WO2024014795A1 - Hydrogel à base de matrice extracellulaire et éponge pour cicatriser des plaies ou régénérer des tissus et procédé de production associé - Google Patents

Hydrogel à base de matrice extracellulaire et éponge pour cicatriser des plaies ou régénérer des tissus et procédé de production associé 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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English (en)
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/ko
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Publication of WO2024014795A1 publication Critical patent/WO2024014795A1/fr

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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

La présente invention concerne : un procédé de production d'un hydrogel préparé à partir d'une matrice extracellulaire sans support polymère séparé ou solution de précurseur d'hydrogel; un hydrogel produit à partir de celui-ci; une éponge produite par lyophilisation de celui-ci; l'utilisation de l'hydrogel pour la cicatrisation des plaies et la régénération tissulaire; et analogues. L'hydrogel à base de matrice extracellulaire et l'éponge de la présente invention présentent une biocompatibilité, une stabilité physique et des propriétés mécaniques élevées et comprennent diverses substances bioactives comprenant des facteurs angiogéniques et sont donc très efficaces dans le traitement des plaies tel que celui de la régénération tissulaire et de la régulation de l'inflammation. Par conséquent, l'hydrogel à base de matrice extracellulaire et l'éponge selon la présente invention sont destinés à être utilisés dans les domaines de la médecine régénérative, du traitement des brûlures et des plaies et analogues.
PCT/KR2023/009718 2022-07-13 2023-07-10 Hydrogel à base de matrice extracellulaire et éponge pour cicatriser des plaies ou régénérer des tissus et procédé de production associé WO2024014795A1 (fr)

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KR10-2022-0086386 2022-07-13
KR1020220086386A KR20240009206A (ko) 2022-07-13 2022-07-13 세포외기질 하이드로겔을 포함하는 상처 치료 또는 조직 재생용 조성물 및 이의 제조방법
KR20230079879 2023-06-21
KR10-2023-0079879 2023-06-21

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102159345B1 (ko) * 2019-09-23 2020-09-23 한국과학기술연구원 상처 치료 또는 조직 재생용 약학적 조성물, 이의 제조 방법, 및 이를 이용한 방법
KR20220019366A (ko) * 2020-08-10 2022-02-17 한국과학기술연구원 폴록사머, 히알루론산, 및 탈세포화된 세포외기질의 혼합물을 포함하는 상처 치료 또는 조직 재생용 조성물 및 이의 용도

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KR102159345B1 (ko) * 2019-09-23 2020-09-23 한국과학기술연구원 상처 치료 또는 조직 재생용 약학적 조성물, 이의 제조 방법, 및 이를 이용한 방법
KR20220019366A (ko) * 2020-08-10 2022-02-17 한국과학기술연구원 폴록사머, 히알루론산, 및 탈세포화된 세포외기질의 혼합물을 포함하는 상처 치료 또는 조직 재생용 조성물 및 이의 용도

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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. *
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