Dispersion solution containing acellular dermal matrix microparticles, filler comprising same, and use thereof

Abstract

The present invention relates to a dispersion solution containing acellular dermal matrix microparticles, a filler comprising same, and use thereof. More specifically, the present invention relates to: a filler comprising a dispersion solution containing acellular dermal matrix microparticles obtained through supercritical fluid extraction and high-pressure dispersion; and a preparation method therefor. The acellular dermal filler according to the present invention has excellent biostability and biocompatibility, and extracellular matrix components such as collagen and elastin are remarkably preserved therein. In addition, excellent biochemical stability without changes in pH during storage or changes in the amounts of collagen and elastin is exhibited, and physical stability in which injectability is continuously maintained is also exhibited. Therefore, the acellular dermal filler can be effectively used as an injectable filler for repair or volume expansion of soft tissue and wrinkle reduction in the plastic and regenerative medicine fields.

Description

Dispersion comprising acellular dermal matrix microparticles, filler comprising the same, and use thereof

[0001] The present invention relates to a dispersion comprising acellular dermal matrix microparticles, a filler comprising the dispersion, and a use thereof. More particularly, the present invention relates to a filler 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] When soft tissue defects in the human skin occur due to external impact, disease, or aging, tissue augmentation techniques, such as soft tissue augmentation, are used for medical and cosmetic purposes. These augmentation techniques can be performed surgically through plastic surgery, or non-surgically by injecting biological tissue or synthetic polymer chemicals into the affected area to increase and expand the volume of soft tissue, restoring and correcting its shape (KR 10-2392812 B1).

[0003] For more efficient repair of damaged soft tissue, the first-generation dermal filler developed was an animal-derived collagen filler. However, its effects lasted only 2 to 4 months after the procedure, and it is rarely used these days due to the inconvenience of having to undergo a skin hypersensitivity test one month before the procedure.

[0004] In addition, collagen-derived substances, such as physiologically active peptides such as KTTKS, are known to inhibit collagenase synthesis, promote extracellular matrix production, and promote the expression of type I and III collagen. However, due to the low stability and skin permeability of peptides in vivo, their use is limited to cosmetics for purposes such as wrinkle improvement and skin regeneration using various derivatives (KR 10-2034872 B1).

[0005] Skin tissue maintains its structure through the extracellular matrix (ECM), which contains proteins such as collagen and elastin, as well as glycosaminoglycans. Various biomaterials utilizing this matrix, such as tissue repair materials and dressing bandages, have been introduced. However, currently commercialized products utilize only a subset of the ECM's components.

[0006] Meanwhile, dermal fillers are desirable to possess sufficient lifting capacity, excellent moldability, and injectability. However, most conventionally developed dermal fillers are manufactured using a crosslinking process, which typically renders them injectable from the moment crosslinking occurs. Therefore, additional processing steps are required to enable injection through microneedles, such as rehydrating the filler composition to the desired concentration and adjusting its size through a homogenization process.

[0007] Accordingly, the inventors of the present invention conducted research to develop an extracellular matrix-containing filler that overcomes the problems of the prior art, and as a result, established conditions and a method for producing an acellular dermal filler mainly composed of skin tissue-derived extracellular matrix 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 present invention was completed by processing the dispersion to produce an injectable acellular dermal filler.

[0008] To achieve the above object, one aspect of the present invention provides an acellular dermal matrix dispersion comprising acellular dermal matrix microparticles.

[0009] Another aspect of the present invention provides an acellular dermal filler comprising the acellular dermal matrix dispersion.

[0010] Another aspect of the present invention provides a method for producing the acellular dermal matrix dispersion.

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

[0012] Another aspect of the present invention provides a prefilled syringe filled with the acellular dermal filler.

[0013] The acellular dermal filler according to the present invention is obtained by decellularizing the skin tissue-derived extracellular matrix used as the main component through a supercritical fluid extraction process without using a surfactant, and does not use a chemical cross-linking agent when manufacturing the filler using the obtained acellular dermal matrix, thereby exhibiting excellent biostability and biocompatibility. In addition, since an acellular dermal matrix dispersion homogenized through a high-pressure dispersion process is applied during the manufacture of the filler, the dispersibility and uniformity of the acellular dermal matrix can be further improved.

[0014] The acellular dermal filler according to the present invention exhibits remarkably high preservation of extracellular matrix components such as collagen and elastin. Furthermore, it exhibits excellent biochemical stability, with no changes in collagen or elastin content, including pH changes during storage. Furthermore, it exhibits physical stability, with sustained injectability over the entire storage period.

[0015] Therefore, the filler containing the acellular dermal matrix dispersion according to the present invention can be usefully utilized in the fields of plastic and regenerative medicine as an injectable filler for repairing or increasing the volume of soft tissue and improving wrinkles.

[0016] Figure 1 schematically illustrates a manufacturing process of a filler 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 3a is a photograph of the appearance of a filler containing an acellular dermal matrix dispersion according to one embodiment of the present invention.

[0019] Figure 3b is a photograph of the appearance of a filler containing an acellular dermal matrix that is currently on the market.

[0020] Figure 4 shows the results of particle size measurement of acellular dermal matrix microparticles according to one embodiment of the present invention.

[0021] Figure 5 shows the results of viscoelasticity measurement according to the concentration of an acellular dermal matrix dispersion according to one embodiment of the present invention.

[0022] Figure 6 shows the results of measuring the discharge pressure of an acellular dermal matrix dispersion according to one embodiment of the present invention and an acellular dermal matrix mixture that was only physically stirred as a comparison group, by each syringe gauge.

[0023] Figure 7 shows the results of collagen content analysis of a filler containing an acellular dermal matrix dispersion according to one embodiment of the present invention.

[0024] Figure 8 shows the results of an analysis of elastin content of a filler containing an acellular dermal matrix dispersion according to one embodiment of the present invention.

[0025] Figure 9 shows the results of measuring the discharge pressure of a filler containing an acellular dermal matrix dispersion according to one embodiment of the present invention for each preservation period.

[0026] Figure 10 shows the results of measuring the pH of a filler containing an acellular dermal matrix dispersion according to one embodiment of the present invention according to the preservation period.

[0027] Figure 11 shows the results of measuring the collagen content of a filler containing an acellular dermal matrix dispersion according to one embodiment of the present invention according to the preservation period.

[0028] Figure 12 shows the results of measuring the elastin content of a filler containing an acellular dermal matrix dispersion according to one embodiment of the present invention according to the preservation period.

[0029] Figures 13a to 13c illustrate the results of analyzing mRNA expression levels in rat skin tissues using qRT-PCR after administration of a filler containing an acellular dermal matrix dispersion according to one embodiment of the present invention. Specifically, graphs illustrating the results of analyzing COL1A1 mRNA expression levels (Figure 13a), COL3A1 mRNA expression levels (Figure 13b), and ACTA2 mRNA expression levels (Figure 13c) are presented. * p<0.05, ** p<0.01, *** p<0.001).

[0030] Figures 14a to 14d show the results of analyzing protein content in rat skin tissue using ELISA after administration of a filler containing an acellular dermal matrix dispersion according to one embodiment of the present invention. Specifically, the graphs show the results of analyzing the content of TNF-α (Figure 14a), IL-6 (Figure 14b), IL-1β (Figure 14c), and IL-8 (Figure 14d). * p<0.05, ** p<0.01, *** p<0.001).

[0031] Figures 15a to 15e illustrate the results of analyzing protein expression levels in rat skin tissues using Western blot after administration of a filler containing an acellular dermal matrix dispersion according to one embodiment of the present invention. Specifically, the graphs illustrate the results of analyzing the protein expression levels of Collagen 1 (Figure 15a), Collagen 3 (Figure 15b), MMP-3 (Figure 15c), MMP-9 (Figure 15d), and MMP-1 (Figure 15e) (*p<0.05, **p<0.01, ***p<0.001).

[0032] Figure 16a is a graph showing the results of analyzing the thickness of the rat membrane through H&E staining after administration of a filler containing an acellular dermal matrix dispersion according to one embodiment of the present invention. * p<0.05, ** p<0.01, *** p<0.001).

[0033] Figure 16b shows a representative image of the histological analysis of the film thickness through H&E staining after administration of a filler containing an acellular dermal matrix dispersion according to one embodiment of the present invention (×200, scale bar: 50 μm).

[0034] Figure 17a is a graph showing the results of analyzing the degree of neovascularization within a rat skin tissue fragment through H&E staining after administration of a filler containing an acellular dermal matrix dispersion according to one embodiment of the present invention. * p<0.05, ** p<0.01, *** p<0.001).

[0035] Figure 17b shows a representative image showing the degree of neovascularization within a skin tissue fragment histologically analyzed through H&E staining after administration of a filler containing an acellular dermal matrix dispersion according to one embodiment of the present invention (×200, scale bar: 50 μm).

[0036] Figure 18a is a graph showing the results of analyzing the degree of fibroblast proliferation within a rat skin tissue fragment through H&E staining after administration of a filler containing an acellular dermal matrix dispersion according to one embodiment of the present invention. * p<0.05, ** p<0.01, *** p<0.001).

[0037] Figure 18b shows a representative image showing the degree of fibroblast proliferation within a skin tissue fragment histologically analyzed through H&E staining after administration of a filler containing an acellular dermal matrix dispersion according to one embodiment of the present invention (×200, scale bar: 50 μm).

[0038] Figure 19a is a graph showing the results of analyzing inflammatory cells within a rat skin tissue fragment through H&E staining after administration of a filler containing an acellular dermal matrix dispersion according to one embodiment of the present invention. * p<0.05, ** p<0.01, *** p<0.001).

[0039] Figure 19b shows a representative image of inflammatory cells in a skin tissue fragment histologically analyzed through H&E staining after administration of a filler containing an acellular dermal matrix dispersion according to one embodiment of the present invention (×200, scale bar: 50 μm).

[0040] Figure 20 shows a representative image of the degree of collagen production in skin tissue analyzed histologically through MT staining after administration of a filler containing an acellular dermal matrix dispersion according to one embodiment of the present invention (×200, scale bar: 50 μm).

[0041] Acellular dermal matrix dispersion

[0042] One aspect of the present invention provides an acellular dermal matrix dispersion comprising acellular dermal matrix microparticles.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] In one specific embodiment of the present invention, the acellular dermal matrix microparticles may have a particle size of, but not limited to, about 50 μm to about 150 μm, and preferably, about 60 μm to about 120 μm.

[0053] By granulating the acellular dermal matrix through the above-described microdifferentiation process, an acellular dermal matrix dispersion can be manufactured at any desired concentration in the subsequent acellular dermal matrix dispersion manufacturing step. Ultimately, by varying the concentration of the acellular dermal matrix dispersion, the physical properties of an acellular dermal filler manufactured using the acellular dermal matrix dispersion can be easily controlled.

[0054] 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.

[0055] The acellular dermal matrix microparticles may be included in an amount of, but not limited to, about 1.0 wt% to 5.0 wt%, about 1.5 wt% to about 4.5 wt%, about 2.0 wt% to about 4.0 wt%, or about 2.5 wt% to about 3.5 wt%, based on the total weight of the acellular dermal matrix dispersion, and preferably, about 3 wt%.

[0056] 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.

[0057] 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 50 μm to about 150 μm at least once.

[0058] 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.

[0059] Additionally, when performing high pressure dispersion, it may pass through a nozzle chamber having a diameter of about 50 μm to about 150 μm, about 60 μm to about 120 μm, or about 100 μm under the above pressure conditions, but is not limited thereto.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] In one specific embodiment of the present invention, the acellular dermal matrix may be decellularized by a supercritical fluid extraction process.

[0064] 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.

[0065] In the present invention, decellularization can be performed by supercritical fluid extraction without surfactant treatment, but is not limited thereto.

[0066] The decellularized dermal tissue of the present invention may be derived from skin tissue isolated from an individual. The skin tissue may be of allogeneic or xenogeneic origin. "Allogeneic" refers to a human, while "xenogeneic" refers to an animal other than a human, such as a mammal such as a pig, cow, or horse.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] Preparation of acellular dermal matrix dispersion

[0071] 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.

[0072] The above “acellular dermal matrix microparticles” and “supercritical fluid” are as described above.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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:

[0089] A step of washing the dermal tissue with a phosphate buffer;

[0090] A step of freeze-drying the washed dermal tissue;

[0091] A step of manufacturing dermal matrix microparticles by micronizing freeze-dried dermal tissue; and

[0092] A step for preparing a dermal matrix dispersion by dispersing dermal matrix microparticles under high pressure in a solution.

[0093] Washing using the above phosphate buffer solution can wash away any remaining solution and impurities present in the dermal tissue after supercritical fluid extraction.

[0094] Freeze-drying can be performed on the above washed dermal tissue.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] The particle size of the acellular dermal tissue granulated through the above-described differentiation process is not limited thereto, but may be about 50 μm to about 150 μm, and preferably about 60 μm to about 120 μm.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] The conditions for performing the above high-pressure dispersion are as described above.

[0105] Acellular dermal filler containing acellular dermal matrix dispersion

[0106] Another aspect of the present invention provides an acellular dermal filler comprising the acellular dermal matrix dispersion.

[0107] As used herein, the term "acellular dermal matrix filler" broadly refers to an injectable material or composition designed to be applied to any part of the body for cosmetic and tissue regeneration purposes, such as for the repair or volume enhancement of soft tissue and the improvement of wrinkles. The filler comprises as its main component an acellular dermal matrix dispersion containing acellular dermal matrix microparticles, and is also used interchangeably as decellularized dermal filler or dermal filler.

[0108] The above filler can be injected into soft tissues such as skin fibrous tissue, muscles, tendons, vocal cords, endometrial tissue, nerves, and synovial tissue, and there are no restrictions on the location (e.g., cheeks, nose, lips, chest, buttocks, legs, and feet) and type of injection.

[0109] The above “acellular dermal matrix”, “acellular dermal matrix microparticles” and “acellular dermal matrix dispersion” are as described above.

[0110] 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 50 μm to about 150 μm, preferably about 60 μm to about 120 μm, are dispersed under high pressure.

[0111] The acellular dermal matrix microparticles may be included in an amount of, but not limited to, about 1.0 wt% to 5.0 wt%, about 1.5 wt% to about 4.5 wt%, about 2.0 wt% to about 4.0 wt%, or about 2.5 wt% to about 3.5 wt%, based on the total weight of the acellular dermal matrix dispersion, and preferably, about 3 wt%.

[0112] 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.

[0113] In one specific embodiment of the present invention, the acellular dermal filler may contain a collagen content of about 100 μg / mg or more based on dry weight. Preferably, the acellular dermal filler may contain about 100 μg / mg to about 500 μg / mg, about 120 μg / mg to about 480 μg / mg, about 140 μg / mg to about 460 μg / mg, about 160 μg / mg to about 440 μg / mg, about 180 μg / mg to about 420 μg / mg, about 200 μg / mg to about 400 μg / mg, about 220 μg / mg to about 380 μg / mg, about 240 μg / mg to about 360 μg / mg, or about 250 μg / mg to about 300 μg / mg, but is not limited thereto.

[0114] In one specific embodiment of the present invention, the acellular dermal filler may contain an elastin content of about 10 μg / mg or more based on dry weight. Preferably, the acellular dermal filler may contain an elastin content of about 10 μg / mg to about 40 μg / mg, about 11 μg / mg to about 38 μg / mg, about 12 μg / mg to about 36 μg / mg, about 13 μg / mg to about 34 μg / mg, about 14 μg / mg to about 32 μg / mg, or about 15 μg / mg to about 30 μg / mg, but is not limited thereto.

[0115] In one specific embodiment of the present invention, the acellular dermal filler is not particularly limited as long as it has a viscosity that can be injected with a syringe, but may have a viscosity in the range of about 80 Pa*s to about 120 Pa*s. Preferably, it may have a viscosity in the range of about 100 Pa*s to about 120 Pa*s.

[0116] In one embodiment of the present invention, the acellular dermal filler is injectable through a syringe needle of at least 18 gauge. Preferably, it is injectable through a syringe needle of at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, or at least 31 gauge.

[0117] In one specific embodiment of the present invention, the acellular dermal filler can be injected through a 27-gauge syringe needle at an injection pressure, i.e., injection force, of about 4 N or less. Additionally, the filler can be injected through a 31-gauge syringe needle at an injection force of about 13 N or less.

[0118] In one specific embodiment of the present invention, the acellular dermal filler does not require separate sizing and / or homogenization prior to injection, and can be directly injected through a syringe needle filled with the acellular dermal filler.

[0119] The acellular dermal filler may further include a local anesthetic. The concentration of the local anesthetic in the acellular dermal filler may be an amount effective to alleviate pain that may be felt upon injection into the body. The concentration of the local anesthetic in the acellular dermal filler may be, but is not limited to, 0.1 to 5.0 (w / v)%, 0.2 to 1.0 (w / v)%, or 0.3 (w / v)%. Specifically, the local anesthetic may be, but is not limited to, one or more selected from the group consisting of lidocaine, etidocaine, bupivacaine, tetracaine, mepivacaine, procaine, prilocaine, ropivacaine, and salts thereof. Preferably, it may be lidocaine and its salts, but is not limited thereto.

[0120] In the present invention, the acellular dermal filler may further comprise other substances or combinations of substances that provide beneficial effects when administered to a subject. Such beneficial substances may include, but are not limited to, anti-scar agents, anti-inflammatory agents, anesthetics, anti-irritant agents, anti-hemorrhagic agents, such as hemostatic agents.

[0121] In exemplary embodiments of the present invention, the acellular dermal matrix microparticle-containing dispersion applied to the manufacture of the acellular dermal filler does not contain a surfactant. Accordingly, the acellular dermal filler manufactured by applying the dispersion has the advantage of minimizing problems such as denaturation of proteins such as collagen and elastin and destruction of growth factors. In addition, the acellular dermal filler has excellent biochemical stability without changes in collagen and elastin content, including changes in pH during the storage period, and also has physical stability that continuously maintains injectability.

[0122] Therefore, the acellular dermal filler according to the present invention can be usefully utilized in the fields of plastic and regenerative medicine as an injectable filler for repairing or increasing the volume of soft tissue, improving wrinkles, or correcting contours.

[0123] Method for preparing an acellular dermal matrix dispersion

[0124] 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:

[0125] a) A step of separating skin tissue separated from an object into an epidermal layer and a dermal layer;

[0126] b) a step of extracting the separated dermal layer with a supercritical fluid;

[0127] c) A step of washing the dermal layer extracted with a supercritical fluid with a phosphate buffer solution;

[0128] d) A step of freeze-drying the washed dermal tissue;

[0129] e) a step of producing dermal matrix microparticles by micronizing freeze-dried dermal tissue; and

[0130] f) A step of preparing a dermal matrix dispersion by dispersing dermal matrix microparticles under high pressure in a solution.

[0131] The above “entity”, “supercritical fluid”, “lyophilization”, “solution”, “high pressure dispersion”, “dermal matrix microparticles”, and “dermal matrix dispersion” are as described above.

[0132] Specific details for each manufacturing step of the above acellular dermal matrix dispersion are as described above in ‘Manufacturing of acellular dermal matrix dispersion’.

[0133] 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.

[0134] 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.

[0135] The dermal matrix microparticles applied in step f) through the above-described differentiation process may have a particle size of about 50 μm to about 150 μm, preferably about 60 μm to about 120 μm, but are not limited thereto.

[0136] 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.

[0137] 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 50 μm to about 150 μm one or more times.

[0138] 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).

[0139] 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.

[0140] Method for manufacturing acellular dermal filler

[0141] Another aspect of the present invention provides a method for producing an acellular dermal filler. The method may comprise the following steps:

[0142] a) A step of separating skin tissue separated from an object into an epidermal layer and a dermal layer;

[0143] b) a step of extracting the separated dermal layer with a supercritical fluid;

[0144] c) A step of washing the dermal layer extracted with a supercritical fluid with a phosphate buffer solution;

[0145] d) A step of freeze-drying the washed dermal tissue;

[0146] e) A step of producing dermal matrix microparticles by micronizing freeze-dried dermal tissue;

[0147] f) a step of preparing a dermal matrix dispersion by dispersing dermal matrix microparticles in a solution under high pressure; and

[0148] g) Step of filling the dermal matrix dispersion into a syringe.

[0149] The above “entity”, “supercritical fluid”, “lyophilization”, “solution”, “high pressure dispersion”, “dermal matrix microparticles”, “dermal matrix dispersion” and “acellular dermal filler” are as described above.

[0150] Specific details of steps a) to f) among the manufacturing steps of the acellular dermal filler 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’.

[0151] In one specific example of the present invention, the acellular dermal filler can be finally obtained by filling a syringe with the acellular dermal matrix dispersion obtained through steps a) to f).

[0152] The above-mentioned acellular dermal filler may contain a collagen content of about 100 μg / mg or more based on dry weight. Preferably, it may contain about 250 μg / mg to about 300 μg / mg, but is not limited thereto.

[0153] In one specific embodiment of the present invention, the acellular dermal filler may contain an elastin content of about 10 μg / mg or more based on dry weight. Preferably, it may contain about 15 μg / mg to about 30 μg / mg, but is not limited thereto.

[0154] In one specific embodiment of the present invention, the acellular dermal filler is not particularly limited as long as it has a viscosity that can be injected with a syringe. The viscosity may be in the range of about 80 Pa*s to about 120 Pa*s, and preferably in the range of about 100 Pa*s to about 120 Pa*s.

[0155] In one specific embodiment of the present invention, the acellular dermal filler does not require separate sizing and / or homogenization prior to injection, and can be directly injected through a syringe needle filled with the acellular dermal filler.

[0156] The above acellular dermal filler can be sterilized after being filled into a syringe in step g).

[0157] 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.

[0158] In one specific example, the acellular dermal filler can be sterilized using gamma rays or eBeam after being filled into a syringe and sealed.

[0159] Another aspect of the present invention provides a prefilled syringe filled with the acellular dermal filler.

[0160] The prefilled syringe may be provided with a syringe needle of, but not limited to, at least 18 gauge. The syringe needle may be, for example, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, or at least 31 gauge.

[0161] In one embodiment of the present invention, the prefilled syringe filled with the acellular dermal filler can be used directly at any application site without separate physical manipulation prior to injection, such as sizing and / or homogenization to enable injection.

[0162] 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.

[0163] I. Preparation of filler containing acellular dermal matrix dispersion

[0164] Example 1. Preparation of acellular dermal filler using dermal matrix dispersion

[0165] A series of processes, including decellularization, micronization, and dispersion preparation, were performed to prepare a filler from a human-derived acellular dermal matrix (ADM). The preparation process of a filler containing an acellular dermal matrix dispersion is schematically illustrated in Fig. 1. The preparation of a filler containing a specific acellular dermal matrix dispersion is as described in Examples 1.1 to 1.3 below.

[0166] Example 1.1. Preparation of acellular dermal matrix using supercritical fluid extraction process

[0167] A supercritical fluid extraction process was performed to decellularize skin tissue.

[0168] 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.

[0169] 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.

[0170] Afterwards, the decellularized dermal tissue was washed with sterilized PBS at room temperature for 24 hours.

[0171] Example 1.2. Preparation of acellular dermal matrix microparticles through a microdifferentiation process.

[0172] In order to process the washed, decellularized, acellular dermal tissue obtained in Example 1.1 above into a filler form, a micronization process was performed.

[0173] 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.

[0174] 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 passed microparticles were collected using a sieve with pores of 100 μm. In other words, acellular dermal matrix microparticles with a size of 100 μm or less were ultimately obtained.

[0175] Example 1.3. Preparation of acellular dermal matrix dispersion

[0176] The acellular dermal matrix microparticles of 100 μm or less in size captured in the above Example 1.2 were physically homogeneously dispersed using a high-pressure disperser (Micronox, MN400BF) (Fig. 2).

[0177] Specifically, acellular dermal matrix microparticles manufactured with a size of 100 μm or less were mixed with sterile distilled water to a final weight ratio of 3%, and physically stirred at 100 RPM for 10 minutes using a magnetic stirrer.

[0178] The above 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 100 μm nozzle. The viscosity of the acellular dermal matrix microparticle-containing dispersion subjected to the primary high-pressure dispersion was visually confirmed to have 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).

[0179] Afterwards, the homogeneous acellular dermal matrix dispersion was packaged in 1 cc syringes and sealed. The packaged acellular dermal matrix dispersion was sterilized using gamma or eBeam sterilization (15 kGy). After sterilization, it was stored at room temperature until use.

[0180] II. Evaluation of the physicochemical properties of fillers containing acellular dermal matrix dispersions

[0181] Experimental Example 1. Observation of the Appearance of Filler Containing Acellular Dermal Matrix Dispersion

[0182] In order to confirm the dispersion stability of the acellular dermal matrix dispersion finally obtained in Example 1.3 above, the appearance of the dispersion packaged in a syringe was observed (Fig. 3a).

[0183] As shown in Fig. 3a, the acellular dermal matrix dispersion filled in a 1 cc syringe was confirmed to be homogeneously dispersed without any segregation. Accordingly, it was confirmed that it can be used immediately without separate physical mixing, such as shaking before use. Furthermore, even after being dispensed from the syringe, a layered structure was observed with a viscosity of approximately 114 Pa*s.

[0184] On the other hand, as shown in Fig. 3b, in the case of a conventional commercially available acellular dermal matrix-containing filler (CG Reallo Inject, CG Bio; 22G needle, 3N injection force), the acellular dermal matrix and the filler solution were confirmed to be phase-separated. Accordingly, it was found that separate physical mixing through shaking was essential before use.

[0185] Experimental Example 2. Particle size measurement of acellular dermal matrix microparticles

[0186] The particle size of the dermal matrix microparticles contained in the acellular dermal matrix dispersion prepared in Example 1.2 was measured using a particle size analyzer (PSA 1190 model (Anton Paar)). Since the dermal matrix microparticles are freeze-dried particles, their particle size changes due to moisture absorption when in a hydrated state. Therefore, since wet analysis is impossible due to the opaque liquid, the particle size was measured through dry analysis.

[0187] As a result of the measurement, it was confirmed that the particles that passed through a sieve with pores of 100 μm after grinding had an average particle size of 72 μm (Fig. 4).

[0188] The values ​​of particle size of the sample measured in triplicate are shown in Table 1 below. In addition, the average value and standard deviation are calculated and shown based on the values ​​of particle size of the sample measured in triplicate in Table 2 below.

[0189] NameD 10 [μm]D 50 [μm]D 90 [μm]Mean size [μm]SpanSample 113.10556.479138.77771.4622.225Sample 212.90956.607139.57371.8672.238Sample 313.13057.122139.08672.1042.205

[0190] * D10 , D 50 , D 90 : Refers to the particle sizes corresponding to 10%, 50%, and 90% of the maximum value in the cumulative distribution of the sample particle size, respectively.

[0191] * Span: The distribution width of the sample, span value = (D 90 - D 10 ) / D 50 Calculated as .

[0192] D 10 [μm]D 50 [μm]D 90 [μm]Mean size [μm]SpanMean value13.04856.736139.14571.8112.223Standard deviation0.121150.34070.40110.32480.016Relative standard deviation0.930.600.290.450.74

[0193] Through the above results, it was found that the acellular dermal matrix microparticles produced by the undifferentiated process were uniformly finely atomized and were suitable for application to injection.

[0194] Experimental Example 3. Viscoelasticity Measurement of Acellular Dermal Matrix Dispersion

[0195] In order to confirm whether the acellular dermal matrix microparticles prepared in the above Example 1.2 were dispersed in a solvent through high-pressure dispersion and had suitable properties as a transplant material, the viscosity was measured. At this time, samples dispersed in sterile distilled water at concentrations of 1 wt% and 3 wt% were used. Meanwhile, as a comparison group, commercially available filler products, Elravie Premier Volumizing (40-80 Pa*s) and Elravie Premier Lite L (40-90 Pa*s) from Humedix were used (results not shown).

[0196] Specifically, the viscosity of the human-derived acellular dermal matrix dispersion dispersed through a high-pressure dispersion process was measured using a rheometer (MCR 302e model (Anton Paar)). The measuring device was a PP50 with a diameter of 5 mm, and the measurement conditions were 0.1 Hz, 25°C, and repeated 4 times per sample.

[0197] As a result of the measurement, it was confirmed that the viscosity difference significantly changed depending on the concentration of the acellular dermal matrix dispersion, as shown in Fig. 5. In particular, among the viscosity measurement samples, the sample dispersed at a concentration of 3 wt% showed a viscosity of 114.45 Pa*s. This is equivalent to the viscosity level of commercially available filler products, which is 80 to 120 Pa*s, and it was found to be suitable for use as a filler for plastic surgery.

[0198] The above results confirmed that the acellular dermal matrix dispersion, a human tissue material that can be used as a grafting material such as a plastic filler, exhibited a viscosity similar to that of commercially available polymer-crosslinked filler materials. Accordingly, it was found that the acellular dermal matrix dispersion can be used as a substitute for filler materials.

[0199] Experimental Example 4. Measurement of the discharge pressure of an acellular dermal matrix dispersion.

[0200] A discharge pressure test using a micro needle was performed on the acellular dermal matrix dispersion prepared in Example 1.3. The discharge pressure test of the human-derived acellular dermal matrix dispersion dispersed through the high-pressure dispersion process was measured using a universal testing machine (AGS-X model (SHIMADZU)). Since the discharge pressure affects the measurement result depending on the thickness of the micro needle, the test was conducted using needles of 27 Gauge and 31 Gauge. The acellular dermal matrix dispersion sample that had undergone high-pressure dispersion treatment was used as the test group. The comparative group was used as the acellular dermal matrix mixture that had been mixed by only physical stirring (not subjected to high-pressure dispersion treatment) for 1 day using a magnetic stirrer as in Experimental Example 1.3.

[0201] As a result of measuring the discharge pressure, it was confirmed that the maximum tensile strength of the acellular dermal matrix dispersion sample was 4 N or less when a 27 Gauge needle was used, and 13 N or less when a 31 Gauge needle was used. On the other hand, in the case of the acellular dermal matrix mixture that was simply physically stirred, it was 11 N or less when a 27 Gauge needle was used, and when a 31 Gauge needle was used, the discharge pressure was confirmed to be such that the needle detached at 55 N and analysis was impossible (Fig. 6).

[0202] Therefore, it was found through the above results that, when the process for manufacturing an acellular dermal matrix dispersion according to the present invention is followed, it is possible to provide an advantage of producing an injection that is easy to inject compared to the case of simply mixing by conventional physical mixing.

[0203] III. Evaluation of biochemical properties of fillers containing acellular dermal matrix dispersions

[0204] Experimental Example 5. Measurement of collagen content in fillers containing acellular dermal matrix dispersion.

[0205] In order to confirm whether the acellular dermal matrix dispersion prepared in Example 1.3 is suitable as a graft material such as a filler, the loss of collagen, an extracellular matrix protein important for tissue regeneration, was measured. The collagen content was measured using the Sircol Insoluble Collagen Assay Kit from Biocolor. The acellular dermal matrix dispersion was used as the test group, and CG Reallo Inject from CG Bio was used as the control group. In addition, each sample was analyzed in a state including hydrated particles that were frozen at -80°C for 2 hours and lyophilized for 1 day to make it suitable for analysis.

[0206] As a result of the analysis, the lyophilized product of the acellular dermal matrix dispersion was measured to have a collagen content of approximately 19 times higher than that of the control group, at a level of 244 μg / mg. The lyophilized product of the control group was measured to have a significantly lower collagen content of 13 μg / mg (Fig. 7).

[0207] Experimental Example 6. Measurement of elastin content in fillers containing acellular dermal matrix dispersion

[0208] In order to confirm the suitability of the acellular dermal matrix dispersion prepared in Example 1.3 as a graft material, the loss of elastin, an extracellular matrix protein important for tissue regeneration, was measured. The elastin content was measured using the Fastin Elastin Assay Kit from Biocolor. The acellular dermal matrix dispersion was used as the test group, and CG Bio's CG Reallo Inject was used as the control group. Each sample was prepared in a state containing hydrated particles in the same manner as in Experimental Example 5 and analyzed.

[0209] As a result of the analysis, the lyophilized product of the acellular dermal matrix dispersion was measured to have an elastin content of approximately 2.6 times higher than that of the control group, at a level of 15.3 μg / mg. The lyophilized product of the control group was measured to have a significantly lower elastin content of 5.9 μg / mg (Fig. 8).

[0210] The above series of results demonstrated that the acellular dermal matrix dispersion, decellularized, micronized, and high-pressure dispersed using a supercritical fluid treatment process, exhibited excellent histological preservation of proteins such as collagen and elastin. Accordingly, it was demonstrated that the acellular dermal matrix dispersion exhibited excellent potential as a grafting material.

[0211] IV. Stability Evaluation of Fillers Containing Acellular Dermal Matrix Dispersion

[0212] Experimental Example 7. Physical Stability Evaluation of Fillers Containing Acellular Dermal Matrix Dispersion

[0213] In order to evaluate the physical stability of the acellular dermal matrix dispersion prepared in Example 1.3, a discharge pressure test using a micro needle was performed on samples at weeks 0, 4, and 8 of the preparation of the acellular dermal matrix dispersion. The discharge pressure test was performed as in Experimental Example 4. At this time, the discharge pressure test was performed using a micro needle with a thickness of 27 Gauge.

[0214] As a result of the discharge pressure measurement, it was confirmed that the discharge pressure, i.e., the injection force, was measured to be 4 N or less in all samples according to the preservation period. It was confirmed that there was no difference between each sample according to the preservation period.

[0215] Through the above results, it was found that the physical stability of the filler containing the acellular dermal matrix dispersion was continuously maintained over the preservation period.

[0216] Experimental Example 8. Biochemical Stability Evaluation of Fillers Containing Acellular Dermal Matrix Dispersion

[0217] Experimental Example 8.1. pH Measurement

[0218] In order to check the oxidation of the filler containing the acellular dermal matrix dispersion prepared as in Example 1.3 above, samples were taken at weeks 0, 1, 2, 3, 4, 6, and 8 of preparation, centrifuged at 13,000 RPM for 5 minutes at 25°C, and the supernatant was separated to measure the pH. The test was conducted using a pH meter (pH Meter; Fiveeasy plus (Mettler Toledo)).

[0219] As a result of pH measurement, it was confirmed that the pH was well maintained at a similar level as immediately after manufacturing without a decrease in the pH in the supernatant of all samples according to the preservation period (Fig. 10).

[0220] Through the above results, it was found that the filler containing the acellular dermal matrix dispersion had excellent preservation stability without corrosion or rancidity during the preservation period.

[0221] Experimental Example 8.2. Measurement of Collagen Content

[0222] In order to confirm the biochemical stability of the filler containing the acellular dermal matrix dispersion prepared as in Example 1.3 according to the storage period, the collagen content of the sample was analyzed using the same method as in Experimental Example 5 at weeks 0, 4, and 8.

[0223] As a result of the analysis, it was confirmed that the collagen content slightly decreased to 15% of the level at week 8 compared to week 0 as the preservation time elapsed, but the collagen content was still 284.6 μg / mg even after 8 weeks (Fig. 11).

[0224] Through the above results, it was found that the filler containing the acellular dermal matrix dispersion preserved the collagen content at a relatively high level without significantly affecting the preservation period (Fig. 11).

[0225] Experimental Example 8.3. Measurement of Elastin Content

[0226] In order to confirm the biochemical stability of the filler containing the acellular dermal matrix dispersion prepared as in Example 1.3 according to the storage period, the elastin content of the sample was analyzed using the same method as in Experimental Example 5 at weeks 0, 4, and 8.

[0227] Analysis results showed no change in elastin content over time (Fig. 12). This indicates that fillers containing acellular dermal matrix dispersion exhibit excellent collagen preservation properties, with no change over time.

[0228] V. In vivo efficacy evaluation of fillers containing acellular dermal matrix dispersions

[0229] The safety and efficacy of the test substance (Table 3) on tissue regeneration were evaluated in vivo in rats. SD (Spargue Dawley, 5-week-old, male, Orient Bio Co., Ltd.) rats were used.

[0230] Group (N=5) Test substance administration Untreated group - Control group 1 Mega fill (L&C Bio) Subcutaneously administered with a volume of 200 μL using a 1 mL syringe Control group 2 CG Reallo Putty (CGBio) Test group Filler containing acellular dermal matrix dispersion (SC Fill)

[0231] The expression levels of cytokines (TNF-α, IL-1β, IL-6, and IL-8) related to inflammation in the body were analyzed using an enzyme-linked immunosorbent assay (ELISA). In addition, the expression levels of collagen (COL1A1, COL3A1), which are synthetic factors for tissue regeneration, and collagen-degrading enzymes (MMP-1, MMP-3, MMP-9) were analyzed using qRT-PCR and / or Western blot. Additionally, myofibroblast protein factor 2 (ACTA2), which is related to tissue regeneration, was analyzed using qRT-PCR (Quantitative Real-Time PCR). The stability of SC Fill and its efficacy on tissue regeneration were observed in cross-sections of rat skin tissue through histological evaluation (H&E (Hematoxyline & Eosin) staining and MT (Masson's Trichrom) staining).

[0232] Experimental Example 9. mRNA Expression Evaluation Test (qRT-PCR)

[0233] As shown in Table 3 above, skin tissues of experimental animals were biopsied at each time point (1, 2, 3, and 6 months) after administration of the test substance, and the tissues were disrupted and reacted with Trizol. Afterwards, RNA was isolated from the tissues using Total RNA extraction reagent (Takara Bio, Inc.) according to the manufacturer's instructions. cDNA synthesis was performed according to the protocol provided by the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Inc.). For quantitative PCR, TB Green®Premix Ex Taq™ II (Takara Bio, Inc.) and QuantStudio™ 3 Real-Time PCR (Thermo Fisher Scientific, USA) were used, and the results were repeated three times (PCR conditions: denaturation at 95°C for 30 s, 45 cycles at 95°C for 5 s, and 60°C for 34 s. The relative mRNA expression value was 2 -ΔΔCt (determined using the method).

[0234] The relative mRNA expression level was expressed as a fold, and the mRNA expression level change rate (%) was calculated according to the following [Mathematical Formula 1].

[0235] [Mathematical Formula 1]

[0236]

[0237] Experimental Example 9.1. Evaluation of COL1A1 mRNA Expression Level

[0238] The expression level of COL1A1 (collagen type I alpha 1) mRNA in rat skin tissue at 6 months was analyzed based on the 1-month point. As a result, the control group 1 (Meagfill), control group 2 (CG putty), and test group (SC Fill) significantly increased by 246.70%, 230.28%, and 250.16%, respectively (Fig. 13a).

[0239] Experimental Example 9.2. Evaluation of COL3A1 mRNA Expression Level

[0240] The expression level of COL3A1 (collagen type Ⅲ alpha 1) mRNA in rat skin tissue at 6 months was analyzed based on the 1-month point. As a result, the control group 1 (Meagfill), control group 2 (CG putty), and test group (SC Fill) showed significant decreases of 65.50%, 61.11%, and 55.29%, respectively (Fig. 13b).

[0241] Experimental Example 9.3. Evaluation of ACTA2 mRNA Expression Level

[0242] The expression level of ACTA2 (actin alpha 2) mRNA in rat skin tissue at 6 months was analyzed based on the 1-month point. As a result, the control group 1 (Meagfill), control group 2 (CG putty), and test group (SC Fill) showed significant increases of 246.32%, 250.05%, and 245.52%, respectively (Fig. 13c).

[0243] qRT-PCR analysis conclusions

[0244] A summary of the results of the above series of qRT-PCR analyses revealed that COL3A1 levels gradually decreased over time in all groups, whereas COL1A1 and ACTA2 levels gradually increased over time, reaching significantly higher levels at 6 months after administration. Control group 2 (CG Putty) and the test group (SC Fill) showed higher levels over time than Control group 1 (Mega Fill).

[0245] Experimental Example 10. Protein Content Evaluation Test (ELISA)

[0246] As shown in Table 3 above, skin tissues of experimental animals were biopsied at each time point (1, 2, 3, and 6 months) after administration of the test substance. After disrupting the tissues, RIPA lysis buffer was added, proteins were extracted, and quantified through BCA assay. According to the manufacturer's protocol, each corresponding ELISA kit was used to measure the absorbance at 450 nm with a Microplate spectrophotometer (BioTek, USA) and analyzed. The contents of each corresponding factor, TNF-α, IL-6, IL-1β, and IL-8, were calculated using the Standard Curve (R 2 ≥0.95) was calculated.

[0247] The rate of change in protein content (%) was calculated according to the following [Mathematical Formula 2].

[0248] [Equation 2]

[0249]

[0250] Experimental Example 10.1. Evaluation of TNF-α Content

[0251] As a result of analyzing the content of TNF-α (tumor necrosis factor-α) in rat skin tissue at 1 month, the control group 1 (Mega fill) showed a significant increase of 62.50% compared to the untreated group, and the control group 2 (CG Putty) and the test group (SC Fill) showed a tendency to increase by 73.08% and 19.23% compared to the untreated group, respectively. Thereafter, as a result of analyzing the content of TNF-α at 6 months, the control group 1 (Mega fill), control group 2 (CG Putty), and the test group (SC Fill) showed a tendency to increase by 0.85%, 9.32%, and 5.08% compared to the untreated group, respectively (Fig. 14a).

[0252] Experimental Example 10.2. IL-6 Content Evaluation

[0253] As a result of analyzing the content of IL-6 (interleukin-6) in rat skin tissue at 6 months, the control group 1 (Mega fill) showed a significant decrease of 22.44% compared to the untreated group, and the control group 2 (CG Putty) and test group (SC Fill) showed a tendency to decrease by 20.42% and 24.25% compared to the untreated group, respectively (Fig. 14b).

[0254] Experimental Example 10.3. Evaluation of IL-1β Content

[0255] As a result of analyzing the content of IL-1β (interleukin-1β) in rat skin tissue, the control group 1 (Mega fill) showed a significant increase of 85.59% compared to the untreated group at 3 months, and a tendency to decrease by 55.49% at 6 months. The control group 2 (CG Putty) and the test group (SC Fill) showed a tendency to decrease by 17.43% and 20.99% compared to the untreated group at 3 months, and a tendency to decrease by 30.82% and 61.04% compared to the untreated group at 6 months (Fig. 14c).

[0256] Experimental Example 10.4. IL-8 Content Evaluation

[0257] As a result of analyzing the content of IL-8 (interleukin-8) in rat skin tissue at 6 months, the control group 1 (Mega fill), control group 2 (CG Putty), and test group (SC Fill) showed a tendency to decrease by 19.55%, 14.49%, and 22.16%, respectively, compared to the untreated group (Fig. 14d).

[0258] ELISA analysis conclusion

[0259] The results of the above series of ELISA analyses showed that TNF-α, IL-6, IL-1β, and IL-8, which are related to inflammation, tended to decrease over time in all groups. The numerical differences between the groups for TNF-α, IL-6, and IL-8 were small, but the test group (SC Fill) showed a relatively low level of IL-1β.

[0260] Experimental Example 11. Protein Expression Level Evaluation Test (Western Blot)

[0261] As shown in Table 3 above, skin tissues of experimental animals were biopsied at each time point (1, 2, 3, and 6 months) after administration of the test substance. The tissues were disrupted, RIPA lysis buffer was added, proteins were extracted, and quantified using BCA assay. Equal amounts of proteins were electrophoresed on Mini Protean Gel (Bio-Rad, USA), transferred to PVDF membranes, and reacted with blocking buffer at room temperature for 1 hour. Primary antibodies against each corresponding factor, Collagen 1, Collagen 3, MMP-3, MMP-9, and MMP-1, were reacted in a refrigerator for 24 hours, and after washing, secondary antibodies were reacted at room temperature for 1 hour, and then washed. ECL TM After reaction with Prime Western Blotting system (GE Healthcare, USA), it was analyzed with ChemiDoc™ Touch Gel Imaging System (Bio-Rad, USA) (relative protein expression level was normalized through β-actin, and phosphorylated form expression level was normalized to total form expression level).

[0262] The rate of change in protein expression (%) was calculated according to the following [Mathematical Formula 3].

[0263] [Equation 3]

[0264]

[0265] Experimental Example 11.1. Evaluation of Collagen 1 Protein Expression

[0266] As a result of analyzing the expression level of Collagen 1 protein in rat skin tissue at 6 months, the untreated group, control group 1 (Mega fill), and control group 2 (CG Putty) showed significant increases of 58.58%, 94.56%, and 73.03%, respectively, compared to 1 month, and the test group (SC Fill) showed a tendency to increase by 98.32% compared to 1 month (Fig. 15a).

[0267] Experimental Example 11.2. Evaluation of Collagen 3 Protein Expression

[0268] As a result of analyzing the expression level of Collagen 3 protein in rat skin tissue at 6 months, the untreated group, control group 2 (CG Putty), and test group showed a tendency to decrease by 62.43%, 66.29%, and 49.64%, respectively, compared to 1 month, and control group 1 (Mega fill) showed a decrease of 49.34% compared to 1 month (Fig. 15b).

[0269] Experimental Example 11.3. Evaluation of MMP-3 Protein Expression

[0270] As a result of analyzing the protein expression level of MMP-3 in rat skin tissue at 6 months, the untreated group showed a significant decrease of 41.51% compared to 1 month, and the control group 1 (Mega fill), control group 2 (CG Putty), and test group (SC Fill) showed a tendency to decrease by 47.47%, 37.00%, and 42.82% compared to 1 month, respectively (Fig. 15c).

[0271] Experimental Example 11.4. Evaluation of MMP-9 Protein Expression

[0272] As a result of analyzing the protein expression level of MMP-9 in rat skin tissue at 6 months, the untreated group, control group 1 (Mega fill), and control group 2 (CG Putty) showed a tendency to decrease by 31.40%, 16.32%, and 26.58%, respectively, compared to 1 month, and in the test group (SC Fill), the content of MMP-9 significantly decreased by 41.51% compared to 1 month (Fig. 15d).

[0273] Experimental Example 11.5. Evaluation of MMP-1 Protein Expression

[0274] As a result of analyzing the protein expression level of MMP-1 in rat skin tissue at 6 months, the untreated group, control group 2 (CG Putty), and test group (SC Fill) showed a tendency to decrease by 15.11%, 39.49%, and 39.13%, respectively, compared to 1 month, and control group 1 (Mega fill) showed a significant decrease by 43.55% compared to 1 month (Fig. 15e).

[0275] Western Blot Analysis Conclusion

[0276] A summary of the results of the above series of Western blot analyses revealed that Collagen III levels gradually decreased over time in all groups, while Collagen I levels tended to increase over time. Levels of MMP-1, MMP-3, and MMP-9 tended to decrease across all groups, with significantly lower levels observed in the test group (SC Fill).

[0277] Experimental Example 12. Histological Evaluation Test (H&E Staining)

[0278] As shown in Table 3 above, skin tissues of experimental animals were biopsied at each time point (1, 2, 3, and 6 months) after administration of the test substance, fixed with 10% formalin solution, and paraffin blocks were made. Afterwards, slides were made by sectioning at 3 μm thickness, and hematoxylin & eosin staining was performed after hydration. Optical microscope (Zeweiss TM , Germany) were used to photograph cross-sections of rat skin tissue (one slide per individual). The thickness of the material film, neovascularization within the material, inflammatory cells, and fibroblasts were analyzed using the Image J (National Institutes of Health, USA) program.

[0279] Experimental Example 12.1. Film Thickness Analysis

[0280] As a result of analyzing the film thickness in rat skin tissue sections using an optical microscope based on the untreated group, the control group 1 (Mega fill) showed a tendency to increase by 1.89% at 3 months compared to the untreated group, but a tendency to decrease by 34.71% at 6 months. The control group 2 (CG Putty) showed a tendency to increase by 12.85% at 3 months compared to the untreated group, but a significant decrease by 50.59% at 6 months. The test group (SC Fill) showed a tendency to increase by 44.51% at 3 months compared to the untreated group, but a tendency to decrease by 16.77% at 6 months (Fig. 16a and Fig. 16b).

[0281] Experimental Example 12.2. Angiogenesis Analysis

[0282] As a result of analyzing the neovascularization within the material using an optical microscope in a rat skin tissue section at 1 month, the control group 1 (Mega fill) showed a significant increase of 166.67% at 6 months, and the control group 2 (CG Putty) showed a tendency to increase by 25.00% at 3 months compared to 1 month, but a tendency to decrease by 8.82% at 6 months. The test group (SC Fill) showed a tendency to increase by 173.02% at 6 months compared to the untreated group (Fig. 17a and Fig. 17b).

[0283] Experimental Example 12.3. Fibroblast Proliferation Analysis

[0284] As a result of analyzing the proliferation of fibroblasts in a rat skin tissue section using an optical microscope at 1 month, the control group 1 (Mega fill) showed a significant increase of 306.36% at 6 months compared to the untreated group, and the control group 2 (CG Putty) and test group (SC Fill) showed an increase of 56.00% and 171.79%, respectively, at 6 months compared to the untreated group (Fig. 18a and Fig. 18b).

[0285] Experimental Example 12.4. Inflammatory Cell Analysis

[0286] As a result of analyzing inflammatory cells in rat skin tissue sections using an optical microscope at 1 month, the untreated group showed a tendency to decrease by 32.59% at 6 months, and the control group 1 (Mega fill) showed a tendency to increase by 30.44% at 2 months and then decreased by 75.61% at 6 months. The control group 2 (CG Putty) showed a tendency to decrease by 89.87% at 6 months, and the test group (SC Fill) showed a significant decrease by 71.74% at 6 months (Fig. 19a and Fig. 19b).

[0287] H&E staining analysis conclusion

[0288] A summary of the results of the above series of H&E staining analyses showed that the film thickness tended to increase in each group up to 3 months and then decreased overall at 6 months. Neovascularization and fibroblast proliferation within the material tended to increase over time, with the test group (SC Fill) showing higher values. Inflammatory cells within the material were confirmed to gradually decrease over time, with the test group (SC Fill) showing lower values ​​at all time points.

[0289] Experimental Example 13. Histological Evaluation Test (MT Staining)

[0290] As shown in Table 3 above, skin tissues of experimental animals were biopsied at each time point (1, 2, 3, and 6 months) after administration of the test substance, fixed with 10% formalin solution, and paraffin blocks were made. Afterwards, 3 μm-thick sections were made into slides, and after hydration, staining and washing with Biebrich Scarlet-Acid Fuchsin solution for 5 minutes, and staining was performed again with phosphotungstic / phosphomolybdic acid for 5 minutes. Optical microscope (Zeweiss TM , Germany) were used to photograph cross-sections of rat skin tissue, one slide per individual.

[0291] As a result of analyzing the degree of collagen production in cross-sections of rat skin tissue using an optical microscope, the collagen production of control group 1 (Mega fill), control group 2 (CG Putty), and test group (SC Fill) all showed a tendency to increase over time compared to the untreated group. In particular, control group 2 (CG Putty) and test group (SC Fill) showed significantly better collagen production ability than control group 1 (Mega Fill) over time (Fig. 20).

[0292] Conclusions on the in vivo efficacy evaluation of fillers containing acellular dermal matrix dispersions

[0293] The results of the above series of in vivo rat experiments confirmed that a cell-free dermal matrix dispersion-containing filler (SC Fill) exhibited tissue regeneration efficacy. Therefore, SC Fill was found to be effective for skin stability and tissue regeneration in an in vivo model.

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 50 μm to 150 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 5.0 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 50 μm to 150 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 co-solvent.

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 filler comprising an acellular dermal matrix dispersion according to any one of claims 1 to 14.

16. In paragraph 15, The above-mentioned acellular dermal matrix dispersion is an acellular dermal filler in which acellular dermal matrix microparticles having a particle size of 50 μm to 150 μm are dispersed under high pressure in a solution.

17. In paragraph 15, The above acellular dermal filler has any one characteristic selected from the following group based on dry weight: Collagen content of 100 μg / mg to 500 μg / mg; and Elastin content of 10 μg / mg to 40 μg / mg.

18. In paragraph 15, The above acellular dermal filler is an acellular dermal filler that can be injected with a syringe.

19. In paragraph 15, The above-mentioned acellular dermal filler has a viscosity of 80 Pa*s to 120 Pa*s.

20. In paragraph 15, The above acellular dermal filler further comprises a local anesthetic.

21. In paragraph 20, An acellular dermal filler, wherein the local anesthetic is at least one selected from the group consisting of lidocaine, etidocaine, bupivacaine, tetracaine, mepivacaine, procaine, prilocaine, ropivacaine, and salts thereof.

22. In paragraph 15, The above acellular dermal filler is an acellular dermal filler for soft tissue repair or volume augmentation, wrinkle improvement or contour correction. 23.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.

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

25. In paragraph 23, A method for producing an acellular dermal matrix dispersion, wherein in the above step f), the dermal matrix microparticles have a particle size of 50 μm to 150 μm.

26. In paragraph 23, A method for producing an acellular dermal matrix dispersion, wherein in the above step f), high-pressure dispersion is performed by applying a pressure of 8,000 psi to 40,000 psi and passing the dispersion through a nozzle chamber of 50 μm to 150 μm at least once. 27.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 manufacturing an acellular dermal filler, comprising the step of filling a dermal matrix dispersion into a syringe.

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

29. In paragraph 27, A method for producing an acellular dermal filler, wherein in the above step f), the dermal matrix microparticles have a particle size of 50 μm to 150 μm.

30. In paragraph 27, A method for producing an acellular dermal filler, wherein in the above step f), high-pressure dispersion is performed by applying a pressure of 8,000 psi to 40,000 psi and passing the filler through a nozzle chamber of 50 μm to 150 μm at least once.

31. In paragraph 27, A method for producing an acellular dermal filler, comprising a step of sterilizing a syringe filled with a dermal matrix dispersion solution after the above step g).

32. A prefilled syringe filled with the acellular dermal filler of Article 15.

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