Acellular dermal tissue prepared by supercritical fluid extraction and application thereof

Abstract

The invention relates to a decellularized acellular dermal tissue prepared by supercritical fluid extraction. More specifically, the present invention relates to a cell-free dermal tissue having improved extracellular matrix retention levels and optimized mechanical properties (e.g., critical loading and elastic restoring force) by extraction using supercritical fluids. The acellular dermal tissue of the present invention is obtained by decellularization, is not treated with a surfactant, thus retains mechanical properties, does not have toxicity caused by a residual surfactant, and minimizes transplant rejection, and thus can be effectively used for treating a patient with damaged skin tissue.

Description

Technical Field

[0001] The present invention relates to an acellular dermal tissue prepared by supercritical fluid extraction. More specifically, the present invention relates to an acellular dermal tissue having an improved extracellular matrix retention level and optimized mechanical properties (such as critical load and elastic recovery force) by using supercritical fluid extraction. Background Art

[0002] The human skin tissue consists of the outermost epidermis, the dermis and subcutaneous tissue. The epidermis is composed of several layers of epithelial cells, melanocytes and immune cells differentiated from the basement membrane that firmly connects the epidermis and dermis. The dermis below the epidermis is mainly composed of fibroblasts and extracellular matrix composed of collagen, elastin and the like.

[0003] Skin tissue or internal organ tissue may be partially damaged due to burns, trauma, ulcers, etc. In this case, the method of transplanting one's own skin tissue or internal organ tissue is used to heal the damaged tissue or for reconstructive plastic surgery. In this case, the recipient must bear the burden of surgery to additionally extract his or her own skin tissue or organ tissue, which may be dangerous if the recipient is in poor health. In addition, there are methods of transplantation using xenografts or synthetic biomaterials. However, these methods may cause immune rejection reactions, resulting in long-term inflammatory reactions after transplantation, resulting in the need for reoperation.

[0004] In order to solve the above problems, Korean Patent Registration Nos. 10-0469661 and 10-0791502 disclose methods for preparing acellular dermis for transplantation from skin tissue extracted from a donor. However, these methods follow a surfactant treatment process. Surfactant treatment may cause problems such as denaturation of proteins such as collagen and destruction of growth factors. In addition, the structural morphology and histological morphology are difficult to maintain, and mechanical properties such as critical load and elasticity may be weakened. Moreover, the residual surfactant is not easy to remove, and if it is not removed thoroughly, it may be toxic.

[0005] In addition, in order to prepare acellular dermal matrix, proteolytic enzymes such as trypsin or dispase have been used as a method for removing immunogenic cells, or repeated freezing and thawing, NaCl and SDS treatment, etc. However, the acellular dermal matrix prepared by these processes contains many antigenic components and is prone to cause immune rejection when transplanted into the recipient site, resulting in a low implantation rate (RJ Walter et al, Burns, 24: 104-113, 1998). Summary of the invention

[0006] Technical issues

[0007] Therefore, the present inventors conducted research to develop an acellular dermal graft material that improves transplant rejection and cost issues, while also improving the problem of reduced mechanical properties caused by chemical decellularization. As a result, an optimized supercritical fluid treatment process without surfactant treatment was established, and acellular dermal tissue with improved mechanical properties and increased extracellular matrix retention levels was obtained. Based on the above, the inventors completed the present invention.

[0008] Solution

[0009] In order to solve the above problems, in one aspect of the present invention, an acellular dermal tissue transplant is provided, wherein the critical load of the acellular dermal tissue is 11 to 19 N, and the Young's modulus of the acellular dermal tissue is 0.5 to 1.2 MPa.

[0010] In another aspect of the present invention, a method for preparing an acellular dermal tissue transplant is provided, the method comprising the following steps: a) separating skin tissue separated from a subject into an epidermis layer and a dermis layer; b) extracting the separated dermis layer with a supercritical fluid; and c) washing the dermis layer extracted with the supercritical fluid with a phosphate buffer.

[0011] Effects of the Invention

[0012] The acellular dermis tissue according to the present invention is obtained by decellularization without being treated with a surfactant, so that the critical load, elasticity and other mechanical properties are retained, and at the same time, the toxicity caused by the residual surfactant is not present, so it is suitable for transplantation to patients with damaged skin tissue such as burns and trauma. Therefore, the acellular dermis tissue obtained by the optimized supercritical fluid treatment process of the present invention does not have toxicity and transplant rejection, so it can be effectively used to treat patients with damaged skin tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is a photograph observing the appearance of an acellular dermal matrix sample according to one embodiment of the present invention. Here, the RTU (Ready-to-use) type is prepared by double-packaging and sterilizing a skin sample including physiological saline. The FD (Freeze-dried) type is prepared by freeze-drying a skin sample and then double-packaging and sterilizing it. In addition, Native is an untreated natural skin tissue sample from which the epidermis is not removed, and SCR, SCF, A, B, C, D, E, and F are shown in Table 1 below. The skin sample size is 1×1 cm 2 .

[0014] Figure 2is a microscope photograph (magnified 100 times) showing the result of H&E (hematoxylin & eosin) staining of an acellular dermal matrix sample obtained after a supercritical fluid extraction process according to one embodiment of the present invention.

[0015] Figure 3 According to one embodiment of the present invention, 1% agarose gel electrophoresis images are used to confirm the residual DNA content in dermal tissue (native) before supercritical fluid extraction process, acellular dermal tissue (SCR and SCF) after supercritical fluid extraction process, and acellular dermal tissue (A, B, C, D, E and F) prepared according to conventional known methods according to the detailed specifications of Table 1. Among them, SCR, A, B, C are RTU (ready-to-use) type samples, and SCF, D, E, F are FD (freeze-dried) type samples.

[0016] Figure 4a is a graph showing the quantitative analysis results of residual DNA of acellular dermal matrix samples obtained after supercritical fluid extraction process according to one embodiment of the present invention. In this example, the quantitative analysis results of residual DNA are displayed based on the dry weight of acellular dermal tissue. Among them, Native represents the original tissue, which is the untreated group; SCR and SCF are the test groups of dermal tissue decellularized by supercritical fluid treatment; A, B, C, D, E and F are the positive control groups of decellularized dermal tissue according to the detailed specifications of Table 1.

[0017] Figure 4b is a diagram showing the quantitative analysis results of residual DNA in acellular dermal matrix samples obtained after supercritical fluid extraction according to one embodiment of the present invention. In this example, the quantitative analysis results of residual DNA are shown based on the original tissue without removing the epidermis.

[0018] Figure 5 is a diagram showing the results of Western blot analysis to confirm the expression level of immunogenic protein MHC1 in acellular dermal matrix samples (SCR and SCF) obtained after a supercritical fluid extraction process according to one embodiment of the present invention.

[0019] Figure 6a is a graph showing the results of collagen content analysis of acellular dermal matrix samples (SCR and SCF) obtained after supercritical fluid extraction process according to one embodiment of the present invention. In this example, the results of collagen content analysis are shown based on the dry weight of acellular dermal tissue.

[0020] Figure 6b is a graph showing the quantitative analysis results (based on original tissue) of the collagen content in acellular dermal matrix samples (SCR and SCF) obtained after a supercritical fluid extraction process according to one embodiment of the present invention.

[0021] Figure 7a is a graph showing the results of elastin content analysis of acellular dermal matrix samples (SCR and SCF) obtained after supercritical fluid extraction process according to one embodiment of the present invention. In this example, the results of elastin content analysis are shown based on the dry weight of acellular dermal tissue.

[0022] Figure 7b is a graph showing the quantitative analysis results (based on original tissue) of the elastin content in acellular dermal matrix samples (SCR and SCF) obtained after a supercritical fluid extraction process according to one embodiment of the present invention.

[0023] Figure 8a is a graph showing the results of a cytokine array confirming the retention levels of growth factors in an acellular dermal matrix sample obtained after a supercritical fluid extraction process, according to one embodiment of the present invention.

[0024] Figure 8b is a graph showing the coordinates of points of a cytokine array confirming the retention level of growth factors in an acellular dermal matrix sample obtained after a supercritical fluid extraction process according to one embodiment of the present invention.

[0025] Figures 8c to 8f is a graph showing results obtained by quantifying cytokine array results confirming the retention level of growth factors in acellular dermal matrix samples obtained after a supercritical fluid extraction process by averaging spot pixel intensity according to one embodiment of the present invention.

[0026] Fig. 9 is a graph showing the cytotoxicity evaluation results of acellular dermal matrix samples obtained after a supercritical fluid extraction process according to one embodiment of the present invention.

[0027] Fig.10a is a graph showing the results of measuring the critical load of an acellular dermal matrix sample obtained after a supercritical fluid extraction process using a universal tensile machine (UTM) according to one embodiment of the present invention.

[0028] Fig.10b is a graph showing the quantitative analysis results of the critical load of the acellular dermal matrix sample obtained after the supercritical fluid extraction process (based on the original tissue) according to one embodiment of the present invention.

[0029] Fig.11a is a graph showing the measurement results of Young's modulus of an acellular dermal matrix sample obtained after a supercritical fluid extraction process according to one embodiment of the present invention.

[0030] Fig.11bis a graph showing the quantitative analysis results (based on the original tissue) of the Young's modulus of the acellular dermal matrix sample obtained after the supercritical fluid extraction process according to one embodiment of the present invention.

[0031] Best Mode for Carrying Out the Invention

[0032] Acellular dermal tissue graft

[0033] In one aspect of the present invention, there is provided an acellular dermal tissue graft having the following characteristics:

[0034] The critical load of acellular dermis tissue is 11 to 19 N, and the Young's modulus of acellular dermis tissue is 0.5 to 1.2 MPa.

[0035] The term "dermis" as used herein refers to a structure in which collagen fibers composed of collagen are the main components and elastic fibers composed of elastin are woven in a mesh shape. The dermis covers most of the skin, provides nutrition to the epidermis, supports the epidermis, and protects the body from external damage. In addition, it has the ability to store water, has the function of regulating body temperature, serves as a receptor for sensation, and has the function of regenerating the skin by interacting with the epidermis.

[0036] The term "acellular dermal matrix (ADM)" used herein refers to the dermal matrix obtained from human or animal skin by acellular technology, and refers to a biologically derived skin substitute in the form of an extracellular matrix (ECM) composed of collagen, elastin, etc.

[0037] Acellular dermal matrix is ​​a biomaterial obtained by removing cells that can cause immune response from the skin isolated from the subject, and can be used to transplant it into patients with skin defects caused by burns, car accidents, ulcers, etc., to repair the skin. In addition, acellular dermal matrix is ​​a skin tissue for human transplantation, which can be widely used in reconstruction of full-thickness skin, ventricular septal defects, brain and spinal cord dura mater defects, as well as reconstruction and cosmetic surgeries such as depressed scar reconstruction, hemifacial atrophy reconstruction, nipple reconstruction and lip augmentation.

[0038] The acellular dermal matrix should selectively remove only cellular antigens that are immune-reactive substances while retaining various structural proteins and components without destroying the three-dimensional structure of the dermis in the skin tissue.

[0039] In addition, the acellular dermal matrix should have appropriate elasticity and critical load so that when applied to damaged tissue or damaged skin to repair the damaged area, it can remain stable even if the applied treatment area moves. The acellular dermal matrix should be a material that does not damage normal tissue near the treatment area and is easy to handle.

[0040] In this specification, acellular dermal matrix may be used interchangeably with acellular dermal tissue graft, acellular dermal tissue, acellular dermis, acellular dermis tissue, or acellular dermal tissue.

[0041] The term "decellularization" as used herein refers to a new method of producing artificial supports by removing cells from whole organs while maintaining the original structure of the intended 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 retained. Therefore, by providing a three-dimensional microenvironment similar to that of intact tissues, the various extracellular matrix components retained in the decellularized tissue, including collagen, fibronectin, elastin, etc., can enhance the survival, proliferation and differentiation of cultured cells.

[0042] In the present invention, decellularization may be performed by supercritical fluid extraction without surfactant treatment, but is not limited thereto.

[0043] The term "supercritical fluid extraction" or "supercritical extraction" as used herein refers to a method of separating substances using a supercritical fluid, which has intermediate properties between gas and liquid and exists above the critical point, i.e., the critical temperature and the critical pressure. The above-mentioned supercritical fluid extraction combines the solvent extraction principle and the distillation principle. The solvent extraction principle is to use the solubility difference between the extracted raw material and the supercritical fluid to dissolve the soluble components contained in the raw material in the supercritical fluid. The distillation principle is the evaporation phenomenon of the supercritical fluid in which the solute molecules contained in the raw material are transformed from a high-density condensed phase to a low-density expanded phase.

[0044] The term "supercritical fluid" as used herein refers to a substance that is a gas under normal conditions but is a fluid above its critical temperature and critical pressure. The supercritical fluid suitable for the present invention is not particularly limited, but includes, for example, carbon dioxide, nitrogen, nitrous oxide, methane, ethylene, propane and propylene. Preferably, carbon dioxide having a critical temperature of 31° C. and a critical pressure of 72.8 atm can be used.

[0045] In the present invention, during supercritical fluid extraction, in addition to the supercritical fluid, decellularization can be performed by adding a "co-solvent". The purpose of adding the above co-solvent is to increase the extraction rate of the supercritical fluid, improve its solubility in the supercritical fluid, etc. Ethanol, methanol, petroleum ether, acetonitrile, hexane, etc. can be used as a co-solvent, but are not limited thereto. In this example, the co-solvent can preferably be ethanol.

[0046] The acellular dermis tissue of the present invention may be derived from skin tissue separated from a subject. The subject may be a subject of the same or different species as the subject to which the dermis tissue is to be transplanted, and may specifically be a mammal, including, but not limited to, humans, mice, rats, rodents, monkeys, chimpanzees, gorillas, horses, cows, pigs, cats, dogs, and rabbits.

[0047] The acellular dermal tissue of the present invention can be a tissue in which 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% of the cells have been removed compared to the original tissue isolated from the subject (i.e., compared to tissue that has not been decellularized).

[0048] The term "critical load" as used herein refers to the limit load when a structure or component is in a state of destruction or excessive deformation, etc. That is, the load at the starting point of deformation or cracking of the specimen when the load is applied by the indenter for indentation evaluation. It refers to the maximum stress until the specimen is deformed or cracked due to the critical load, and represents the value obtained by dividing the maximum load at fracture by the original cross-sectional area of ​​the specimen. The larger the critical load, the greater the force required to break the support, which may also mean that the dermis tissue can maintain its shape well when applied to damaged skin or damaged tissue, and during distribution and storage.

[0049] The critical load measured in the present invention can be measured using a universal tensile machine. In addition, in the present invention, the critical load of the acellular dermis tissue can be a wet critical load measured under the condition of being wetted with sterile distilled water, that is, in a wet state, but is not limited thereto.

[0050] In the present invention, the acellular dermis tissue is prepared under wet conditions based on 1×1 cm 2 For the size of the dermal tissue sample, the critical load can be between 11 and 19N.

[0051] In one embodiment, the critical load of the RTU (ready-to-use) type dermal tissue sample that has not been freeze-dried can be 11.0N, 11.1N, 11.2N, 11.3N, 11.4N, 11.5N, 11.6N, 11.7N, 11.8N, 11.9N or 12.0N, but is not limited thereto. In this case, the critical load is based on 1×1 cm 2 The dimensions of the dermal tissue sample are measured.

[0052] In addition, in one embodiment, the critical load of the freeze-dried FD (freeze-dried) type dermal tissue sample can be 16.0 to 19.0 N, 16.5 to 19.0 N, 17.0 to 19.0 N, 17.5 to 19.0 N, 18.0 to 19.0 N, 18.1 N, 18.2 N, 18.3 N, 18.4 N, 18.5 N, 18.6 N, 18.7 N or 18.8 N, but is not limited thereto. In this case, the critical load is based on 1×1 cm 2 The dimensions of the dermal tissue sample are measured.

[0053] The term "Young's modulus" used in this article is also called "elastic modulus" or "elastic coefficient", which refers to the ratio of the pressure (stress) of an object to the deformation of the object. Young's modulus is an elastic modulus that indicates the degree to which an object deforms when subjected to pressure. The basic principle of measuring Young's modulus is to use the property that an object will deform elastically when compressed or expanded, and return to its original shape when the load is removed. For example, a flexible object will deform more than a rigid object, and a high Young's modulus value means no elasticity or rigidity.

[0054] The Young's modulus measured in the present invention can be measured using a universal tensile machine. In addition, the Young's modulus of the dermis tissue in the present invention can be measured under wet conditions, but is not limited thereto.

[0055] In the present invention, the acellular dermis tissue is prepared under wet conditions based on 1×1 cm 2 The size of the dermal tissue sample can have a Young's modulus of 0.5 to 1.2 MPa.

[0056] In one embodiment, the non-freeze-dried RTU (ready-to-use) type dermal tissue sample may have a Young's modulus of 0.50 MPa, 0.51 MPa, 0.52 MPa, 0.53 MPa, 0.54 MPa, 0.55 MPa, 0.56 MPa, 0.57 MPa, 0.58 MPa, 0.59 MPa or 0.60 MPa, but is not limited thereto. In this case, the Young's modulus is based on 1×1 cm 2 The dimensions of the dermal tissue sample are measured.

[0057] In addition, in one embodiment, the Young's modulus of the freeze-dried FD (freeze-dried) type dermal tissue sample may be 1.00 MPa, 1.01 MPa, 1.02 MPa, 1.03 MPa, 1.04 MPa, 1.05 MPa, 1.06 MPa, 1.07 MPa, 1.08 MPa, 1.09 MPa or 1.10 MPa, but is not limited thereto. In this case, the Young's modulus is based on 1×1 cm 2 The dimensions of the dermal tissue sample are measured.

[0058] The acellular dermal tissue of the present invention may have any of the following characteristics based on dry weight, but is not limited thereto:

[0059] The residual DNA content was 80 to 110 ng / mg;

[0060] Collagen content of 400 to 700 μg / mg; and

[0061] The elastin content is 13 to 19 μg / mg.

[0062] In the present invention, the dermal tissue may contain 110 ng / mg or less of residual DNA based on dry weight. Preferably, it may contain 80 to 110 ng / mg, 81 to 109 ng / mg, 82 to 108 ng / mg, 83 to 107 ng / mg, 84 to 106 ng / mg, 85 to 105 ng / mg, 86 to 104 ng / mg or 87 to 103 ng / mg of residual DNA, but is not limited thereto.

[0063] In the present invention, based on dry weight, dermal tissue can include 400 μ g / mg or more collagen content. Preferably, it can include 400 to 700 μ g / mg, 402 to 698 μ g / mg, 404 to 696 μ g / mg, 406 to 694 μ g / mg, 408 to 692 μ g / mg, 410 to 690 μ g / mg, 411 to 688 μ g / mg, 412 to 686 μ g / mg, 413 to 684 μ g / mg, 414 to 682 μ g / mg, 415 to 680 μ g / mg, 416 to 679 μ g / mg, 417 to 678 μ g / mg or 418 to 677 μ g / mg collagen content, but is not limited thereto.

[0064] In the present invention, based on dry weight, dermal tissue can include 13 μ g / mg or more elastin content. Preferably, it can include 13.0 to 19.0 μ g / mg, 13.1 to 18.9 μ g / mg, 13.2 to 18.8 μ g / mg, 13.3 to 18.7 μ g / mg, 13.4 to 18.6 μ g / mg, 13.5 to 18.5 μ g / mg, 13.6 to 18.4 μ g / mg, 13.7 to 18.3 μ g / mg, 13.8 to 18.2 μ g / mg or 13.9 to 18.1 μ g / mg elastin content, but is not limited thereto.

[0065] Compared with the original tissue, the acellular dermal tissue of the present invention may have any of the following characteristics, but is not limited thereto:

[0066] DNA residues ranged from 5% to 8%;

[0067] Collagen content ranges from 55% to 99%;

[0068] The elastin content is 65% to 95%;

[0069] Critical loads of 90% to 165%; and

[0070] Young's modulus is 80% to 200%.

[0071] In the present invention, the dermal tissue may contain 8% or less residual DNA compared to the original tissue, i.e., compared to the dermal tissue before decellularization or the dermal tissue without decellularization. Preferably, it may include 5 to 8%, 5.1 to 7.8%, 5.2 to 7.6%, 5.3 to 7.6%, 5.4 to 7.4%, 5.5 to 7.4%, 5.6 to 7.2%, 5.7 to 7.0% or 5.8 to 6.8% residual DNA, but is not limited thereto.

[0072] In other words, compared with the original tissue, the dermis tissue may be a tissue in which 93% or more of the cells are removed. Preferably, it may be a tissue in which 93% to 98%, 94% to 98%, 95% to 98%, 96% to 98% or 97% to 98% of the cells are removed, but is not limited thereto.

[0073] The dermal tissue according to the present invention may be a tissue in which extracellular matrix components (eg, collagen and elastin) are maintained at a predetermined content compared to the original tissue.

[0074] Specifically, in the present invention, the dermis tissue may contain 55% or more collagen content compared to the original tissue. Preferably, it may contain 55 to 99%, 56 to 98%, 57 to 97% or 58 to 96% collagen content, but is not limited thereto.

[0075] In the present invention, compared with the original tissue, the dermis tissue may contain 65% or more elastin content. Preferably, it may contain 65 to 95%, 66 to 94%, 67 to 93%, 68 to 92%, 69 to 91% or 70 to 90% elastin content, but is not limited thereto.

[0076] In the present invention, the dermis tissue may show a critical load of 90% to 165% compared to the original tissue, but is not limited thereto.

[0077] In the present invention, the dermis tissue may show 80% to 200% of Young's modulus compared to the original tissue, but is not limited thereto.

[0078] Compared with the original tissue, the acellular dermal tissue of the present invention may have any of the following characteristics, but is not limited thereto:

[0079] Adiponectin content ranged from 22% to 44%;

[0080] Apolipoprotein A1 content ranged from 84 to 94%;

[0081] Angiogenin content ranged from 19% to 38%;

[0082] Angiopoietin-2 content is 90% to 99%;

[0083] Brain-derived neurotrophic factor (BDNF) content ranges from 73% to 99%;

[0084] The complement component C5 / C5a content is 9 to 19%;

[0085] CD30 content ranged from 59 to 99%;

[0086] CD40 ligand content ranged from 6% to 28%;

[0087] CD26 content is 2 to 5%;

[0088] Emmprin content ranges from 36% to 44%;

[0089] CD105 content ranged from 41% to 99%;

[0090] Fas ligand content ranged from 2% to 45%;

[0091] FGF-2 content ranges from 15 to 64%;

[0092] FGF19 content ranged from 56% to 98%;

[0093] IFN-γ content ranged from 63 to 99%;

[0094] IL-1A(α) content ranged from 65 to 99%;

[0095] IL-1RA(α) content ranged from 12 to 43%;

[0096] IL-17 content ranged from 82 to 99%;

[0097] Kallikrein (KLK) 3 content is 92% to 99%;

[0098] MIF (macrophage migration inhibitory factor) content ranges from 2 to 9%;

[0099] MMP (matrix metalloproteinase) 9 content ranged from 83 to 99%;

[0100] Pentraxin (PTX) 3 content ranged from 44% to 91%;

[0101] Retinol binding protein (RBP) 4 content ranged from 8% to 36%;

[0102] Vitamin D binding protein (VDBP) content of 6 to 16%; and

[0103] CD31 content ranged from 67% to 99%.

[0104] In an exemplary embodiment of the present invention, the dermis tissue does not contain a surfactant. In the case of a decellularized dermis tissue treated with a conventional surfactant, there are problems such as denaturation of proteins such as collagen and destruction of growth factors due to the surfactant treatment. In addition, the residual surfactant is not easy to remove, and if it is not completely removed, toxicity problems may occur.

[0105] In addition, there is a difficulty that the mechanical properties of the dermis, such as critical load and elasticity, are weakened by surfactants, and when surfactants remain in the dermis, they can cause transplant rejection due to toxic substances.

[0106] However, the acellular dermis obtained by the supercritical fluid process of the present invention has the same level of acellular characteristics as the dermis decellularized by conventional surfactant treatment, and the tissue morphology and mechanical properties are maintained. In particular, compared with the dermis treated with a surfactant, the acellular dermis obtained by the supercritical fluid process has the advantage of significantly excellent retention of extracellular matrix (ECM) substances such as collagen and elastin in the dermis.

[0107] Therefore, the acellular dermal tissue of the present invention can be used as a transplant by decellularization without losing the extracellular matrix while maintaining tissue morphology and mechanical properties.

[0108] Preparation of acellular dermal tissue grafts

[0109] The acellular dermal tissue graft of the present invention can be prepared by a preparation method including the step of extracting dermal tissue separated from a subject with a supercritical fluid.

[0110] The above-mentioned "decellularization", "object", "dermal tissue" and "supercritical fluid" are the same as described above.

[0111] In the present invention, the supercritical fluid can extract lipid components from the dermal tissue separated from the object based on solubility, specifically, extract phospholipid components that are the main components of cell membranes, and perform decellularization to prepare acellular dermal tissue.

[0112] The supercritical fluid can be selected from carbon dioxide gas, ammonia, nitrogen, nitric oxide (NO) gas, nitrogen dioxide (NO2) gas, nitrous oxide (N2O) gas, sulfur dioxide gas, hydrogen, water vapor, saturated hydrocarbons, unsaturated hydrocarbons, aromatic compounds and mixed gases thereof. Specifically, the supercritical fluid can be carbon dioxide gas, but the type is not limited thereto, as long as it is a supercritical fluid that can remove most of the cells of the dermis while maintaining the mechanical properties and structural morphology of the dermis. When carbon dioxide gas is used as a supercritical fluid, the critical temperature (31°C) and critical pressure (73bar) of carbon dioxide are low, so it is easy to adjust to a supercritical state, and it has the advantages of being widely present in nature, colorless, odorless, harmless to the human body, and chemically stable.

[0113] In the present invention, the supercritical extraction step may be performed under a pressure condition 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.

[0114] Specifically, the pressure in the supercritical extraction step can be 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, but is not limited to this.

[0115] In addition, the pressure of the supercritical extraction step 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, but is not limited thereto.

[0116] The pressure conditions of the supercritical extraction step are not limited thereto, as long as the conditions are such that the dermis tissue can be efficiently prepared by removing most of the cells of the dermis tissue while retaining the mechanical properties of the dermis tissue and the structural morphology of the tissue.

[0117] In the above-mentioned supercritical extraction step, in addition to the supercritical fluid, a co-solvent may also be included. The co-solvent may be at least one solvent selected from ethanol, water, methanol, hexane, petroleum ether, acetonitrile, acetone, ethyl acetate, and dichloromethane. Preferably, ethanol may also be included as a co-solvent.

[0118] The co-solvent is added for the purpose of increasing the extraction rate of the supercritical fluid, improving its solubility in the supercritical fluid, etc., extracting lipids in the separated dermal tissue, specifically phospholipids of the cell membrane, thereby removing most of the cells of the dermal tissue. However, its type is not particularly limited as long as the co-solvent retains the mechanical properties of the dermal tissue and the structural morphology of the tissue.

[0119] In the present invention, the supercritical extraction step may be performed at a temperature of 31° C. to 40° C., 31° C. to 39° C., 32° C. to 38° C., 33° C. to 37° C., 34° C. to 36° C. or 35° C., but is not limited thereto.

[0120] In the present invention, the supercritical extraction step can be performed for less than 3 hours, but is not limited thereto. Preferably, it can be performed for 60 minutes to 180 minutes, 70 minutes to 170 minutes, 80 minutes to 160 minutes, 90 minutes to 150 minutes, 100 minutes to 140 minutes, 110 minutes to 130 minutes or 120 minutes, but is not limited thereto.

[0121] The acellular dermal tissue of the present invention can be prepared by, including but not limited to, a step of separating the epidermis layer and the dermis layer before the step of extracting with a supercritical fluid.

[0122] The separation of the epidermis and the dermis can be carried out by methods known in the art. Generally speaking, the separation of the epidermis and the dermis can be carried out using various proteolytic enzymes, such as dispase, thermolysin, trypsin, and the like.

[0123] Furthermore, the epidermis and dermis can be separated by changing the ionic strength of the solution. Specifically, the epidermis and dermis 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.

[0124] In one embodiment, the separation of the epidermis layer and the dermis layer may be performed by treating with 1 M NaCl at a temperature of 37° C. for 24 hours, but is not limited thereto.

[0125] The acellular dermal tissue of the present invention can be prepared by further including, but not limited to, any of the following steps after the supercritical fluid extraction step:

[0126] The dermal tissue was washed with phosphate-buffered saline;

[0127] Freeze-drying dermal tissue; and

[0128] Disinfect the dermis tissue.

[0129] Residual solution and impurities present in the dermal tissue after supercritical fluid extraction can be washed by washing with phosphate buffered saline.

[0130] In one embodiment of the present invention, after supercritical fluid extraction, the decellularized dermal tissue is washed with PBS (phosphate buffered saline) at room temperature for 16 hours to remove residual solution and impurities.

[0131] The dermal tissue obtained after supercritical fluid extraction can be stored by freeze-drying until use.

[0132] During the freeze-drying process, a cryoprotectant may be added, but is not limited thereto. The cryoprotectant may prevent structural changes in the dermis tissue, as well as physical and chemical damage to the dermis tissue.

[0133] As a cryoprotectant, a cryoprotectant known in the art can be used. Exemplary cryoprotectants can be sugars and corresponding sugar alcohols. Specific examples of sugars and corresponding sugar alcohols include sucrose, maltitol, glucitol, lactitol and isomaltulose. In addition, currently widely used cryoprotectants also include DMSO, dextran, propylene glycol, glycerol, trehalose, polyethylene glycol, serum albumin, etc.

[0134] The dermal tissue may be treated with a cryoprotectant so that the cryoprotectant can fully penetrate into the dermal tissue before freeze drying, but is not limited thereto. Specifically, the tissue treated with the cryoprotectant can be stored under ultra-low temperature freezing conditions of about -70°C or lower, preferably -40°C to -70°C. In addition, the tissue treated with the cryoprotectant can be stored for 4 hours or longer, preferably 4 to 48 hours.

[0135] Freeze drying may be performed using a freeze dryer for 24 to 48 hours, but is not limited thereto.

[0136] In one embodiment of the present invention, the dermal tissue obtained after supercritical fluid extraction is treated and infiltrated with maltitol for freeze drying, and then frozen and stored at a temperature of about -70°C or lower for at least 4 hours. After freezing, freeze drying is performed using a freeze dryer for about 24 to 48 hours and stored until use.

[0137] The dermal tissue obtained after supercritical fluid extraction can be sterilized by irradiation. The irradiation range can be 10 to 30 kGy, but is not limited thereto.

[0138] In one embodiment of the present invention, after packaging, the dermal tissue obtained after supercritical fluid extraction is sterilized by irradiating 10 to 30 kGy of gamma rays.

[0139] Uses of acellular dermal tissue grafts

[0140] The acellular dermis tissue according to the present invention is obtained by decellularization, without being treated with a surfactant, and thus retains mechanical properties such as critical load and elasticity, and does not have toxicity caused by residual surfactants. Therefore, the dermis tissue obtained by the optimized supercritical fluid treatment process of the present invention does not have toxicity and transplant rejection, and can therefore be effectively used in reconstructive plastic surgery and cosmetic plastic surgery, including correction of severe burns, trauma, depressed scars, correction of hemifacial atrophy, nipple reconstruction, lip augmentation, etc.

[0141] Method for preparing acellular dermal tissue graft

[0142] In another aspect of the present invention, a method for preparing an acellular dermal tissue transplant is provided, the method comprising the following steps: a) separating skin tissue separated from a subject into an epidermis layer and a dermis layer; b) extracting the separated dermis layer with a supercritical fluid; and c) washing the dermis layer extracted with the supercritical fluid with a phosphate buffer.

[0143] The above-mentioned "object" and "supercritical fluid" are the same as described above.

[0144] The above-mentioned step a) is a step of separating the skin tissue separated from the object into the epidermis and the dermis. Specifically, the separation of the epidermis and the dermis can be carried out by methods known in the art. In general, the separation of the epidermis and the dermis can be carried out using various proteolytic enzymes, such as dispase, thermolysin, trypsin, etc. In addition, the epidermis and the dermis can be separated by changing the ionic strength of the solution. Specifically, the epidermis and the dermis can be separated by treating with 1M or higher sodium chloride (NaCl) solution or 20mM EDTA solution at 37°C for 14 to 32 hours.

[0145] The above step b) is a step of extracting the dermis layer separated from the epidermis layer using a supercritical fluid. Specifically, the cell components other than the extracellular matrix of the dermis layer, such as phospholipid components (the main component of the cell membrane that induces immune response), are extracted using a supercritical fluid to perform decellularization.

[0146] The type of supercritical fluid, pressure conditions, temperature conditions, and execution time of supercritical fluid extraction are the same as those described in the above-mentioned 'Preparation of acellular dermal tissue graft'.

[0147] In addition, the supercritical fluid in step b) may further contain a cosolvent, but is not limited thereto. The cosolvent is added for the purpose of increasing the extraction rate of the supercritical fluid, improving its solubility in the supercritical fluid, etc., and the cosolvent can extract the cell components other than the extracellular matrix in the separated dermis layer, thereby removing most of the cells of the dermis tissue.

[0148] The type of co-solvent is not particularly limited, as long as the co-solvent retains the mechanical properties of the dermal tissue and the structural morphology of the tissue, and the co-solvent can be, for example, at least one solvent selected from ethanol, water, methanol, hexane, petroleum ether, acetonitrile, acetone, ethyl acetate, dichloromethane, etc. Preferably, ethanol can also be included as a co-solvent.

[0149] The above step c) is a step of washing the dermis extracted with the supercritical fluid with a phosphate buffer. The residual solution and impurities present in the dermis tissue after the supercritical fluid extraction can be washed by washing with a phosphate buffer.

[0150] In addition, the cleaned dermal tissue may be subjected to any one of freeze-drying, sterilization, and sealed packaging, but is not limited thereto. DETAILED DESCRIPTION

[0151] The present invention is described in more detail by the following examples. However, the following examples are only used to illustrate the present invention, and the scope of the present invention is not limited to these examples.

[0152] I. Preparation of acellular dermal matrix using supercritical fluid extraction process

[0153] Example 1. Preparation of acellular dermal matrix using supercritical fluid extraction process

[0154] To decellularize the skin tissue, a supercritical fluid extraction process was performed.

[0155] First, the fat layer was removed from human skin tissue of donor patients (IRB No. 20201305, Asan Medical Center, Seoul). After the fat layer was removed, the epidermis was treated with 1M NaCl (Sigma Aldrich, Cat No. S9888) at 37°C for 24 hours to remove the epidermis. After the epidermis was removed, the separated dermis was washed with sterile PBS (biowest, Cat No. L0615-500) for 1 hour.

[0156] After washing, the obtained dermal tissue is placed in the extraction tank of the supercritical extraction system (SES), and supercritical fluid carbon dioxide and co-solvent ethanol are injected into the extraction tank. Then, the dermal tissue is decellularized by supercritical treatment at a pressure of 72.8 bar and a temperature of 31°C for 1 to 3 hours.

[0157] Then, the acellular dermis tissue is washed with sterile PBS at room temperature for 24 hours. For the RTU (ready-to-use) type, the washed acellular dermis tissue is sealed and packaged with sterile saline. In addition, for the FD (freeze-dried) type, the acellular dermis tissue is treated and infiltrated with maltitol and frozen at -80°C for 4 hours or more. After that, freeze-drying is performed using a freeze dryer for 24 hours and then sealed and packaged. The packaged acellular dermis tissue is gamma sterilized (Greenpia, 15kGy) and then stored at room temperature until use.

[0158] Preparation Example 1. Preparation and appearance observation of acellular dermal matrix samples

[0159] As a comparison group of the acellular dermal matrix (ADM) samples prepared in the above Example 1, human dermal ADM samples were prepared according to the detailed specifications in Table 1 below according to conventional known methods.

[0160] The thickness of each prepared sample was measured (Table 2), and the appearance was observed ( Figure 1 ). At this time, the thickness of the commercially available product was measured using a vernier caliper to measure the thickness at three different locations.

[0161] [Table 1]

[0162]

[0163] [Table 2]

[0164] serial number Sample name Thickness(mm) 1 Native 3.0 2 SCR 3.0 3 SCF 2.8 4 A 3.0 5 B 2.0 6 C 3.0 7 D 3.0 8 E 1.0 9 F 3.0

[0165] Specifically, all dermal tissues (including native tissues) were treated with gamma ray sterilization (15 kGy). At this time, FD (freeze-dried) type native samples were not included, and RTU type native tissues were used as untreated control samples for all analyses. In addition, the native values ​​contained in the analysis results of FD type samples were expressed as the values ​​of RTU (ready-to-use) type native samples.

[0166] The RTU type was prepared by double-packing a dermis sample (including saline) and then sterilizing it. In addition, the FD type was prepared by freeze-drying and double-packing a dermis sample and then sterilizing it. The FD type acellular dermal matrix sample was immersed in saline for about 100 minutes to hydrate it, and its appearance was observed ( Figure 1 ).

[0167] like Figure 1 As shown, it was confirmed that, in terms of appearance, the color of the acellular dermal matrix samples (SCR and SCF) prepared in Example 1 above was similar to the color of the human dermal ADM product prepared according to conventional known methods.

[0168] II. Evaluation of the biochemical properties of acellular dermal matrix prepared by supercritical fluid extraction

[0169] Experimental Example 1. Histological Analysis of Acellular Dermal Tissue

[0170] For the histological analysis of the decellularized dermal tissue prepared in Example 1 above, H&E (hematoxylin & eosin) staining was performed to analyze the histological morphology and decellularization degree of the tissue. That is, the presence or absence of cell nuclei was confirmed by staining with hematoxylin (blue) and eosin (red), which is an indicator of decellularization performance. Since hematoxylin bound to cellular DNA is correlated with the DNA quantification results, the decellularization level can be analyzed.

[0171] For H&E staining, the original tissue sample (native) treated with supercritical fluid without removing the epidermis was used as a negative control group. The acellular dermal matrix samples (SCR and SCF) treated with supercritical fluid and removing the epidermis were used as test groups. In addition, human dermal ADM samples (A, B, C, D, E, and F) were used as comparison groups according to the detailed specifications of Table 1.

[0172] The staining results confirmed that the cell nuclei in the acellular dermis samples SCR and SCF were completely removed by the supercritical fluid process ( Figure 2 ). In addition, in the human dermal ADM samples, no nuclei were observed in samples C and F. In contrast, hematoxylin-stained sections were confirmed in samples A, B, D, and E, indicating that decellularization was not completely achieved.

[0173] At the same time, it was confirmed that the retention level of the internal structure of samples A and D was the best. Specifically, in the case of RTU type, the retention level of the internal structure was A>SCR (samples processed by supercritical fluid process)>C>B. In addition, in the case of FD type, the retention level of the internal structure was D>SCF (samples processed by supercritical fluid process)>E=F.

[0174] Experimental Example 2. Confirmation of the amount of residual DNA in acellular dermal tissue

[0175] In order to confirm the decellularization level of the decellularized dermis tissue (SCR, SCF) prepared in Example 1 above, the DNA content in the decellularized dermis samples was measured. At this time, the original tissue (native) not treated with supercritical fluid was used as a negative control group, and the acellular dermis tissue sample in Table 1 prepared by a conventional method was used as a positive control group. The DNA content of the RTU (ready-to-use) type and FD (freeze-dried) type acellular dermal matrix samples was measured.

[0176] Specifically, for each sample, the gDNA of each dermal tissue was extracted using the Dneasy Blood & Tissue Kit (QIAGEN, Cat#69506). Subsequently, 1% agarose gel electrophoresis was performed ( Figure 3 ). During electrophoresis, use ExcelBand TM 1KB Plus (0.1-10 kb) DNA Ladder (SMOBIO, Cat# DM3200) was used as a size marker.

[0177] The electrophoresis results confirmed that the residual dsDNA in the decellularized dermis samples (SCR and SCF) according to the supercritical fluid treatment process was lower than that in the commercially available acellular dermal matrix samples (A, B, C, D, E, and F) for both RTU and FD types. Among the acellular dermal matrix samples prepared by chemical or enzyme treatment according to conventional methods, a faint DNA band was observed in B, but clear DNA bands were confirmed in the remaining acellular dermal matrix samples A, D, and E. In contrast, it was confirmed that no DNA bands were observed at all in SCR and SCF ( Figure 3 ).

[0178] In addition, the residual dsDNA in the acellular dermal matrix samples was quantified using the Qubit dsDNA BR Assay Kit (Thermo, Cat# Q32853), which measures fluorescence intensity by adding a fluorescent dye that directly binds to specific regions of dsDNA to the extracted DNA ( Figure 4a and 4b ). At this point, all samples were freeze-dried and analyzed based on solid mass.

[0179] As a result of the analysis, the measured DNA quantitative value was similar to the sum of the fragmented DNA obtained by electrophoresis. That is, it was confirmed that, similar to the electrophoresis results, the residual dsDNA in the acellular dermis samples (SCR and SCF) according to the supercritical fluid treatment process was lower than that in the acellular dermal matrix samples (A, B, C, D, E, and F) prepared according to the conventional method for RTU type and FD type.

[0180] It was found that the supercritical fluid treatment was superior in DNA removal compared to the chemical treatment or enzyme treatment process using surfactants used in the conventional acellular dermal matrix production. In addition, it was confirmed that the original tissue had 90% or more of the cells removed, indicating that it had excellent effects as a transplant without causing transplant rejection.

[0181] Experimental Example 3. Confirmation of the expression level of immunogenic proteins in acellular dermal tissue

[0182] In order to confirm the transplant rejection level of the acellular dermal tissue (SCR and SCF) prepared in Example 1 above, the expression level of the immunogenic protein MHC1 in the acellular dermal matrix samples was confirmed. At this time, the acellular dermal tissue samples (A, B, C, D, E, F) prepared by conventional chemical treatment or enzyme treatment process were used as positive control groups, and the RTU type and FD type acellular dermal matrix samples were tested.

[0183] Specifically, for each sample, the protein of each dermal tissue was extracted using RIPA lysis buffer. Subsequently, protein quantification was performed using the Pierce BCA protein assay kit (Thermo, Cat#23225) using the BCA (bicinchoninic acid) method. After protein quantification, Western blot analysis was performed using MHC1 (SANTA CRUZ Biotechnology, Inc., Cat#sc-55582) antibody ( Figure 5 ).

[0184] The experimental results confirmed that the immunogenic protein MHC1, a cell membrane protein, was negative in all acellular dermal matrix samples.

[0185] It was confirmed that the expression of the immunogenic protein MHC1 was negative after treatment with supercritical fluid, which was similar to the acellular dermal tissue sample prepared according to the conventional method, indicating that it has excellent effect as a transplant and will not cause transplant rejection.

[0186] Experimental Example 4. Confirmation of the extracellular matrix (ECM) retention level of decellularized dermal tissue

[0187] The retention levels of the extracellular matrix components, namely collagen, elastin, and sGAG, in the acellular dermal tissues (SCR and SCF) prepared in Example 1 above were confirmed.

[0188] Experimental Example 4.1. Determination of collagen content in acellular dermal tissue

[0189] In order to confirm that there is no loss of protein in the decellularized dermis tissue (SCR and SCF) prepared in Example 1 above, the collagen content was measured using the Sircol Insoluble Collagen Assay Kit (Biocolor, Cat#S2000). At this time, the original tissue (native) that has not been treated with supercritical fluid was used as a negative control group. The dermis tissue samples (A, B, C, D, E and F) that have been treated with enzymes or chemically treated with surfactants were used as positive controls. Each sample was freeze-dried and about 5 mg was taken for measuring the collagen content.

[0190] The measurement results confirmed that for RTU and FD samples, based on the original tissue, the collagen retention rate in the decellularized dermis tissue prepared according to the supercritical fluid process was the best compared to the acellular dermis tissue sample prepared according to the conventional method. Specifically, based on the dry weight of the sample, it was confirmed that the collagen content of the RTU sample was 418μg / mg and the collagen content of the FD sample was 677μg / mg. Compared with the original tissue, they were 58.8% and 95.2%, respectively, indicating that the protein loss in the decellularized dermis tissue according to the supercritical fluid process was minimized ( Figure 6a and 6b ).

[0191] In comparison, it has been confirmed that the collagen content in the dermal tissue decellularized by conventional chemical treatment or enzyme treatment process is 16μg / mg, 23μg / mg, and 164μg / mg, respectively, based on the dry weight of the sample, for RTU type A, B, and C samples, and 317μg / mg, 370μg / mg, and 375μg / mg, respectively, for FD type D, E, and F samples. Compared with the original tissue, the RTU type A, B, and C samples were only 2.25%, 3.23%, and 23.1%, respectively, while the FD type D, E, and F samples were 44.6%, 52.0%, and 52.7%, respectively. In other words, it was found that when decellularization is performed by conventional chemical treatment or enzyme treatment, although the decellularization effect is excellent, significant protein loss will occur in the dermal tissue.

[0192] The above results show that the decellularized dermis treated by the supercritical fluid process maintains its histological morphology and achieves effective decellularization while minimizing protein loss. Therefore, it is found that it has excellent effects that can be used as a transplant.

[0193] Experimental Example 4.2. Determination of elastin content in acellular dermal tissue

[0194] In order to confirm that there is no elastin loss in the acellular dermis tissue (SCR, SCF) prepared in Example 1 above, elastin analysis was performed using the Fastin Elastin detection kit (Biocolor, Cat#F2000). At this time, the original tissue (native) that has not been treated with supercritical fluid was used as a negative control group. The dermis tissue samples (A, B, C, D, E and F) that have been treated with enzymes or chemically treated with surfactants were used as positive controls. Each sample was freeze-dried and about 5 mg was taken for measuring the elastin content.

[0195] The analysis results confirmed that for RTU and FD products, based on the original tissue, the elastin retention rate in the decellularized dermis tissue prepared according to the supercritical fluid process was the same or higher than that of the acellular dermis tissue sample prepared according to the conventional method. Specifically, it was confirmed that the elastin content of the RTU sample was 18 μg / mg and the elastin content of the FD sample was 14 μg / mg, based on the dry weight of the sample. Compared with the original tissue, it was 90% and 70%, respectively, and it was found that no elastin loss occurred in the decellularized dermis tissue according to the supercritical fluid process ( Figure 7a and 7b ).

[0196] In comparison, it was confirmed that the elastin content in the dermal tissue decellularized by conventional chemical treatment or enzyme treatment process was 10μg / mg, 19μg / mg, and 9μg / mg, respectively, based on the dry weight of the sample, for RTU type A, B, and C samples, and 15μg / mg, 11μg / mg, and 8μg / mg, respectively, for FD type D, E, and F samples. Compared with the original tissue, the RTU type A, B, and C samples were 50%, 95%, and 45%, respectively, and the FD type D, E, and F samples were 75%, 55%, and 40%, respectively. In other words, it was found that significant elastin loss occurred except for samples B and D.

[0197] The above results show that the acellular dermal tissue treated by supercritical fluid technology minimizes the loss of elastin while maintaining its histological morphology, and therefore has excellent effects as a transplant.

[0198] Experimental Example 5. Confirmation of Growth Factor Retention Levels in Acellular Dermal Tissue

[0199] In order to confirm the retention level of growth factors in the acellular dermis tissue (SCR and SCF) prepared in Example 1 above, the number and expression level of significantly expressed cytokines were confirmed using Proteome Profiler Array, Human XL Cytokine Array Kit, 105 Human CytokineArray (ARY022B; R&D Systems USA) ( Figures 8a to 8f ). At this time, the original tissue (native) that was not treated with supercritical fluid was used as the negative control group. The dermal tissue samples (A, B, C, and D) that were treated with enzymes or chemically treated with surfactants were used as the positive control group.

[0200] Specifically, the protein of each sample was extracted, separated and purified from the acellular dermal tissue sample using RIPA lysis buffer. Subsequently, protein quantification was performed using the Pierce BCA protein assay kit (Thermo, Cat#23225) using the BCA method. 100 mg of protein from each sample was evenly loaded onto each array membrane [Proteome ProfilerArray, Human XL Cytokine Array Kit (ARY022B), R&D Systems USA] and then analyzed.

[0201] The spot intensity values ​​were measured using ImageJ software, and the number of cytokines with any intensity of 20 or higher was then counted. The analysis results are summarized in Tables 3 and 4 below.

[0202] [Table 3]

[0203] Sample name <![CDATA[Cytokine #(intensity > 20 * )]]> Native 13 SCR 5 SCF 7 A 2 B 4 C 3 D 6

[0204] * Average pixel intensity

[0205] [Table 4]

[0206]

[0207]

[0208] The results of the cytokine array confirmed that the growth factor retention rate was comparable to that of the acellular dermal tissue D sample prepared according to the conventional method. In particular, it was found that among the RTU type samples, the growth factor retention rate of the acellular dermal matrix sample (SCR) prepared by the supercritical fluid process of the present invention was the best.

[0209] At the same time, the growth factor retention rates of the D sample prepared according to the conventional freeze-dried method and the SCF sample according to the present invention were compared. The results showed that although there were differences in the cases of gamma sterilization or not, the growth factor retention rate of the SCF acellular dermal matrix of the present invention decellularized by the supercritical fluid treatment process was superior even in the same freeze-dried samples.

[0210] Experimental Example 6. Cytotoxicity Evaluation

[0211] The cytotoxicity of the acellular dermal tissues (SCR and SCF) prepared in Example 1 was evaluated. Specifically, the L929 mouse fibroblast cell line known in the art was used to culture 1×10 4The number of cells / well was used to evaluate cytotoxicity. In addition, cytotoxicity was evaluated according to the tissue elution method (using the KCL-approved testing agency protocol), in which the tissue was immersed in a cell culture medium at 37°C for 24 hours and the internal tissue substances were eluted into the culture medium. At this time, if the survival rate was reduced to less than 70% of the blank test solution according to the above-mentioned approved testing agency standards, it was evaluated as having potential cytotoxicity.

[0212] The results of cytotoxicity evaluation showed that the cytotoxicity of the acellular dermal matrix samples (SCR and SCF) prepared by the supercritical fluid process of the present invention was significantly lower than that of the acellular dermal tissue samples (A, B, C, D, E and F) prepared by conventional chemical treatment or enzyme treatment process ( Fig. 9 ).

[0213] The above results show that the acellular dermal tissue treated by supercritical fluid technology maintains its histological morphology and has no cytotoxicity, so it has excellent effect as a graft material.

[0214] III. Evaluation of the physicochemical properties of acellular dermal matrix by supercritical fluid extraction process

[0215] Experimental Example 7. Measurement of critical load and elasticity of acellular dermal tissue by supercritical fluid extraction process

[0216] The critical load and elasticity of the acellular dermis (SCR and SCF) prepared in Example 1 were measured. The critical load and elasticity of the acellular dermis obtained by supercritical fluid extraction were measured using a universal tensile tester (EZ-x model, Shimadzu Co.). 2 For the test samples, the original tissue (native) that was not treated with supercritical fluid was used as a negative control group. The dermal tissue samples (A, B, C, D, E, and F) that were treated with enzymes or chemically treated with surfactants were used as positive controls. At the same time, before measuring the critical load and elasticity, each sample was rehydrated with PBS for 100 minutes, and the water on the tissue surface was removed with gauze before measurement.

[0217] The results confirmed that the critical load of the decellularized dermis tissue (SCR) according to the RTU type supercritical fluid process was 11.6 N, which was almost completely maintained compared to the original tissue, which was different from the acellular dermis tissue sample A (9.8 N), sample B (8.9 N), and sample C (6.8 N) prepared according to the conventional method. In particular, it was confirmed that the critical load of the decellularized dermis tissue (SCF) according to the FD type supercritical fluid process was 18.8 N, which was about 1.6 times that of the original tissue (11.6 N), and showed a critical load level comparable to or higher than that of the acellular dermis tissue sample D (15.6 N), sample E (8.5 N), and sample F (17.4 N) prepared according to the conventional method ( Fig.10a and 10b ).

[0218] The Young's modulus of the RTU-type SCR sample according to the present invention was also measured to be 0.6 MPa, which was equal to the Young's modulus of the original tissue sample, confirming that its elasticity was almost completely maintained compared to the original tissue. In addition, it was confirmed that the Young's modulus of the FD-type SCF sample was 1.1 MPa, which was about 1.8 times the Young's modulus of the original tissue sample (0.6 MPa), showing that its Young's modulus level was comparable to or higher than that of the acellular dermis tissue sample D (1.0 MPa), sample E (0.2 MPa) and sample F (1.1 MPa) prepared according to the conventional method ( Fig.11a and 11b ).

Claims

1. An acellular dermal tissue graft, wherein: The critical load of the acellular dermis tissue is 11N to 19N, and the Young's modulus of the acellular dermis tissue is 0.5MPa to 1.2MPa.

2. The acellular dermal tissue graft according to claim 1, wherein: The acellular dermal tissue has any one of the following characteristics based on dry weight: The residual DNA content was 80 ng / mg to 110 ng / mg; A collagen content of 400 μg / mg to 700 μg / mg; and The elastin content is 13μg / mg to 19μg / mg.

3. The acellular dermal tissue graft according to claim 1, wherein: The acellular dermal tissue has any one of the following characteristics compared to the original tissue: DNA residues ranged from 5% to 8%; Collagen content ranges from 55% to 99%; The elastin content is 65% to 95%; 90% to 165% critical load; and Young's modulus is 80% to 200%.

4. The acellular dermal tissue graft according to claim 1, wherein: The acellular dermal tissue has any one of the following characteristics compared to the original tissue: Adiponectin content ranged from 22% to 44%; Apolipoprotein A1 content ranges from 84% to 94%; Angiogenin content ranged from 19% to 38%; Angiopoietin-2 content is 90% to 99%; Brain-derived neurotrophic factor (BDNF) content ranges from 73% to 99%; The complement component C5 / C5a content is 9% to 19%; CD30 content ranged from 59% to 99%; CD40 ligand content ranged from 6% to 28%; CD26 content is 2% to 5%; Emmprin content ranges from 36% to 44%; CD105 content ranged from 41% to 99%; Fas ligand content ranged from 2% to 45%; FGF-2 content ranges from 15% to 64%; FGF19 content ranged from 56% to 98%; IFN-γ content ranged from 63% to 99%; IL-1A(α) content ranges from 65% to 99%; IL-1RA(α) content ranged from 12% to 43%; IL-17 content ranged from 82% to 99%; Kallikrein (KLK) 3 content is 92% to 99%; MIF (macrophage migration inhibitory factor) content ranges from 2% to 9%; MMP (matrix metalloproteinase) 9 content ranged from 83% to 99%; Pentraxin (PTX) 3 content ranged from 44% to 91%; Retinol binding protein (RBP) 4 content ranged from 8% to 36%; Vitamin D binding protein (VDBP) content of 6% to 16%; and CD31 content ranged from 67% to 99%.

5. The acellular dermal tissue graft according to claim 1, wherein: The acellular dermal tissue is prepared by a preparation method including the step of extraction with supercritical fluid.

6. The acellular dermal tissue graft according to claim 5, wherein: The solvent used as the supercritical fluid is carbon dioxide.

7. The acellular dermal tissue graft according to claim 5, wherein: The supercritical fluid also contains ethanol as a co-solvent.

8. The acellular dermal tissue graft according to claim 5, wherein: The extraction is performed at a temperature of 30°C to 40°C.

9. The acellular dermal tissue graft according to claim 5, wherein: The extraction is carried out under pressure conditions of 50 bar to 400 bar.

10. The acellular dermal tissue graft according to claim 5, wherein: The extraction is carried out for 1 to 3 hours.

11. The acellular dermal tissue graft according to claim 5, wherein: The step of separating the epidermis layer and the dermis layer is included before the step of extracting with supercritical fluid.

12. The acellular dermal tissue graft according to claim 5, wherein: The preparation method comprises any one of the following steps after the step of extracting with a supercritical fluid: Washing the dermis tissue with phosphate buffered saline; freeze-drying the dermal tissue; as well as The dermis tissue is sterilized.

13. A method for preparing an acellular dermal tissue transplant, comprising the following steps: a) separating skin tissue separated from a subject into an epidermis layer and a dermis layer; b) extracting the separated dermis layer using a supercritical fluid; as well as c) The dermis extracted with the supercritical fluid was washed with phosphate buffer.

14. The preparation method according to claim 13, wherein: The supercritical fluid in step b) further comprises a co-solvent.

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