Dispersion comprising acellular dermal matrix microparticles, film comprising same, and uses thereof

Abstract

The present invention relates to a dispersion comprising acellular dermal matrix microparticles, a film comprising same, and uses thereof. More specifically, the present invention relates to a film containing a dispersion comprising acellular dermal matrix microparticles obtained through supercritical fluid extraction and high-pressure dispersion operations, and a manufacturing method therefor. The acellular dermal film according to the present invention exhibits excellent biostability and biocompatibility, and demonstrates significantly superior preservation of extracellular matrix components such as collagen. In addition, the present invention has excellent mechanical properties and thus can be easily applied to graft surgery and suturing, and has a porous structure that facilitates the movement of body fluids, and thus may support tissue regeneration. Accordingly, the acellular dermal film can be usefully employed in the field of regenerative medicine as as wound dressings, adhesion barriers, and as implantable biomaterials such as drug delivery implants and artificial skin.

Description

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

[0001] The present invention relates to a dispersion comprising acellular dermal matrix microparticles, a film comprising the dispersion, and uses thereof. More particularly, the present invention relates to a film containing a dispersion comprising acellular dermal matrix microparticles obtained through a supercritical fluid extraction process and a high-pressure dispersion process, and a method for producing the dispersion.

[0002] Human skin tissue consists of the epidermis, the outermost layer, the dermis, and the subcutaneous tissue. The epidermis is composed of epithelial cells, differentiated into multiple layers from the basement membrane, which firmly connects the epidermis and dermis, as well as melanocytes and immune cells. Beneath the epidermis, the dermis is primarily composed of fibroblasts and the extracellular matrix, which is composed of collagen and elastin.

[0003] The extracellular matrix is ​​a crucial substance that forms the structure of the human body and regulates vital functions. Various biomaterials utilizing it, such as tissue repair materials and dressing bandages, have been introduced. However, currently commercialized products utilize only a subset of the extracellular matrix components.

[0004] Biomaterials can be defined as artificial substances that come into contact with surrounding tissues and body fluids intermittently or continuously in the body to replace the functions of damaged or dysfunctional human tissues and organs.

[0005] Biomaterials used in the human body must be similar to the tissue to be repaired in terms of size and shape, and their mechanical and physical properties must be as similar as possible to the original tissue to maintain a lasting effect.

[0006] Accordingly, they are manufactured in various forms, such as solutions, films, sponges, fibers, and hydrogels, to suit the application site and intended use. Among these, film-type biomaterials are used to function as wound dressings or to strengthen the epithelial cell layer and increase the rate of tissue engraftment in the affected area. Furthermore, they can be used as drug delivery agents, anti-adhesion barriers, and artificial organs for medical purposes (WO 2016-195152 A1).

[0007] Meanwhile, Korean Patent No. 10-1434041 discloses a 200-300 μm thick collagen membrane for inducing regeneration manufactured from biotissue-derived materials. However, the manufacturing of the collagen-containing membrane involves the use of cross-linking agents that can remain in the body and exhibit toxicity, making it difficult to ensure the safety of the biomaterial. Furthermore, the multi-step cross-linking process, including freeze-drying, re-dissolution, and subsequent physical cross-linking, necessitates a complex, time-consuming, and energy-intensive production process.

[0008] Accordingly, the inventors of the present invention conducted research to develop a film-type extracellular matrix-containing biomaterial that overcomes the problems of the prior art. As a result, they established conditions and a method for producing an acellular dermal film without a chemical decellularization process or a chemical cross-linking process. Through a series of processes including a supercritical fluid extraction process and a high-pressure dispersion process, a dispersion containing acellular dermal matrix microparticles was obtained, and the acellular dermal film was manufactured by processing the obtained dispersion, thereby completing the present invention.

[0009] To achieve the above object, one aspect of the present invention provides an acellular dermal matrix dispersion comprising acellular dermal matrix microparticles.

[0010] Another aspect of the present invention provides an acellular dermal film comprising the acellular dermal matrix dispersion.

[0011] Another aspect of the present invention provides a method for producing the acellular dermal matrix dispersion.

[0012] Another aspect of the present invention provides a method for producing the acellular dermal film.

[0013] The acellular dermal film according to the present invention is obtained by decellularizing the skin tissue-derived extracellular matrix used as a main component through a supercritical fluid extraction process without using a surfactant, and since no chemical cross-linking agent is used in the film production using the obtained acellular dermal matrix, it exhibits excellent biostability and biocompatibility. Furthermore, since an acellular dermal matrix dispersion homogenized through a high-pressure dispersion process is applied during film production, the dispersibility and uniformity of the acellular dermal matrix can be further improved.

[0014] The acellular dermal film according to the present invention not only exhibits remarkably high preservation of extracellular matrix components such as collagen, but also exhibits high tensile strength, resulting in superior mechanical properties, making it suitable for transplantation and suturing. Furthermore, the film possesses a porous structure that facilitates the movement of body fluids, thereby contributing to tissue regeneration.

[0015] Therefore, the film containing the acellular dermal matrix dispersion according to the present invention can be usefully utilized in the field of regenerative medicine as a biomaterial for transplantation, such as a wound dressing, an anti-adhesion agent, a drug delivery transplant material, and artificial skin.

[0016] Figure 1 schematically illustrates a manufacturing process of a film containing a human-derived acellular dermal matrix dispersion according to one embodiment of the present invention.

[0017] FIG. 2 is a photograph of an acellular dermal matrix dispersion obtained after high-pressure dispersion in sterile distilled water containing acellular dermal matrix microparticles according to one embodiment of the present invention.

[0018] Figure 3 is a photograph taken after sealing and sterilizing a film-shaped product manufactured by vacuum-drying an acellular dermal matrix dispersion according to one embodiment of the present invention.

[0019] FIG. 4a is a photograph of the appearance of a film containing an acellular dermal matrix dispersion according to one embodiment of the present invention.

[0020] Figure 4b is a photograph taken to observe whether a film containing an acellular dermal matrix dispersion according to one embodiment of the present invention can be applied to a desired area after hydration.

[0021] Figures 4c and 4d are enlarged photographs of pattern images of the outer appearance of a film containing an acellular dermal matrix dispersion according to one embodiment of the present invention. Specifically, the photographs are enlarged photographs of images of a circular punching pattern (Figure 4c) and a linear punching pattern (Figure 4d).

[0022] FIG. 5a is a photograph showing the results of measuring the thickness of a film containing an acellular dermal matrix dispersion according to one embodiment of the present invention using Nikon (MFC-101A) digimicro equipment.

[0023] Figure 5b is a photograph showing the results of confirming the homogenization characteristics of a film containing an acellular dermal matrix dispersion according to one embodiment of the present invention.

[0024] Figure 5c is a comparative photograph showing the homogenization characteristics of a film containing an acellular dermal matrix dispersion according to one embodiment of the present invention and a conventional commercially available dermal matrix film.

[0025] Figure 6a shows the results of a tensile test of a film containing a dried acellular dermal matrix dispersion according to one embodiment of the present invention.

[0026] Figure 6b shows the results of a tensile test of a film containing a hydrated acellular dermal matrix dispersion according to one embodiment of the present invention.

[0027] Figure 6c shows the results of a comparative tensile test for a film containing a hydrated acellular dermal matrix dispersion according to one embodiment of the present invention and a conventional commercially available dermal matrix film.

[0028] Figure 7a is a microscopic photograph showing the result of examining the surface morphology of a film containing an acellular dermal matrix dispersion according to one embodiment of the present invention, which was hydrated and freeze-dried, using a scanning electron microscope (SEM) at a magnification of 3,000 times.

[0029] Figure 7b is a microscopic photograph showing the results of confirming the surface morphology of a film containing an acellular dermal matrix dispersion according to one embodiment of the present invention after hydrating and freeze-drying it, and then using a scanning electron microscope (SEM) at magnifications of 120x and 1,000x, respectively.

[0030] FIG. 8 is a photograph showing the results of confirming the surgical applicability of a film containing a dried or hydrated acellular dermal matrix dispersion according to one embodiment of the present invention.

[0031] Figure 9 shows the results of collagen content analysis of a film (SC Derm Matrix) containing an acellular dermal matrix dispersion according to one embodiment of the present invention.

[0032] FIG. 10 is a photograph of a surgical process of transplanting and suturing a film containing an acellular dermal matrix dispersion according to one embodiment of the present invention into the subcutaneous tissue of a pig animal model.

[0033] Figure 11 shows the results of H&E staining to confirm whether an inflammatory response is induced in surrounding tissues and the regenerative effect at 4 weeks after transplantation of a film containing an acellular dermal matrix dispersion according to one embodiment of the present invention.

[0034] Figure 12 is a schematic diagram schematically showing a schedule for evaluating the wound healing efficacy of a film containing an acellular dermal matrix dispersion (SC derm matrix) according to one embodiment of the present invention using a rat wound model.

[0035] Figure 13 shows a representative image of a rat wound model according to one embodiment of the present invention.

[0036] Figure 14a shows the results of an evaluation of wound healing efficacy according to attachment of CGderm matrix (positive control group 1), MatriDerm (positive control group 2), and SC derm matrix (test group, film containing acellular dermal matrix dispersion) to a rat wound model. Specifically, this graph shows the results of a visual evaluation and comparative analysis of the wound areas of the untreated group (negative control group), positive control group 1, positive control group 2, and test group.

[0037] Figures 14b to 14e are graphs showing the results of analyzing the wound area of ​​the untreated group (Figure 14b), positive control group 1 (Figure 14c), positive control group 2 (Figure 14d), and test group (Figure 14e) applied to the rat wound model through visual evaluation. † p<0.05, †† p<0.01, ††† p<0.001).

[0038] Figure 14f shows representative images of the wound area analysis results of the untreated group, positive control group 1 (CGderm matrix), positive control group 2 (MatriDerm), and test group (SC derm matrix) applied to the rat wound model.

[0039] Figure 15a shows a graph showing the results of 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 derm matrix) applied to the rat wound model using H&E staining. * p<0.05, ** p<0.01, *** p<0.001).

[0040] Figure 15b shows representative images of the wound widths of the untreated group, positive control group 1 (CGderm matrix), positive control group 2 (MatriDerm), and test group (SC derm matrix) applied to the rat wound model, histologically analyzed using H&E staining.

[0041] Figure 16a shows a graph showing the results of histological analysis of the number of inflammatory cells through H&E staining after application of untreated, CGderm matrix (positive control group 1), MatriDerm (positive control group 2), and SC derm matrix (test group) to a rat wound model ( * p<0.05, ** p<0.01, *** p<0.001).

[0042] Figures 16b and 16c show representative images of inflammatory cells in each group analyzed histologically at 7 days (Figure 16b) and 14 days (Figure 16c) after application of untreated, CGderm matrix (positive control group 1), MatriDerm (positive control group 2), and SC derm matrix (test group) to a rat wound model using H&E staining.

[0043] Figure 17a shows a graph showing the results of histological analysis of the re-epithelialization thickness through H&E staining after application of untreated, CGderm matrix (positive control group 1), MatriDerm (positive control group 2), and SC derm matrix (test group) to a rat wound model ( * p<0.05, ** p<0.01, *** p<0.001).

[0044] Figures 17b and 17c show representative images of the reepithelialization thickness of each group analyzed histologically through H&E staining at 7 days (Figure 17b) and 14 days (Figure 17c) after application of no treatment, CGderm matrix (positive control group 1), MatriDerm (positive control group 2), and SC derm matrix (test group) to a rat wound model.

[0045] Figure 18a shows a graph showing the results of histological analysis of collagen density through MT staining after application of untreated, CGderm matrix (positive control group 1), MatriDerm (positive control group 2), and SC derm matrix (test group) to a rat wound model ( * p<0.05, ** p<0.01, *** p<0.001).

[0046] Figure 18b shows representative images (×1.25 magnification) of the collagen density of each group analyzed histologically through MT staining after application of untreated, CGderm matrix (positive control group 1), MatriDerm (positive control group 2), and SC derm matrix (test group) to a rat wound model.

[0047] Figures 18c and 18d show representative images (×200 magnification) of the collagen density of each group analyzed histologically through MT staining at 7 days (Figure 18c) and 14 days (Figure 18d) after application of untreated, CGderm matrix (positive control group 1), MatriDerm (positive control group 2), and SC derm matrix (test group) to a rat wound model.

[0048] Figure 19a shows a graph showing the results of histological analysis of the fluorescence intensity of Vimentin through IF staining after application of untreated, CGderm matrix (positive control group 1), MatriDerm (positive control group 2), and SC derm matrix (test group) to a rat wound model ( * p<0.05, ** p<0.01, *** p<0.001).

[0049] Figures 19b and 19c show representative images (×200 magnification) of Vimentin fluorescence intensity of each group analyzed histologically through IF staining at 7 days (Figure 19b) and 14 days (Figure 19c) after application of untreated, CGderm matrix (positive control group 1), MatriDerm (positive control group 2), and SC derm matrix (test group) to a rat wound model.

[0050] Figure 20a shows a graph showing the results of histological analysis of the fluorescence intensity of α-SMA through IF staining after application of untreated, CGderm matrix (positive control group 1), MatriDerm (positive control group 2), and SC derm matrix (test group) to a rat wound model ( * p<0.05, ** p<0.01, *** p<0.001).

[0051] Figures 20b and 20c show representative images (×200 magnification) of the α-SMA fluorescence intensity of each group analyzed histologically through IF staining at 7 days (Figure 20b) and 14 days (Figure 20c) after application of untreated, CGderm matrix (positive control group 1), MatriDerm (positive control group 2), and SC derm matrix (test group) to a rat wound model.

[0052] Acellular dermal matrix dispersion

[0053] One aspect of the present invention provides an acellular dermal matrix dispersion comprising acellular dermal matrix microparticles.

[0054] As used herein, the term "dermis" refers to a layer of skin composed primarily of collagen fibers, interwoven with elastic fibers made of elastin. The dermis comprises the majority of the skin, providing nutrients and support to the epidermis and protecting it from external damage. It also has the ability to store moisture, regulate body temperature, and act as a sensory receptor, as well as regenerate the skin by interacting with the epidermis.

[0055] The term "acellular dermal matrix (ADM)" as used herein refers to an extracellular matrix derived from decellularized skin tissue, and is used interchangeably with 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 through acellularization technology, and refers to a bio-derived skin substitute in the form of an extracellular matrix (ECM) composed of collagen, elastin, etc.

[0056] The above acellular dermal matrix is ​​a biomaterial obtained by removing cells that can cause an immune response from skin separated from an individual, and can be used to restore skin by transplanting it to patients with skin defects caused by burns, traffic accidents, ulcers, etc.

[0057] As used herein, the term "microparticle", also referred to as a microparticle, particulate, or microsphere, means a spherical substance on the micrometer scale having a particle diameter typically between 1 and 1,000 μm.

[0058] The term "acellular dermal matrix microparticle" as used herein refers to a spherical material in powder form obtained by processing acellular dermal matrix into micrometer-sized particles through a freeze-drying and micronization process.

[0059] The above freeze-drying is a method of rapidly cooling a frozen acellular dermal matrix and then absorbing moisture through a vacuum. By controlling the moisture within the dermal matrix according to the freeze-drying process, particle formation can be easily performed.

[0060] The above freeze-drying is not limited thereto, but can be performed using a freeze dryer for 12 to 72 hours, 24 to 60 hours, 36 to 55 hours, 40 to 50 hours, or 48 hours.

[0061] Prior to the freeze-drying, the acellular dermal matrix may be frozen under ultra-low temperature freezing conditions of about -80°C or lower or freezing conditions of about -40°C or lower for at least 4 hours, preferably 12 to 24 hours, but is not limited thereto.

[0062] The freeze-dried acellular dermal matrix can be granulated through a micronization process. The micronization process can be performed using a grinding device known in the art, such as a grinder such as a cryo-grinder or an ultrasonic grinder, a mixer, a homogenizer, a hand blender, a plunger mill, a cutting mill, etc.

[0063] In one specific embodiment of the present invention, the acellular dermal matrix microparticles may have a particle size of, but not limited to, about 100 μm to about 500 μm, and preferably, about 150 μm to about 300 μm.

[0064] By granulating the acellular dermal matrix through the above-described microdifferentiation process, an acellular dermal matrix dispersion can be produced at any desired concentration in the subsequent acellular dermal matrix dispersion production step. Ultimately, by varying the concentration of the acellular dermal matrix dispersion, the physical properties of the acellular dermal film produced using the acellular dermal matrix dispersion can be easily controlled.

[0065] In one specific example of the present invention, the acellular dermal matrix dispersion can be prepared by high-pressure dispersing acellular dermal matrix microparticles in a solution.

[0066] The acellular dermal matrix microparticles may be included 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%, based on the total weight of the acellular dermal matrix dispersion, and preferably about 2 wt%.

[0067] The solution for manufacturing the above-mentioned acellular dermal matrix dispersion is not particularly limited as long as it is a physiologically acceptable solution, and includes distilled water, normal saline, phosphate buffer solution (PBS), HBSS (Hank's balanced salt solution), TBS (Tris buffered saline), TAPS (N-Tris(hydroxy-methyl)methyl-3-aminopropanesulfonic acid) buffer solution, Bicine (N,N-Bis(2-hydroxyethyl) glycine) buffer solution, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer solution, TES (NTris(hydroxymethyl)methyl-2-aminoethanesulfonicd acid) buffer solution, PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid) buffer solution, cacodylate buffer solution, MES (2-(N-morpholino)ethanesulfonic acid) buffer solution, MEM (Minimum Essential Media), DMEM (Dulbecco's Modified Eagle Media), RPMI1640, IMDM (Iscove's Modified Dulbecco's Media), Defined Keratinocyte-SFM (without BPE (bovine pituitary extract)), Keratinocyte-SFM (with BPE), KnockOut D-MEM, AmnioMAX-II Complete Medium, AmnioMAX-C100 Complete Medium, and mixtures thereof.

[0068] The high-pressure dispersion conditions for producing the above-mentioned acellular dermal matrix dispersion are not limited thereto, but may be a pressure of about 8,000 psi to about 40,000 psi applied to pass the dispersion through a nozzle chamber of about 100 μm to about 300 μm at least once.

[0069] In one embodiment of the present invention, the high pressure dispersion conditions may be, but are not limited to, 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.

[0070] Additionally, when performing high pressure dispersion, it may pass through 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 under the above pressure conditions, but is not limited thereto.

[0071] In addition, when performing high-pressure dispersion, the number of times the nozzle chamber passes may be 1 or more, 2 or more, or 3 or more times, but is not particularly limited as long as acellular dermal matrix microparticles of a more homogeneous size can be uniformly dispersed in the solution.

[0072] Additionally, high pressure dispersion 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.

[0073] The above high-pressure dispersion can be performed using a high-pressure disperser. For example, by applying strong pressure to the solution using a high-pressure disperser such as a high-pressure homogenizer, the size of the particles contained in the solution is reduced, thereby enabling the particles within the solution to be evenly dispersed.

[0074] In one specific embodiment of the present invention, the acellular dermal matrix may be decellularized by a supercritical fluid extraction process.

[0075] As used herein, the term "decellularization" refers to the removal of cellular components, such as the nucleus, cell membrane, and nucleic acids, from a tissue, excluding the extracellular matrix. Decellularization is a novel method for producing artificial scaffolds by removing cells from an entire organ while maintaining the original structure of the desired transplanted tissue or organ. During the decellularization process, cellular components are removed from the tissue, but the extracellular matrix and some growth factor proteins are preserved. Therefore, various extracellular matrix components, including collagen, glycosaminoglycans (GAGs), fibronectin, and elastin, preserved in the decellularized tissue provide a three-dimensional microenvironment similar to that of intact tissue, thereby enhancing the survival, proliferation, and differentiation of cultured cells.

[0076] In the present invention, decellularization can be performed by supercritical fluid extraction without surfactant treatment, but is not limited thereto.

[0077] The decellularized dermal tissue of the present invention may be derived from skin tissue isolated from an individual. The skin tissue may be allogeneic or xenogeneic. "Allogeneic" refers to a human, while "xenogeneic" refers to a non-human animal, such as a mammal such as a pig, cow, or horse.

[0078] The term "supercritical fluid extraction" or "supercritical extraction" as used herein refers to a method of separating substances using a supercritical fluid that has properties intermediate between those of a gas and a liquid that exist above its critical point, i.e., its critical temperature and pressure. The supercritical fluid extraction utilizes the principle of solvent extraction, in which soluble components contained in a raw material are dissolved in a supercritical fluid due to the difference in solubility between the raw material to be extracted and the supercritical fluid, and the principle of distillation, in which solute molecules contained in the raw material move from a high-density condensed phase to a low-density expanded phase, the supercritical fluid, as an evaporation phenomenon.

[0079] The term "supercritical fluid," as used herein, refers to a gaseous substance under normal conditions but a fluid at a critical temperature and pressure above the critical temperature. Suitable supercritical fluids for use in the present invention are not particularly limited, but include, for example, carbon dioxide, nitrogen, nitrous oxide, methane, ethylene, propane, and propylene. Preferably, carbon dioxide, which has a critical temperature of 31°C and a critical pressure of 72.8 atm, can be used.

[0080] In the present invention, decellularization may be performed by adding a "co-solvent" in addition to the supercritical fluid during supercritical fluid extraction. The co-solvent may be added for purposes such as increasing the extractability and improving the solubility of the supercritical fluid, and may include, but is not limited to, ethanol, methanol, petroleum ether, acetonitrile, hexane, and the like as the co-solvent. In this case, the co-solvent may preferably be ethanol.

[0081] Preparation of acellular dermal matrix dispersion

[0082] The acellular dermal matrix dispersion according to the present invention can be manufactured using acellular dermal matrix microparticles as a main component. The acellular dermal matrix microparticles can be manufactured by a manufacturing method including a step of extracting skin tissue separated from an individual using a supercritical fluid.

[0083] The above “acellular dermal matrix microparticles” and “supercritical fluid” are as described above.

[0084] In the present invention, a supercritical fluid can produce an extracellular matrix derived from decellularized skin tissue by extracting lipid components, specifically, phospholipid components, which are the main components of cell membranes, from skin tissue separated from an individual based on solubility and decellularizing them.

[0085] The above supercritical fluid may be selected from the group consisting of carbon dioxide gas, ammonia gas, nitrogen gas, nitrogen monoxide (NO) gas, nitrogen dioxide (NO2) gas, nitrous oxide (N2O) gas, sulfur dioxide gas, hydrogen gas, water vapor, saturated hydrocarbons, unsaturated hydrocarbons, aromatic compounds, and mixed gases thereof. Preferably, it may be carbon dioxide gas. When carbon dioxide gas is used as the above supercritical fluid, carbon dioxide has a low critical temperature (31°C) and critical pressure (73 bar), so it can be easily adjusted to supercritical conditions, and has the advantages of being widely present in nature, colorless, odorless, harmless to the human body, and chemically stable.

[0086] In the present invention, the supercritical extraction step may be performed under pressure conditions of 0 to 1000 bar, 30 to 900 bar, 60 to 800 bar, 90 to 700 bar, 120 to 600 bar, 150 to 500 bar, or 200 to 400 bar.

[0087] In the present invention, the supercritical extraction step may be performed under pressure conditions of 0 to 1000 bar, 30 to 900 bar, 60 to 800 bar, 90 to 700 bar, 120 to 600 bar, 150 to 500 bar, or 200 to 400 bar.

[0088] Specifically, the pressure of the supercritical extraction step may be, but is not limited to, 0 bar or more, 50 bar or more, 100 bar or more, 150 bar or more, 200 bar or more, 250 bar or more, 300 bar or more, 350 bar or more, 400 bar or more, 450 bar or more, 500 bar or more, 550 bar or more, 600 bar or more, 650 bar or more, 700 bar or more, 750 bar or more, 800 bar or more, 850 bar or more, 900 bar or more, or 950 bar or more.

[0089] Additionally, the pressure of the supercritical extraction step is not limited thereto, but may be 1000 bar or less, 950 bar or less, 900 bar or less, 850 bar or less, 800 bar or less, 750 bar or less, 700 bar or less, 650 bar or less, 600 bar or less, 550 bar or less, 500 bar or less, 450 bar or less, 400 bar or less, 350 bar or less, 300 bar or less, 250 bar or less, 200 bar or less, 150 bar or less, 100 bar or less, or 50 bar or less.

[0090] The pressure conditions of the above supercritical extraction step are not limited to the range of conditions that can efficiently produce decellularized skin tissue-derived extracellular matrix by removing most of the cells of the skin tissue while maintaining the components and growth factors of the skin tissue-derived extracellular matrix.

[0091] In the above supercritical extraction step, in addition to the supercritical fluid, a cosolvent may be further included. The cosolvent may be one or more solvents selected from the group consisting of ethanol, water, methanol, hexane, petroleum ether, acetonitrile, acetone, ethyl acetate, and methylene chloride. Preferably, ethanol may be further included as a cosolvent.

[0092] The above co-solvent is added for the purpose of increasing the extractability and improving the solubility of the supercritical fluid, and removing most of the lipids and cells in the separated skin tissue, but its type is not particularly limited as long as the components and growth factors of the extracellular matrix derived from the skin tissue are preserved.

[0093] In the present invention, the supercritical extraction step may be performed under temperature conditions of, but not limited to, 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.

[0094] In the present invention, the supercritical extraction step may be performed for, but is not limited to, 3 hours or less. Preferably, it may be performed for, but is not limited to, 60 to 180 minutes, 70 to 170 minutes, 80 to 160 minutes, 90 to 150 minutes, 100 to 140 minutes, 110 to 130 minutes, or 120 minutes.

[0095] When manufacturing an acellular dermal matrix dispersion containing acellular dermal matrix microparticles as a main component according to the present invention, the process may include, but is not limited to, a step of separating the epidermal layer and the dermal layer of skin tissue before the step of extracting with the supercritical fluid.

[0096] The separation of the epidermis and dermis can be performed using methods known in the art. Typically, the separation of the epidermis and dermis can be performed using various proteolytic enzymes, such as dispase, thermolysin, and trypsin.

[0097] Additionally, the epidermal and dermal layers can be separated by varying the ionic strength of the solution. Specifically, the epidermal and dermal layers can be separated by treating with a 1 M or higher sodium chloride (NaCl) solution or a 20 mM EDTA solution at 37°C for 14 to 32 hours.

[0098] In one specific example, the separation of the epidermal layer and the dermal layer can be performed by treating with 1 M NaCl for 24 hours under temperature conditions of 37°C, but is not limited thereto.

[0099] When manufacturing an acellular dermal matrix dispersion containing acellular dermal matrix microparticles as a main component according to the present invention, it may be manufactured by further including, but not limited to, any one of the following steps after the step of extracting with the supercritical fluid:

[0100] A step of washing the dermal tissue with a phosphate buffer;

[0101] A step of freeze-drying the washed dermal tissue;

[0102] A step of manufacturing dermal matrix microparticles by micronizing freeze-dried dermal tissue; and

[0103] A step for preparing a dermal matrix dispersion by dispersing dermal matrix microparticles under high pressure in a solution.

[0104] Washing using the above phosphate buffer solution can wash away any remaining solution and impurities present in the dermal tissue after supercritical fluid extraction.

[0105] Freeze-drying can be performed on the above washed dermal tissue.

[0106] The above freeze-drying is a method of rapidly cooling frozen dermal tissue and then absorbing moisture through a vacuum. The moisture content within the dermal tissue can be controlled according to the freeze-drying process, and granulation can be easily performed.

[0107] The above freeze-drying is not limited thereto, but can be performed using a freeze dryer for 12 to 72 hours, 24 to 60 hours, 36 to 55 hours, 40 to 50 hours, or 48 hours.

[0108] Prior to the freeze-drying, the acellular dermal tissue may be frozen under ultra-low temperature freezing conditions of about -80°C or lower or freezing conditions of about -40°C or lower for at least 4 hours, preferably 12 to 24 hours, but is not limited thereto.

[0109] The above freeze-dried acellular dermal tissue can be granulated through a micronization process. The micronization process can be performed using a grinding device known in the art, such as a grinder such as a cryo-grinder or an ultrasonic grinder, a mixer, a homogenizer, a hand blender, a plunger mill, a cutting mill, etc.

[0110] The particle size of the acellular dermal tissue granulated through the above-described differentiation process is not limited thereto, but may be from about 100 μm to about 500 μm, and preferably from about 150 μm to about 300 μm.

[0111] By micro-particleizing the acellular dermal tissue through the above-described differentiation process, the dissolution efficiency in the solution can be increased in the subsequent acellular dermal matrix dispersion manufacturing step.

[0112] The granulated dermal tissue obtained through the above-described micronization process can be easily suspended or dissolved in any solution. Any physiologically acceptable solution capable of dissolving the granulated dermal tissue can be used without limitation. For example, one or more solutions selected from the group consisting of various aqueous solutions, such as distilled water, buffer solutions, and saline solutions, can be used.

[0113] By performing physical mixing, such as high-pressure dispersion, pipetting, vortexing, shaking, or agitation, on the mixture of the above solution and the granulated dermal tissue, the dermal matrix microparticles can be uniformly suspended or dissolved in the solution. Through this, an acellular dermal matrix dispersion in which the dermal matrix microparticles are homogeneously suspended or dissolved in the solution can be obtained.

[0114] In one specific example of the present invention, the acellular dermal matrix dispersion is preferably prepared by physically mixing a solution and granulated dermal tissue using a high-pressure dispersion method, but is not limited thereto.

[0115] The conditions for performing the above high-pressure dispersion are as described above.

[0116] Acellular dermal film comprising an acellular dermal matrix dispersion

[0117] Another aspect of the present invention provides an acellular dermal film comprising the acellular dermal matrix dispersion.

[0118] The term "acellular dermal matrix film" as used herein refers to a thin film having a predetermined thickness obtained by applying an acellular dermal matrix dispersion prepared using acellular dermal matrix microparticles as a main component to any flat plate shape. The acellular dermal film is used interchangeably with acellular dermal matrix film and acellular dermal tissue film, and the film also comprehensively includes thin film forms such as membranes, sheets, patches, etc.

[0119] The above “acellular dermal matrix”, “acellular dermal matrix microparticles” and “acellular dermal matrix dispersion” are as described above.

[0120] In one specific example of the present invention, the acellular dermal matrix dispersion may be a solution in which acellular 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.

[0121] The acellular dermal matrix microparticles may be included 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%, based on the total weight of the acellular dermal matrix dispersion, and preferably about 2 wt%.

[0122] The solution in which the above-mentioned acellular dermal matrix microparticles are dispersed under high pressure and the conditions for performing the high pressure dispersion are as described above.

[0123] In one specific embodiment of the present invention, the acellular dermal film including the acellular dermal matrix dispersion may have a sheet or membrane shape that is transparent or translucent. The acellular dermal film may have a thickness of about 0.01 mm to about 1 mm. The thickness of the acellular dermal film may be, for example, about 10 μm to about 1,000 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.

[0124] In one specific example of the present invention, the tensile strength, which is a mechanical property of the acellular dermal film, is not limited thereto, but may be from about 0.5 N to about 30 N. Specifically, under hydrated conditions, the film may have a tensile strength of from about 0.5 N to about 3 N, and preferably, a tensile strength of from about 1.3 N to about 1.5 N. In addition, under dry conditions, the film may have a tensile strength of from about 25 N to about 30 N, and preferably, a tensile strength of from about 27 N.

[0125] The term "tensile strength" as used herein refers to the maximum stress at which a specimen breaks under a tensile load, and is defined as the maximum load to rupture divided by the original cross-sectional area of ​​the specimen. A higher tensile strength indicates a greater force required to fracture the support, which may also imply ease of transplantation and suturing when applying acellular dermal films as biomaterials.

[0126] The tensile strength measured in the present invention can be measured using a universal testing machine. Furthermore, the tensile strength of the acellular dermal film in the present invention can be measured under conditions of hydrated or dried states, such as when wet with a physiologically acceptable solution.

[0127] In one specific embodiment of the present invention, the acellular dermal film may contain a collagen content of about 200 μg / mg or more based on dry weight. Preferably, the content may 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.

[0128] In one specific embodiment of the present invention, the acellular dermal film may have a porous structure. The acellular dermal film, due to its porous structure, facilitates the movement of body fluids, thereby providing the advantage of assisting in tissue regeneration.

[0129] In exemplary embodiments of the present invention, the acellular dermal matrix microparticle-containing dispersion used in the manufacture of the acellular dermal film does not contain a surfactant. Accordingly, the acellular dermal film manufactured using the acellular dermal film has the advantage of minimizing problems such as denaturation of proteins such as collagen and destruction of growth factors. Furthermore, the acellular dermal film possesses excellent mechanical properties, making it easy to suture when applied surgically as a biomaterial for transplantation.

[0130] Therefore, the acellular dermal film according to the present invention possesses excellent mechanical properties, a porous structural characteristic, and excellent preservation of extracellular matrix such as collagen and growth factors, making it suitable for use as a biomaterial for transplantation. For example, it can be usefully used as a biomaterial for transplantation such as a wound dressing, an anti-adhesion agent, a drug delivery graft, or artificial skin.

[0131] Meanwhile, when used as the above-mentioned biomaterial for transplantation, the acellular dermal film may contain skin or pharmaceutically active ingredients. The skin and pharmaceutically active ingredients can be delivered through the area to which the acellular dermal film is applied.

[0132] The above skin-related effective ingredients may include any cosmetic ingredient or nutritional ingredient that can deliver an effective effect to the skin, and examples thereof include retinol, retinoic acid, hyaluronic acid, tocopherol, adenosine, vitamins, etc.

[0133] In addition, the above pharmaceutically active ingredients may include, but are not limited to, analgesics, antihistamines, anti-inflammatory agents, etc.

[0134] Method for preparing an acellular dermal matrix dispersion

[0135] Another aspect of the present invention provides a method for preparing an acellular dermal matrix dispersion. The method for preparing the acellular dermal matrix dispersion may comprise the following steps:

[0136] a) A step of separating skin tissue separated from an object into an epidermal layer and a dermal layer;

[0137] b) a step of extracting the separated dermal layer with a supercritical fluid;

[0138] c) A step of washing the dermal layer extracted with a supercritical fluid with a phosphate buffer solution;

[0139] d) A step of freeze-drying the washed dermal tissue;

[0140] e) a step of producing dermal matrix microparticles by micronizing freeze-dried dermal tissue; and

[0141] f) A step of preparing a dermal matrix dispersion by dispersing dermal matrix microparticles under high pressure in a solution.

[0142] The above “entity”, “supercritical fluid”, “lyophilization”, “solution”, “high pressure dispersion”, “dermal matrix microparticles”, and “dermal matrix dispersion” are as described above.

[0143] Specific details for each manufacturing step of the above acellular dermal matrix dispersion are as described above in ‘Manufacturing of acellular dermal matrix dispersion’.

[0144] In the above step e), micronization can be performed using a grinding means known in the art, for example, a grinder such as a cryogenic grinder, an ultrasonic grinder, a mixer, a homogenizer, a hand blender, a plunger mill, a cutting mill, etc.

[0145] The above-described micronization process may further include, but is not limited to, a step of passing the micronized particles through a sieve having an arbitrary pore size to obtain dermal matrix microparticles of a desired size.

[0146] The dermal matrix microparticles applied in step f) through the above-described differentiation process may have a particle size of about 100 μm to about 500 μm, preferably about 150 μm to about 300 μm, but are not limited thereto.

[0147] The dermal matrix microparticles of the desired size obtained above can be added to the solution at any desired concentration in step f). An acellular dermal matrix dispersion can be obtained by homogeneously suspending or dissolving the dermal matrix microparticles added to the solution into a uniform size using a high-pressure dispersion method.

[0148] The above high pressure dispersion conditions are not limited thereto, but may be applied at a pressure of about 8,000 psi to about 40,000 psi to pass the nozzle chamber of about 100 μm to about 300 μm one or more times.

[0149] In one specific embodiment of the present invention, a step of mixing and stirring dermal matrix microparticles with a solution may be further included before step f).

[0150] The above agitation mixing can be replaced with any physical mixing method known to those skilled in the art, such as pipetting, vortexing, shaking, etc., without limitation. As long as an acellular dermal matrix dispersion in which dermal matrix microparticles are homogeneously suspended or dissolved in the solution can be easily obtained, there is no particular limitation.

[0151] Method for manufacturing acellular dermal film

[0152] Another aspect of the present invention provides a method for producing an acellular dermal film. The method may comprise the following steps:

[0153] a) A step of separating skin tissue separated from an object into an epidermal layer and a dermal layer;

[0154] b) a step of extracting the separated dermal layer with a supercritical fluid;

[0155] c) A step of washing the dermal layer extracted with a supercritical fluid with a phosphate buffer solution;

[0156] d) A step of freeze-drying the washed dermal tissue;

[0157] e) A step of producing dermal matrix microparticles by micronizing freeze-dried dermal tissue;

[0158] f) a step of preparing a dermal matrix dispersion by dispersing dermal matrix microparticles in a solution under high pressure; and

[0159] g) A step of applying a dermal matrix dispersion and drying it to produce a dermal film.

[0160] The above “entity”, “supercritical fluid”, “lyophilization”, “solution”, “high pressure dispersion”, “dermal matrix microparticles”, “dermal matrix dispersion” and “acellular dermal film” are as described above.

[0161] Specific details of steps a) to f) among the manufacturing steps of the acellular dermal film using the above acellular dermal matrix dispersion are as described above in ‘Manufacturing of acellular dermal matrix dispersion’ and ‘Method for manufacturing acellular dermal matrix dispersion’.

[0162] In one specific example of the present invention, the acellular dermal film can be obtained by applying or dispensing the acellular dermal matrix dispersion obtained through steps a) to f) onto any flat plate shape and drying it.

[0163] When manufacturing the above-described acellular dermal film, a step of removing air bubbles from the acellular dermal matrix dispersion may be further included, but is not limited thereto. As long as an acellular dermal film having any uniform thickness and uniform surface can be manufactured, the step of removing air bubbles may be omitted.

[0164] In the above step g), drying may be performed under a temperature condition of about 5°C to about 50°C under vacuum, but is not limited thereto. As an example, the drying may be performed under 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 under vacuum, but is not limited thereto.

[0165] In the above step g), drying should be performed under vacuum to minimize problems that may occur during the film manufacturing process. When manufacturing a film under vacuum, it can have advantages such as preventing surface deterioration of the film due to evaporation or partial evaporation, such as preventing wrinkle formation on the film surface, preventing transparency degradation, and preventing formation of uneven film thickness.

[0166] Additionally, the drying may be performed for, but is not limited to, about 6 to about 72 hours. As an example, the drying may be performed for, but is not limited to, about 6 to about 72 hours, about 12 to about 36 hours, about 18 to about 30 hours, about 20 to about 28 hours, or about 24 hours.

[0167] In one specific embodiment of the present invention, the acellular dermal film may have a porous structure and may have a thickness of about 0.01 mm to about 1 mm. The thickness of the acellular dermal film may be, for example, about 10 μm to about 1,000 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.

[0168] Additionally, the acellular dermal film may have a tensile strength of, but not limited to, about 0.5 N to about 30 N.

[0169] In addition, the acellular dermal film may contain a collagen content of about 200 μg / mg or more based on dry weight. Preferably, it may contain 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.

[0170] The above acellular dermal film can be sterilized after step g).

[0171] As used herein, the term "sterilization" means killing or eliminating all vegetative cells and spores of fungi, bacteria, viruses, and protozoa present on and inside a target object, thereby rendering it sterile. 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 target object or its composition.

[0172] In one specific example, the acellular dermal film can be sterilized using gamma rays or eBeam.

[0173] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended only to illustrate the present invention, and the scope of the present invention is not limited to these examples.

[0174] I. Preparation of a film containing acellular dermal matrix dispersion

[0175] Example 1. Preparation of an acellular dermal film using a dermal matrix dispersion.

[0176] A series of processes, including decellularization, micronization, dispersion preparation, and drying, were performed to produce a film from a human-derived acellular dermal matrix (ADM). The manufacturing process of a film containing an acellular dermal matrix dispersion is schematically illustrated in Fig. 1. The specific manufacturing of a film containing an acellular dermal matrix dispersion is as described in Examples 1.1 to 1.4 below.

[0177] Example 1.1. Preparation of acellular dermal matrix using supercritical fluid extraction process

[0178] A supercritical fluid extraction process was performed to decellularize skin tissue.

[0179] First, the fat layer was removed from the donated human skin tissue (IRB No. 20201305, Asan Medical Center, Seoul). After removal of the fat layer, the epidermis was removed by treating the tissue with 1 M NaCl (Sigma Aldrich, Cat No. S9888) at 37°C for 24 hours. After removal of the epidermis, the separated dermis was washed with sterile PBS (biowest, Cat No. L0615-500) for 1 hour.

[0180] After washing, the obtained dermal matrix was placed in the extraction tank of a supercritical extraction system (SES), and supercritical fluid carbon dioxide and co-solvent ethanol were injected together into the extraction tank. Thereafter, the dermal tissue was decellularized through supercritical treatment for 1 to 3 hours under pressure conditions of 72.8 bar and temperature conditions of 31°C.

[0181] Afterwards, the decellularized dermal tissue was washed with sterilized PBS at room temperature for 24 hours.

[0182] Example 1.2. Preparation of acellular dermal matrix microparticles through a microdifferentiation process.

[0183] In order to process the washed, decellularized, acellular dermal tissue obtained in Example 1.1 above into a film form, a micronization process was performed.

[0184] Specifically, acellular homologous dermis decellularized through a supercritical fluid treatment process was frozen at -80°C for 1 day to facilitate pulverization before particleization. After freezing for 1 day, it was freeze-dried for 2 days using a freeze dryer.

[0185] The freeze-dried acellular dermal tissue was first granulated using a cutting mill (Taemyung Science, FRITSCH Universal Cutting Mill). At this time, the particles that passed through a sieve with pores of 500 μm were collected. Afterwards, to prevent the material from denaturing due to the heat generated during granulation, the tissue was secondarily crushed for 5 minutes using a freeze crusher (SPEX, 6875D Freezer / Mill) using liquid nitrogen. At this time, the microparticles that did not pass through were collected using a sieve with pores of 100 μm. In other words, acellular dermal matrix microparticles with a size of 100 μm to 500 μm were ultimately obtained.

[0186] Example 1.3. Preparation of acellular dermal matrix dispersion

[0187] The acellular dermal matrix microparticles having a size of 100 μm or more and 500 μm or less, i.e., 100 μm to 500 μm, captured in the above Example 1.2 were physically homogeneously dispersed using a high-pressure disperser (Micronox, MN400BF) (Fig. 2).

[0188] Specifically, acellular dermal matrix microparticles manufactured to have a size of 100 μm to 500 μm were mixed with sterile distilled water to a final weight ratio of 2%, and physically stirred at 100 RPM for 10 minutes using a magnetic stirrer.

[0189] The physically stirred and mixed acellular dermal matrix microparticle-containing solution was injected into a high-pressure disperser and then subjected to primary dispersion at 15,000 psi using a 200 μm nozzle chamber. The viscosity of the acellular dermal matrix microparticle-containing dispersion subjected to the primary high-pressure dispersion was visually confirmed to be increased, and phase separation was observed, indicating that the dispersion was not sufficiently dispersed. Accordingly, high-pressure dispersion was performed twice more, repeating the process three times in total (Fig. 2). Afterwards, air bubbles were removed using a vacuum oven (LK Lab Korea, LO-VS380N) at -0.9 bar for 1 hour.

[0190] Example 1.4. Drying of acellular dermal matrix dispersion

[0191] In order to process the homogeneous acellular dermal matrix dispersion obtained in Example 1.3 above into a film formulation, a vacuum drying process was performed.

[0192] Specifically, a 2 wt% acellular dermal matrix dispersion was dispersed at 100 cm 2 50 mL was added to a dish of the size of 100 ml, and the dish was tilted to ensure even distribution. Afterwards, it was placed in a vacuum oven at 40°C and dried at -0.9 bar for 1 day.

[0193] After drying, the thickness of the film separated from the dish was measured to be 96 μm, which was standardized, placed in a sterile packaging material, and sealed. The dried acellular dermal matrix dispersion, i.e., the film containing the acellular dermal matrix dispersion, after the sealing was completed, was sterilized by performing gamma or eBeam sterilization (15 kGy). After sterilization, it was stored at room temperature until use (Fig. 3).

[0194] II. Evaluation of the physicochemical properties of films containing acellular dermal matrix dispersions

[0195] Experimental Example 1.1. Observation of the Appearance of a Film Containing Acellular Dermal Matrix Dispersion

[0196] The appearance of the acellular dermal film manufactured using the dermal matrix dispersion in Example 1 was observed (Fig. 4a), and the applicability of the sample after hydration was examined (Fig. 4b). The observation results confirmed that the tissue had properties suitable for transfer using forceps or application to wound sites.

[0197] Experimental Example 1.2. Observation of the external pattern image of a film containing acellular dermal matrix dispersion.

[0198] A circular punching pattern (Fig. 4c) or a straight punching pattern (Fig. 4d) was designed for the acellular dermal film obtained in Example 1 using a scalpel or punch.

[0199] It was confirmed that a film containing an acellular dermal matrix dispersion having a pattern as described above improved the homogeneity and hydration speed as exudate passed through, and also prevented the phenomenon of the film's ends curling compared to commercially available dermal film products.

[0200] Experimental Example 2.1. Observation of homogenization of a film containing acellular dermal matrix dispersion.

[0201] The thickness of the film containing the acellular dermal matrix dispersion prepared in Example 1 was measured using a Nikon (MFC-101A) Digimicro device. The film thickness was confirmed to be 96 μm (Fig. 5a). To confirm the homogenization characteristics of the product, transparency was observed on the printed letters. Transparency was confirmed to be sufficient to fully display the letters, indicating that the acellular dermal matrix dispersion was uniformly dispersed during film preparation (Fig. 5b).

[0202] Experimental Example 2.2. Comparative observation of homogenization between films containing acellular dermal matrix dispersion and commercial films.

[0203] The homogenization properties of the film containing the acellular dermal matrix dispersion prepared in Example 1 above were compared with those of a commercially available dermal matrix film (CGderm from CG Bio) TM Matrix) and compared with each other.

[0204] Observation results confirmed that the dermal matrix film according to the present invention exhibited superior transparency compared to conventional commercially available dermal matrix films under both pre- and post-hydration conditions (Fig. 5c). Therefore, it was found that the dermal matrix film according to the present invention, as a film in which an acellular dermal matrix is ​​uniformly dispersed, can offer the advantage of improved efficacy and usability compared to conventional commercially available dermal matrix films.

[0205] Experimental Example 3.1. Tensile strength test of a film containing acellular dermal matrix dispersion

[0206] The tensile strength of the film containing the acellular dermal matrix dispersion prepared in Example 1 was measured. The tensile strength test was performed using a universal testing machine (AGS-X model (SHIMADZU)). At this time, the tensile strength is affected by the thickness and width of the specimen, so the specimen was first dried and cut into 1×5 cm pieces. 2 It was prepared by cutting. The tensile strength of the specimens prepared as above was measured in the dried and hydrated states, respectively. However, the length of the hydrated specimen hydrated with sterilized distilled water was 3 cm, and the thickness was 150 μm.

[0207] As a result of the measurement, it was confirmed that the film containing the dried acellular dermal matrix dispersion broke at a maximum load of approximately 27 N (Fig. 6a), and the film containing the hydrated acellular dermal matrix dispersion broke at a maximum load of approximately 1.42 N (Fig. 6b).

[0208] Experimental Example 3.2. Tensile strength comparison test between a film containing an acellular dermal matrix dispersion and a commercially available film.

[0209] A film containing the acellular dermal matrix dispersion prepared in Example 1 above and a commercially available dermal matrix film (CGderm from CG Bio) TM A fracture test was performed on the matrix to compare the tensile strength difference between the two films. At this time, the specimens were 1×5 cm in a dry state. 2 The cut product was used. The tensile strength was measured at a speed of 4 mm / min using a universal testing machine as in Experimental Example 3.1 above. However, the tensile strength of the specimen was measured in a hydrated state, and after hydration with sterile distilled water for 10 minutes, the length of the specimen was 3 cm, and the thickness was 150 μm for the film containing the acellular dermal matrix dispersion and 500 μm for the commercially available dermal matrix film.

[0210] As a result of the measurement, it was confirmed that the film containing the acellular dermal matrix dispersion (SC Derm Matrix) broke at a maximum load of approximately 1.4 N, and the commercially available dermal matrix film (CG Derm Matrix) broke at a maximum load of approximately 0.14 N (Fig. 6c). Therefore, it was found that the dermal matrix film according to the present invention can provide the advantage of improved efficacy and usability as a film with a tensile strength that is approximately 10 times improved compared to conventional commercially available dermal matrix films.

[0211] Experimental Example 4. Surface Morphology Analysis of Films Containing Acellular Dermal Matrix Dispersion

[0212] A surface morphology analysis was performed on the film containing the acellular dermal matrix dispersion prepared in Example 1. The surface analysis was performed using a scanning electron microscope (SEM; IM-150 model (ETS)). The sample was sputtered with inorganic gold for surface analysis of organic matter (SEM) and then photographed at 5 kV. To confirm the morphology of the tissue after hydration, the sample was hydrated for 1 minute using sterile distilled water, then frozen at -80°C for 1 hour, and freeze-dried for 1 day to reproduce the morphology of the hydrated tissue. The dried cross-section of the film containing the acellular dermal matrix dispersion was confirmed to have a dense structure when examined at 3,000x magnification (Fig. 7a). In addition, when magnified 120x and 1,000x, respectively, it was confirmed that the cross-section of the film freeze-dried after hydration had a porous structure (Fig. 7b). Through the above results, it was found that the film containing the acellular dermal matrix dispersion formed a porous structure after hydration, and thus had the characteristic of facilitating the movement of body fluids, and thus could help in tissue regeneration.

[0213] Experimental Example 5. Confirmation of the Surgical Applicability of a Film Containing Acellular Dermal Matrix Dispersion

[0214] In order to confirm the applicability of the film containing the acellular dermal matrix dispersion prepared in Example 1 above for surgical operations, the film containing the acellular dermal matrix dispersion was applied after surgical incision and sutured using a suture or surgical stapler.

[0215] Specifically, films containing acellular dermal matrix dispersions in both dried and hydrated states were sutured with gauze using polyamide suture. The film was then checked for detachment from the gauze and for any damage during suturing.

[0216] To determine whether films containing acellular dermal matrix dispersions in both dried and hydrated states are applicable to surgical staplers, they were fixed to gauze using a standard office stapler. It was confirmed that there were no particular problems in fixing the films containing acellular dermal matrix dispersions under all of the above conditions (Fig. 8).

[0217] Therefore, it was found that the film containing the acellular dermal matrix dispersion according to the present invention has excellent efficacy and usability when applied as a transplant material.

[0218] III. Evaluation of biochemical properties of films containing acellular dermal matrix dispersions

[0219] Experimental Example 6. Confirmation of the degree of preservation of the extracellular matrix (ECM) in a film containing an acellular dermal matrix dispersion.

[0220] In order to confirm whether the film containing the acellular dermal matrix dispersion prepared in Example 1 above is suitable as a graft material, the loss of extracellular matrix proteins important for tissue regeneration was measured. Specifically, the collagen content was measured using the Sircol Insoluble Collagen Assay Kit from Biocolor. At this time, the film containing the acellular dermal matrix dispersion was used as the test group, and the CGderm™ Matrix from CG Bio and Matriderm from MedSkin Solution Dr. Suwelack Headquarters were used as the comparison group. ® was used.

[0221] As a result of the measurement, among the samples used as a comparison group, CGderm™ Matrix preserved collagen at the level of 59 μg / mg, and Matriderm ® It was confirmed that collagen was preserved at the level of 178 μg / mg. In contrast, the film containing the acellular dermal matrix dispersion, which was the test group, was confirmed to preserve collagen at a significantly higher level of 452 μg / mg (Fig. 9).

[0222] The above results demonstrate that acellular dermal tissue films fabricated using acellular dermal matrix dispersion exhibit excellent histological preservation of proteins such as collagen. Consequently, this demonstrates excellent potential as a potential graft material.

[0223] Experimental Example 7. Confirmation of the feasibility of biotransplantation of a film containing acellular dermal matrix dispersion.

[0224] In order to apply the film containing the acellular dermal matrix dispersion prepared in Example 1 above to the subcutaneous tissue, the acellular dermal film was first cut into 40 cm 2 The film was processed to size and applied to three sites in an animal model constructed to allow injection. After applying the film as a graft material to subcutaneous tissue, the cell growth and structural maintenance period were confirmed.

[0225] Specifically, for subcutaneous implantation of a film containing an acellular dermal matrix dispersion, a large area of ​​the back was designated as the experimental site in a pig animal model weighing 45 kg or more, an incision was made, and an acellular dermal film was inserted. The incised skin tissue was sutured to secure the inserted acellular dermal film (Fig. 10).

[0226] Experimental Example 8. Verification of the degree of engraftment and effectiveness of films containing acellular dermal matrix dispersion in living tissue through histological analysis.

[0227] In the above Experimental Example 7, a film containing an acellular dermal matrix dispersion was transplanted into the subcutaneous tissue and sutured. Four weeks later, the transplanted area was incised and tissue containing the entire skin layer and the film was obtained. The obtained tissue was stained with H&E and subjected to histological analysis.

[0228] H&E staining confirmed that the area where the acellular dermal film was injected as a graft within the fat layer was present, and staining was confirmed without an inflammatory reaction in the area after 4 weeks. This confirmed that the acellular dermal film did not cause inflammation or contamination of the surrounding tissue. In addition, numerous cell nuclei were observed within the graft, indicating good engraftment (Fig. 11).

[0229] IV. Evaluation of the efficacy of a film containing acellular dermal matrix dispersion in a rat wound model

[0230] The wound healing efficacy of the test substances (Table 1) was evaluated in an in vivo rat wound model. SD rats (Spargue Dawley, 6 weeks old, male, Orient Bio Co., Ltd.) were used. After a 1-week acclimatization period, the animals were anesthetized with 3.0% isoflurane and their backs were shaved. Then, a biopsy punch (10 mm, Kai sterile dermal biopsy punch, Kai Ind., Japan) was used to create a circular defect (full thickness; epidermis and dermis) on the backs of all groups to induce wounds. The test substances were attached to the wound sites of positive control group 1 (CGderm matrix), positive control group 2 (MatriDerm), and test group (SC derm matrix), excluding the untreated group (Figs. 12 and 13).

[0231] Group (N=5) Wound-inducing test substance untreated group + - Positive control group 1 + CGderm matrix (CBBio) Positive control group 2 + MatriDerm (MedSkin Solutions Dr. Suwelack AG) Test group + Film containing acellular dermal matrix dispersion (SC derm matrix)

[0232] Changes in wound area were measured through visual evaluation, and wound width, inflammatory cell count, and re-epithelialization thickness were analyzed through H&E (Hematoxylin & Eosin) staining in cross-sections of rat skin tissue. In addition, collagen production was evaluated by measuring collagen density through MT (Masson's Trichrome) staining, and the expression levels of vimentin, a fibroblast marker, and α-SMA (alpha-smooth muscle actin), a myofibroblast marker, were evaluated using immunofluorescence (IF) staining.

[0233] Experimental Example 9. Visual Evaluation

[0234] Experimental Example 9.1. Analysis of Wound Area

[0235] On days 0, 1, 3, 5, 7, and 14 after wound induction, the wound sites of rats were photographed at a fixed distance for each group using a digital camera (CAMEDIATM, Olympus, Japan). Afterwards, the wound healing process was visually observed, and the change in wound area (%) was measured and analyzed using the Image J program (The National Institutes of Health, USA).

[0236] The rate of change in wound area (%) was calculated according to [Mathematical Formula 1] or [Mathematical Formula 2] below.

[0237] [Mathematical Formula 1]

[0238]

[0239] [Equation 2]

[0240]

[0241] As a result of the wound area analysis, in the case of positive control group 1 (CGderm matrix), the wound area tended to decrease by 4.95%, 9.34%, 10.67%, and 53.33% on days 1, 3, 5, and 14, respectively, compared to the untreated group, and significantly decreased (improved) by 29.62% compared to the untreated group on day 7.

[0242] In the case of positive control group 2 (MatriDerm), the wound area showed a tendency to decrease (improve) by 6.74%, 4.09%, 9.22%, and 62.66% on days 1, 3, 5, and 14, compared to the untreated group, and showed a significant decrease (improvement) of 31.97% on day 7.

[0243] In the case of the test group (SC derm matrix), the wound area showed a tendency to decrease by 15.40% and 81.96% at 5 and 14 days, respectively, compared to the untreated group, and significantly decreased (improved) by 20.75%, 15.20%, and 43.76% at 1, 3, and 7 days, respectively (Figs. 14a to 14f).

[0244] Experimental Example 10. Histological Evaluation (H&E Staining)

[0245] At 7 and 14 days after wound induction, the wound site and surrounding area of ​​the rats were biopsied and fixed with 10% formalin solution to create paraffin blocks. Afterwards, slides were prepared by sectioning at 3 μm thickness, and staining with H&E solution was performed after hydration. Afterwards, the images were observed under an optical microscope (Zeweiss TM , Germany) were used to photograph cross-sections of rat skin tissue, and wound width, inflammatory cells, and re-epithelialization thickness were analyzed using the Image J (The National Institutes of Health, USA) program. At this time, the wound width change rate (%), inflammatory cell change rate (%), and re-epithelialization change rate (%) were calculated according to [Mathematical Formula 3], [Mathematical Formula 4], and [Mathematical Formula 5], respectively.

[0246] [Equation 3]

[0247]

[0248] [Equation 4]

[0249]

[0250] [Equation 5]

[0251]

[0252] Experimental Example 10.1. Wound Width Analysis

[0253] Analysis of the wound width through H&E staining of skin tissue sections of rats at 7 and 14 days after wound induction showed that at 7 days, the positive control group 1 (CGderm matrix) showed a tendency to decrease by 14.71% compared to the untreated group, and the positive control group 2 (MatriDerm) and test group (SC derm matrix) showed significant decreases (improvements) by 18.20% and 38.80%, respectively, compared to the untreated group. At 14 days, the positive control group 2 (MatriDerm) showed a tendency to decrease by 19.04% compared to the untreated group, and the positive control group 1 (CGderm matrix) and test group (SC derm matrix) showed significant decreases (improvements) by 26.19% and 38.67%, respectively, compared to the untreated group (Fig. 15a and Fig. 15b).

[0254] Experimental Example 10.2. Inflammatory Cell Analysis

[0255] As a result of analyzing inflammatory cells through H&E staining in skin tissue sections of rats at 7 and 14 days after wound induction, the positive control group 1 (CGderm matrix) showed a tendency to decrease by 27.64% compared to the untreated group at 7 days, and the positive control group 2 (MatriDerm) and test group (SC derm matrix) showed a significant decrease (improvement) by 29.82% and 45.41%, respectively, compared to the untreated group. At the 14th day, positive control group 1 (CGderm matrix) and positive control group 2 (MatriDerm) showed a tendency to decrease by 38.69% and 32.16%, respectively, compared to the untreated group, and positive control group 1 (CGderm matrix) and test group (SC derm matrix) showed a significant decrease (improvement) by 38.69% and 44.82%, respectively, compared to the untreated group (Figs. 16a to 16c).

[0256] Experimental Example 10.3. Re-epithelialization Analysis

[0257] Analysis of the re-epithelialization thickness through H&E staining of skin tissue sections of rats at 7 and 14 days after wound induction showed that at 7 days, the re-epithelialization thickness of positive control group 1 (CGderm matrix) and positive control group 2 (MatriDerm) tended to increase by 33.11% and 16.76%, respectively, compared to the untreated group, and in the test group (SC derm matrix), the re-epithelialization thickness significantly increased (improved) by 56.25% compared to the untreated group. At 14 days, the re-epithelialization thickness of positive control group 1 (CGderm matrix), positive control group 2 (MatriDerm), and test group (SC derm matrix) tended to decrease by 9.74%, 15.23%, and 21.78%, respectively, compared to the untreated group (Figs. 17a to 17c).

[0258] H&E staining analysis conclusion

[0259] When the above series of H&E staining analysis results are summarized, the wound width analysis results showed that the test group significantly decreased (improved) by 38.80% and 38.67% compared to the untreated group at 7 and 14 days. The inflammatory cell count analysis results showed that the test group significantly decreased (improved) by 45.41% and 44.82% compared to the untreated group at 7 and 14 days. In addition, the reepithelialization thickness analysis results showed that the test group significantly increased (improved) by 56.25% compared to the untreated group at 7 days.

[0260] Experimental Example 11. Histological Evaluation (MT Staining)

[0261] At 7 and 14 days after wound induction, the wound site and surrounding area of ​​the rats were biopsied and fixed with 10% formalin solution to create paraffin blocks. After that, 3 μm-thick sections were made into slides, and after hydration, staining was performed with Biebrich Scarlet-Acid Fuchsin solution for 5 minutes. After washing, staining was performed again with phosphotungstic / phosphomolybdic acid for 5 minutes, followed by staining with aniline blue and acetic acid solution. After that, the images were observed under an optical microscope (Zeweiss TM , Germany) were used to photograph cross-sections of rat skin tissue, and collagen density was analyzed using the Image J (The National Institutes of Health, USA) program.

[0262] The collagen density change rate (%) was calculated according to the following [Mathematical Formula 6].

[0263] [Equation 6]

[0264]

[0265] Experimental Example 11.1. Collagen Density Analysis

[0266] As a result of analyzing the collagen density through MT staining in the skin tissue cross-sections of rats at 14 days after wound induction, 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 at 7 days, and the collagen density of the test group (SC derm matrix) significantly increased (improved) by 75.42% compared to the untreated group. At 14 days, the collagen density of positive control group 1 (CGderm matrix), positive control group 2 (MatriDerm), and test group (SC derm matrix) significantly increased (improved) by 65.55%, 64.79%, and 81.57%, respectively, compared to the untreated group (Figs. 18a to 18d).

[0267] Experimental Example 12. Histological Evaluation (IF Staining)

[0268] At 7 and 14 days after wound induction, biopsies were taken from the wound site and surrounding area of ​​the rats, and fixed in 10% formalin to prepare tissue slides. After that, 3 μm-thick sections were prepared to prepare slides, and after hydration, antigens were exposed using 10 mM sodium citrate buffer. After treatment with the primary antibodies vimentin and α-SMA (Abcam, USA), respectively, fluorescence staining was performed using the secondary antibody Rabbit Anti-mouse IgG H&L (Alexa Fluor®488). After DAPI staining (VECTASHIELD® Antifade Mounting Medium with DAPI), cross-sections of rat skin tissues were photographed using a fluorescence microscope (Zeiss Axio Observer 7, Germany). Fluorescence intensity analysis was performed using the Image J (The National Institutes of Health, USA) program. At this time, the Vimentin change rate (%) and α-SMA change rate (%) were calculated according to [Mathematical Formula 7] and [Mathematical Formula 8], respectively.

[0269] [Equation 7]

[0270]

[0271] [Equation 8]

[0272]

[0273] Experimental Example 12.1. Fibroblast Analysis (Vimentin)

[0274] The fluorescence intensity of vimentin, a fibroblast biomarker, was analyzed through IF staining in cross-sections of rat skin tissues at 7 and 14 days after wound induction. As a result, 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 at 7 days, and the fluorescence intensity of vimentin in the test group (SC derm matrix) significantly increased (improved) by 40.40% compared to the untreated group. In addition, at the 14th day, 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, and in the test group (SC derm matrix), the fluorescence intensity of vimentin significantly increased (improved) by 39.14% compared to the untreated group (Figs. 19a to 19c).

[0275] Experimental Example 12.2. Myofibroblast Analysis (α-SMA)

[0276] The fluorescence intensity of α-SMA, a biomarker of myofibroblasts, was analyzed through IF staining in cross-sections of rat skin tissues at 7 and 14 days after wound induction. As a result, the fluorescence intensity of α-SMA in the positive control group 1 (CGderm matrix) showed a tendency to increase by 32.16% compared to the untreated group at 7 days, and the fluorescence intensity of α-SMA in the positive control group 2 (MatriDerm) and test group (SC derm matrix) significantly increased (improved) by 46.21% and 60.23%, respectively, compared to the untreated group. At the 14th day, the fluorescence intensity of α-SMA in positive control group 1 (CGderm matrix), positive control group 2 (MatriDerm), and test group (SC derm matrix) significantly increased (improved) by 36.80%, 41.52%, and 55.75%, respectively, compared to the untreated group (Figs. 20a to 20c).

[0277] IF staining analysis conclusion

[0278] When the results of the above series of immunofluorescence staining (IF staining) analyses are summarized, the fluorescence density of Vimentin, a biomarker of fibroblasts, was significantly increased (improved) by 40.40% and 39.14% in the test group compared to the untreated group at 7 and 14 days after wound induction, and the analysis results of α-SMA, a biomarker of myofibroblasts, were significantly increased (improved) by 60.23% and 55.75% in the test group compared to the untreated group at 7 and 14 days.

[0279] Conclusions on the wound healing efficacy of films containing acellular dermal matrix dispersions.

[0280] Macroscopic and histological analyses using the above series of in vivo rat wound models confirmed that a film containing an acellular dermal matrix dispersion (SC derm Matrix) exhibited wound healing efficacy. Therefore, SC derm Matrix could be usefully utilized in the field of regenerative medicine as a biomaterial for transplantation, such as wound dressings and artificial skin.

Claims

1. An acellular dermal matrix dispersion containing acellular dermal matrix microparticles.

2. In paragraph 1, An acellular dermal matrix dispersion, wherein the acellular dermal matrix microparticles have a particle size of 100 μm to 500 μm.

3. In paragraph 1, The above-mentioned acellular dermal matrix dispersion is an acellular dermal matrix dispersion prepared by high-pressure dispersing acellular dermal matrix microparticles in a solution.

4. In paragraph 1, An acellular dermal matrix dispersion, wherein the acellular dermal matrix microparticles are contained in an amount of 1.0 wt% to 3.0 wt% based on the total weight of the acellular dermal matrix dispersion.

5. In paragraph 1, An acellular dermal matrix dispersion, wherein the acellular dermal matrix is ​​decellularized by a supercritical fluid extraction process.

6. In paragraph 3, The above high pressure dispersion condition is an acellular dermal matrix dispersion solution that passes through a nozzle chamber of 100 μm to 300 μm at least once by applying a pressure of 8,000 psi to 40,000 psi.

7. In paragraph 3, The above solutions are distilled water, normal saline, phosphate buffer solution (PBS), Hank's balanced salt solution (HBSS), Tris buffered saline (TBS), N-Tris(hydroxy-methyl)methyl-3-aminopropanesulfonic acid (TAPS) buffer solution, N,N-Bis(2-hydroxyethyl) glycine (Bicine) buffer solution, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer solution, TES (NTris(hydroxymethyl)methyl-2-aminoethanesulfonic acid) buffer solution, PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid) buffer solution, cacodylate buffer solution, MES (2-(N-morpholino)ethanesulfonic acid) buffer solution, MEM (Minimum Essential Media), DMEM (Dulbecco's Modified Eagle Media), An acellular dermal matrix dispersion selected from the group consisting of RPMI1640, IMDM (Iscove's Modified Dulbecco's Media), Defined Keratinocyte-SFM (without BPE (bovine pituitary extract)), Keratinocyte-SFM (with BPE), KnockOut D-MEM, AmnioMAX-II Complete Medium, AmnioMAX-C100 Complete Medium, and mixtures thereof.

8. In paragraph 5, An acellular dermal matrix dispersion, wherein the solvent used as the supercritical fluid is carbon dioxide.

9. In paragraph 5, An acellular dermal matrix dispersion, wherein the supercritical fluid further contains ethanol as a cosolvent.

10. In paragraph 5, An acellular dermal matrix dispersion, wherein the above extraction is performed under temperature conditions of 30°C to 40°C.

11. In paragraph 5, An acellular dermal matrix dispersion, wherein the above extraction is performed under pressure conditions of 50 bar to 400 bar.

12. In paragraph 5, An acellular dermal matrix dispersion, wherein the above extraction is performed for 1 to 3 hours.

13. In paragraph 5, An acellular dermal matrix dispersion comprising a step of separating the epidermal layer and the dermal layer before the step of extracting with the supercritical fluid.

14. In paragraph 5, An acellular dermal matrix dispersion, comprising one of the following steps after the step of extracting with the supercritical fluid: A step of washing the dermal tissue with a phosphate buffer; A step of freeze-drying the washed dermal tissue; A step of manufacturing dermal matrix microparticles by micronizing freeze-dried dermal tissue; and A step for preparing a dermal matrix dispersion by dispersing dermal matrix microparticles under high pressure in a solution.

15. An acellular dermal film comprising an acellular dermal matrix dispersion of any one of claims 1 to 14.

16. In paragraph 15, The above-mentioned acellular dermal matrix dispersion is an acellular dermal film in which acellular dermal matrix microparticles having a particle size of 100 μm to 500 μm are dispersed under high pressure in a solution.

17. In paragraph 15, An acellular dermal film having a thickness of 0.01 mm to 1 mm.

18. In paragraph 15, An acellular dermal film having a tensile strength of 0.5 N to 30 N.

19. In paragraph 15, An acellular dermal film having a collagen content of 200 μg / mg to 600 μg / mg based on dry weight.

20. In paragraph 15, The above-mentioned acellular dermal film has a porous structure.

21. In paragraph 15, The above acellular dermal film is used as a wound dressing, an anti-adhesion agent, a drug delivery graft or artificial skin. 22.a) A step of separating skin tissue separated from an object into an epidermal layer and a dermal layer; b) a step of extracting the separated dermal layer with a supercritical fluid; c) A step of washing the dermal layer extracted with a supercritical fluid with a phosphate buffer solution; d) A step of freeze-drying the washed dermal tissue; e) a step of producing dermal matrix microparticles by micronizing freeze-dried dermal tissue; and f) A method for producing an acellular dermal matrix dispersion, comprising the step of producing a dermal matrix dispersion by dispersing dermal matrix microparticles under high pressure in a solution.

23. In paragraph 22, A method for producing an acellular dermal matrix dispersion, further comprising a step of mixing dermal matrix microparticles with a solution before step f).

24. In paragraph 22, A method for producing an acellular dermal matrix dispersion, wherein in the above step f), the dermal matrix microparticles have a particle size of 100 μm to 500 μm.

25. In paragraph 22, A method for producing an acellular dermal matrix dispersion, wherein in the above step f), high-pressure dispersion is performed by passing the dispersion through a nozzle chamber of 100 μm to 300 μm at least once while applying a pressure of 8,000 psi to 40,000 psi. 26.a) A step of separating skin tissue separated from an object into an epidermal layer and a dermal layer; b) a step of extracting the separated dermal layer with a supercritical fluid; c) A step of washing the dermal layer extracted with a supercritical fluid with a phosphate buffer solution; d) A step of freeze-drying the washed dermal tissue; e) A step of producing dermal matrix microparticles by micronizing freeze-dried dermal tissue; f) a step of preparing a dermal matrix dispersion by dispersing dermal matrix microparticles in a solution under high pressure; and g) A method for producing an acellular dermal film, comprising the step of applying a dermal matrix dispersion and drying the same to produce a dermal film.

27. In paragraph 26, A method for producing an acellular dermal film, further comprising a step of mixing dermal matrix microparticles with a solution before step f).

28. In paragraph 26, A method for producing an acellular dermal film, wherein in the above step f), the dermal matrix microparticles have a particle size of 100 μm to 500 μm.

29. In paragraph 26, A method for producing an acellular dermal film, wherein in the above step f), high-pressure dispersion is performed by passing the film through a nozzle chamber of 100 μm to 300 μm at least once while applying a pressure of 8,000 psi to 40,000 psi.

30. In paragraph 26, A method for producing an acellular dermal film, wherein in the above step g), the dermal film is produced to a thickness of 0.01 mm to 1 mm.

31. In paragraph 26, A method for producing an acellular dermal film, comprising a step of sterilizing the dermal film after the above step g).

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