WO2024014795A1 - Extracellular matrix-based hydrogel and sponge for wound healing or tissue regeneration, and production method therefor - Google Patents

Extracellular matrix-based hydrogel and sponge for wound healing or tissue regeneration, and production method therefor Download PDF

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WO2024014795A1
WO2024014795A1 PCT/KR2023/009718 KR2023009718W WO2024014795A1 WO 2024014795 A1 WO2024014795 A1 WO 2024014795A1 KR 2023009718 W KR2023009718 W KR 2023009718W WO 2024014795 A1 WO2024014795 A1 WO 2024014795A1
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extracellular matrix
hydrogel
wound
sponge
regeneration
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PCT/KR2023/009718
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French (fr)
Korean (ko)
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박귀덕
하상수
권재원
시닌타사비트리
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한국과학기술연구원
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Priority claimed from KR1020220086386A external-priority patent/KR20240009206A/en
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Publication of WO2024014795A1 publication Critical patent/WO2024014795A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like

Definitions

  • the present invention relates to a method for producing a hydrogel produced from an extracellular matrix without a separate polymer support or hydrogel precursor solution, a hydrogel produced therefrom, an ECM sponge produced by freeze-drying the same, and its use in wound treatment and tissue regeneration. It's about.
  • Materials used in wound treatment require mechanical properties that can be fixed to the wound area, the ability to maintain appropriate moisture on the surface in contact with the wound, the ability to control wound secretions, and non-toxicity to the human body.
  • Secondary infection rate rate must be low, it must be easy to manufacture and store, it must be easy to manipulate and use in vitro, and the delivery of the active substance must be easy.
  • Existing wound treatments are mostly wound coating materials that are primarily intended to absorb wound exudates, prevent external infection, and provide moisture, and most of them utilize biocompatible polymers such as collagen and alginate.
  • biocompatible polymers such as collagen and alginate.
  • hydrogel refers to a material that absorbs a large amount of water or body fluid into a crosslinked lattice and swells, and maintains a three-dimensional structure without dispersing even in water. Even after swelling, it remains thermodynamically stable and has mechanical and physicochemical properties corresponding to an intermediate form between a liquid and a solid.
  • These hydrogels usually exhibit excellent biocompatibility, high porosity and oxygen permeability, exhibit physical properties similar to living soft tissues, and are safe for the human body, so they are used in various fields such as moist wound healing, tissue regeneration, and drug delivery. is being used.
  • extracellular matrix has excellent regenerative treatment efficacy
  • ECM extracellular matrix
  • the present inventors demonstrated that the hydrogel produced by adding ultrapure distilled water to decellularized extracellular matrix without a separate synthetic polymer support or hydrogel precursor solution and the ECM sponge produced by freeze-drying the hydrogel produced excellent tissue regeneration and wound healing.
  • the present invention was completed by confirming effects such as hair follicle regeneration.
  • the purpose of the present invention is to provide an extracellular matrix hydrogel composition, an ECM sponge manufactured using the same, and a method for manufacturing the same.
  • Another object of the present invention is to provide a hydrogel composition for wound treatment or tissue regeneration comprising the hydrogel composition.
  • Another object of the present invention is to provide an ECM sponge for wound treatment or tissue regeneration manufactured using the above composition.
  • the present invention provides a method for producing a hydrogel composition without a separate polymer support or hydrogel precursor solution including the following steps.
  • the present invention provides a method for producing a hydrogel composition for wound treatment or tissue regeneration without a separate polymer support or hydrogel precursor solution including steps (1) to (3) above.
  • the present invention provides a method of producing an extracellular matrix (ECM) sponge for wound treatment or tissue regeneration, comprising the step of freeze-drying the hydrogel produced by the above production method.
  • ECM extracellular matrix
  • the distilled water in step (1) may be sterile ultrapure distilled water, and preferably 3 to 8 ml may be added based on a 100 mm culture vessel.
  • step (2) the mixture prepared in step (1) is compressed, frozen at -60 to 90°C for more than a day, thawed at 20 to 40°C, and then additionally processed at high speed. It may be stirring.
  • step (2) may include the following steps (a) to (d):
  • step (a) is performed by rotating the mixture of step (1) at a speed of 3000 to 4000 rpm for 5 to 15 minutes, preferably 8 to 12 minutes, more preferably 10 minutes. It may be compressed by stirring.
  • step (d) may be high-speed stirring at a speed of 3000 to 4000 rpm for 3 to 8 minutes, preferably 4 to 6 minutes, and more preferably 5 minutes.
  • the decellularized extracellular matrix may be obtained from cells cultured in vitro.
  • the cells include fibroblasts, chondrocytes, osteoblasts, vascular endothelial cells, myocytes, smooth muscle cells, hepatocytes, nerve cells, cardiomyocytes, spinal disc cells, mesenchymal stem cells, and their It may be one or more cells selected from the group consisting of combinations, and the cells may be isolated from humans, but are not limited thereto.
  • the hydrogel composition may contain growth factors related to neovascularization.
  • the growth factors related to angiogenesis include amphiregulin, coagulation factor III, dipeptidyl peptidase IV (DPPIV), endostatin (endostatin, collagen XVIII), and FGF.
  • acidic Fibroblast growth factor-acidic
  • FGF-7 Fibroblast Growth Factor 7
  • HGF Hepatocyte growth factor
  • Pentraxin 3 plasminogen activator inhibitor-1 (serpin E1), serpin E1 ( It may be one or more selected from the group consisting of PEDF), TIMP-1 (Tissue inhibitor of metalloproteinase), thrombospondin-1, uPA (urokinase-type plasminogen activator), and combinations thereof. It is not limited.
  • the hydrogel composition may include type I collagen (Collagen I) and/or fibronectin.
  • the hydrogel composition may exhibit a wound recovery effect, a wound healing effect, a tissue regeneration effect, an epidermal regeneration effect, a dermal regeneration effect, a neovascularization regeneration effect, or a skin appendage regeneration effect.
  • the wound may be a wound, burn wound, abrasion, laceration, stab wound, ulcer, or a combination thereof. However, it is not limited to this.
  • the hydrogel composition may stably maintain its shape in a moisture environment.
  • the present invention provides a hydrogel composition and ECM sponge prepared by the above production method.
  • the present invention provides the use of the ECM hydrogel composition and extracellular matrix sponge for wound treatment or tissue regeneration.
  • the prepared hydrogel may be in a three-dimensional form.
  • the prepared three-dimensional hydrogel may have a thickness of 7 to 12 mm, preferably 10 mm.
  • the present invention provides a wound treatment or tissue regeneration method comprising administering to an individual a hydrogel composition prepared by the above production method or an extracellular matrix sponge prepared by freeze-drying the hydrogel composition.
  • the subject may be a mammal with a wound on the skin, and the administration may be applying or transplanting the hydrogel composition or extracellular matrix sponge to the affected area.
  • the present invention provides the use of the hydrogel composition and extracellular matrix sponge prepared by the above production method for the production of drugs for wound treatment or tissue regeneration.
  • the present invention provides a method for producing a three-dimensional hydrogel or hydrogel sheet (or sponge) under neutral conditions without a separate synthetic polymer support or hydrogel precursor solution, and the extracellular matrix hydrogel and ECM sponge produced by the above method. to provide.
  • the extracellular matrix hydrogel and ECM sponge of the present invention are implanted in the body with excellent biocompatibility and appropriate physical properties and maintain their shape without decomposition for a certain period of time, thereby facilitating cell movement and wound healing through secretion of bioactive substances. It can be induced.
  • the extracellular matrix hydrogel and sponge of the present invention induce regeneration of the epidermis, dermis, and skin appendages, and have excellent wound recovery speed, so they are used in regenerative medicine, wounds, burns, abrasions, lacerations, cuts, ulcers, etc. It can be used in the treatment of. Additionally, the extracellular matrix hydrogel and ECM sponge of the present invention can be used in treatments such as soft tissue reconstruction.
  • Figure 1 shows a method for producing decellularized fibroblast-derived matrix (FDM) according to an embodiment of the present invention.
  • FIG 2 is a schematic diagram of a method for manufacturing a fibroblast-derived extracellular matrix hydrogel (FDM-gel) according to an embodiment of the present invention.
  • Figure 2a shows the collection process
  • Figure 2b shows the compression process
  • Figure 2c shows the freezing and thawing process. It represents.
  • Figure 3a shows the results of evaluating the physical stability of the FDM-gel of the present invention compared to collagen-gel, a positive control, in PBS at 37°C.
  • Figure 3b shows the elastic moduli of collagen-gel and FDM-gel measured with a rheometer.
  • Figure 4 shows the results of angiogenesis-related cytokine analysis of the FDM-gel of the present invention.
  • Figure 5 shows the results of evaluating the biocompatibility of the FDM-gel of the present invention.
  • Figures 5a and 5c show the hydrogel 3 days after subcutaneous implantation of collagen-gel and FDM-gel, respectively.
  • Figures 5b and 5d show the results of H&E staining after subcutaneous implantation of collagen-gel and FDM-gel, respectively.
  • Figure 6 shows the results confirming the effect of inducing tissue regeneration when dressing (control group), collagen-gel, and FDM-gel were applied to the wound area.
  • Figure 6a shows the results of H&E staining on tissue sections obtained from each experimental group.
  • Figure 6b shows the thickness of the regenerated epidermis in each experimental group.
  • Figure 6c shows the number of new hair follicles in the regenerated dermal tissue in each experimental group.
  • Figure 7 shows the results of confirming the wound healing effect when dressing (control group), collagen-gel, and FDM-gel were treated on the burn site.
  • Figure 7a shows the degree of wound recovery in each experimental group on days 0, 12, and 18.
  • Figure 7b shows the size of the wound area in each experimental group on the 12th and 18th days.
  • Figure 8a shows the results of H&E staining on tissue sections obtained from each experimental group after treating the burn area with dressing (control group), collagen-gel, and FDM-gel.
  • Figure 8b shows the results of cyto-keratin 10 immunofluorescence staining for each experimental group.
  • Figure 8c shows the results of cyto-keratin 14 immunofluorescence staining for each experimental group.
  • Figure 9 shows the results of comparing the wound treatment effect between the hydrogel (PHF) containing extracellular matrix (FDM) derived from human lung fibroblasts reported in the prior art (KR 10-2020-0099706) and the FDM-gel of the present invention.
  • Figure 9a shows the degree of wound repair in the PHF-treated group and the FDM-gel-treated group on days 0, 7, and 14.
  • Figure 9b shows the size of the wound area in each experimental group on day 0, day 7, and day 14.
  • Figure 9c shows the results of H&E staining on tissue sections obtained from each experimental group.
  • Figure 9d shows the thickness of the regenerated epidermis in each experimental group.
  • Figure 9e shows the average size of new blood vessels in each experimental group.
  • Figure 9f shows the number of new hair follicles regenerated in each experimental group.
  • Figure 10 shows the results of biomarker fluorescence staining by obtaining a tissue section after treating the wound area with a dressing (control group) and FDM-gel, or by obtaining a section from normal skin tissue without a wound.
  • Figures 10a, 10c, and 10e show the epidermal areas of dressing (control group), FDM-gel treatment group, and normal skin tissue, respectively.
  • Figures 10b, 10d, and 10f show the dermal regions of dressing (control group), FDM-gel treated group, and normal skin tissue, respectively (here, (left) ⁇ -SMA, ⁇ -catenin, (right) K10, CD34, size bar is 20 ⁇ m).
  • Figure 11a shows the degree of adhesion of human hair follicle cells cultured in TCP and FDM.
  • Figure 11b shows the degree of proliferation of human hair follicle cells cultured in TCP and FDM on the 3rd and 7th days.
  • Figure 11c shows the expression levels of ⁇ -SMA and ⁇ -catenin in human hair follicle cells cultured in TCP and FDM.
  • Figure 11d shows ⁇ -catenin positive nuclei of human hair follicle cells cultured in TCP and FDM.
  • Figure 12 shows the results of Coomassie blue staining of decellularized extracellular matrix derived from human mesenchymal stem cells and the results of immunofluorescence staining of type I collagen (Col I) and fibronectin (FN).
  • Figure 13 is a photograph of a hydrogel produced by adding ultrapure distilled water to decellularized extracellular matrix derived from mesenchymal stem cells and performing compression, freezing, and thawing, and an ECM sponge obtained by freeze-drying the same.
  • Figure 14a is a photograph showing an example of applying collagen-based PELNAC TM sheet and ECM sponge to a burn wound
  • Figure 14b shows the size of the burn wound when only dressing is treated and when PELNAC TM sheet or ECM sponge is treated.
  • Figure 15 shows an H&E staining photograph of tissue obtained 18 days after dressing a burn wound, treating a PELNAC TM sheet or ECM sponge, and based on this, quantifying the thickness of the epidermis, the production of blood vessels, the number of hair follicles, and the level of mature collagen. This is one graph.
  • Figure 16 shows the results of immunofluorescence staining of tissue obtained 18 days after treating the burn wound with dressing, PELNACTM sheet, or ECM sponge.
  • an extracellular matrix hydrogel composition prepared under neutral conditions without a separate synthetic polymer support or hydrogel precursor solution has excellent physical stability and mechanical properties, and has a tissue regeneration inducing effect, thereby stably treating wounds. It was confirmed that this is possible.
  • the present invention includes the steps of (1) adding distilled water to the decellularized extracellular matrix; (2) compressing, freezing, and thawing the mixture prepared in step (1); and (3) preparing a hydrogel. It provides a method of producing a hydrogel composition without a separate polymer support or hydrogel precursor solution.
  • hydrogel composition composed of extracellular matrix is provided according to the above manufacturing method.
  • the present invention provides a hydrogel sheet obtained by freeze-drying the hydrogel prepared by the above method.
  • the hydrogel sheet is referred to as an extracellular matrix sponge (ECM sponge).
  • ECM sponge extracellular matrix sponge
  • hydrogel refers to water-soluble polymers forming three-dimensional crosslinks through physical (hydrogen bonds, van der Waals forces, hydrophobic interactions, or polymer crystals) or chemical (covalent bonds) bonds. It refers to a material that maintains its network structure. The hydrogel swells by absorbing a large amount of water or body fluid into the crosslinked lattice, and can maintain a three-dimensional structure without dispersing even in water. Therefore, even after swelling, the hydrogel remains thermodynamically stable and can have mechanical and physicochemical properties corresponding to intermediate forms between liquid and solid.
  • extracellular matrix refers to a complex assembly of biopolymers that fills intratissue or extracellular space.
  • the extracellular matrix is composed of various types of molecules synthesized by cells and secreted and accumulated outside the cells, such as fibrous proteins, complex proteins such as proteoglycans, and cell adhesion proteins such as fibronectin and laminin. Therefore, the composition of the extracellular matrix may vary depending on the type of cell from which it is derived or the degree of cell differentiation.
  • decellularization refers to the removal of other cellular components, such as nuclei, cell membranes, and nucleic acids, other than the extracellular matrix, from cells or tissues.
  • the term “decellularized extracellular matrix” refers to the extracellular matrix remaining after cellular components such as nuclei, cell membranes, and nucleic acids are removed from tissues or cells.
  • the decellularized extracellular matrix can utilize all extracellular matrix components by removing only the cell nucleus and cell membrane from the cell population, thereby providing a more natural biomimetic microenvironment for cells to grow and differentiate.
  • the decellularized extracellular matrix may also be referred to as “extracellular matrix (ECM)” or “extracellular matrix matrix.”
  • the decellularized extracellular matrix may be obtained from cells cultured in vitro.
  • the decellularized extracellular matrix may be more preferably obtained from a cell population cultured in vitro than an extracellular matrix derived from tissue formed within the subject.
  • the supply problem which is a disadvantage of tissue-derived extracellular matrix, can be solved, and since autologous cells can be used, there is a low possibility of immune rejection.
  • Cells for obtaining the extracellular matrix include, for example, fibroblasts, chondrocytes, osteoblasts, vascular endothelial cells, myocytes, smooth muscle cells, hepatocytes, nerve cells, cardiomyocytes, spinal disc cells, and stem cells. It may be one or more selected cells, but is not limited thereto, and is preferably fibroblasts or stem cells, and more preferably human lung fibroblasts or mesenchymal stem cells.
  • the decellularization method for obtaining the decellularized extracellular matrix may be performed by a known method or an appropriate modification thereof.
  • intracellular components can be removed by breaking the cell membrane of a fibroblast or mesenchymal stem cell population cultured in vitro using Triton X-100, a non-ionic detergent.
  • the decellularized extracellular matrix may contain growth factors related to neovascularization.
  • the decellularized extracellular matrix may include type I collagen (Collagen I) and/or fibronectin.
  • composition for wound treatment or tissue regeneration comprising a hydrogel composition composed of an extracellular matrix is provided according to the above manufacturing method.
  • wound or wound refers to a state in which a living body is damaged, and includes tissues forming the internal or external surface of the living body, such as skin, muscle, nervous tissue, bone, soft tissue, internal organs, or blood vessels. It can encompass pathological conditions in which tissues are divided or destroyed.
  • the wound or wound is a contusion or bruise, laceration, avulsion, penetrating wound, non-healing traumatic wound, destruction of tissue by irradiation, abrasion, Bone gangrene, gun shot wound, cut, burn wound, frostbite, skin ulcer, dry skin, keratosis, cracking, rupture, dermatitis, pain due to dermatophytosis, surgical wound, vascular disease wound, corneal wound. Covers damage to any part of the entity, such as back wounds, bedsores, flat sores, diabetes or conditions related to poor circulation, chronic ulcers, suture sites after plastic surgery, spinal injury wounds, gynecological wounds, chemical wounds, or acne. It can mean:
  • tissue regeneration refers to creating new blood vessels in damaged tissues and organs, regenerating or restoring cells constituting tissues and organs, or regenerating tissues responsible for the inherent functions of the tissues and organs. It may mean restoring function.
  • the tissue regeneration may include epidermal regeneration, dermal regeneration, new blood vessel regeneration, or skin appendage regeneration.
  • treatment refers to any action in which a wound or wound condition is improved or beneficially changed by the composition.
  • composition for wound treatment or tissue regeneration may further include one or more active ingredients having a wound healing effect such as wound healing or tissue regeneration.
  • composition for wound treatment or tissue regeneration may further include pharmaceutically acceptable additives.
  • pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, and phosphoric acid.
  • Calcium hydrogen, lactose, mannitol, taffy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba lead, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate. , calcium stearate, white sugar, etc. can be used.
  • Carriers, excipients, and diluents that may be included in the composition for wound treatment or tissue regeneration include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, and gelatin. , calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, mineral oil, etc.
  • composition for wound treatment or tissue regeneration can be prepared to be suitable for parenteral administration.
  • the composition for wound treatment or tissue regeneration may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, etc., and the suspensions include propylene glycol, polyethylene glycol, and olive. Vegetable oils such as oil, injectable esters such as ethyl oleate, etc. can be used.
  • the parenteral administration may be performed externally on the skin or by intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection.
  • composition for wound treatment or tissue regeneration may be prepared in the form of an external skin preparation.
  • composition for wound treatment or tissue regeneration is not particularly limited in formulation depending on the target body part, and may be manufactured in any commonly manufactured formulation with reference to known techniques in the art.
  • it may be used in the form of a liquid, ointment, cream, lotion, spray, patch, oil, wax, emulsion, suspension, gel, or aerosol, but is not limited thereto.
  • composition for wound treatment or tissue regeneration may include conventional additives, such as preservatives, solvents that assist drug penetration, emollients in the case of ointments and creams, and may contain a conventional carrier such as ethanol or oleyl alcohol. You can.
  • composition for wound treatment or tissue regeneration is not limited to the above-mentioned ingredients, and may include other ingredients blended in conventional cosmetic compositions or pharmaceutical compositions as needed.
  • fat ingredients moisturizers, emollients, surfactants, organic or inorganic pigments, organic powders, ultraviolet absorbers, preservatives, disinfectants, antioxidants, plant extracts, pH adjusters, alcohol, pigments, fragrances, blood circulation promoters, It may contain cooling agents, limiting agents, purified water, etc.
  • the hydrogel composition composed of the extracellular matrix may be provided in the form of a sponge.
  • an extracellular matrix sponge (ECM sponge) to distinguish it from that provided in a gel form.
  • the ECM sponge of the present invention can be manufactured by freeze-drying the above-described extracellular matrix-based hydrogel composition. It was experimentally confirmed that the extracellular matrix sponge of the present invention is much more effective in treating burns than the commercially available collagen-based Pelnac TM .
  • first, second, A, B, (a), and (b) may be used. These terms are only used to distinguish the component from other components, and the nature, sequence, or order of the component is not limited by the term.
  • a component is described as being “connected,” “coupled,” or “connected” to another component, that component may be directly connected or connected to that other component, but there is no need for another component between each component. It should be understood that may be “connected,” “combined,” or “connected.”
  • Human lung fibroblast WI-38 cell line (ATCC, CCL-75) was inoculated into a 100 mm cell culture vessel at an amount of 2x10 4 cells/cm 2 .
  • FBS fetal bovine serum
  • penicillin 100 U/ml bovine serum
  • 50 ⁇ g/ml ascorbic acid 50 ⁇ g/ml ascorbic acid
  • streptomycin 100 ⁇ g/ml streptomycin.
  • This added Dulbecco's modified Eagle's medium (DMEM) was added, and cultured under normal culture conditions (5% CO2, 37°C) for about 10 days while changing the medium at intervals of about 2 to 3 days.
  • DMEM Dulbecco's modified Eagle's medium
  • Sterilized ultrapure distilled water (5 ml/100 mm culture vessel) was added to the decellularized ECM, and FDM was scraped off using a cell scraper and transferred to a 50 ml tube (a in Figure 2).
  • the tube was stirred at high speed at 3500 rpm for 10 minutes to perform a compression process to collect FDM at the bottom of the tube, and then the supernatant was removed (b in FIG. 2).
  • 5 ml distilled water was added to the tube and stored at -80°C for more than one day.
  • the frozen tube was completely thawed at room temperature or 37°C, and then stirred at high speed for 5 minutes at a speed of 3500 rpm or higher.
  • FDM-gel fibroblast-derived extracellular matrix hydrogel
  • Figure 2c Sterilized PBS or ultrapure distilled water was added to the FDM-gel and stored at -20°C.
  • ECM sponge mesenchymal stem cell-derived extracellular matrix sponge
  • FDM-gel showed an elastic modulus (G') that was about half that of collagen-gel ( Figure 3b). This means that, despite the FDM-gel of the present invention having a lower elastic modulus compared to collagen-gel, it has much higher physical stability in a moisture environment due to mutual bonding or cross-linking between internal extracellular matrix proteins.
  • the FDM-gel prepared according to Example 1 described above contains amphiregulin, coagulation factor III, DPPIV (Dipeptidyl Peptidase IV), and endostatin. , collagen E1), serpin E1 (PEDF), TIMP-1 (Tissue inhibitor of metalloproteinase), thrombospondin-1, and uPA (urokinase-type plasminogen activator). It was confirmed that it contained cytokines.
  • Example 1-1 In order to evaluate the biocompatibility of the FDM-gel prepared in Example 1-1 compared to the collagen-gel, which is a positive control, the back of an experimental rat was incised and collagen-gel and FDM-gel were made, respectively. It was implanted subcutaneously.
  • the subcutaneously implanted FDM-gel does not decompose quickly, maintains physical stability, and has significantly better cell penetration into the hydrogel than collagen-gel.
  • new blood vessel formation around the FDM-gel is excellent, suggesting that the FDM-gel of the present invention has excellent biocompatibility in the body.
  • Example 1-1 To evaluate the tissue regenerative ability of the FDM-gel prepared in Example 1-1, an Excisional full-thickness wound with a diameter of 6 mm was induced in an experimental rat, and then a dressing (control group), collagen-gel, and FDM were applied. -gel was applied to each affected area, and on the 14th day, tissue from the wound area of each affected area was obtained. Paraffin fixation was performed on the secured tissue to obtain very thin tissue sections. H&E staining was performed on the tissue sections to compare and analyze histological characteristics.
  • the number of new hair follicles in the regenerated dermal tissue was quantified and shown in c in FIG. 6.
  • the FDM-gel treatment group had the highest number of new hair follicles per unit area ( 9 It was.
  • the FDM-gel of the present invention has an excellent tissue regeneration inducing effect, so it can be seen that it is a promising material that can be used for various indications.
  • Example 1-1 To evaluate the tissue regenerative ability of the FDM-gel prepared in Example 1-1, burn wounds were induced in experimental rats using an aluminum rod with a diameter of 6 mm, and then dressing (control group), collagen-gel, and FDM-gel were applied. was applied to each affected area, and on the 14th day, tissue from the wound area of each affected area was obtained. Afterwards, it was covered with a commercially available wound dressing to prevent hydrogel detachment and external contamination.
  • the size of the wound area in the FDM-gel treated group was reduced by about 60% compared to the control group and by about 40% compared to the collagen-gel treated group on day 12.
  • the size of the wound area decreased by approximately 220% compared to the control group and by approximately 120% compared to the collagen-gel treated group.
  • the wound area of the FDM-gel treated group had almost recovered to 5%.
  • tissue from the burn wound area of the rats treated in each experimental group was obtained, tissue sections were obtained from this, and H&E staining was performed.
  • cyto-keratin 10 (CK10) immunofluorescence staining was performed, and as a result, it was observed that more hair follicles were newly formed from the epidermis in the FDM-gel treated group compared to the other experimental groups ( Figure 8b). .
  • the wound treatment effect was compared between the hydrogel (PHF) containing extracellular matrix (FDM) derived from human lung fibroblasts reported in the prior art (KR 10-2020-0099706) and the FDM-gel of the present invention.
  • PPF hydrogel
  • FDM extracellular matrix
  • FDM was obtained in the same manner as in Example 1, and then mixed with a mixture of poloxamer and hyaluronic acid to prepare hydrogel (PHF) as a comparative example.
  • Hair follicles are one of the important skin appendages in wound healing.
  • dressing control group
  • FDM-gel were applied to the affected area, respectively, and on the 14th day, each affected area was treated. Tissue from the wound area was obtained. Normal skin tissue was prepared for comparison. From this, tissue sections were obtained, fluorescence staining for biomarkers was performed, and the results of comparing the epidermis and dermis in each experimental group are shown in Figure 10 (here, the dotted line is the interface between the epidermis and dermis).
  • human follicle cells human follicle dermal papilla cells, HFDPC
  • TCP tissue culture plastic
  • FDM cell growth, marker expression, etc.
  • the extracellular matrix hydrogel composition (FDM-gel) not only has excellent effects such as wound healing and hair follicle regeneration by containing a number of angiogenic factors, but also has excellent physical stability even when implanted in the body. It has high biocompatibility and allows stable tissue regeneration, so it can be used in fields such as regenerative medicine, burns, and wound treatment.
  • the level of mature collagen increased in the PELNAC-treated group and the ECM sponge-treated group compared to the control group, but the ECM sponge-treated group showed significantly higher levels of collagen production and collagen production compared to the PELNAC-treated group. It was confirmed that maturation can be induced.
  • the ECM sponge treatment group was able to confirm a relatively high level of normalization of epidermal tissue in the dermis compared to the PELNAC treatment group.

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Abstract

The present invention relates to: a method for producing a hydrogel prepared from an extracellular matrix without a separate polymer support or a hydrogel precursor solution; a hydrogel produced therefrom; a sponge produced by freeze-drying same; use of the hydrogel for wound healing and tissue regeneration; and the like. The extracellular matrix-based hydrogel and sponge of the present invention have high biocompatibility, physical stability and mechanical properties and include various bioactive substances including angiogenic factors, and thus are very effective in wound treatment such as that of tissue regeneration and inflammation control. Therefore, the extracellular matrix-based hydrogel and sponge of the present invention are expected to be used in the fields of regenerative medicine, burns and wound treatment, and the like.

Description

상처 치료 또는 조직 재생용 세포외 기질 기반의 하이드로겔 및 스펀지와 이의 제조방법 Extracellular matrix-based hydrogels and sponges for wound treatment or tissue regeneration and their manufacturing method
본 발명은 별도의 고분자 지지체 또는 하이드로겔 전구체 용액 없이 세포외 기질로부터 제조된 하이드로겔 제조방법과 이로부터 제조된 하이드로겔, 이를 동결건조하여 제조된 ECM 스펀지, 및 이의 상처 치료 및 조직 재생의 용도 등에 관한 것이다.The present invention relates to a method for producing a hydrogel produced from an extracellular matrix without a separate polymer support or hydrogel precursor solution, a hydrogel produced therefrom, an ECM sponge produced by freeze-drying the same, and its use in wound treatment and tissue regeneration. It's about.
창상 치료에 있어서 사용되는 재료는 상처 부위에 고정될 수 있는 기계적 물성, 상처와의 접촉면에서 적당한 습기의 유지 능력, 상처 분비물의 조절 능력, 및 인체에 대한 무독성이 요구되며, 2차 감염률(secondary infection rate)이 낮고 제조 및 저장이 용이하고, 체외에서 쉽게 조작 및 이용할 수 있어야 하며, 활성물질의 전달이 용이하여야 한다.Materials used in wound treatment require mechanical properties that can be fixed to the wound area, the ability to maintain appropriate moisture on the surface in contact with the wound, the ability to control wound secretions, and non-toxicity to the human body. Secondary infection rate rate) must be low, it must be easy to manufacture and store, it must be easy to manipulate and use in vitro, and the delivery of the active substance must be easy.
기존 창상 치료제는 1차적으로 상처 침출물 흡수, 외부 감염 방지와 습윤 제공을 주 목적으로 한 창상 피복재가 다수이며, 대부분 콜라겐, 알지네이트 등 생체 적합 고분자를 활용하고 있다. 그러나 기존 창상 치료제는 깊은 창상이나 궤양 환자를 위해 진피층을 포함하는 온전한 조직 재생 효과를 기대하기 어렵다. 따라서, 피부 부속기관, 신생혈관 형성을 포함하는 재생 효능 유도를 위한 신규 창상 치료제 개발이 필요한 실정이다.Existing wound treatments are mostly wound coating materials that are primarily intended to absorb wound exudates, prevent external infection, and provide moisture, and most of them utilize biocompatible polymers such as collagen and alginate. However, it is difficult to expect the effect of existing wound treatments on regenerating intact tissues, including the dermal layer, for patients with deep wounds or ulcers. Therefore, there is a need to develop new wound treatments to induce regenerative effects, including the formation of skin appendages and new blood vessels.
한편, 하이드로겔(hydrogel)은 물 또는 체액 내에서 가교된 격자 안으로 많은 양의 물 또는 체액을 흡수하여 팽윤되며, 물속에서도 흩어지지 않고 3차원 구조를 유지하는 재료를 의미한다. 팽윤된 이후에도, 열역학적으로 안정하게 존재하여 액체와 고체의 중간 형태에 해당하는 기계적 및 물리화학적 특성을 갖는다. 이러한 하이드로겔은 대개 우수한 생체 적합성, 높은 다공성 및 산소 투과도를 보이며, 생체 연조직과 비슷한 물리적 특성을 나타내고 인체에 안전하여, 습윤 창상 치유(moist wound healing)를 비롯하여 조직 재생분야, 약물전달분야 등 다양한 분야에 사용되고 있다.Meanwhile, hydrogel refers to a material that absorbs a large amount of water or body fluid into a crosslinked lattice and swells, and maintains a three-dimensional structure without dispersing even in water. Even after swelling, it remains thermodynamically stable and has mechanical and physicochemical properties corresponding to an intermediate form between a liquid and a solid. These hydrogels usually exhibit excellent biocompatibility, high porosity and oxygen permeability, exhibit physical properties similar to living soft tissues, and are safe for the human body, so they are used in various fields such as moist wound healing, tissue regeneration, and drug delivery. is being used.
다만, 기존의 일반적인 하이드로겔 제제는 제한적인 물리적 특성으로 인해 체내에 적용하기 위해서는 외과적 수술이 요구되고, 창상 또는 상처 부위와 같은 피부에 적용하는 경우에도, 하이드로겔 제제의 제조, 보관, 체외에서 조작 등에 있어서 용이하지 않다는 문제점이 있다.However, due to the limited physical properties of existing general hydrogel preparations, surgical operations are required for application to the body, and even when applied to skin such as wounds or wounds, the preparation, storage, and in vitro testing of hydrogel preparations are necessary. There is a problem that it is not easy to operate.
세포외기질(extracellular matrix, ECM)은 뛰어난 재생 치료 효능에도 불구하고 일정한 형태를 갖춘 제형으로 만드는데 기술적 어려움이 존재한다. 구체적으로 배양 용기에서 자란 세포들로부터 탈세포화 후 수득할 수 있는 세포외기질의 양이 제한적이고 기계적 물성이 취약하기 때문이다.Although extracellular matrix (ECM) has excellent regenerative treatment efficacy, there are technical difficulties in making it into a formulation with a certain shape. Specifically, this is because the amount of extracellular matrix that can be obtained after decellularization from cells grown in a culture vessel is limited and the mechanical properties are weak.
이런 배경 하에서, 본 발명자들은 별도의 합성 고분자 지지체 또는 하이드로겔 전구체 용액 없이 탈세포화된 세포외기질에 초순수 증류수를 첨가하여 제조된 하이드로겔과 이를 동결건조하여 제조된 ECM 스펀지의 우수한 조직 재생, 창상 치료, 모낭 재생 등의 효과를 확인함으로써 본 발명을 완성하였다.Under this background, the present inventors demonstrated that the hydrogel produced by adding ultrapure distilled water to decellularized extracellular matrix without a separate synthetic polymer support or hydrogel precursor solution and the ECM sponge produced by freeze-drying the hydrogel produced excellent tissue regeneration and wound healing. The present invention was completed by confirming effects such as hair follicle regeneration.
본 발명의 목적은 세포외기질 하이드로겔 조성물, 이를 이용하여 제조된 ECM 스펀지, 및 이의 제조방법을 제공하는 것이다.The purpose of the present invention is to provide an extracellular matrix hydrogel composition, an ECM sponge manufactured using the same, and a method for manufacturing the same.
본 발명의 다른 목적은 상기 하이드로겔 조성물을 포함하는 상처 치료 또는 조직 재생용 하이드로겔 조성물을 제공하는 것이다.Another object of the present invention is to provide a hydrogel composition for wound treatment or tissue regeneration comprising the hydrogel composition.
본 발명의 다른 목적은 상기 조성물을 이용하여 제조된 상처 치료 또는 조직 재생용 ECM 스펀지를 제공하는 것이다.Another object of the present invention is to provide an ECM sponge for wound treatment or tissue regeneration manufactured using the above composition.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당해 기술분야의 통상의 기술자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.
상기 과제를 해결하기 위하여, 본 발명은 하기 단계를 포함하는 별도의 고분자 지지체 또는 하이드로겔 전구체 용액 없이 하이드로겔 조성물을 제조하는 방법을 제공한다.In order to solve the above problem, the present invention provides a method for producing a hydrogel composition without a separate polymer support or hydrogel precursor solution including the following steps.
(1) 탈세포화된 세포외기질에 증류수를 첨가하는 단계;(1) adding distilled water to the decellularized extracellular matrix;
(2) 상기 (1)단계에서 제조된 혼합물을 압축, 동결, 및 해동하는 단계; 및(2) compressing, freezing, and thawing the mixture prepared in step (1); and
(3) 하이드로겔을 제조하는 단계.(3) Step of producing hydrogel.
또한, 본 발명은 상기 (1) 내지 (3)단계를 포함하는 별도의 고분자 지지체 또는 하이드로겔 전구체 용액 없이 상처 치료 또는 조직 재생용 하이드로겔 조성물을 제조하는 방법을 제공한다.In addition, the present invention provides a method for producing a hydrogel composition for wound treatment or tissue regeneration without a separate polymer support or hydrogel precursor solution including steps (1) to (3) above.
또한, 본 발명은 상기 제조방법으로 제조된 하이드로겔을 동결건조하는 단계를 포함하는 상처 치료 또는 조직 재생용 세포외기질(ECM) 스펀지를 제조하는 방법을 제공한다.In addition, the present invention provides a method of producing an extracellular matrix (ECM) sponge for wound treatment or tissue regeneration, comprising the step of freeze-drying the hydrogel produced by the above production method.
본 발명의 일 구현예로서, 상기 (1)단계에서 증류수는 멸균된 초순수 증류수(sterile ultrapure distilled water)일 수 있으며, 바람직하게는 100 mm 배양용기 기준으로 3~8 ml를 첨가하는 것일 수 있다.As an embodiment of the present invention, the distilled water in step (1) may be sterile ultrapure distilled water, and preferably 3 to 8 ml may be added based on a 100 mm culture vessel.
본 발명의 다른 구현예로서, 상기 (2)단계는 상기 (1)단계에서 제조된 혼합물을 압축한 후, 영하 60~90℃에서 하루 이상 동결하고, 20~40℃에서 해동한 다음, 추가적으로 고속 교반하는 것일 수 있다.In another embodiment of the present invention, in step (2), the mixture prepared in step (1) is compressed, frozen at -60 to 90°C for more than a day, thawed at 20 to 40°C, and then additionally processed at high speed. It may be stirring.
구체적으로 상기 (2) 단계는 하기 (a) 내지 (d) 단계를 포함할 수 있다:Specifically, step (2) may include the following steps (a) to (d):
(a) 상기 (1) 단계에서 제조된 혼합물을 압축하는 단계;(a) compressing the mixture prepared in step (1);
(b) 상기 압축된 혼합물을 영하 60~90℃에서 하루 이상 동결하는 단계;(b) freezing the compressed mixture at -60 to 90°C for more than one day;
(c) 상기 동결된 혼합물을 20~40℃에서 해동하는 단계; 및(c) thawing the frozen mixture at 20-40°C; and
(d) 상기 해동된 혼합물을 고속 교반하는 단계.(d) stirring the thawed mixture at high speed.
본 발명의 일 구현예로서, 상기 (a) 단계는 상기 (1) 단계의 혼합물을 3000~4000 rpm의 속도로 5~15 분, 바람직하게는 8~12분, 더욱 바람직하게는 10분 동안 고속 교반하여 압축하는 것일 수 있다.In one embodiment of the present invention, step (a) is performed by rotating the mixture of step (1) at a speed of 3000 to 4000 rpm for 5 to 15 minutes, preferably 8 to 12 minutes, more preferably 10 minutes. It may be compressed by stirring.
본 발명의 다른 구현예로서, 상기 (d) 단계는 3000~4000 rpm의 속도로 3~8분, 바람직하게는 4~6분, 더욱 바람직하게는 5분 동안 고속 교반하는 것일 수 있다.As another embodiment of the present invention, step (d) may be high-speed stirring at a speed of 3000 to 4000 rpm for 3 to 8 minutes, preferably 4 to 6 minutes, and more preferably 5 minutes.
본 발명의 다른 구현예로서, 상기 탈세포화된 세포외기질은 생체 외(in vitro)에서 배양된 세포로부터 수득된 것일 수 있다.As another embodiment of the present invention, the decellularized extracellular matrix may be obtained from cells cultured in vitro.
본 발명의 또 다른 구현예로서, 상기 세포는 섬유아세포, 연골세포, 조골세포, 혈관내피세포, 근세포, 평활근세포, 간세포, 신경세포, 심근세포, 척추 추간판세포, 중간엽 줄기세포, 및 이들의 조합으로 이루어진 군으로부터 선택되는 어느 하나 이상인 것일 수 있으며, 상기 세포는 인간으로부터 분리된 것일 수 있으나 이에 제한되는 것은 아니다.In another embodiment of the present invention, the cells include fibroblasts, chondrocytes, osteoblasts, vascular endothelial cells, myocytes, smooth muscle cells, hepatocytes, nerve cells, cardiomyocytes, spinal disc cells, mesenchymal stem cells, and their It may be one or more cells selected from the group consisting of combinations, and the cells may be isolated from humans, but are not limited thereto.
본 발명의 다른 구현예로서, 상기 하이드로겔 조성물은 신생혈관 형성 관련 성장 인자를 포함하는 것일 수 있다.As another embodiment of the present invention, the hydrogel composition may contain growth factors related to neovascularization.
본 발명의 또 다른 구현예로서, 상기 신생혈관 형성 관련 성장 인자는 엠피레귤린(amphiregulin), 혈액 응고 인자 Ⅲ(Coagulation Factor Ⅲ), DPPⅣ(Dipeptidyl Peptidase Ⅳ), 엔도스타틴(Endostatin, collagen XVⅢ), FGF acidic(Fibroblast growth factor-acidic), FGF-7(Fibroblast Growth Factor 7), HGF(Hepatocyte growth factor), 펜트락신 3(Pentraxin 3), 플라스미노겐 활성인자 억제제-1(serpin E1), serpin E1(PEDF), TIMP-1(Tissue inhibitor of metalloproteinase), 트롬보스폰딘-1(thrombospondin-1), uPA(urokinase-type plasminogen activator), 및 이들의 조합으로 이루어진 군으로부터 선택되는 어느 하나 이상인 것일 수 있으나 이에 제한되는 것은 아니다.In another embodiment of the present invention, the growth factors related to angiogenesis include amphiregulin, coagulation factor Ⅲ, dipeptidyl peptidase Ⅳ (DPPⅣ), endostatin (endostatin, collagen XVⅢ), and FGF. acidic (Fibroblast growth factor-acidic), FGF-7 (Fibroblast Growth Factor 7), HGF (Hepatocyte growth factor), Pentraxin 3, plasminogen activator inhibitor-1 (serpin E1), serpin E1 ( It may be one or more selected from the group consisting of PEDF), TIMP-1 (Tissue inhibitor of metalloproteinase), thrombospondin-1, uPA (urokinase-type plasminogen activator), and combinations thereof. It is not limited.
본 발명의 또 다른 구현예로서, 상기 하이드로겔 조성물은 Ⅰ형 콜라겐(Collagen Ⅰ) 및/또는 피브로넥틴(Fibronectin)을 포함하는 것일 수 있다.As another embodiment of the present invention, the hydrogel composition may include type I collagen (Collagen I) and/or fibronectin.
본 발명의 다른 구현예로서, 상기 하이드로겔 조성물은 창상 회복 효과, 상처 치료 효과, 조직 재생 효과, 표피 재생 효과, 진피 재생 효과, 신생혈관 재생 효과, 또는 피부 부속기관 재생 효과를 나타내는 것일 수 있다.As another embodiment of the present invention, the hydrogel composition may exhibit a wound recovery effect, a wound healing effect, a tissue regeneration effect, an epidermal regeneration effect, a dermal regeneration effect, a neovascularization regeneration effect, or a skin appendage regeneration effect.
본 발명의 또 다른 구현예로서, 상기 상처는 창상(wound), 화상(burn wound), 찰과상(abrasion), 열상(laceration), 자상(stab wound), 궤양(ulcer), 또는 이들의 조합일 수 있으나, 이에 제한되는 것은 아니다.In another embodiment of the present invention, the wound may be a wound, burn wound, abrasion, laceration, stab wound, ulcer, or a combination thereof. However, it is not limited to this.
본 발명의 다른 구현예로서, 상기 하이드로겔 조성물은 수분 환경에서 안정적으로 형태를 유지하는 것일 수 있다.As another embodiment of the present invention, the hydrogel composition may stably maintain its shape in a moisture environment.
또한, 본 발명은 상기 제조방법으로 제조된 하이드로겔 조성물 및 ECM 스펀지를 제공한다.Additionally, the present invention provides a hydrogel composition and ECM sponge prepared by the above production method.
또한, 본 발명은 ECM하이드로겔 조성물 및 세포외기질 스펀지의 상처 치료 또는 조직 재생의 용도를 제공한다.Additionally, the present invention provides the use of the ECM hydrogel composition and extracellular matrix sponge for wound treatment or tissue regeneration.
본 발명의 일 구현예로서, 상기 제조된 하이드로겔은 3차원 형태일 수 있다.As an embodiment of the present invention, the prepared hydrogel may be in a three-dimensional form.
본 발명의 다른 구현예로서, 상기 제조된 3차원 하이드로겔은 7 ~ 12 mm, 바람직하게는 10 mm의 두께를 가지는 것일 수 있다.As another embodiment of the present invention, the prepared three-dimensional hydrogel may have a thickness of 7 to 12 mm, preferably 10 mm.
또한, 본 발명은 상기 제조방법으로 제조된 하이드로겔 조성물 또는 상기 하이드로겔 조성물을 동결건조하여 제조된 세포외기질 스펀지를 개체에 투여하는 단계를 포함하는 상처 치료 또는 조직 재생방법을 제공한다.In addition, the present invention provides a wound treatment or tissue regeneration method comprising administering to an individual a hydrogel composition prepared by the above production method or an extracellular matrix sponge prepared by freeze-drying the hydrogel composition.
본 발명의 일 구현예로서, 상기 개체는 피부에 상처를 가진 포유류일 수 있으며, 상기 투여는 상기 하이드로겔 조성물 또는 세포외기질 스펀지를 환부에 도포 또는 이식하는 것일 수 있다.In one embodiment of the present invention, the subject may be a mammal with a wound on the skin, and the administration may be applying or transplanting the hydrogel composition or extracellular matrix sponge to the affected area.
또한, 본 발명은 상처 치료 또는 조직 재생용 약제의 제조를 위한 상기 제조방법으로 제조된 하이드로겔 조성물과 세포외기질 스펀지의 용도를 제공한다.In addition, the present invention provides the use of the hydrogel composition and extracellular matrix sponge prepared by the above production method for the production of drugs for wound treatment or tissue regeneration.
본 발명은 별도의 합성 고분자 지지체 또는 하이드로겔 전구체 용액 없이 중성 조건에서 3차원 형태의 하이드로겔 또는 하이드로겔 시트(또는 스펀지)를 제조하는 방법과 상기 방법으로 제조된 세포외기질 하이드로겔 및 ECM 스펀지를 제공한다. 본 발명의 제공으로 비교적 간단한 방법으로 다양한 생리활성 인자를 포함하는 세포외기질 기반의 생체재료를 제조할 수 있다. 또한, 본 발명의 세포외기질 하이드로겔과 ECM 스펀지는 우수한 생체 적합성과 적절한 물성으로 체내 이식되어 일정한 시간 동안 분해되지 않고 그 형태를 유지하여 생리활성물질의 분비를 통한 세포의 이동과 상처의 치료를 유도할 수 있다. 또한, 본 발명의 세포외기질 하이드로겔 및 스펀지는 표피, 진피, 및 피부 부속기관 등의 재생을 유도하여 상처 회복 속도가 매우 우수하므로, 재생 의료, 창상, 화상, 찰과상, 열상, 자상, 궤양 등의 치료에 활용될 수 있다. 또한 본 발명의 세포외기질 하이드로겔 및 ECM 스펀지는 연조직 재건 등의 치료에 활용될 수 있다.The present invention provides a method for producing a three-dimensional hydrogel or hydrogel sheet (or sponge) under neutral conditions without a separate synthetic polymer support or hydrogel precursor solution, and the extracellular matrix hydrogel and ECM sponge produced by the above method. to provide. With the provision of the present invention, it is possible to manufacture extracellular matrix-based biomaterials containing various bioactive factors in a relatively simple method. In addition, the extracellular matrix hydrogel and ECM sponge of the present invention are implanted in the body with excellent biocompatibility and appropriate physical properties and maintain their shape without decomposition for a certain period of time, thereby facilitating cell movement and wound healing through secretion of bioactive substances. It can be induced. In addition, the extracellular matrix hydrogel and sponge of the present invention induce regeneration of the epidermis, dermis, and skin appendages, and have excellent wound recovery speed, so they are used in regenerative medicine, wounds, burns, abrasions, lacerations, cuts, ulcers, etc. It can be used in the treatment of. Additionally, the extracellular matrix hydrogel and ECM sponge of the present invention can be used in treatments such as soft tissue reconstruction.
도 1은 본 발명의 일 실시예에 따른 탈세포화된 섬유아세포 유래 세포외기질(fibroblast-derived matrix, FDM)의 제조방법을 나타낸 것이다.Figure 1 shows a method for producing decellularized fibroblast-derived matrix (FDM) according to an embodiment of the present invention.
도 2는 본 발명의 일 실시예에 따른 섬유아세포 유래 세포외기질 하이드로겔(FDM-gel)의 제조방법의 모식도로서, 도 2a는 수집 공정, 도 2b는 압축 공정, 도 2c는 동결 및 해동 공정을 나타낸 것이다.Figure 2 is a schematic diagram of a method for manufacturing a fibroblast-derived extracellular matrix hydrogel (FDM-gel) according to an embodiment of the present invention. Figure 2a shows the collection process, Figure 2b shows the compression process, and Figure 2c shows the freezing and thawing process. It represents.
도 3a는 37℃, PBS에서 양성대조군인 collagen-gel과 비교하여 본 발명의 FDM-gel의 물리적 안정성을 평가한 결과이다. 도 3b는 rheometer로 측정된 collagen-gel과 FDM-gel의 탄성 계수를 나타낸 것이다.Figure 3a shows the results of evaluating the physical stability of the FDM-gel of the present invention compared to collagen-gel, a positive control, in PBS at 37°C. Figure 3b shows the elastic moduli of collagen-gel and FDM-gel measured with a rheometer.
도 4는 본 발명의 FDM-gel의 신생혈관형성 관련 사이토카인 분석을 수행한 결과를 나타낸 것이다.Figure 4 shows the results of angiogenesis-related cytokine analysis of the FDM-gel of the present invention.
도 5는 본 발명의 FDM-gel의 생체적합성을 평가한 결과이다. 도 5a 및 도 5c는 각각 collagen-gel과 FDM-gel을 피하 이식 후 3일째 하이드로겔의 모습이다. 도 5b 및 도 5d는 각각 collagen-gel과 FDM-gel을 피하 이식 후 H&E 염색 결과를 나타낸 것이다.Figure 5 shows the results of evaluating the biocompatibility of the FDM-gel of the present invention. Figures 5a and 5c show the hydrogel 3 days after subcutaneous implantation of collagen-gel and FDM-gel, respectively. Figures 5b and 5d show the results of H&E staining after subcutaneous implantation of collagen-gel and FDM-gel, respectively.
도 6은 창상 부위에 드레싱(대조군), collagen-gel, FDM-gel을 처리한 경우 조직재생 유도 효과를 확인한 결과이다. 도 6a는 각 실험군으로부터 조직 절편을 수득하여 H&E 염색을 수행한 결과를 나타낸 것이다. 도 6b는 각 실험군에서 재생된 표피의 두께를 나타낸 것이다. 도 6c는 각 실험군에서 재생된 진피 조직 내 신생 모낭 수를 나타낸 것이다.Figure 6 shows the results confirming the effect of inducing tissue regeneration when dressing (control group), collagen-gel, and FDM-gel were applied to the wound area. Figure 6a shows the results of H&E staining on tissue sections obtained from each experimental group. Figure 6b shows the thickness of the regenerated epidermis in each experimental group. Figure 6c shows the number of new hair follicles in the regenerated dermal tissue in each experimental group.
도 7은 화상 부위에 드레싱(대조군), collagen-gel, FDM-gel을 처리한 경우 상처 치료 효과를 확인한 결과이다. 도 7a는 0일째, 12일째, 18일째 각 실험군의 상처 수복 정도를 나타낸 것이다. 도 7b는 12일째, 18일째 각 실험군에서 상처 부위의 크기를 나타낸 것이다.Figure 7 shows the results of confirming the wound healing effect when dressing (control group), collagen-gel, and FDM-gel were treated on the burn site. Figure 7a shows the degree of wound recovery in each experimental group on days 0, 12, and 18. Figure 7b shows the size of the wound area in each experimental group on the 12th and 18th days.
도 8a는 화상 부위에 드레싱(대조군), collagen-gel, FDM-gel을 처리한 후, 각 실험군으로부터 조직 절편을 수득하여 H&E 염색을 수행한 결과를 나타낸 것이다. 도 8b는 각 실험군에 대해 cyto-keratin 10 면역형광염색을 수행한 결과를 나타낸 것이다. 도 8c는 각 실험군에 대해 cyto-keratin 14 면역형광염색을 수행한 결과를 나타낸 것이다.Figure 8a shows the results of H&E staining on tissue sections obtained from each experimental group after treating the burn area with dressing (control group), collagen-gel, and FDM-gel. Figure 8b shows the results of cyto-keratin 10 immunofluorescence staining for each experimental group. Figure 8c shows the results of cyto-keratin 14 immunofluorescence staining for each experimental group.
도 9는 선행기술(KR 10-2020-0099706)에서 보고된 인간 폐 섬유아세포 유래 세포외기질(FDM)을 포함하는 하이드로겔(PHF)과 본 발명의 FDM-gel간의 창상 치료 효과를 비교한 결과이다. 도 9a는 0일째, 7일째, 14일째 PHF 처리군과 FDM-gel 처리군의 상처 수복 정도를 나타낸 것이다. 도 9b는 0일째, 7일째, 14일째 각 실험군에서 상처 부위의 크기를 나타낸 것이다. 도 9c는 각 실험군으로부터 조직 절편을 수득하여 H&E 염색을 수행한 결과를 나타낸 것이다. 도 9d는 각 실험군에서 재생된 표피의 두께를 나타낸 것이다. 도 9e는 각 실험군에서 신생혈관의 평균 크기를 나타낸 것이다. 도 9f는 각 실험군에서 재생된 신생 모낭 수를 나타낸 것이다.Figure 9 shows the results of comparing the wound treatment effect between the hydrogel (PHF) containing extracellular matrix (FDM) derived from human lung fibroblasts reported in the prior art (KR 10-2020-0099706) and the FDM-gel of the present invention. am. Figure 9a shows the degree of wound repair in the PHF-treated group and the FDM-gel-treated group on days 0, 7, and 14. Figure 9b shows the size of the wound area in each experimental group on day 0, day 7, and day 14. Figure 9c shows the results of H&E staining on tissue sections obtained from each experimental group. Figure 9d shows the thickness of the regenerated epidermis in each experimental group. Figure 9e shows the average size of new blood vessels in each experimental group. Figure 9f shows the number of new hair follicles regenerated in each experimental group.
도 10은 창상 부위에 드레싱(대조군), FDM-gel을 처리한 후, 조직 절편을 수득하거나 창상이 없는 정상 피부 조직으로부터 절편을 수득하여 바이오마커 형광 염색을 수행한 결과를 나타낸 것이다. 도 10a, 10c, 및 10e는 각각 드레싱(대조군), FDM-gel 처리군, 및 정상 피부 조직의 표피 영역을 나타낸 것이다. 도 10b, 10d, 및 10f는 각각 드레싱(대조군), FDM-gel 처리군, 및 정상 피부 조직의 진피 영역을 나타낸 것이다(이 때, (좌) α-SMA, β-catenin, (우) K10, CD34이고, 크기 막대는 20 μm이다).Figure 10 shows the results of biomarker fluorescence staining by obtaining a tissue section after treating the wound area with a dressing (control group) and FDM-gel, or by obtaining a section from normal skin tissue without a wound. Figures 10a, 10c, and 10e show the epidermal areas of dressing (control group), FDM-gel treatment group, and normal skin tissue, respectively. Figures 10b, 10d, and 10f show the dermal regions of dressing (control group), FDM-gel treated group, and normal skin tissue, respectively (here, (left) α-SMA, β-catenin, (right) K10, CD34, size bar is 20 μm).
도 11에 a는 TCP와 FDM에서 배양한 인간 모낭세포의 부착 정도를 나타낸 것이다. 도 11에 b는 3일째, 7일째 TCP와 FDM에서 배양한 인간 모낭세포의 증식 정도를 나타낸 것이다. 도 11에 c는 TCP와 FDM에서 배양한 인간 모낭세포의 α-SMA, β-catenin 발현 정도를 나타낸 것이다. 도 11에 d는 TCP와 FDM에서 배양한 인간 모낭세포의 β-catenin positive nuclei을 나타낸 것이다.Figure 11a shows the degree of adhesion of human hair follicle cells cultured in TCP and FDM. Figure 11b shows the degree of proliferation of human hair follicle cells cultured in TCP and FDM on the 3rd and 7th days. Figure 11c shows the expression levels of α-SMA and β-catenin in human hair follicle cells cultured in TCP and FDM. Figure 11d shows β-catenin positive nuclei of human hair follicle cells cultured in TCP and FDM.
도 12는 인간 중간엽 줄기세포 유래의 탈세포화된 세포외기질의 쿠마시 블루(Coomassie blue) 염색 결과와 Ⅰ형 콜라겐(Col Ⅰ) 및 피브로넥틴(FN)의 면역형광염색 결과이다.Figure 12 shows the results of Coomassie blue staining of decellularized extracellular matrix derived from human mesenchymal stem cells and the results of immunofluorescence staining of type I collagen (Col I) and fibronectin (FN).
도 13은 중간엽 줄기세포 유래의 탈세포화된 세포외기질에 초순수 증류수를 첨가하고, 압축, 동결, 및 해동을 수행하여 제작된 하이드로겔과 이를 동결건조하여 획득된 ECM 스펀지의 사진이다.Figure 13 is a photograph of a hydrogel produced by adding ultrapure distilled water to decellularized extracellular matrix derived from mesenchymal stem cells and performing compression, freezing, and thawing, and an ECM sponge obtained by freeze-drying the same.
도 14a는 화상 창상에 콜라겐 기반의 PELNACTM 시트와 ECM sponge를 적용의 예시를 나타낸 사진이이고, 도 14b는 드래싱만 처리하는 경우와 PELNACTM 시트 또는 ECM sponge를 처리한 경우에 화상 창상의 크기를 창상 유도 0, 7, 14, 및 18일에 창상의 사진과 창상의 크기를 정량한 그래프이다.Figure 14a is a photograph showing an example of applying collagen-based PELNAC TM sheet and ECM sponge to a burn wound, and Figure 14b shows the size of the burn wound when only dressing is treated and when PELNAC TM sheet or ECM sponge is treated. These are photographs of wounds and graphs quantifying the size of wounds on days 0, 7, 14, and 18 of wound induction.
도 15는 화상 창상에 드래싱, PELNACTM 시트 또는 ECM sponge를 처리하고 18일째에 획득된 조직의 H&E 염색 사진과 이를 기반으로 표피의 두께, 혈관의 생성, 모낭의 수, 및 성숙한 콜라겐의 수준을 정량한 그래프이다.Figure 15 shows an H&E staining photograph of tissue obtained 18 days after dressing a burn wound, treating a PELNAC TM sheet or ECM sponge, and based on this, quantifying the thickness of the epidermis, the production of blood vessels, the number of hair follicles, and the level of mature collagen. This is one graph.
도 16은 화상 창상에 드래싱, PELNACTM 시트 또는 ECM sponge를 처리하고 18일째에 획득된 조직의 면역형광염색 결과이다.Figure 16 shows the results of immunofluorescence staining of tissue obtained 18 days after treating the burn wound with dressing, PELNACTM sheet, or ECM sponge.
본 발명자들은 별도의 합성 고분자 지지체 또는 하이드로겔 전구체 용액 없이 중성 조건에서 제조된 세포외기질 하이드로겔 조성물(FDM-gel)이 물리적 안정성, 기계적 물성이 우수하고, 조직재생 유도 효과를 가져 안정적으로 상처 치료 등이 가능하다는 점을 확인하였다.The present inventors have found that an extracellular matrix hydrogel composition (FDM-gel) prepared under neutral conditions without a separate synthetic polymer support or hydrogel precursor solution has excellent physical stability and mechanical properties, and has a tissue regeneration inducing effect, thereby stably treating wounds. It was confirmed that this is possible.
따라서, 본 발명은 (1) 탈세포화된 세포외기질에 증류수를 첨가하는 단계; (2) 상기 (1)단계에서 제조된 혼합물을 압축, 동결, 및 해동하는 단계; 및 (3) 하이드로겔을 제조하는 단계를 포함하는 별도의 고분자 지지체 또는 하이드로겔 전구체 용액 없이 하이드로겔 조성물을 제조하는 방법을 제공한다.Therefore, the present invention includes the steps of (1) adding distilled water to the decellularized extracellular matrix; (2) compressing, freezing, and thawing the mixture prepared in step (1); and (3) preparing a hydrogel. It provides a method of producing a hydrogel composition without a separate polymer support or hydrogel precursor solution.
또한, 상기 제조방법에 따라 세포외기질로 구성된 하이드로겔 조성물을 제공한다.In addition, a hydrogel composition composed of extracellular matrix is provided according to the above manufacturing method.
또한, 본 발명은 상기 방법으로 제조된 하이드로겔을 동결건조하여 획득된 하이드로겔 시트를 제공한다.Additionally, the present invention provides a hydrogel sheet obtained by freeze-drying the hydrogel prepared by the above method.
본 명세서에서 상기 하이드로겔 시트는 세포외기질 스펀지(ECM sponge)라고 명명한다.In this specification, the hydrogel sheet is referred to as an extracellular matrix sponge (ECM sponge).
본 명세서의 용어, "하이드로겔(hydrogel)"은 수용성 고분자가 물리적(수소결합, 반데르발스 힘, 소수성 상호작용, 혹은 고분자의 결정) 혹은 화학적(공유결합)인 결합으로 3차원의 가교를 형성하고 있는 망상 구조를 유지하는 재료를 의미한다. 상기 하이드로겔은 물 또는 체액 내에서 가교된 격자 안으로 많은 양의 물 또는 체액을 흡수하여 팽윤되며, 물속에서도 흩어지지 않고 삼차원 구조를 유지할 수 있다. 따라서, 상기 하이드로겔은 팽윤된 이후에도, 열역학적으로 안정하게 존재하여 액체와 고체의 중간 형태에 해당하는 기계적 및 물리화학적 특성을 가질 수 있다.As used herein, the term “hydrogel” refers to water-soluble polymers forming three-dimensional crosslinks through physical (hydrogen bonds, van der Waals forces, hydrophobic interactions, or polymer crystals) or chemical (covalent bonds) bonds. It refers to a material that maintains its network structure. The hydrogel swells by absorbing a large amount of water or body fluid into the crosslinked lattice, and can maintain a three-dimensional structure without dispersing even in water. Therefore, even after swelling, the hydrogel remains thermodynamically stable and can have mechanical and physicochemical properties corresponding to intermediate forms between liquid and solid.
본 명세서의 용어, "세포외기질(extracellular matrix, ECM)"은 조직내 또는 세포외의 공간을 채우고 있는 생체고분자의 복잡한 집합체를 의미한다. 세포외기질은 섬유성 단백질, 프로테오글리칸과 같은 복합 단백질, 피브로넥틴, 라미닌 등의 세포 부착성 단백질 등 세포에 의해 합성되고 세포외에 분비 축적된 다양한 종류의 분자로 구성된다. 따라서 세포외기질은 유래 세포의 종류 또는 세포의 분화 정도에 따라 그 성분이 달라질 수 있다.As used herein, the term “extracellular matrix (ECM)” refers to a complex assembly of biopolymers that fills intratissue or extracellular space. The extracellular matrix is composed of various types of molecules synthesized by cells and secreted and accumulated outside the cells, such as fibrous proteins, complex proteins such as proteoglycans, and cell adhesion proteins such as fibronectin and laminin. Therefore, the composition of the extracellular matrix may vary depending on the type of cell from which it is derived or the degree of cell differentiation.
본 명세서의 용어, "탈세포화(decellularization)"는 세포 또는 조직으로부터 세포외기질을 제외한 다른 세포 성분, 예를 들면 핵, 세포막, 핵산 등을 제거하는 것을 의미한다.As used herein, the term “decellularization” refers to the removal of other cellular components, such as nuclei, cell membranes, and nucleic acids, other than the extracellular matrix, from cells or tissues.
본 명세서의 용어, "탈세포화된 세포외기질"은 조직 또는 세포로부터 핵, 세포막, 핵산과 같은 세포 성분이 제거되고 남은 세포외기질을 의미한다. 상기 탈세포화된 세포외기질은 세포 집단으로부터 세포의 핵 및 세포막 등만을 제거하여, 세포외기질 성분 전체를 활용할 수 있어, 세포가 성장 및 분화하기에 더욱 자연스러운 생체모방 미세환경을 제공할 수 있다. 본 명세서에서 상기 탈세포화된 세포외기질은 "세포외기질(extracellular matrix, ECM)" 또는 "세포외기질 메트릭스"라고도 명명될 수 있다.As used herein, the term “decellularized extracellular matrix” refers to the extracellular matrix remaining after cellular components such as nuclei, cell membranes, and nucleic acids are removed from tissues or cells. The decellularized extracellular matrix can utilize all extracellular matrix components by removing only the cell nucleus and cell membrane from the cell population, thereby providing a more natural biomimetic microenvironment for cells to grow and differentiate. As used herein, the decellularized extracellular matrix may also be referred to as “extracellular matrix (ECM)” or “extracellular matrix matrix.”
상기 탈세포화된 세포외기질은 생체 외(in vitro)에서 배양된 세포로부터 수득된 것일 수 있다. 상기 탈세포화된 세포외기질은 개체내에서 형성된 조직으로부터 유래된 세포외기질보다 생체 외에서 배양된 세포 집단으로부터 수득한 세포외기질이 더 바람직할 수 있다. 상기 탈세포화된 세포외기질은 생체 외에서 배양된 세포로부터 얻을 경우, 조직 유래 세포외기질의 단점인 공급 문제를 해결할 수 있으며, 자가 세포를 사용할 수 있어 면역거부반응이 일어날 가능성이 낮다.The decellularized extracellular matrix may be obtained from cells cultured in vitro. The decellularized extracellular matrix may be more preferably obtained from a cell population cultured in vitro than an extracellular matrix derived from tissue formed within the subject. When the decellularized extracellular matrix is obtained from cells cultured in vitro, the supply problem, which is a disadvantage of tissue-derived extracellular matrix, can be solved, and since autologous cells can be used, there is a low possibility of immune rejection.
상기 세포외기질을 수득하기 위한 세포는 예를 들면 섬유아세포, 연골세포, 조골세포, 혈관내피세포, 근세포, 평활근세포, 간세포, 신경세포, 심근세포, 척추 추간판세포, 및 줄기세포로 이루어진 군으로부터 선택되는 하나 이상일 수 있으나, 이에 제한되지 않으며, 바람직하게는 섬유아세포 또는 줄기세포일 수 있으며, 더욱 바람직하게는 인간 폐 섬유아세포 또는 중간엽 줄기세포일 수 있다.Cells for obtaining the extracellular matrix include, for example, fibroblasts, chondrocytes, osteoblasts, vascular endothelial cells, myocytes, smooth muscle cells, hepatocytes, nerve cells, cardiomyocytes, spinal disc cells, and stem cells. It may be one or more selected cells, but is not limited thereto, and is preferably fibroblasts or stem cells, and more preferably human lung fibroblasts or mesenchymal stem cells.
상기 탈세포화된 세포외기질을 수득하기 위한 탈세포화 방법은 공지된 방법 또는 이의 적절한 변형에 의해 이루어질 수 있다. 일 구체예에서 생체 외에서 배양된 섬유아세포 또는 중간엽 줄기세포 집단을 비이온성 세척제(Non-ionic detergent)인 Triton X-100을 이용해서 세포막을 깨뜨려서 세포 내 성분을 제거할 수 있다.The decellularization method for obtaining the decellularized extracellular matrix may be performed by a known method or an appropriate modification thereof. In one embodiment, intracellular components can be removed by breaking the cell membrane of a fibroblast or mesenchymal stem cell population cultured in vitro using Triton X-100, a non-ionic detergent.
상기 탈세포화된 세포외기질은 신생혈관 형성 관련 성장 인자를 포함하는 것일 수 있다.The decellularized extracellular matrix may contain growth factors related to neovascularization.
상기 탈세포화된 세포외기질은 Ⅰ형 콜라겐(Collagen Ⅰ) 및/또는 피브로넥틴(Fibronectin)을 포함하는 것일 수 있다.The decellularized extracellular matrix may include type I collagen (Collagen I) and/or fibronectin.
또한, 상기 제조방법에 따라 세포외기질로 구성된 하이드로겔 조성물을 포함하는 상처 치료 또는 조직 재생용 조성물을 제공한다.In addition, a composition for wound treatment or tissue regeneration comprising a hydrogel composition composed of an extracellular matrix is provided according to the above manufacturing method.
본 명세서의 용어, “상처 또는 창상(wound)”은 생체가 손상된 상태를 말하는 것으로서, 생체 내부 또는 외부 표면을 이루는 조직, 예를 들면 피부, 근육, 신경조직, 뼈, 연조직, 내부기관, 또는 혈관조직이 분단 또는 파괴된 병리학적 상태를 포괄할 수 있다. 구체적으로 상기 상처 또는 창상은 좌상(contusion or bruise), 열상(laceration), 결출상(avulsion), 관통상(penetrated wound), 비-치유 외상성 창상, 방사선조사에 의한 조직의 파괴, 찰과상(abrasion), 골괴저, 총상(gun shot wound), 절상, 화상(burn wound), 동상, 피부궤양, 피부 건조, 피부각화증, 갈라짐, 터짐, 피부염, 피부사상균증에 의한 통증, 수술상, 혈관질환 창상, 각막창상 등의 창상, 욕창, 와창, 당뇨병, 또는 순환불량에 관련된 상태, 만성궤양, 성형수술 후 봉합부위, 척추상해성 창상, 부인과적 창상, 화학적 창상, 또는 여드름 등의 개체의 일부에 대한 손상을 포괄적으로 의미할 수 있다.As used herein, the term “wound or wound” refers to a state in which a living body is damaged, and includes tissues forming the internal or external surface of the living body, such as skin, muscle, nervous tissue, bone, soft tissue, internal organs, or blood vessels. It can encompass pathological conditions in which tissues are divided or destroyed. Specifically, the wound or wound is a contusion or bruise, laceration, avulsion, penetrating wound, non-healing traumatic wound, destruction of tissue by irradiation, abrasion, Bone gangrene, gun shot wound, cut, burn wound, frostbite, skin ulcer, dry skin, keratosis, cracking, rupture, dermatitis, pain due to dermatophytosis, surgical wound, vascular disease wound, corneal wound. Covers damage to any part of the entity, such as back wounds, bedsores, flat sores, diabetes or conditions related to poor circulation, chronic ulcers, suture sites after plastic surgery, spinal injury wounds, gynecological wounds, chemical wounds, or acne. It can mean:
본 명세서의 용어, “조직 재생”은 손상된 조직과 장기의 신생혈관을 생성하거나, 조직 및 장기를 구성하는 세포를 재생 혹은 복원시키거나, 그 조직 및 장기의 고유기능을 담당하는 조직을 재생시켜 그 기능을 복원하는 것을 의미할 수 있다. 상기 조직 재생은 표피 재생, 진피 재생, 신생혈관 재생, 또는 피부 부속기관 재생 등을 포함할 수 있다.As used herein, the term “tissue regeneration” refers to creating new blood vessels in damaged tissues and organs, regenerating or restoring cells constituting tissues and organs, or regenerating tissues responsible for the inherent functions of the tissues and organs. It may mean restoring function. The tissue regeneration may include epidermal regeneration, dermal regeneration, new blood vessel regeneration, or skin appendage regeneration.
본 명세서의 용어, "치료 (treatment)"는 상기 조성물에 의해 상처 또는 창상 상태가 호전되거나 이롭게 변경되는 모든 행위를 의미한다.As used herein, the term “treatment” refers to any action in which a wound or wound condition is improved or beneficially changed by the composition.
상기 상처 치료 또는 조직 재생용 조성물은 상처 치료 또는 조직 재생 등의 창상 회복 효과를 가지는 유효성분을 1 종 이상 더 포함할 수 있다.The composition for wound treatment or tissue regeneration may further include one or more active ingredients having a wound healing effect such as wound healing or tissue regeneration.
상기 상처 치료 또는 조직 재생용 조성물은 약학적으로 허용 가능한 첨가제를 더 포함할 수 있으며, 이때 약학적으로 허용 가능한 첨가제로는 전분, 젤라틴화 전분, 미결정셀룰로오스, 유당, 포비돈, 콜로이달실리콘디옥사이드, 인산수소칼슘, 락토스, 만니톨, 엿, 아라비아고무, 전호화전분, 옥수수전분, 분말셀룰로오스, 히드록시프로필셀룰로오스, 오파드라이, 전분글리콜산나트륨, 카르나우바납, 합성규산알루미늄, 스테아린산, 스테아린산마그네슘, 스테아린산알루미늄, 스테아린산칼슘, 백당 등이 사용될 수 있다.The composition for wound treatment or tissue regeneration may further include pharmaceutically acceptable additives. In this case, the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, and phosphoric acid. Calcium hydrogen, lactose, mannitol, taffy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba lead, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate. , calcium stearate, white sugar, etc. can be used.
상기 상처 치료 또는 조직 재생용 조성물에 포함될 수 있는 담체, 부형제, 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 올리고당, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐 피롤리돈, 물, 메틸히드록시 벤조에이트, 프로필히드록시 벤조에이트, 탈크, 마그네슘 스테아레이트, 광물유 등이 있다.Carriers, excipients, and diluents that may be included in the composition for wound treatment or tissue regeneration include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, and gelatin. , calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, mineral oil, etc.
상기 상처 치료 또는 조직 재생용 조성물은 비경구투여에 적합하도록 제조될 수 있다.The composition for wound treatment or tissue regeneration can be prepared to be suitable for parenteral administration.
따라서, 상기 상처 치료 또는 조직 재생용 조성물은 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제등을 포함할 수 있고, 현탁용제로는 프로필렌글리콜 (Propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다.Therefore, the composition for wound treatment or tissue regeneration may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, etc., and the suspensions include propylene glycol, polyethylene glycol, and olive. Vegetable oils such as oil, injectable esters such as ethyl oleate, etc. can be used.
상기 비경구투여는 피부 외용 또는 복강 내 주사, 직장 내 주사, 피하주사, 정맥주사, 근육 내 주사 또는 흉부 내 주사 주입방식을 사용하여 이루어질 수 있다.The parenteral administration may be performed externally on the skin or by intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection.
상기 상처 치료 또는 조직 재생용 조성물은 피부 외용제의 형태로 제조될 수 있다.The composition for wound treatment or tissue regeneration may be prepared in the form of an external skin preparation.
상기 상처 치료 또는 조직 재생용 조성물은 목적하는 신체 부위에 따라 그 제형이 특별히 한정되지 않으며, 당업계의 공지기술을 참조하여 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있다. 예를 들어, 액제, 연고제, 크림제, 로션제, 스프레이제, 패취제, 오일제, 왁스제, 유탁액제, 현탁액제, 겔제 또는 에어로졸제 등 형태로 사용될 수 있으나, 이에 한정되는 것은 아니다.The composition for wound treatment or tissue regeneration is not particularly limited in formulation depending on the target body part, and may be manufactured in any commonly manufactured formulation with reference to known techniques in the art. For example, it may be used in the form of a liquid, ointment, cream, lotion, spray, patch, oil, wax, emulsion, suspension, gel, or aerosol, but is not limited thereto.
상기 상처 치료 또는 조직 재생용 조성물은 통상적인 첨가제, 예를 들어 보존제, 의약 침투를 보조하는 용매, 연고 및 크림의 경우 연화제 등을 포함할 수 있으며, 에탄올 또는 올레일 알코올과 같은 통상적 담체를 함유할 수 있다.The composition for wound treatment or tissue regeneration may include conventional additives, such as preservatives, solvents that assist drug penetration, emollients in the case of ointments and creams, and may contain a conventional carrier such as ethanol or oleyl alcohol. You can.
상기 상처 치료 또는 조직 재생용 조성물은 상술한 성분에 한정되지 않고, 필요에 따라 통상의 화장료 조성물 또는 약제학적 조성물에 배합되는 다른 성분을 포함할 수 있다. 예를 들어, 유지 성분, 보습제, 에몰리엔트제, 계면활성제, 유기 또는 무기 안료, 유기 분체, 자외선 흡수제, 방부제, 살균제, 산화 방지제, 식물 추출물, pH 조정제, 알콜, 색소, 향료, 혈행 촉진제, 냉감제, 제한(制汗)제, 정제수 등을 포함할 수 있다.The composition for wound treatment or tissue regeneration is not limited to the above-mentioned ingredients, and may include other ingredients blended in conventional cosmetic compositions or pharmaceutical compositions as needed. For example, fat ingredients, moisturizers, emollients, surfactants, organic or inorganic pigments, organic powders, ultraviolet absorbers, preservatives, disinfectants, antioxidants, plant extracts, pH adjusters, alcohol, pigments, fragrances, blood circulation promoters, It may contain cooling agents, limiting agents, purified water, etc.
상처 치료 및/또는 피부 재생의 용도에 보다 적합하도록 본 발명의 구현예로서, 상기 세포외기질로 구성된 하이드로겔 조성물은 스펀지 형태로 제공될 수 있다. 본 발명에서 상기 스펀지 형태로 제공되는 경우 겔 형태로 제공되는 것과 구분하기 위하여 세포외기질 스펀지(ECM sponge)로 명명한다. 본 발명의 상기 ECM sponge는 상술한 세포외기질 기반의 하이드로겔 조성물을 동결건조하여 제조될 수 있다. 본 발명의 세포외기질 스펀지는 상용화된 콜라겐 기반의 PelnacTM 보다 화상 치료에 훨씬 더 효과적임이 실험으로 확인되었다.As an embodiment of the present invention to be more suitable for use in wound treatment and/or skin regeneration, the hydrogel composition composed of the extracellular matrix may be provided in the form of a sponge. In the present invention, when provided in the form of a sponge, it is called an extracellular matrix sponge (ECM sponge) to distinguish it from that provided in a gel form. The ECM sponge of the present invention can be manufactured by freeze-drying the above-described extracellular matrix-based hydrogel composition. It was experimentally confirmed that the extracellular matrix sponge of the present invention is much more effective in treating burns than the commercially available collagen-based Pelnac TM .
실시예에서 사용한 용어는 단지 설명을 목적으로 사용된 것으로, 한정하려는 의도로 해석되어서는 안 된다. 단수의 표현은 문맥상 명백하게 다르게 뜻하지 않는 한, 복수의 표현을 포함한다. 본 명세서에서, "포함하다" 또는 "가지다" 등의 용어는 명세서 상에 기재된 특징, 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것이 존재함을 지정하려는 것이지, 하나 또는 그 이상의 다른 특징들이나 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것들의 존재 또는 부가 가능성을 미리 배제하지 않는 것으로 이해되어야 한다.The terms used in the examples are for descriptive purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as “comprise” or “have” are intended to designate the presence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, but are not intended to indicate the presence of one or more other features. It should be understood that this does not exclude in advance the possibility of the existence or addition of elements, numbers, steps, operations, components, parts, or combinations thereof.
다르게 정의되지 않는 한, 기술적이거나 과학적인 용어를 포함해서 여기서 사용되는 모든 용어들은 실시예가 속하는 기술 분야에서 통상의 지식을 가진 자에 의해 일반적으로 이해되는 것과 동일한 의미를 가지고 있다. 일반적으로 사용되는 사전에 정의되어 있는 것과 같은 용어들은 관련 기술의 문맥 상 가지는 의미와 일치하는 의미를 가지는 것으로 해석되어야 하며, 본 출원에서 명백하게 정의하지 않는 한, 이상적이거나 과도하게 형식적인 의미로 해석되지 않는다.Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by a person of ordinary skill in the technical field to which the embodiments belong. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and unless explicitly defined in the present application, should not be interpreted in an ideal or excessively formal sense. No.
실시예의 구성 요소를 설명하는 데 있어서, 제1, 제2, A, B, (a), (b) 등의 용어를 사용할 수 있다. 이러한 용어는 그 구성 요소를 다른 구성 요소와 구별하기 위한 것일 뿐, 그 용어에 의해 해당 구성 요소의 본질이나 차례 또는 순서 등이 한정되지 않는다. 어떤 구성 요소가 다른 구성요소에 "연결", "결합" 또는 "접속"된다고 기재된 경우, 그 구성 요소는 그 다른 구성요소에 직접적으로 연결되거나 접속될 수 있지만, 각 구성 요소 사이에 또 다른 구성 요소가 "연결", "결합" 또는 "접속"될 수도 있다고 이해되어야 할 것이다.In describing the components of the embodiment, terms such as first, second, A, B, (a), and (b) may be used. These terms are only used to distinguish the component from other components, and the nature, sequence, or order of the component is not limited by the term. When a component is described as being "connected," "coupled," or "connected" to another component, that component may be directly connected or connected to that other component, but there is no need for another component between each component. It should be understood that may be “connected,” “combined,” or “connected.”
이하에서, 첨부된 도면을 참조하여 실시예들을 상세하게 설명한다. 그러나, 실시예들에는 다양한 변경이 가해질 수 있어서 특허출원의 권리범위가 이러한 실시예들에 의해 제한되거나 한정되는 것은 아니다. 실시예들에 대한 모든 변경, 균등물 내지 대체물이 권리 범위에 포함되는 것으로 이해되어야 한다.Hereinafter, embodiments will be described in detail with reference to the attached drawings. However, since various changes can be made to the embodiments, the scope of the patent application is not limited or limited by these embodiments. It should be understood that all changes, equivalents, or substitutes for the embodiments are included in the scope of rights.
또한, 첨부 도면을 참조하여 설명함에 있어, 도면 부호에 관계없이 동일한 구성 요소는 동일한 참조부호를 부여하고 이에 대한 중복되는 설명은 생략하기로 한다. 실시예를 설명함에 있어서 관련된 공지 기술에 대한 구체적인 설명이 실시예의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우 그 상세한 설명을 생략한다.In addition, when describing with reference to the accompanying drawings, identical components will be assigned the same reference numerals regardless of the reference numerals, and overlapping descriptions thereof will be omitted. In describing the embodiments, if it is determined that detailed descriptions of related known technologies may unnecessarily obscure the gist of the embodiments, the detailed descriptions are omitted.
본 발명은 다양한 변환을 가할 수 있고 여러 가지 실시예를 가질 수 있는 바, 이하 특정 실시예들을 도면에 예시하고 상세한 설명에 상세하게 설명하고자 한다. 그러나, 이는 본 발명을 특정한 실시 형태에 대해 한정하려는 것이 아니며, 본 발명의 사상 및 기술 범위에 포함되는 모든 변환, 균등물 내지 대체물을 포함하는 것으로 이해되어야 한다. 본 발명을 설명함에 있어서 관련된 공지 기술에 대한 구체적인 설명이 본 발명의 요지를 흐릴 수 있다고 판단되는 경우 그 상세한 설명을 생략한다.Since the present invention can be modified in various ways and can have various embodiments, specific embodiments will be illustrated in the drawings and explained in detail in the detailed description below. However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all transformations, equivalents, and substitutes included in the spirit and technical scope of the present invention. In describing the present invention, if it is determined that a detailed description of related known technologies may obscure the gist of the present invention, the detailed description will be omitted.
실시예 1. 세포외기질 하이드로겔 및 ECM 스펀지의 제조 Example 1. Preparation of extracellular matrix hydrogel and ECM sponge
1-1. 인간 폐 섬유아세포 유래 세포외기질 하이드로겔(FDM-gel) 제조1-1. Manufacture of extracellular matrix hydrogel (FDM-gel) derived from human lung fibroblasts
인간 폐 섬유아세포 WI-38 세포주(ATCC, CCL-75)를 100 mm 세포배양 용기에 2x104 세포/cm2의 양으로 접종하였다. 접종된 플레이트에 10%(v/v)의 소태아혈청(Fetal bovine serum, FBS), 100 U/ml의 페니실린, 50 μg/ml의 아스코르브산(Ascorbic acid), 및 100 μg/ml의 스트렙토마이신이 첨가된 둘베코 변형 이글 배지(Dulbecco's modified Eagle's medium, DMEM)를 가하고, 일반 배양 조건(5% CO2, 37℃)에서 약 2 내지 3일 간격으로 배지를 교체하면서 약 10일 동안 배양하였다.Human lung fibroblast WI-38 cell line (ATCC, CCL-75) was inoculated into a 100 mm cell culture vessel at an amount of 2x10 4 cells/cm 2 . To the inoculated plate, 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin, 50 μg/ml ascorbic acid, and 100 μg/ml streptomycin. This added Dulbecco's modified Eagle's medium (DMEM) was added, and cultured under normal culture conditions (5% CO2, 37°C) for about 10 days while changing the medium at intervals of about 2 to 3 days.
배양된 세포를 인산염 완충 식염수(Phosphate-buffered saline, PBS)로 세척하였다. 이후, 세척된 세포에 0.25%(v/v) Triton-X 100과 50 mM NH4OH(Sigma)를 가하고, 뒤이어 50 U/ml의 Dnase Ⅰ(Invitrogen) 및 2.5 μl/ml의 Rnase A(Invitrogen)을 가하고 37℃에서 2시간 동안 인큐베이션하여 세포를 탈세포화하였다. 탈세포화된 세포외기질(Extracellular matrix, ECM)를 PBS로 여러 번 세척하여 섬유아세포 유래 세포외기질(Fibroblast-derived matrix, FDM)을 수득하였다(도 1).Cultured cells were washed with phosphate-buffered saline (PBS). Afterwards, 0.25% (v/v) Triton- X 100 and 50 mM NH4OH (Sigma) were added to the washed cells, followed by 50 U/ml of Dnase I (Invitrogen) and 2.5 μl/ml of Rnase A (Invitrogen). and incubated at 37°C for 2 hours to decellularize the cells. The decellularized extracellular matrix (ECM) was washed several times with PBS to obtain fibroblast-derived matrix (FDM) (Figure 1).
탈세포화된 ECM에 멸균된 초순수 증류수(5 ml/100 mm 배양용기)를 넣고 cell scraper를 이용해 FDM을 긁어모아 50 ml 튜브로 옮겼다(도 2에 a). 상기 해당 튜브를 3500 rpm 속도에서 10분 동안 고속 교반하여 FDM을 튜브 바닥에 모으는 압축 공정을 수행한 후, 상층액을 제거하였다(도 2에 b). 상기 튜브에 5 ml 증류수를 넣고 영하 80℃에서 하루 이상 보관하였다. 상기 동결된 튜브를 상온 또는 37℃에서 완전히 녹인 후, 다시 3500 rpm 이상의 속도에서 5분 동안 고속 교반하였다. 이후 상층액을 완전히 제거하여 순수한 세포외기질로 구성된 섬유아세포 유래 세포외기질 하이드로겔(fibroblast-derived matrix hydrogel, FDM-gel)을 수득하였다(도 2에 c). 상기 FDM-gel에 멸균된 PBS 또는 초순수 증류수를 넣어 영하 20℃에서 보관하였다.Sterilized ultrapure distilled water (5 ml/100 mm culture vessel) was added to the decellularized ECM, and FDM was scraped off using a cell scraper and transferred to a 50 ml tube (a in Figure 2). The tube was stirred at high speed at 3500 rpm for 10 minutes to perform a compression process to collect FDM at the bottom of the tube, and then the supernatant was removed (b in FIG. 2). 5 ml distilled water was added to the tube and stored at -80°C for more than one day. The frozen tube was completely thawed at room temperature or 37°C, and then stirred at high speed for 5 minutes at a speed of 3500 rpm or higher. Afterwards, the supernatant was completely removed to obtain a fibroblast-derived extracellular matrix hydrogel (FDM-gel) composed of pure extracellular matrix (Figure 2c). Sterilized PBS or ultrapure distilled water was added to the FDM-gel and stored at -20°C.
1-2. 중간엽 줄기세포 유래 세포외기질 스펀지(ECM sponge)의 제조1-2. Manufacturing of mesenchymal stem cell-derived extracellular matrix sponge (ECM sponge)
인간 중간엽 줄기세포를 배양하여 실시예 1-1과 동일한 방법으로 탈세포화된 세포외기질을 수득하였다. 수득된 탈세포화된 세포외기질을 형광염색결과 Ⅰ형 콜라겐과 피브로넥틴이 포함됨을 확인하였다(도 12). 이어서, 상기 탈세포화된 세포외기질에 멸균된 초순수 증류수를 첨가하고, 고속 교반하고, 압축 공정 수행한 후 상층액이 제거된 혼합물을 동결, 해동, 및 추가적인 고속교반을 수행하여 하이드로겔을 제작하였다. 이어서, 상기 하이드로겔을 영하 80℃에서 24시간 동안 동결건조하여 세포외기질 기반의 스펀지(ECM sponge)를 제작하였다(도 13).Human mesenchymal stem cells were cultured to obtain decellularized extracellular matrix in the same manner as in Example 1-1. As a result of fluorescent staining of the obtained decellularized extracellular matrix, it was confirmed that it contained type I collagen and fibronectin (FIG. 12). Then, sterilized ultrapure distilled water was added to the decellularized extracellular matrix, stirred at high speed, and a compression process was performed. Then, the mixture from which the supernatant was removed was frozen, thawed, and further stirred at high speed to produce a hydrogel. . Subsequently, the hydrogel was freeze-dried at -80°C for 24 hours to produce an extracellular matrix-based sponge (ECM sponge) (FIG. 13).
실험예 1. FDM-gel의 물리적 안정성 및 기계적 물성Experimental Example 1. Physical stability and mechanical properties of FDM-gel
양성대조군(positive control)인 collagen-gel과 비교하여 실시예 1-1에서 제조한 FDM-gel의 체내 이식 시, 물리적 안정성 및 기계적 물성을 평가하였다.The physical stability and mechanical properties of the FDM-gel prepared in Example 1-1 were evaluated when implanted in the body in comparison with the positive control collagen-gel.
그 결과, collagen-gel은 37℃, PBS에 담긴 상태에서 5일이 경과하면서 급격히 형태가 무너지고 7일차에는 거의 사라진 반면, FDM-gel은 14일까지도 비교적 초기 형태를 유지하고 있음을 확인하였다(도 3에 a). 이는 본 발명의 FDM-gel은 체내 이식 시, 비교적 안정적으로 형태를 유지할 수 있다는 점을 시사한다.As a result, it was confirmed that the collagen-gel rapidly lost its shape after 5 days in PBS at 37°C and almost disappeared on the 7th day, whereas the FDM-gel maintained its relatively initial shape even up to 14 days ( Figure 3 a). This suggests that the FDM-gel of the present invention can maintain its shape relatively stably when implanted in the body.
또한, rheometer를 이용한 기계적 물성 측정 결과, FDM-gel은 collagen-gel 대비 절반 정도의 탄성 계수(G')를 나타내었다(도 3에 b). 이는 본 발명의 FDM-gel이 collagen-gel 대비 낮은 탄성 계수에도 불구하고 내부 세포외기질 단백질 간 상호결합이나 가교 등으로 인해 수분 환경에서의 물리적 안정성이 훨씬 높음을 의미한다.In addition, as a result of measuring mechanical properties using a rheometer, FDM-gel showed an elastic modulus (G') that was about half that of collagen-gel (Figure 3b). This means that, despite the FDM-gel of the present invention having a lower elastic modulus compared to collagen-gel, it has much higher physical stability in a moisture environment due to mutual bonding or cross-linking between internal extracellular matrix proteins.
실험예 2. FDM-gel의 신생혈관인자 분석Experimental Example 2. Analysis of angiogenic factors in FDM-gel
실시예 1-1에서 제조한 FDM-gel에 포함되어 있는 신생혈관 형성(angiogenesis) 관련 사이토카인 분석을 수행하였다.Analysis of cytokines related to angiogenesis contained in the FDM-gel prepared in Example 1-1 was performed.
그 결과, 도 4에 나타난 바와 같이, 상술한 실시예 1에 따라 제조된 FDM-gel은 엠피레귤린(amphiregulin), 혈액 응고 인자 Ⅲ(Coagulation Factor Ⅲ), DPPⅣ (Dipeptidyl Peptidase Ⅳ), 엔도스타틴(Endostatin, collagen XVⅢ), FGF acidic(Fibroblast growth factor-acidic), FGF-7(Fibroblast Growth Factor 7), HGF(Hepatocyte growth factor), 펜트락신 3(Pentraxin 3), 플라스미노겐 활성인자 억제제-1(serpin E1), serpin E1(PEDF), TIMP-1(Tissue inhibitor of metalloproteinase), 트롬보스폰딘-1(thrombospondin-1), uPA(urokinase-type plasminogen activator) 등 다수의 신생혈관 형성 및 조직재생과 관련된 주요 사이토카인들을 포함하고 있음을 확인하였다.As a result, as shown in Figure 4, the FDM-gel prepared according to Example 1 described above contains amphiregulin, coagulation factor Ⅲ, DPPⅣ (Dipeptidyl Peptidase Ⅳ), and endostatin. , collagen E1), serpin E1 (PEDF), TIMP-1 (Tissue inhibitor of metalloproteinase), thrombospondin-1, and uPA (urokinase-type plasminogen activator). It was confirmed that it contained cytokines.
실험예 3. FDM-gel의 생체적합성 평가Experimental Example 3. Evaluation of biocompatibility of FDM-gel
양성대조군(positive control)인 collagen-gel과 비교하여 실시예 1-1에서 제조한 FDM-gel의 생체적합성을 평가하기 위해, 실험 쥐의 등 부위를 절개한 후 collagen-gel과 FDM-gel을 각각 피하 이식하였다.In order to evaluate the biocompatibility of the FDM-gel prepared in Example 1-1 compared to the collagen-gel, which is a positive control, the back of an experimental rat was incised and collagen-gel and FDM-gel were made, respectively. It was implanted subcutaneously.
이식 후 3일이 지난 시점에 피부를 개방하였을 때, collagen-gel과 FDM-gel이 각각 피하 내에 안정적으로 자리잡고 있음을 확인하였다(도 5에 ac).When the skin was opened 3 days after transplantation, it was confirmed that the collagen-gel and FDM-gel were stably located within the subcutaneous area (ac) in Figure 5.
또한, H&E 염색을 통해 해당 조직을 고배율로 관찰한 결과, collagen-gel 내부의 경우 세포(검은 점)의 침투가 거의 없는 반면(도 5에 b), FDM-gel 내부의 경우 많은 수의 세포들이 내부에 자리잡고 있음을 확인하였다(도 5에 d).In addition, as a result of observing the tissue at high magnification through H&E staining, it was found that there was almost no infiltration of cells (black dots) inside the collagen-gel (b in Figure 5), whereas a large number of cells were present inside the FDM-gel. It was confirmed that it was located inside (d in Figure 5).
즉, 피하 이식한 FDM-gel은 빠르게 분해되지 않고, 물리적 안정성을 유지하고 있으며, collagen-gel에 비해 하이드로겔 내부로 세포 침투가 현저히 우수하게 이루어짐을 알 수 있다. 또한 FDM-gel 주변으로 신생혈관형성도 우수하므로, 본 발명의 FDM-gel은 체내에서 우수한 생체적합성을 가지고 있음을 시사한다.In other words, it can be seen that the subcutaneously implanted FDM-gel does not decompose quickly, maintains physical stability, and has significantly better cell penetration into the hydrogel than collagen-gel. In addition, new blood vessel formation around the FDM-gel is excellent, suggesting that the FDM-gel of the present invention has excellent biocompatibility in the body.
실험예 4. FDM-gel의 조직재생 유도 효과Experimental Example 4. Tissue regeneration inducing effect of FDM-gel
실시예 1-1에서 제조한 FDM-gel의 조직재생능을 평가하기 위해, 실험 쥐에 직경 6 mm의 창상(Excisional full-thickness wound)을 유도한 후, 드레싱(대조군), collagen-gel, FDM-gel을 각각 환부에 적용하고, 14일차에 각 환부의 창상 부위 조직을 확보하였다. 상기 확보된 조직에 대하여 파라핀 고정을 수행하여 매우 얇은 조직 절편을 수득하였다. 상기 조직 절편에 대하여 H&E 염색을 수행하여 조직학적 특성을 비교 분석하였다.To evaluate the tissue regenerative ability of the FDM-gel prepared in Example 1-1, an Excisional full-thickness wound with a diameter of 6 mm was induced in an experimental rat, and then a dressing (control group), collagen-gel, and FDM were applied. -gel was applied to each affected area, and on the 14th day, tissue from the wound area of each affected area was obtained. Paraffin fixation was performed on the secured tissue to obtain very thin tissue sections. H&E staining was performed on the tissue sections to compare and analyze histological characteristics.
그 결과, 도 6에 a에 나타낸 바와 같이, 모든 실험군에서 표피와 진피의 재생이 확인되었다. 특히 FDM-gel 처리군은 다른 실험군 대비 모낭, 땀샘과 같은 피부 부속기관(skin adnexa)의 형성이 더욱 뚜렷하게 관찰되었다.As a result, as shown in a in Figure 6, regeneration of the epidermis and dermis was confirmed in all experimental groups. In particular, the formation of skin appendages such as hair follicles and sweat glands was observed more clearly in the FDM-gel treated group compared to the other experimental groups.
또한, 각 실험군을 처리한 후 재생된 표피 두께를 정량적으로 분석한 결과를 도 6에 b에 나타내었다. FDM-gel 처리군에서 다른 실험군 대비 가장 얇은 표피 재생이 관찰되었으며, 이는 정상 피부의 표피 두께(약 24.14 μm)와 가장 근접한 수준이었다.In addition, the results of quantitative analysis of the regenerated epidermal thickness after treatment of each experimental group are shown in Figure 6b. The thinnest epidermal regeneration was observed in the FDM-gel treatment group compared to the other experimental groups, which was closest to the epidermal thickness of normal skin (approximately 24.14 μm).
조직염색 결과를 토대로, 재생된 진피 조직내 신생 모낭의 수를 정량화 후 도 6에 c에 나타내었다. 그 결과, FDM-gel 처리군이 다른 실험군 대비 재생된 진피에서 단위면적 (9 x 104 μm2)당 신생 모낭의 수가 가장 많았으며, 이는 정상 피부의 평균 모낭 수(약 11.7개)와 유사한 수준이었다.Based on the tissue staining results, the number of new hair follicles in the regenerated dermal tissue was quantified and shown in c in FIG. 6. As a result, the FDM-gel treatment group had the highest number of new hair follicles per unit area ( 9 It was.
즉, 본 발명의 FDM-gel은 뛰어난 조직 재생 유도 효과를 가지므로, 다양한 적응증에 활용될 수 있는 유망한 소재임을 알 수 있다.In other words, the FDM-gel of the present invention has an excellent tissue regeneration inducing effect, so it can be seen that it is a promising material that can be used for various indications.
실험예 5. FDM-gel의 상처 치료 효과Experimental Example 5. Wound healing effect of FDM-gel
실시예 1-1에서 제조한 FDM-gel의 조직재생능을 평가하기 위해, 실험 쥐에 6 mm 직경의 알루미늄 로드를 이용한 화상 창상을 유도한 후, 드레싱(대조군), collagen-gel, FDM-gel을 각각 환부에 적용하고, 14일차에 각 환부의 창상 부위 조직을 확보하였다. 이후 시판용 창상피복제로 덮어 하이드로겔 탈착 및 외부 오염을 방지하였다.To evaluate the tissue regenerative ability of the FDM-gel prepared in Example 1-1, burn wounds were induced in experimental rats using an aluminum rod with a diameter of 6 mm, and then dressing (control group), collagen-gel, and FDM-gel were applied. was applied to each affected area, and on the 14th day, tissue from the wound area of each affected area was obtained. Afterwards, it was covered with a commercially available wound dressing to prevent hydrogel detachment and external contamination.
창상 유도 후 12일차 및 18일차에 각 실험군의 상처 수복(wound closure) 정도를 확인하였다. 도 7에 a에 나타낸 바와 같이, 다른 실험군과 비교하여 FDM-gel 처리군에서 더 빠른 화상 창상 회복이 나타났다.The degree of wound closure of each experimental group was checked on the 12th and 18th days after wound induction. As shown in Figure 7 a, faster burn wound recovery was observed in the FDM-gel treated group compared to the other experimental groups.
상처 회복 정도를 정량적으로 분석한 결과(도 7에 b), FDM-gel 처리군은 12일차에 대조군 대비 약 60%, collagen-gel 처리군 대비 약 40% 상처 부위의 크기가 감소했으며, 18일차에 대조군 대비 약 220%, collagen-gel 처리군 대비 약 120% 상처 부위의 크기가 감소하였다. 특히, 18일차에 FDM-gel 처리군의 상처 부위는 5% 수준으로 거의 회복이 이루어졌음을 알 수 있다.As a result of quantitative analysis of the degree of wound recovery (b in Figure 7), the size of the wound area in the FDM-gel treated group was reduced by about 60% compared to the control group and by about 40% compared to the collagen-gel treated group on day 12. The size of the wound area decreased by approximately 220% compared to the control group and by approximately 120% compared to the collagen-gel treated group. In particular, it can be seen that on the 18th day, the wound area of the FDM-gel treated group had almost recovered to 5%.
또한, 18일차 각 실험군을 처리한 실험 쥐의 화상 창상 부위 조직을 확보하고, 이로부터 조직 절편을 수득하여 H&E 염색을 수행하였다.In addition, on the 18th day, tissue from the burn wound area of the rats treated in each experimental group was obtained, tissue sections were obtained from this, and H&E staining was performed.
그 결과, 도 8에 a에 나타낸 바와 같이, 모든 실험군에서 피부 재생이 확인되었다. 특히 FDM-gel 처리군에서 다른 실험군 대비 재생된 진피에서 비교적 높은 수준의 피부 부속기관의 재생을 확인하였다.As a result, as shown in a in Figure 8, skin regeneration was confirmed in all experimental groups. In particular, a relatively high level of regeneration of skin appendages was confirmed in the regenerated dermis in the FDM-gel treatment group compared to the other experimental groups.
표피 형성을 확인하기 위해, cyto-keratin 10 (CK10) 면역형광염색을 수행한 결과, FDM-gel 처리군에서 다른 실험군 대비 표피로부터 모낭이 새롭게 더 많이 형성되고 있음을 관찰하였다(도 8에 b).To confirm epidermal formation, cyto-keratin 10 (CK10) immunofluorescence staining was performed, and as a result, it was observed that more hair follicles were newly formed from the epidermis in the FDM-gel treated group compared to the other experimental groups (Figure 8b). .
피부 부속기관의 형성을 확인하기 위해, cyto-keratin 14 (CK14)으로 면역형광염색을 수행한 결과, collagen-gel 처리군은 피부 부속기관의 재생이 일부에서만 이루어진 반면, FDM-gel 처리군은 피부 부속기관의 재생이 매우 활발하게 이루어짐을 알 수 있다(도 8에 c).To confirm the formation of skin appendages, immunofluorescence staining with cyto-keratin 14 (CK14) was performed. As a result, the collagen-gel treatment group showed only partial regeneration of skin appendages, whereas the FDM-gel treatment group showed skin regeneration. It can be seen that regeneration of appendages occurs very actively (c in Figure 8).
추가적으로, 선행기술(KR 10-2020-0099706)에서 보고된 인간 폐 섬유아세포 유래 세포외기질(FDM)을 포함하는 하이드로겔(PHF)과 본 발명의 FDM-gel간의 창상 치료 효과를 비교하였다.Additionally, the wound treatment effect was compared between the hydrogel (PHF) containing extracellular matrix (FDM) derived from human lung fibroblasts reported in the prior art (KR 10-2020-0099706) and the FDM-gel of the present invention.
구체적으로 실시예 1와 동일한 방법으로 FDM을 수득한 후, 폴록사머(poloxamer)와 히알루론산(hyaluronic acid)의 혼합물과 혼합하여 비교예로서 하이드로겔(PHF)을 제조하였다.Specifically, FDM was obtained in the same manner as in Example 1, and then mixed with a mixture of poloxamer and hyaluronic acid to prepare hydrogel (PHF) as a comparative example.
창상 유도 후 14일차에 각 실험군의 상처 수복 정도를 확인하였다. 도 9에 a 및 b에 나타낸 바와 같이, PHF 처리군 대비 FDM-gel 처리군에서 보다 빠른 상처 회복이 나타났다. 특히 14일차의 경우 통계적으로 유의한 수준에서 FDM-gel 처리군이 PHF 처리군보다 상처 부위가 현저하게 많이 감소했음을 알 수 있다.On the 14th day after wound induction, the degree of wound recovery in each experimental group was confirmed. As shown in Figure 9 a and b, faster wound recovery was observed in the FDM-gel treated group compared to the PHF treated group. In particular, on the 14th day, it can be seen that the wound area was significantly reduced in the FDM-gel treated group compared to the PHF treated group at a statistically significant level.
또한, 7일차의 진피와 14일차의 표피 조직을 확보하여 H&E 염색을 수행한 결과, FDM-gel 처리군에서 PHF 처리군에 비해 신생혈관 형성 및 모낭 재생이 눈에 띄게 증가하였다(도 9에 c). FDM-gel 처리군과 PHF 처리군의 재생된 표피의 두께는 비슷한 수준이었으나(도 9에 d), 신생혈관형성 정도는 FDM-gel 처리군이 약 2.5배 우수하였으며(도 9에 e), 재생된 모낭의 수 역시 FDM-gel 처리군이 약 4배 정도 더 우세하였다(도 9에 f). 이는 통계적으로 두 군간 유의미한 차이가 있음을 보여준다.In addition, dermis on day 7 and epidermis on day 14 were obtained for H&E staining, and as a result, neovascularization and hair follicle regeneration were noticeably increased in the FDM-gel treated group compared to the PHF treated group (Figure 9c) ). The thickness of the regenerated epidermis of the FDM-gel treated group and the PHF treated group was similar (d in Figure 9), but the degree of neovascularization was about 2.5 times better in the FDM-gel treated group (e), and the degree of regeneration The number of hair follicles formed was also approximately 4 times higher in the FDM-gel treated group (f in Figure 9). This shows that there is a statistically significant difference between the two groups.
상술한 결과들은 본 발명의 FDM-gel를 처리하는 경우, 치료를 하지 않거나(드레싱), collagen-gel 처리군은 물론, 기존에 알려진 세포외기질 유래 하이드로겔 처리군 보다 표피, 진피, 및 피부 부속기관의 재생이 활발하게 이루어져 상처 회복 능력이 월등히 우수함을 시사한다.The above-described results show that when treated with the FDM-gel of the present invention, the epidermis, dermis, and skin appendages were better than those treated with no treatment (dressing) or collagen-gel, as well as the previously known extracellular matrix-derived hydrogel treated group. This suggests that organ regeneration is active and wound recovery ability is significantly superior.
실험예 6. FDM-gel의 모낭 재생 효과Experimental Example 6. Hair follicle regeneration effect of FDM-gel
모낭(hair follicle)은 창상 치료에서 중요한 피부 부속기관 중 하나이다. 실시예 1-1에서 제조한 FDM-gel의 모낭재생능을 평가하기 위해, 실험 쥐에 창상을 유도한 후, 드레싱(대조군), FDM-gel을 각각 환부에 적용하고, 14일차에 각 환부의 창상 부위 조직을 확보하였다. 비교를 위해 정상 피부 조직을 준비하였다. 이로부터 조직 절편을 수득하여 바이오마커 형광 염색을 수행하고, 각 실험군에서 표피와 진피를 비교한 결과를 도 10에 나타내었다(이 때, 점선은 표피와 진피의 경계면이다).Hair follicles are one of the important skin appendages in wound healing. To evaluate the hair follicle regeneration ability of the FDM-gel prepared in Example 1-1, after inducing a wound in an experimental rat, dressing (control group) and FDM-gel were applied to the affected area, respectively, and on the 14th day, each affected area was treated. Tissue from the wound area was obtained. Normal skin tissue was prepared for comparison. From this, tissue sections were obtained, fluorescence staining for biomarkers was performed, and the results of comparing the epidermis and dermis in each experimental group are shown in Figure 10 (here, the dotted line is the interface between the epidermis and dermis).
드레싱만 처리했을 때, 모낭 재생 효과는 거의 관찰되지 않았으나, FDM-gel 처리군은 매우 활발한 모낭 재생의 근거로서 관련된 바이오 마커(K10, K14, AE15 등)를 발현하였고, 정상 피부 조직 대비 상당히 높은 형태학적 유사성을 나타내었다(도 10). 특히, 표피에서 강한 β-catenin(a Wnt ligand) 신호는 모낭 성장과 재생에 중요한 단서로 판단된다. 즉, 본 발명의 FDM-gel은 모낭 성장과 재생을 촉진할 수 있다는 점을 시사한다.When only the dressing was treated, little hair follicle regeneration effect was observed, but the FDM-gel treated group expressed relevant biomarkers (K10, K14, AE15, etc.) as evidence for very active hair follicle regeneration, and had significantly higher morphology compared to normal skin tissue. showed academic similarity (Figure 10). In particular, strong β-catenin (a Wnt ligand) signal in the epidermis is considered an important cue for hair follicle growth and regeneration. In other words, this suggests that the FDM-gel of the present invention can promote hair follicle growth and regeneration.
또한, FDM의 모낭재생능을 세포 수준에서 검증하기 위해, 인간 모낭세포(human follicle dermal papilla cell, HFDPC)를 TCP(tissue culture plastic)와 FDM 위에 이식하여 7일간 배양하면서, 세포 성장과 마커 발현 등의 비교 분석을 진행하였다.In addition, to verify the hair follicle regeneration ability of FDM at the cellular level, human follicle cells (human follicle dermal papilla cells, HFDPC) were transplanted onto TCP (tissue culture plastic) and FDM and cultured for 7 days, and cell growth, marker expression, etc. A comparative analysis was conducted.
그 결과, FDM에서 더 많은 세포 부착을 관찰하였고(도 11에 a), 7일간 세포 증식의 정량적 분석을 통해 FDM에서 모낭세포의 증식이 유의미하게 활발한 것을 확인하였다(도 11에 b).As a result, more cell adhesion was observed in FDM (a in Figure 11), and through quantitative analysis of cell proliferation for 7 days, it was confirmed that the proliferation of hair follicle cells in FDM was significantly active (b in Figure 11).
또한 모낭세포의 주요한 마커로 알려진 α-SMA과 β-catenin 단백질에 대해 TCP 대비 FDM에서 훨씬 우세한 발현이 관찰되었고(도 11에 c), 정량적 비교 결과에서 양 군간 통계적으로도 유의미하게 다른 β-catenin 발현 수준을 보여주었다(도 11에 d).In addition, much more dominant expression of α-SMA and β-catenin proteins, known as major markers of hair follicle cells, was observed in FDM compared to TCP (c in Figure 11), and the quantitative comparison results showed that β-catenin was statistically significantly different between the two groups. The expression level was shown (d in Figure 11).
상술한 결과들은 실험예 4 및 5와 일치하는 결과로서 본 발명의 FDM-gel이 우수한 피부 부속기관, 특히 모낭 재생 효과를 가짐을 의미한다.The above-described results are consistent with Experimental Examples 4 and 5, meaning that the FDM-gel of the present invention has excellent skin appendage organ regeneration effects, especially hair follicle regeneration effects.
따라서, 본 발명의 일 실시예에 따른 세포외기질 하이드로겔 조성물(FDM-gel)은 다수의 신생혈관인자를 포함하여 창상 치료, 모낭 재생 등 효과가 우수할 뿐만 아니라 체내 이식 시에도 물리적 안정성이 우수하고 생체적합성이 높아 안정적으로 조직재생이 가능하므로 재생 의료, 화상, 창상 치료 등의 분야에 활용될 수 있다.Therefore, the extracellular matrix hydrogel composition (FDM-gel) according to an embodiment of the present invention not only has excellent effects such as wound healing and hair follicle regeneration by containing a number of angiogenic factors, but also has excellent physical stability even when implanted in the body. It has high biocompatibility and allows stable tissue regeneration, so it can be used in fields such as regenerative medicine, burns, and wound treatment.
실험예 7. ECM sponge의 화상 치료 효과Experimental Example 7. Burn treatment effect of ECM sponge
실시예 1-2에서 제조한 ECM sponge의 조직재생능을 평가하기 위해, 실험예 4와 동일하게실험 쥐에 화상 창상을 유도하고 드레싱(대조군), PELNACTM(양성대조군), ECM sponge(실험군)을 각각 환부에 적용하고, 18일차에 각 환부의 창상 부위 조직을 확보하였다(도 14a).To evaluate the tissue regeneration ability of the ECM sponge prepared in Example 1-2, burn wounds were induced in experimental rats in the same manner as in Experimental Example 4, and dressings (control group), PELNACTM (positive control group), and ECM sponge (experimental group) were applied. It was applied to each affected area, and the wound tissue of each affected area was obtained on the 18th day (Figure 14a).
창상 유도 후 12일차 및 18일차에 각 대조군과 실험군의 상처 수복(wound closure) 정도를 확인하였다. 도 14b에 나타낸 바와 같이, 대조군과 비교하여 ECM sponge 처리군에서 더 빠른 화상 창상 회복이 나타났다.The degree of wound closure of each control group and experimental group was confirmed on the 12th and 18th days after wound induction. As shown in Figure 14b, faster burn wound recovery was observed in the ECM sponge treated group compared to the control group.
상처 회복 정도를 정량적으로 분석한 결과(도 14b), ECM sponge 처리군은 7일차에 PELNAC 처리군 대비 약 60% 상처 부위의 크기가 감소했으며, 14일차에 PELNAC 처리군 대비 약 150% 상처 부위의 크기가 감소하였다. 특히, 18일차에 ECM sponge 처리군의 상처 부위는 거의 회복이 이루어졌음을 알 수 있다.As a result of quantitative analysis of the degree of wound recovery (Figure 14b), the size of the wound area in the ECM sponge-treated group was reduced by about 60% compared to the PELNAC-treated group on the 7th day, and the size of the wound area was reduced by about 150% compared to the PELNAC-treated group on the 14th day. The size decreased. In particular, it can be seen that on the 18th day, the wound area of the ECM sponge treatment group had almost completely recovered.
또한, 18일차 각 실험군을 처리한 실험 쥐의 화상 창상 부위 조직을 확보하고, 이로부터 조직 절편을 수득하여 H&E 염색을 수행하였다(도 15).In addition, on day 18, tissue from the burn wound area of mice treated in each experimental group was obtained, tissue sections were obtained from this, and H&E staining was performed (FIG. 15).
구체적으로, 재생된 표피 두께를 정량적으로 분석한 결과, PELNAC 처리군과 과 ECM sponge 처리군은 대조군 대비 얇은 표피 재생이 관찰되었으며, ECM sponge 처리군의 표피 두께는 정상 피부의 표피 두께(약 24.14 μm)와 가장 근접한 수준으로 회복됨을 확인하였다.Specifically, as a result of quantitative analysis of the regenerated epidermal thickness, thinner epidermal regeneration was observed in the PELNAC-treated group and the ECM sponge-treated group compared to the control group, and the epidermal thickness of the ECM sponge-treated group was similar to the epidermal thickness of normal skin (approximately 24.14 μm). ) was confirmed to have recovered to the level closest to that of ).
이어서, 재생된 진피 조직내 신생 모낭의 수를 정량한 결과, 대조군의 경우 모낭이 거의 회복되지 아니하였으며, PELNAC 처리군은 적은 수의 모낭이 회복되었으나, ECM sponge 처리군은 현저하게 높은 수준으로 모낭이 회복됨을 확인할 수 있었다.Subsequently, as a result of quantifying the number of new hair follicles in the regenerated dermal tissue, in the control group, almost no hair follicles were recovered, and in the PELNAC-treated group, a small number of hair follicles were recovered, but in the ECM sponge-treated group, a significantly higher level of hair follicles was recovered. This recovery was confirmed.
또한, 진피에서 성숙한 콜라을 정량적으로 분석한 결과, PELNAC 처리군과 ECM sponge 처리군은 대조군 대비 성숙한 콜라겐의 수준이 증가하였으나, ECM sponge 처리군은 PELNAC 처리군과 비교하여 현저하게 높은 수준으로 콜라겐 생성과 성숙을 유도할 수 있음을 확인하였다.In addition, as a result of quantitative analysis of mature collagen in the dermis, the level of mature collagen increased in the PELNAC-treated group and the ECM sponge-treated group compared to the control group, but the ECM sponge-treated group showed significantly higher levels of collagen production and collagen production compared to the PELNAC-treated group. It was confirmed that maturation can be induced.
상기로부터, ECM sponge 처리군은 PELNAC 처리군과 비교하여 진피에서 비교적 높은 수준의 표피 조직의 정상화를 확인할 수 있었다.From the above, the ECM sponge treatment group was able to confirm a relatively high level of normalization of epidermal tissue in the dermis compared to the PELNAC treatment group.
이어서, 각 대조군과 실험군의 조직 절편의 면역형광염색을 수행하였다(도 16). 케라틴10(K10)과 비멘틴(vimentin)의 분포와 수준으로 보아 ECM sponge 처리군은 PELNAC 처리군과 비교되지 않을 수준으로 표피 조직과 그 부속기관의 재생을 활발하게 유도함을 알 수 있었다(도 16에 상부 패널). 또한, 혈관 성숙화 마커인 CD31 및 a-SMA의 염색 결과 ECM sponge는 표피의 정상화 뿐만 아니라 혈관 성숙화를 통한 피부 조직의 정상화를 유도하였으며(도 16에 중간 패널), 나아가 염증반응도 감소시킴을 확인할 수 있었다(도 16에 하부 패널).Subsequently, immunofluorescence staining was performed on tissue sections of each control and experimental group (FIG. 16). Based on the distribution and levels of keratin 10 (K10) and vimentin, it was found that the ECM sponge treatment group actively induced regeneration of epidermal tissue and its appendages at a level that was incomparable to the PELNAC treatment group (Figure 16 in upper panel). In addition, as a result of staining CD31 and a-SMA, which are vascular maturation markers, it was confirmed that ECM sponge not only normalized the epidermis, but also induced normalization of skin tissue through vascular maturation (middle panel in Figure 16), and further reduced inflammatory response. (lower panel in Figure 16).
이상과 같이 실시예들이 비록 한정된 도면에 의해 설명되었으나, 해당 기술분야에서 통상의 지식을 가진 자라면 상기를 기초로 다양한 기술적 수정 및 변형을 적용할 수 있다. 예를 들어, 설명된 기술들이 설명된 방법과 다른 순서로 수행되거나, 및/또는 설명된 시스템, 구조, 장치, 회로 등의 구성요소들이 설명된 방법과 다른 형태로 결합 또는 조합되거나, 다른 구성요소 또는 균등물에 의하여 대치되거나 치환되더라도 적절한 결과가 달성될 수 있다.Although the embodiments have been described with limited drawings as described above, those skilled in the art can apply various technical modifications and variations based on the above. For example, the described techniques are performed in a different order than the described method, and/or components of the described system, structure, device, circuit, etc. are combined or combined in a different form than the described method, or other components are used. Alternatively, appropriate results may be achieved even if substituted or substituted by an equivalent.
그러므로, 다른 구현들, 다른 실시예들 및 특허청구범위와 균등한 것들도 후술하는 청구범위의 범위에 속한다.Therefore, other implementations, other embodiments, and equivalents of the claims also fall within the scope of the following claims.

Claims (20)

  1. (1) 탈세포화된 세포외기질에 증류수를 첨가하는 단계;(1) adding distilled water to the decellularized extracellular matrix;
    (2) 상기 (1)단계에서 제조된 혼합물을 압축, 동결, 및 해동하는 단계; 및(2) compressing, freezing, and thawing the mixture prepared in step (1); and
    (3) 하이드로겔을 제조하는 단계;(3) preparing a hydrogel;
    를 포함하는,Including,
    별도의 고분자 지지체 또는 하이드로겔 전구체 용액 없이 하이드로겔 조성물을 제조하는 방법.A method of producing a hydrogel composition without a separate polymer support or hydrogel precursor solution.
  2. 제1항에 있어서,According to paragraph 1,
    상기 (2)단계는 상기 (1)단계에서 제조된 혼합물을 압축한 후, 영하 60~90℃에서 동결하고, 20~40℃에서 해동한 다음, 추가적으로 고속 교반하는 것을 특징으로 하는, 하이드로겔 조성물을 제조하는 방법.In step (2), the mixture prepared in step (1) is compressed, frozen at -60 to 90 ° C, thawed at 20 to 40 ° C, and then additionally stirred at high speed. A hydrogel composition. How to manufacture.
  3. 제1항에 있어서,According to paragraph 1,
    상기 (3)단계에서 제조된 하이드로겔은 7 ~ 12 mm의 두께를 가지는 것을 특징으로 하는, 하이드로겔 조성물을 제조하는 방법.A method of producing a hydrogel composition, characterized in that the hydrogel prepared in step (3) has a thickness of 7 to 12 mm.
  4. 제1항에 있어서,According to paragraph 1,
    상기 탈세포화된 세포외기질은 생체 외(in vitro)에서 배양된 세포로부터 수득된 것인, 하이드로겔 조성물을 제조하는 방법.A method of producing a hydrogel composition, wherein the decellularized extracellular matrix is obtained from cells cultured in vitro.
  5. 제4항에 있어서,According to paragraph 4,
    상기 세포는 섬유아세포, 연골세포, 조골세포, 혈관내피세포, 근세포, 평활근세포, 간세포, 신경세포, 심근세포, 척추 추간판세포, 중간엽 줄기세포, 유전자 조작 세포 및 이들의 조합으로 이루어진 군으로부터 선택되는 어느 하나 이상인 것인, 하이드로겔 조성물을 제조하는 방법.The cells are selected from the group consisting of fibroblasts, chondrocytes, osteoblasts, vascular endothelial cells, myocytes, smooth muscle cells, hepatocytes, nerve cells, cardiomyocytes, spinal disc cells, mesenchymal stem cells, genetically modified cells, and combinations thereof. A method of producing a hydrogel composition, which is one or more of the following.
  6. 제1항 내지 제5항의 방법으로 제조된 하이드로겔 조성물.A hydrogel composition prepared by the method of claims 1 to 5.
  7. 제6항에 있어서,According to clause 6,
    상기 하이드로겔 조성물은 수분 환경에서 안정적으로 형태를 유지하는 것을 특징으로 하는, 하이드로겔 조성물.A hydrogel composition, characterized in that the hydrogel composition stably maintains its shape in a moisture environment.
  8. 제6항에 있어서,According to clause 6,
    상기 하이드로겔 조성물은 신생혈관형성 관련 성장 인자를 포함하는 것인, 하이드로겔 조성물.The hydrogel composition is a hydrogel composition comprising growth factors related to angiogenesis.
  9. 제6항에 있어서,According to clause 6,
    상기 하이드로겔 조성물은 창상 회복용, 상처 치료용, 조직 재생용, 표피 재생용, 진피 재생용, 신생혈관 재생용, 또는 피부 부속기관 재생용인 것을 특징으로 하는, 하이드로겔 조성물.The hydrogel composition is characterized in that it is used for wound recovery, wound treatment, tissue regeneration, epidermis regeneration, dermis regeneration, new blood vessel regeneration, or skin appendage regeneration.
  10. 제9항에 있어서,According to clause 9,
    상기 상처는 창상(wound), 화상(burn wound), 찰과상(abrasion), 열상(laceration), 자상(stab wound), 궤양(ulcer), 또는 이들의 조합인 것인, 하이드로겔 조성물.The hydrogel composition wherein the wound is a wound, burn wound, abrasion, laceration, stab wound, ulcer, or a combination thereof.
  11. (1) 탈세포화된 세포외기질에 증류수를 첨가하는 단계;(1) adding distilled water to the decellularized extracellular matrix;
    (2) 상기 (1)단계에서 제조된 혼합물을 압축, 동결, 및 해동하는 단계;(2) compressing, freezing, and thawing the mixture prepared in step (1);
    (3) 하이드로겔을 제조하는 단계; 및(3) preparing a hydrogel; and
    (4) 상기 하이드로겔을 동결 건조하는 단계;를 포함하는,(4) freeze-drying the hydrogel; including,
    세포외기질 스펀지 제조 방법.Method for manufacturing extracellular matrix sponge.
  12. 제11항에 있어서,According to clause 11,
    상기 (2)단계는 상기 (1)단계에서 제조된 혼합물을 압축한 후, 영하 60~90℃에서 동결하고, 20~40℃에서 해동한 다음, 추가적으로 고속 교반하는 것을 특징으로 하는, 세포외기질 스펀지 제조방법.Step (2) is an extracellular matrix characterized in that the mixture prepared in step (1) is compressed, frozen at -60 to 90°C, thawed at 20 to 40°C, and then additionally stirred at high speed. Sponge manufacturing method.
  13. 제11항에 있어서, According to clause 11,
    상기 (3)단계에서 제조된 하이드로겔은 7 ~ 12 mm의 두께를 가지는 것을 특징으로 하는, 세포외기질 스펀지 제조방법.A method for producing an extracellular matrix sponge, characterized in that the hydrogel prepared in step (3) has a thickness of 7 to 12 mm.
  14. 제11항에 있어서, According to clause 11,
    상기 탈세포화된 세포외기질은 생체 외(in vitro)에서 세포를 배양하여 수득된 것인, 세포외기질 스펀지 제조방법.A method of producing an extracellular matrix sponge, wherein the decellularized extracellular matrix is obtained by culturing cells in vitro.
  15. 제11항에 있어서,According to clause 11,
    상기 제조방법은 하이드로겔 제조를 위한 별도의 고분자 지지체 또는 하이드로겔 전구체를이용하지 않는 것을 특징으로 하는, 세포외기질 스펀지 제조방법.The method of producing an extracellular matrix sponge is characterized in that it does not use a separate polymer support or hydrogel precursor for hydrogel production.
  16. 제11항 내지 제15항 중 어느 한 항의 제조방법에 따라 제조된 세포외기질 스펀지.An extracellular matrix sponge manufactured according to the manufacturing method of any one of claims 11 to 15.
  17. 제16항에 있어서, According to clause 16,
    상기 세포외기질 스펀지는 Ⅰ형 콜라겐 및/또는 피브로넥틴을 포함하는 것인, 세포외기질 스펀지.The extracellular matrix sponge includes type I collagen and/or fibronectin.
  18. 제16항에 있어서,According to clause 16,
    상기 세포외기질 스펀지는 신생혈관형성 관련 성장 인자를 포함하는 것인, 세포외기질 스펀지.The extracellular matrix sponge is an extracellular matrix sponge containing growth factors related to angiogenesis.
  19. 제16항에 있어서,According to clause 16,
    상기 세포외기질 스펀지는 창상 회복용, 상처 치료용, 조직 재생용, 표피 재생용, 진피 재생용, 신생혈관 재생용, 또는 피부 부속기관 재생용인 것을 특징으로 하는, 세포외기질 스펀지.The extracellular matrix sponge is characterized in that it is used for wound recovery, wound treatment, tissue regeneration, epidermis regeneration, dermis regeneration, new blood vessel regeneration, or skin appendage regeneration.
  20. 제19항에 있어서,According to clause 19,
    상기 상처는 창상(wound), 화상(burn wound), 찰과상(abrasion), 열상(laceration), 자상(stab wound), 궤양(ulcer), 또는 이들의 조합인 것인, 세포외기질 스펀지.The wound is a wound, burn wound, abrasion, laceration, stab wound, ulcer, or a combination thereof, extracellular matrix sponge.
PCT/KR2023/009718 2022-07-13 2023-07-10 Extracellular matrix-based hydrogel and sponge for wound healing or tissue regeneration, and production method therefor WO2024014795A1 (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102159345B1 (en) * 2019-09-23 2020-09-23 한국과학기술연구원 Pharmaceutical composition for wound healing or tissue regeneration, preparation method thereof, and use thereof
KR20220019366A (en) * 2020-08-10 2022-02-17 한국과학기술연구원 Composition for wound treatment or tissue regeneration comprising a mixture of poloxamer, hyaluronic acid, and extracellular matrix and use thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102159345B1 (en) * 2019-09-23 2020-09-23 한국과학기술연구원 Pharmaceutical composition for wound healing or tissue regeneration, preparation method thereof, and use thereof
KR20220019366A (en) * 2020-08-10 2022-02-17 한국과학기술연구원 Composition for wound treatment or tissue regeneration comprising a mixture of poloxamer, hyaluronic acid, and extracellular matrix and use thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
"Ph.D. thesis", 1 January 2022, UNIVERSITY OF SCIENCE AND TECHNOLOGY (UST), Korea, article SAVITRI, C.: "Investigation of Macrophage-Extracellular Matrix Interactions and the Impact on Wound Healing", pages: 1 - 133, XP009552173 *
KIM HYO-SUNG, HWANG HYUN-JEONG, KIM HAN-JUN, CHOI YEJI, LEE DAEHYUNG, JUNG HONG-HEE, DO SUN HEE: "Effect of Decellularized Extracellular Matrix Bioscaffolds Derived from Fibroblasts on Skin Wound Healing and Remodeling", FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY, FRONTIERS RESEARCH FOUNDATION, CH, vol. 10, 29 June 2022 (2022-06-29), CH , pages 865545, XP093128489, ISSN: 2296-4185, DOI: 10.3389/fbioe.2022. *
WANG FANGFANG; ZHANG RUI; GAO NIUNIU; CHANG CHUNYU; WANG ZONGHUAN; ZHOU YINGJIE; ZHANG CHI; MA JIANWEI; JIN YONG; WEI PENG; MEI JI: "Coagulation/anticoagulation-regulable and tough extracellular matrix hydrogels", COMPOSITES PART B, ELSEVIER, AMSTERDAM, NL, vol. 239, 26 April 2022 (2022-04-26), AMSTERDAM, NL, XP087082799, ISSN: 1359-8368, DOI: 10.1016/j.compositesb.2022.109938 *

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