WO2018048047A1 - 생착률을 증가시킨 이식용 진피층 및 이의 제조방법 - Google Patents

생착률을 증가시킨 이식용 진피층 및 이의 제조방법 Download PDF

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Publication number
WO2018048047A1
WO2018048047A1 PCT/KR2017/003070 KR2017003070W WO2018048047A1 WO 2018048047 A1 WO2018048047 A1 WO 2018048047A1 KR 2017003070 W KR2017003070 W KR 2017003070W WO 2018048047 A1 WO2018048047 A1 WO 2018048047A1
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Prior art keywords
chitosan
solution
dermal
layer
dna fragment
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PCT/KR2017/003070
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English (en)
French (fr)
Inventor
김익수
김한규
홍철암
이수연
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주식회사 파마리서치프로덕트
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Application filed by 주식회사 파마리서치프로덕트 filed Critical 주식회사 파마리서치프로덕트
Priority to JP2019534614A priority Critical patent/JP6765540B2/ja
Priority to CN201780053891.9A priority patent/CN109641082B/zh
Priority to US16/331,226 priority patent/US11109955B2/en
Priority to EP17848932.4A priority patent/EP3511029B1/en
Publication of WO2018048047A1 publication Critical patent/WO2018048047A1/ko
Priority to US17/466,415 priority patent/US20210393396A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/10—Hair or skin implants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/10—Hair or skin implants
    • A61F2/105—Skin implants, e.g. 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • 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
    • 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
    • A61L27/3604—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
    • A61L27/3633—Extracellular matrix [ECM]
    • 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
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/258—Genetic materials, DNA, RNA, genes, vectors, e.g. plasmids
    • 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
    • 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
    • A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking

Definitions

  • the present invention relates to a grafted dermal layer with an increased engraftment rate and a method of manufacturing the same. More specifically, it relates to a grafted dermal layer which increased the engraftment rate during transplantation by filling a layered solution containing DNA fragment complex and chitosan in a biologically inactivated dermal layer.
  • Skin is the largest organ covering the entire human body surface, preventing the loss of body fluids, preventing the influx of harmful substances and microorganisms from the outside, and protecting our body from physical stimulation, radiation and ultraviolet rays.
  • the skin possesses several appendages such as hair follicles, hair, sweat glands, and sebaceous glands, and is an important complex organ that performs various functions in addition to the protective film function (Kim Cheon Ho, et al., 2002)
  • the epidermal layer is a thin layer outside the skin, consisting of newly differentiated cells.
  • the dermal layer is the layer below the epidermis, which contains various structures such as blood vessels and nerves.
  • It is composed of a papillary layer rich in fibroblasts and microvascular distribution, and a retinal connective tissue rich in thick collagen fibers.
  • Subcutaneous tissue is the area between the dermis, the muscles, and the skeleton, composed of adipose tissue and connective tissue, including blood vessels, lymphatic vessels, and nerve endings.
  • the fatty tissue in the subcutaneous tissue protects against external pressure and provides a padding effect on the friction force, making it easier to move the skin over deep structures such as bones and muscles. It not only retains, but also absorbs strata, protecting the body and accumulating extra nutrients.
  • autograft which transplants your own skin tissue for the purpose of healing damaged tissue or for reconstructive shaping
  • Allografts homografts, allografts
  • xenograft methods for transplanting the skin of animals are used, of which the most suitable are those where the transplantation is the most ideal but the area to be treated is limited to areas where tissue can be secured. It is difficult for the harvest site to remain as a new wound, where cognate transplantation helps to move and heal the cells around the wound rather than permanent engraftment.
  • a scaffold is needed, and artificial skin is being developed for this. Artificial skin should act as a protective barrier to prevent infections and loss of fluids and to prevent scars in the wounds of patients, and in principle to prevent serious contractions that may occur during healing (Yannas IV, 1995). .
  • the temporary coating wound wound protects the affected area temporarily until the skin is damaged by burns or trauma.It prevents exposure to moisture from the body, absorbs exudates, and prevents invasion and infection of microorganisms from the outside.
  • permanently grafted artificial skin is composed of synthetic polymers and natural polymers, and the role of wound skin cloning, of course, provides an easy path for human cells to penetrate and actively helps regenerate into the original skin tissue.
  • the bioartifical skin is one of the permanent grafted artificial skins, providing a protective layer as well as growth factors and extracellular matrix necessary for the regeneration of damaged tissues. Remarkable effects such as the reduction of scars have been proven.
  • artificial skin is affected by burns, bedsores, trauma, plastic surgery, intractable ulcers, diabetic skin necrosis and pressure erosion. It is the application.
  • chitosan (poly (3- (l ⁇ 4) -2-amino-2-deoxy-D-glucan) is a linear polymer in which D-glucosamine binds ⁇ - (1 ⁇ 4), chitin. ) (poly
  • inorganic acids such as hydrochloric acid, sulfuric acid, formic acid, acetic acid, acetic acid, propionic acid, oxalic acid, succinic acid, etc. oxy acid, such as gluconic acid), lactic acid, malic acid,
  • Acid salts are formed and dissolved in dilute aqueous solutions of acidic amino acids, such as glutamic acid (Mussarelli R.A.A., 1997; Brine C.J., et al., 1992).
  • Chitosan solutions have high viscosity and vary according to deacetylation, ion strength, pH, temperature, etc. For example, when the pH of the solvent is low, the viscosity of the chitosan solution decreases and the pH is high. Viscosity increases, and in the case of temperature, the viscosity of the chitosan solution decreases as the degree of silver increases, and when the temperature reaches the original temperature, the viscosity becomes the first.
  • Chitosan is widely used and widely used in a wide range of fields because it has physiological activity on animals and plants, including humans.
  • the most widely used chitosan is antibacterial activity. It is known to be expressed by the oxidized amino group. A silver bond occurs between the amino group of the cationized chitosan and acidic groups such as sialic acid and phospholipid, which make up the bacterial cell wall.
  • acidic groups such as sialic acid and phospholipid
  • the present inventors in the study of skin edible compositions, increased the rate of engraftment at the site of transplantation of the grafted dermal layer prepared by filling the biologically inactivated dermal layer with a layered solution containing DNA fragment complex and chitosan. I was able to complete the present invention by confirming that it was.
  • Korean Patent Nos. 0469661 and 1362402 describe a cell-free dermal layer for transplantation, which is similar to the composition of the present invention.However, a method of layering a filling solution containing a DNA fragment complex and chitosan is disclosed. Although not described, there is a difference in the composition of the present invention.
  • US Patent Publication No. 2004-0031067 describes a method for administering a nucleic acid encoding an angiogenesis-inducing factor during skin transplantation. The composition is different from the composition of the present invention because it is not mentioned at all and is not described as a drug-derived dermal composition. No.
  • 1,534,276 is a composition containing polydioxyribonucleotide for enhancing the engraftment rate of fat cells after fat transplant surgery, but is similar to the composition of the present invention, but chitosan is not mentioned at all, and the drug is a dermal composition with a layered drug. There is no difference in the composition of the present invention because it is not described.
  • the purpose of the present invention is to provide a grafted dermal layer with an increased engraftment rate and a manufacturing method thereof.
  • the present invention relates to a method for producing a transplanted dermal layer, which stores DNA fragment complexes.
  • the fourth step relates to a method for manufacturing a grafted dermal layer comprising a.
  • the manufacturing method comprises the first step of preparing a DNA fragment mixture storage solution by dissolving the DNA fragment mixture in a complete solution and dissolving at 50 o C to 70 o C for 1 hour to 3 hours; A second step of preparing a chitosan storage solution by dissolving in a complete solution; mixing the DNA fragment mixture storage solution and the chitosan storage solution so that a weight ratio of the DNA fragment mixture and the chitosan is 20: 1-10,000: 1 ° C to 75, stirred for 1 to 2 hours at 0 C sterilized by a third step of producing a layer wholly containing the DNA fragments common compound and chitosan; a, and the charge amount by using values vacuum drug layer battlefield acellular Layer 4 charge to the dermal graft.
  • the DNA fragment complex can be isolated from testis or semen of fish.
  • the fish may be salmon
  • the DNA fragment complex may have a molecular weight of 50 kDa to 10,000 kDa.
  • the DNA fragment complex is 0.01% by weight based on the total amount of the filling solution.
  • the chitosan may have a molecular weight of 3kDa to l, 000kDa.
  • the chitosan may be 1 wt% to 0.15 wt% based on the total weight of the total layer liquid.
  • the acellular dermal graft of the fourth step contains cells and impurities from the dermis.
  • It may be a collagen-used tissue retainer that has been removed and freeze-dried after virus inactivation.
  • the dermis may be autologous, homologous or heterologous.
  • the fourth stage of filling is viscous into the acellular dermal graft using pressure. It may be to fill in the empty space between collagen by injecting the filling charge.
  • the vacuum drug filling apparatus of the fourth step connects the layer solution in one direction and generates positive or negative pressure so that the connected layer solution fills the space between collagen in the acellular dermal graft.
  • the present invention also relates to a edible dermal layer prepared by the above-mentioned manufacturing method.
  • the present invention also relates to a skin damage treatment agent comprising the dermal layer for transplantation.
  • the skin damage is abrasion, incision, laceration,
  • At least one selected from the group of skin avulsion, skin bruise, puncture wound, and surgical skin resection may be the cause.
  • the present invention relates to a first step of preparing a stock solution of DNA fragments; A second step of preparing a chitosan storage solution; a third step of preparing a layered solution containing a DNA fragment mixture and a chitosan by mixing and stirring the DNA fragment mixture storage solution and the chitosan storage solution; and the layer solution Acellular dermis
  • a layer charge on the graft is directed to a method for producing an edible dermal layer comprising;
  • the manufacturing method of the dermal layer for transplantation comprises the first step of preparing a DNA fragment mixture storage solution by dissolving the DNA fragment mixture in a buffer solution and dissolving for 1 hour to 3 hours while stirring at 50 ° C. to 70 ° C .; A second step of preparing a chitosan storage solution by dissolving chitosan in an acidic complete solution; the weight ratio of the DNA fragment mixture and the chitosan storage solution to the DNA fragment complex and chitosan is 20: 1-10,000: 1 A third step of preparing a filling solution containing a DNA fragment complex and chitosan by stirring and stirring at 65 0 C to 75 0 C for 1 hour to 2 hours; and using the vacuum drug layering apparatus as the layer solution. By four steps of layering on acellular dermal grafts.
  • the first step can be used to prepare the DNA fragment complex storage solution
  • sodium phosphate dibasic dodecahy drate sodium chloride
  • HEPES N- (2-hydroxyethyl) -piperazine-N'-2-ethanesulfonic acid
  • Glycerol-3-phosphate buffer solution preferably sodium phosphate dibasic dodecahydrate (sodium phosphate dibasic)
  • dodecahydrate can be used but is not limited to this.
  • the DNA fragment complex may be isolated from testis or semen of fish.
  • the fish may be salmon, preferably salmon or trout, most preferably salmon.
  • the DNA fragment complexes include phosphoric acid, four kinds of bases,
  • DNA corresponding to a living polymer composed of deoxyribose which refers to the presence of a fragment of reduced molecular weight.
  • the DNA fragment mixture refers to a substance in which DNA fragments of a specific size are mixed to act as a pharmacological effect or a polymer support.
  • It may be a polynucleotide.
  • the polydeoxyribonucleotide plays a role in cell regeneration and wound healing.
  • the polynucleotide may play a role in constructing a cell regeneration environment through the support structure.
  • the DNA fragment complex may have a molecular weight of 50 kDa to 10,00 () kDa.
  • Buffer solutions include acetic acid, hydrochloric acid,
  • Ascorbic acid ascorbic acid
  • lactic acid lactic acid
  • nitric acid nitric acid
  • ace ti c add it is not limited to this.
  • the chitosan may have a molecular weight of 3 kDa to l, 000 kDa.
  • the weight ratio of the DNA fragment complex and the chitosan may be 20: 1-10,000: 1, preferably 20: 1-1,000: 1, and more preferably 50: 1. -200: 1.
  • the DNA fragment complex has an increase of 0 ⁇ 1 ⁇ 3/4 based on the total amount of the total layer liquid
  • the DNA fragment mixture is less than 0.01% by weight, it is difficult to expect the effect of increasing the engraftment rate, and when the DNA fragment mixture exceeds 3% by weight, problems in the preparation process of the filling solution may be undesirable.
  • the chitosan may be added in an amount of lxlO- 6 % to 0.15% by weight based on the total weight of the filling liquid, preferably lxlO- 5 % to 0.15% by weight, and preferably lxlO- 4 % to 0.075% by weight. If the chitosan is less than 1 ⁇ 10- ⁇ weight% or more than 0.15% by weight, the viscosity of the filling solution decreases the filling rate during filling of acellular dermal grafts, which is undesirable.
  • the chitosan can be mixed with DNA fragment complexes to increase the viscosity of the whole layer solution. This increase in viscosity results in an increase in the layer transfer efficiency that fills the voids between the collagen in the layered acellular dermal grafts of the filling solution. DNA fragments introduced into the acellular dermal graft may be present in the graft.
  • the chitosan is composed of negatively charged collagen, which is a major component of acellular dermal grafts. Ion bonding can increase the charging efficiency of the layer charge filler.
  • sterilization is high temperature and high pressure steam sterilization, dry heat sterilization, and EO (ethylene).
  • Oxygen sterilization can be used, preferably high temperature steam sterilization, but not limited to this.
  • the acellular dermal matrix removes cells and impurities from the dermis, freeze-dried after virus inactivation, and biologically inactivated tissue supplements and scaffolds. Can be.
  • the supplement may cover the wound and may be artificial skin.
  • the acellular dermal grafts can be manufactured from a series of processes from the dermis and can be used commercially available.
  • the dermis may be autologous, homologous or heterologous.
  • the fourth step of layering is to fill the void space between collagen by injecting a viscous filling solution into the acellular dermal graft by using pressure, which can be filled so that the active ingredient can be present in a large amount inside the graft.
  • pressure which can be filled so that the active ingredient can be present in a large amount inside the graft.
  • negative or positive pressure may increase the inflow of the layered fluid into the acellular dermal graft, thereby increasing the filling efficiency of filling the empty spaces between collagen.
  • the layer change can be made using a vacuum drug layering device.
  • the vacuum drug filling device may be a device that introduces a positive or negative pressure into the acellular dermal graft so that the filling solution is introduced into the acellular dermal graft.
  • the vacuum drug filling device includes a portion for fixing an acellular dermal graft.
  • It can consist of the part where the layer solution enters, or where the vacuum pump for positive or negative pressure is connected.
  • the fixing part should be packed to prevent the drug from leaking under the pressure during drug filling.
  • the present invention also relates to a edible dermal layer prepared by the above-mentioned manufacturing method.
  • the dermal layer for implantation can increase the engraftment rate by facilitating the fusion with the surrounding tissues by allowing the cells to move rapidly from the transplanted peripheral tissues due to the filler containing the laminated DNA fragment complex and chitosan. It can relieve early inflammation and speed recovery after transplantation.
  • the present invention relates to a therapeutic agent for skin damage, including a transplanted dermal layer.
  • the skin injury may include abrasion, incision, laceration, One or more of the skin avulsions, skin bruises, puncture wounds, and surgical skin resections may be the cause.
  • the skin damage treatment may be performed by supplementing or supplementing a portion of the skin that is seriously damaged.
  • the scaffold is implanted to cover the wound so that fluid loss at the wound is reduced.
  • the present invention relates to a grafted dermal layer with an increased engraftment rate and a method for manufacturing the same, wherein a filler containing a DNA fragment complex and chitosan can be layered on a biologically activated dermal graft. Due to the layered solution containing the DNA fragment complex and chitosan layered inside, the grafted dermal layer reduces the initial inflammatory response and promotes fusion with the surrounding tissues by speeding up the introduction and fixation of cells from the transplanted peripheral tissues. Confirmed that.
  • the implantable dermal layer of the present invention is implanted into the damaged area.
  • FIG. 1 shows the results of confirming the filling of the layered solution containing the DNA fragment complex and the chitosan.
  • FIG. 2 shows the change in size of the dermis over time after transplantation.
  • control 1 On which acellular dermal grafts were immersed in PBS solution, control 2 (B) on 30 minutes in layer 1-1, and control 3 (C) on 0.005% by weight of chitosan. While the size of the acellular dermal grafts transplanted over time was not significant, transplanted dermal layers filled with 1-1 of the present invention (D) were similar to the control group for up to 3 weeks, but 4 weeks later. Afterwards, it can be seen that the border of the transplanted dermal layer is decomposed to reduce the size of the cortical layer.
  • FIG 3 shows the degree of inflammation caused by transplantation of the transplanted dermal layer of the present invention.
  • the transplantation dermal layer (B) of the present invention is transplanted than that of control group 1 (A).
  • the number of cells in the dermal layer for transplantation increased.
  • a layer solution containing the mixture and chitosan was prepared.
  • DNA fragment mixture was dissolved in dodecahydrate (sodium phosphate dibasic dodecahydrate) in a complete solution and dissolved for at least 2 hours using a heat stirrer at 60 ° C.
  • dodecahydrate sodium phosphate dibasic dodecahydrate
  • a stock solution was prepared. Chitosan was added to 90 mM acetic acid and stirred for 3 hours at room temperature to prepare a chitosan stock solution.
  • the DNA fragment complex storage solution and chitosan storage solution prepared above were mixed and stirred for 2 hours in a heat stirrer at 70 ° C. At this time, the concentration and the mixing ratio of the DNA fragment complex and chitosan were Conditions were mixed.
  • the layered solution containing the prepared DNA fragment mixture and chitosan was sterilized by a high-temperature autoclave.
  • Acellular dermal matrix (trade name: Nature-Derma) was purchased from Hans Biomed Co., Ltd. (Seoul, Korea). This product removes cells and impurities from pork dermis by chemical treatment process and removes viruses. Freeze-dried collagen-based tissue repair material after inactivation Or it is a collagen-containing product used to replenish damaged bones, cartilage, etc.
  • Example 1-1 After fixing the acellular dermal grafts obtained from Hans Biomed Co., Ltd. to a vacuum drug filling apparatus, visual inspection of the inventors in the layered solution containing the DNA fragment complex and chitosan prepared in Example 1-1 was performed.
  • the epidermal layer 1-1 which was excellent in the sensation, was filled with acellular dermal grafts to produce a transplanted dermal layer of the present invention.
  • H & E staining was performed to confirm that acellular dermal grafts were filled with DNA fragment complexes and chitosan-filled fillers.
  • hematoxylin combines with a nucleic acid with a negative charge to give a purple color
  • eosin combines with a protein in the cytoplasm with a positive charge to give red color. In combination with eosin, it is dyed red.
  • H & E staining was carried out using a transplanted dermal layer filled with the layered solution 1-1 of Table 1 prepared in Example 1-2, wherein the cell-free dermis without layering the layered solution with the control group. H & E staining was performed with the graft (control 1), the graft with the acellular dermal graft (control 2) in the fill solution for 1 hour, and the results are shown in FIG.
  • a filler containing a DNA fragment complex and chitosan was prepared.
  • the feed was used as a solid feed material for laboratory animals (Harlan laboratories, Inc., USA), and the feed was freely fed with solid feed.
  • Ultraviolet rays were irradiated after filtration and freely ingested by an automatic water supply system.
  • the environment of the experimental animal breeding room was maintained at 23 ⁇ 3 ° C, relative humidity 55 ⁇ 15%, lighting time 12 hours and illuminance 150 ⁇ 300Lux.
  • Example 2-1 Transplantation of Dermal Dermal Layer
  • the grafted dermal layer prepared in Example 1-2 was further immersed in a solution containing the same concentration of layer solution for about 30 minutes. At this time, the cell-free dermal graft without filling the control solution with PBS was used. (phosphate buffer saline) solution (control 1) and layered solution 1-1 for 30 minutes (control 2) and 0.005% by weight of chitosan (control 3) were used.
  • control 1 phosphate buffer saline
  • control 2 layered solution 1-1 for 30 minutes
  • control 3 0.005% by weight of chitosan
  • the rats of Experimental Example 1 were anesthetized with isofluran, and then the sites to be transplanted were sterilized with povidone and ethanol.
  • the dermal layer or control group of the present invention prepared in the above graft was inserted, and then the incision site was closed.
  • the grafted dermal layers of the present invention were transplanted.
  • One operated rat was placed in a cage and reared in a stable environment.
  • the tissues of the site were separated and visually observed for changes in the grafted dermal layer, and the results are shown in Table 2 and Fig. 2.
  • Table 2 the change in the size of the transplanted dermal layer after 4 weeks after transplantation was confirmed.
  • the size of the transplanted dermis compared to the size of the transplanted dermis was used as a percentage ( ⁇ 3 ⁇ 4).
  • Control 1 Acellular Dermal Graft Support in PBS Solution
  • Control 2 Acellular Dermal Graft Support in Filler 1-1)
  • Control 3 Acellular dermal grafts with 0.005% by weight of chitosan
  • mice were collected after transplantation of the transplanted dermal layer of the present invention in which the layered solution 1-1 was layered and the acellular dermal graft (control group 1) supported on the PBS solution. After H & E staining, biocompatibility and engraftment rate were confirmed.
  • FIGS. 3 and 4 show biocompatibility by observing the fibrous tissue capsule caused by inflammation. 4 shows the result of confirming the cell engraftment rate by observing the change of the number of cells in the transplanted dermal layer.
  • the fibrous tissue caused by inflammation is present.
  • the thickness of the capsule is thin when transplanting the transplantable dermal layer (B) of the present invention as compared to the control group 1 (A), and at week 4 both the fibrous tissues when the control group 1 (A) and the implantable dermal layer (B) of the present invention were implanted. The thickness of the capsule became thin.
  • the empty space between the control group 1 (A) and the collagen of the transplanted dermal layer (B) of the present invention was observed at 1 week after transplantation, while at 4 weeks.
  • the voids between collagen were observed to be filled with cells.
  • the transplantation of the transplanted dermal layer (B) of the present invention showed that the number of cells increased more than that of the control group 1 (A).

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Abstract

본 발명은 생착률을 증가시킨 이식용 진피층 및 이의 제조방법에 관한 것으로, 세포가 제거된 무세포성 진피 이식편에 DNA 단편 흔합물 및 키토산을 포함하는 충전액을 충전시켜 제조할 수 있다. 이렇게 제조된 이식용 진피층이 내부에 충전되어 있는 DNA 단편 흔합물 및 키토산을 포함하는 충전액으로 인해, 이식된 주변 조직으로부터 세포들 유입과 고착을 빠르게 함으로써 초기 염증반응을 완화하고 주변 조직과의 융합이 촉진되는 것을 확인하였다.

Description

명세서
발명의 명칭:생착률을증가시킨이식용진피층및이의제조방법 기술분야
[1] 본발명은생착률을증가시킨이식용진피층및이의제조방법에관한것이다. 더욱상세하게는，생물학적으로비활성화된진피층에 DNA단편흔합물및 키토산을포함하는층전액을충전시켜이식시생착률을증가시킨이식용 진피층에관한것이다.
배경기술
[2] 피부는인체표면전체를덮고있는가장큰장기로서，체액의유실을막아주고, 외부로부터유해물질과미생물의유입을막고，물리적자극，방사선과자외선 등으로부터우리몸을보호하는기능을수행하고있다.피부는모낭，털，땀샘, 피지선등여러부속기관을보유하고있어보호막기능의에도다양한기능을 수행하고있는중요한복합기관이다 (김천호， et al., 2002).피부는크게
표피증 (epidermis),진피증 (dermis)및피하조직 (subcutaneous tissue,
hypodermis)으로나 다.
[3] 표피층은피부외부의얇은층으로서，새로분화하는세포들로구성되어
있으며，이들은외부환경으로부터피부를보호하는세포들을계속새로운 세포로교체해준다.제일하단부로부터기저층 (stratum basalis, basal cell layer), 유극증 (stratum spinosum, spinous cell layer),과립증 (stratum granulosum, granular cell layer),각질층 (stratum corneum)으로계층화되어 있어피부표면에는최종 분화된죽은세포가여러겹으로쌓여보호막기능을수행하게된다.
[4] 진피층은표피의아래에 있는층으로혈관，신경등의다양한구조물들을
지지해주는기질을공급하는층으로콜라겐,탄성섬유및세포외
기질 (extracellular matrix)들로이루어져 있다.진피층은크게상층부의
섬유아세포 (fibroblast)가풍부하고미세혈관이분포하는유두층 (papillary layer)과두꺼운콜라겐섬유가풍부한세망결합조직 (reticular connective tissue)으로구성되어있다.
[5] 피하조직은진피와근육，골격사이에있는부분으로지방조직 (adipose tissue)과 결합조직으로구성되어 있고，혈관과림프관및신경말단부를포함한다.
피하조직내의지방조직은외부의압력으로부터보호해주며,마찰력에대한 패딩효과를제공하여뼈와근육과같은심부구조위에서피부의 이동을쉽게 하도록한다.또한,지방조직이많아서열을생산하고생산한열을보유할뿐만 아니라층격을흡수함으로써신체를보호하며여분의영양소를축적한다.
[6] 피부조직은화상，외상및피부질환등으로인해조직의일부가손상될수
있는데，이경우손상된조직의치유를목적으로하거나재건성형을목적으로 본인피부조직을이식하는자가이식 (autograft),다른사람의피부를이삭하는 동종이식 (homograft, allograft),동물의피부를이식하는이종이식 (heterograft, xenograft)방법을사용한다.이들방법중자가이식이가장이상적이지만 치료부위가광범위한경우조직을확보할수있는부위에제한이따르며，채취 부위가새로운상처부위로남게되는어려움이 있다.동족이식은영구적인 생착보다는상처주변부의세포의이동과치유를돕는역할을한다.
[7] 피부의손실이심각한상태의환자를위해서는상처를덮을수있는
지지체 (scaffold)가필요하며,이를위해인공피부 (artificial skin)가개발되고있다. 인공피부는손실부위의감염및체액의손실을막아주는보호막의 역할과， 환자의상처부위에흉터가남지않고，치유과정에서발생될수있는심각한 수축을원칙적으로막아줄수있어야한다 (Yannas I.V., 1995).
[8] 인공피부는크게창상피복제 (wound dressing)라고하는일시적
피복형 (temporary skin equivalent)과배양피부또는체내이식용인
영구생착형 (permanent skin equivalent)이라고하는두가지로크게나뉠수있다. 일시적피복형인창상피복제는화상이나외상등에의해손상을입은피부가 회복할때까지일시적으로환부를보호하는것으로，체내로부터의수분노출을 방지하고삼출액을흡수하며,외부로부터미생물의침입및감염을방지하는 역할을한다.반면，영구생착형인공피부는합성고분자와천연고분자로 이루어지며，창상피복제역할은물론인체의세포가쉽게침투할수있는통로를 제공하며본래의피부조직으로재생되는것을적극적으로돕고，그자체는인체 내에서분해，흡수된다.영구생착형인공피부중하나인생인공피부 (bioartifical skin)는보호막기능은물론손상조직의재생에필요한성장인자와세포외 기질올제공하여신속한상처치유와상흔의감소등그탁월한효과가입증되고 있다.현재생인공피부는화상，욕창,외상,성형,난치성궤양，당뇨성피부괴사, 압력미란 (erosion)둥에임상적용되고있다.
[9] 피부이식은이식하는외식편 (explant)의두께와이식부위관리에따라성공 여부가다양하게나타난다 (Gallico G.G. 3rd., 1990; Kunert P., 1991).성공적인 피부이식을위해피부이식의기전에대한연구,기술적발달과수술후공여부 관리，수술부위관리등에대한연구도활발히이루어져왔다 (Johnson C.S., et al, 2001; McDowell F., 1977).일반적으로알려진피부이식의기전은이식피부가 착상배지로부터혈류를공급받아살게되며이식성공여부는 24-48시간에 발생—하는영-양—및산소의확산효과언혈장흡수 (plasmatic irabibitio^
신생혈관형성 (revascularization)에의해결정된다 (Johnson C.S., et al., 2001).피부 이식의실패원인으로가장흔한것은이식피부하혈종이나체액의 저류로 보고되고있다.두번째로흔한원인은감염으로알려져 있다 (Barret J.P., et al, 2002; McGergor A.D., et al., 2000).그외피부이식의성공에 영향을미치는 다양한인자들아나연관인자들이 있을것으로생각되나이에대한연구결과는 부족하며，조절가능한적절한피부이식의전제조건이나기준은제시된바가 없다 (안기찬, et al, 2012). [10] 키토산 (chitosan)(poly (3-(l→4)-2-amino-2-deoxy-D-glucan)은 D-글루코사민이 β-(1→4)결합한선형고분자로，키틴 (chitin)(poly
(3-(l→4)-2-acetamined-2-deoxy-D-glucan)의 C2위치에 있는아세트아미드기 중에서아세틸기를제거시킴으로써얻어지는천연다당류이다.키토산은 키틴에비해결정성이낮기때문에염산 (hydrochloric acid),황산 (sulfuric acid) 등의무기산，포름산 (formic acid),아세트산 (acetic acid),프로피온산 (propionic acid),옥살산 (oxalic acid),숙신산 (succinic acid)등의유기산，글루콘산 (gluconic acid),락트산 (lactic acid),말산 (malic acid)등의옥시산 (oxy acid),
글루타민산 (glutamic acid)등의산성아미노산의묽은수용액에서산염을 형성하여녹는다 (Mussarelli R.A.A., 1997; Brine C.J., et al., 1992).
[11] 키토산용액은점도가높은특징을가지고있으며，탈아세틸화도，이온강도, pH,온도등에따라차이가있다.예를들면,용매의 pH가낮으면키토산용액의 점도는감소하고 pH가높아지면점도는증가하며,온도의경우에는은도가 높아지면키토산용액의 점도가감소하고다시원래의온도가되면처음과같은 점도가된다.
[12] 키토산은사람을포함한동물,식물에대한생리활성을가지기때문에넓은 분야에서웅용및이용되고있다.키토산의생리작용을활용하여가장넓게 웅용되고있는것은항균작용이다.키토산의항균작용은양이온화된 아미노기에의해발현되는것으로알려져 있다.양이온화된키토산의 아미노기와세균의세포벽을구성하는시알산 (sialic acid),인지질 (phospholipid) 등의산성기 (acidic group)간에아은결합이 일어나고분자전해질복합체를 형성한결과，세포막중인지질의극성화로접촉면반대측의세포막조직이 파괴되고그로인해세포내의원형질이누출되어세균이죽게된다.또한, 생체의료공학분야에서우수한인체적합성과거부반웅이최소인고분자 소재로서키토산을이용하려는연구들이지속되어왔으며,창상보호재，창상 연고，치과용재,정형외과재료，수술용봉합사등의인체적합성 생체고분자 소재로서각광받고있다 (고병열, et al., 2002).
[13] 이에,본발명자들은피부이식용조성물을연구하는과정에서，생물학적으로 비활성화된진피층에 DNA단편흔합물및키토산을포함하는층전액으로 충전시켜제조한이식용진피층이 이식한부위에서생착률이증가된것을 확인함으로쩌본발명을완성할수았었 -다.
[14] 종래선행기술로서한국등록특허제 0469661호및제 1362402호에는이식용 무세포진피층이기재되어본발명의구성과유사하나, DNA단편흔합물및 키토산을포함하는충전액을층전시키는방법은기재되어 있지않아본발명의 구성과차이가있다.또한，미국공개특허제 2004-0031067호에는피부이식시 신생혈관형성유도인자를암호화하고있는핵산을투여하는방법을기재하고 있으나，본발명의키토산은전혀언급되어 있지않고,약물아층전된진피 조성물이기재되어 있지않아본발명의구성과상이하다.한국등록특허 제 1534276호에는지방이식수술후지방세포의생착률증진을위한 폴리디옥시리보뉴클리오티드를성분으로하는조성물이기재되어 있어본 발명의구성과유사하나，키토산은전혀언급되어 있지않고,약물이층전된 진피조성물이기재되어 있지않아본발명의구성과차이가있다.
발명의상세한설명
기술적과제
[15] 본발명의목적은생착률을증가시킨이식용진피층및이의제조방법을
제공하는데있다.
과제해결수단
[16] 본발명은이식용진피층제조방법에관한것으로, DNA단편흔합물저장
용액 (stock solution)을제조하는제 1단계;키토산저장용액을제조하는제 2단계; 상기 DNA단편흔합물저장용액및상기키토산저장용액을흔합하여교반한 후멸균하여 DNA단편흔합물및키토산을포함하는층전액을제조하는 제 3단계;및상기충전액을무세포성진피이식편에충전하는제 4단계;를 포함하는이식용진피층의제조방법에관한것이다.
[17] 상기제조방법은 DNA단편흔합물을완층용액에 넣고 50oC내지 70oC에서 교반하면서 1시간내지 3시간동안용해시켜 DNA단편흔합물저장용액을 제조하는제 1단계;키토산을산성완층용액에용해시켜키토산저장용액을 제조하는제 2단계;상기 DNA단편흔합물저장용액및상기키토산저장용액을 DNA단편흔합물및키토산의중량비율이 20: 1-10,000:1이되도록혼합하여 65°C내지 750C에서 1시간내지 2시간동안교반한후멸균하여 DNA단편 흔합물및키토산을포함하는층전액을제조하는제 3단계;및상기충전액을 진공약물층전장치를이용하여무세포성진피이식편에층전하는제 4단계;를 포함할수있다.
[18] 상기 DNA단편흔합물은어류의정소또는정액에서분리할수있다.
[19] 상기어류는연어과일수있다ᅳ
[20] 상기 DNA단편흔합물은분자량이 50kDa내지 10,000kDa일수있다.
[21] 상기 DNA단편흔합물은충전액총증량을기준으로 0.01중량 %내지
3중량%일수있다.
[22] 상기키토산은분자량이 3kDa내지 l ,000kDa일수있다.
[23] 상기키토산은층전액총중량을기준으로 lxlO-6중량 %내지 0.15증량 %일수 있다.
[24] 상기제 4단계의무세포성진피이식편은진피로부터세포및불순물이
제거되고，바이러스불활화처리후동결건조처리하여생물학적으로비활성화 된콜라겐사용조직보층재일수있다.
[25] 상기진피는자가，동종또는이종에서분리한것일수있다ᅳ
[26] 상기제 4단계의충전은압력을이용해무세포성진피이식편내부로점성이 있는충전액을유입시켜콜라겐사이의빈공간을채우는것일수있다.
[27] 상기제 4단계의진공약물충전장치는층전액을일방향에연결하고，양압또는 음압을발생시켜연결된층전액이무세포진피이식편내부의콜라겐사이의 공간을채워층전되도록할수있다.
[28] 또한，본발명은상기제조방법으로제조된이식용진피층에관한것이다.
[29] 또한，본발명은상기 이식용진피층을포함하는피부손상치료제에관한
것이다.
[30] 상기피부손상은찰과상 (abrasion),절개 (incision),열상 (laceration),
피부결출 (skin avulsion),피부타박 (skin bruise),천자부상 (puncture wound)및 수술에의한피부절제군에서선택되는 1종이상이원인될수있다.
[31] 이하본발명을상세하게설명한다.
[32] 본발명은 DNA단편혼합물저장용액 (stock solution)을제조하는제 1단계 ; 키토산저장용액을제조하는제 2단계;상기 DNA단편혼합물저장용액및상기 키토산저장용액을혼합하여교반한후멸균하여 DNA단편혼합물및키토산을 포함하는층전액을제조하는제 3단계;및상기층전액을무세포성진피
이식편에층전하는제 4단계;를포함하는이식용진피층의제조방법에관한 것이다.
[33] 상기이식용진피층의제조방법은 DNA단편흔합물을완충용액에넣고 50oC 내지 70°C에서교반하면서 1시간내지 3시간동안용해시켜 DNA단편흔합물 저장용액을제조하는제 1단계;키토산을산성완층용액에용해시켜키토산 저장용액을제조하는제 2단계;상기 DNA단편흔합물저장용액및상기키토산 저장용액을 DNA단편흔합물및키토산의중량비율이 20:1-10,000:1이되도록 흔합하여 650C내지 750C에서 1시간내지 2시간동안교반한후멸균하여 DNA 단편흔합물및키토산을포함하는충전액을제조하는제 3단계;및상기 층전액을진공약물층전장치를이용하여무세포성진피이식편에층전하는 계 4단계;를포함할수있다.
[34] 상기제 1단계에서 DNA단편흔합물저장용액제조시 이용할수있는
완충용액으로는소듐포스페이트디베이직도데카하이드레이트 (sodium phosphate dibasic dodecahy drate) ,염화나트륨 (sodium chloride),
염화마그네슘 (magnesium chloride),염화칼륨 (potassium chloride),인산완충 식염수 (phosphate buffer sline),
HEPES(N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid)또는
글리세를 -3-인산 (glycerol-3-phosphate)완충용액이 있으며,바람직하게는소듐 포스페이트디베이직도데카하이드레이트 (sodium phosphate dibasic
dodecahydrate)를이용할수있으나，이에한정하는것은아니다.
[35] 상기 DNA단편흔합물은어류의정소또는정액으로부터분리한것일수있다.
[36] .상기어류는연어과일수있고,바람직하게는연어또는송어일수있고，가장 바람직하게는연어일수있다. [37] 상기 DNA단편흔합물이란,인산, 4종류의염기,
데옥시리보오스 (deoxyribose)로이루어진생체고분자에해당하는 DNA로， 분자량이 저감된단편형태로흔합되어존재하는것을지칭한다.
[38] 상기 DNA단편흔합물은특정한크기의 DNA단편이혼합되어약리효과또는 고분자지지체역할을하는물질을말한다.
[39] 상기 DNA단편흔합물은
폴리데옥시리보뉴클레오티드 (Polydeoxyribonucleotide)및
폴리뉴클레오티드 (Polynucleotide)일수있다.
[40] 상기폴리데옥시리보뉴클레오티드는세포재생，상처치유의역할을하는
물질로알려져 있으며，상기폴리뉴클레오티드는지지체구조를통한세포재생 환경을구성하는역할을할수있다.
[41] 상기 DNA단편흔합물은분자량이 50kDa내지 10,00()kDa일수있다.
바람직하게는 50kDa내지 5,000kDa이고,가장바람직하게는 50kDa내지
3,500kDa이다.
[42] 상기제 2단계에서키토산저장용액제조시이용할수있는산성
완충용액으로는아세트산 (acetic acid),염산 (hydrochloric acid),
아스코르브산 (ascorbic acid),젖산 (lactic acid),질산 (nitric acid)이 있으며， 바람직하게는아세트산 (acetic add)을이용할수있으나,이에한정하는것은 아니다.
[43] 상기키토산은분자량이 3kDa내지 l,000kDa일수있다.
[44] 상기제 3단계에서 DNA단편흔합물및키토산의중량비율이 20: 1~ 10,000:1일 수있으며，바람직하게는 20:1~1，000:1이고，더욱바람직하게는 50: 1-200:1이다.
[45] 상기 DNA단편흔합물은층전액총증량을기준으로 0Ό1증량 <¾내지
3중량 %일수있고，바람직하게는 0.1중량 %내지 2중량%이며，가장
바람직하게는 0.5중량 %내지 1.5중량 <¾이다. DNA단편혼합물이 0.01중량 % 미만일경우에는생착률증진효과를기대하기어렵고， 3중량 %초과인경우에는 충전액제조공정상의문제점이발생할수있어바람직하지못하다.
[46] 상기키토산은충전액총중량을기준으로 lxlO-6증량 %내지 0.15증량 %일수 있고，바람직하게는 lxlO-5중량 %내지 0.15증량%이며，가장바람직하게는 lxlO-4 중량 %내지 0.075중량 %이다.키토산이 1χ10-δ중량 %미만또는 0.15중량 % 초과인경우에는충전액의점성에의해무세포진피이식편충전시충전율이 낮아져바람직하지못하다.
[47] 상기키토산은 DNA단편흔합물과흔합됨으로써층전액의 점성을증가시킬 수있다.이러한점성의증가는충전액의층전시무세포성진피이식편내부의 콜라겐사이의빈공간을채우는층전효율을증가시킬수있으며,무세포성 진피이식편내부로유입된 DNA단편흔합물이 이식편내부에존재하도록할수 있다.
[48] 상기키토산은무세포진피이식편의주성분인음전하를띄는콜라겐과 이온결합을통해층전시충전액의충전효율을증가시킬수있다.
[49] 상기제 3단계에서멸균은고온고압증기멸균，건열멸균, EO(ethylene
oxide)가스멸균등을이용할수있으며,바람직하게는고온고압증기멸균을 이용하지만,이에한정하는것은아니다.
[50] 상기제 4단계에서무세포진피 이식편 (Acellular dermal matrix)은진피로부터 세포및불순물이제거되고,바이러스불활화처리후동결건조처리하여 생물학적으로비활성화된콜라겐사용조직보충재및지지체 (scaffold)일수 있다.
[51] 상기보충재는상처를덮을수있는것으로，인공피부 (artificial skin)일수있다.
[52] 상기무세포진피이식편은진피로부터일련의과정을통해제조할수있으며 또한，상용화되어판매되는것을이용할수있다.
[53] 상기진피는자가,동종또는이종에서분리한진피일수있다.
[54] 상기제 4단계의층전은압력을이용해무세포성진피이식편내부로점성이 있는충전액을유입시켜콜라겐사이의빈공간을채우는것으로，유효성분이 이식편내부에다량으로존재할수있도록채우는것일수있다.특히，층전액이 점성을띠어무세포성진피이식편내부로의유입이어려운경우,음압또는 양압을가함으로써무세포성진피이식편내부로층전액유입을증가시켜 콜라겐사이의빈공간을채우는충전효율을증가시킬수있다.또한，층전액의 이식편내부로의유입을증가시켜층전액에포함되어 있는물질과무세포성 진피이식편의주요성분인콜라겐과의접촉면적을증가시킴으로써，층전액에 포함되어 있는물질과콜라겐과의이온결합기회를높여두물질간의결합을 촉진시킴으로써충전효율이증가될수있다.
[55] 상기층전은진공약물층전장치를이용하여이루어질수있다.
[56] 상기진공약물충전장치는무세포성진피이식편에양압 (positive pressure) 또는음압 (negative pressure)을가함으로써충전액이무세포성진피이식편 내부로유입되도록하는장치일수있다ᅳ바람직하게는음압을이용한다.
[57] 상기진공약물충전장치는무세포성진피이식편을고정하는부위와
층전액이유입되는부위，양압또는음압을위한진공펌프가연결된부위로 이루어질수있다.
[58] 상기고정하는부위는약물충전시압력에의해약물이새어나가지못하도록 패킹 (packing)되어야한다.
[59] 또한，본발명은상기제조방법으로제조된이식용진피층에관한것이다.
[60] 상기 이식용진피층은층전되어 있는 DNA단편흔합물및키토산을포함하는 충전액으로인해，이식된주변조직으로부터세포들이빠르게이동하도록 함으로써주변조직과의융합을촉진시켜생착률을증가시킬수있으며,초기 염증을완화시켜이식후회복을빠르게할수있다.
[61] 또한，본발명은이식용진피층을포함하는피부손상치료제에관한것이다.
[62] 상기피부손상은찰과상 (abrasion),절개 (incision),열상 (laceration), 피부결출 (skin avulsion),피부타박 (skin bruise),천^ 부상 (puncture wound)및 수술에의한피부절제군에서선택되는 1종이상이원인이되어나타나는것일 수있다ᅳ
[63] 상기피부손상치료는피부의손상이심각한부위에보충재또는
지지체 (scaffold)를이식하여상처부위를덮어손상부위의체액손실을
막아주고，상처부위에흉터가남지않도록하는것이다.
발명의효과
[64] 본발명은생착률을증가시킨이식용진피층및이의제조방법에관한것으로, 생물학적으로바활성화된진피이식편에 DNA단편흔합물및키토산을 포함하는충전액을층전시켜제조할수있디-.이렇게제조된이식용진피층이 내부에층전되어 있는 DNA단편흔합물및키토산을포함하는층전액으로인해， 이식된주변조직으로부터세포들유입과고착을빠르게함으로써초기 염증 반응을줄이고주변조직과의융합을촉진하는것을확인하였다.
[65] 따라서，본발명의이식용진피층을피부가손상된부위에 이식함으로써
생착률이높고，빠른피부손상치료효과를얻을수있을것으로기대된다.
도면꾀간단한설명
[66] 도 1은 DNA단편흔합물및키토산을포함하는층전액의충전여부를확인한 결과를보여주고있다.대조군 1인충전액을층전하지않은무세포성진피 이식편 (A)및대조군 2인충전액에담지시킨무세포성진피이식편 (B)의 경우에는헤마톡실린에의해자색으로염색된부분이없으나，본발명의이식용 진피층 (C)의경우에는 '붉은색으로염색된콜라겐사이사이에자색으로염색된 부분이다수존재하는것으로관찰되었다.검은색화살표는헤마톡실린에의해 자색으로염색된，충전액이층전된부위를나타낸다.
[67] 도 2는이식용진피층이식후시간에따른진피층의크기변화를나타내고
있다.무세포성진피이식편을 PBS용액에담지시킨대조군 1(A)과층전액 1-1에 30분정도담지시킨대조군 2(B)및 0.005중량 %의키토산을층전시킨 대조군 3(C)의경우에는시간이지나도이식한무세포성진피이식편의크기 변화가크게나타나지않는반면에，본발명의충전액 1-1을충전시킨이식용 진피층을이식한경우 (D)에는 3주까지는대조군과비슷하였지만 4주가지난 후에는이식한진피층의테두리가분해되어진피층의크기가작아지는것을알 수있다.
[68] 도 3은본발명의이식용진피층의이식에의한염증유발정도를보여주고
있다.염증에의해나타나는섬유조직캡슐의두께를관찰한결과대조군 1을 이식한경우 (A)에비해,본발명의이식용진피층을이식한경우 (B)가초기염증 반웅을완화시킨다는것을알수있다.
[69] 도 4는본발명의이식용진피층의 이식에따른세포생착률을보여주고있다. 본발명의이식용진피층 (B)을이식한경우가대조군 1(A)을이식한경우보다 이식용진피층내의세포수가더많이증가하였다.
발명의실시를위한최선의형태
[70] 이하본발명의바람직한실시예를상세히설명하기로한다.그러나，본발명은 여기서설명되는실시예에한정되지않고다른형태로구체화될수도있다. 오히려，여기서소개되는내용이철저하고완전해지고，당업자에게본발명의 사상을층분히전달하기위해제공하는것이다.
[71] <실시예 1.생착률을증가시킨이식용진피층제조>
[72] 실시예 1-1. DNA단편혼합물및키토산을포함하는충전액제조
[73] 본발명의생착률을증가시킨이식용진피층을제조하기위해 DNA단편
흔합물및키토산을포함하는층전액을제조하였다.
[74] DNA단편흔합물을 190mM소듐포스페이트디베이직
도데카하이드레이트 (sodium phosphate dibasic dodecahydrate)완층용액에 넣고 60°C의열교반기를이용하여 2시간이상용해시켜 DNA단편흔합물
저장용액 (stock solution)을제조하였다.키토산은 90mM아세트산 (acetic acid)에 넣고상온에서 3시간동안교반시켜용해하여키토산저장용액을제조하였다.
[75] 상기에서제조한 DNA단편흔합물저장용액및키토산저장용액을흔합하여 70°C의열교반기에서 2시간동안교반하였다.이때, DNA단편흔합물및 키토산의농도및흔합비율이하기표 1의조건이되도록흔합하였다.제조된 DNA단편흔합물및키토산을포함하는층전액을고온고압증기멸균기로멸균 처뫼하였다.
[76] [표 1]
Figure imgf000011_0001
[77]
[78] 실시예 1-2.생착률을증가시킨이식용진피층제조
[79] 본발명의생착률을증가시킨이식용진피층을제조하기위해
한스바이오메드사 (서울， Korea)로부터무세포성진피이식편 (Acellular dermal matrix) (상품명: Nature-Derma)을구입하여사용하였다.이제품은화학처리 공정에의한돼지진피로부터세포및불순물을제거하고,바이러스불활화처리 후동결건조처리된콜라겐사용조직보층재로내외과적처리및수술시결손 또는손상된뼈，연골등을보충하기위하여사용하는콜라겐함유제품이다.
[80] 상기한스바이오메드사로부터구입한무세포성진피이식편을진공약물충전 장치에고정시킨후，상기실시예 1-1에서제조한 DNA단편흔합물및키토산을 포함하는층전액중에서발명자의육안검사와층전테스트를통해사용감이 우수한층전액 1—1을무세포성진피이식편에충전시켜본발명의 이식용 진피층을제조하였다.
[81] <실시예 2. DNA단편혼합물및키토산을포함하는층전액의층전확인>
[82] 무세포성진피이식편에 DNA단편흔합물및키토산을포함하는충전액이 충전되었는지확인하기위해 H&E(haematoxylin and eosin)염색을진행하였다. H&E염색과정에서헤마톡실린 (haematoxylin)이음전하를갖는핵산과결합하여 자색을나타내고,에오신 (eosin)이양전하를갖는세포질내단백질과결합하여 붉은색을나타내는데，무세포성진피이식편의주성분인콜라겐이에오신과 결합하여붉은색으로염색된다.
[83] 상기실시예 1-2에서제조한이식용진피층중표 1의층전액 1-1을충전시킨 이식용진피층을이용해 H&E염색을진행하였다.이때，대조군으로층전액을 층전하지않은무세포성진피이식편 (대조군 1),무세포성진피이식편을 충전액에담가 1시간정도담지시킨이식편 (대조군 2)및가지고 H&E염색을 진행하였고，그결과를도 1에나타내었다.
[84] 도 1에서보여주듯이， DNA단편흔합물및키토산을포함하는충전액을
층전하지않은무세포성진피 이식편 (대조군 1)(A)및층전액에담지시킨 무세포성진피이식편 (대조군 2)(B)의경우에는에오신에의해콜라겐부분이 붉은색으로염색되었으나,헤마톡실린에의해자색으로염색된부분이없는 것을알수있다.반면,상기실시예 1-2에서제조한이식용진피층 (C)의경우에는 붉은색으로염색된콜라겐사이사이에자색으로염색된부분 (검은색화살표)이 나타나는것을알수있다.
[85] 이를통해，충전액에담지하는방법은무세포성진피이식편내부로의
충전액이유입되지않는다는것을알수있으며,무세포성진피이식편에 DNA 단편흔합물및키토산을포함하는층전액을충전하는방법으로진공약물층전 장치를이용한방법이가장효율적이라는것을알수있었다.
[86] <실험예 1.실험동물의준비>
[87] 동물실험은 7주령의무게가 230~280g인 Sprague-Dawley계수컷쥐 (Rat)를
영바이오 (성남시,대한민국)로부터구입하여사용하였다.사료는실험동물용 고형사료 (Harlan laboratories, Inc., USA)를사용하였고급이기에고형사료를넣어 자유섭취시켰다.음수는수듯물을필터유수살균기로여과한후자외선을 조사하였고,자동급수장치로자유섭취시켰다.실험동물사육실의환경은온도 23±3°C,상대습도 55±15%，조명시간 12시간및조도 150~300Lux로유지하였다.
[88] <실험예 2.생착률을증가시킨이식용진피층의생착률확인 >
[89] 실험예 2-1.이식용진피층의이식 [90] 상기실시예 1-2에서제조한이식용진피층을동일한농도의층전액이담긴 용액에 30분정도추가담지하여사용하였다.이때，대조군으로충전액을 충전시키지않은무세포성진피이식편을 PBS(phosphate buffer saline)용액에 담지시킨것 (대조군 1)과층전액 1-1에 30분정도담지시킨것 (대조군 2), 0.005중량 %의키토산을충전시킨것 (대조군 3)을이용하였다.
[91] 상기실험예 1의쥐를이소플루란 (isofluran)으로호흡마취시킨후，이식하고자 하는부위를포비돈과에탄올로소독하였다.소독후쥐의척추를따라등 부위를절개하여핀셋을이용하여외피를들어 이식공간을확보하였다.확보된 이식공간에상기에서준비한본발명의 이식용진피층또는대조군을넣은후 절개부위를봉합하였다.이때，척추를중심으로쥐의왼쪽등피하에는 대조군을，오른쪽둥피하에는본발명의이식용진피층을이식하였다.수술한 쥐를케이지에 1마리씩넣고안정한환경속에서사육하였다.
[92] 실험예 2-2.육안관찰을통한생착률확인
[93] 상기실험예 2-1의이식수술후 1주마다쥐를 1마리씩희생시킨후이식
부위의조직을분리하여이식용진피층의변화여부를육안으로관찰하였고，그 결과를표 2및도 2에나타내었다.표 2의경우에는이식후 4주가지난쥐의 이식용진피층의크기변화를확인한결과로，이식시이용한이식용진피층 크기대비이식후크기를백분율(<¾)로나타내었다.
[94] [표 2]
Figure imgf000013_0001
상기표 2및도 2에서보여주듯이,대조군 1(PBS용액에담지한무세포성진피 이식편 )(A),대조군 2(충전액 1-1에담지한무세포성진피이식편 )(B)및대조군 3(0.005중량 %키토산을층전시킨무세포성진피이식편 )(C)을이식한경우에는 시간이지나도이식한진피 이식편의크기가변화가거의없는반면에，본 발명의이식용진피층을이식한경우 (D)에는 3주까지는크기변화가거의 없었으나, 4주가지난후에는이식한진피층의테두리가분해되어진피층의 크기가작아지는것을알수있다.
이를통해，대조군 2와같이무세포성진피이식편을층전액에담지하여외부 표면에만흡착시킨경우및대조군 3과같이키토산만이포함된층전액을 층전시킨경우에는생착률이증가되지않으며， DNA단편흔합물및키토산을 포함하는층전액을층전시켜제조한본발명의이식용진피층에서생착를이 증가한것을알수있었다.
[98] 실험예 2-3.조직염색을통한세포생착률및생체적합성확인
[99] 상기실험예 2-1에서층전액 1-1을층전시킨본발명의이식용진피층및 PBS 용액에담지한무세포성진피이식편 (대조군 1)을이식한후 1주차와 4주차의 조직을수거하아 H&E염색한후생체적합성및생착률을확인하였다.
[100] 1주차와 4주차의이식부위의조직을수거하여포르말린으로고정하였고, 일련의탈수작업을거쳐파라핀에심어파라핀블록을제작하였다.제작한 파라핀블록을 5/ΛΠ두께로조직 .절편하고,조직절편을 Mayer's
hematoxyline(Sigma, USA)용액에 1초간반웅시킨후，흐르는물에서 10분간 세척하였다.이후 eosin(Sigma, USA)용액에 3초간반응시켰다.염색이완료된 조직은탈수과정을거쳐 Permount (Fischer scientific, USA)로봉입하였다. H&E 염색한조직절편올현미경을이용하여조직병리학적변화를관찰하였고,그 결과를도 3및도 4에나타내었다.도 3은염증에의해나타나는섬유조직 캡슐 (fibrous tissue capsule)을관찰함으로써생체적합성을확인한결과를 보여주고있으며，도 4는이식한진피층내의세포수의변화를관찰함으로써 세포생착률을확인한결과를보여주고있다.
[101] 도 3에서보여주듯이，이식후 1주차에는염증에의해나타나는섬유조직
캡슬의두께가대조군 1(A)에비해본발명의이식용진피층 (B)을이식한경우에 얇은것을알수있고, 4주차에는대조군 1(A)및본발명의이식용진피층 (B)을 이식한경우에모두섬유조직캡슐의두께가얇아졌다.
[102] 또한，세포생착률의경우에는，도 4에서보여주듯이，이식후 1주차에는대조군 1(A)및본발명의이식용진피층 (B)의콜라겐사이의빈공간이다수관찰된 반면, 4주차에는콜라겐사이의빈공간들이세포들로채워진것을관찰하였다. 특히나본발명의이식용진피층 (B)을이식한경우가대조군 1(A)을이식한 경우보다세포수가더많이증가한것을알수있다.
[103] 이를통해,본발명의이식용진피층에층전된 DNA단편흔합물및키토산을 포함하는충전액쎄의해초기염증이완화되었음을알수있었으며，주변 조직으로부터세포들이빠르게이동하도록하여주변조직과의융합이촉진되어 세포생착률이증가되었음을알수있었다.

Claims

청구범위
[청구항 1] DNA단편흔합물저장용액 (stock solution)을제조하는제 1단계;
키토산저장용액을제조하는제 2단계;
상기 DNA단편흔합물저장용액및상기키토산저장용액을혼합하여 교반한후멸균하여 DNA단편흔합물및키토산을포함하는층전액을 제조하는제 3단계;및
상기층전액을무세포성진피이식편에층전하는제 4단계;
를포함하는것을특징으로하는생착률을증가시킨이식용진피층의 제조방법.
[청구항 2] 제 1항에 있어서，
상기제조방법은,
DNA단편흔합물을완충용액에넣고 50oC내지 70°C에서교반하면서 1시간내지 3시간동안용해시켜 DNA단편흔합물저장용액을제조하는 ^ll l단계;
키토산을산성완충용액에용해시켜키토산저장용액을제조하는 제 2단계;
상기 DNA단편흔합물저장용액및상가키토산저장용액을 DNA단편 흔합물및키토산의증량비율이 20: 1-10,000:1이되도록흔합하여 65°C 내지 75°C에서 1시간내지 2시간동안교반한후멸균하여 DNA단편 흔합물및키토산을포함하는충전액을제조하는제 3단계;및
상기층전액을진공약물충전장치를이용하여무세포성진피이식편에 층전하는제 4단계;
를포함하는것을특징으로하는생착률을증가시킨이식용진피층의 제조방법. .
[청구항 3] 제 1항또는제 2항에 있어서，
상기 DNA단편흔합물은어류의정소또는정액에서분리한것임을 특징으로하는생착률을증가시킨이식용진피층의제조방법.
[청구항 4] 제 3항에있어서,
상기어류는연어과인갓을특징으로하는생착률을증가시킨이식용 진피층의제조방법.
[청구항 5] 제 1항또는제 2항에 있어서,
상기 DNA단편혼합물은분차량이 50kDa내지 10,000kDa인것을 특징으로하는생착률을증가시킨이식용진피층의제조방법.
[청구항 6] 제 1항또는제 2항에 있어서，
상기 DNA단편흔합물은충전액총중량을기준으로 0Ό1증량 %내지
3중량 %인것을특징으로하는생착를을증가시킨이식용진피층의제조 방법. [청구항 7] 제 1항또는제 2항에 있어서,
상기키토산은분자량이 3kDa내지 l,000kDa인것을특징으로하는 생착를을증가시킨이식용진피층의제조방법.
[청구항 8] 제 1항또는제 2항에 있어서,
상기키토산은충전액총중량을기준으로 lxlO-6중량 %내지 αΐ5중량 %인것을특징으로하는생착률을증가시킨이식용진피층의 제조방법.
[청구항 9] 제 1항또는제 2항에 있어서，
상기제 4단계와무세포성진피이식편은진피로부터세포및불순물이 제거되고，바이러스불활화처리후동결건조처리하여생물학적으로 비활성화된콜라겐사용조직보충재인것을특징으로하는생착률을 증가시킨이식용진피층의제조방법.
[청구항 10] 제 9항에 있어서，
상기진피는자가,동종또는이종에서분리한진피인것을특징으로하는 생착률을증가시킨이식용진피층의제조방법.
[청구항 11] 제 1항또는제 2항에 있어서,
상기제 4단계의층전은압력을이용해무세포성진피이식편내부로 점성이 있는층전액을유입시켜콜라겐사이의빈공간을채우도록하는 것을특징으로하는생착률을증가시킨이식용진피층의제조방법.
[청구항 1 제 2항에 있어서,
상기제 4단계의진공약물층전장치는층전액을일방향에연결하고, 양압또는음압을발생시켜연결된층전액이무세포진피이식편내부의 콜라겐사이의공간을채워층전되도록하는것을특징으로하는 생착률을증가시킨이식용진피층의제조방법.
[청구항 13] 제 1항또는제 2항의방법으로제조된생착률을증가시킨이식용진피층.
[청구항 14] 제 13항의이식용진피층을포함하는피부손상치료제ᅳ
[청구항 15] 제 14항에 있어서,
상가피부손상은찰과상 (abrasion),절개 (incision),열상 (laceration), 피부결출 (skin avulsion),피부타박 (skin bruise),천자부상 (puncture wound) 및수술에의한피부절제군에서선택되는 1종이상이원인이되는것을 특징으로하는피부손상치료제 .
PCT/KR2017/003070 2016-09-07 2017-03-22 생착률을 증가시킨 이식용 진피층 및 이의 제조방법 WO2018048047A1 (ko)

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