Dispersion comprising acellular dermal matrix microparticles, membrane comprising same and use thereof

Abstract

The present invention relates to a dispersion comprising acellular dermal matrix microparticles, a membrane comprising the same, and uses thereof. More specifically, the present invention relates to a membrane comprising a dispersion containing acellular dermal matrix microparticles obtained by supercritical fluid extraction and high pressure dispersion operations, and a method for preparing the same. The acellular dermal membrane according to the present invention exhibits excellent biological stability and biocompatibility, and shows significantly better preservation of extracellular matrix components such as collagen. In addition, the material has excellent mechanical properties, so that the material is easily applied to transplantation operation and suture, and has a porous structure, so that body fluid flow is facilitated, and tissue regeneration can be supported. Thus, the acellular dermal membrane can be applied as wound dressing, adhesion barrier and implantable biomaterials such as drug delivery grafts and artificial skin in the field of regenerative medicine.

Description

TECHNICAL FIELD

[0001] The present invention relates to a dispersion comprising acellular dermal matrix microparticles, a film comprising the same and use thereof. More particularly, the present invention relates to a film comprising a dispersion of acellular dermal matrix microparticles obtained by a supercritical fluid extraction and high pressure dispersion process and a method for preparing the same. BACKGROUND

[0002] The skin tissue of a human body is composed of an epidermis layer as the outermost layer, a dermis layer under the epidermis layer, and a subcutaneous tissue. Among them, the epidermis layer is composed of epithelial cells differentiated into several layers starting from a basal layer which tightly binds the epidermis layer to the dermis layer, melanocytes, immune cells. The dermis layer under the epidermis layer is mainly composed of extracellular matrix composed of fibroblasts, collagen, elastin, etc.

[0003] The extracellular matrix is a key substance that constitutes the structure of the human body and regulates important functions. Various biomaterials using this matrix, such as tissue repair materials and dressing bandages, have been developed. However, the currently marketed products use only a part of the extracellular matrix components.

[0004] A biomaterial can be defined as an artificial substance that is intermittently or continuously in contact with the surrounding tissue and is exposed to body fluids in the body, for the purpose of replacing the function of damaged or dysfunctional human tissues and organs.

[0005] A biomaterial for the human body must be similar in size and shape to the tissue site to be repaired, and its mechanical and physical properties must be as similar as possible to those of the natural tissue to maintain a lasting effect.

[0006] Therefore, biomaterials are prepared in various forms such as solutions, films, sponges, fibers, and hydrogels to adapt to different application sites and intended uses. Among them, biomaterials in the form of films are used to achieve the role of wound dressing or to enhance the epithelial cell layer, thereby improving its implantation rate in the affected tissue. In addition, they are also used in medical applications such as drug delivery formulations, adhesion barriers, and artificial organs (WO2016-195152A1).

[0007] Meanwhile, Korean Patent Registration No. 10-1434041 discloses a collagen film for inducing regeneration having a thickness of 200 to 300 μm made of a material derived from a biological tissue. However, the cross-linking substance used in the preparation process of the collagen film can remain in the body and have toxicity, which makes it difficult to ensure the safety of the biomaterial. In addition, since a cross-linking process that requires a plurality of steps such as freeze-drying, re-dissolution, and re-physical cross-linking, the production process is complex, time-consuming, and consumes a large amount of energy. SUMMARY

[0008] TECHNICAL PROBLEM

[0009] Accordingly, the inventors have conducted research to develop an extracellular matrix-containing biomaterial in the form of a membrane that overcomes the problems of the prior art. As a result, conditions and methods for preparing an acellular dermal membrane were established without a chemical decellularization process or a chemical crosslinking process. Through a series of processes, including a supercritical fluid extraction process and a high-pressure dispersion process, a dispersion liquid containing acellular dermal matrix microparticles was obtained, and the dispersion liquid containing acellular dermal matrix microparticles was further processed to prepare an acellular dermal membrane. Based on the above research, the inventors completed the present invention.

[0010] Solution to the technical problem

[0011] To achieve the above object, in one aspect of the present invention, there is provided an acellular dermal matrix dispersion liquid containing acellular dermal matrix microparticles.

[0012] In another aspect of the present invention, there is provided an acellular dermal membrane containing the acellular dermal matrix dispersion liquid.

[0013] In another aspect of the present invention, there is provided a method of preparing the acellular dermal matrix dispersion liquid.

[0014] In another aspect of the present invention, there is provided a method of preparing the acellular dermal membrane.

[0015] Effects of the invention

[0016] The acellular dermal membrane according to the present invention is prepared by decellularization through a supercritical fluid extraction method, in which a surfactant is not used in obtaining the extracellular matrix derived from skin tissue used as a main ingredient. Since a chemical crosslinking agent is not used in preparing a membrane using the resulting acellular dermal matrix, the membrane has excellent biostability and biocompatibility. In addition, the use of an acellular dermal matrix dispersion liquid homogenized through a high-pressure dispersion process during membrane preparation can further improve the dispersibility and uniformity of the acellular dermal matrix.

[0017] The acellular dermal membrane according to the present invention not only exhibits excellent preservation of extracellular matrix components such as collagen, but also exhibits high tensile strength, thereby having excellent mechanical properties, making it easy to perform transplantation and suturing. In addition, the membrane has a porous structure, which is advantageous for the flow of body fluids, thereby enabling promotion of tissue regeneration.

[0018] Accordingly, the membrane containing the acellular dermal matrix dispersion liquid according to the present invention can be effectively used in the field of regenerative medicine as a biomaterial for transplantation, such as a wound dressing, an adhesion barrier, a drug delivery graft, and an artificial skin. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a schematic diagram showing a method of preparing a film containing a dispersion liquid of acellular dermal matrix derived from a human body according to one embodiment of the present application.

[0020] Figure 2 is a photograph of a dispersion liquid of acellular dermal matrix obtained by high-pressure dispersion of sterile distilled water containing acellular dermal matrix microparticles according to one embodiment of the present application.

[0021] Figure 3 is a photograph of a film-shaped product prepared by drying a dispersion liquid of acellular dermal matrix according to one embodiment of the present application under vacuum after a sealing, packaging and sterilization process.

[0022] Figure 4a is a photograph of the appearance of a film containing a dispersion liquid of acellular dermal matrix according to one embodiment of the present application.

[0023] Figure 4b is a photograph taken for observing whether a film containing a dispersion liquid of acellular dermal matrix according to one embodiment of the present application can be applied to a desired area after hydration.

[0024] Figure 4c and 4d are enlarged photographs of pattern images of the appearance of a film containing a dispersion liquid of acellular dermal matrix according to one embodiment of the present application. Specifically, these photographs are enlarged photographs of a circular punch pattern image Figure 4c ) and a linear punch pattern image Figure 4d ), respectively.

[0025] Figure 5a is a photograph showing the results of measuring the thickness of a film containing a dispersion liquid of acellular dermal matrix according to one embodiment of the present application using a Nikon (MFC-101A) Digimicro device.

[0026] Figure 5b is a photograph showing the results of confirming the homogenization characteristics of a film containing a dispersion liquid of acellular dermal matrix according to one embodiment of the present application.

[0027] Figure 5c is a photograph comparing the homogenization characteristics of a film containing a dispersion liquid of acellular dermal matrix according to one embodiment of the present application with those of a conventional dermal matrix film commercially available.

[0028] Figure 6a is a graph showing the results of a tensile force test of a dried film containing a dispersion liquid of acellular dermal matrix according to one embodiment of the present application.

[0029] Figure 6bis a graph showing the results of a tensile force test of a hydrated film containing a cell-free dermal matrix dispersion according to one embodiment of the present application.

[0030] Figure 6c is a comparison of the results of a tensile force test of a hydrated film containing a cell-free dermal matrix dispersion according to one embodiment of the present application and a commercially available conventional dermal matrix film.

[0031] Figure 7a is a microscope photograph showing the surface morphology of a film containing a cell-free dermal matrix dispersion according to one embodiment of the present application, which has been hydrated and freeze-dried, observed using a scanning electron microscope (SEM) at a magnification of 3,000 times.

[0032] Figure 7b is a microscope photograph showing the surface morphology of a film containing a cell-free dermal matrix dispersion according to one embodiment of the present application, which has been hydrated and freeze-dried, observed using a scanning electron microscope (SEM) at a magnification of 120 times and 1,000 times, respectively.

[0033] Figure 8 is a photograph showing the results of confirmation of surgical applicability of a dried film or a hydrated film containing a cell-free dermal matrix dispersion according to one embodiment of the present application.

[0034] Figure 9 is a graph showing the results of analysis of the collagen content of a film (SC dermal matrix) containing a cell-free dermal matrix dispersion according to one embodiment of the present application.

[0035] Figure 10 is a photograph showing the surgical procedure of implanting and suturing a film containing a cell-free dermal matrix dispersion according to one embodiment of the present application into the subcutaneous tissue of a pig animal model.

[0036] Figure 11 is a graph showing the results of H&E staining for confirming whether the surrounding tissue of a film containing a cell-free dermal matrix dispersion according to one embodiment of the present application induces an inflammatory response and a regeneration effect 4 weeks after implantation.

[0037] Figure 12 is a schematic diagram showing a wound healing efficacy evaluation scheme of a film (SC dermal matrix) containing a cell-free dermal matrix dispersion according to one embodiment of the present application in a rat wound model.

[0038] Figure 13 is a representative image of a rat wound model according to one embodiment of the present application.

[0039] Figure 14ais a graph showing the results of the visual evaluation of the wound area of the untreated group (negative control group), positive control group 1 (CGderm Matrix), positive control group 2 (MatriDerm), and test group (SC Dermal Matrix) in a rat wound model.

[0040] Figures 14b to 14e are graphs showing the results of the visual evaluation of the wound area of the untreated group (negative control group), Figure 14b positive control group 1 (CGderm Matrix), Figure 14c positive control group 2 (MatriDerm), and Figure 14d test group (SC Dermal Matrix) in a rat wound model. Figure 14e † p<0.05, †† p<0.01, ††† p<0.001).

[0041] Figure 14f is a representative image showing the results of the wound area analysis of the untreated group, positive control group 1 (CGderm Matrix), positive control group 2 (MatriDerm), and test group (SC Dermal Matrix) in a rat wound model.

[0042] Figure 15a is a graph showing the results of the histological analysis of the wound width of the untreated group, positive control group 1 (CGderm Matrix), positive control group 2 (MatriDerm), and test group (SC Dermal Matrix) in a rat wound model using H&E staining. * p<0.05, ** p<0.01, *** p<0.001).

[0043] Figure 15b is a representative image showing the histological analysis of the wound width of the untreated group, positive control group 1 (CGderm Matrix), positive control group 2 (MatriDerm), and test group (SC Dermal Matrix) in a rat wound model using H&E staining.

[0044] Figure 16a is a graph showing the results of the histological analysis of the inflammatory cell count of the untreated group, CGderm Matrix (positive control group 1), MatriDerm (positive control group 2), and SC Dermal Matrix (test group) in a rat wound model using H&E staining. * p<0.05, ** p<0.01, *** p<0.001). ​

[0045] Figure 16b and 16c Representative images showing histological analysis of inflammatory cells using H&E staining at day 7 (A) and day 14 (B) after application of untreated, CGderm matrix (positive control group 1), MatriDerm (positive control group 2) and SC dermal matrix (test group) in a rat wound model. Figure 16b Figure 16c

[0046] Figure 17a Graph showing results of histological analysis of re-epithelization thickness using H&E staining after application of untreated, CGderm matrix (positive control group 1), MatriDerm (positive control group 2) and SC dermal matrix (test group) in a rat wound model. * p<0.05, ** p<0.01, *** p<0.001).

[0047] Figure 17b and 17c Representative images showing histological analysis of re-epithelization thickness using H&E staining at day 7 (A) and day 14 (B) after application of untreated, CGderm matrix (positive control group 1), MatriDerm (positive control group 2) and SC dermal matrix (test group) in a rat wound model. Figure 17b Figure 17c

[0048] Figure 18a Graph showing results of histological analysis of collagen density using MT staining after application of untreated, CGderm matrix (positive control group 1), MatriDerm (positive control group 2) and SC dermal matrix (test group) in a rat wound model. * p<0.05, ** p<0.01, *** p<0.001).

[0049] Figure 18b Representative images showing histological analysis of collagen density using MT staining of each group after application of untreated, CGderm matrix (positive control group 1), MatriDerm (positive control group 2) and SC dermal matrix (test group) in a rat wound model (x 1.25 magnification).

[0050] Figure 18c and 18d ​​​​The results showed that after application of untreated, CGderm matrix (positive control group 1), MatriDerm (positive control group 2), and SC dermal matrix (test group) in a rat wound model, the MT staining method was used to analyze the results of each group on day 7. Figure 18c ) and the 14th day ( Figure 18d Representative images (×200x magnification) of collagen density for histological analysis.

[0051] Figure 19a This figure shows the histological analysis results of vimentin fluorescence intensity using IF staining after application of untreated, CGderm matrix (positive control group 1), MatriDerm (positive control group 2), and SC dermal matrix (test group) in a rat wound model. * p<0.05, ** p<0.01, *** p<0.001).

[0052] Figure 19b and 19c The results showed that after application of untreated, CGderm matrix (positive control group 1), MatriDerm (positive control group 2), and SC dermal matrix (test group) in a rat wound model, the IF staining method was used to analyze the results of each group on day 7. Figure 19b ) and the 14th day ( Figure 19c Representative images (×200x magnification) of the fluorescence intensity of the vimentin in histological analysis.

[0053] Figure 20a This figure shows the histological analysis results of α-SMA fluorescence intensity using IF staining after application of untreated, CGderm matrix (positive control group 1), MatriDerm (positive control group 2), and SC dermal matrix (test group) in a rat wound model. * p<0.05, ** p<0.01, *** p<0.001).

[0054] Figure 20b and 20c The results showed that after application of untreated, CGderm matrix (positive control group 1), MatriDerm (positive control group 2), and SC dermal matrix (test group) in a rat wound model, the IF staining method was used to analyze the results of each group on day 7. Figure 20b ) and the 14th day ( Figure 20c Representative images (×200x magnification) of α-SMA fluorescence intensity for histological analysis. Detailed Implementation

[0055] The best embodiment of the present invention

[0056] Cell-free dermal matrix dispersion liquid

[0057] In one aspect of the present application, a cell-free dermal matrix dispersion liquid comprising cell-free dermal matrix microparticles is provided.

[0058] As used herein, the term "dermis" refers to a layer in which collagen fibers composed of collagen are the main component, and elastin fibers composed of elastin are interwoven in a network in the collagen fibers. The dermis covers most of the skin, provides nutrition to the epidermis, supports the epidermis, and protects the body from external damage. In addition, the dermis has a water storage capacity, a function of regulating body temperature, serves as a receptor for sensation, and exerts a skin regeneration function by interacting with the epidermis.

[0059] As used herein, the term "acellular dermal matrix (ADM)" refers to an extracellular matrix derived from decellularized skin tissue, which can be used interchangeably with terms such as decellularized dermal matrix, acellular dermal tissue, and decellularized dermal tissue. The acellular dermal matrix is a dermal layer matrix obtained from human or animal skin by decellularization technology, and refers to a biologically derived skin substitute in the form of extracellular matrix (ECM) composed of collagen, elastin, etc.

[0060] The acellular dermal matrix is a biomaterial obtained by removing cells on the skin separated from a subject that can cause an immune response, and can repair the skin by being transplanted to a patient having a skin defect due to a burn, a traffic accident, an ulcer, etc.

[0061] As used herein, the term "microparticle", also referred to as fine particle, particulate matter, or microsphere, generally refers to a micron-sized spherical material having a particle size of 1 μm to 1000 μm.

[0062] As used herein, the term "acellular dermal matrix microparticle" refers to a spherical material in the form of a powder obtained by processing an acellular dermal matrix into a micron-sized particle through a freeze-drying process and a micronization process.

[0063] The freeze-drying process includes rapidly cooling the frozen acellular dermal matrix and then absorbing moisture under vacuum. By controlling the moisture within the dermal matrix through the freeze-drying process, granulation can be facilitated.

[0064] The freeze-drying process can be performed using a freeze-dryer for 12 hours to 72 hours, 24 hours to 60 hours, 36 hours to 55 hours, 40 hours to 50 hours, or 48 hours, but is not limited thereto.

[0065] The acellular dermal matrix can be frozen for at least 4 hours, preferably 12 hours to 24 hours, at a supercooling temperature of about -80°C or less or a freezing temperature of about -40°C or less, prior to the freeze-drying process, but is not limited thereto.

[0066] The freeze-dried acellular dermal matrix can be particulated by a micronization process. The micronization process can be performed using any grinding apparatus known in the art, for example, a grinder such as a freeze grinder and an ultrasonic grinder, a blender, a homogenizer, a hand blender, a plunger grinder, a cutting grinder, etc.

[0067] In one embodiment of the present application, the particle size of the acellular dermal matrix microparticles can be, but is not limited to, about 100 μm to about 500 μm, preferably about 150 μm to about 300 μm.

[0068] By particulating the acellular dermal matrix through the micronization process, an acellular dermal matrix dispersion of any desired concentration can be prepared in a subsequent preparation step of the acellular dermal matrix dispersion. Ultimately, by varying the concentration of the acellular dermal matrix dispersion, its physical properties can be easily controlled during the preparation of an acellular dermal matrix film using the acellular dermal matrix dispersion.

[0069] In one embodiment of the present application, the acellular dermal matrix dispersion can be prepared by high-pressure dispersion of the acellular dermal matrix microparticles in a solution.

[0070] The acellular dermal matrix microparticles can be added in an amount of, but not limited to, about 1.0 wt% to about 3.0 wt%, or about 1.5 wt% to about 2.5 wt%, preferably about 2 wt%, based on the total weight of the acellular dermal matrix dispersion.

[0071] The solution for preparing the dispersion of acellular dermal matrix is not particularly limited as long as it is a physiologically acceptable solution, and can be selected from the group consisting of distilled water, physiological saline, a phosphate buffered solution (PBS), HBSS (Hanks' Balanced Salt Solution), TBS (Tris Buffered Salt Solution), a TAPS (N-tris(hydroxymethyl)-3-aminopropanesulfonic acid) buffered solution, a Bicine (N,N-bis(2-hydroxyethyl)glycine) buffered solution, a HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffered solution, a TES (N-tris(hydroxymethyl)-2-aminoethanesulfonic acid) buffered solution, a PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)) buffered solution, a cacodylate buffered solution, a MES (2-(N-morpholino)ethanesulfonic acid) buffered solution, MEM (Minimum Essential Medium), DMEM (Dulbecco's Modified Eagle's Medium), RPMI 1640, IMDM (Iscove's Modified Dulbecco's Medium), 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.

[0072] The conditions for high pressure dispersion for preparing the dispersion of acellular dermal matrix can be achieved by applying a pressure of about 8,000 psi to about 40,000 psi, and passing once or more through a nozzle chamber of about 100 μm to about 300 μm, but are not limited thereto.

[0073] In one embodiment of the present application, the conditions for high pressure dispersion can be achieved by applying a pressure of about 8,000 psi or more, about 8,000 psi to about 40,000 psi, about 9,000 psi to about 35,000 psi, about 10,000 psi to about 30,000 psi, about 11,000 psi to about 25,000 psi, about 12,000 psi to about 20,000 psi, about 13,000 psi to about 18,000 psi, about 14,000 psi to about 16,000 psi, or about 15,000 psi, but are not limited thereto.

[0074] Further, in the high pressure dispersion process, under the above pressure conditions, a nozzle chamber having a diameter of about 100 μm to about 300 μm, about 150 μm to about 250 μm, or about 200 μm can be used, but is not limited thereto.

[0075] Further, the number of times of passing through the nozzle chamber in the high-pressure dispersion process can be one or more, two or more, or three or more. However, the number of times is not particularly limited as long as the microparticles of the acellular dermal matrix having a relatively uniform particle size can be uniformly dispersed in the solution.

[0076] Further, the high-pressure dispersion process can be performed under temperature conditions of about 10°C to about 40°C, about 11°C to about 39°C, about 12°C to about 38°C, about 13°C to about 37°C, about 14°C to about 36°C, or about 15°C to about 35°C.

[0077] The high-pressure dispersion process can be performed using a high-pressure disperser. For example, by applying a strong pressure to the solution using a high-pressure disperser (e.g., a high-pressure homogenizer), the size of the particles contained in the solution can be reduced, thereby uniformly dispersing the particles in the solution.

[0078] In one embodiment of the present application, the acellular dermal matrix can be decellularized by a supercritical fluid extraction process.

[0079] As used herein, the term "decellularization" refers to the removal of cellular components other than extracellular matrix, such as nuclei, cell membranes, nucleic acids, etc., from a tissue. Decellularization is a new method of preparing an artificial scaffold by removing cells in the entire organ while maintaining the original structure of the target transplanted tissue or organ. In the decellularization process, cellular components are removed from the tissue, but the extracellular matrix and some growth factor proteins are retained. Thus, the various extracellular matrix components, including collagen, glycosaminoglycan (GAG), fibronectin, and elastin, which are retained in the decellularized tissue, can enhance the survival, proliferation, and differentiation of cultured cells by providing a three-dimensional microenvironment similar to that in the intact tissue.

[0080] In the present application, decellularization can be performed by supercritical fluid extraction without surfactant treatment, but is not limited thereto.

[0081] The decellularized dermal tissue of the present application can be derived from a skin tissue isolated from a subject. The skin tissue can be allogeneic or xenogeneic. The term "allogeneic" refers to a human, and the term "xenogeneic" refers to a non-human animal, such as a mammal, e.g., a pig, a cow, or a horse.

[0082] As used herein, the term "supercritical fluid extraction" or "SFE" refers to a method of separating substances using a supercritical fluid, which has intermediate properties between gas and liquid existing above the critical point (i.e., critical temperature and critical pressure). The above-mentioned supercritical fluid extraction is a combination of a solvent extraction principle in which a useful component contained in a raw material is dissolved into a supercritical fluid due to a difference in solubility between the raw material and the supercritical fluid, and a distillation principle in which a solute molecule contained in the raw material is transferred from a high-density condensed phase to a low-density expanded phase (i.e., supercritical fluid) as a distillation phenomenon.

[0083] As used herein, the term "supercritical fluid" refers to a substance that is a gas under normal conditions, but is a fluid above its critical temperature and critical pressure. Suitable supercritical fluids for use in the present application are not particularly limited, but include, 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.

[0084] In the present application, decellularization can be achieved by adding a "co-solvent" other than a supercritical fluid during supercritical fluid extraction. The purpose of adding the above-mentioned co-solvent is to improve extractability and solubility of the supercritical fluid, and a co-solvent that can be used is, for example, but not limited to, ethanol, methanol, petroleum ether, acetonitrile, hexane, etc. Herein, ethanol is preferably used as the co-solvent.

[0085] Preparation of acellular dermal matrix dispersion

[0086] The acellular dermal matrix dispersion according to the present application can be prepared using an acellular dermal matrix microparticle as a main ingredient. The acellular dermal matrix microparticle can be prepared by a preparation method including a step of extracting a skin tissue separated from a subject with a supercritical fluid.

[0087] The above-mentioned "acellular dermal matrix microparticle" and "supercritical fluid" are the same as described above.

[0088] In the present application, a supercritical fluid can extract and remove a lipid component (particularly, a phospholipid component, which is a main component of a cell membrane) in a skin tissue separated from a subject based on solubility to prepare an extracellular matrix derived from the skin tissue.

[0089] The supercritical fluid can 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 hydrocarbon, unsaturated hydrocarbon, aromatic compound, and a mixture gas thereof. Preferably, the supercritical fluid is carbon dioxide gas. When carbon dioxide gas is used as the supercritical fluid, carbon dioxide has a low critical temperature (31°C) and a low critical pressure (73 bar), and thus it is easy to adjust to a supercritical state, and has the advantages of being widely present in nature, colorless, odorless, harmless to the human body, and chemically stable.

[0090] In the present application, the supercritical extraction step can be performed under a pressure condition of 0 bar to 1,000 bar, 30 bar to 900 bar, 60 bar to 800 bar, 90 bar to 700 bar, 120 bar to 600 bar, 150 bar to 500 bar, or 200 bar to 400 bar.

[0091] In the present application, the supercritical extraction step can be performed under a pressure condition of 0 bar to 1,000 bar, 30 bar to 900 bar, 60 bar to 800 bar, 90 bar to 700 bar, 120 bar to 600 bar, 150 bar to 500 bar, or 200 bar to 400 bar.

[0092] Specifically, the pressure of 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 thereto.

[0093] Further, the pressure of the supercritical extraction step can be 1,000 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.

[0094] The pressure condition of the supercritical extraction step is not limited thereto, as long as the condition can effectively prepare the extracellular matrix derived from the decellularized skin tissue by removing most of the cells of the skin tissue while retaining components of the extracellular matrix, growth factors, etc. derived from the skin tissue.

[0095] In the supercritical extraction step described above, a co-solvent can be additionally added, in addition to the supercritical fluid. The co-solvent can be at least one solvent selected from the group consisting of ethanol, water, methanol, hexane, petroleum ether, acetonitrile, acetone, ethyl acetate, and dichloromethane. Preferably, ethanol can be additionally added as a co-solvent.

[0096] The purpose of adding the co-solvent is to improve extractability of the supercritical fluid, improve solubility, etc. The type of the co-solvent is not particularly limited as long as it removes most of the lipids and cells in the separated skin tissue while preserving components of the extracellular matrix, growth factors, etc. derived from the skin tissue.

[0097] In the present application, the supercritical extraction step can be performed at a temperature condition of 28°C to 38°C, 28°C to 37°C, 29°C to 36°C, 29°C to 35°C, 30°C to 34°C, 30°C to 33°C, or 31°C, but is not limited thereto.

[0098] In the present application, the supercritical extraction step can be performed for less than 3 hours, but is not limited thereto. Preferably, the step 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.

[0099] The method for preparing a cell-free dermal matrix dispersion containing cell-free dermal matrix microparticles as a main ingredient according to the present application can further include, but is not limited to, a step of separating an epidermis layer and a dermis layer of a skin tissue, before the step of extracting with a supercritical fluid.

[0100] The separation of the epidermis layer and the dermis layer can be performed using any method known in the art. In general, the separation of the epidermis layer and the dermis layer can be performed using various proteolytic enzymes, such as dispersin, thermitase, trypsin, etc.

[0101] In addition, the epidermis layer and the dermis layer can be separated by changing the ionic strength of a solution. Specifically, the epidermis layer and the dermis layer can be separated by treating with a 1M or higher sodium chloride (NaCl) solution or a 20mM EDTA solution at 37°C for 14 hours to 32 hours.

[0102] In one embodiment, the separation of the epidermis layer and the dermis layer can be performed by treating with 1M NaCl at a temperature condition of 37°C for 24 hours, but is not limited thereto.

[0103] The method for preparing a cell-free dermal matrix dispersion containing cell-free dermal matrix microparticles as a main ingredient according to the present application can further include, but is not limited to, any one of the following steps performed after the supercritical fluid extraction step:

[0104] the step of washing the dermal tissue with a phosphate buffered solution;

[0105] the step of freeze-drying the washed dermal tissue;

[0106] the step of micronizing the freeze-dried dermal tissue to prepare dermal matrix microparticles;

[0107] the step of high pressure dispersing the dermal matrix microparticles in a solution to prepare a dermal matrix dispersion.

[0108] Residual solution and impurities present in the dermal tissue after supercritical fluid extraction can be removed by washing with a phosphate buffered solution.

[0109] The washed dermal tissue can be subjected to a freeze-drying process.

[0110] The freeze-drying process includes rapid cooling of the frozen dermal tissue followed by absorption of moisture under vacuum. The freeze-drying process can control the moisture within the dermal tissue and facilitate its particulation.

[0111] The freeze-drying process can be performed using a freeze-dryer for a period of time ranging from, but not limited to, 12 hours to 72 hours, 24 hours to 60 hours, 36 hours to 55 hours, 40 hours to 50 hours, or 48 hours.

[0112] The acellular dermal tissue can be frozen for a period of at least 4 hours, preferably 12 hours to 24 hours, but not limited to, at a super-cold freezing condition of about -80°C or lower or a cold freezing condition of about -40°C or lower prior to the freeze-drying process.

[0113] The freeze-dried acellular dermal tissue can be particulated by a micronization process. The micronization process can be performed using any milling equipment known in the art, for example, a mill such as a cryomill and an ultrasonic mill, a blender, a homogenizer, a hand-held blender, a plunger mill, a cutting mill, and the like.

[0114] The particulate size of the acellular dermal tissue particulate by the micronization process can range from about 100 pm to about 500 pm, but not limited to. Preferably, the particulate size can range from about 150 pm to about 300 pm.

[0115] Micronization of the acellular dermal tissue by the micronization process can improve the dissolution efficiency in a solution during a subsequent step of preparing an acellular dermal matrix dispersion.

[0116] The dermal tissue granulated by the micronization process can be easily suspended or dissolved in any solution. The solution can be used without limitation as long as it is a physiologically acceptable solution capable of dissolving the granulated dermal tissue. For example, one or more solutions selected from various aqueous solutions (i.e., distilled water, buffer solution, and physiological saline) can be used.

[0117] The mixture of the solution and the granulated dermal tissue can be physically mixed, for example, high-pressure dispersion, whipping, vortexing, shaking, or stirring, to uniformly suspend or dissolve the dermal matrix microparticles in the solution. By this method, a cell-free dermal matrix dispersion can be obtained in which the dermal matrix microparticles are uniformly suspended or dissolved in the solution.

[0118] In one embodiment of the present application, the cell-free dermal matrix dispersion is preferably prepared by physically mixing the solution with the granulated dermal tissue using a high-pressure dispersion method, but is not limited thereto.

[0119] The conditions for performing the high-pressure dispersion are the same as described above.

[0120] Cell-free dermal film comprising the cell-free dermal matrix dispersion

[0121] In another aspect of the present application, a cell-free dermal film is provided, which comprises the cell-free dermal matrix dispersion.

[0122] As used herein, the term "cell-free dermal film (cell-free dermal matrix film)" refers to a thin film having a predetermined thickness obtained by applying a cell-free dermal matrix dispersion prepared with cell-free dermal matrix microparticles as a main component to any flat plate shape. The above-mentioned cell-free dermal film can be used interchangeably with a cell-free dermal matrix film and a cell-free dermal tissue film, and the film also encompasses a thin film form such as a film, a sheet, a patch, and the like.

[0123] The above-mentioned "cell-free dermal matrix", "cell-free dermal matrix microparticle", and "cell-free dermal matrix dispersion" are the same as described above.

[0124] In one embodiment of the present application, the cell-free dermal matrix dispersion can be a solution in which the cell-free dermal matrix microparticles having a particle size of, but not limited to, about 100 μm to about 500 μm, preferably about 150 μm to about 300 μm, are dispersed under high pressure.

[0125] The cell-free dermal matrix microparticles can be added in an amount of, but not limited to, about 1.0 wt% to about 3.0 wt%, or about 1.5 wt% to about 2.5 wt%, preferably about 2 wt%, based on the total weight of the cell-free dermal matrix dispersion.

[0126] The solution in which the cell-free dermal matrix microparticles are dispersed under high pressure and the conditions for performing the high-pressure dispersion are the same as described above.

[0127] In one embodiment of the present application, the acellular dermal film comprising the acellular dermal matrix dispersion can be in the shape of a transparent or translucent sheet or film. The acellular dermal film can have a thickness of about 0.01 mm to about 1 mm. The acellular dermal film can have a thickness of, for example, about 10 μm to about 1000 μm, about 20 μm to about 900 μm, about 30 μm to about 800 μm, about 40 μm to about 700 μm, about 50 μm to about 600 μm, about 60 μm to about 500 μm, about 70 μm to about 400 μm, about 80 μm to about 300 μm, about 85 μm to about 200 μm, or about 90 μm to about 100 μm.

[0128] In one embodiment of the present application, the acellular dermal film can have a mechanical property, i.e., tensile strength, of about 0.5 N to about 30 N, but is not limited thereto. Specifically, in the case of a hydrated state, it can have a tensile strength of about 0.5 N to about 3 N, and preferably a tensile strength of about 1.3 N to about 1.5 N. Further, in the case of a dry state, it can have a tensile strength of about 25 N to about 30 N, and preferably a tensile strength of about 27 N.

[0129] As used herein, the term "tensile strength" refers to the maximum stress a test specimen can withstand without rupture under tensile loading, calculated by dividing the maximum load at rupture by the original cross-sectional area of the specimen. The higher the tensile strength, the greater the force required to break the support material, which also means that the acellular dermal film can be easily applied as a biomaterial to a transplantation surgery and suturing.

[0130] The tensile strength measured in the present application can be measured using a universal testing machine. Further, the tensile strength of the acellular dermal film in the present application can be measured in a hydrated state (e.g., wetted with a physiologically acceptable solution) or a dry state.

[0131] In one embodiment of the present application, the acellular dermal film can comprise a collagen content of about 200 μg / mg or more on a dry weight basis. Preferably, the content can be about 200 μg / mg to about 600 μg / mg, about 250 μg / mg to about 600 μg / mg, about 300 μg / mg to about 600 μg / mg, about 350 μg / mg to about 600 μg / mg, about 400 μg / mg to about 600 μg / mg, or about 450 μg / mg to about 600 μg / mg, but is not limited thereto.

[0132] In one embodiment of the present application, the acellular dermal film can have a porous structure. The acellular dermal film has an advantage of being able to promote tissue regeneration because it has the characteristics of a porous structure, thereby having a property of facilitating the flow of body fluids.

[0133] In an exemplary embodiment of the present application, the dispersion liquid containing the acellular dermal matrix microparticles for producing the acellular dermal membrane does not contain a surfactant. Thus, the acellular dermal membrane prepared by applying the dispersion liquid has an advantage of minimizing problems such as denaturation of proteins such as collagen and destruction of growth factors. In addition, the acellular dermal membrane has excellent mechanical properties, making it easy to suture when applied as a biomaterial for transplantation in a surgical operation.

[0134] Thus, the acellular dermal membrane according to the present application has excellent mechanical properties, a porous structure feature, and excellent extracellular matrix (e.g., collagen and growth factors) preservation ability, making it suitable for use as a biomaterial for transplantation. For example, it can be effectively used as a biomaterial for transplantation, such as a wound dressing, an adhesion barrier, a drug delivery graft, or an artificial skin.

[0135] Meanwhile, when used as a biomaterial for transplantation, the acellular dermal membrane can contain skin- or drug-related active ingredients. These skin- or drug-related active ingredients can be delivered through the site to which the acellular dermal membrane is applied.

[0136] The skin-related active ingredients can include any cosmetic or nutritional ingredients capable of having a beneficial effect on the skin. Examples thereof can include retinol, tretinoin, hyaluronic acid, tocopherol, adenosine, and vitamins.

[0137] In addition, the drug-related active ingredients can include, but are not limited to, analgesics, antihistamines, anti-inflammatory drugs, and the like.

[0138] Method of preparing an acellular dermal matrix dispersion liquid

[0139] In another aspect of the present application, a method of preparing an acellular dermal matrix dispersion liquid is provided. At this time, the method of preparing an acellular dermal matrix dispersion liquid can include the following steps:

[0140] a) a step of separating skin tissue separated from a subject into an epidermal layer and a dermal layer;

[0141] b) a step of extracting the separated dermal layer with a supercritical fluid;

[0142] c) a step of washing the dermal layer extracted with a supercritical fluid with a phosphate buffer solution;

[0143] d) a step of freeze-drying the washed dermal tissue;

[0144] e) a step of micronizing the freeze-dried dermal tissue to prepare dermal matrix microparticles; and

[0145] f) The step of dispersing dermal matrix microparticles under high pressure in a solution to prepare a dermal matrix dispersion.

[0146] The terms "object", "supercritical fluid", "freeze-drying", "solution", "high-pressure dispersion", "dermal matrix microparticles" and "dermal matrix dispersion solution" are the same as those described above.

[0147] The specific details of each step in preparing the cell-free dermal matrix dispersion are the same as those described in the section "Preparation of Cell-Free Dermal Matrix Dispersion" above.

[0148] In step e), the micronization process can be carried out using any grinding equipment known in the art, such as grinders like cryogenic grinders and ultrasonic grinders, mixers, homogenizers, hand mixers, plunger grinders, cutting grinders, etc.

[0149] The micronization process may also include, but is not limited to, the step of passing through a sieve of any aperture to obtain dermal matrix microparticles of the desired size.

[0150] The dermal matrix microparticles applied in step f) after the micronization process can have a particle size of about 100 μm to about 500 μm, preferably about 150 μm to 300 μm, but are not limited thereto.

[0151] In step f), the desired size of dermal matrix microparticles can be added to the solution at any desired concentration. High-pressure dispersion can be used to uniformly suspend or dissolve the added dermal matrix microparticles into a homogeneous size, thereby obtaining a cell-free dermal matrix dispersion.

[0152] High-pressure dispersion conditions can be achieved by applying pressures of approximately 8,000 psi to approximately 40,000 psi and passing through a nozzle chamber of approximately 100 μm to approximately 300 μm once or multiple times, but are not limited thereto.

[0153] In one embodiment of the present invention, the method may further include a step of stirring and mixing the dermal matrix microparticles with the solution before step f).

[0154] Agitation mixing can be replaced by any physical mixing method known to those skilled in the art, such as blowing, vortexing, or shaking. The method is not particularly limited as long as a cell-free dermal matrix dispersion in which dermal matrix microparticles are readily and uniformly suspended or dissolved in the solution can be obtained.

[0155] Methods for preparing cell-free dermal membranes

[0156] In another aspect of the present invention, a method for preparing a cell-free dermal membrane is provided. In this method, the preparation of a cell-free dermal membrane may include the following steps:

[0157] a) a step of separating skin tissue separated from a subject into an epidermis layer and a dermis layer;

[0158] b) a step of extracting the separated dermis layer with a supercritical fluid;

[0159] c) a step of washing the dermis layer extracted with the supercritical fluid with a phosphate buffer solution;

[0160] d) a step of freeze-drying the washed dermis tissue;

[0161] e) a step of micronizing the freeze-dried dermis tissue to prepare dermis matrix microparticles;

[0162] f) a step of high-pressure dispersing the dermis matrix microparticles in a solution to prepare a dermis matrix dispersion; and

[0163] g) a step of applying the dermis matrix dispersion and drying it to prepare a dermis film.

[0164] The above "subject", "supercritical fluid", "freeze-drying", "solution", "high-pressure dispersion", "dermis matrix microparticles", "dermis matrix dispersion", and "acellular dermis film" are the same as described above.

[0165] The specific details of each of the steps a) to f) of preparing an acellular dermis film using an acellular dermis matrix dispersion are the same as described above in "Preparation of an acellular dermis matrix dispersion" and "Method of preparing an acellular dermis matrix dispersion".

[0166] In one embodiment of the present application, an acellular dermis film can be obtained by applying or dispensing the acellular dermis matrix dispersion obtained from steps a) to f) onto any flat plate shape and drying it.

[0167] The method of preparing an acellular dermis film can further include a step of removing air bubbles in the acellular dermis matrix dispersion, but is not limited thereto. As long as an acellular dermis film having any uniform thickness and uniform surface can be prepared, the step of removing air bubbles can be omitted.

[0168] The drying in step g) can be performed under vacuum at a temperature condition of about 5°C to about 50°C, but is not limited thereto. For example, the drying process can be performed under vacuum at a temperature condition of about 5°C to about 50°C, about 10°C to about 48°C, about 20°C to about 46°C, about 30°C to about 44°C, about 35°C to about 42°C, or about 40°C, but is not limited thereto.

[0169] The drying in step g) should be performed under vacuum to minimize problems that can occur during the film preparation. There are many advantages in preparing the film under vacuum, for example, it can prevent changes in the film surface due to evaporation or partial evaporation, such as wrinkles in the film surface, reduction in transparency, and uneven film thickness.

[0170] Further, the drying can be performed for about 6 hours to about 72 hours, but is not limited thereto. For example, the drying can be performed for about 6 hours to about 72 hours, about 12 hours to about 36 hours, about 18 hours to about 30 hours, about 20 hours to about 28 hours, or about 24 hours, but is not limited thereto.

[0171] In one embodiment of the present application, the acellular dermal film can have a porous structure, and the thickness thereof can be about 0.01 mm to about 1 mm. For example, the thickness of the acellular dermal film can be about 10 μm to about 1000 μm, about 20 μm to about 900 μm, about 30 μm to about 800 μm, about 40 μm to about 700 μm, about 50 μm to about 600 μm, about 60 μm to about 500 μm, about 70 μm to about 400 μm, about 80 μm to about 300 μm, about 85 μm to about 200 μm, or about 90 μm to about 100 μm.

[0172] Further, the acellular dermal film can have a tensile strength of about 0.5 N to about 30 N, but is not limited thereto.

[0173] Further, the acellular dermal film can include a collagen content of about 200 μg / mg or more based on the dry weight. Preferably, the content can be about 200 μg / mg to about 600 μg / mg, about 250 μg / mg to about 600 μg / mg, about 300 μg / mg to about 600 μg / mg, about 350 μg / mg to about 600 μg / mg, about 400 μg / mg to about 600 μg / mg, or about 450 μg / mg to about 600 μg / mg, but is not limited thereto.

[0174] After step g), the acellular dermal film can be sterilized.

[0175] As used herein, the term "sterilization" refers to killing or removing all fungi, bacteria, viruses, and vegetative cells and spores of protozoa on and inside the surface of a target object, thereby making the object aseptic. Sterilization methods include physical, chemical, and mechanical methods, and an appropriate sterilization method known in the art can be selected and implemented depending on the characteristics of the object to be sterilized or the composition thereof.

[0176] In one embodiment, the acellular dermal film can be sterilized using gamma rays or eBeam.

[0177] Embodiments of the present application

[0178] Hereinafter, the present application will be described in more detail through the following examples. However, the following examples are only for illustrating the present application, and the scope of the present application is not limited to these examples.

[0179] I. Preparation of a film containing a dispersion of acellular dermal matrix

[0180] Example 1. Preparation of an acellular dermal film using a dermal matrix dispersion

[0181] A series of processes, including decellularization, micronization, dispersion preparation, and drying, were performed to process an acellular dermal matrix (ADM) derived from a human body into a film. Figure 1 A series of steps of a method of preparing a film containing a dispersion of acellular dermal matrix are schematically shown. The specific preparation of a film containing a dispersion of acellular dermal matrix is described below in Examples 1.1 to 1.4.

[0182] Example 1.1. Preparation of an acellular dermal matrix using a supercritical fluid extraction method

[0183] In order to decellularize the skin tissue, a supercritical fluid extraction process was performed.

[0184] First, the adipose layer was removed from the human skin tissue of a donor patient (Ethics Committee Approval No: 20201305, Seoul Gasan Medical Center). After removing the adipose layer, the epidermis was removed by treating with a 1M sodium chloride solution (Sigma Aldrich, Catalog No: S9888) for 24 hours at a temperature condition of 37℃. After removing the epidermis, the separated dermal layer was washed with sterile PBS (biowest, Catalog No: L0615-500) for 1 hour.

[0185] After washing, the obtained dermal matrix was placed in an extraction tank of a supercritical extraction system (SES), and supercritical fluid carbon dioxide and a cosolvent ethanol were injected into the extraction tank. Subsequently, the dermal tissue was decellularized by supercritical treatment for 1 to 3 hours at a pressure condition of 72.8 bar and a temperature condition of 31℃.

[0186] After that, the decellularized dermal tissue was washed with sterile PBS for 24 hours at room temperature.

[0187] Example 1.2. Preparation of an acellular dermal matrix microparticle using a micronization process

[0188] A micronization process was performed to process the washed and decellularized acellular dermal tissue obtained in the above Example 1.1 into a film.

[0189] Specifically, the homogenized acellular dermis after decellularization by the supercritical fluid treatment process was frozen at -80°C for 1 day in order to be ground before granulation. After being frozen for 1 day, freeze-drying was performed using a freeze dryer for 2 days.

[0190] The freeze-dried acellular dermis tissue was subjected to primary granulation using a cutting mill (Taemyong Scientific, FRITSCH Universal Cutting Mill). At this time, the particles were passed through a screen having a pore diameter of 500 μm, and the particles passing through the screen were collected. Thereafter, in order to prevent denaturation of the material due to heat generated during the granulation process, secondary grinding was performed using a cryogenic mill (SPEX, 6875D Freezer / Mill) for 5 minutes using liquid nitrogen. At this time, the particles not passing through the screen were collected using a screen having a pore diameter of 100 μm. Finally, acellular dermis matrix particles having a particle size of 100 μm or more and 500 μm or less were obtained.

[0191] Example 1.3. Preparation of an acellular dermis matrix dispersion

[0192] The acellular dermis matrix particles having a particle size of 100 μm or more and 500 μm or less (i.e., a particle size of 100 μm to 500 μm) were physically homogenously dispersed using a high-pressure disperser (Micronox, MN400BF) at 15,000 psi for 30 minutes. Figure 2

[0193] Specifically, the acellular dermis matrix particles having a particle size of 100 μm to 500 μm, which had been prepared, were mixed with sterile distilled water to a final weight ratio of 2%, and physically stirred using a magnetic stirrer at 100 RPM for 10 minutes.

[0194] The solution containing the acellular dermis matrix particles mixed by physical stirring was injected into the high-pressure disperser, and then subjected to primary dispersion using a 200 μm nozzle chamber at 15,000 psi. The dispersion liquid containing the acellular dermis matrix particles after the primary high-pressure dispersion process was visually inspected for an increase in viscosity. As a result, phase separation was observed, confirming that the dispersion liquid was not sufficiently dispersed. Therefore, high-pressure dispersion was performed twice more, for a total of three repetitions. Figure 2 Thereafter, a vacuum oven (LK LabKorea, LO-VS380N) was used to remove air bubbles at -0.9 bar for 1 hour.

[0195] Example 1.4. Drying of the acellular dermis matrix dispersion

[0196] A vacuum drying process was performed to process the homogenously dispersed acellular dermis matrix dispersion obtained in the above Example 1.3 into a film preparation.

[0197] ​Specifically, 50 ml of acellular dermal matrix dispersion dispersed at 2 wt % was added to a 100 cm 2 sized petri dish and the petri dish was tilted to ensure even application. Then, the petri dish was put into a vacuum oven at 40°C and dried for 1 day at -0.9 bar.

[0198] After drying, the thickness of the film separated from the petri dish was measured to be 96 pm. This was standardized, put into a sterilized packaging bag and sealed. The sealed packaged dried acellular dermal matrix dispersion (i.e. film containing acellular dermal matrix dispersion) was sterilized using gamma ray or eBeam sterilization (15 kGy). After sterilization, it was stored at room temperature until use ( Figure 3 ).

[0199] II. Evaluation of the physico-chemical properties of the film containing acellular dermal matrix dispersion

[0200] Experimental Example 1.1. Observation of the appearance of the film containing acellular dermal matrix dispersion

[0201] The external morphology of the acellular dermal film prepared using the dermal matrix dispersion in the above Example 1 was observed Figure 4a , and the applicability of the sample after hydration was observed Figure 4b . The observation results confirmed that the tissue had properties suitable for transfer or application to a wound site using forceps.

[0202] Experimental Example 1.2. Observation of the appearance pattern image of the film containing acellular dermal matrix dispersion

[0203] Using a scalpel or a puncher, a circular punch pattern Figure 4c or a linear punch pattern Figure 4d was designed on the acellular dermal film obtained in the above Example 1.

[0204] It was confirmed that the film containing acellular dermal matrix dispersion having the above type of pattern improved the uniformity and hydration speed of the film upon passage of exudate, and also prevented the phenomenon of curling of the film end compared to the commercially available dermal film product.

[0205] Experimental Example 2.1. Observation of the homogenization of the film containing acellular dermal matrix dispersion

[0206] The thickness of the film containing acellular dermal matrix dispersion prepared in the above Example 1 was measured using a Nikon (MFC-101A) Digimicro device. The film thickness was confirmed to be 96 pm Figure 5a). To verify the homogenization property of the product, the transparency on the printed letters was observed. It was confirmed that the transparency was sufficient to clearly see all the printed letters, indicating that the acellular dermal matrix dispersion liquid was uniformly dispersed in the film preparation process Figure 5b ).

[0207] Experimental Example 2.2. Comparative observation of homogenization of the film containing the acellular dermal matrix dispersion liquid and the commercially available film

[0208] The homogenization property of the film containing the acellular dermal matrix dispersion liquid prepared in the above Example 1 was observed, and compared with that of the commercially available dermal matrix film (CG Bio's CGderm™ Matrix).

[0209] The observation result confirmed that the transparency of the dermal matrix film of the present application was superior to that of the conventional commercially available dermal matrix film, both before and after hydration Figure 5c . Thus, it was found that the dermal matrix film of the present application, as a film containing an acellular dermal matrix uniformly dispersed therein, has better efficacy and usability compared to the conventional commercially available dermal matrix film.

[0210] Experimental Example 3.1. Tensile force test of the film containing the acellular dermal matrix dispersion liquid

[0211] The tensile strength of the film containing the acellular dermal matrix dispersion liquid prepared in the above Example 1 was measured. The tensile strength test was performed using a universal testing machine (AGS-X type (SHIMADZU)). Since the tensile strength is affected by the thickness and width of the sample, the sample was first prepared by cutting into a 1 x 5 cm 2 strip in a dry state. The tensile strength of the above-prepared sample was tested in a dry state and a hydrated state. Among them, the hydrated sample hydrated with sterile distilled water had a length of 3 cm and a thickness of 150 µm.

[0212] The measurement result confirmed that the dry film containing the acellular dermal matrix dispersion liquid was broken at a maximum load of about 27 N Figure 6a , and the hydrated film containing the acellular dermal matrix dispersion liquid was broken at a maximum load of about 1.42 N Figure 6b .

[0213] Experimental Example 3.2. Comparative test of the tensile force of the film containing the acellular dermal matrix dispersion liquid and the commercially available film

[0214] The breaking test was performed on the film containing the acellular dermal matrix dispersion liquid prepared in the above Example 1 and the commercially available dermal matrix film (CG Bio's CGderm™ Matrix), and the tensile strength of the two films was compared. At this time, the sample in a dry state was cut into a 1 x 5 cm 2The tensile strength was measured using a universal testing machine at a speed of 4 mm / min, in the same manner as in Experimental Example 3.1 above. However, the tensile strength of the sample was measured in a hydrated state. After hydration for 10 minutes with sterile distilled water, the length of the sample was 3 cm, and the thickness of the film containing the acellular dermal matrix dispersion was 150 μm, while the thickness of the commercially available dermal matrix film was 500 μm.

[0215] The measurement results confirmed that the film containing the acellular dermal matrix dispersion (SC Derm Matrix) was ruptured at a maximum load of about 1.4 N, while the commercially available dermal matrix film (CG Derm Matrix) was ruptured at a maximum load of about 0.14 N Figure 6c ). Thus, it was found that the dermal matrix film of the present application can provide the advantages of better efficacy and usability, since the tensile strength thereof is about 10 times higher than that of the conventional commercially available dermal matrix film.

[0216] Experimental Example 4. Surface topography analysis of the film containing the acellular dermal matrix dispersion

[0217] The film containing the acellular dermal matrix dispersion prepared in Example 1 above was subjected to surface topography analysis. The surface analysis was performed using a scanning electron microscope (SEM; Model IM-150 (ETS)). At this time, the sample was inorganically gold-sputtered for surface analysis (SEM) of organic materials, and then photographed at 5 kV. In order to confirm the topography of the tissue after hydration, the sample was hydrated in sterile distilled water for 1 minute, then frozen at -80°C for 1 hour, and finally freeze-dried for 1 day to reproduce the topography of the hydrated tissue. When observed at a magnification of 3000 times, it was confirmed that the dried cross-section of the film containing the acellular dermal matrix dispersion had a dense structure Figure 7a ). In addition, when magnified at 120 times and 1000 times, respectively, it was confirmed that the cross-section of the freeze-dried film after hydration had a porous structure Figure 7b ). The above results indicate that it was found that the prepared film containing the acellular dermal matrix dispersion had a porous structure after hydration, and thus had the characteristics of promoting the flow of body fluids, thereby being useful for tissue regeneration.

[0218] Experimental Example 5. Confirmation of the applicability of the film containing the acellular dermal matrix dispersion to surgical operations

[0219] In order to confirm the applicability of the film containing the acellular dermal matrix dispersion prepared in Example 1 above to surgical operations, the film containing the acellular dermal matrix dispersion was applied to a surgical incision, and then sutured with a suture or a surgical stapler.

[0220] Specifically, in order to sew the film containing the acellular dermal matrix dispersion (whether in a dry state or a hydrated state) with gauze, sewing was performed using a polyamide suture. Thereafter, it was confirmed whether the film was separated from the gauze and whether the film was damaged during the sewing process.

[0221] In order to verify whether the film containing the acellular dermal matrix dispersion is applicable to a stapler in a dry state and a hydrated state, the film was fixed to gauze using a general office stapler. It was confirmed that the film containing the acellular dermal matrix dispersion did not have any special problems in all of the above conditions Figure 8 ).

[0222] Accordingly, it was found that the film containing the acellular dermal matrix dispersion according to the present application has excellent efficacy and usability when applied as a graft material.

[0223] III. Evaluation of biochemical properties of the film containing the acellular dermal matrix dispersion

[0224] Experimental Example 6. Confirmation of extracellular matrix (ECM) preservation in the film containing the acellular dermal matrix dispersion

[0225] In order to confirm whether the film containing the acellular dermal matrix dispersion prepared in the above Example 1 is suitable as a graft material, the loss of extracellular matrix proteins essential for tissue regeneration was measured. Specifically, the collagen content was measured using a Sircol insoluble collagen assay kit by Biocolor. At this time, the film containing the acellular dermal matrix dispersion was used as a test group, and CGderm™ matrix from CG Bio and Matriderm® from MedSkin Solutions Dr. Suwelack Headquarters were used as comparison groups.

[0226] The measurement results confirmed that, in the samples used as comparison groups, the collagen level preserved by the CGderm™ matrix was 59 pg / mg, and the collagen level preserved by Matriderm® was 178 pg / mg. In contrast, it was confirmed that the collagen level preserved by the film containing the acellular dermal matrix dispersion, i.e., the test group, was significantly high at 452 pg / mg Figure 9 ).

[0227] From the above results, it was found that the acellular dermal tissue film produced using the acellular dermal matrix dispersion has excellent histological preservation rates for proteins such as collagen. Accordingly, it was found that the acellular dermal tissue film has excellent effects that can be used as a graft material.

[0228] Experimental Example 7. Confirmation of biological graft applicability of the film containing the acellular dermal matrix dispersion

[0229] To apply the film containing the cell-free dermal matrix dispersion prepared in Example 1 above to the subcutaneous tissue, the cell-free dermal film was first processed to a size of 40 cm 2 in length, 20 cm in width, and 0.1 mm in thickness, and applied to three sites in an animal model constructed to allow infusion of the film. After applying the film as a grafting material to the subcutaneous tissue, the cell growth and structure maintenance time were confirmed.

[0230] Specifically, for the subcutaneous implantation of the film containing the cell-free dermal matrix dispersion, a large area of the back was selected as the experimental site in a pig animal model of 45 kg or more, the skin was incised, and the cell-free dermal film was inserted. The incised skin tissue was sutured to fix the implanted cell-free dermal film Figure 10 ).

[0231] Experimental Example 8. Verification of the degree of implantation and effectiveness of the film containing the cell-free dermal matrix dispersion in biological tissue through histological analysis

[0232] In Experimental Example 7 above, the film containing the cell-free dermal matrix dispersion was implanted into the subcutaneous tissue and sutured. Four weeks later, the implanted area was incised, and tissue containing the entire skin layer and the film was obtained. The resulting tissue was subjected to histological analysis using H&E staining.

[0233] As a result of H&E staining, the area into which the cell-free dermal film determined as a grafting material was infused into the adipose layer was confirmed. It was confirmed that, after 4 weeks, the area was well-stained, and no inflammatory reaction was observed. As such, the cell-free dermal film did not cause any effects such as inflammation or contamination of the surrounding tissue. In addition, a large number of cell nuclei were observed within the graft, indicating successful transplantation Figure 11 ).

[0234] IV. Evaluation of the efficacy of the film containing the cell-free dermal matrix dispersion in a rat wound model

[0235] The wound healing efficacy of the test substances (Table 1) was evaluated in an in vivo rat wound model. At this time, SD rats (Spargue Dawley, 6 weeks old, male, Orient Bio Inc.) were used. After a one-week adaptation period after the experimental animals were caged, the animals were anesthetized with 3.0% isoflurane, and the hair on the back of the animals was shaved. Then, using a biopsy punch (10 mm, Kai Sterile Dermal Biopsy Punch, Kai Ind., Japan), a circular defect (full-thickness; epidermis and dermis) was made on the back of all groups to induce a wound. Except for the untreated group, for the positive control group 1 (CGderm Matrix), the positive control group 2 (MatriDerm), and the test group (SCderm Matrix), the test substances were applied to the wound site Figure 12 and Figure 13 ).

[0236] [Table 1]

[0237]

[0238] The change in wound area was evaluated by visual assessment, and the wound width on the cross-section of rat skin tissue, inflammatory cell count, and re-epithelialization thickness were analyzed using hematoxylin-eosin (H&E) staining. In addition, collagen production was evaluated by measuring collagen density using Masson's trichrome staining (MT), and the expression levels of fibroblast marker vimentin and myofibroblast marker a-smooth muscle actin (a-SMA) were evaluated using immunofluorescence (IF) staining.

[0239] Experimental Example 9. Visual assessment

[0240] Experimental Example 9.1. Wound area analysis

[0241] On day 0, day 1, day 3, day 5, day 7, and day 14 after wound induction, photographs of the wound sites of the rats in each group were taken at a fixed distance using a digital camera (CAMEDIA™, Olympus, Japan). Thereafter, the wound changes and wound healing process were observed by visual observation, and the percentage change (%) in wound area was measured and analyzed using the ImageJ program (National Institutes of Health, USA).

[0242] The following mathematical formula 1 or mathematical formula 2 was used to calculate the percentage change (%) in wound area.

[0243] [Mathematical Formula 1]

[0244] Percentage change in wound area (%) = ((wound area on day 0 - wound area on the day of analysis) / wound area on day 0) x 100

[0245] [Mathematical Formula 2]

[0246] Percentage change in wound area (%) = ((wound area of the untreated group - wound area of the test group) / wound area of the untreated group) x 100

[0247] The results of the wound area analysis showed that in the positive control group 1 (CGderm matrix), compared to the untreated group, the wound area showed a decreasing trend on day 1, day 3, day 5, and day 14, decreasing by 4.95%, 9.34%, 10.67%, and 53.33%, respectively, and significantly decreasing (i.e., improving) by 29.62% on day 7 compared to the untreated group.

[0248] In the positive control group 2 (MatriDerm), the wound area showed a tendency of reduction (i.e. improvement) compared to the untreated group at day 1, day 3, day 5 and day 14 by 6.74%, 4.09%, 9.22% and 62.66% respectively, and significantly reduced (i.e. improved) by 31.97% at day 7.

[0249] In the test group (SC Dermatogrid), the wound area showed a tendency of reduction compared to the untreated group at day 5 and day 14 by 15.40% and 81.96% respectively, and significantly reduced (i.e. improved) by 20.75%, 15.20% and 43.76% at day 1, day 3 and day 7 respectively. Figures 14a to 14f

[0250] Experimental Example 10. Histological evaluation (H&E staining)

[0251] At day 7 and day 14 after wound induction, biopsy tissues were taken from the wound site and its surrounding area of the rats, fixed with 10% formalin solution, and made into paraffin blocks. Thereafter, the paraffin blocks were cut into 3 pm-thick sections, subjected to hydration treatment, and stained with H&E solution. Subsequently, cross-sectional photographs of the rat skin tissues were taken using an optical microscope (Zeweiss™, Germany), and the wound width, inflammatory cells and re-epithelialization thickness were analyzed using ImageJ software (National Institutes of Health, USA). At this time, the percentage change (%) of the wound width, the percentage change (%) of the inflammatory cells and the percentage change (%) of the re-epithelialization were calculated using Mathematical Formula 3, Mathematical Formula 4 and Mathematical Formula 5, respectively.

[0252] [ Mathematical Formula 3 ]

[0253] Percentage change (%) of wound width = ((Untreated group wound width - Test group wound width) / Untreated group wound width) x 100

[0254] [ Mathematical Formula 4 ]

[0255] Percentage change (%) of inflammatory cells = ((Untreated group inflammatory cell count - Test group inflammatory cell count) / Untreated group inflammatory cell count) x 100

[0256] [ Mathematical Formula 5 ]

[0257] Percentage change (%) of re-epithelialization = ((Test group skin thickness - Untreated group skin thickness) / Untreated group skin thickness) x 100

[0258] Experimental Example 10.1. Wound width analysis

[0259] ​At day 7 and day 14 after wound induction, the wound width of the rat skin tissue cross-sections was analyzed using H&E staining. The results show that at day 7, the wound width of positive control group 1 (CGderm Matrix) shows a decreasing tendency compared to the untreated group, decreasing by 14.71 %; the wound width of positive control group 2 (MatriDerm) and the test group (SCderm Matrix) significantly decreases (i.e. improves) by 18.20 % and 38.80 %, respectively, compared to the untreated group. At day 14, the wound width of positive control group 2 (MatriDerm) shows a decreasing tendency compared to the untreated group, decreasing by 19.04 %; the wound width of positive control group 1 (CGderm Matrix) and the test group (SCderm Matrix) significantly decreases (i.e. improves) by 26.19 % and 38.67 %, respectively, compared to the untreated group. Figure 15a and 15b ).

[0260] Experimental Example 10.2. Inflammatory cell analysis

[0261] At day 7 and day 14 after wound induction, the inflammatory cells on the rat skin tissue cross-sections were analyzed using H&E staining. The results show that at day 7, the inflammatory cell count of positive control group 1 (CGderm Matrix) shows a decreasing tendency compared to the untreated group, decreasing by 27.64 %, while the inflammatory cell count of positive control group 2 (MatriDerm) and the test group (SCderm Matrix) significantly decreases by 29.82 % and 45.41 %, respectively, compared to the untreated group. At day 14, the inflammatory cell count of positive control group 1 (CGderm Matrix) and positive control group 2 (MatriDerm) shows a decreasing tendency compared to the untreated group, decreasing by 38.69 % and 32.16 %, respectively, while the inflammatory cell count of positive control group 1 (CGderm Matrix) and the test group (SCderm Matrix) significantly decreases (i.e. improves) by 38.69 % and 44.82 %, respectively, compared to the untreated group. Figures 16a to 16c ).

[0262] Experimental Example 10.3. Re-epithelialization analysis

[0263] At day 7 and day 14 post-wound induction, re-epithelialization thickness on the cross-section of rat skin tissue was analyzed using H&E staining. The results showed that at day 7, re-epithelialization thickness of positive control group 1 (CGderm Matrix) and positive control group 2 (MatriDerm) showed a trend of increase, with an increase of 33.11% and 16.76% respectively, compared to the untreated group; while in the test group (SCderm Matrix), re-epithelialization thickness was significantly increased (i.e. improved) by 56.25% compared to the untreated group. At day 14, re-epithelialization thickness of positive control group 1 (CGderm Matrix), positive control group 2 (MatriDerm) and test group (SCderm Matrix) showed a trend of decrease, with a decrease of 9.74%, 15.23% and 21.78% respectively, compared to the untreated group. Figures 17a to 17c .

[0264] Conclusion of H&E staining analysis

[0265] Summarizing the results of the series of H&E staining analysis above, wound width analysis showed that the wound width of the test group was significantly reduced (i.e. improved) by 38.80% and 38.67% at day 7 and day 14 respectively, compared to the untreated group. Inflammatory cell count analysis showed that the inflammatory cell count of the test group was significantly reduced (i.e. improved) by 45.41% and 44.82% at day 7 and day 14 respectively, compared to the untreated group. In addition, re-epithelialization thickness analysis showed that the re-epithelialization thickness of the test group was significantly increased (i.e. improved) by 56.25% at day 7, compared to the untreated group.

[0266] Experimental Example 11. Histological evaluation (MT staining)

[0267] At day 7 and day 14 post-wound induction, biopsy tissues were taken from the wound site and its surrounding area of the rats, respectively, and fixed with 10% formalin solution and paraffin-embedded. Thereafter, the paraffin blocks were cut into 3 pm-thick sections, hydrated and stained with Biebrich Scarlet-Acid Fuchsin solution for 5 minutes. After washing, they were again stained with phosphotungstic / phosphomolybdic acid for 5 minutes, followed by staining with aniline blue and acetic acid solution. Thereafter, photographs of the cross-section of rat skin tissue were taken using an optical microscope (Zeweiss™, Germany) and collagen density was analyzed using ImageJ software (National Institutes of Health, USA).

[0268] The percentage (%) change in collagen density was calculated using the following mathematical formula 6.

[0269] [mathematical formula 6]

[0270] Collagen density change percentage (%) = ((test group collagen density - untreated group collagen density) / untreated group collagen density) x 100

[0271] Experimental Example 11.1. Collagen density analysis

[0272] At day 14 after wound induction, the collagen density in the cross-section of rat skin tissue was analyzed using the MT staining method. The results showed that, at day 7, the collagen density of positive control group 1 (CGderm Matrix) and positive control group 2 (MatriDerm) showed a tendency to increase by 17.09% and 32.87%, respectively, compared to the untreated group, while the collagen density of the test group (SCderm Matrix) was significantly increased (i.e., improved) by 75.42% compared to the untreated group. At day 14, the collagen density of positive control group 1 (CGderm Matrix), positive control group 2 (MatriDerm), and the test group (SCderm Matrix) was significantly increased (i.e., improved) by 65.55%, 64.79%, and 81.57%, respectively, compared to the untreated group. Figures 18a to 18d .

[0273] Experimental Example 12. Histological evaluation (immunofluorescence staining)

[0274] At day 7 and day 14 after wound induction, biopsy tissues were taken from the wound site and its surrounding area of the rats, respectively, and the biopsy tissues were fixed with 10% formalin and prepared into tissue sections. Subsequently, they were cut into 3 pm-thick sections, subjected to hydration treatment, and subjected to antigen exposure using 10 mM sodium citrate buffer. Thereafter, the sections were treated with primary antibodies, vimentin and a-SMA (Abcam, USA), respectively, and subjected to fluorescent staining using secondary antibody, rabbit anti-mouse IgG H&L (Alexa Fluor® 488). After DAPI staining (DAPI-containing VECTASHIELD® anti-fluorescence quenching mounting medium), cross-sectional photographs of rat skin tissue were taken using a fluorescence microscope (Zeiss AxioObserver 7, Germany). Fluorescent intensity analysis was performed using ImageJ software (National Institutes of Health, USA). At this time, the vimentin change percentage (%) and a-SMA change percentage (%) were calculated using mathematical formula 7 and mathematical formula 8, respectively.

[0275]

Mathematical Formula 7

[0276] Vimentin change percentage (%) = ((test group vimentin - untreated group vimentin) / untreated group vimentin) x 100

[0277]

Mathematical Formula 8

[0278] Alpha-SMA % change (%) = ((Test Group Alpha-SMA - Untreated Group Alpha-SMA) / Untreated Group Alpha-SMA) x 100

[0279] Experimental Example 12.1. Fibroblast (Vimentin) Analysis

[0280] At day 7 and day 14 post-wound induction, the fluorescence intensity of the fibroblast marker vimentin in cross-sections of rat skin tissue was analyzed using immunofluorescence staining. The results showed that at day 7, the fluorescence intensity of vimentin in positive control group 1 (CGderm Matrix) and positive control group 2 (MatriDerm) showed a tendency to increase, by 27.62% and 30.37%, respectively, compared to the untreated group, while the fluorescence intensity of vimentin in the test group (SCderm Matrix) was significantly increased (i.e. improved) by 40.40% compared to the untreated group. Furthermore, at day 14, the fluorescence intensity of vimentin in positive control group 1 (CGderm Matrix) and positive control group 2 (MatriDerm) showed a tendency to increase, by 22.60% and 24.17%, respectively, compared to the untreated group, while the fluorescence intensity of vimentin in the test group (SCderm Matrix) was significantly increased (i.e. improved) by 39.14% compared to the untreated group. Figures 19a to 19c .

[0281] Experimental Example 12.2. Myofibroblast (Alpha-SMA) Analysis

[0282] At day 7 and day 14 post-wound induction, the fluorescence intensity of the myofibroblast marker alpha-SMA in cross-sections of rat skin tissue was analyzed using immunofluorescence staining. The results showed that at day 7, the fluorescence intensity of alpha-SMA in positive control group 1 (CGderm Matrix) showed a tendency to increase, by 32.16%, compared to the untreated group; while the fluorescence intensity of alpha-SMA in positive control group 2 (MatriDerm) and the test group (SCderm Matrix) was significantly increased (i.e. improved) by 46.21% and 60.23%, respectively, compared to the untreated group. At day 14, the fluorescence intensity of alpha-SMA in positive control group 1 (CGderm Matrix), positive control group 2 (MatriDerm) and the test group (SCderm Matrix) was significantly increased (i.e. improved) by 36.80%, 41.52% and 55.75%, respectively, compared to the untreated group. Figures 20a to 20c .

[0283] Conclusion of immunofluorescence staining analysis

[0284] The results of the immunofluorescence staining (IF staining) analysis were summarized, and at 7 days and 14 days after the wound induction, the fluorescence density of the fibroblast biomarker vimentin in the test group was significantly increased (i.e., improved) by 40.40% and 39.14%, respectively, compared to the untreated group; at 7 days and 14 days, the analysis of the myofibroblast biomarker a-SMA in the test group showed that the fluorescence density of a-SMA was significantly increased (i.e., improved) by 60.23% and 55.75%, respectively, compared to the untreated group.

[0285] Conclusion of wound healing efficacy evaluation of films containing acellular dermal matrix dispersion Conclusion of wound healing efficacy evaluation of films containing acellular dermal matrix dispersion

[0286] The above series of visual observation and histological analysis using the in vivo rat wound model confirmed that the film containing the acellular dermal matrix dispersion (SC dermal matrix) showed a wound healing effect. Therefore, it was found that the SC dermal matrix can be effectively applied to the regenerative medicine field as a biomaterial for transplantation, for example, as a wound dressing and artificial skin.

Claims

1. A cell-free dermal matrix dispersion liquid comprising cell-free dermal matrix microparticles.

2. The cell-free dermal matrix dispersion liquid according to claim 1, wherein the particle size of the cell-free dermal matrix microparticles is 100 μm to 500 μm.

3. The cell-free dermal matrix dispersion liquid according to claim 1, wherein the cell-free dermal matrix dispersion liquid is prepared by high pressure dispersion of the cell-free dermal matrix microparticles in a solution.

4. The cell-free dermal matrix dispersion liquid according to claim 1, wherein the cell-free dermal matrix microparticles are contained in an amount of 1.0 wt% to 3.0 wt% based on the total weight of the cell-free dermal matrix dispersion liquid.

5. The cell-free dermal matrix dispersion liquid according to claim 1, wherein the cell-free dermal matrix is decellularized by a supercritical fluid extraction process.

6. The cell-free dermal matrix dispersion liquid according to claim 3, wherein the conditions of high pressure dispersion are performed by applying a pressure of 8,000 psi to 40,000 psi and passing through a nozzle chamber of 100 μm to 300 μm once or more.

7. The cell-free dermal matrix dispersion liquid according to claim 3, wherein the solution is selected from the group consisting of distilled water, physiological saline, phosphate buffered solution (PBS), HBSS (Hanks Balanced Salt Solution), TBS (Tris Buffered Salt Solution), TAPS (N-tris(hydroxymethyl)-3-aminopropanesulfonic acid) buffered solution, Bicine (N,N-bis(2-hydroxyethyl)glycine) buffered solution, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffered solution, TES (N-tris(hydroxymethyl)-2-aminethanesulfonic acid) buffered solution, PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)) buffered solution, arsenite buffered solution, MES (2-(N-morpholino)ethanesulfonic acid) buffered solution, MEM (Minimum Essential Medium), DMEM (Dulbecco's Modified Eagle Medium), RPMI1640, IMDM (Iscove's Modified Dulbecco's Medium), defined keratinocyte serum-free culture without BPE (bovine pituitary extract), keratinocyte serum-free culture with BPE, KnockOut D-MEM, AmnioMAX-II complete medium, AmnioMAX-C100 complete medium, and mixtures thereof.

8. The cell-free dermal matrix dispersion liquid according to claim 5, wherein the solvent used as a supercritical fluid is carbon dioxide.

9. The cell-free dermal matrix dispersion liquid according to claim 5, wherein the supercritical fluid further comprises ethanol as a co-solvent.

10. The cell-free dermal matrix dispersion liquid according to claim 5, wherein the extraction is performed under temperature conditions of 30°C to 40°C.

11. The cell-free dermal matrix dispersion liquid according to claim 5, wherein the extraction is performed under pressure conditions of 50 bar to 400 bar.

12. The acellular dermal matrix dispersion liquid of claim 5, wherein the extraction is performed for 1 hour to 3 hours.

13. The acellular dermal matrix dispersion liquid of claim 5, wherein a step of separating an epidermis layer and a dermis layer is included prior to the step of extracting with a supercritical fluid.

14. The acellular dermal matrix dispersion liquid of claim 5, wherein any one of the following steps is further included after the step of extracting with a supercritical fluid: a step of washing the dermis tissue with a phosphate buffer solution; a step of freeze-drying the washed dermis tissue; a step of micronizing the freeze-dried dermis tissue to prepare dermal matrix microparticles; and a step of high-pressure dispersing the dermal matrix microparticles in a solution to prepare the dermal matrix dispersion liquid.

15. An acellular dermal membrane comprising the acellular dermal matrix dispersion liquid of any one of claims 1 to 14.

16. The acellular dermal membrane of claim 15, wherein the acellular dermal matrix dispersion liquid is a solution in which dermal matrix microparticles having a particle size of 100 pm to 500 pm are dispersed under high pressure.

17. The acellular dermal membrane of claim 15, wherein the acellular dermal membrane has a thickness of 0.01 mm to 1 mm.

18. The acellular dermal membrane of claim 15, wherein the acellular dermal membrane has a tensile strength of 0.5 N to 30 N.

19. The acellular dermal membrane of claim 15, wherein the acellular dermal membrane has a collagen content of 200 pg / mg to 600 pg / mg based on dry weight.

20. The acellular dermal membrane of claim 15, wherein the acellular dermal membrane has a porous structure.

21. The acellular dermal membrane of claim 15, wherein the acellular dermal membrane is used as a wound dressing, an adhesion barrier, a drug delivery graft, or an artificial skin.

22. A method of preparing an acellular dermal matrix dispersion liquid, comprising: a) a step of separating a skin tissue isolated from a subject into an epidermis layer and a dermis layer; b) a step of extracting the separated dermis layer with a supercritical fluid; c) a step of washing the dermis layer after the supercritical fluid extraction with a phosphate buffer solution; d) a step of freeze-drying the washed dermis tissue; e) a step of micronizing the freeze-dried dermis tissue to prepare dermal matrix microparticles; and f) a step of high-pressure dispersing the dermal matrix microparticles in a solution to prepare the dermal matrix dispersion liquid.

23. The method of preparing an acellular dermal matrix dispersion liquid of claim 22, wherein the method further comprises a step of stirring the dermal matrix microparticles with a solution prior to step f).

24. The method of preparing an acellular dermal matrix dispersion liquid of claim 22, wherein the dermal matrix microparticles in step f) have a particle size of 100 pm to 500 pm. ​ ​ 25. The method of claim 22, wherein the high pressure dispersion in step f) is performed by applying a pressure of 8,000 psi to 40,000 psi and passing one or more times through a nozzle chamber of 100 pm to 300 pm.

26. A method of preparing an acellular dermal membrane, comprising: a) a step of separating a skin tissue isolated from a subject into an epidermis layer and a dermis layer; b) a step of extracting the separated dermis layer with a supercritical fluid; c) a step of washing the supercritical fluid extracted dermis layer with a phosphate buffered solution; d) a step of freeze-drying the washed dermis tissue; e) a step of micronizing the freeze-dried dermis tissue to prepare dermis matrix microparticles; f) a step of high pressure dispersing the dermis matrix microparticles in a solution to prepare a dermis matrix dispersion; and g) a step of applying the dermis matrix dispersion and drying the dermis matrix dispersion to prepare a dermis membrane.

27. The method of claim 26, wherein the method further comprises a step of mixing the dermis matrix microparticles with a solution by stirring before step f).

28. The method of claim 26, wherein the particle size of the dermis matrix microparticles in step f) is 100 pm to 500 pm.

29. The method of claim 26, wherein the high pressure dispersion in step f) is performed by applying a pressure of 8,000 psi to 40,000 psi and passing one or more times through a nozzle chamber of 100 pm to 300 pm.

30. The method of claim 26, wherein the dermis membrane prepared in step g) has a thickness of 0.01 mm to 1 mm.

31. The method of claim 26, wherein the method comprises a step of sterilizing the dermis membrane after step g).

Images