoration agent for burns, traffic accidents, ulcers, etc., a full-thickness skin reconstruction agent, a nasal septum reconstruction agent, a reconstruction agent for dural defects of the brain and spinal cord, a depression correction agent, a scar correction agent, and a hemifacial atrophy correction agent.

[0137] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended only to illustrate the present invention, and the scope of the present invention is not limited to these examples.

[0138] Example 1. Preparation of acellular dermal tissue coated with gelatin

[0139] The manufacturing process of gelatin-coated acellular dermal tissue is schematically illustrated in a series of steps in FIGS. 1 and 2. Specifically, the manufacturing of gelatin-coated acellular dermal tissue (also referred to as 'acellular dermal matrix (ADM) mimicking basement membrane' or 'acellular dermal tissue including a basement membrane-mimicking layer') is as illustrated in Examples 1.1 to 1.3 below.

[0140] Example 1.1. Preparation of acellular dermal tissue using a supercritical fluid extraction process.

[0141] A supercritical fluid extraction process was performed to decellularize skin tissue. At this time, human skin tissue without the basement membrane (BM) that exists between the epidermal layer and the dermal layer, i.e. dermal tissue with the basement membrane and epidermal layer removed, was used as the raw material for the product (Solvita; CTS, Community Blood Center and Community Tissue Services, USA) (Fig. 2a). The dermal tissue was placed in the extraction tank of a supercritical extraction system (SES), and supercritical fluid carbon dioxide and co-solvent ethanol were injected together into the extraction tank. Then, the dermal tissue was decellularized by supercritical treatment for 1 to 3 hours under pressure conditions of 72.8 atmospheres (bar) and temperature conditions of 31°C.

[0142] Afterwards, the decellularized dermal tissue was washed with sterilized PBS at room temperature for 24 hours. After washing, acellular dermal tissue of the ready-to-use (RTU) type was obtained.

[0143] Example 1.2. Preparation of gelatin derived from human skin tissue

[0144] Human skin tissue-derived gelatin was prepared using a portion (159 g) of the acellular dermal tissue prepared in Example 1.1 above. Specifically, the acellular dermal tissue was treated with an acid or alkali solution, such as 4% hydrochloric acid (HCl; pH <1.5) as an acidic solution or a saturated calcium hydroxide solution (pH >10) as a basic solution, at room temperature for 24 hours to denature the collagen component in the dermal tissue (Fig. 2b).

[0145] Acid- or alkali-treated skin dermis was neutralized. At this time, neutralization was performed using an alkaline solution for acid treatment, and an acidic solution for alkaline treatment. The neutralization was adjusted so that the pH value of the raw material was 6.5 to 7.5. After neutralization, the remaining acidic or alkaline solution was removed by washing. Next, in the presence of 5 mL of distilled water per 1 mg of skin tissue, the collagen component in the dermis was hydrolyzed and gelatin was extracted by heat treatment at stages of 50°C, 60°C, 70°C, 80°C, and 90°C for approximately 1 day in order to hydrolyze the collagen component in the dermis and extract gelatin (Fig. 2c). The gelatin solution extracted by heat treatment at each temperature stage was collected in a standby tank and completely dried in a vacuum desiccator or hot air desiccator (70°C) to obtain gelatin in solid form (29 g). Afterwards, it was crushed into a size of approximately 0.5 cm or less and stored until use (left side of Fig. 2d and e).

[0146] Example 1.3. Preparation of basement membrane-mimicking acellular dermis

[0147] The gelatin powder obtained in Example 1.2 was dissolved in distilled water at a concentration of 20% (w / w), and then 1% formaldehyde was added as a cross-linking agent. Immediately after addition, it was evenly applied to one or both sides of the acellular dermal tissue prepared in Example 1.1. Thereafter, cross-linking treatment was performed at room temperature for approximately 30 seconds to 1 minute, followed by washing three times with distilled water, sealing, and E-beam sterilization (15 kGy) (right side of e in Fig. 2 and sample ③ in Fig. 3).

[0148] Preparation Example 1. Preparation of samples for analysis

[0149] To evaluate the physicochemical and biochemical properties of the gelatin-coated acellular dermal tissue prepared in Example 1, analytical samples including a control group were prepared (Table 1). Specifically, acellular dermis with the basement membrane intact (ADM sample with BM, control group 1), acellular dermis without a basement membrane and without gelatin cross-linking (ADM sample without BM, control group 2), and acellular dermis without a basement membrane and cross-linked with gelatin (ADM sample without BM + gelatin cross-linking, test group) samples were prepared.

[0150] Control group 1Control group 2Test groupBasement membrane (BM)○××Gelatin××○

[0151] Experimental Example 1. Observation of the appearance of acellular dermal tissue coated with gelatin.

[0152] Experimental Example 1.1. Thickness Measurement

[0153] The thickness of the gelatin-coated acellular dermal tissue (ADM without BM + gelatin cross-linked sample) prepared in Example 1 was measured using a thickness gauge (Dial Indicator Thickness Gauge, Mitutoyo). As positive controls, acellular dermal tissue that had been decellularized but had the basement membrane layer not removed (Control Group 1; ADM sample with BM) and acellular dermal tissue from which the basement membrane layer had been removed (Control Group 2; ADM sample without BM) were used.

[0154] Specifically, the smooth movement of the thickness gauge's needle, short needle, and spindle was verified, and a space for the measurement sample was created using a lever. Each sample to be measured was then placed in the space, and the value was recorded at the position where the needle stopped, paying attention to parallax. Using the same method, measurements were taken 10 times at different locations on each sample. The average of the measured values ​​was calculated to determine the thickness of each sample. The results are shown in Figure 4 and Table 2 below.

[0155] Sample thickness (mm) [n=10] Control group 13.3 Control group 22.3 Test group 4.5

[0156] Experimental Example 1.2. Visual Elasticity Measurement

[0157] The elasticity of the gelatin-coated acellular dermal tissue (ADM without BM + gelatin cross-linked sample) manufactured in Example 1 was visually confirmed using a self-made protractor. At this time, acellular dermal tissue in which decellularization was performed but the basement membrane layer was not removed (Control Group 1: ADM sample with BM) and acellular dermal tissue in which the basement membrane layer was removed (Control Group 2: ADM sample without BM) were used as positive controls.

[0158] As a result of the verification, as shown in Fig. 5, the test group (ADM without BM + gelatin cross-linked sample) was visually confirmed to have the highest elasticity. This is expected to be due to the difference in thickness, i.e., it was found that a basement membrane-mimicking layer was well formed on the acellular dermal tissue without a basement membrane layer.

[0159] Through the above confirmation results, it was found that it showed an excellent level of elasticity compared to control group 1 in which a basement membrane exists, and thus could be usefully used as a graft material that is easy to manipulate during transplantation.

[0160] Experimental Example 2. Cytotoxicity Evaluation of Gelatin-Coated Acellular Dermal Tissue

[0161] The cytotoxicity of the gelatin-coated acellular dermal tissue (ADM without BM + gelatin cross-linked sample) prepared in Example 1 was evaluated. Specifically, the cytotoxicity evaluation was performed using the L929 mouse fibroblast cell line (Korea Cell Line Bank), which is known in the art, and 1 × 10 4The cells were cultured for one day at a cell count of 10 cells / well. In addition, the cytotoxicity evaluation was performed according to the tissue dissolution method (applied by the KCL accredited testing agency protocol, ISO10993-5), which involves immersing the tissue in a cell culture medium at 37°C for 24 hours to dissolve internal tissue substances into the culture medium. Specifically, after 24 hours of culture after treatment with 0% (control), 100%, 50%, 25%, and 12.5% ​​of the dissolution medium, the cells were evaluated using the MTT assay. At this time, if the viability decreased to less than 70% of the blank test solution according to the standards of the accredited testing agency, it was evaluated as potentially cytotoxic.

[0162] Sample survival rate (%), coefficient of variation (CV), blank test solution 1000.6, control group 1950.4, control group 2950.3, test group 930.7

[0163] As a result of the cytotoxicity evaluation, all samples treated with 100% concentration of the three test substances showed a cell viability rate of over 90%. Accordingly, in the test group sample manufactured by applying gelatin extracted from human skin tissue to acellular dermal tissue (ADM) from which the basement membrane layer (BM) had been removed and then cross-linking it, i.e., the BM-mimetic ADM sample, it was confirmed that there was no cytotoxicity caused by cross-linked gelatin (Fig. 6 and Table 3).

[0164] Accordingly, it was found that acellular dermal tissue (ADM) with the basement membrane layer (BM) removed and cross-linked with gelatin extracted from human skin tissue is non-cytotoxic and can be usefully used as a graft material.

[0165] Experimental Example 3. Viscoelasticity Measurement of Gelatin-Coated Acellular Dermal Tissue

[0166] In order to confirm whether the gelatin-coated acellular dermal tissue (ADM without BM + gelatin cross-linked sample) manufactured in the above Example 1 had suitable properties as a graft material, viscosity measurements were performed. The viscosity measurements were performed using a rheometer (MCR 302e model (Anton Paar)). The measuring device was a PP50 with a diameter of 5 mm, and the measurement conditions were 0.1 Hz, 25℃, and repeated 4 times per sample. At this time, acellular dermal tissue that had been decellularized but had the basement membrane layer not removed (Control group 1: ADM sample with BM) and acellular dermal tissue from which the basement membrane layer had been removed (Control group 2: ADM sample without BM) were used as positive controls.

[0167] The homogeneity of physical properties was confirmed for Control Group 1 (ADM sample with BM), Control Group 2 (ADM sample without BM), and Test Group (ADM without M + gelatin cross-linked sample). As a result, it was confirmed that the test group sample, i.e., the BM-mimicking ADM sample, which was prepared by applying gelatin extracted from human skin tissue to acellular dermal tissue (ADM) from which the basement membrane layer (BM) had been removed and then cross-linking it, exhibited viscoelasticity similar to that of the acellular dermal tissue sample from which the basement membrane layer had not been removed (Fig. 7a, Fig. 7b, and Table 4).

[0168] Accordingly, the gelatin-coated acellular dermal tissue has a certain level of viscoelasticity or higher, and thus can be usefully used as a graft material that is easy to manipulate during transplantation.

[0169] Evaluation items Control group 1 Control group 2 Test group Complex viscosity [1Hz, Pa.s] 9,643 1,985 8,540 % compared to Control group 1 100 2189

[0170] Experimental Example 4. Measurement of collagen content in gelatin-coated acellular dermal tissue.

[0171] To determine the content of extracellular matrix proteins important for tissue regeneration, collagen content was measured using Biocolor's Sircol Insoluble Collagen Assay Kit. Native tissue that had not been decellularized or gelatin-crosslinked was used as a negative control. Acellular dermal tissue that had been decellularized but had the basement membrane layer not removed (Control Group 1: ADM sample with BM) and acellular dermal tissue with the basement membrane layer removed (Control Group 2: ADM sample without BM) were used as positive controls. Gelatin-coated acellular dermal tissue (ADM sample without BM + gelatin-crosslinked sample) was used as a test group. Collagen content was determined for each sample.

[0172] As a result of measuring collagen content, it was confirmed that the test group sample, i.e., the BM-mimicking ADM sample, which was prepared by applying gelatin extracted from human skin tissue to acellular dermal tissue (ADM) from which the basement membrane layer (BM) had been removed and then cross-linking, had a significantly higher collagen content than the control group 1 and control group 2 (Fig. 8 and Table 5).

[0173] Sample average (AV) Standard deviation (SD) % (compared to original tissue) Native tissue 49324100 Control group 12971260 Control group 21501530 Test group 4346988

[0174] Experimental Example 5. Histological Analysis of Gelatin-Coated Acellular Dermal Tissue

[0175] Experimental Example 5.1. Histological Analysis of Acellular Dermal Tissue Using H&E Staining

[0176] To perform histological analysis on the gelatin-coated acellular dermal tissue (BM-free ADM + gelatin cross-linked sample) prepared in Example 1 above, H&E staining (Hematoxylin & Eosin staining) was performed.

[0177] As a result of H&E staining, it was confirmed that the BM-mimicking ADM test group sample, which was prepared by applying gelatin derived from human skin tissue to acellular dermal tissue with the basement membrane layer removed and then cross-linking, exhibited a basement membrane morphology similar to that of the ADM sample (control group 1) in which decellularization was performed but the basement membrane layer was not removed (Fig. 9).

[0178] Experimental Example 5.2. Histological Analysis of Acellular Dermal Tissue Using MT Staining

[0179] To perform a histological analysis on the gelatin-coated acellular dermal tissue (ADM without BM + gelatin cross-linked sample) prepared in Example 1 above, MT staining (Masson trichrom straining) was performed.

[0180] MT staining results showed that the BM-mimicking ADM test group samples, which were prepared by applying human skin tissue-derived gelatin to acellular dermal tissue with the basement membrane layer removed and then cross-linking, exhibited a basement membrane morphology similar to that of the ADM sample (control group 1) in which decellularization was performed but the basement membrane layer was not removed. Meanwhile, it was confirmed that decellularization was also well achieved in both the control and test group samples (Fig. 10).

[0181] Through the above series of results, it was found that the gelatin-coated acellular dermal tissue not only showed a histologically similar morphology to the acellular dermal tissue without the basement membrane layer removed, but also had certain physical properties and could exclude the induction of an immune rejection response through excellent decellularization, so it could be usefully used as a transplant material with both ease of handling and safety.

Claims

1. Acellular dermal tissue coated with gelatin.

2. In paragraph 1, The above gelatin is an acellular dermal tissue coated on one or both sides of the acellular dermal tissue.

3. In paragraph 1, An acellular dermal tissue, wherein the gelatin is cross-linked with any one cross-linking agent selected from the group consisting of formaldehyde, glutaraldehyde, glyceraldehyde, dialdehyde cellulose (DAC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM), spermine, putrescine, spermidine, 1,4-butandiol diglycidyl ether (BDDE), and ethylene glycol diglycidyl ether (EGDGE).

4. In paragraph 1, The above gelatin is an acellular dermal tissue that mimics the basement membrane layer.

5. In paragraph 1, The above gelatin is an acellular dermal tissue derived from homologous or heterologous skin tissue.

6. In paragraph 1, The above gelatin is an acellular dermal tissue obtained by hydrolyzing collagen components within skin tissue.

7. In paragraph 1, The above gelatin is an acellular dermal tissue obtained by a method comprising the following steps: i) A step of treating decellularized dermal tissue with acid or alkali by supercritical fluid extraction; ii) a step of neutralizing acid or alkali-treated dermal tissue; and iii) A step of heat-treating the neutralized dermal tissue.

8. In paragraph 1, An acellular dermal tissue, wherein the gelatin is contained in an amount of 4% to 30% by weight based on the total weight of the physiologically acceptable solvent in which it is dissolved.

9. In paragraph 1, The above acellular dermal tissue has a thickness of 0.3 mm to 5.5 mm.

10. In paragraph 1, The above acellular dermal tissue has a viscosity of 8,000 Pa·s to 9,500 Pa·s.

11. In paragraph 1, The above acellular dermal tissue has a collagen content of 350 μg / mg to 500 μg / mg based on dry weight.

12. In paragraph 1, The above acellular dermal tissue is an acellular dermal tissue having a collagen content of 70% to 100% compared to the original tissue.

13. In paragraph 1, The above acellular dermal tissue is acellular dermal tissue that has been decellularized by supercritical fluid extraction.

14. A method for producing acellular dermal tissue coated with gelatin, comprising the following steps: a) A step of decellularizing dermal tissue with a supercritical fluid to obtain decellularized dermal tissue; b) a step of hydrolyzing collagen components in decellularized dermal tissue to obtain gelatin; and c) A step of coating the decellularized dermal tissue obtained in step a) with the gelatin obtained in step b).

15. In paragraph 14, A manufacturing method in which the coating of the above step c) is performed by cross-linking gelatin by applying it to one or both sides of the dermal tissue.

16. In paragraph 15, A manufacturing method wherein the above cross-linked gelatin mimics a basement membrane layer.

17. In paragraph 14, d) A manufacturing method further comprising a step of sterilizing the gelatin-coated dermal tissue.

18. In paragraph 14, A manufacturing method, wherein the above-mentioned acellular dermal tissue has a thickness of 0.3 mm to 5.5 mm.

19. In paragraph 14, A manufacturing method wherein the above-mentioned acellular dermal tissue has a viscosity of 8,000 Pa·s to 9,500 Pa·s.

20. In paragraph 14, A manufacturing method, wherein the above acellular dermal tissue has a collagen content of 350 μg / mg to 500 μg / mg based on dry weight.

21. In paragraph 14, A manufacturing method wherein the above acellular dermal tissue has a collagen content of 70% to 100% compared to the original tissue.

22. A skin damage treatment agent comprising acellular dermal tissue coated with gelatin according to Article 1 or acellular dermal tissue manufactured according to the manufacturing method of Article 14.

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