WO2018199698A1 - Composition comprising fibrous acellular dermal matrix and biocompatible polymer, and method for producing same - Google Patents

Composition comprising fibrous acellular dermal matrix and biocompatible polymer, and method for producing same Download PDF

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Publication number
WO2018199698A1
WO2018199698A1 PCT/KR2018/004955 KR2018004955W WO2018199698A1 WO 2018199698 A1 WO2018199698 A1 WO 2018199698A1 KR 2018004955 W KR2018004955 W KR 2018004955W WO 2018199698 A1 WO2018199698 A1 WO 2018199698A1
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composition
dermal matrix
acellular dermal
weight
cell
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PCT/KR2018/004955
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French (fr)
Korean (ko)
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박희준
유현승
한진욱
이지혜
장학수
이선이
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(주)시지바이오
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Priority to KR1020197037462A priority Critical patent/KR102232847B1/en
Publication of WO2018199698A1 publication Critical patent/WO2018199698A1/en

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    • 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
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
    • A61L15/32—Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
    • 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
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/40—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing ingredients of undetermined constitution or reaction products thereof, e.g. plant or animal extracts
    • 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
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/42—Use of materials characterised by their function or physical properties
    • A61L15/44—Medicaments
    • 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
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • 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
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/222—Gelatin
    • 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
    • A61L27/36—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
    • A61L27/3604—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 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/362—Skin, e.g. dermal papillae
    • 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
    • A61L27/3683—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3687—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
    • 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
    • A61L27/3683—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3691—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
    • 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
    • A61L27/60—Materials for use in artificial skin
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/80—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special chemical form
    • A61L2300/802—Additives, excipients, e.g. cyclodextrins, fatty acids, surfactants
    • 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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/34—Materials or treatment for tissue regeneration for soft tissue reconstruction

Definitions

  • the present invention relates to a composition
  • a composition comprising a human-derived acellular dermal matrix and gelatin, and more particularly, the degree of crosslinking, viscoelasticity, hardening and / or extruding force values after sterilization of the fibrous acellular dermal matrix and gelatin composition. Is 30% or less, preferably 20% or less, most preferably 10% or less, compared to the value before sterilization, and relates to a composition, a method for preparing the same, and a sheet production using the same.
  • Wound is a state in which the continuity of the tissue is broken by breaking or deficiency of the skin or other tissues by external pressure, and usually means damage to the dermal layer of the skin and opening of the skin. Wound surgery basically prevents infection and suppresses inflammatory reactions by suturing and dressing wound sites to block exposure to the outside environment.
  • the material of the wound coating material can be largely divided into allogeneic and heterogeneous dermal materials.
  • the wound coating material can be manufactured by extracting a specific polymer from the material and by using a dermal matrix.
  • Dermal tissue is composed of 80-90% collagen, elastin and glycosaminoglycan (GAG).
  • LifeCell Inc acemented and lyophilized skin tissues obtained from dead bodies in 1994 to commercialize Acellular Dermal Matrix (ADM) for use in burn treatment and skin reconstruction. It was. Alloderm (allograft) is safer than heterogeneous products and has significantly better engraftment and healing effects. Similar products include Bard Dabol's AlloMax, which is used as an implant, and Ethicon's FlexHD with increased adhesion.
  • ADM Acellular Dermal Matrix
  • the coating material of the sheet-shaped acellular dermal matrix as described above is difficult to be used in the wound or deeply cut wounds, and the efficiency of the sheet-shaped dermal matrix is poor because the tissue sheet must be cut or used in accordance with the size and shape of the wound.
  • the composition which crosslinked the cell-free dermal matrix particles and the biocompatible polymer as described above has high structural stability but also has high crosslinking degree, viscoelasticity, hardenability, and extruding force, making it suitable for fillers, implants, etc.
  • the micronized acellular dermal matrix has a disadvantage in that it is not evenly distributed on the wound, such as flowing out of the dressing and not fixed to the wound site in liquid form.
  • crosslinking occurs when the E-beam is irradiated for sterilizing purposes even without artificially adding a crosslinking agent to the cell-free dermal matrix particles and the biocompatible polymer composition. Changes in physical properties or dosage form occur. Compositions with increased curability have poor applicability and are not suitable for use as injections for wound coating pastes, gels, ointments, emulsions, creams and suspensions.
  • the present inventors studied a composition in the form of a paste by combining a cell-free dermal matrix and a biocompatible polymer having excellent hemostatic properties, that is, gelatin. However, after the radiation sterilization for a long time (more than 1 year) or at low temperatures, the hardness and the extrusion force was increased to confirm an embodiment of the stability of the composition.
  • the present inventors have made efforts to develop a composition of a paste formulation that can be used regardless of the curvature, size and shape of the wound site, is smooth and has good spreadability, and has high stability.
  • the sterilization of the composition in which the fibrous acellular dermal matrix and the biocompatible polymer, preferably alginate, fibrin, collagen, fibronectin, polyglycolic acid, polylactic acid and hyaluronic acid, more preferably gelatin are mixed preferably By constant heat treatment before and / or after the radiation sterilization, the degree of crosslinking was suppressed, so that the physical properties of the soft paste were maintained and the storage stability was improved, thereby completing the present invention.
  • the present inventors have made diligent efforts to develop a wound coating sheet capable of adjusting the width, length and thickness of the wound suitable for the wound site, and then paste the cell-free dermal matrix and gelatin composition into a mold (mold or frame).
  • the sheet was prepared by coating and lyophilization, and it was confirmed that the sheet is stable even in a long time hydration (rehydration) process, has excellent bioadhesiveness and excellent therapeutic effect, and completed the present invention.
  • It is an object of the present invention to provide a composition comprising a cell-free dermal matrix and gelatin that is sterile, ready-to-use and has improved stability as an effective coating material on curved or deeply cut wounds.
  • Another object of the present invention is to provide a method for preparing the composition.
  • Another object of the present invention is a fibrosis, characterized in that the ratio of the short axis and the long axis of the acellular dermal matrix is 1:30 to 1: 2,000 is 70%, preferably 80%, most preferably 90% or more.
  • a composition comprising a cell-free dermal matrix and a biocompatible polymer.
  • Still another object of the present invention is to provide a method for preparing a composition comprising the fibrized cell-free dermal matrix and a biocompatible polymer.
  • Another object of the present invention is a sheet prepared using a cell-free dermal substrate in the form of a paste containing 75 to 85% by weight of the cell-free dermal substrate, 15 to 25% by weight of the biocompatible polymer and 3 to 8% by weight of water It is to provide a sheet for wound covering, characterized in that the horizontal length is 4 ⁇ 20 cm, the vertical length is 4 ⁇ 20 cm and the thickness is 0.5 ⁇ 3 mm.
  • Another object of the present invention is to provide a method for producing the sheet.
  • the composition is sterile and ready-to-use, after sterilization crosslinking, viscoelasticity, degree of cure and And / or a change in extrusion force value is 30% or less compared to the value before sterilization.
  • Ready-to-use herein is intended to dissolve solutes, eg, solid particles and lyophilized ingredients, in a solvent immediately prior to use, or proteins that are susceptible to modification to certain ingredients, such as room temperature. For the purpose of mixing other components, etc., it means that the reconstitute step is not included. In the case of a sheet, it is meant to be used immediately after rehydration.
  • Ready-to-use compositions of the present invention include solutions, emulsions, creams, ointments, pastes or gels in the form of sterile injectables or aerosols which can be administered immediately without a reconstitute step.
  • the sheet includes a form that can be used immediately after the rehydration step without addition of other substances.
  • the degree of crosslinking of the present invention refers to the degree of crosslinking, that is, the degree of crosslinking, and cross-linking means that the polymer chains are chemically linked to each other directly or through several bonds at any position other than the terminal. it means.
  • the degree of crosslinking increases, the values of viscoelasticity, curing degree, and extrusion force increase, thereby improving structural stability such as heat resistance, durability, and workability.
  • the degree of crosslinking decreases, the values of viscoelasticity, hardenability and extrusion force decrease, thereby improving free deformation, flexibility, and adhesiveness.
  • the degree of crosslinking, viscoelasticity, hardenability, and extruding force may be too high or too low, resulting in a hardening of physical properties or an increase in decomposition rate in the body. Accordingly, in the present invention, the optimum physical state of the composition is confirmed, and the change in the physical properties, that is, the degree of change in the crosslinking degree, the viscoelastic curing degree and the extruding force value is 30% or less, preferably during processing or storage such as sterilization. Provides a composition that is at most 20%, most preferably at most 10%.
  • the composition is 5-20% by weight of acellular dermal matrix, 0.5-5% by weight of gelatin and 75-94.5% by weight of water, preferably 6-16% by weight of acellular dermal matrix, 1-3% by weight of gelatin and water 81- 93% by weight, more preferably 8-12% by weight of acellular dermal matrix, 1-2% by weight of gelatin and 86-91% by weight of water.
  • the cell-free dermal matrix is characterized in that the fibrous cell-free dermal matrix.
  • fibrous acellular dermal matrix refers to a fibrous, elongated fibrous form in which the individual individual forms of the granulated acellular dermal matrix are not spherical or streamlined particles.
  • the fibrous acellular dermal matrix of the present invention has a long axis length of 500 to 3,000 ⁇ m, preferably 500 to 2,000 ⁇ m, and a ratio of short axis and long axis is 1:30 to 1: 2,000 ⁇ m, preferably 1:30 to 1 It is characterized by that it is 70% or more, preferably 80%, most preferably 90% or more: 1: 1,000 micrometers, Most preferably, 1: 30-1: 70 micrometers.
  • the strength of the gelatin is at least 250 Bloom, preferably at least 270 Bloom and the viscosity is 30 to 45 mps, preferably 33 to 42 mps, more preferably 35 to 40 mps and the pH is 5 to 6.5, preferably 5.5 It is characterized in that from 6 to.
  • the sterilization may include dry heat sterilization, pasteurization sterilization, radiation sterilization, gas sterilization and the like, and preferably radiation sterilization, more preferably radiation sterilization using E-beam.
  • the heat treatment is carried out for 1 to 180 minutes, preferably 30 to 150 minutes, more preferably 60 to 120 minutes.
  • composition is an aqueous solution, suspension, emulsion, paste, cream, balm, ointment, foam, sheet, gel It is prepared in the form of gel, gum, spray, slurry, film, granule, patch, powder, or the like.
  • preferred embodiments of the present invention include paste or sheet form.
  • the sheet is characterized in that the paste composition is applied to a mold (mold or frame) and dried or lyophilized to contain 3 to 8, preferably 4 to 5% of moisture.
  • composition may further comprise an antimicrobial agent, excipient and additives used pharmaceutically.
  • Suitable antimicrobials include biocides such as short-chain alcohols, benzoalkonium chloride (“BAC”), didecyl dimethyl ammonium chloride (“DDAC”) and zeolites (“CWT-A”). Include.
  • antibacterial agents are isothiazolones, alkyl dimethyl ammonium chlorides, triazines, 2-thiocyanomethylthio benzothiazoles, methylene bis thiocyanates, acrolein, dodecylguanidine hydrogen chloride, chlorophenols, quaternary ammonium salts, gluter Aldehyde, dithiocarbamate, 2-mercaptobenzothiazole, para-chloro-meth-xyllenol, silver, chlorhexidine, polyhexamethylene biguanide, n-halamine, triclosan, phospholipids, alpha hydroxy acids, 2 , 2-dibromo-3-nitrilopropionamide, 2-bromo-2-nitro-1,3-propanediol, farnesol, iodine, bromine, hydrogen peroxide, chlorine dioxide, vegetable oils, plant extracts, benzal Cornium chloride, chlorine, sodium hypochlorite, or combinations thereof
  • Suitable excipients are starch, cellulose, glucose, lactose, sucrose, gelatin, corn, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, as stabilizers, antioxidants, osmotic-modulating agents, buffers and pH-adjusting agents.
  • Suitable additives are physiologically biocompatible buffers (eg tromethamine hydrochloride), chelating agents (eg DTPA or DTPA-bisamide) or calcium chelate complexes (eg calcium DTPA, CaNaDTPA-bisamide) ) Or optionally calcium or sodium salts (eg, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate).
  • physiologically biocompatible buffers eg tromethamine hydrochloride
  • chelating agents eg DTPA or DTPA-bisamide
  • calcium chelate complexes eg calcium DTPA, CaNaDTPA-bisamide
  • calcium or sodium salts eg, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate.
  • compositions can be used as wound coatings, adhesives, surgical and medical devices, artificial skin, bandages, foaming agents, films, antiadsorbents and implants, preferably wound coatings.
  • the present invention also comprises the steps of a) pulverizing the cell-free dermal matrix, b) mixing the pulverized acellular dermal matrix, gelatin-dissolved polymer aqueous solution and water to form a mixture; c) heat-treating the mixture at 30-60 ° C. for 1-180 minutes; d) sterilizing the heat-treated mixture using an E-beam and e) sterilizing and ready-to-use, including heat-treating the sterilized mixture at 30-60 ° C. for 1-180 minutes. And a degree of change in crosslinking degree, viscoelasticity, curing degree and / or extrusion force value after sterilization is 30% or less than a value before sterilization.
  • the degree of change is calculated by the following equation.
  • Pi degree of crosslinking, viscoelasticity, cure and / or extrusion force before sterilization
  • Pd degree of crosslinking, viscoelasticity, curing and / or extrusion force after sterilization
  • the mixture of step b) is 5-20% by weight of acellular dermal matrix, 0.5-5% by weight of gelatin and 75-94.5% by weight of water, preferably 6-16% by weight of acellular dermal matrix, 1-3% by weight of gelatin And 81-93% by weight of water, more preferably 8-12% by weight of acellular dermal substrate, 1-2% by weight of gelatin and 86-91% by weight of water.
  • the cell-free dermal matrix is characterized in that the fibrous cell-free dermal matrix.
  • the fibrous acellular dermal matrix of the present invention has a long axis length of 500 to 3,000 ⁇ m, preferably 500 to 2,000 ⁇ m, and a ratio of short axis and long axis is 1:30 to 1: 2,000 ⁇ m, preferably 1:30 to 1 It is characterized by that it is 70% or more, preferably 80%, most preferably 90% or more: 1: 1,000 micrometers, Most preferably, 1: 30-1: 70 micrometers.
  • the cell-free dermal matrix of step a) of pulverizing the cell-free dermal matrix is characterized in that it is used to select a thickness of 1 mm or more, preferably 1.5 mm or more.
  • the cell-free dermal substrate crushing is a cutting mill, a food processor, agate grinder, freeze grinder, micronizer, Vibratory micro mill, Jaw crusher, Mortar grinder, Planetary mill, Disk mill, Ball mill, Variable speed rotor mill, preferably using a cutting mill (Cutting mill).
  • the cutting mill preferably uses a rotational speed of 500 rpm and operating conditions of 750 ⁇ m sieve size.
  • the cell-free dermal matrix comprises a) removing the epidermis of allogeneic skin; b) removing the cells in the dermis; c) preparing a cryoprotectant by mixing glycerol, propylene glycol, and a base solvent or solution and dissolving in the solution a final concentration of maltitol, sucrose or sorbitol, preferably maltitol, at 20-40% by weight; d) infiltrating the cryoprotectant into the skin from which the cells in the epidermis and dermis have been removed, and e) freeze drying the skin in which the cryoprotectant has infiltrated.
  • the maltitol was confirmed to prevent the browning phenomenon of the dermal tissue, it is characterized by using maltitol accordingly.
  • Mixing ratio of the glycerol, propylene glycol and the basic solvent or solution is characterized in that 0.5 to 2: 0.5 to 2: 6 to 10 by weight.
  • the basic solvent or solution is distilled water, physiological saline (normal saline), phosphate buffered solution (PBS), HBSS (Hank's balanced salt solution), TBS (Tris buffered saline), TAPS (N-Tris (hydroxy-methyl) methyl-3 -aminopropanesulfonic acid) buffer, Bicine (N, N-Bis (2-hydroxyethyl) glycine) buffer, HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) buffer, TES (NTris (hydroxymethyl) methyl- 2-aminoethanesulfonicd acid buffer, piperazine-N, N'-bis (2-ethanesulfonic acid) buffer, cacodylate buffer, MES (2- (N-morpholino) ethanesulfonic acid) buffer , Minimum Essential Media (MEM), Dulbecco's Modified Eagle Media (DMEM), RPMI1640, Iscove
  • the present invention is a composition comprising a fibrous acellular dermal matrix and a biocompatible polymer, the cell-free dermal matrix is 1: 30 ⁇ 1: 2,000 ⁇ m, preferably 1: 30 ⁇ 1 It is characterized by that it is 70% or more, preferably 80%, most preferably 90% or more: 1: 1,000 micrometers, Most preferably, 1: 30-1: 70 micrometers.
  • the composition is characterized in that it is a sterile, ready-to-use composition.
  • the ready-to-use composition may be a solution, emulsion, cream, ointment, paste or sterile injectable or aerosol form that can be administered immediately without the need for a reconstitute step to dissolve the solute in a solvent or to mix certain ingredients. Gels.
  • the composition is 5 to 20% by weight of the fibrous acellular dermal substrate, 0.5 to 5% by weight of the biocompatible polymer and 75 to 99.5% by weight of water, preferably 6 to 16% by weight of the fibrous acellular dermal substrate, biocompatible polymer 1 ⁇ 3% by weight and 81-93% by weight of water, more preferably 8-12% by weight of fibrized acellular dermal substrate, 1-2% by weight of biocompatible polymer and 86-91% by weight of water.
  • the long axis length of the fibrous acellular dermal matrix is characterized in that 500 to 3,000 ⁇ m, preferably 500 to 2,000 ⁇ m.
  • the biocompatible polymer includes gelatin, hyaluronic acid, collagen, poloxamer or mixtures thereof.
  • the preferred biocompatible polymer is gelatin.
  • the strength of the gelatin is at least 250 Bloom, preferably at least 270 Bloom and the viscosity is 30 to 45 mps, preferably 33 to 42 mps, more preferably 35 to 40 mps and the pH is 5 to 6.5, preferably 5.5 It is characterized in that from 6 to.
  • composition is an aqueous solution, suspension, emulsion, paste, cream, balm, ointment, foam, sheet, gel It is prepared in the form of gel, gum, spray, slurry, film, granule, patch, powder, or the like.
  • preferred embodiments of the present invention include paste or sheet form.
  • the sheet is characterized in that the paste composition is applied to a mold (mold or frame) and dried or lyophilized to contain 3 to 8%, preferably 4 to 5% of water.
  • the sterilization may include dry heat sterilization, pasteurization sterilization, radiation sterilization, gas sterilization and the like, and preferably radiation sterilization, more preferably radiation sterilization using E-beam.
  • the heat treatment is carried out for 1 to 180 minutes, preferably 30 to 150 minutes, more preferably 60 to 120 minutes.
  • the fibrous acellular dermal matrix is characterized in that it is used to select a thickness of 1 mm or more, preferably 1.5 mm or more.
  • the cell-free dermal substrate crushing is a cutting mill, a food processor, agate grinder, freeze grinder, micronizer, Vibratory micro mill, Jaw crusher, Mortar grinder, Planetary mill, Disk mill, Ball mill, Variable speed rotor mill, preferably using a cutting mill (Cutting mill).
  • the cutting mill preferably uses a rotational speed of 500 rpm and operating conditions of 750 ⁇ m sieve size.
  • composition may further comprise an antimicrobial agent, excipient and additives used pharmaceutically.
  • Suitable antimicrobials include biocides such as short-chain alcohols, benzoalkonium chloride (“BAC”), didecyl dimethyl ammonium chloride (“DDAC”) and zeolites (“CWT-A”). Include.
  • antibacterial agents are isothiazolones, alkyl dimethyl ammonium chlorides, triazines, 2-thiocyanomethylthio benzothiazoles, methylene bis thiocyanates, acrolein, dodecylguanidine hydrogen chloride, chlorophenols, quaternary ammonium salts, gluter Aldehyde, dithiocarbamate, 2-mercaptobenzothiazole, para-chloro-meth-xyllenol, silver, chlorhexidine, polyhexamethylene biguanide, n-halamine, triclosan, phospholipids, alpha hydroxy acids, 2 , 2-dibromo-3-nitrilopropionamide, 2-bromo-2-nitro-1,3-propanediol, farnesol, iodine, bromine, hydrogen peroxide, chlorine dioxide, vegetable oils, plant extracts, benzal Cornium chloride, chlorine, sodium hypochlorite, or combinations thereof
  • Suitable excipients are starch, cellulose, glucose, lactose, sucrose, gelatin, corn, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, as stabilizers, antioxidants, osmotic-modulating agents, buffers and pH-adjusting agents.
  • Suitable additives are physiologically biocompatible buffers (eg tromethamine hydrochloride), chelating agents (eg DTPA or DTPA-bisamide) or calcium chelate complexes (eg calcium DTPA, CaNaDTPA-bisamide) ) Or optionally calcium or sodium salts (eg, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate).
  • physiologically biocompatible buffers eg tromethamine hydrochloride
  • chelating agents eg DTPA or DTPA-bisamide
  • calcium chelate complexes eg calcium DTPA, CaNaDTPA-bisamide
  • calcium or sodium salts eg, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate.
  • compositions can be used as wound coatings, adhesives, surgical and medical devices, artificial skin, bandages, foaming agents, films, antiadsorbents and implants, preferably wound coatings.
  • the invention also comprises the steps of a) fibrosis of acellular dermal matrix; b) mixing the fibrous acellular dermal matrix, a biocompatible polymer aqueous solution and water to form a mixture; c) heat treating the mixture at 30 to 60 ° C. for 1 to 30 minutes; d) sterilizing the heat-treated mixture using an E-beam, and e) sterilizing and ready-to-use, including heat-treating the sterilized mixture at 30 to 60 ° C. for 1 to 30 minutes.
  • the ratio of the short axis and the long axis of the fibrous acellular dermal matrix is 1:30 to 1: 2,000 ratio of the manufacturing method of the composition, characterized in that 70%, preferably 80%, most preferably 90% or more. to provide.
  • the mixture of step b) is 5-20% by weight of the fibrous acellular dermal substrate, 0.5-5% by weight of the biocompatible polymer and 75-94.5% by weight of water, preferably 6-16% by weight of the fibrous acellular dermal substrate, 1 to 3% by weight of biocompatible polymer and 81 to 93% by weight of water, more preferably 8 to 12% by weight of fibrized acellular dermal substrate, 1 to 2% by weight of biocompatible polymer and 86 to 91% by weight of water. .
  • the fibrous, cell-free dermal substrate has a long axis length of 500 to 3,000 ⁇ m, preferably 500 to 2,000 ⁇ m, and cuts the cell-free dermal substrate into a cutting mill, a food processor, an agate grinder, a freeze grinder, It is prepared by grinding with a micronizer, Vibratory micro mill, Jaw crusher, Mortar grinder, Planetary mill, Disk mill, Ball mill, Variable speed rotor mill, preferably Cutting mill.
  • the cell-free dermal matrix comprises a) removing the epidermis of allogeneic skin; b) removing the cells in the dermis; c) preparing a cryoprotectant by mixing glycerol, propylene glycol, and a base solvent or solution and dissolving in the solution such that the final concentration of maltitol, sucrose or sorbitol is 20 to 40% by weight; d) infiltrating the cryoprotectant into the skin from which the cells in the epidermis and dermis have been removed, and e) freeze drying the skin in which the cryoprotectant has infiltrated.
  • the maltitol was confirmed to prevent the browning phenomenon of the dermal tissue, it is characterized by using maltitol accordingly.
  • Mixing ratio of the glycerol, propylene glycol and the basic solvent or solution is characterized in that 0.5 to 2: 0.5 to 2: 6 to 10 by weight.
  • the basic solvent or solution is distilled water, physiological saline (normal saline), phosphate buffered solution (PBS), HBSS (Hank's balanced salt solution), TBS (Tris buffered saline), TAPS (N-Tris (hydroxy-methyl) methyl-3 -aminopropanesulfonic acid) buffer, Bicine (N, N-Bis (2-hydroxyethyl) glycine) buffer, HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) buffer, TES (NTris (hydroxymethyl) methyl- 2-aminoethanesulfonicd acid buffer, piperazine-N, N'-bis (2-ethanesulfonic acid) buffer, cacodylate buffer, MES (2- (N-morpholino) ethanesulfonic acid) buffer , Minimum Essential Media (MEM), Dulbecco's Modified Eagle Media (DMEM), RPMI1640, Iscove
  • the present invention provides a wound coating sheet comprising 75 to 85% by weight of cell-free dermal substrate, 15 to 25% by weight of biocompatible polymer and 3 to 8% by weight of water, having a length of 4 to 20 cm And a length of 4 to 20 cm and a thickness of 1.5 to 3 mm to provide a sheet for wound covering material.
  • the cell-free dermal matrix is characterized in that the fibrous cell-free dermal matrix.
  • the fibrous acellular dermal matrix of the present invention has a long axis length of 500 to 3,000 ⁇ m, preferably 500 to 2,000 ⁇ m, and a ratio of a short axis and a long axis of 1:30 to 1: 2,000 ⁇ m, preferably 1:30 to It is characterized in that 1: 1000 ⁇ m, most preferably 1:30 ⁇ 1:70 ⁇ m is 70% or more, preferably 80%, most preferably 90% or more.
  • the biocompatible polymer includes gelatin, hyaluronic acid, collagen, poloxamer or mixtures thereof.
  • the preferred biocompatible polymer is gelatin.
  • the strength of the gelatin is at least 250 Bloom, preferably at least 270 Bloom and the viscosity is 30 to 45 mps, preferably 33 to 42 mps, more preferably 35 to 40 mps and the pH is 5 to 6.5, preferably 5.5 It is characterized in that from 6 to.
  • the sheet may further comprise a pharmaceutically used antimicrobial agent, excipient and additive.
  • Suitable antimicrobials include biocides such as short-chain alcohols, benzoalkonium chloride (“BAC”), didecyl dimethyl ammonium chloride (“DDAC”) and zeolites (“CWT-A”). Include.
  • antibacterial agents are isothiazolones, alkyl dimethyl ammonium chlorides, triazines, 2-thiocyanomethylthio benzothiazoles, methylene bis thiocyanates, acrolein, dodecylguanidine hydrogen chloride, chlorophenols, quaternary ammonium salts, gluter Aldehyde, dithiocarbamate, 2-mercaptobenzothiazole, para-chloro-meth-xyllenol, silver, chlorhexidine, polyhexamethylene biguanide, n-halamine, triclosan, phospholipids, alpha hydroxy acids, 2 , 2-dibromo-3-nitrilopropionamide, 2-bromo-2-nitro-1,3-propanediol, farnesol, iodine, bromine, hydrogen peroxide, chlorine dioxide, vegetable oils, plant extracts, benzal Cornium chloride, chlorine, sodium hypochlorite, or combinations thereof
  • Suitable excipients are starch, cellulose, glucose, lactose, sucrose, gelatin, corn, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, as stabilizers, antioxidants, osmotic-modulating agents, buffers and pH-adjusting agents.
  • Suitable additives are physiologically biocompatible buffers (eg tromethamine hydrochloride), chelating agents (eg DTPA or DTPA-bisamide) or calcium chelate complexes (eg calcium DTPA, CaNaDTPA-bisamide) ) Or optionally calcium or sodium salts (eg, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate).
  • physiologically biocompatible buffers eg tromethamine hydrochloride
  • chelating agents eg DTPA or DTPA-bisamide
  • calcium chelate complexes eg calcium DTPA, CaNaDTPA-bisamide
  • calcium or sodium salts eg, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate.
  • the invention also comprises the steps of a) grinding the cell free dermal matrix; b) mixing the ground cell free dermal matrix, a biocompatible polymer aqueous solution and water to form a mixture, and c) applying the mixture to a mold (mold or frame) and freeze drying to prepare a sheet.
  • a method for producing a wound coating sheet is provided.
  • the mixture of step b) is 5-20% by weight of acellular dermal matrix, 0.5-5% by weight of biocompatible polymer and 75-94.5% by weight of water, preferably 6-16% by weight of acellular dermal matrix, biocompatible 1- 3 wt% and 81-93 wt% water, more preferably 8-12 wt% acellular dermal substrate, 1-2 wt% biocompatible polymer and 86-91 wt% water.
  • the sheet of step c) is 75 to 85% by weight of the cell-free dermal matrix, 15 to 25% by weight of the biocompatible polymer and 3 to 8% by weight of water, preferably 78 to 82% by weight of the cell free dermal matrix, biocompatible 18 22 wt% and 4-5 wt% water.
  • the cell-free dermal matrix is characterized in that the fibrous cell-free dermal matrix.
  • the fibrous acellular dermal matrix of the present invention has a long axis length of 500 to 3,000 ⁇ m, preferably 500 to 2,000 ⁇ m, and a ratio of a short axis and a long axis of 1:30 to 1: 2,000 ⁇ m, preferably 1:30 to It is characterized in that 1: 1000 ⁇ m, most preferably 1:30 ⁇ 1:70 ⁇ m is 70% or more, preferably 80%, most preferably 90% or more.
  • the cell-free dermal matrix of step a) of pulverizing the cell-free dermal matrix is characterized in that it is used to select a thickness of 1 mm or more, preferably 1.5 mm or more.
  • the cell-free dermal substrate crushing is a cutting mill, a food processor, agate grinder, freeze grinder, micronizer, Vibratory micro mill, Jaw crusher, Mortar grinder, Planetary mill, Disk mill, Ball mill, Variable speed rotor mill, preferably using a cutting mill (Cutting mill).
  • the cutting mill preferably uses a rotational speed of 500 rpm and operating conditions of 750 ⁇ m sieve size.
  • the cell-free dermal matrix comprises a) removing the epidermis of allogeneic skin; b) removing the cells in the dermis; c) preparing a cryoprotectant by mixing glycerol, propylene glycol, and a base solvent or solution and dissolving in the solution such that the final concentration of maltitol, sucrose or sorbitol is 20 to 40% by weight; d) infiltrating the cryoprotectant into the skin from which the cells in the epidermis and dermis have been removed, and e) freeze drying the skin in which the cryoprotectant has infiltrated.
  • the maltitol was confirmed to prevent the browning phenomenon of the dermal tissue, it is characterized by using maltitol accordingly.
  • Mixing ratio of the glycerol, propylene glycol and the basic solvent or solution is characterized in that 0.5 to 2: 0.5 to 2: 6 to 10 by weight.
  • the basic solvent or solution is distilled water, physiological saline (normal saline), phosphate buffered solution (PBS), HBSS (Hank's balanced salt solution), TBS (Tris buffered saline), TAPS (N-Tris (hydroxy-methyl) methyl-3 -aminopropanesulfonic acid) buffer, Bicine (N, N-Bis (2-hydroxyethyl) glycine) buffer, HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) buffer, TES (NTris (hydroxymethyl) methyl- 2-aminoethanesulfonicd acid buffer, piperazine-N, N'-bis (2-ethanesulfonic acid) buffer, cacodylate buffer, MES (2- (N-morpholino) ethanesulfonic acid) buffer , Minimum Essential Media (MEM), Dulbecco's Modified Eagle Media (DMEM), RPMI1640, Iscove
  • FIG. 1 is a photograph comparing the appearance of the fibrotic cell-free dermal matrix (A) and the cell-free dermal matrix (B) in the form of beads.
  • Figure 2 shows the long axis length and the distribution of the fibrous acellular dermal matrix particles.
  • Figure 3 shows the uniaxial length of the fibrotic acellular dermal matrix particles and their distribution.
  • Figure 4A shows the long, short axis ratio of the fibrotic acellular dermal matrix particles
  • Figure 4B shows the long, short axis ratio of the bead-shaped acellular dermal matrix particles
  • Figures 4C and 4D are fibrous acellular dermal matrix Micrograph of a bead-type acellular dermal matrix.
  • FIG. 5 is a photograph of the appearance of a paste containing fibrized acellular dermal matrix and gelatin.
  • Figure 6 shows the viscosity of the paste containing the fibrillated cell-free dermal substrate and gelatin, comparing the paste viscosity at the time of completion of production and 3 months after storage.
  • Figure 7 shows the extrusion force of the paste according to the temperature conditions at the time of sterilization and E-beam sterilization (times before and after sterilization of the E-beam paste at 45 °C temperature reaction is not E-beam sterilization This resulted in a lower extruding force value than the non-paste paste, which is expected to show lower extruding force values due to no cross-linking of the paste without E-beam sterilization. It is interpreted that the degree of hardening was slightly increased during the abnormal storage, thereby increasing the extrusion force value.
  • Figure 8 is a measurement of the gel fraction of the paste (crosslinked) according to the temperature conditions at the time of sterilization and E-beam sterilization over time.
  • FIG. 9 is a photograph showing the clinical effect of a wide and deep wound site of a paste containing fibrized acellular dermal matrix and gelatin.
  • Figure 10 is a photograph showing the clinical effect of the foot wound of the paste containing the fibrous acellular dermal matrix and gelatin.
  • FIG. 11 is a photograph showing the clinical effect of chronicized wound of a paste comprising fibrized acellular dermal matrix and gelatin.
  • FIG. 12 is a photograph showing the clinical effect of cut tissue of a paste containing fibrized acellular dermal matrix and gelatin.
  • Figure 13 is a photograph showing the clinical effect of the refractory wound of the paste containing the fibrous acellular dermal matrix and gelatin.
  • FIG. 14 is a photograph of the appearance of a sheet prepared using a paste containing fibrous acellular dermal matrix and gelatin.
  • 15 is a photograph observing the physical properties and shape of the sheet prepared by using a paste containing the fibrous acellular dermal matrix and gelatin in the saline solution for 1 hour.
  • Figure 16 confirms the storage stability according to the heat treatment before sterilization in terms of viscosity.
  • Figure 17 shows the storage stability of the heat treatment before sterilization in terms of gel fraction.
  • TPA texture profile analysis
  • a human-derived cell-free cell characterized by being sterilized and ready-to-use, applicable to curved or deeply cut wounds, and maintaining soft properties even during long-term or low temperature storage.
  • a composition comprising the dermis and gelatin.
  • the composition changes the physical properties such as cross-linking degree, curing degree, extrusion force, etc. by radiation sterilization, and accordingly minimize the change of composition properties by including a constant heat treatment step before and / or after radiation sterilization And stability could be improved.
  • the present invention is a composition
  • a composition comprising a human-derived acellular dermal matrix and gelatin, wherein the composition is sterile and ready-to-use, crosslinking degree after sterilization, Viscoelasticity, degree of cure, and / or change in extruding force value is 30% or less compared to the value before sterilization, and a composition and a method for producing the same.
  • the dermal tissue of the present invention may be bone, ligament, tendon or skin, and may be a dermal tissue derived from heterogeneous or allogeneic, preferably human dermal tissue.
  • the raw material strength of the gelatin is 250 Bloom or more, the viscosity is 30 ⁇ 45 mps, the pH is 5 ⁇ 6.5, it can be characterized in that the drying loss is 5 ⁇ 15%.
  • the gelatin, alginate, agarose, fibrin, collagen, fibronectin, polyglycolic acid, polylactic acid, hyaluronic acid, polyethylene glycol, chondroital Biocompatible polymers of, dermatan, polysaccharides, mucopolysaccharides, hydrogels, dextran, amylose, proteins, glycoproteins and derivatives thereof may be further included.
  • the constituent content of the composition may be 5 to 20% by weight of the acellular dermal matrix, 0.5 to 5% by weight of gelatin and 75 to 99.5% by weight of water, preferably 8 to 12% by weight of the acellular dermal matrix, gelatin 1 ⁇ 2 weight percent and 86-91 weight percent water.
  • the sterilization process may include a dry heating method, pasteurization method, radiation sterilization method, gas sterilization method and the like, and may preferably include a radiation sterilization method performed by E-beam irradiation.
  • E-beam irradiation was found to induce crosslinking in the cell-free dermis and gelatin compositions, resulting in changes in the viscoelasticity, degree of cure, and extruding force values correlated therewith.
  • the cross-linking by E-beam irradiation increased with longer storage period after manufacture of the finished product, especially when cross-linking increased at low temperature.
  • Measurement of physical property changes such as the degree of crosslinking, viscoelasticity, curing degree, and extrusion force is not limited to a specific experimental method. In the case of the degree of crosslinking, the gel fraction, swelling ratio, etc., which are indirect indicators, may be used.
  • the present invention provides a composition comprising a fibrous Acellular Dermal Matrix and a biocompatible polymer, wherein the ratio of a short axis and a long axis is 1:30 to 1: 2,000. It relates to a composition and a method for producing the same, characterized in that the proportion of the thing is 70%, preferably 80%, most preferably 90% or more.
  • the cell free dermal matrix of the present invention is in fibrotic form.
  • the dermis having a thickness of 1 mm or more was used in order to prepare a fibrous acellular dermal matrix.
  • the dermal matrix having a thickness of 1 mm or more was acellularized and then dried or lyophilized to contain an appropriate moisture content, for example, 5 to 10% of moisture.
  • the cell-free dermal substrate was pulverized and fibrized using a mill such as a cutting mill, a food processor, agate grinder, or freeze grinder.
  • the length ratio of the short axis and the long axis of the fibrous acellular dermal matrix of the present invention may be 70% or more of the ratio of 1:30 to 1: 2,000, preferably the ratio of the short axis and the long axis is 1:30 to 1 Fiber particles of: 2,000 can account for 80% of the total composition.
  • the fibrous cell-free dermal matrix under the above conditions coexists in the form of a bundle of thread and a single, long and long fiber, and is excellent in preservation of the body when used as a wound coating material, and well maintained in a humid environment. It shows a structure that is stably supported with a polymer even without bonding.
  • the present invention provides a wound coating sheet comprising 75 to 85% by weight of acellular dermal matrix (ADM), 15 to 25% by weight of biocompatible polymer and 3 to 8% by weight of water, having a transverse length of 4 to It relates to a sheet for wound covering, characterized in that it is 20 cm in length and 4 to 20 cm in length and 1.5 to 3 mm in thickness.
  • ADM acellular dermal matrix
  • the paste-free dermal matrix and the biocompatible polymer-containing composition are applied to a specific mold (mold or frame) and lyophilized to prepare a wound coating sheet.
  • the lyophilization may be performed until the moisture content of the sheet is 3 to 8%, preferably 4 to 5%.
  • the skin tissues isolated from the carcasses were purchased from EURO skin bank, Allosource, CTS, and then tissues having a thickness of 1 mm or more were selected and used for the preparation of acellular dermal matrix.
  • the skin tissue was reacted at 38 ° C. for 6 hours to 24 hours using 1M NaCl solution, and then the epidermis was removed using forceps.
  • the epidermis from which the epidermis was removed was washed with phosphate buffer solution, and then reacted with 0.5% SDS (Sodium dodecyl sulfate) at room temperature for 1 hour to remove the cells in the dermis.
  • SDS Sodium dodecyl sulfate
  • the cryoprotectant was infiltrated into the dermis for 12 hours in a 4 ° C. low temperature reactor.
  • the penetrated dermis was placed in a taibag (Korea Advanced Materials, Korea) and placed in a lyophilizer with a vacuum of 5 torr for 24 hours to prepare a freeze-dried acellular dermal matrix.
  • Lyophilized acellular dermal substrates were triturated using a cutting mill (Pulverisette 19, FRITSCH, Germany) to fiberize.
  • Figure 1 compares the appearance of the fibroblast-free dermal matrix (A) and the bead-shaped acellular dermal matrix (B) prepared by the above method.
  • the fibrous cell-free dermal matrix was in the form of a cotton, ie, entangled fibers, and had a larger volume (surface area) and a more stable structural shape compared to the bead type.
  • the bead type was selected from acellular dermal substrates with a thickness of 1 mm or less, and then acellularized, lyophilized, ground, and prepared in the same manner as described above.
  • the long axis length and distribution of the fibrous acellular dermal matrix are about 33% for 500-1,000 ⁇ m, about 30% for 1,000-1,500 ⁇ m, about 27% for 1,500-2,000 ⁇ m, and 2,000-2,500 ⁇ m.
  • the length and distribution of the fibrous acellular dermal matrix are 10-20 ⁇ m, about 20%, 20-30 ⁇ m, about 39%, 30-40 ⁇ m, about 28%, and 40-50 ⁇ m.
  • About 9% and 50-60 ⁇ m were about 4%. That is, it was confirmed that about 70% of the fiber particles have a short axis length of 20 to 40 ⁇ m. That is, fiber particles having a short axis length of 20 to 40 ⁇ m accounted for 70% of the whole particles.
  • the intestinal and shortening ratios of the fibroblasted dermal matrix were about 3% for 20-30: 1, about 23% for 30-40: 1, about 33% for 50-50: 1, and 50%.
  • ⁇ 60: 1 was measured at about 34% and 60-70: 1 at about 7%. That is, the fiber particles having a long and short axis ratio of 40 to 60: 1 accounted for 70% of the total particles, and the fiber particles having 40 to 70: 1 accounted for about 75%.
  • the length and shortening ratio of the bead-type acellular dermal matrix about 90% of the particles showed a ratio of 1-2: 1.
  • the bead form was found to be in the form of particles close to the sphere.
  • FIG. 4C shows a micrograph of a fibrous acellular dermal matrix
  • FIG. 4D shows a micrograph of a bead-shaped acellular dermal matrix.
  • the paste of five samples was prepared by the method of Example 3, and the paste viscosity after initial storage and after 3 months storage was measured.
  • the viscosity of the paste was measured using the torque generated by the viscosity of the fluid by rotating the disk in the fluid according to the rotational viscometer method (KEPCO general test method) and the circulator (DV2THB Viscometer, Brookfield, USA) circulator) conditions were repeatedly measured at a viscosity of 15 g at 15 rpm and 25 ° C.
  • both the initial state and the paste after 3 months storage showed a viscosity between 20,000 and 25,000 cps. That is, it was confirmed that the viscosity physical properties of the paste remained unchanged for three months.
  • the extrusion force is a measure of the load value generated for a given time, force, and force when the work is applied to the specimen in a constant direction.
  • the extruding force of the paste was compared according to the radiation sterilization in the paste preparation step or the reaction temperature conditions before and after the radiation sterilization.
  • UPM universal testing machine
  • the extrusion force of the paste without E-beam sterilization was maintained at about 10 to 15 N (average 12.1 N), and did not show any increase or decrease with time.
  • the paste having a temperature reaction of 10 ° C. increased the magnitude of the extrusion force with time.
  • the extruding force value reached about 40 N, and the average value was 26.82 N.
  • the paste with temperature reaction at 25 °C showed an extrusion force value of about 15-20 N, an average value of 16.06 N and did not show any increase or decrease with time.
  • the paste having a temperature reaction at 45 ° C. exhibited an extruding force value of about 6 to 8 N, an average of 6.645 N, and the change in extruding force over time showed the most gentle pattern among the experimental groups (FIG. 7). This means that the paste having a temperature reaction at 45 ° C. does not gel and the particles are evenly distributed.
  • pastes not treated with E-beam and pastes with temperature reaction at 10 °C and 25 °C together with E-beam sterilization showed that the extrusion force curve was not smooth but sharply bent because the particles were recognized by gelation. It became.
  • the average extruding force of the paste without e-beam sterilization and the paste with e-beam sterilization at 45 ° C. was about 12.1 N and 6.645 N, respectively, and the change of the extrusion force value was about 45%.
  • the gel fraction (crosslinking degree) of the cell-free dermal matrix paste was measured according to the reaction temperature before and after radiation sterilization (FIG. 8).
  • the gel fraction After measuring the initial weight of the paste reacted at each temperature, it was immersed in tertiary distilled water (Deionized water) and treated at room temperature for 48 hours or 72 hours in a constant temperature shaker (60rpm). After the reaction, the insoluble portion was filtered through a mesh and dried in a 50 ° C. dry oven.
  • the gel fractions of the pastes reacted at 5, 15, 25, 35, 45 and 55 ° C. were analyzed to be 99, 96, 78, 18, 6 and 3%, respectively. Accordingly, the higher the reaction temperature before and after the sterilization of the paste, the lower the gel fraction, that is, the degree of crosslinking was confirmed.
  • the gel fraction of the paste without E-beam sterilization was about 2%, which was analyzed as not crosslinking.
  • the gel fraction was about 79%.
  • the gel fractions of the paste without e-beam sterilization and the pastes subjected to the e-beam sterilization at 45 ° C. and 55 ° C. were about 2%, 6.3%, and 3%, respectively. Analyzed in%.
  • the gel fraction was increased by crosslinking, but when the heat was applied at 45 ° C or 55 ° C during sterilization of the E-beam, it was confirmed that the gel fraction was reduced by inhibiting the crosslinking. .
  • Bone and tendon wounds were treated with 2 cc of paste of the present invention for 2 weeks with Curacao bag. After two weeks, it was confirmed that granulation tissue of the site where the paste was grafted was reshaped to cover the bone exposure (FIG. 9).
  • the paste of the present invention was treated with a yugotool barrier and a curabag on a pocket wound due to foot trauma. As a result, it was confirmed that granulation tissue was formed on the 7th day of application, so that the depth of the pocket wound was shallow, and after that, it was confirmed that the foot ulcer was treated without surgery (FIG. 10).
  • the paste and curabag of the present invention were treated in parallel in the deeply dug wounds of patients who had cut toes due to diabetic complications. As a result, it was confirmed that blood vessels were introduced into the paste on the 9th day of treatment to promote granulation tissue formation and to be cured after 44 days (FIG. 12). Since the cure of the toe cut wound is known to take an average of about 90 days, it was confirmed that the healing period is reduced by half when the paste of the present invention is applied.
  • Flap was performed on the exposed bone of soft tissue, but the tunneling wound was reopened, and granulation tissue was not formed.
  • the paste of the present invention is applied to a case that is difficult to be cured by the existing treatment and is difficult to operate, it was confirmed that blood vessels rise and granulation tissue is formed and cured by the final suture (FIG. 13).
  • the paste prepared according to Example 3 was lyophilized after the horizontal, vertical and thickness values were applied to 10 cm, 10 cm and 2.5 mm molds.
  • FIG. 14 is an external photograph of a sheet manufactured by the above method
  • FIG. 15 is a photograph taken after hydration of the sheet in a saline solution for 1 hour. After 1 hour of hydration, it was confirmed that the form of the sheet was maintained without a great change in appearance.
  • the viscosity of the paste was measured using the torque generated by the viscosity of the fluid by rotating the disk in the fluid according to the Rotational Viscometer Method (KEPCO General Test Method) and the circulator of the DVK2THB Viscometer (Brookfield, USA) ) The conditions were 15 rpm and 25 ° C., and the viscosity of the sample 1 g was repeatedly measured. Samples were subjected to comparative testing of samples that were processed at optimized temperature (45 ° C.) and those that were not before sterilization.
  • the sample heat-treated as shown in FIG. 16 showed a viscosity of 20,000-3,000 cps similar to the initial state even after radiation sterilization, while in other samples, crosslinking occurred after radiation sterilization, and thus the viscosity was measured to be higher than 3 times. .
  • the gel fraction (crosslinked degree) of the cell-free dermal matrix paste with or without heat treatment before radiation sterilization was measured.
  • the gel fraction was immersed in tertiary distilled water (Deionized water) and treated at room temperature for 48 hours or 72 hours in a constant temperature shaker (60rpm). After the reaction, the insoluble portion was filtered through a mesh and dried in a 50 ° C. dry oven.
  • the gel fraction after radiation sterilization was maintained at about 6% over time, whereas in other samples, the gel fraction was 70-85% after radiation sterilization, resulting in crosslinking. It was confirmed.
  • the paste was measured using a texture profile analysis (TPA) to compare the adhesion in preparing ADM pulverized into fibrous and ADM pulverized into beads (FIG. 18).
  • TPA texture profile analysis
  • the sample was pressed with a compression plate at a rate of 50 mm / min, and the force required to drop the sample and the compression plate was expressed as a negative force area.
  • the paste prepared from ADM pulverized into fibrous form appeared to be about 26,000 g.s, while the paste prepared from ADM pulverized into bead form was found to have low adhesion at about 3,000 g.s.
  • the wound coating paste according to the present invention can be easily applied to a difficult or topical area where the conventional sheet-shaped dermal matrix is difficult to apply, and is a ready-to-use product that can be used immediately and is easy to use and has a serious wound. Quick cure is possible without surgery.
  • the wound coating sheet according to the present invention is excellent in bioadhesiveness and does not release the polymer structure well even in a long rehydration (excellent) process, it is excellent in shape retention ability during the professional procedure.
  • the sheet can adjust the width, length and thickness size required by the wound site or surgical characteristics can be improved quality of the wound treatment.

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Abstract

The present invention relates to a composition comprising a human-derived acellular dermal matrix and gelatin. More specifically, the present invention relates to: a composition in which the degree of crosslinking, viscoelasticity, degree of cure and/or change in the extrusion load value of the composition of the fibrous acellular dermal matrix and the gelatin after sterilization is 30% or less, preferably 20% or less, most preferably 10% or less compared to the composition before sterilization; a method for producing the composition; and a sheet using the same. A wound dressing paste of the present invention is easily applicable to areas on which a conventional sheet-type acellular dermal matrix is difficult to apply, or to localized areas, is easy to use, being an out-of-the-box ready-to-use product, and is capable of quickly and fully curing even severe wounds without surgery. Also, the wound dressing paste is excellent in stably maintaining properties even in long-term or low-temperature storage, softly spreading and tightly adhering when applied to localized areas. Therefore, the wound dressing paste protects the wound area from external contaminants and maintains a moist environment, helping the wound area to be efficiently treated. In addition, a wound dressing sheet of the present invention has excellent bioadhesive properties and excellently retains shape during specialist procedures since the polymer structure hardly breaks down even during a long rehydration process. Also, the size of the wound dressing sheet can be adjusted as required in terms of length, width, and thickness for the wound area or specific features of surgery, thereby improving the quality of wound treatment.

Description

섬유화 무세포 진피 기질 및 생체적합성 고분자를 포함하는 조성물 및 이의 제조 방법Compositions comprising the fibroblast-free dermal matrix and biocompatible polymers and methods for preparing the same
본 발명은 인체 유래 무세포 진피 기질 및 젤라틴을 포함하는 조성물에 관한 것으로, 보다 자세하게는 섬유화된 무세포 진피 기질과 젤라틴 조성물이 멸균 후 가교도, 점탄성, 경화도 및/또는 압출력 값의 변화도가 멸균 전 값에 비해 30% 이하, 바람직하게는 20% 이하, 가장 바람직하게는 10% 이하인 것을 특징으로 하는 조성물, 이의 제조방법 및 이를 이용한 시트 제조에 관한 것이다.The present invention relates to a composition comprising a human-derived acellular dermal matrix and gelatin, and more particularly, the degree of crosslinking, viscoelasticity, hardening and / or extruding force values after sterilization of the fibrous acellular dermal matrix and gelatin composition. Is 30% or less, preferably 20% or less, most preferably 10% or less, compared to the value before sterilization, and relates to a composition, a method for preparing the same, and a sheet production using the same.
창상(Wound)은 외부의 압력에 의하여 피부 또는 다른 조직이 끊기거나 결손 되어 조직의 연속성이 파괴되는 상태로, 통상 피부의 진피층까지 손상되어 피부가 열리는 현상을 말한다. 창상 수술은 기본적으로 창상 부위를 봉합 및 드레싱 하여 외부 환경에 노출되는 것을 차단함으로써 감염을 예방하고 염증 반응을 억제한다.Wound is a state in which the continuity of the tissue is broken by breaking or deficiency of the skin or other tissues by external pressure, and usually means damage to the dermal layer of the skin and opening of the skin. Wound surgery basically prevents infection and suppresses inflammatory reactions by suturing and dressing wound sites to block exposure to the outside environment.
창상 피복재의 재료는 크게 동종진피와 이종진피 재료로 나눌 수 있으며, 이의 재료에서 특정 고분자를 추출하는 방법과 진피 기질(matrix)을 이용하는 방법으로 창상 피복재를 제조할 수 있다. 진피 조직은 80~90%의 콜라겐(Collagen), 엘라스틴(Elastin) 및 글리코사미노글리칸(Glycosaminoglycan; GAG)으로 구성되어 있다.The material of the wound coating material can be largely divided into allogeneic and heterogeneous dermal materials. The wound coating material can be manufactured by extracting a specific polymer from the material and by using a dermal matrix. Dermal tissue is composed of 80-90% collagen, elastin and glycosaminoglycan (GAG).
한편, LifeCell사는 1994년에 사체에서 채취한 피부조직을 무세포화 및 동결건조하여 무세포 진피 기질(Acellular Dermal Matrix; ADM)을 제품화(Alloderm(allograft))하여 화상치료, 피부재건 등의 용도로 사용하였다. Alloderm(allograft)은 이종 제품보다 안전하며 월등히 나은 생착률과 치유 효과를 보였다. 이와 유사한 제품으로 이식재로 사용되는 Bard Dabol사의 AlloMax, 접착성이 증대된 Ethicon사의 FlexHD 등의 제품이 개발되었다. On the other hand, LifeCell Inc. acemented and lyophilized skin tissues obtained from dead bodies in 1994 to commercialize Acellular Dermal Matrix (ADM) for use in burn treatment and skin reconstruction. It was. Alloderm (allograft) is safer than heterogeneous products and has significantly better engraftment and healing effects. Similar products include Bard Dabol's AlloMax, which is used as an implant, and Ethicon's FlexHD with increased adhesion.
하지만, 상기와 같은 시트형 무세포 진피 기질의 피복재는 굴곡지거나 깊게 파인 창상부위에 사용하기에 어려움이 있으며 창상 부위의 크기와 모양에 따라 조직 시트를 자르거나, 여러 장을 붙여서 사용해야 하므로 효율성이 떨어진다.However, the coating material of the sheet-shaped acellular dermal matrix as described above is difficult to be used in the wound or deeply cut wounds, and the efficiency of the sheet-shaped dermal matrix is poor because the tissue sheet must be cut or used in accordance with the size and shape of the wound.
이에 따라, LifeCell사는 1999년에 무세포 진피 기질을 입자화 하는 기술을 출원하였고(US6933326B1), 2008년에 입자화된 무세포 진피 기질과 산성 용액이 혼합된 조성물에 관한 특허를 출원(US9382422B2)하였으며 미립자성 주사제제(injectable micronized) 형태의 AlloDerm(Cymetra) 제품을 출시하였다. 이 외 Wright Medical Group사가 무세포 진피 기질의 시트형인 Graftjacket 제품을 주사제제(injectable) 형태로 개발하여 Graftjacket Xpress 제품을 출시하였다.Accordingly, LifeCell applied for a technique for granulating a cell-free dermal matrix in 1999 (US6933326B1), and in 2008 a patent for a composition containing a mixture of granulated cell-free dermal substrate and an acidic solution (US9382422B2). AlloDerm (Cymetra) products in the form of injectable micronized are introduced. Wright Medical Group Inc. has also developed Graftjacket Xpress, an injectable form of Graftjacket, a sheet of cell-free dermal matrix.
또한, 한국의 엘앤씨바이오사는 2014년에 무세포 진피 기질을 입자화 한 후, 히알루론산을 가교 결합시킨 조성물에 관한 특허를 출원하였다(KR10-1523878B1).In addition, L & C Bio of Korea has applied for a patent on a composition in which the cell-free dermal matrix was granulated in 2014, and then cross-linked hyaluronic acid (KR10-1523878B1).
하지만, 상기와 같이 무세포 진피 기질 입자와 생체적합성 고분자를 가교결합시킨 조성물은 구조적 안정성은 높으나 더불어 가교도, 점탄성, 경화도 및 압출력이 높아져 필러, 보형물 등의 용도로는 적합하나 창상 피복재로는 부족한 측면이 있으며, 미립자성(micronized) 형태의 무세포 진피 기질은 액상 형태로 창상 부위에 고정되지 못하고 드레싱 밖으로 흘러나오는 등 창상에 골고루 분포되지 못한다는 단점이 있다. However, the composition which crosslinked the cell-free dermal matrix particles and the biocompatible polymer as described above has high structural stability but also has high crosslinking degree, viscoelasticity, hardenability, and extruding force, making it suitable for fillers, implants, etc. There is a disadvantage in that the micronized acellular dermal matrix has a disadvantage in that it is not evenly distributed on the wound, such as flowing out of the dressing and not fixed to the wound site in liquid form.
또한, 무세포 진피 기질 입자와 생체적합성 고분자 조성물에 인위적으로 가교제를 첨가하지 않더라도 멸균 용도로 E-beam 등을 조사할 시, 가교결합이 일어나며 이의 조성물은 저장기간이 길어 질수록 경화도가 증가되는 등의 물성 혹은 제형 변화가 일어난다. 경화도가 증가된 조성물은 발림성이 떨어져 창상 피복재용 페이스트, 젤, 연고, 에멀젼, 크림 및 현탁액의 주사제로 사용하기에 적합하지 않다.In addition, crosslinking occurs when the E-beam is irradiated for sterilizing purposes even without artificially adding a crosslinking agent to the cell-free dermal matrix particles and the biocompatible polymer composition. Changes in physical properties or dosage form occur. Compositions with increased curability have poor applicability and are not suitable for use as injections for wound coating pastes, gels, ointments, emulsions, creams and suspensions.
본 발명자들은 창상 피복재로 적합한 무세포 진피 기질을 개발하기 위하여, 무세포 진피 기질과 지혈성능이 우수한 생체적합성 고분자 즉, 젤라틴을 조합하여 페이스트 형태의 조성물을 연구하였다. 하지만, 방사선 멸균 후 장기간(1년 이상) 또는 저온에서 보관 시 경화도와 압출력이 증가하여 조성물의 안정성이 떨어지는 양태를 확인하였다.In order to develop a cell-free dermal matrix suitable as a wound coating material, the present inventors studied a composition in the form of a paste by combining a cell-free dermal matrix and a biocompatible polymer having excellent hemostatic properties, that is, gelatin. However, after the radiation sterilization for a long time (more than 1 year) or at low temperatures, the hardness and the extrusion force was increased to confirm an embodiment of the stability of the composition.
이에, 본 발명자들은 창상 부위의 굴곡, 크기 및 모양에 구애받지 않고 사용 가능하며 부드럽고(Smooth) 발림성이 좋으며 안정성이 높은 페이스트(Paste) 제형의 조성물을 개발하고자 예의 노력한 결과, 무세포 진피 기질, 바람직하게는 섬유화된 무세포 진피 기질과 생체적합성 고분자, 바람직하게는 알지네이트, 피브린, 콜라겐, 피브로넥틴, 폴리글리콜산, 폴리락틱산 및 히알루론산, 더욱 바람직하게는 젤라틴이 혼합된 조성물의 멸균, 바람직하게는 방사선 멸균 전 및/또는 후 단계에 일정한 열 처리를 함으로써 가교도가 억제되어 부드러운 페이스트의 물성이 유지되고 보관 안정성이 향상되는 것을 확인하고, 본 발명을 완성하게 되었다. Accordingly, the present inventors have made efforts to develop a composition of a paste formulation that can be used regardless of the curvature, size and shape of the wound site, is smooth and has good spreadability, and has high stability. Preferably, the sterilization of the composition in which the fibrous acellular dermal matrix and the biocompatible polymer, preferably alginate, fibrin, collagen, fibronectin, polyglycolic acid, polylactic acid and hyaluronic acid, more preferably gelatin are mixed, preferably By constant heat treatment before and / or after the radiation sterilization, the degree of crosslinking was suppressed, so that the physical properties of the soft paste were maintained and the storage stability was improved, thereby completing the present invention.
또한, 본 발명자들은 창상 부위에 적합한 가로, 세로 및 두께 길이를 조절할 수 있는 창상 피복재용 시트를 개발하고자 예의 노력한 결과, 상기 무세포 진피 기질과 젤라틴 조성물을 페이스트화한 후 금형(mold 또는 frame)에 도포 및 동결건조하여 시트를 제조하고, 상기 시트가 장시간 수화(rehydration) 과정에서도 안정하며 생체 접착성이 뛰어나고 치료 효과가 우수한 것을 확인하고, 본 발명을 완성하게 되었다.In addition, the present inventors have made diligent efforts to develop a wound coating sheet capable of adjusting the width, length and thickness of the wound suitable for the wound site, and then paste the cell-free dermal matrix and gelatin composition into a mold (mold or frame). The sheet was prepared by coating and lyophilization, and it was confirmed that the sheet is stable even in a long time hydration (rehydration) process, has excellent bioadhesiveness and excellent therapeutic effect, and completed the present invention.
발명의 요약Summary of the Invention
본 발명의 목적은 굴곡지거나 깊이 파인 창상 부위에 효과적인 피복재로서, 멸균되고 즉시 사용 가능하며(ready-to-use) 안정성이 향상된 무세포 진피 기질과 젤라틴을 포함하는 조성물을 제공하는 데 있다.It is an object of the present invention to provide a composition comprising a cell-free dermal matrix and gelatin that is sterile, ready-to-use and has improved stability as an effective coating material on curved or deeply cut wounds.
본 발명의 다른 목적은 상기 조성물의 제조 방법을 제공하는데 있다.Another object of the present invention is to provide a method for preparing the composition.
본 발명의 또 다른 목적은 무세포 진피 기질의 단축과 장축의 비율이 1:30 내지 1:2,000인 것의 비율이 70%, 바람직하게는 80%, 가장 바람직하게는 90% 이상인 것을 특징으로 하는 섬유화된 무세포 진피 기질과 생체적합성 고분자를 포함하는 조성물을 제공하는 데 있다.Another object of the present invention is a fibrosis, characterized in that the ratio of the short axis and the long axis of the acellular dermal matrix is 1:30 to 1: 2,000 is 70%, preferably 80%, most preferably 90% or more. To provide a composition comprising a cell-free dermal matrix and a biocompatible polymer.
본 발명의 또 다른 목적은 상기 섬유화된 무세포 진피 기질과 생체적합성 고분자를 포함하는 조성물의 제조 방법을 제공하는데 있다.Still another object of the present invention is to provide a method for preparing a composition comprising the fibrized cell-free dermal matrix and a biocompatible polymer.
본 발명의 또 다른 목적은 페이스트 형태의 무세포 진피 기질을 이용하여 제조한 시트로서 무세포 진피 기질 75 내지 85 중량%, 생체적합 고분자 15 내지 25 중량% 및 3 내지 8 중량%의 물을 포함하며 가로 길이가 4~2O cm 이며 세로 길이가 4~20 cm이며 두께가 0.5~3 mm인것을 특징으로 하는 창상 피복재용 시트를 제공하는데 있다.Another object of the present invention is a sheet prepared using a cell-free dermal substrate in the form of a paste containing 75 to 85% by weight of the cell-free dermal substrate, 15 to 25% by weight of the biocompatible polymer and 3 to 8% by weight of water It is to provide a sheet for wound covering, characterized in that the horizontal length is 4 ~ 20 cm, the vertical length is 4 ~ 20 cm and the thickness is 0.5 ~ 3 mm.
본 발명의 또 다른 목적은 상기 시트의 제조 방법을 제공하는데 있다.Another object of the present invention is to provide a method for producing the sheet.
상기 목적을 달성하기 위하여, 본 발명에서는, 무세포 진피 기질과 젤라틴을 포함하는 조성물로서, 상기 조성물은 멸균되고 즉시 사용 가능한(ready-to-use) 것이며, 멸균 후 가교도, 점탄성, 경화도 및/또는 압출력 값의 변화도가 멸균 전 값에 비해 30% 이하인 것을 특징으로 하는 조성물을 제공한다.In order to achieve the above object, in the present invention, as a composition comprising a cell-free dermal matrix and gelatin, the composition is sterile and ready-to-use, after sterilization crosslinking, viscoelasticity, degree of cure and And / or a change in extrusion force value is 30% or less compared to the value before sterilization.
본 명세서에서 "즉시 사용 가능한(ready-to-use)"은 사용 직전 용질 예를 들어, 고체 입자 및 동결 건조된 성분을 용매에 용해할 목적, 또는 특정 성분 예를 들어, 상온에서 변형되기 쉬운 단백질, 기타 성분 등을 혼합할 목적으로, 수행하는 재수화(reconstitute)하는 단계가 포함되지 않는 것을 의미한다. 시트의 경우, 수화(rehydration) 후 바로 사용하는 것을 의미한다.“Ready-to-use” herein is intended to dissolve solutes, eg, solid particles and lyophilized ingredients, in a solvent immediately prior to use, or proteins that are susceptible to modification to certain ingredients, such as room temperature. For the purpose of mixing other components, etc., it means that the reconstitute step is not included. In the case of a sheet, it is meant to be used immediately after rehydration.
본 발명의 즉시 사용 가능한 조성물은 재수화(reconstitute) 단계 없이 즉시 투여 가능한 멸균성 주사제제 또는 에어로졸 형태의 용액, 에멀젼, 크림, 연고, 페이스트 또는 겔을 포함한다. 또한, 시트의 경우에는 기타 물질의 첨가 없이 수화(rehydration) 단계 후 즉시 사용 가능한 형태를 포함한다.Ready-to-use compositions of the present invention include solutions, emulsions, creams, ointments, pastes or gels in the form of sterile injectables or aerosols which can be administered immediately without a reconstitute step. In addition, the sheet includes a form that can be used immediately after the rehydration step without addition of other substances.
본 발명의 가교도는 가교의 정도 즉, 교차결합의 정도를 의미하는 것으로, 교차결합(cross-linking)은 고분자 사슬이 말단 이외의 임의 위치에서 서로 직접 또는 수개의 결합을 매개하여 화학적으로 연결하는 것을 의미한다. 일반적으로 가교도가 증가하면 점탄성, 경화도 및 압출력의 수치가 증가하며 이로 인해 내열성, 내구성, 가공성 등의 구조적 안정성 등이 증진된다. 반면에, 가교도가 감소하면 점탄성, 경화도 및 압출력의 수치가 감소하여 자유로운 변형성, 유연성, 접착성이 등이 증진된다. 한편, 가교도, 점탄성, 경화도 및 압출력은 너무 높거나 낮아지는 것에 따라 물성이 딱딱하게 굳는 현상이 일어나거나 체내 분해율이 증가되는 문제를 초래하기도 한다. 따라서, 본 발명에서는 조성물의 최적의 물성 상태를 확인하고, 멸균 등의 가공 과정 또는 저장 과정에서도 상기 물성의 변화 즉, 가교도, 점탄성 경화도 및 압출력 값의 변화도가 30% 이하, 바람직하게는 20% 이하, 가장 바람직하게는 10% 이하인 조성물을 제공한다. The degree of crosslinking of the present invention refers to the degree of crosslinking, that is, the degree of crosslinking, and cross-linking means that the polymer chains are chemically linked to each other directly or through several bonds at any position other than the terminal. it means. In general, as the degree of crosslinking increases, the values of viscoelasticity, curing degree, and extrusion force increase, thereby improving structural stability such as heat resistance, durability, and workability. On the other hand, when the degree of crosslinking decreases, the values of viscoelasticity, hardenability and extrusion force decrease, thereby improving free deformation, flexibility, and adhesiveness. On the other hand, the degree of crosslinking, viscoelasticity, hardenability, and extruding force may be too high or too low, resulting in a hardening of physical properties or an increase in decomposition rate in the body. Accordingly, in the present invention, the optimum physical state of the composition is confirmed, and the change in the physical properties, that is, the degree of change in the crosslinking degree, the viscoelastic curing degree and the extruding force value is 30% or less, preferably during processing or storage such as sterilization. Provides a composition that is at most 20%, most preferably at most 10%.
상기 조성물은 무세포 진피 기질 5~20 중량%, 젤라틴 0.5~5 중량% 및 물 75~94.5 중량%, 바람직하게는 무세포 진피 기질 6~16 중량%, 젤라틴 1~3 중량% 및 물 81~93 중량%, 더욱 바람직하게는 무세포 진피 기질 8~12 중량%, 젤라틴 1~2 중량% 및 물 86~91 중량%을 포함한다.The composition is 5-20% by weight of acellular dermal matrix, 0.5-5% by weight of gelatin and 75-94.5% by weight of water, preferably 6-16% by weight of acellular dermal matrix, 1-3% by weight of gelatin and water 81- 93% by weight, more preferably 8-12% by weight of acellular dermal matrix, 1-2% by weight of gelatin and 86-91% by weight of water.
상기 무세포 진피 기질은 섬유화된 무세포 진피 기질인 것을 특징으로 한다. The cell-free dermal matrix is characterized in that the fibrous cell-free dermal matrix.
용어 "섬유화된 무세포 진피 기질"은, 입자화된 무세포 진피 기질의 개별 낱개 형태가 구형 또는 유선형의 입자가 아닌 실처럼 가늘고 긴 섬유 형태를 의미한다.The term "fibrillated acellular dermal matrix" refers to a fibrous, elongated fibrous form in which the individual individual forms of the granulated acellular dermal matrix are not spherical or streamlined particles.
본 발명의 상기 섬유화된 무세포 진피 기질은 장축 길이가 500 내지 3,000 ㎛, 바람직하게는 500 내지 2,000 ㎛, 단축과 장축의 비율이 1:30~1:2,000 ㎛, 바람직하게는 1:30~1:1,000 ㎛, 가장 바람직하게는 1:30~1:70 ㎛인 것이 70% 이상, 바람직하게는 80%, 가장 바람직하게는 90% 이상인 것을 특징으로 한다.The fibrous acellular dermal matrix of the present invention has a long axis length of 500 to 3,000 μm, preferably 500 to 2,000 μm, and a ratio of short axis and long axis is 1:30 to 1: 2,000 μm, preferably 1:30 to 1 It is characterized by that it is 70% or more, preferably 80%, most preferably 90% or more: 1: 1,000 micrometers, Most preferably, 1: 30-1: 70 micrometers.
상기 젤라틴의 강도는 250 Bloom 이상, 바람직하게는 270 Bloom 이상이며 점도는 30 내지 45 mps, 바람직하게는 33 내지 42 mps, 더욱 바람직하게는 35 내지 40 mps이고 pH가 5 내지 6.5, 바람직하게는 5.5 내지 6인 것을 특징으로 한다.The strength of the gelatin is at least 250 Bloom, preferably at least 270 Bloom and the viscosity is 30 to 45 mps, preferably 33 to 42 mps, more preferably 35 to 40 mps and the pH is 5 to 6.5, preferably 5.5 It is characterized in that from 6 to.
상기 멸균은 건조가열 멸균, 저온살균 멸균, 방사선 멸균, 가스 멸균 등을 포함할 수 있으며 바람직하게는 방사선 멸균, 더욱 바람직하게는 E-beam을 이용한 방사선 멸균이다.The sterilization may include dry heat sterilization, pasteurization sterilization, radiation sterilization, gas sterilization and the like, and preferably radiation sterilization, more preferably radiation sterilization using E-beam.
상기 E-beam 이용한 방사선 멸균 전 및/또는 후 단계에 30 내지 60℃, 바람직하게는 35 내지 55℃, 더욱 바람직하게는 40 내지 50℃의 열처리 공정을 포함한다. 또한, 멸균 중에도 상기 온도를 유지하는 것이 바람직하다.The heat treatment step of 30 to 60 ℃, preferably 35 to 55 ℃, more preferably 40 to 50 ℃ before and / or after the sterilization of the radiation using the E-beam. Moreover, it is preferable to maintain the said temperature also during sterilization.
상기 열처리는 1 내지 180 분간, 바람직하게는 30 내지 150 분간, 더욱 바람직하게는 60 내지 120 분간 수행된다.The heat treatment is carried out for 1 to 180 minutes, preferably 30 to 150 minutes, more preferably 60 to 120 minutes.
상기 조성물은 수용액(aqueous solution), 현탁액(suspention), 유화액(emulsion), 페이스트(paste), 크림(cream), 밤(balm), 연고(ointment), 거품(foam), 시트(sheet), 겔(gel), 검(gum), 스프레이(spray), 슬러리(slurry), 필름(film), 과립(granule), 패치(patch), 분말(powder) 등의 형태로 제조된다. 특히, 본 발명의 바람직한 양태는 페이스트 또는 시트 형태를 포함한다.The composition is an aqueous solution, suspension, emulsion, paste, cream, balm, ointment, foam, sheet, gel It is prepared in the form of gel, gum, spray, slurry, film, granule, patch, powder, or the like. In particular, preferred embodiments of the present invention include paste or sheet form.
상기 시트는 페이스트 형태의 조성물을 금형(mold 또는 frame)에 도포 및 건도 또는 동결건조하여 3 내지 8, 바람직하게는 4 내지 5%의 수분을 포함하는 것을 특징으로 한다.The sheet is characterized in that the paste composition is applied to a mold (mold or frame) and dried or lyophilized to contain 3 to 8, preferably 4 to 5% of moisture.
상기 조성물은 약제학적으로 사용되는 항균제, 부형제 및 첨가제를 더 포함할 수 있다. 적절한 항균제는 단쇄 알코올, 벤즈알코늄 염화물(benzoalkonium chloride) ("BAC"), 디데실 디메틸 암모늄 염화물(didecyl dimethyl ammonium chloride) ("DDAC"), 제올라이트 ("CWT-A")와 같은 살생제를 포함한다. 다른 가능한 항균제는, 이소티아졸론, 알킬 디메틸 암모늄 염화물, 트리아진, 2-티오시아노메틸티오 벤조티아졸, 메틸렌 비스 티오시아네이트, 아크롤레인, 도데실구아니딘 염화수소, 클로로페놀, 4급 암모늄염, 글루테르알데히드, 디티오카바메이트, 2-메르캅토벤조티아졸, 파라-클로로-메타-크실레놀, 은, 클로르헥시딘, 폴리헥사메틸렌 비구아나이드, n-할라민, 트리클로산, 인지질, 알파 히드록시산, 2,2-디브로모-3-니트릴로프로피온아미드, 2-브로모-2-니트로-1,3-프로판디올, 파네솔, 요오드, 브롬, 과산화수소, 이산화 염소, 식물성 오일, 식물 추출물, 벤즈알코늄 염화물, 염소, 차아염소산 나트륨, 또는 이들의 조합을 포함한다. 적절한 부형제는 안정화제, 산화방지제, 삼투압-조정제, 완충제 및 pH-조정제로 전분, 셀룰로스, 글루코스, 락토스, 수크로스, 젤라틴, 옥수수, 쌀, 밀가루, 백악, 실리카 겔, 스테아르산마그네슘, 스테아르산나트륨, 글리세롤 모노스테아레이트, 염화나트륨, 글리세롤, 프로필렌 글리콜, 물, 에탄올 등을 포함한다. 적절한 첨가제는 생리학적으로 생체 친화성인 완충제(예를 들어, 트로메타민 하이드로클로라이드), 킬레이트제(예컨대, DTPA 또는 DTPA-비스아마이드) 또는 칼슘 킬레이트 착체(예를 들어, 칼슘 DTPA, CaNaDTPA-비스아마이드) 또는 임의적으로는 칼슘 또는 나트륨 염(예를 들어, 염화칼슘, 아스코르브산칼슘, 글루콘산칼슘 또는 락트산칼슘)을 포함한다. The composition may further comprise an antimicrobial agent, excipient and additives used pharmaceutically. Suitable antimicrobials include biocides such as short-chain alcohols, benzoalkonium chloride ("BAC"), didecyl dimethyl ammonium chloride ("DDAC") and zeolites ("CWT-A"). Include. Other possible antibacterial agents are isothiazolones, alkyl dimethyl ammonium chlorides, triazines, 2-thiocyanomethylthio benzothiazoles, methylene bis thiocyanates, acrolein, dodecylguanidine hydrogen chloride, chlorophenols, quaternary ammonium salts, gluter Aldehyde, dithiocarbamate, 2-mercaptobenzothiazole, para-chloro-meth-xyllenol, silver, chlorhexidine, polyhexamethylene biguanide, n-halamine, triclosan, phospholipids, alpha hydroxy acids, 2 , 2-dibromo-3-nitrilopropionamide, 2-bromo-2-nitro-1,3-propanediol, farnesol, iodine, bromine, hydrogen peroxide, chlorine dioxide, vegetable oils, plant extracts, benzal Cornium chloride, chlorine, sodium hypochlorite, or combinations thereof. Suitable excipients are starch, cellulose, glucose, lactose, sucrose, gelatin, corn, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, as stabilizers, antioxidants, osmotic-modulating agents, buffers and pH-adjusting agents. Glycerol monostearate, sodium chloride, glycerol, propylene glycol, water, ethanol and the like. Suitable additives are physiologically biocompatible buffers (eg tromethamine hydrochloride), chelating agents (eg DTPA or DTPA-bisamide) or calcium chelate complexes (eg calcium DTPA, CaNaDTPA-bisamide) ) Or optionally calcium or sodium salts (eg, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate).
상기 조성물은 창상 피복재, 접착제, 수술용 및 의료용 장치, 인공 피부, 붕대, 발포제, 필름, 흡착방지제 및 이식재, 바람직하게는 창상 피복재로 사용 가능하다.The compositions can be used as wound coatings, adhesives, surgical and medical devices, artificial skin, bandages, foaming agents, films, antiadsorbents and implants, preferably wound coatings.
본 발명은 또한, a) 무세포 진피 기질을 분쇄하는 단계, b) 상기 분쇄된 무세포 진피 기질, 젤라틴이 용해된 고분자 수용액과 물을 혼합하여 혼합물을 형성하는 단계; c) 상기 혼합물을 30~60℃에서 1~180분간 열처리하는 단계; d) E-beam을 이용하여 열처리된 혼합물을 멸균하는 단계 및 e) 상기 멸균된 혼합물을 30~60℃에서 1~180분간 열처리하는 단계를 포함하는 멸균되고 즉시 사용 가능(ready-to-use)하며, 멸균 후 가교도, 점탄성, 경화도 및/또는 압출력 값의 변화도가 멸균 전 값에 비해 30% 이하인 것을 특징으로 하는 조성물의 제조 방법을 제공한다.The present invention also comprises the steps of a) pulverizing the cell-free dermal matrix, b) mixing the pulverized acellular dermal matrix, gelatin-dissolved polymer aqueous solution and water to form a mixture; c) heat-treating the mixture at 30-60 ° C. for 1-180 minutes; d) sterilizing the heat-treated mixture using an E-beam and e) sterilizing and ready-to-use, including heat-treating the sterilized mixture at 30-60 ° C. for 1-180 minutes. And a degree of change in crosslinking degree, viscoelasticity, curing degree and / or extrusion force value after sterilization is 30% or less than a value before sterilization.
상기 변화도는 아래와 같은 식으로 계산한다.The degree of change is calculated by the following equation.
변화도(%) = │Pd-Pi│ / Pi * 100% Gradient = │Pd-Pi│ / Pi * 100
Pi: 멸균 전 가교도, 점탄성, 경화도 및/또는 압출력Pi: degree of crosslinking, viscoelasticity, cure and / or extrusion force before sterilization
Pd: 멸균 후 가교도, 점탄성, 경화도 및/또는 압출력Pd: degree of crosslinking, viscoelasticity, curing and / or extrusion force after sterilization
상기 b) 단계의 혼합물은 무세포 진피 기질 5~20 중량%, 젤라틴 0.5~5 중량% 및 물 75~94.5 중량%, 바람직하게는 무세포 진피 기질 6~16 중량%, 젤라틴 1~3 중량% 및 물 81~93 중량%, 더욱 바람직하게는 무세포 진피 기질 8~12 중량%, 젤라틴 1~2 중량% 및 물 86~91 중량%을 포함한다.The mixture of step b) is 5-20% by weight of acellular dermal matrix, 0.5-5% by weight of gelatin and 75-94.5% by weight of water, preferably 6-16% by weight of acellular dermal matrix, 1-3% by weight of gelatin And 81-93% by weight of water, more preferably 8-12% by weight of acellular dermal substrate, 1-2% by weight of gelatin and 86-91% by weight of water.
상기 무세포 진피 기질은 섬유화된 무세포 진피 기질인 것을 특징으로 한다.The cell-free dermal matrix is characterized in that the fibrous cell-free dermal matrix.
본 발명의 상기 섬유화된 무세포 진피 기질은 장축 길이가 500 내지 3,000 ㎛, 바람직하게는 500 내지 2,000 ㎛이며 단축과 장축의 비율이 1:30~1:2,000 ㎛, 바람직하게는 1:30~1:1,000 ㎛, 가장 바람직하게는 1:30~1:70 ㎛인 것이 70% 이상, 바람직하게는 80%, 가장 바람직하게는 90% 이상인 것을 특징으로 한다.The fibrous acellular dermal matrix of the present invention has a long axis length of 500 to 3,000 μm, preferably 500 to 2,000 μm, and a ratio of short axis and long axis is 1:30 to 1: 2,000 μm, preferably 1:30 to 1 It is characterized by that it is 70% or more, preferably 80%, most preferably 90% or more: 1: 1,000 micrometers, Most preferably, 1: 30-1: 70 micrometers.
상기 무세포 진피 기질을 분쇄하는 a) 단계의 무세포 진피 기질은 두께 1 mm 이상, 바람직하게는 1.5 mm 이상인 것을 선별하여 이용되는 것을 특징으로 한다. The cell-free dermal matrix of step a) of pulverizing the cell-free dermal matrix is characterized in that it is used to select a thickness of 1 mm or more, preferably 1.5 mm or more.
상기 무세포 진피 기질의 분쇄는 커팅밀(Cutting mill), 푸드프로세서(Food processor), 마노 분쇄기, 동결 분쇄기, 초미 분쇄기(Micronizer), Vibratory micro mill, Jaw crusher, Mortar grinder, Planetary mill, Disk mill, Ball mill, Variable speed rotor mill, 바람직하게는 커팅밀(Cutting mill)을 사용하여 수행한다. 상기 커팅밀에서는 500 rpm의 회전 속도, 750 μm 체(sieve) 사이즈의 작동 조건을 이용하는 것이 바람직하다.The cell-free dermal substrate crushing is a cutting mill, a food processor, agate grinder, freeze grinder, micronizer, Vibratory micro mill, Jaw crusher, Mortar grinder, Planetary mill, Disk mill, Ball mill, Variable speed rotor mill, preferably using a cutting mill (Cutting mill). The cutting mill preferably uses a rotational speed of 500 rpm and operating conditions of 750 μm sieve size.
상기 무세포 진피 기질은 a) 동종 피부의 표피를 제거하는 단계; b) 진피 내 세포를 제거하는 단계; c) 글리세롤, 프로필렌 글리콜, 및 기본 용매 또는 용액을 혼합하고 상기 용액에 말티톨, 수크로스 또는 솔비톨, 바람직하게는 말티톨의 최종 농도가 20 내지 40 중량%가 되도록 용해하여 동결 보호제를 제조하는 단계; d) 상기 동결 보호제를 상기 표피 및 진피 내 세포가 제거된 피부에 침투시키는 단계 및 e) 상기 동결 보호제가 침투된 피부를 동결 건조하는 단계를 포함하는 방법에 의해서 제조된다.The cell-free dermal matrix comprises a) removing the epidermis of allogeneic skin; b) removing the cells in the dermis; c) preparing a cryoprotectant by mixing glycerol, propylene glycol, and a base solvent or solution and dissolving in the solution a final concentration of maltitol, sucrose or sorbitol, preferably maltitol, at 20-40% by weight; d) infiltrating the cryoprotectant into the skin from which the cells in the epidermis and dermis have been removed, and e) freeze drying the skin in which the cryoprotectant has infiltrated.
상기 말티톨이 진피 조직의 갈변 현상을 방지하는 것을 확인하였고, 이에 따라 말티톨을 사용하는 것을 특징으로 한다.The maltitol was confirmed to prevent the browning phenomenon of the dermal tissue, it is characterized by using maltitol accordingly.
상기 글리세롤, 프로필렌 글리콜 및 기본 용매 또는 용액의 혼합비가 중량 기준으로 0.5~2:0.5~2:6~10인 것을 특징으로 한다.Mixing ratio of the glycerol, propylene glycol and the basic solvent or solution is characterized in that 0.5 to 2: 0.5 to 2: 6 to 10 by weight.
상기 기본 용매 또는 용액이 증류수, 생리 식염수(normal saline), 인산완충용액(PBS), HBSS(Hank's balanced salt solution), TBS(Tris buffered saline), TAPS(N-Tris(hydroxy- methyl)methyl-3-aminopropanesulfonic acid) 완충용액, Bicine(N,N-Bis(2-hydroxyethyl) glycine) 완충용액, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) 완충용액, TES(NTris(hydroxymethyl)methyl-2-aminoethanesulfonicd acid) 완충용액, PIPES(piperazine- N,N'-bis(2-ethanesulfonic acid) 완충용액, 카코딜레이트(cacodylate) 완충용액, MES(2-(N-morpholino)ethanesulfonic acid) 완충용액, MEM(Minimum Essential Media), DMEM(Dulbecco's Modified Eagle Media), RPMI1640, IMDM(Iscove's Modified Dulbecco's Media), Defined Keratinocyte-SFM(without BPE(bovine pituitary extract)), Keratinocyte-SFM(with BPE), KnockOut D-MEM, AmnioMAX-II Complete Medium, AmnioMAX-C100 Complete Medium 및 이의 혼합물로 이루어지는 그룹 중에서 선택되는 것을 특징으로 한다.The basic solvent or solution is distilled water, physiological saline (normal saline), phosphate buffered solution (PBS), HBSS (Hank's balanced salt solution), TBS (Tris buffered saline), TAPS (N-Tris (hydroxy-methyl) methyl-3 -aminopropanesulfonic acid) buffer, Bicine (N, N-Bis (2-hydroxyethyl) glycine) buffer, HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) buffer, TES (NTris (hydroxymethyl) methyl- 2-aminoethanesulfonicd acid buffer, piperazine-N, N'-bis (2-ethanesulfonic acid) buffer, cacodylate buffer, MES (2- (N-morpholino) ethanesulfonic acid) buffer , Minimum Essential Media (MEM), Dulbecco's Modified Eagle Media (DMEM), RPMI1640, Iscove's Modified Dulbecco's Media (IMDM), defined Keratinocyte-without bovine pituitary extract (BPE), Keratinocyte-SFM (with BPE), KnockOut D -MEM, AmnioMAX-II Complete Medium, AmnioMAX-C100 Complete Medium, and mixtures thereof.
본 발명은 다른 측면에서, 섬유화된 무세포 진피 기질과 생체적합성 고분자를 포함하는 조성물로서 무세포 진피 기질은 단축과 장축의 비율이 1:30~1:2,000 ㎛, 바람직하게는 1:30~1:1,000 ㎛, 가장 바람직하게는 1:30~1:70 ㎛인 것이 70% 이상, 바람직하게는 80%, 가장 바람직하게는 90% 이상인 것을 특징으로 한다.In another aspect, the present invention is a composition comprising a fibrous acellular dermal matrix and a biocompatible polymer, the cell-free dermal matrix is 1: 30 ~ 1: 2,000 ㎛, preferably 1: 30 ~ 1 It is characterized by that it is 70% or more, preferably 80%, most preferably 90% or more: 1: 1,000 micrometers, Most preferably, 1: 30-1: 70 micrometers.
상기 조성물은 멸균되고 즉시 사용 가능한(ready-to-use) 조성물인 것을 특징으로 한다.The composition is characterized in that it is a sterile, ready-to-use composition.
상기 즉시 사용 가능한 조성물은 용질을 용매에 용해할 목적 또는 특정 성분을 혼합할 목적으로 수행하는 재수화(reconstitute) 단계 없이 즉시 투여 가능한 멸균 주사제제 또는 에어로졸 형태의 용액, 에멀젼, 크림, 연고, 페이스트 또는 겔을 포함한다.The ready-to-use composition may be a solution, emulsion, cream, ointment, paste or sterile injectable or aerosol form that can be administered immediately without the need for a reconstitute step to dissolve the solute in a solvent or to mix certain ingredients. Gels.
상기 조성물은 섬유화된 무세포 진피 기질 5~20 중량%, 생체적합성 고분자 0.5~5 중량% 및 물 75~94.5 중량%, 바람직하게는 섬유화된 무세포 진피 기질 6~16 중량%, 생체적합성 고분자 1~3 중량% 및 물 81~93 중량%, 더욱 바람직하게는 섬유화된 무세포 진피 기질 8~12 중량%, 생체적합성 고분자 1~2 중량% 및 물 86~91 중량%을 포함한다.The composition is 5 to 20% by weight of the fibrous acellular dermal substrate, 0.5 to 5% by weight of the biocompatible polymer and 75 to 99.5% by weight of water, preferably 6 to 16% by weight of the fibrous acellular dermal substrate, biocompatible polymer 1 ˜3% by weight and 81-93% by weight of water, more preferably 8-12% by weight of fibrized acellular dermal substrate, 1-2% by weight of biocompatible polymer and 86-91% by weight of water.
상기 섬유화된 무세포 진피 기질의 장축 길이는 500 내지 3,000 ㎛, 바람직하게는 500 내지 2,000 ㎛인 것을 특징으로 한다.The long axis length of the fibrous acellular dermal matrix is characterized in that 500 to 3,000 ㎛, preferably 500 to 2,000 ㎛.
상기 생체적합성 고분자는 젤라틴, 히알루론산, 콜라겐, 폴록사머 또는 이의 혼합물인 것을 포함한다. The biocompatible polymer includes gelatin, hyaluronic acid, collagen, poloxamer or mixtures thereof.
상기 바람직한 생체적합성 고분자는 젤라틴이다. 상기 젤라틴의 강도는 250 Bloom 이상, 바람직하게는 270 Bloom 이상이며 점도는 30 내지 45 mps, 바람직하게는 33 내지 42 mps, 더욱 바람직하게는 35 내지 40 mps이고 pH가 5 내지 6.5, 바람직하게는 5.5 내지 6인 것을 특징으로 한다.The preferred biocompatible polymer is gelatin. The strength of the gelatin is at least 250 Bloom, preferably at least 270 Bloom and the viscosity is 30 to 45 mps, preferably 33 to 42 mps, more preferably 35 to 40 mps and the pH is 5 to 6.5, preferably 5.5 It is characterized in that from 6 to.
상기 조성물은 수용액(aqueous solution), 현탁액(suspention), 유화액(emulsion), 페이스트(paste), 크림(cream), 밤(balm), 연고(ointment), 거품(foam), 시트(sheet), 겔(gel), 검(gum), 스프레이(spray), 슬러리(slurry), 필름(film), 과립(granule), 패치(patch), 분말(powder) 등의 형태로 제조된다. 특히, 본 발명의 바람직한 양태는 페이스트 또는 시트 형태를 포함한다.The composition is an aqueous solution, suspension, emulsion, paste, cream, balm, ointment, foam, sheet, gel It is prepared in the form of gel, gum, spray, slurry, film, granule, patch, powder, or the like. In particular, preferred embodiments of the present invention include paste or sheet form.
상기 시트는 페이스트 형태의 조성물을 금형(mold 또는 frame)에 도포 및 건조 또는 동결건조하여 3 내지 8%, 바람직하게는 4 내지 5% 함량의 수분을 포함하는 것을 특징으로 한다.The sheet is characterized in that the paste composition is applied to a mold (mold or frame) and dried or lyophilized to contain 3 to 8%, preferably 4 to 5% of water.
상기 멸균은 건조가열 멸균, 저온살균 멸균, 방사선 멸균, 가스 멸균 등을 포함할 수 있으며 바람직하게는 방사선 멸균, 더욱 바람직하게는 E-beam을 이용한 방사선 멸균이다.The sterilization may include dry heat sterilization, pasteurization sterilization, radiation sterilization, gas sterilization and the like, and preferably radiation sterilization, more preferably radiation sterilization using E-beam.
상기 E-beam 이용한 방사선 멸균 전 및/또는 후 단계에 30 내지 60 ℃, 바람직하게는 35 내지 55 ℃, 더욱 바람직하게는 40 내지 50 ℃의 열처리 공정을 포함한다.The heat treatment step of 30 to 60 ℃, preferably 35 to 55 ℃, more preferably 40 to 50 ℃ before and / or after the sterilization of the radiation using the E-beam.
상기 열처리는 1 내지 180 분간, 바람직하게는 30 내지 150 분간, 더욱 바람직하게는 60 내지 120 분간 수행된다.The heat treatment is carried out for 1 to 180 minutes, preferably 30 to 150 minutes, more preferably 60 to 120 minutes.
상기 섬유화된 무세포 진피 기질은 두께 1 mm 이상, 바람직하게는 1.5 mm 이상인 것을 선별하여 이용되는 것을 특징으로 한다.The fibrous acellular dermal matrix is characterized in that it is used to select a thickness of 1 mm or more, preferably 1.5 mm or more.
상기 무세포 진피 기질의 분쇄는 커팅밀(Cutting mill), 푸드프로세서(Food processor), 마노 분쇄기, 동결 분쇄기, 초미 분쇄기(Micronizer), Vibratory micro mill, Jaw crusher, Mortar grinder, Planetary mill, Disk mill, Ball mill, Variable speed rotor mill, 바람직하게는 커팅밀(Cutting mill)을 사용하여 수행한다. 상기 커팅밀에서는 500 rpm의 회전 속도, 750 μm 체(sieve) 사이즈의 작동 조건을 이용하는 것이 바람직하다.The cell-free dermal substrate crushing is a cutting mill, a food processor, agate grinder, freeze grinder, micronizer, Vibratory micro mill, Jaw crusher, Mortar grinder, Planetary mill, Disk mill, Ball mill, Variable speed rotor mill, preferably using a cutting mill (Cutting mill). The cutting mill preferably uses a rotational speed of 500 rpm and operating conditions of 750 μm sieve size.
상기 조성물은 약제학적으로 사용되는 항균제, 부형제 및 첨가제를 더 포함할 수 있다. 적절한 항균제는 단쇄 알코올, 벤즈알코늄 염화물(benzoalkonium chloride) ("BAC"), 디데실 디메틸 암모늄 염화물(didecyl dimethyl ammonium chloride) ("DDAC"), 제올라이트 ("CWT-A")와 같은 살생제를 포함한다. 다른 가능한 항균제는, 이소티아졸론, 알킬 디메틸 암모늄 염화물, 트리아진, 2-티오시아노메틸티오 벤조티아졸, 메틸렌 비스 티오시아네이트, 아크롤레인, 도데실구아니딘 염화수소, 클로로페놀, 4급 암모늄염, 글루테르알데히드, 디티오카바메이트, 2-메르캅토벤조티아졸, 파라-클로로-메타-크실레놀, 은, 클로르헥시딘, 폴리헥사메틸렌 비구아나이드, n-할라민, 트리클로산, 인지질, 알파 히드록시산, 2,2-디브로모-3-니트릴로프로피온아미드, 2-브로모-2-니트로-1,3-프로판디올, 파네솔, 요오드, 브롬, 과산화수소, 이산화 염소, 식물성 오일, 식물 추출물, 벤즈알코늄 염화물, 염소, 차아염소산 나트륨, 또는 이들의 조합을 포함한다. 적절한 부형제는 안정화제, 산화방지제, 삼투압-조정제, 완충제 및 pH-조정제로 전분, 셀룰로스, 글루코스, 락토스, 수크로스, 젤라틴, 옥수수, 쌀, 밀가루, 백악, 실리카 겔, 스테아르산마그네슘, 스테아르산나트륨, 글리세롤 모노스테아레이트, 염화나트륨, 글리세롤, 프로필렌 글리콜, 물, 에탄올 등을 포함한다. 적절한 첨가제는 생리학적으로 생체 친화성인 완충제(예를 들어, 트로메타민 하이드로클로라이드), 킬레이트제(예컨대, DTPA 또는 DTPA-비스아마이드) 또는 칼슘 킬레이트 착체(예를 들어, 칼슘 DTPA, CaNaDTPA-비스아마이드) 또는 임의적으로는 칼슘 또는 나트륨 염(예를 들어, 염화칼슘, 아스코르브산칼슘, 글루콘산칼슘 또는 락트산칼슘)을 포함한다.The composition may further comprise an antimicrobial agent, excipient and additives used pharmaceutically. Suitable antimicrobials include biocides such as short-chain alcohols, benzoalkonium chloride ("BAC"), didecyl dimethyl ammonium chloride ("DDAC") and zeolites ("CWT-A"). Include. Other possible antibacterial agents are isothiazolones, alkyl dimethyl ammonium chlorides, triazines, 2-thiocyanomethylthio benzothiazoles, methylene bis thiocyanates, acrolein, dodecylguanidine hydrogen chloride, chlorophenols, quaternary ammonium salts, gluter Aldehyde, dithiocarbamate, 2-mercaptobenzothiazole, para-chloro-meth-xyllenol, silver, chlorhexidine, polyhexamethylene biguanide, n-halamine, triclosan, phospholipids, alpha hydroxy acids, 2 , 2-dibromo-3-nitrilopropionamide, 2-bromo-2-nitro-1,3-propanediol, farnesol, iodine, bromine, hydrogen peroxide, chlorine dioxide, vegetable oils, plant extracts, benzal Cornium chloride, chlorine, sodium hypochlorite, or combinations thereof. Suitable excipients are starch, cellulose, glucose, lactose, sucrose, gelatin, corn, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, as stabilizers, antioxidants, osmotic-modulating agents, buffers and pH-adjusting agents. Glycerol monostearate, sodium chloride, glycerol, propylene glycol, water, ethanol and the like. Suitable additives are physiologically biocompatible buffers (eg tromethamine hydrochloride), chelating agents (eg DTPA or DTPA-bisamide) or calcium chelate complexes (eg calcium DTPA, CaNaDTPA-bisamide) ) Or optionally calcium or sodium salts (eg, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate).
상기 조성물은 창상 피복재, 접착제, 수술용 및 의료용 장치, 인공 피부, 붕대, 발포제, 필름, 흡착방지제 및 이식재, 바람직하게는 창상 피복재로 사용 가능하다.The compositions can be used as wound coatings, adhesives, surgical and medical devices, artificial skin, bandages, foaming agents, films, antiadsorbents and implants, preferably wound coatings.
본 발명은 또한, a) 무세포 진피 기질을 섬유화하는 단계; b) 상기 섬유화된 무세포 진피 기질, 생체 적합성 고분자 수용액과 물을 혼합하여 혼합물을 형성하는 단계; c) 상기 혼합물을 30~60 ℃에서 1~30분간 열처리하는 단계; d) E-beam을 이용하여 열처리된 혼합물을 멸균하는 단계 및 e) 상기 멸균된 혼합물을 30~60 ℃에서 1~30분간 열처리하는 단계를 포함하는 멸균되고 즉시 사용 가능(ready-to-use)하며, 상기 섬유화된 무세포 진피 기질의 단축과 장축의 비율이 1:30~1:2,000인 것이 70%, 바람직하게는 80%, 가장 바람직하게는 90% 이상인 것을 특징으로 하는 조성물의 제조 방법을 제공한다.The invention also comprises the steps of a) fibrosis of acellular dermal matrix; b) mixing the fibrous acellular dermal matrix, a biocompatible polymer aqueous solution and water to form a mixture; c) heat treating the mixture at 30 to 60 ° C. for 1 to 30 minutes; d) sterilizing the heat-treated mixture using an E-beam, and e) sterilizing and ready-to-use, including heat-treating the sterilized mixture at 30 to 60 ° C. for 1 to 30 minutes. In addition, the ratio of the short axis and the long axis of the fibrous acellular dermal matrix is 1:30 to 1: 2,000 ratio of the manufacturing method of the composition, characterized in that 70%, preferably 80%, most preferably 90% or more. to provide.
상기 b) 단계의 혼합물은 섬유화된 무세포 진피 기질 5~20 중량%, 생체적합성 고분자 0.5~5 중량% 및 물 75~94.5 중량%, 바람직하게는 섬유화된 무세포 진피 기질 6~16 중량%, 생체적합성 고분자 1~3 중량% 및 물 81~93 중량%, 더욱 바람직하게는 섬유화된 무세포 진피 기질 8~12 중량%, 생체적합성 고분자 1~2 중량% 및 물 86~91 중량%을 포함한다.The mixture of step b) is 5-20% by weight of the fibrous acellular dermal substrate, 0.5-5% by weight of the biocompatible polymer and 75-94.5% by weight of water, preferably 6-16% by weight of the fibrous acellular dermal substrate, 1 to 3% by weight of biocompatible polymer and 81 to 93% by weight of water, more preferably 8 to 12% by weight of fibrized acellular dermal substrate, 1 to 2% by weight of biocompatible polymer and 86 to 91% by weight of water. .
상기 섬유화된 무세포 진피 기질은 장축 길이가 500 내지 3,000 ㎛, 바람직하게는 500 내지 2,000 ㎛이며, 무세포 진피 기질을 커팅밀(Cutting mill), 푸드프로세서(Food processor), 마노 분쇄기, 동결 분쇄기, 초미 분쇄기(Micronizer), Vibratory micro mill, Jaw crusher, Mortar grinder, Planetary mill, Disk mill, Ball mill, Variable speed rotor mill, 바람직하게는 커팅밀(Cutting mill)로 분쇄하여 제조한다.The fibrous, cell-free dermal substrate has a long axis length of 500 to 3,000 μm, preferably 500 to 2,000 μm, and cuts the cell-free dermal substrate into a cutting mill, a food processor, an agate grinder, a freeze grinder, It is prepared by grinding with a micronizer, Vibratory micro mill, Jaw crusher, Mortar grinder, Planetary mill, Disk mill, Ball mill, Variable speed rotor mill, preferably Cutting mill.
상기 무세포 진피 기질은 a) 동종 피부의 표피를 제거하는 단계; b) 진피 내 세포를 제거하는 단계; c) 글리세롤, 프로필렌 글리콜, 및 기본 용매 또는 용액을 혼합하고 상기 용액에 말티톨, 수크로스 또는 솔비톨의 최종 농도가 20 내지 40 중량%가 되도록 용해하여 동결 보호제를 제조하는 단계; d) 상기 동결 보호제를 상기 표피 및 진피 내 세포가 제거된 피부에 침투시키는 단계 및 e) 상기 동결 보호제가 침투된 피부를 동결 건조하는 단계를 포함하는 방법에 의해서 제조된다.The cell-free dermal matrix comprises a) removing the epidermis of allogeneic skin; b) removing the cells in the dermis; c) preparing a cryoprotectant by mixing glycerol, propylene glycol, and a base solvent or solution and dissolving in the solution such that the final concentration of maltitol, sucrose or sorbitol is 20 to 40% by weight; d) infiltrating the cryoprotectant into the skin from which the cells in the epidermis and dermis have been removed, and e) freeze drying the skin in which the cryoprotectant has infiltrated.
상기 말티톨이 진피 조직의 갈변 현상을 방지하는 것을 확인하였고, 이에 따라 말티톨을 사용하는 것을 특징으로 한다.The maltitol was confirmed to prevent the browning phenomenon of the dermal tissue, it is characterized by using maltitol accordingly.
상기 글리세롤, 프로필렌 글리콜 및 기본 용매 또는 용액의 혼합비가 중량 기준으로 0.5~2:0.5~2:6~10인 것을 특징으로 한다. Mixing ratio of the glycerol, propylene glycol and the basic solvent or solution is characterized in that 0.5 to 2: 0.5 to 2: 6 to 10 by weight.
상기 기본 용매 또는 용액이 증류수, 생리 식염수(normal saline), 인산완충용액(PBS), HBSS(Hank's balanced salt solution), TBS(Tris buffered saline), TAPS(N-Tris(hydroxy- methyl)methyl-3-aminopropanesulfonic acid) 완충용액, Bicine(N,N-Bis(2-hydroxyethyl) glycine) 완충용액, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) 완충용액, TES(NTris(hydroxymethyl)methyl-2-aminoethanesulfonicd acid) 완충용액, PIPES(piperazine- N,N'-bis(2-ethanesulfonic acid) 완충용액, 카코딜레이트(cacodylate) 완충용액, MES(2-(N-morpholino)ethanesulfonic acid) 완충용액, MEM(Minimum Essential Media), DMEM(Dulbecco's Modified Eagle Media), RPMI1640, IMDM(Iscove's Modified Dulbecco's Media), Defined Keratinocyte-SFM(without BPE(bovine pituitary extract)), Keratinocyte-SFM(with BPE), KnockOut D-MEM, AmnioMAX-II Complete Medium, AmnioMAX-C100 Complete Medium 및 이의 혼합물로 이루어지는 그룹 중에서 선택되는 것을 사용한다.The basic solvent or solution is distilled water, physiological saline (normal saline), phosphate buffered solution (PBS), HBSS (Hank's balanced salt solution), TBS (Tris buffered saline), TAPS (N-Tris (hydroxy-methyl) methyl-3 -aminopropanesulfonic acid) buffer, Bicine (N, N-Bis (2-hydroxyethyl) glycine) buffer, HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) buffer, TES (NTris (hydroxymethyl) methyl- 2-aminoethanesulfonicd acid buffer, piperazine-N, N'-bis (2-ethanesulfonic acid) buffer, cacodylate buffer, MES (2- (N-morpholino) ethanesulfonic acid) buffer , Minimum Essential Media (MEM), Dulbecco's Modified Eagle Media (DMEM), RPMI1640, Iscove's Modified Dulbecco's Media (IMDM), defined Keratinocyte-without bovine pituitary extract (BPE), Keratinocyte-SFM (with BPE), KnockOut D Use one selected from the group consisting of MEM, AmnioMAX-II Complete Medium, AmnioMAX-C100 Complete Medium, and mixtures thereof.
본 발명은 또 다른 측면에서, 무세포 진피 기질 75 내지 85 중량%, 생체적합 고분자 15 내지 25 중량% 및 3 내지 8 중량%의 물을 포함하는 창상 피복재용 시트로서, 가로 길이가 4 내지 20 cm 이며 세로 길이가 4 내지 20 cm이며 두께가 1.5 내지 3 mm인 것을 특징으로 하는 창상 피복재용 시트를 제공한다.In another aspect, the present invention provides a wound coating sheet comprising 75 to 85% by weight of cell-free dermal substrate, 15 to 25% by weight of biocompatible polymer and 3 to 8% by weight of water, having a length of 4 to 20 cm And a length of 4 to 20 cm and a thickness of 1.5 to 3 mm to provide a sheet for wound covering material.
상기 무세포 진피 기질은 섬유화된 무세포 진피 기질인 것을 특징으로 한다. The cell-free dermal matrix is characterized in that the fibrous cell-free dermal matrix.
본 발명의 상기 섬유화된 무세포 진피 기질은 장축 길이가 500 내지 3,000 ㎛, 바람직하게는 500 내지 2,000 ㎛이며, 단축과 장축의 비율이 1:30~1:2,000 ㎛, 바람직하게는 1:30~1:1,000 ㎛, 가장 바람직하게는 1:30~1:70 ㎛인 것이 70% 이상, 바람직하게는 80%, 가장 바람직하게는 90% 이상인 것을 특징으로 한다.The fibrous acellular dermal matrix of the present invention has a long axis length of 500 to 3,000 μm, preferably 500 to 2,000 μm, and a ratio of a short axis and a long axis of 1:30 to 1: 2,000 μm, preferably 1:30 to It is characterized in that 1: 1000 ㎛, most preferably 1:30 ~ 1:70 ㎛ is 70% or more, preferably 80%, most preferably 90% or more.
상기 생체적합성 고분자는 젤라틴, 히알루론산, 콜라겐, 폴록사머 또는 이의 혼합물인 것을 포함한다. The biocompatible polymer includes gelatin, hyaluronic acid, collagen, poloxamer or mixtures thereof.
상기 바람직한 생체적합성 고분자는 젤라틴이다. 상기 젤라틴의 강도는 250 Bloom 이상, 바람직하게는 270 Bloom 이상이며 점도는 30 내지 45 mps, 바람직하게는 33 내지 42 mps, 더욱 바람직하게는 35 내지 40 mps이고 pH가 5 내지 6.5, 바람직하게는 5.5 내지 6인 것을 특징으로 한다.The preferred biocompatible polymer is gelatin. The strength of the gelatin is at least 250 Bloom, preferably at least 270 Bloom and the viscosity is 30 to 45 mps, preferably 33 to 42 mps, more preferably 35 to 40 mps and the pH is 5 to 6.5, preferably 5.5 It is characterized in that from 6 to.
상기 시트는 약제학적으로 사용되는 항균제, 부형제 및 첨가제를 더 포함할 수 있다. 적절한 항균제는 단쇄 알코올, 벤즈알코늄 염화물(benzoalkonium chloride) ("BAC"), 디데실 디메틸 암모늄 염화물(didecyl dimethyl ammonium chloride) ("DDAC"), 제올라이트 ("CWT-A")와 같은 살생제를 포함한다. 다른 가능한 항균제는, 이소티아졸론, 알킬 디메틸 암모늄 염화물, 트리아진, 2-티오시아노메틸티오 벤조티아졸, 메틸렌 비스 티오시아네이트, 아크롤레인, 도데실구아니딘 염화수소, 클로로페놀, 4급 암모늄염, 글루테르알데히드, 디티오카바메이트, 2-메르캅토벤조티아졸, 파라-클로로-메타-크실레놀, 은, 클로르헥시딘, 폴리헥사메틸렌 비구아나이드, n-할라민, 트리클로산, 인지질, 알파 히드록시산, 2,2-디브로모-3-니트릴로프로피온아미드, 2-브로모-2-니트로-1,3-프로판디올, 파네솔, 요오드, 브롬, 과산화수소, 이산화 염소, 식물성 오일, 식물 추출물, 벤즈알코늄 염화물, 염소, 차아염소산 나트륨, 또는 이들의 조합을 포함한다. 적절한 부형제는 안정화제, 산화방지제, 삼투압-조정제, 완충제 및 pH-조정제로 전분, 셀룰로스, 글루코스, 락토스, 수크로스, 젤라틴, 옥수수, 쌀, 밀가루, 백악, 실리카 겔, 스테아르산마그네슘, 스테아르산나트륨, 글리세롤 모노스테아레이트, 염화나트륨, 글리세롤, 프로필렌 글리콜, 물, 에탄올 등을 포함한다. 적절한 첨가제는 생리학적으로 생체 친화성인 완충제(예를 들어, 트로메타민 하이드로클로라이드), 킬레이트제(예컨대, DTPA 또는 DTPA-비스아마이드) 또는 칼슘 킬레이트 착체(예를 들어, 칼슘 DTPA, CaNaDTPA-비스아마이드) 또는 임의적으로는 칼슘 또는 나트륨 염(예를 들어, 염화칼슘, 아스코르브산칼슘, 글루콘산칼슘 또는 락트산칼슘)을 포함한다.The sheet may further comprise a pharmaceutically used antimicrobial agent, excipient and additive. Suitable antimicrobials include biocides such as short-chain alcohols, benzoalkonium chloride ("BAC"), didecyl dimethyl ammonium chloride ("DDAC") and zeolites ("CWT-A"). Include. Other possible antibacterial agents are isothiazolones, alkyl dimethyl ammonium chlorides, triazines, 2-thiocyanomethylthio benzothiazoles, methylene bis thiocyanates, acrolein, dodecylguanidine hydrogen chloride, chlorophenols, quaternary ammonium salts, gluter Aldehyde, dithiocarbamate, 2-mercaptobenzothiazole, para-chloro-meth-xyllenol, silver, chlorhexidine, polyhexamethylene biguanide, n-halamine, triclosan, phospholipids, alpha hydroxy acids, 2 , 2-dibromo-3-nitrilopropionamide, 2-bromo-2-nitro-1,3-propanediol, farnesol, iodine, bromine, hydrogen peroxide, chlorine dioxide, vegetable oils, plant extracts, benzal Cornium chloride, chlorine, sodium hypochlorite, or combinations thereof. Suitable excipients are starch, cellulose, glucose, lactose, sucrose, gelatin, corn, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, as stabilizers, antioxidants, osmotic-modulating agents, buffers and pH-adjusting agents. Glycerol monostearate, sodium chloride, glycerol, propylene glycol, water, ethanol and the like. Suitable additives are physiologically biocompatible buffers (eg tromethamine hydrochloride), chelating agents (eg DTPA or DTPA-bisamide) or calcium chelate complexes (eg calcium DTPA, CaNaDTPA-bisamide) ) Or optionally calcium or sodium salts (eg, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate).
본 발명은 또한, a) 무세포 진피 기질을 분쇄하는 단계; b) 상기 분쇄된 무세포 진피 기질, 생체적합 고분자 수용액과 물을 혼합하여 혼합물을 형성하는 단계 및 c) 상기 혼합물을 금형(mold 또는 frame)에 도포 및 동결 건조하여 시트를 제조하는 단계를 포함하는 창상 피복재용 시트의 제조 방법을 제공한다.The invention also comprises the steps of a) grinding the cell free dermal matrix; b) mixing the ground cell free dermal matrix, a biocompatible polymer aqueous solution and water to form a mixture, and c) applying the mixture to a mold (mold or frame) and freeze drying to prepare a sheet. A method for producing a wound coating sheet is provided.
상기 b) 단계의 혼합물은 무세포 진피 기질 5~20 중량%, 생체적합성 고분자 0.5~5 중량% 및 물 75~94.5 중량%, 바람직하게는 무세포 진피 기질 6~16 중량%, 생체적합성 1~3 중량% 및 물 81~93 중량%, 더욱 바람직하게는 무세포 진피 기질 8~12 중량%, 생체적합성 고분자 1~2 중량% 및 물 86~91 중량%을 포함한다.The mixture of step b) is 5-20% by weight of acellular dermal matrix, 0.5-5% by weight of biocompatible polymer and 75-94.5% by weight of water, preferably 6-16% by weight of acellular dermal matrix, biocompatible 1- 3 wt% and 81-93 wt% water, more preferably 8-12 wt% acellular dermal substrate, 1-2 wt% biocompatible polymer and 86-91 wt% water.
상기 c)단계의 시트는 무세포 진피 기질 75 내지 85 중량%, 생체적합성 고분자 15 내지 25 중량% 및 3 내지 8 중량%의 물, 바람직하게는 무세포 진피 기질 78~82 중량%, 생체적합성 18~22 중량% 및 물 4~5 중량%을 포함한다.The sheet of step c) is 75 to 85% by weight of the cell-free dermal matrix, 15 to 25% by weight of the biocompatible polymer and 3 to 8% by weight of water, preferably 78 to 82% by weight of the cell free dermal matrix, biocompatible 18 22 wt% and 4-5 wt% water.
상기 무세포 진피 기질은 섬유화된 무세포 진피 기질인 것을 특징으로 한다. The cell-free dermal matrix is characterized in that the fibrous cell-free dermal matrix.
본 발명의 상기 섬유화된 무세포 진피 기질은 장축 길이가 500 내지 3,000 ㎛, 바람직하게는 500 내지 2,000 ㎛이며, 단축과 장축의 비율이 1:30~1:2,000 ㎛, 바람직하게는 1:30~1:1,000 ㎛, 가장 바람직하게는 1:30~1:70 ㎛인 것이 70% 이상, 바람직하게는 80%, 가장 바람직하게는 90% 이상인 것을 특징으로 한다.The fibrous acellular dermal matrix of the present invention has a long axis length of 500 to 3,000 μm, preferably 500 to 2,000 μm, and a ratio of a short axis and a long axis of 1:30 to 1: 2,000 μm, preferably 1:30 to It is characterized in that 1: 1000 ㎛, most preferably 1:30 ~ 1:70 ㎛ is 70% or more, preferably 80%, most preferably 90% or more.
상기 무세포 진피 기질을 분쇄하는 a) 단계의 무세포 진피 기질은 두께 1 mm 이상, 바람직하게는 1.5 mm 이상인 것을 선별하여 이용되는 것을 특징으로 한다. The cell-free dermal matrix of step a) of pulverizing the cell-free dermal matrix is characterized in that it is used to select a thickness of 1 mm or more, preferably 1.5 mm or more.
상기 무세포 진피 기질의 분쇄는 커팅밀(Cutting mill), 푸드프로세서(Food processor), 마노 분쇄기, 동결 분쇄기, 초미 분쇄기(Micronizer), Vibratory micro mill, Jaw crusher, Mortar grinder, Planetary mill, Disk mill, Ball mill, Variable speed rotor mill, 바람직하게는 커팅밀(Cutting mill)을 사용하여 수행한다. 상기 커팅밀에서는 500 rpm의 회전 속도, 750 μm 체(sieve) 사이즈의 작동 조건을 이용하는 것이 바람직하다.The cell-free dermal substrate crushing is a cutting mill, a food processor, agate grinder, freeze grinder, micronizer, Vibratory micro mill, Jaw crusher, Mortar grinder, Planetary mill, Disk mill, Ball mill, Variable speed rotor mill, preferably using a cutting mill (Cutting mill). The cutting mill preferably uses a rotational speed of 500 rpm and operating conditions of 750 μm sieve size.
상기 무세포 진피 기질은 a) 동종 피부의 표피를 제거하는 단계; b) 진피 내 세포를 제거하는 단계; c) 글리세롤, 프로필렌 글리콜, 및 기본 용매 또는 용액을 혼합하고 상기 용액에 말티톨, 수크로스 또는 솔비톨의 최종 농도가 20 내지 40 중량%가 되도록 용해하여 동결 보호제를 제조하는 단계; d) 상기 동결 보호제를 상기 표피 및 진피 내 세포가 제거된 피부에 침투시키는 단계 및 e) 상기 동결 보호제가 침투된 피부를 동결 건조하는 단계를 포함하는 방법에 의해서 제조된다.The cell-free dermal matrix comprises a) removing the epidermis of allogeneic skin; b) removing the cells in the dermis; c) preparing a cryoprotectant by mixing glycerol, propylene glycol, and a base solvent or solution and dissolving in the solution such that the final concentration of maltitol, sucrose or sorbitol is 20 to 40% by weight; d) infiltrating the cryoprotectant into the skin from which the cells in the epidermis and dermis have been removed, and e) freeze drying the skin in which the cryoprotectant has infiltrated.
상기 말티톨이 진피 조직의 갈변 현상을 방지하는 것을 확인하였고, 이에 따라 말티톨을 사용하는 것을 특징으로 한다.The maltitol was confirmed to prevent the browning phenomenon of the dermal tissue, it is characterized by using maltitol accordingly.
상기 글리세롤, 프로필렌 글리콜 및 기본 용매 또는 용액의 혼합비가 중량 기준으로 0.5~2:0.5~2:6~10인 것을 특징으로 한다.Mixing ratio of the glycerol, propylene glycol and the basic solvent or solution is characterized in that 0.5 to 2: 0.5 to 2: 6 to 10 by weight.
상기 기본 용매 또는 용액이 증류수, 생리 식염수(normal saline), 인산완충용액(PBS), HBSS(Hank's balanced salt solution), TBS(Tris buffered saline), TAPS(N-Tris(hydroxy- methyl)methyl-3-aminopropanesulfonic acid) 완충용액, Bicine(N,N-Bis(2-hydroxyethyl) glycine) 완충용액, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) 완충용액, TES(NTris(hydroxymethyl)methyl-2-aminoethanesulfonicd acid) 완충용액, PIPES(piperazine- N,N'-bis(2-ethanesulfonic acid) 완충용액, 카코딜레이트(cacodylate) 완충용액, MES(2-(N-morpholino)ethanesulfonic acid) 완충용액, MEM(Minimum Essential Media), DMEM(Dulbecco's Modified Eagle Media), RPMI1640, IMDM(Iscove's Modified Dulbecco's Media), Defined Keratinocyte-SFM(without BPE(bovine pituitary extract)), Keratinocyte-SFM(with BPE), KnockOut D-MEM, AmnioMAX-II Complete Medium, AmnioMAX-C100 Complete Medium 및 이의 혼합물로 이루어지는 그룹 중에서 선택되는 것을 사용한다.The basic solvent or solution is distilled water, physiological saline (normal saline), phosphate buffered solution (PBS), HBSS (Hank's balanced salt solution), TBS (Tris buffered saline), TAPS (N-Tris (hydroxy-methyl) methyl-3 -aminopropanesulfonic acid) buffer, Bicine (N, N-Bis (2-hydroxyethyl) glycine) buffer, HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) buffer, TES (NTris (hydroxymethyl) methyl- 2-aminoethanesulfonicd acid buffer, piperazine-N, N'-bis (2-ethanesulfonic acid) buffer, cacodylate buffer, MES (2- (N-morpholino) ethanesulfonic acid) buffer , Minimum Essential Media (MEM), Dulbecco's Modified Eagle Media (DMEM), RPMI1640, Iscove's Modified Dulbecco's Media (IMDM), defined Keratinocyte-without bovine pituitary extract (BPE), Keratinocyte-SFM (with BPE), KnockOut D Use one selected from the group consisting of MEM, AmnioMAX-II Complete Medium, AmnioMAX-C100 Complete Medium, and mixtures thereof.
도 1은 섬유화된 무세포 진피 기질(A)과 비드형으로 된 무세포 진피 기질(B)의 외형을 비교한 사진이다.1 is a photograph comparing the appearance of the fibrotic cell-free dermal matrix (A) and the cell-free dermal matrix (B) in the form of beads.
도 2는 섬유화된 무세포 진피 기질 입자의 장축 길이와 이의 분포도를 나타낸 것이다.Figure 2 shows the long axis length and the distribution of the fibrous acellular dermal matrix particles.
도 3은 섬유화된 무세포 진피 기질 입자의 단축 길이와 이의 분포도를 나타낸 것이다.Figure 3 shows the uniaxial length of the fibrotic acellular dermal matrix particles and their distribution.
도 4A는 섬유화된 무세포 진피 기질 입자의 장, 단축 비율을 나타낸 것이고, 도4B는 비드형 무세포 진피 기질 입자의 장, 단축 비율을 나타낸 것이며, 도 4C 및 도 4D는 섬유화된 무세포 진피 기질과 비드형 무세포 진피 기질의 현미경 사진이다.Figure 4A shows the long, short axis ratio of the fibrotic acellular dermal matrix particles, Figure 4B shows the long, short axis ratio of the bead-shaped acellular dermal matrix particles, Figures 4C and 4D are fibrous acellular dermal matrix Micrograph of a bead-type acellular dermal matrix.
도 5는 섬유화된 무세포 진피 기질과 젤라틴을 포함한 페이스트의 외형 사진이다.5 is a photograph of the appearance of a paste containing fibrized acellular dermal matrix and gelatin.
도 6은 섬유화된 무세포 진피 기질과 젤라틴을 포함한 페이스트의 점도를 나타낸 것으로, 제조 완료 시점과 저장 3개월 이후의 페이스트 점도를 비교한 것이다.Figure 6 shows the viscosity of the paste containing the fibrillated cell-free dermal substrate and gelatin, comparing the paste viscosity at the time of completion of production and 3 months after storage.
도 7은 E-beam 멸균 여부 및 멸균 시 온도조건에 따른 페이스트의 압출력을 시간에 따라 측정한 것이다(E-beam 멸균 전, 후 단계에서 45 ℃ 온도반응을 가진 페이스트는 E-beam 멸균을 하지 않은 페이스트보다 낮은 압출력 값을 보였다. 이는, E-beam 멸균을 하지 않은 페이스트는 가교 결합이 일어나지 않아 보다 낮은 수준의 압출력 값을 보일 것으로 예상되는 바에 반하는 결과로, 페이스트 제조 후 상온에서 24시간 이상 저장 시 경화도가 다소 증가되어 압출력 값이 증가한 것으로 해석된다.)Figure 7 shows the extrusion force of the paste according to the temperature conditions at the time of sterilization and E-beam sterilization (times before and after sterilization of the E-beam paste at 45 ℃ temperature reaction is not E-beam sterilization This resulted in a lower extruding force value than the non-paste paste, which is expected to show lower extruding force values due to no cross-linking of the paste without E-beam sterilization. It is interpreted that the degree of hardening was slightly increased during the abnormal storage, thereby increasing the extrusion force value.)
도 8은 E-beam 멸균 여부 및 멸균 시 온도조건에 따른 페이스트의 겔 분율(가교도)을 시간에 따라 측정한 것이다.Figure 8 is a measurement of the gel fraction of the paste (crosslinked) according to the temperature conditions at the time of sterilization and E-beam sterilization over time.
도 9는 섬유화된 무세포 진피 기질과 젤라틴을 포함한 페이스트의 넓고 깊은 창상 부위의 임상적 효과를 나타낸 사진이다.9 is a photograph showing the clinical effect of a wide and deep wound site of a paste containing fibrized acellular dermal matrix and gelatin.
도 10은 섬유화된 무세포 진피 기질과 젤라틴을 포함한 페이스트의 족부 창상의 임상적 효과를 나타낸 사진이다.Figure 10 is a photograph showing the clinical effect of the foot wound of the paste containing the fibrous acellular dermal matrix and gelatin.
도 11은 섬유화된 무세포 진피 기질과 젤라틴을 포함한 페이스트의 만성화된 창상의 임상적 효과를 나타낸 사진이다.FIG. 11 is a photograph showing the clinical effect of chronicized wound of a paste comprising fibrized acellular dermal matrix and gelatin.
도 12는 섬유화된 무세포 진피 기질과 젤라틴을 포함한 페이스트의 조직이 절단된 창상의 임상적 효과를 나타낸 사진이다.12 is a photograph showing the clinical effect of cut tissue of a paste containing fibrized acellular dermal matrix and gelatin.
도 13은 섬유화된 무세포 진피 기질과 젤라틴을 포함한 페이스트의 난치성 창상의 임상적 효과를 나타낸 사진이다.Figure 13 is a photograph showing the clinical effect of the refractory wound of the paste containing the fibrous acellular dermal matrix and gelatin.
도 14는 섬유화된 무세포 진피 기질과 젤라틴을 포함한 페이스트 이용하여 제조한 시트의 외형 사진이다.14 is a photograph of the appearance of a sheet prepared using a paste containing fibrous acellular dermal matrix and gelatin.
도 15는 섬유화된 무세포 진피 기질과 젤라틴을 포함한 페이스트 이용하여 제조한 시트를 saline 용액에서 1시간 수화(rehydration)한 후 물성 및 형태를 관찰한 사진이다.15 is a photograph observing the physical properties and shape of the sheet prepared by using a paste containing the fibrous acellular dermal matrix and gelatin in the saline solution for 1 hour.
도 16은 멸균전 열처리에 따른 보관안정성을 점도 측면에서 확인한 것이다.Figure 16 confirms the storage stability according to the heat treatment before sterilization in terms of viscosity.
도 17은 멸균전 열처리에 따른 보관안정성을 겔분율 측면에서 확인한 것이다.Figure 17 shows the storage stability of the heat treatment before sterilization in terms of gel fraction.
도 18은 부착성 측정을 위한 TPA (texture profile analysis) 실험에 대한 개념도이다.18 is a conceptual diagram for a texture profile analysis (TPA) experiment for measuring adhesion.
도 19는 분쇄된 ADM 형사에 따른 부착성을 확인한 결과이다.19 is a result confirming the adhesion according to the crushed ADM detective.
발명의 상세한 설명 및 바람직한 구현예Detailed Description of the Invention and Preferred Embodiments
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술 분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로 본 명세서에서 사용된 명명법은 본 기술 분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.
본 발명에서는 멸균되고 즉시 사용 가능하며(ready-to-use), 굴곡지거나 깊이 파인 창상 부위에 적용 가능하고, 장기간 또는 저온 저장 시에도 부드러운(soft) 물성이 유지되는 것을 특징으로 하는 인체 유래 무세포 진피와 젤라틴을 포함하는 조성물을 제조하고자 하였다. In the present invention, a human-derived cell-free cell characterized by being sterilized and ready-to-use, applicable to curved or deeply cut wounds, and maintaining soft properties even during long-term or low temperature storage. To prepare a composition comprising the dermis and gelatin.
본 발명에서는, 상기 조성물이 방사선 멸균에 의해 가교도, 경화도, 압출력 등의 물성이 변하는 것을 확인하고 이에 따라 방사선 멸균 전 및/또는 후 단계에 일정한 열처리 공정을 포함하여 조성물 물성의 변화를 최소화하며 안정성을 향상 시킬 수 있었다. In the present invention, it is confirmed that the composition changes the physical properties such as cross-linking degree, curing degree, extrusion force, etc. by radiation sterilization, and accordingly minimize the change of composition properties by including a constant heat treatment step before and / or after radiation sterilization And stability could be improved.
따라서, 본 발명은 일 관점에서, 인체 유래 무세포 진피 기질(Acellular Dermal Matrix)과 젤라틴을 포함하는 조성물로서, 상기 조성물은 멸균되고 즉시 사용 가능한(ready-to-use) 것이며, 멸균 후 가교도, 점탄성, 경화도 및/또는 압출력 값의 변화도가 멸균 전 값에 비해 30% 이하인 것을 특징으로 하는 조성물 및 이의 제조 방법에 관한 것이다.Thus, in one aspect, the present invention is a composition comprising a human-derived acellular dermal matrix and gelatin, wherein the composition is sterile and ready-to-use, crosslinking degree after sterilization, Viscoelasticity, degree of cure, and / or change in extruding force value is 30% or less compared to the value before sterilization, and a composition and a method for producing the same.
본 발명의 상기 진피 조직체는 뼈, 인대, 건 또는 피부일 수 있으며, 이종 또는 동종 유래의 진피 조직일 수 있으나, 바람직하게는 인체 유래 진피 조직이다.The dermal tissue of the present invention may be bone, ligament, tendon or skin, and may be a dermal tissue derived from heterogeneous or allogeneic, preferably human dermal tissue.
본 발명에 있어서, 상기 젤라틴의 원재료 강도는 250 Bloom 이상이며 점도가 30~45 mps이며 pH가 5~6.5, 건조 감량이 5~15%인 것을 특징으로 할 수 있다. 본 발명에서는 상기 젤라틴 외 알지네이트, 아가로즈, 피브린, 콜라겐, 피브로넥틴, 폴리글리콜산(polyglycolic acid), 폴리락틱산(polylactic acid), 히알루론산(hyaluronic acid), 폴리에틸렌클리콜, 콘드로이탈(chondroital), 덜마탄(dermatan), 폴리사카라이드, 뮤코폴리사카라이드, 하이드로겔, 덱스트란, 아밀로즈, 프로테인, 글리코프로테인 및 그 유도체의 생체적합 고분자가 추가로 포함될 수 있다.In the present invention, the raw material strength of the gelatin is 250 Bloom or more, the viscosity is 30 ~ 45 mps, the pH is 5 ~ 6.5, it can be characterized in that the drying loss is 5 ~ 15%. In the present invention, the gelatin, alginate, agarose, fibrin, collagen, fibronectin, polyglycolic acid, polylactic acid, hyaluronic acid, polyethylene glycol, chondroital Biocompatible polymers of, dermatan, polysaccharides, mucopolysaccharides, hydrogels, dextran, amylose, proteins, glycoproteins and derivatives thereof may be further included.
한편, 조성물의 구성 함량은 무세포 진피 기질 5~20 중량%, 젤라틴 0.5~5 중량% 및 물 75~94.5 중량%일 수 있으나, 바람직하게는 무세포 진피 기질 8~12 중량%, 젤라틴 1~2 중량% 및 물 86~91 중량%일 수 있다.On the other hand, the constituent content of the composition may be 5 to 20% by weight of the acellular dermal matrix, 0.5 to 5% by weight of gelatin and 75 to 99.5% by weight of water, preferably 8 to 12% by weight of the acellular dermal matrix, gelatin 1 ~ 2 weight percent and 86-91 weight percent water.
본 발명의 일 실시예에서는 상기 조성물의 멸균 전 및/또는 후 단계에 30~60 ℃, 바람직하게는 35~55 ℃, 가장 바람직하게는 40~50 ℃에서 1~180분간, 바람직하게는 60~120분간 열처리를 수행함으로써 물성의 안정성이 유지되는 것을 확인하였다. In one embodiment of the present invention before and / or after sterilization of the composition at 30 to 60 ℃, preferably 35 to 55 ℃, most preferably 40 to 50 ℃ 1 to 180 minutes, preferably 60 ~ It was confirmed that stability of physical properties was maintained by performing heat treatment for 120 minutes.
상기 멸균 공정은 건조가열법, 저온살균법, 방사선 멸균법, 가스멸균법 등을 포함할 수 있으며 바람직하게는 E-beam 조사로 수행되는 방사선 멸균법을 포함 할 수 있다. E-beam 조사는 상기 무세포 진피와 젤라틴 조성물 내 가교결합을 유도하여 이와 상관관계가 있는 점탄성, 경화도 및 압출력 수치에 변화를 일으키는 것으로 확인되었다. 또한, E-beam 조사에 의한 가교결합은 완제품 제조 후 저장기간이 길어 질수록 증가하였고 특히, 저온에서 보관할 시 가교결합이 더욱 증가하였다. 상기 가교도, 점탄성, 경화도, 압출력 등 물성 변화의 측정은 특정 실험법으로 한정하지 않는다. 가교도의 경우, 간접지표인 겔 분율(Gel fraction), 팽윤도(Swelling ratio) 등을 활용할 수 있다. The sterilization process may include a dry heating method, pasteurization method, radiation sterilization method, gas sterilization method and the like, and may preferably include a radiation sterilization method performed by E-beam irradiation. E-beam irradiation was found to induce crosslinking in the cell-free dermis and gelatin compositions, resulting in changes in the viscoelasticity, degree of cure, and extruding force values correlated therewith. In addition, the cross-linking by E-beam irradiation increased with longer storage period after manufacture of the finished product, especially when cross-linking increased at low temperature. Measurement of physical property changes such as the degree of crosslinking, viscoelasticity, curing degree, and extrusion force is not limited to a specific experimental method. In the case of the degree of crosslinking, the gel fraction, swelling ratio, etc., which are indirect indicators, may be used.
본 발명은 다른 관점에서, 섬유화된 무세포 진피 기질(Acellular Dermal Matrix)과 생체적합 고분자를 포함하는 조성물로서, 상기 섬유화된 무세포 진피 기질은 단축과 장축의 비율이 1:30 내지 1:2,000인 것의 비율이 70%, 바람직하게는 80%, 가장 바람직하게는 90% 이상인 것을 특징으로 하는 조성물 및 이의 제조 방법에 관한 것이다.In another aspect, the present invention provides a composition comprising a fibrous Acellular Dermal Matrix and a biocompatible polymer, wherein the ratio of a short axis and a long axis is 1:30 to 1: 2,000. It relates to a composition and a method for producing the same, characterized in that the proportion of the thing is 70%, preferably 80%, most preferably 90% or more.
본 발명의 무세포 진피 기질은 섬유화 형태이다. 본 발명에서는 섬유화 무세포 진피 기질을 제조하기 위하여, 두께가 1 mm 이상인 진피를 선별하여 사용하였다. 두께 1 mm 이상의 진피 기질을 무세포화 한 후 건조 또는 동결건조하여 적당한 수분 함량 예를 들어, 5 내지 10% 정도의 수분을 함유할 수 있도록 하였다. 이후 상기 무세포 진피 기질을 커팅밀(Cutting mill), 푸드프로세서(Food processor), 마노 분쇄기, 동결 분쇄기 등의 분쇄기를 이용하여 분쇄, 섬유화 하였다.The cell free dermal matrix of the present invention is in fibrotic form. In the present invention, in order to prepare a fibrous acellular dermal matrix, the dermis having a thickness of 1 mm or more was used. The dermal matrix having a thickness of 1 mm or more was acellularized and then dried or lyophilized to contain an appropriate moisture content, for example, 5 to 10% of moisture. Thereafter, the cell-free dermal substrate was pulverized and fibrized using a mill such as a cutting mill, a food processor, agate grinder, or freeze grinder.
본 발명의 섬유화된 무세포 진피 기질의 단축과 장축의 길이 비율은 1:30 내지 1:2,000인 것의 비율이 70% 이상을 차지할 수 있으며, 바람직하게는 단축과 장축의 비율이 1:30 내지 1:2,000인 섬유 입자가 전체 조성물의 80%를 차지할 수 있다.The length ratio of the short axis and the long axis of the fibrous acellular dermal matrix of the present invention may be 70% or more of the ratio of 1:30 to 1: 2,000, preferably the ratio of the short axis and the long axis is 1:30 to 1 Fiber particles of: 2,000 can account for 80% of the total composition.
상기와 같은 조건의 섬유화된 무세포 진피 기질은 실뭉치 처럼 뭉친 형태와 실처럼 가늘고 긴 단일 섬유 형태가 공존하며, 창상 피복재로 사용될 시 체내 보존성이 우수하고 습윤 환경의 유지가 잘되며, 별도의 화학적 결합 없이도 고분자와 안정하게 지탱되는 구조를 보인다. The fibrous cell-free dermal matrix under the above conditions coexists in the form of a bundle of thread and a single, long and long fiber, and is excellent in preservation of the body when used as a wound coating material, and well maintained in a humid environment. It shows a structure that is stably supported with a polymer even without bonding.
본 발명은 다른 관점에서, 무세포 진피 기질(ADM) 75 내지 85 중량%, 생체적합 고분자 15 내지 25 중량% 및 3 내지 8 중량%의 물을 포함하는 창상 피복재용 시트로서, 가로 길이가 4~2O cm 이며 세로 길이가 4~2O cm이며 두께가 1.5~3 mm인것을 특징으로 하는 창상 피복재용 시트에 관한 것이다.In another aspect, the present invention provides a wound coating sheet comprising 75 to 85% by weight of acellular dermal matrix (ADM), 15 to 25% by weight of biocompatible polymer and 3 to 8% by weight of water, having a transverse length of 4 to It relates to a sheet for wound covering, characterized in that it is 20 cm in length and 4 to 20 cm in length and 1.5 to 3 mm in thickness.
본 발명에서는 상기 페이스트 형태의 무세포 진피 기질과 생체적합 고분자 포함 조성물을 특정 금형(mold 또는 frame)에 도포 및 동결건조하여 창상 피복재용 시트를 제조하였다. In the present invention, the paste-free dermal matrix and the biocompatible polymer-containing composition are applied to a specific mold (mold or frame) and lyophilized to prepare a wound coating sheet.
상기 동결건조는 시트의 수분함량이 3~8%, 바람직하게는 4~5%가 될 때까지 수행할 수 있다.The lyophilization may be performed until the moisture content of the sheet is 3 to 8%, preferably 4 to 5%.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.
실시예 1: 인체 유래 무세포 진피 기질의 섬유화 방법Example 1 Fibrosis Method of Human Cell-Free Dermal Matrix
사체에서 분리한 피부조직을 EURO skin bank, Allosource, CTS에서 구입한 후, 두께가 1 mm 이상인 조직을 선별하여 무세포 진피 기질 제조에 사용하였다. The skin tissues isolated from the carcasses were purchased from EURO skin bank, Allosource, CTS, and then tissues having a thickness of 1 mm or more were selected and used for the preparation of acellular dermal matrix.
상기 피부조직을 1M NaCl 용액을 이용하여 38℃에서 6시간 내지 24시간 동안 반응시킨 후 겸자(Forceps)를 이용하여 표피를 제거하였다. 표피가 제거된 진피를 인산완충용액으로 세척한 후, 0.5% SDS(Sodium dodecyl sulfate)와 상온에서 1시간 동안 반응시켜 진피 내 세포를 제거하였다.The skin tissue was reacted at 38 ° C. for 6 hours to 24 hours using 1M NaCl solution, and then the epidermis was removed using forceps. The epidermis from which the epidermis was removed was washed with phosphate buffer solution, and then reacted with 0.5% SDS (Sodium dodecyl sulfate) at room temperature for 1 hour to remove the cells in the dermis.
글리세롤(Sigma, 미국), 프로필렌 글리콜(Sigma, 미국) 및 인산완충용액(Gibco, 미국)을 1:1:8의 중량비로 혼합한 용액에 말티톨, 수크로스 또는 솔비톨의 최종 농도를 30%로 조절하여 동결 보호제를 만들었다. 상기 말티톨은 진피 조직의 갈변 현상을 방지하는 것으로 확인하였고, 이에 말티톨을 사용하였다. Final concentration of maltitol, sucrose or sorbitol is adjusted to 30% in a solution of glycerol (Sigma, USA), propylene glycol (Sigma, USA) and phosphate buffer solution (Gibco, USA) at a weight ratio of 1: 1: 8. To make a cryoprotectant. The maltitol was confirmed to prevent browning of the dermal tissue, maltitol was used for this.
4℃ 저온 반응기에서 상기 진피에 상기 동결 보호제를 12시간 동안 침투시켰다. 침투가 완료된 진피를 타이백(고려신소재, 한국)에 넣고 진공 5 torr인 동결건조기에 넣어 24시간 동안 건조시켜 동결건조된 무세포 진피 기질을 만들었다.The cryoprotectant was infiltrated into the dermis for 12 hours in a 4 ° C. low temperature reactor. The penetrated dermis was placed in a taibag (Korea Advanced Materials, Korea) and placed in a lyophilizer with a vacuum of 5 torr for 24 hours to prepare a freeze-dried acellular dermal matrix.
동결건조된 무세포 진피 기질을 커팅밀(Pulverisette19, FRITSCH, 독일)을 사용하여 분쇄하여 섬유화하였다.Lyophilized acellular dermal substrates were triturated using a cutting mill (Pulverisette 19, FRITSCH, Germany) to fiberize.
도 1은 상기 방법을 통해 제조된 섬유화된 무세포 진피 기질(A)과 비드형으로된 무세포 진피 기질(B)의 외형을 비교한 것이다.Figure 1 compares the appearance of the fibroblast-free dermal matrix (A) and the bead-shaped acellular dermal matrix (B) prepared by the above method.
섬유화된 무세포 진피 기질은 솜(cotton) 형태 즉, 섬유들이 엉키어 붙어 있는 형태로, 비드형과 비교하여 부피(표면적)가 크고 분쇄된 기질의 구조 형상이 안정적이었다. The fibrous cell-free dermal matrix was in the form of a cotton, ie, entangled fibers, and had a larger volume (surface area) and a more stable structural shape compared to the bead type.
상기 비드형은 두께 1 mm 이하의 무세포 진피 기질을 선별하여 상기와 동일한 방법으로 무세포화, 동결건조, 분쇄 및 제조하였다.The bead type was selected from acellular dermal substrates with a thickness of 1 mm or less, and then acellularized, lyophilized, ground, and prepared in the same manner as described above.
실시예 2: 섬유화된 무세포 진피 기질의 장, 단축 길이, 이의 분포도 (distribution) 및 장, 단축 길이 비율의 측정Example 2 Determination of the Longitudinal, Shortening Length, Distribution, and Longitudinal, Shortening Ratio of Fibrous Cellular Dermal Matrix
상기 실시예 1을 통해 획득한 섬유화된 무세포 진피 기질 입자의 장, 단축 길이를 Zoom Stereomicroscope(SMZ745, Nikon, Japan) 현미경으로 사진 촬영한 후, Image Analyzer 프로그램(i-SolutionTM IMT i-Solution Inc., 한국)을 이용하여 측정하였다. After photographing the long and short length of the fibrous acellular dermal matrix particles obtained through Example 1 with a Zoom Stereomicroscope (SMZ745, Nikon, Japan) microscope, the Image Analyzer program (i-SolutionTM IMT i-Solution Inc.). , Korea).
섬유화된 무세포 진피 기질의 장축 길이와 분포도는 도 2에 나타난 바와 같이, 500~1,000 μm가 약 33%, 1,000~1,500 μm가 약 30%, 1,500~2,000 μm가 약 27%, 2,000~2,500 μm가 약 7% 및 2,500~3,000 μm가 약 3%로 나타났다. 즉, 장축 길이가 1,000에서 2,000 μm인 섬유 입자가 전체 입자 중 약 60%를 차지하였으며, 500에서 2,000 μm인 섬유 입자가 전체 입자 중 약 90%를 차지하였다. As shown in FIG. 2, the long axis length and distribution of the fibrous acellular dermal matrix are about 33% for 500-1,000 μm, about 30% for 1,000-1,500 μm, about 27% for 1,500-2,000 μm, and 2,000-2,500 μm. Was about 7% and 2,500 ~ 3,000 μm was about 3%. That is, fiber particles having a major axis length of 1,000 to 2,000 μm accounted for about 60% of the total particles, and fiber particles having 500 to 2,000 μm accounted for about 90% of the total particles.
섬유화된 무세포 진피 기질의 단축 길이와 분포도는 도 3에 나타난 바와 같이, 10~20 μm가 약 20%, 20~30 μm가 약 39%, 30~40 μm가 약 28%, 40~50 μm가 약 9% 및 50~60 μm가 약 4%로 나타났다. 즉, 약 70%의 섬유 입자가 20에서 40 μm의 단축 길이를 가지는 것으로 확인하였다. 즉, 단축 길이가 20에서 40 μm인 섬유 입자가 전체 입자 중 70%를 차지하였다.As shown in FIG. 3, the length and distribution of the fibrous acellular dermal matrix are 10-20 μm, about 20%, 20-30 μm, about 39%, 30-40 μm, about 28%, and 40-50 μm. About 9% and 50-60 μm were about 4%. That is, it was confirmed that about 70% of the fiber particles have a short axis length of 20 to 40 μm. That is, fiber particles having a short axis length of 20 to 40 μm accounted for 70% of the whole particles.
섬유화된 무세포 진피 기질의 장, 단축 비율은 도 4A에 나타난 바와 같이, 20~30:1이 약 3%, 30~40:1이 약 23%, 40~50:1이 약 33%, 50~60:1이 약 34% 및 60~70:1이 약 7%로 측정되었다. 즉, 장, 단축 비율이 40~60:1인 섬유 입자가 전체 입자 중 70%를 차지하였으며, 40~70:1인 섬유 입자가 약 75%를 차지하였다.As shown in FIG. 4A, the intestinal and shortening ratios of the fibroblasted dermal matrix were about 3% for 20-30: 1, about 23% for 30-40: 1, about 33% for 50-50: 1, and 50%. ˜60: 1 was measured at about 34% and 60-70: 1 at about 7%. That is, the fiber particles having a long and short axis ratio of 40 to 60: 1 accounted for 70% of the total particles, and the fiber particles having 40 to 70: 1 accounted for about 75%.
반면에 비드형 무세포 진피 기질의 장, 단축 비율은 도 4B에 나타난 바와 같이, 약 90%의 입자가 1~2:1의 비율을 나타내었다. 섬유화된 입자와 비교하여 비드형은 구형에 가까운 입자 형태인 것으로 확인하였다.On the other hand, as shown in Figure 4B, the length and shortening ratio of the bead-type acellular dermal matrix, about 90% of the particles showed a ratio of 1-2: 1. Compared with the fiberized particles, the bead form was found to be in the form of particles close to the sphere.
도 4C는 섬유화된 무세포 진피 기질의 현미경 사진, 도 4D는 비드형 무세포 진피 기질의 현미경 사진을 나타낸 것이다. FIG. 4C shows a micrograph of a fibrous acellular dermal matrix, and FIG. 4D shows a micrograph of a bead-shaped acellular dermal matrix.
실시예 3: 무세포 진피 기질을 이용한 페이스트의 제조Example 3: Preparation of Paste Using Cell-Free Dermal Substrate
돈피 젤라틴(삼미, 한국) 1.25 g에 물을 첨가하여 100 ml의 혼합액을 만든 후, 60℃에서 용해시켜 젤라틴 수용액을 제조하였다. 상기 젤라틴의 강도는 299 bloom, 점도는 38.3 mps, pH는 5.83, 건조 감량은 11.4%으로 이취, 이물, 불용물이 없으며 SO2, 중금속, 비소, 강열잔분, 크롬, 납, 일반 박테리아가 기준 수치 이하로 측정되었다. 상기 젤라틴 수용액 89g과 상기 실시예 1을 통해 획득한 섬유화된 무세포 진피 기질 11 g을 60 ℃에서 혼합하여 페이스트(Paste)를 제조하였다.Water was added to 1.25 g of pork skin gelatin (Sammi, Korea) to make a 100 ml mixed solution, and then dissolved at 60 ℃ to prepare a gelatin aqueous solution. The gelatin has a strength of 299 bloom, a viscosity of 38.3 mps, a pH of 5.83, and a loss of dryness of 11.4%. There is no off-flavor, foreign matter, or insoluble matter, and SO2, heavy metals, arsenic, ignition residue, chromium, lead, and general bacteria are below the standard value Was measured. 89 g of the gelatin aqueous solution and 11 g of the fibrous acellular dermal substrate obtained in Example 1 were mixed at 60 ° C. to prepare a paste.
상기 페이스트를 시린지에 담아 P.E.T tray 및 타이백 이중 포장 후 E-beam 방사선 멸균을 실시하였다. 멸균 전 및/또는 후 단계에 45 ℃ incubation에서 2시간 또는 1시간 열처리를 하여 방사선 멸균에 의한 페이스트의 가교반응을 억제하였다. 도 5는 페이스트의 제형을 나타낸 사진이다. The paste was placed in a syringe and subjected to E-beam radiation sterilization after P.E.T tray and tie bag double packaging. The crosslinking reaction of the paste by radiation sterilization was inhibited by heat treatment at 45 ° C. incubation for 2 hours or 1 hour before and / or after sterilization. Figure 5 is a photograph showing the formulation of the paste.
실시예 4: 페이스트의 점도 측정Example 4: Viscosity Measurement of Pastes
상기 실시예 3의 방법으로 5개 샘플의 페이스트를 제조하여 초기 상태와 3개월 보관 후의 페이스트 점도를 측정하였다.The paste of five samples was prepared by the method of Example 3, and the paste viscosity after initial storage and after 3 months storage was measured.
상기 페이스트의 점도는 회전점도계법(대한약전 일반시험법)에 따라 유체 내에서 원판을 회전시켜 유체의 점성으로 인해서 생기는 토크를 이용하여 측정하였으며 회전점도계(DV2THB Viscometer, Brookfield, 미국)의 써큘레이터(circulator) 조건은 15rpm 및 25℃로 맞추어 시료 1 g의 점도를 반복 측정하였다. The viscosity of the paste was measured using the torque generated by the viscosity of the fluid by rotating the disk in the fluid according to the rotational viscometer method (KEPCO general test method) and the circulator (DV2THB Viscometer, Brookfield, USA) circulator) conditions were repeatedly measured at a viscosity of 15 g at 15 rpm and 25 ° C.
그 결과, 도 6과 같이 초기 상태와 3개월 보관 후의 페이스트 모두 20,000에서 25,000 cps 사이의 점도를 나타내었다. 즉, 페이스트의 점도 물성이 3개월간 변하지 않고 유지되는 것을 확인하였다. As a result, as shown in Figure 6, both the initial state and the paste after 3 months storage showed a viscosity between 20,000 and 25,000 cps. That is, it was confirmed that the viscosity physical properties of the paste remained unchanged for three months.
실시예 5: 페이스트의 압출력 측정Example 5: Measurement of Extrusion Force of Paste
압출력은 일정한 시간과 힘 그리고 일정한 방향으로 시편에 대해 일을 가해줄 때, 그 힘에 대해 발생한 load값을 측정한 것이다. 페이스트 제조 단계에서 방사선 멸균 여부 또는 방사선 멸균 전, 후 단계의 반응 온도 조건에 따른 페이스트의 압출력을 비교하였다. The extrusion force is a measure of the load value generated for a given time, force, and force when the work is applied to the specimen in a constant direction. The extruding force of the paste was compared according to the radiation sterilization in the paste preparation step or the reaction temperature conditions before and after the radiation sterilization.
E-beam 멸균을 하지 않은 페이스트와 E-beam 멸균 전, 후 단계에 반응 온도를 각기 10, 25 및 45 ℃로 조절한 페이스트를 3cc 주사기에 넣고 UTM(Universal Testing Machine)을 이용하여 일정한 힘으로 주사기로부터 시료를 압출하였다. Paste without E-beam sterilization and paste with the reaction temperature adjusted to 10, 25, and 45 ° C before and after E-beam sterilization were put in a 3cc syringe, and the syringe was applied with a constant force using a universal testing machine (UTM). The sample was extruded from.
그 결과, E-beam 멸균을 하지 않은 페이스트의 압출력은 약 10에서 15 N 사이를 유지하였고(평균 12.1 N), 시간에 따른 증가 또는 감소의 양상을 보이지 않았다. E-beam 멸균을 수행한 페이스트 중 10 ℃ 온도반응을 가진 페이스트는 시간에 따라 압출력의 크기가 정비례하게 증가하였다. 압출을 가한 지 30초 후에는 약 40 N에 달하는 압출력 수치를 보였고, 평균 값은 26.82 N으로 측정되었다. 25 ℃에서 온도반응을 가진 페이스트는 약 15~20 N 사이의 압출력 값, 평균 16.06 N 값을 보이며 시간에 따른 증가 또는 감소의 양상을 보이지 않았다. As a result, the extrusion force of the paste without E-beam sterilization was maintained at about 10 to 15 N (average 12.1 N), and did not show any increase or decrease with time. Among the pastes subjected to the E-beam sterilization, the paste having a temperature reaction of 10 ° C. increased the magnitude of the extrusion force with time. Thirty seconds after the extrusion, the extruding force value reached about 40 N, and the average value was 26.82 N. The paste with temperature reaction at 25 ℃ showed an extrusion force value of about 15-20 N, an average value of 16.06 N and did not show any increase or decrease with time.
45 ℃에서 온도반응을 가진 페이스트는 약 6~8 N 사이의 압출력 값, 평균 6.645 N 값을 보이며 시간에 따른 압출력의 변화도가 상기 실험군들 중 가장 완만한 양상을 보였다(도 7). 이는 45 ℃에서 온도반응을 가진 페이스트가 겔화되지 않고 입자가 고르게 분포되어 있다는 것을 의미한다. 반면에, E-beam을 처리하는 않은 페이스트, E-beam 멸균과 함께 10 ℃ 및 25 ℃에서 온도반응을 가진 페이스트는 겔화에 의한 입자가 인지되어 압출력 곡선이 완만하지 않고 급격하게 꺾이는 양상이 확인되었다. The paste having a temperature reaction at 45 ° C. exhibited an extruding force value of about 6 to 8 N, an average of 6.645 N, and the change in extruding force over time showed the most gentle pattern among the experimental groups (FIG. 7). This means that the paste having a temperature reaction at 45 ° C. does not gel and the particles are evenly distributed. On the other hand, pastes not treated with E-beam and pastes with temperature reaction at 10 ℃ and 25 ℃ together with E-beam sterilization showed that the extrusion force curve was not smooth but sharply bent because the particles were recognized by gelation. It became.
E-beam 멸균을 하지 않은 페이스트와 45 ℃에서 E-beam 멸균을 수행한 페이스트의 평균 압출력은 각기 약 12.1 N과 6.645 N으로, 압출력 값의 변화가 약 45%로 나타내었다. The average extruding force of the paste without e-beam sterilization and the paste with e-beam sterilization at 45 ° C. was about 12.1 N and 6.645 N, respectively, and the change of the extrusion force value was about 45%.
페이스트 제형에 E-beam 멸균을 할 경우, 가교 결합에 의해 압출력이 증가하는 양상을 보였다. 다만, E-beam 멸균 시 45 ℃의 열을 가할 경우, 가교 결합이 억제되어 압출력이 감소되었고, 다른 실험군에 비해 안정적인 양상의 압출력 값을 보였다.When the paste formulation was sterilized by E-beam, the extrusion force was increased by crosslinking. However, when heat was applied at 45 ° C. during sterilization of E-beam, the cross-linking was suppressed and the extrusion force was decreased, and the extrusion force value was more stable than other experimental groups.
실시예 6: 겔 분율(가교도) 측정Example 6: Determination of gel fraction (crosslinking degree)
상기 실시예 3의 페이스트 제조 단계에서 방사선 멸균 전, 후 단계의 반응 온도에 따른 무세포 진피 기질 페이스트의 겔 분율(가교도)을 측정하였다(도 8). 겔 분율을 분석하기 위하여, 각 온도에서 반응시킨 페이스트의 초기 무게를 측정한 후, 3차 증류수(Deionized water)에서 침지하여 항온진탕수조(60rpm)에서 48시간 또는 72시간 상온 처리 하였다. 상기 반응 후, 불용성 부분을 메시로 필터링 하여 50 ℃ 드라이 오븐에서 건조하였다.In the paste preparation step of Example 3, the gel fraction (crosslinking degree) of the cell-free dermal matrix paste was measured according to the reaction temperature before and after radiation sterilization (FIG. 8). In order to analyze the gel fraction, after measuring the initial weight of the paste reacted at each temperature, it was immersed in tertiary distilled water (Deionized water) and treated at room temperature for 48 hours or 72 hours in a constant temperature shaker (60rpm). After the reaction, the insoluble portion was filtered through a mesh and dried in a 50 ° C. dry oven.
Gel Fraction(%) = Wd/Wi * 100Gel Fraction (%) = Wd / Wi * 100
Wi: 최초 시료무게Wi: Initial sample weight
Wd: 추출 후 건조무게Wd: dry weight after extraction
5, 15, 25, 35, 45 및 55 ℃에서 반응시킨 페이스트의 겔 분율은 각기 99, 96, 78, 18, 6 및 3%로 분석되었다. 이에 따라, 페이스트의 방사선 멸균 전, 후 단계의 반응 온도가 높을수록 겔 분율이 낮아지는 즉, 가교도가 낮아지는 현상을 확인하였다. The gel fractions of the pastes reacted at 5, 15, 25, 35, 45 and 55 ° C. were analyzed to be 99, 96, 78, 18, 6 and 3%, respectively. Accordingly, the higher the reaction temperature before and after the sterilization of the paste, the lower the gel fraction, that is, the degree of crosslinking was confirmed.
또한, E-beam 멸균을 하지 않은 페이스트의 겔 분율은 약 2%로, 가교가 이루어지지 않은 것으로 분석되었다. 반면에, 상온에서 E-beam 멸균을 처리할 경우 겔 분율의 값은 약 79%로 나타났다.In addition, the gel fraction of the paste without E-beam sterilization was about 2%, which was analyzed as not crosslinking. On the other hand, when the E-beam sterilization at room temperature, the gel fraction was about 79%.
E-beam 멸균을 하지 않은 페이스트와 45 ℃ 및 55 ℃에서 E-beam 멸균을 수행한 페이스트의 겔 분율은 각기 약 2%와 6.3% 및 3%로, 겔 분율의 변화도가 약 68% 및 33%로 분석되었다.The gel fractions of the paste without e-beam sterilization and the pastes subjected to the e-beam sterilization at 45 ° C. and 55 ° C. were about 2%, 6.3%, and 3%, respectively. Analyzed in%.
페이스트 제형에 E-beam 멸균을 할 경우, 가교 결합에 의해 겔 분율이 증가하지만, E-beam 멸균 시 45 ℃ 또는 55 ℃의 열을 가할 경우, 가교 결합이 억제되어 겔 분율이 감소되는 것을 확인하였다.When the paste formulation was sterilized by E-beam, the gel fraction was increased by crosslinking, but when the heat was applied at 45 ° C or 55 ° C during sterilization of the E-beam, it was confirmed that the gel fraction was reduced by inhibiting the crosslinking. .
실시예 7: 페이스트의 임상 치료 효과Example 7: Clinical Treatment Effect of Paste
7-1: 넓고 깊은 창상 부위의 임상 치료 효과7-1: Clinical treatment effect for wide and deep wounds
뼈와 건이 노출된 창상 부위에 본 발명의 페이스트 2cc를 2주 동안 2번, 큐라백과 함께 처리하였다. 2주 후, 페이스트를 생착시킨 부위의 육아조직이 재형성되어 뼈의 노출을 덮는 것을 확인하였다(도 9). Bone and tendon wounds were treated with 2 cc of paste of the present invention for 2 weeks with Curacao bag. After two weeks, it was confirmed that granulation tissue of the site where the paste was grafted was reshaped to cover the bone exposure (FIG. 9).
7-2: 족부 창상의 임상 치료 효과7-2: Clinical treatment effect of foot wound
족부 외상으로 인한 포켓 창상에 본 발명의 페이스트를 유고툴 베리어, 큐라백과 함께 처리하였다. 그 결과, 적용 7일차에 육아조직이 형성되어 포켓 창상의 깊이가 얕아 진 것을 확인하였고, 이후 반복 처리하여 수술 없이 족부궤양이 치료되는 것을 확인하였다(도 10).The paste of the present invention was treated with a yugotool barrier and a curabag on a pocket wound due to foot trauma. As a result, it was confirmed that granulation tissue was formed on the 7th day of application, so that the depth of the pocket wound was shallow, and after that, it was confirmed that the foot ulcer was treated without surgery (FIG. 10).
7-3: 만성화된 창상의 임상 치료 효과7-3: Clinical treatment effect of chronicized wound
흉부 수술 후 감염으로 인해 수술창이 벌어진 케이스로, 환자의 상태가 좋지 않아 재수술이 어렵고 큐라백 단독 사용으로도 6개월간 육아조직이 형성되지 않아 만성화된 창상에, 본 발명의 페이스트를 처리하여 수술 없이 창상이 완치된 것을 확인하였다(도 11)Injury after thoracic surgery due to infection, the operation window is open, it is difficult to reoperate because the patient's condition is not good, and the granulation tissue is not formed for 6 months by using Cura Bag alone. It was confirmed that this was cured (FIG. 11).
7-4: 조직이 절단된 창상의 임상 치료 효과7-4: Clinical treatment effect of tissue cuts
당뇨 합병증으로 인해 발가락을 절단한 환자의 깊게 패인 창상에 본 발명의 페이스트와 큐라백을 병행 처리하였다. 그 결과, 치료 9일차에 페이스트 안으로 혈관이 차 들어와 육아조직 형성이 촉진되어 44일만에 완치된 것을 확인하였다(도 12). 상기와 같은 발가락 절단 창상의 완치는 평균 90일 정도 소요되는 것으로 알려져 있으므로 본 발명의 페이스트 적용 시 치유 기간이 절반으로 단축되는 것으로 확인할 수 있었다.The paste and curabag of the present invention were treated in parallel in the deeply dug wounds of patients who had cut toes due to diabetic complications. As a result, it was confirmed that blood vessels were introduced into the paste on the 9th day of treatment to promote granulation tissue formation and to be cured after 44 days (FIG. 12). Since the cure of the toe cut wound is known to take an average of about 90 days, it was confirmed that the healing period is reduced by half when the paste of the present invention is applied.
7-5: 난치성 창상의 임상 치료 효과7-5: Clinical treatment effect of refractory wound
연조직 손상에 있는 뼈가 노출된 창상에 피판형성술(Flap 시술)을 하였으나, 창상이 다시 벌어진 터널링 창상으로 육아조직이 형성되지 않아 난치성 창상으로 발전하였다. 이와 같이 기존 치료로 치유되지 않고 수술하기도 어려운 케이스에 본 발명의 페이스트를 적용하자 혈관이 차 오르고 육아조직이 형성되어 최종 봉합으로 완치된 것을 확인하였다(도 13).Flap was performed on the exposed bone of soft tissue, but the tunneling wound was reopened, and granulation tissue was not formed. Thus, when the paste of the present invention is applied to a case that is difficult to be cured by the existing treatment and is difficult to operate, it was confirmed that blood vessels rise and granulation tissue is formed and cured by the final suture (FIG. 13).
실시예 8: 창상 피복재용 시트의 제조 Example 8: Preparation of wound covering sheet
상기 실시예 3에 의해 제조된 페이스트를 가로, 세로 및 두께 수치가 10 cm, 10 cm 및 2.5 mm 금형에 도포한 후 동결건조하였다.The paste prepared according to Example 3 was lyophilized after the horizontal, vertical and thickness values were applied to 10 cm, 10 cm and 2.5 mm molds.
도 14는 상기 방법으로 제조된 시트의 외형 사진이며, 도 15는 상기 시트를 1시간 동안 saline 용액에서 수화(rehydaration)한 후 촬영한 사진이다. 1시간 수화 후에도 외형의 큰 변화 없이 시트의 형태가 유지되는 것을 확인하였다.FIG. 14 is an external photograph of a sheet manufactured by the above method, and FIG. 15 is a photograph taken after hydration of the sheet in a saline solution for 1 hour. After 1 hour of hydration, it was confirmed that the form of the sheet was maintained without a great change in appearance.
실시예 9: 멸균전 열처리에 따른 보관 안정성 확인Example 9: Confirmation of storage stability by heat treatment before sterilization
9-1. 점도 확인9-1. Check viscosity
페이스트의 점도는 회전점도계법(대한약전 일반시험법)에 따라 유체 내에서 원판을 회전시켜 유체의 점성으로 인해서 생기는 토크를 이용하여 측정하였으며 회전점도계(DV2THB Viscometer, Brookfield, 미국)의 써큘레이터(circulator) 조건은 15rpm 및 25℃로 맞추어 시료 1 g의 점도를 반복 측정하였다. 시료는 멸균 전 최적화된 온도 (45℃)에서 처리한 것과 그렇지 않은 시료를 비교 테스트 진행하였다. The viscosity of the paste was measured using the torque generated by the viscosity of the fluid by rotating the disk in the fluid according to the Rotational Viscometer Method (KEPCO General Test Method) and the circulator of the DVK2THB Viscometer (Brookfield, USA) ) The conditions were 15 rpm and 25 ° C., and the viscosity of the sample 1 g was repeatedly measured. Samples were subjected to comparative testing of samples that were processed at optimized temperature (45 ° C.) and those that were not before sterilization.
그 결과, 도 16에 개시된 바와 같이 열처리 한 시료에서는 방사선 멸균 후에도 초기 상태와 비슷한 점도 20,000-3,000cp가 나타나는 반면, 그렇지 않은 시료에서는 방사선 멸균 후 가교가 일어나 점도가 3배 보다 높게 측정되는 것을 확인하였다.As a result, the sample heat-treated as shown in FIG. 16 showed a viscosity of 20,000-3,000 cps similar to the initial state even after radiation sterilization, while in other samples, crosslinking occurred after radiation sterilization, and thus the viscosity was measured to be higher than 3 times. .
9-2. 겔분율 확인9-2. Gel fraction check
페이스트 제조 단계에서 방사선 멸균 전 열처리 유무에 따른 무세포 진피 기질 페이스트의 겔 분율(가교도)을 측정하였다. 겔 분율을 분석하기 위하여, 각 온도에서 반응시킨 페이스트의 초기 무게를 측정한 후, 3차 증류수(Deionized water)에서 침지하여 항온진탕수조(60rpm)에서 48시간 또는 72시간 상온 처리 하였다. 상기 반응 후, 불용성 부분을 메시로 필터링 하여 50 ℃ 드라이 오븐에서 건조하였다.In the paste preparation step, the gel fraction (crosslinked degree) of the cell-free dermal matrix paste with or without heat treatment before radiation sterilization was measured. In order to analyze the gel fraction, after measuring the initial weight of the paste reacted at each temperature, it was immersed in tertiary distilled water (Deionized water) and treated at room temperature for 48 hours or 72 hours in a constant temperature shaker (60rpm). After the reaction, the insoluble portion was filtered through a mesh and dried in a 50 ° C. dry oven.
Gel Fraction(%) = Wd/Wi * 100Gel Fraction (%) = Wd / Wi * 100
Wi: 최초 시료무게Wi: Initial sample weight
Wd: 추출 후 건조무게Wd: dry weight after extraction
그 결과, 도 17에 개시된 바와 같이 열처리 한 시료에서는 시간이 지나도 방사선 멸균 후 겔 분율은 약 6%를 유지하는 반면, 그렇지 않은 시료에서는 방사선 멸균 후 가교가 일어나 70-85%의 겔 분율이 측정되는 것을 확인하였다.As a result, in the heat-treated sample as shown in FIG. 17, the gel fraction after radiation sterilization was maintained at about 6% over time, whereas in other samples, the gel fraction was 70-85% after radiation sterilization, resulting in crosslinking. It was confirmed.
실시예 10: 분쇄된 ADM 형상에 따른 부착성 확인Example 10: Confirmation of adhesion according to the crushed ADM shape
페이스트를 섬유형으로 분쇄한 ADM과 비드형으로 분쇄한 ADM 제조시 부착성을 비교 측정하기 위해 TPA (texture profile analysis)를 이용하여 측정하였다(도 18). 50 mm/min의 속도로 시료를 압축판으로 눌러 측정하며 시료와 압축판이 떨어지는데 까지 필요한 힘을 negative force area로 나타내었다. The paste was measured using a texture profile analysis (TPA) to compare the adhesion in preparing ADM pulverized into fibrous and ADM pulverized into beads (FIG. 18). The sample was pressed with a compression plate at a rate of 50 mm / min, and the force required to drop the sample and the compression plate was expressed as a negative force area.
그 결과, 도 19에 개시된 바와 같이 섬유형으로 분쇄한 ADM으로 제조한 페이스트는 약 26,000g.s로 나타난 반면 비드형으로 분쇄한 ADM으로 제조한 페이스트는 약 3,000g.s로 부착성이 낮은 것을 확인하였다.As a result, as shown in FIG. 19, the paste prepared from ADM pulverized into fibrous form appeared to be about 26,000 g.s, while the paste prepared from ADM pulverized into bead form was found to have low adhesion at about 3,000 g.s.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
본 발명에 따른 창상 피복재용 페이스트는 종래의 시트형 무세포 진피 기질의 적용이 어려운 부위나 국소 부위에도 용이하게 적용이 가능하며, 즉시 사용 가능한 ready-to-use 제품으로 사용이 편리하며 심각한 수준의 창상도 수술 없이 빠른 완치가 가능하다. The wound coating paste according to the present invention can be easily applied to a difficult or topical area where the conventional sheet-shaped dermal matrix is difficult to apply, and is a ready-to-use product that can be used immediately and is easy to use and has a serious wound. Quick cure is possible without surgery.
또한, 장기간 또는 저온 보관시에도 물성의 안정성이 우수하여 국소 부위에 도포 시 발림성이 부드럽고 접착성이 우수하다. 이에 따라, 창상 부위를 외부 오염원으로부터 보호하고 습윤 환경을 유지함으로써 효율적으로 창상 부위의 치료에 도움을 줄 수 있다. In addition, it is excellent in the stability of physical properties even during long-term storage or low temperature, and when applied to a topical area, the application is smooth and excellent adhesion. Accordingly, it is possible to effectively treat the wound site by protecting the wound site from external contaminants and maintaining a wet environment.
또한, 본 발명에 따른 창상 피복재용 시트는 생체 접착성이 우수하고 오랜 수화(rehydration) 과정에서도 고분자 구조가 잘 풀어지지 않아 전문의 시술 시 형태 유지능이 우수하다. 또한, 상기 시트는 창상 부위 또는 수술 특성에 의해 요구되는 가로, 세로 및 두께 사이즈를 조절할 수 있어 창상 치료가 질적으로 향상 될 수 있다.In addition, the wound coating sheet according to the present invention is excellent in bioadhesiveness and does not release the polymer structure well even in a long rehydration (excellent) process, it is excellent in shape retention ability during the professional procedure. In addition, the sheet can adjust the width, length and thickness size required by the wound site or surgical characteristics can be improved quality of the wound treatment.

Claims (63)

  1. 인체 유래 무세포 진피 기질(Acellular Dermal Matrix)과 젤라틴을 포함하는 조성물로서, 상기 조성물은 멸균되고 즉시 사용 가능한(ready-to-use) 것이며, 멸균 후 가교도, 점탄성, 경화도 및/또는 압출력 값의 변화도가 멸균 전 값에 비해 30% 이하인 것을 특징으로 하는 조성물. A composition comprising a human-derived Acellular Dermal Matrix and gelatin, wherein the composition is sterile and ready-to-use, and is cross-linked, viscoelastic, curable and / or extrudable after sterilization. The composition characterized in that the change in value is 30% or less compared to the value before sterilization.
  2. 제1항에 있어서, 상기 조성물은 5 내지 20 중량%의 무세포 진피 기질, 0.5 내지 5 중량%의 젤라틴 및 75 내지 94.5 중량%의 물을 포함하는 조성물.The composition of claim 1, wherein the composition comprises 5-20 wt% acellular dermal matrix, 0.5-5 wt% gelatin and 75-94.5 wt% water.
  3. 제1항에 있어서, 상기 무세포 진피 기질은 섬유화된 무세포 진피 기질인 것을 특징으로 하는 조성물.The composition of claim 1, wherein the cell-free dermal matrix is a fibrotic cell-free dermal matrix.
  4. 제3항에 있어서, 상기 섬유화된 무세포 진피 기질은 장축 길이가 500 내지 2,000 ㎛인 것을 특징으로 하는 조성물.4. The composition of claim 3, wherein the fibrous acellular dermal matrix has a long axis length of 500 to 2,000 μm.
  5. 제3항에 있어서, 상기 섬유화된 무세포 진피 기질은 단축과 장축의 비율이 1:30 내지 1:2,000인 것의 비율이 70% 이상인 것을 특징으로 하는 조성물. 4. The composition of claim 3, wherein the fibrous acellular dermal matrix has a ratio of 1:30 to 1: 2,000 of short axis and long axis of 70% or more.
  6. 제1항에 있어서, 상기 젤라틴의 강도가 250 Bloom 이상이며 점도가 30~45 mps이며 pH가 5~6.5인것을 특징으로 하는 조성물.The composition of claim 1, wherein the gelatin has a strength of at least 250 Bloom, a viscosity of 30 to 45 mps, and a pH of 5 to 6.5.
  7. 제1항에 있어서, E-beam을 이용한 방사선 멸균 단계를 포함하는 공정을 통해 제조된 것을 특징으로 하는 조성물. According to claim 1, wherein the composition characterized in that it is prepared through a process comprising a radiation sterilization step using E-beam.
  8. 제7항에 있어서, 상기 E-beam을 이용한 방사선 멸균 단계 전 및/또는 후에, 30~60 ℃의 열처리 단계를 포함하는 것을 특징으로 하는 조성물. According to claim 7, wherein before and / or after the radiation sterilization step using the E-beam, the composition comprising a heat treatment step of 30 ~ 60 ℃.
  9. 제8항에 있어서, 상기 열처리는 1~180 분간 수행되는 것을 특징으로 하는 조성물. The composition of claim 8, wherein the heat treatment is performed for 1 to 180 minutes.
  10. 제1항에 있어서, 상기 조성물은 페이스트(paste) 형태 또는 시트(sheet) 형태인 것을 특징으로 하는 조성물.The composition of claim 1, wherein the composition is in the form of a paste or a sheet.
  11. 제10항에 있어서, 상기 시트는 페이스트 형태의 조성물을 금형에 도포 및 동결 건조하여 4 내지 8% 함량의 수분을 포함하는 것을 특징으로 하는 조성물.The composition as claimed in claim 10, wherein the sheet contains a moisture content of 4 to 8% by applying a paste-like composition to a mold and freeze drying.
  12. 제1항에 있어서, 상기 조성물은 약제학적으로 사용되는 항균제, 부형제 및 첨가제를 더 포함하는 것을 특징으로 하는 조성물.The composition of claim 1, wherein the composition further comprises a pharmaceutically used antimicrobial agent, excipient and additive.
  13. 제1항에 있어서, 상기 조성물을 창상 피복재, 접착제, 수술용 및 의료용 장치, 인공 피부, 붕대, 발포제, 필름, 흡착방지제, 이식재에 사용하는 것을 특징으로 하는 조성물.The composition of claim 1, wherein the composition is used in wound coatings, adhesives, surgical and medical devices, artificial skin, bandages, foaming agents, films, antiadhesion agents, implants.
  14. a) 인체 유래 무세포 진피 기질을 분쇄하는 단계;a) milling the acellular dermal matrix derived from the human body;
    b) 상기 분쇄된 무세포 진피 기질, 젤라틴이 용해된 고분자 수용액과 물을 혼합하여 혼합물을 형성하는 단계;b) mixing the ground cell free dermal matrix, gelatin-dissolved polymer aqueous solution and water to form a mixture;
    c) 상기 혼합물을 30~60 ℃에서 1~180분간 열처리하는 단계;c) heat-treating the mixture at 30-60 ° C. for 1-180 minutes;
    d) E-beam을 이용하여 열처리된 혼합물을 멸균하는 단계 및 d) sterilizing the heat-treated mixture using E-beam and
    e) 상기 멸균된 혼합물을 30~60 ℃에서 1~180분간 열처리하는 단계를 포함하는,e) heat-treating the sterilized mixture at 30 to 60 ℃ for 1 to 180 minutes,
    멸균되고 즉시 사용 가능(ready-to-use)하며, 멸균 후 가교도, 점탄성, 경화도 및/또는 압출력 값의 변화도가 멸균 전 값에 비해 30% 이하인 것을 특징으로 하는 조성물의 제조 방법.A method for the preparation of a composition, characterized in that it is sterile and ready-to-use and the degree of change in crosslinking, viscoelasticity, curing and / or extrusion force values after sterilization is 30% or less compared to the value before sterilization.
  15. 제14항에 있어서, 상기 b) 단계의 혼합물은 5 내지 20 중량%의 무세포 진피 기질, 0.5 내지 5 중량%의 젤라틴 및 75 내지 94.5 중량%의 물을 포함하는 것을 특징으로 하는 조성물의 제조 방법.The method of claim 14, wherein the mixture of step b) comprises 5 to 20% by weight of acellular dermal matrix, 0.5 to 5% by weight of gelatin and 75 to 94.5% by weight of water. .
  16. 제14항에 있어서, 상기 무세포 진피 기질은 섬유화된 무세포 진피 기질인 것을 특징으로 하는 조성물의 제조 방법.15. The method of claim 14, wherein said cell free dermal matrix is a fibrous cell free dermal matrix.
  17. 제16항에 있어서, 상기 섬유화된 무세포 진피 기질의 장축 길이가 500 내지 2,000 ㎛인 것을 특징으로 하는 조성물의 제조 방법.17. The method of claim 16, wherein the long axis length of the fibrized acellular dermal matrix is 500-2,000 μm.
  18. 제16항에 있어서, 상기 섬유화된 무세포 진피 기질은 단축과 장축의 비율이 1:30 내지 1:2,000인것의 비율이 70% 이상인 것을 특징으로 하는 조성물의 제조 방법.17. The method of claim 16, wherein the fibrous acellular dermal matrix has a ratio of 1:30 to 1: 2,000 of short axis and long axis of 70% or more.
  19. 제14항에 있어서, 상기 무세포 진피 기질을 분쇄하는 단계의 무세포 진피 기질은 두께 1 mm 이상인 것을 선별하여 이용되는 것을 특징으로 하는 조성물의 제조 방법. The method for producing a composition according to claim 14, wherein the cell-free dermal matrix of the step of grinding the cell-free dermal matrix is selected and used having a thickness of 1 mm or more.
  20. 제14항에 있어서, 상기 무세포 진피 기질의 분쇄는 커팅밀(cutting mill)을 이용하여 수행되는 것을 특징으로 하는 조성물의 제조 방법.15. The method of claim 14, wherein the grinding of the cell free dermal matrix is performed using a cutting mill.
  21. 제14항에 있어서, 상기 무세포 진피 기질은The method of claim 14, wherein the cell-free dermal matrix is
    a) 동종 피부의 표피를 제거하는 단계;a) removing the epidermis of allogeneic skin;
    b) 진피 내 세포를 제거하는 단계;b) removing the cells in the dermis;
    c) 글리세롤, 프로필렌 글리콜, 및 기본 용매 또는 용액을 혼합하고 상기 용액에 말티톨, 수크로스 또는 솔비톨의 최종 농도가 20 내지 40 중량%가 되도록 용해하여 동결 보호제를 제조하는 단계;c) preparing a cryoprotectant by mixing glycerol, propylene glycol, and a base solvent or solution and dissolving in the solution such that the final concentration of maltitol, sucrose or sorbitol is 20 to 40% by weight;
    d) 상기 동결 보호제를 상기 표피 및 진피 내 세포가 제거된 피부에 침투시키는 단계 및d) infiltrating the cryoprotectant into the skin from which the cells in the epidermis and dermis have been removed, and
    e) 상기 동결 보호제가 침투된 피부를 동결 건조하는 단계를 포함하는 방법에 의해서 제조되는 것을 특징으로 하는 조성물의 제조 방법.e) a process for producing a composition, characterized in that it is prepared by a method comprising the step of freeze drying the skin in which the cryoprotectant has penetrated.
  22. 제21항에 있어서, 상기 글리세롤, 프로필렌 글리콜 및 기본 용매 또는 용액의 혼합비가 중량 기준으로 0.5~2:0.5~2:6~10인 것을 특징으로 하는 조성물의 제조 방법.The method of claim 21, wherein the mixing ratio of the glycerol, propylene glycol and the base solvent or solution is 0.5 to 2: 0.5 to 2: 6 to 10 by weight.
  23. 제22항에 있어서, 상기 기본 용매 또는 용액이 증류수, 생리 식염수(normal saline), 인산완충용액(PBS), HBSS(Hank's balanced salt solution), TBS(Tris buffered saline), TAPS(N-Tris(hydroxy- methyl)methyl-3-aminopropanesulfonic acid) 완충용액, Bicine(N,N-Bis(2-hydroxyethyl) glycine) 완충용액, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) 완충용액, TES(NTris(hydroxymethyl)methyl-2-aminoethanesulfonicd acid) 완충용액, PIPES(piperazine- N,N'-bis(2-ethanesulfonic acid) 완충용액, 카코딜레이트(cacodylate) 완충용액, MES(2-(N-morpholino)ethanesulfonic acid) 완충용액, MEM(Minimum Essential Media), DMEM(Dulbecco's Modified Eagle Media), RPMI1640, IMDM(Iscove's Modified Dulbecco's Media), Defined Keratinocyte-SFM(without BPE(bovine pituitary extract)), Keratinocyte-SFM(with BPE), KnockOut D-MEM, AmnioMAX-II Complete Medium, AmnioMAX-C100 Complete Medium 및 이의 혼합물로 이루어지는 그룹 중에서 선택되는 것을 특징으로 하는 조성물의 제조 방법.The method of claim 22, wherein the basic solvent or solution is distilled water, physiological saline (normal saline), phosphate buffer (PBS), HBSS (Hank's balanced salt solution), TBS (Tris buffered saline), TAPS (N-Tris (hydroxy) -methyl) methyl-3-aminopropanesulfonic acid) buffer, Bicine (N, N-Bis (2-hydroxyethyl) glycine) buffer, HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) buffer, TES ( NTris (hydroxymethyl) methyl-2-aminoethanesulfonicd acid (PIPES) buffer, piperazine-N, N'-bis (2-ethanesulfonic acid) buffer, cacodylate buffer, MES (2- (N-morpholino) ) ethanesulfonic acid) buffer, Minimum Essential Media (MEM), Dulbecco's Modified Eagle Media (DMEM), RPMI1640, Iscove's Modified Dulbecco's Media (IMDM), defined Keratinocyte-SFM (without bovine pituitary extract (BPE), Keratinocyte-SFM with BPE), KnockOut D-MEM, AmnioMAX-II Complete Medium, AmnioMAX-C100 Complete Medium and mixtures thereof Process for producing a composition as described.
  24. 섬유화된 인체 유래 무세포 진피 기질(Acellular Dermal Matrix)과 생체적합 고분자를 포함하는 조성물로서, 상기 섬유화된 무세포 진피 기질은 단축과 장축의 비율이 1:30 내지 1:2,000인 것의 비율이 70% 이상인 것을 특징으로 하는 조성물. A composition comprising a fibrous human-derived acellular dermal matrix and a biocompatible polymer, wherein the fibrous acellular dermal matrix has a ratio of 1:30 to 1: 2,000 of short axis and long axis of 70%. The composition characterized by the above.
  25. 제24항에 있어서, 상기 조성물은 멸균되고 즉시 사용 가능한(ready-to-use) 것임을 특징으로 하는 조성물. 25. The composition of claim 24, wherein the composition is sterile and ready-to-use.
  26. 제24항에 있어서, 상기 조성물은 5 내지 20 중량%의 섬유화된 무세포 진피 기질, 0.5 내지 5 중량%의 생체적합 고분자 및 75 내지 94.5 중량%의 물을 포함하는 조성물.The composition of claim 24, wherein the composition comprises 5-20 wt% of the fibrized acellular dermal matrix, 0.5-5 wt% of the biocompatible polymer, and 75-94.5 wt% of water.
  27. 제24항에 있어서, 상기 섬유화된 무세포 진피 기질의 장축 길이가 500 내지 2,000 ㎛인 것을 특징으로 하는 조성물.25. The composition of claim 24, wherein the long axis length of the fibrized acellular dermal matrix is between 500 and 2,000 μm.
  28. 제24항에 있어서, 상기 생체적합 고분자가 젤라틴, 히알루론산, 콜라겐, 폴록사머 또는 이의 혼합물인 것을 특징으로 하는 조성물.The composition of claim 24, wherein the biocompatible polymer is gelatin, hyaluronic acid, collagen, poloxamer or mixtures thereof.
  29. 제28항에 있어서, 상기 생체적합 고분자가 젤라틴인 것을 특징으로 하는 조성물.29. The composition of claim 28, wherein said biocompatible polymer is gelatin.
  30. 제24항에 있어서, 상기 조성물은 페이스트(paste) 형태 또는 시트(sheet) 형태인 것을 특징으로 하는 조성물.25. The composition of claim 24, wherein the composition is in the form of a paste or sheet.
  31. 제30항에 있어서, 상기 시트는 페이스트 형태의 조성물을 금형에 도포 및 동결 건조하여 4 내지 8% 함량의 수분을 포함하는 것을 특징으로 하는 조성물.31. The composition of claim 30, wherein the sheet comprises 4-8% moisture by applying a paste-like composition to the mold and freeze drying.
  32. 제24항에 있어서, E-beam을 이용한 방사선 멸균 단계를 포함하는 공정을 통해 제조된 것을 특징으로 하는 조성물. The method of claim 24, wherein the composition is prepared through a process comprising the step of sterilizing radiation using E-beam.
  33. 제32항에 있어서, 상기 E-beam을 이용한 멸균 단계 전 및/또는 후에, 30~60 ℃에서 열처리 단계를 포함하는 것을 특징으로 하는 조성물. 33. The composition of claim 32, comprising a heat treatment step at 30 to 60 ° C. before and / or after sterilization with the E-beam.
  34. 제33항에 있어서, 상기 열처리는 1~180 분간 수행되는 것을 특징으로 조성물. The composition of claim 33, wherein the heat treatment is performed for 1 to 180 minutes.
  35. 제24항에 있어서, 상기 섬유화된 무세포 진피 기질은 두께 1 mm 이상의 무세포 진피 기질을 선별하여 섬유화로 제조된 것을 특징으로 하는 조성물.25. The composition of claim 24, wherein the fibrous acellular dermal matrix is prepared by fibrosis by selecting acellular dermal matrix of at least 1 mm in thickness.
  36. 제24항에 있어서, 상기 섬유화된 무세포 진피 기질은 커팅밀(cutting mill)을 이용하여 섬유화로 제조된 것을 특징으로 하는 조성물.25. The composition of claim 24, wherein the fibrous, acellular dermal matrix is prepared by fibrosis using a cutting mill.
  37. 제24항에 있어서, 상기 조성물은 약제학적으로 사용되는 항균제, 부형제 및 첨가제를 더 포함하는 것을 특징으로 하는 조성물.The composition of claim 24, wherein the composition further comprises a pharmaceutically used antimicrobial agent, excipient and additive.
  38. 제24항에 있어서, 상기 조성물을 창상 피복재, 접착제, 수술용 및 의료용 장치, 인공 피부, 붕대, 발포제, 필름, 흡착방지제, 이식재에 사용하는 것을 특징으로 하는 조성물.25. The composition of claim 24, wherein the composition is used in wound dressings, adhesives, surgical and medical devices, artificial skin, bandages, foaming agents, films, anti-adsorption agents, implants.
  39. a) 인체 유래 무세포 진피 기질을 섬유화하는 단계;a) fibrating the acellular dermal matrix derived from a human body;
    b) 상기 섬유화된 무세포 진피 기질, 생체 적합성 고분자 수용액과 물을 혼합하여 혼합물을 형성하는 단계;b) mixing the fibrous acellular dermal matrix, a biocompatible polymer aqueous solution and water to form a mixture;
    c) 상기 혼합물을 30~60 ℃에서 1~30분간 열처리하는 단계;c) heat treating the mixture at 30 to 60 ° C. for 1 to 30 minutes;
    d) E-beam을 이용하여 열처리된 혼합물을 멸균하는 단계 및 d) sterilizing the heat-treated mixture using E-beam and
    e) 상기 멸균된 혼합물을 30~60 ℃에서 1~30분간 열처리하는 단계를 포함하는,e) heat-treating the sterilized mixture at 30 to 60 ℃ for 1 to 30 minutes,
    멸균되고 즉시 사용 가능(ready-to-use)하며, 상기 섬유화된 무세포 진피 기질의 단축과 장축의 비율이 1:30 내지 1:70인 것의 비율이 70% 이상인 것을 특징으로 하는 조성물의 제조 방법.Sterile, ready-to-use, wherein the ratio of the short axis and the long axis of the fibrous acellular dermal substrate is from 1:30 to 1:70, wherein the ratio of the composition is 70% or more. .
  40. 제39항에 있어서, 상기 b) 단계의 혼합물은 5 내지 20 중량%의 섬유화된 무세포 진피 기질, 0.5 내지 5 중량%의 생체적합 고분자 및 75 내지 94.5 중량%의 물을 포함하는 것을 특징으로 하는 조성물의 제조 방법.40. The method of claim 39, wherein the mixture of step b) comprises from 5 to 20% by weight of a fibrous acellular dermal matrix, from 0.5 to 5% by weight of a biocompatible polymer and from 75 to 94.5% by weight of water. Method of Preparation of the Composition.
  41. 제39항에 있어서, 상기 섬유화된 무세포 진피 기질의 장축 길이가 500 내지 2,000 ㎛인 것을 특징으로 하는 조성물의 제조 방법.40. The method of claim 39, wherein the fibrous acellular dermal matrix has a long axis length of 500-2,000 μm.
  42. 제39항에 있어서, 상기 a) 단계의 무세포 진피 기질의 섬유화는 무세포 진피 기질을 커팅밀(cutting mill)을 이용하여 수행되는 것을 특징으로 하는 조성물의 제조 방법.40. The method of claim 39, wherein the fibrosis of the cell-free dermal matrix of step a) is carried out using a cutting mill of the cell-free dermal matrix.
  43. 제39항에 있어서, 상기 무세포 진피 기질은40. The method of claim 39, wherein the cell free dermal matrix is
    a) 동종 피부의 표피를 제거하는 단계;a) removing the epidermis of allogeneic skin;
    b) 진피 내 세포를 제거하는 단계;b) removing the cells in the dermis;
    c) 글리세롤, 프로필렌 글리콜, 및 기본 용매 또는 용액을 혼합하고 상기 용액에 말티톨, 수크로스 또는 솔비톨의 최종 농도가 20 내지 40 중량%가 되도록 용해하여 동결 보호제를 제조하는 단계;c) preparing a cryoprotectant by mixing glycerol, propylene glycol, and a base solvent or solution and dissolving in the solution such that the final concentration of maltitol, sucrose or sorbitol is 20 to 40% by weight;
    d) 상기 동결 보호제를 상기 표피 및 진피 내 세포가 제거된 피부에 침투시키는 단계 및d) infiltrating the cryoprotectant into the skin from which the cells in the epidermis and dermis have been removed, and
    e) 상기 동결 보호제가 침투된 피부를 동결 건조하는 단계를 포함하는 방법에 의해서 제조되는 것을 특징으로 하는 조성물의 제조 방법.e) a process for producing a composition, characterized in that it is prepared by a method comprising the step of freeze drying the skin in which the cryoprotectant has penetrated.
  44. 제39항에 있어서, 상기 글리세롤, 프로필렌 글리콜 및 기본 용매 또는 용액의 혼합비가 중량 기준으로 0.5~2:0.5~2:6~10인 것을 특징으로 하는 조성물의 제조 방법.40. The method of claim 39, wherein the mixing ratio of the glycerol, propylene glycol and the base solvent or solution is 0.5-2: 0.5-2: 6-10 by weight.
  45. 제44항에 있어서, 상기 기본 용매 또는 용액이 증류수, 생리 식염수(normal saline), 인산완충용액(PBS), HBSS(Hank's balanced salt solution), TBS(Tris buffered saline), TAPS(N-Tris(hydroxy- methyl)methyl-3-aminopropanesulfonic acid) 완충용액, Bicine(N,N-Bis(2-hydroxyethyl) glycine) 완충용액, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) 완충용액, TES(NTris(hydroxymethyl)methyl-2-aminoethanesulfonicd acid) 완충용액, PIPES(piperazine- N,N'-bis(2-ethanesulfonic acid) 완충용액, 카코딜레이트(cacodylate) 완충용액, MES(2-(N-morpholino)ethanesulfonic acid) 완충용액, MEM(Minimum Essential Media), DMEM(Dulbecco's Modified Eagle Media), RPMI1640, IMDM(Iscove's Modified Dulbecco's Media), Defined Keratinocyte-SFM(without BPE(bovine pituitary extract)), Keratinocyte-SFM(with BPE), KnockOut D-MEM, AmnioMAX-II Complete Medium, AmnioMAX-C100 Complete Medium 및 이의 혼합물로 이루어지는 그룹 중에서 선택되는 것을 특징으로 하는 조성물의 제조 방법.45. The method of claim 44, wherein the basic solvent or solution is distilled water, physiological saline (normal saline), phosphate buffered solution (PBS), HBSS (Hank's balanced salt solution), TBS (Tris buffered saline), TAPS (N-Tris (hydroxy) -methyl) methyl-3-aminopropanesulfonic acid) buffer, Bicine (N, N-Bis (2-hydroxyethyl) glycine) buffer solution, HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) buffer solution, TES ( NTris (hydroxymethyl) methyl-2-aminoethanesulfonicd acid (PIPES) buffer, piperazine-N, N'-bis (2-ethanesulfonic acid) buffer, cacodylate buffer, MES (2- (N-morpholino) ) ethanesulfonic acid) buffer, Minimum Essential Media (MEM), Dulbecco's Modified Eagle Media (DMEM), RPMI1640, Iscove's Modified Dulbecco's Media (IMDM), defined Keratinocyte-SFM (without bovine pituitary extract (BPE), Keratinocyte-SFM with BPE), KnockOut D-MEM, AmnioMAX-II Complete Medium, AmnioMAX-C100 Complete Medium and mixtures thereof Process for producing a composition as described.
  46. 인체 유래 무세포 진피 기질 75 내지 85 중량%, 생체적합 고분자 15 내지 25 중량% 및 3 내지 8 중량%의 물을 포함하는 창상 피복재용 시트로서, 가로 길이가 4 내지 20 cm 이며 세로 길이가 4 내지 20 cm이며 두께가 0.5 내지 3 mm인 것을 특징으로 하는 창상 피복재용 시트.A wound coating sheet comprising 75 to 85% by weight of human-derived cell-free dermal substrate, 15 to 25% by weight of biocompatible polymer, and 3 to 8% by weight of water, having a length of 4 to 20 cm and a length of 4 to 4 A wound covering sheet, characterized in that it is 20 cm and has a thickness of 0.5 to 3 mm.
  47. 제46항에 있어서, 상기 무세포 진피 기질은 섬유화 형태인 것을 특징으로 하는 창상 피복재용 시트.47. The wound covering sheet according to claim 46, wherein said cell free dermal matrix is in fibrous form.
  48. 제47항에 있어서, 상기 섬유화된 무세포 진피 기질은 장축 길이가 500 내지 2,000 ㎛인 것을 특징으로 하는 창상 피복재용 시트.48. The wound coating sheet according to claim 47, wherein the fibrous acellular dermal matrix has a long axis length of 500 to 2,000 µm.
  49. 제47항에 있어서, 상기 섬유화된 무세포 진피 기질은 단축과 장축의 비율이 1:30 내지 1:2,000인 것의 비율이 70% 이상인 것을 특징으로 하는 창상 피복재용 시트.48. The wound covering sheet according to claim 47, wherein the fibrous acellular dermal matrix has a ratio of 1:30 to 1: 2,000 of a short axis and a long axis of 70% or more.
  50. 제46항에 있어서, 상기 생체적합성 고분자가 젤라틴, 히알루론산, 콜라겐, 폴록사머 또는 이의 혼합물인 것을 특징으로 하는 창상 피복재용 시트.47. The wound covering sheet according to claim 46, wherein the biocompatible polymer is gelatin, hyaluronic acid, collagen, poloxamer or a mixture thereof.
  51. 제50항에 있어서, 상기 생체적합성 고분자가 젤라틴인 것을 특징으로 하는 창상 피복재용 시트.51. The wound covering sheet according to claim 50, wherein the biocompatible polymer is gelatin.
  52. 제46항에 있어서, 상기 창상 피복재용 시트는 약제학적으로 사용되는 항균제, 부형제 및 첨가제를 더 포함하는 것을 특징으로 하는 창상 피복재용 시트.47. The wound covering sheet according to claim 46, wherein the wound covering sheet further comprises an antimicrobial agent, excipient and additive used in the pharmaceutical.
  53. a) 인체 유래 무세포 진피 기질을 분쇄하는 단계;a) milling the acellular dermal matrix derived from the human body;
    b) 상기 분쇄된 무세포 진피 기질, 생체적합성 고분자 수용액과 물을 혼합하여 혼합물을 형성하는 단계 및b) mixing the ground cell free dermal matrix, a biocompatible polymer aqueous solution and water to form a mixture, and
    c) 상기 혼합물을 금형에 도포 및 동결 건조하여 시트를 제조하는 단계를 포함하는 창상 피복재용 시트의 제조 방법.c) applying the mixture to a mold and freeze-drying to produce a sheet.
  54. 제53항에 있어서, 상기 b) 단계의 혼합물은 5 내지 20 중량%의 무세포 진피 기질, 0.5 내지 5 중량%의 생체적합성 고분자 및 75 내지 94.5 중량%의 물을 포함하는 것을 특징으로 하는 창상 피복재용 시트의 제조 방법.55. The wound coating of claim 53, wherein the mixture of step b) comprises 5-20 wt% of acellular dermal matrix, 0.5-5 wt% of a biocompatible polymer and 75-94.5 wt% of water. Method for producing a sheet for
  55. 제53항에 있어서, 상기 c) 단계의 시트는 무세포 진피 기질 75 내지 85 중량%, 생체적합 고분자 15 내지 25 중량% 및 3 내지 8 중량%의 물을 포함하는 것을 특징으로 하는 창상피복재용 시트의 제조 방법.54. The wound dressing of claim 53, wherein the sheet of step c) comprises 75 to 85% by weight of acellular dermal substrate, 15 to 25% by weight of biocompatible polymer and 3 to 8% by weight of water. Method of preparation.
  56. 제53항에 있어서, 상기 무세포 진피 기질은 섬유화된 무세포 진피 기질인 것을 특징으로 하는 창상 피복재용 시트의 제조 방법.54. The method of claim 53, wherein the cell-free dermal matrix is a fibrotic cell-free dermal matrix.
  57. 제56항에 있어서, 상기 섬유화된 무세포 진피 기질의 장축 길이가 500 내지 2,000 ㎛인 것을 특징으로 하는 창상 피복재용 시트의 제조 방법.57. The method of claim 56, wherein the fibrous acellular dermal matrix has a long axis length of 500 to 2,000 µm.
  58. 제57항에 있어서, 상기 섬유화된 무세포 진피 기질은 단축과 장축의 비율이 1:30 내지 1:2,000인 것의 비율이 70% 이상인 것을 특징으로 하는 창상 피복재용 시트의 제조 방법.59. The method of claim 57, wherein the fibrous acellular dermal matrix has a ratio of 1:30 to 1: 2,000 of short axis and long axis of 70% or more.
  59. 제53항에 있어서, 상기 무세포 진피 기질을 분쇄하는 단계의 무세포 진피 기질은 두께 1 mm 이상인 것을 선별하여 이용되는 것을 특징으로 하는 창상 피복재용 시트의 제조 방법. 54. The method of claim 53, wherein the cell-free dermal matrix in the step of pulverizing the cell-free dermal matrix is used by selecting one having a thickness of 1 mm or more.
  60. 제53항에 있어서, 상기 무세포 진피 기질의 분쇄는 커팅밀(cutting mill)을 이용하여 수행되는 것을 특징으로 하는 창상 피복재용 시트의 제조 방법.54. The method of claim 53, wherein grinding of the cell-free dermal substrate is performed using a cutting mill.
  61. 제53항에 있어서, 상기 무세포 진피 기질은54. The method of claim 53, wherein the cell free dermal matrix is
    a) 동종 피부의 표피를 제거하는 단계;a) removing the epidermis of allogeneic skin;
    b) 진피 내 세포를 제거하는 단계;b) removing the cells in the dermis;
    c) 글리세롤, 프로필렌 글리콜, 및 기본 용매 또는 용액을 혼합하고 상기 용액에 말티톨, 수크로스 또는 솔비톨의 최종 농도가 20 내지 40 중량%가 되도록 용해하여 동결 보호제를 제조하는 단계;c) preparing a cryoprotectant by mixing glycerol, propylene glycol, and a base solvent or solution and dissolving in the solution such that the final concentration of maltitol, sucrose or sorbitol is 20 to 40% by weight;
    d) 상기 동결 보호제를 상기 표피 및 진피 내 세포가 제거된 피부에 침투시키는 단계 및d) infiltrating the cryoprotectant into the skin from which the cells in the epidermis and dermis have been removed, and
    e) 상기 동결 보호제가 침투된 피부를 동결 건조하는 단계를 포함하는 방법에 의해서 제조되는 것을 특징으로 하는 창상 피복재용 시트의 제조 방법.e) a method for producing a sheet for wound covering material, which is prepared by a method comprising the step of freeze-drying the skin in which the freeze protection agent has penetrated.
  62. 제61항에 있어서, 상기 글리세롤, 프로필렌 글리콜 및 기본 용매 또는 용액의 혼합비가 중량 기준으로 0.5~2:0.5~2:6~10인 것을 특징으로 하는 창상 피복재용 시트의 제조 방법.The method for producing a wound coating sheet according to claim 61, wherein the mixing ratio of the glycerol, propylene glycol and the basic solvent or solution is 0.5 to 2: 0.5 to 2: 6 to 10 by weight.
  63. 제62항에 있어서, 상기 기본 용매 또는 용액이 증류수, 생리 식염수(normal saline), 인산완충용액(PBS), HBSS(Hank's balanced salt solution), TBS(Tris buffered saline), TAPS(N-Tris(hydroxy- methyl)methyl-3-aminopropanesulfonic acid) 완충용액, Bicine(N,N-Bis(2-hydroxyethyl) glycine) 완충용액, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) 완충용액, TES(NTris(hydroxymethyl)methyl-2-aminoethanesulfonicd acid) 완충용액, PIPES(piperazine- N,N'-bis(2-ethanesulfonic acid) 완충용액, 카코딜레이트(cacodylate) 완충용액, MES(2-(N-morpholino)ethanesulfonic acid) 완충용액, MEM(Minimum Essential Media), DMEM(Dulbecco's Modified Eagle Media), RPMI1640, IMDM(Iscove's Modified Dulbecco's Media), Defined Keratinocyte-SFM(without BPE(bovine pituitary extract)), Keratinocyte-SFM(with BPE), KnockOut D-MEM, AmnioMAX-II Complete Medium, AmnioMAX-C100 Complete Medium 및 이의 혼합물로 이루어지는 그룹 중에서 선택되는 것을 특징으로 하는 창상 피복재용 시트의 제조 방법.63. The method of claim 62, wherein the basic solvent or solution is distilled water, physiological saline (normal saline), phosphate buffered solution (PBS), HBSS (Hank's balanced salt solution), TBS (Tris buffered saline), TAPS (N-Tris (hydroxy) -methyl) methyl-3-aminopropanesulfonic acid) buffer, Bicine (N, N-Bis (2-hydroxyethyl) glycine) buffer solution, HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) buffer solution, TES ( NTris (hydroxymethyl) methyl-2-aminoethanesulfonicd acid (PIPES) buffer, piperazine-N, N'-bis (2-ethanesulfonic acid) buffer, cacodylate buffer, MES (2- (N-morpholino) ) ethanesulfonic acid) buffer, Minimum Essential Media (MEM), Dulbecco's Modified Eagle Media (DMEM), RPMI1640, Iscove's Modified Dulbecco's Media (IMDM), defined Keratinocyte-SFM (without bovine pituitary extract (BPE), Keratinocyte-SFM with BPE), KnockOut D-MEM, AmnioMAX-II Complete Medium, AmnioMAX-C100 Complete Medium and mixtures thereof Method of producing a sheet for wound dressing as claimed.
PCT/KR2018/004955 2017-04-28 2018-04-27 Composition comprising fibrous acellular dermal matrix and biocompatible polymer, and method for producing same WO2018199698A1 (en)

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