WO2014038885A1 - Biocompatible small intestinal submucosa sheet having adjustable in vivo degradation time and method for preparing same - Google Patents

Biocompatible small intestinal submucosa sheet having adjustable in vivo degradation time and method for preparing same Download PDF

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WO2014038885A1
WO2014038885A1 PCT/KR2013/008067 KR2013008067W WO2014038885A1 WO 2014038885 A1 WO2014038885 A1 WO 2014038885A1 KR 2013008067 W KR2013008067 W KR 2013008067W WO 2014038885 A1 WO2014038885 A1 WO 2014038885A1
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small intestinal
intestinal submucosa
sheet
biocompatible
crosslinking agent
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PCT/KR2013/008067
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French (fr)
Korean (ko)
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김다연
진링메이
박지훈
김문석
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아주대학교산학협력단
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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
    • 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
    • A61L27/38—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 containing added animal cells
    • 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
    • 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
    • A61F13/00—Bandages or dressings; Absorbent pads
    • A61F13/02—Adhesive plasters or dressings
    • 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
    • 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
    • A61L15/64—Use of materials characterised by their function or physical properties specially adapted to be resorbable inside the body
    • 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
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/0005—Ingredients of undetermined constitution or reaction products thereof
    • 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
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/001—Use of materials characterised by their function or physical properties
    • A61L24/0015—Medicaments; Biocides
    • 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
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/001—Use of materials characterised by their function or physical properties
    • A61L24/0042—Materials resorbable by the body
    • 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
    • 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
    • A61L27/3629—Intestinal tissue, e.g. small intestinal submucosa
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61L27/58—Materials at least partially resorbable by the body
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
    • C12N2533/40—Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers

Definitions

  • the present invention relates to a biocompatible small intestinal submucosa sheet capable of controlling the degradation period in vivo, and a method of manufacturing the same.
  • Natural materials are derived from natural materials, animals and the human body and have very good biocompatibility.
  • a representative example is an extracellular matrix (ECM), which is a biomaterial extracted from a complex human body and an animal, and has characteristics that can control the function of cells.
  • ECM extracellular matrix
  • the support made of natural material is evaluated as an ideal tissue engineering support material because it can provide excellent bio-functionality and biodegradability as well as less inflammatory response after implantation into a living body.
  • the small intestinal submucosa of pigs is a cell-free tissue and almost no immune response. More than 90% of the components are composed of type I and type II collagen in the skin. Consisting of small amounts of type V and VI collagen.
  • the small intestinal submucosa of pigs is glycoaminoglycan and fibronectin, chondroitin sulfate, heparin, heparin sulfate, hyaluronic acid and basic fibroblast growth factor-2 (FGF-) as extracellular matrix (ECM).
  • NGF nerve growth factor
  • TGF- ⁇ transforming growth factor- ⁇
  • EGF epidermal growth factor
  • vascular endothelial growth factor vascular
  • cytokines such as endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1), so that the cell functions in terms of cell adhesion, growth, migration and differentiation. By helping, it can be applied to many fields besides tissue regeneration.
  • Tissue engineering is a field that requires mutual cooperation between disciplines applied to the regeneration of biological substitutes for the purpose of restoring or preserving lost organ function using the principles of bioscience and engineering. Artificial organs can be developed and applied as a tissue engineering support for regenerating damaged tissue.
  • small intestinal submucosa The study of the small intestinal submucosa was initiated by Badylak at "Purdue University" in the United States, and is being actively researched by many leading pharmaceutical companies such as "COOK". As a result of this study, small intestinal submucosa is currently commercialized as a vein or artery, and a graft of the dermis, epithelium, bone, and graft regeneration of the bile duct and tightening the bladder of the incontinence patient. Therefore, many studies are expected as a small intestinal submucosa as a biocompatible material that can be variously applied as various bio substitutes.
  • the present inventors formed a crosslink by reacting with a crosslinking agent in the small intestinal submucosa, which hardly caused an immune reaction while studying biocompatible materials, and thus the mechanical properties were improved in proportion to the presence or absence of crosslinking and the crosslinking reaction time. This delay was confirmed and the present invention was completed.
  • the present invention is to provide a biocompatible small intestinal submucosa sheet capable of controlling the in vivo degradation period in which crosslinking is formed by reacting the small intestinal submucosa of animals other than humans with a crosslinking agent.
  • the present invention is to provide a method for producing a biocompatible small intestinal submucosa sheet capable of controlling the degradation period in vivo.
  • the present invention provides a biocompatible small intestinal submucosa sheet capable of controlling in vivo degradation period in which crosslinking is formed by reacting small intestinal submucosa of animals other than humans with a crosslinking agent.
  • the present invention also provides a method for producing a biocompatible small intestinal submucosa sheet capable of controlling the degradation period in vivo.
  • the biocompatible small intestinal submucosa sheet according to the present invention can delay the degradation period in vivo by forming a crosslink with a crosslinking agent, and can control the degradation period according to the type of crosslinking agent and the degree of crosslinking formation according to the crosslinking time. It can be used very usefully as a support for tissue engineering, drug carrier, wound dressing or hemostatic agent.
  • FIG. 1 is a view showing the chemical structure of the crosslinking agent that can be used as a crosslinking agent of the small intestinal submucosal tissue sheet of the present invention.
  • Figure 2 is a diagram showing the chemical structure of the crosslinking agent prepared by reacting with EDC-NHS after introducing a carboxyl group at the PEG terminal according to Example 2-1 of the present invention and its NMR spectrum.
  • Figure 3 is a diagram showing the chemical structure of the crosslinking agent prepared by introducing a carboxyl group in the terminal and / or side chain of the PCLA polymer according to Example 2-2 of the present invention and reacted with DCC and its NMR spectrum thereof.
  • Figure 4 is a diagram schematically showing the degradation behavior of the biocompatible small intestinal submucosa sheet through crosslinking of the present invention.
  • Example 5 is a view illustrating the degradation behavior of the biocompatible small intestinal submucosa sheet prepared in Example 1 of the present invention.
  • Figure 6 is a diagram showing the change in weight with time of the biocompatible small intestinal submucosa sheet prepared in Example 1 of each cross-linking agent.
  • Figure 7 is a diagram showing the decomposition behavior of the biocompatible small intestinal submucosa sheet prepared in Example 2 according to the crosslinking reaction time.
  • Example 8 is a view showing the weight change with time of the biocompatible small intestinal submucosa sheet prepared in Example 2 according to the type of crosslinking agent.
  • the present invention provides a biocompatible small intestinal submucosa sheet capable of controlling in vivo degradation period in which crosslinking is formed by reacting small intestinal submucosa of animals other than humans with a crosslinking agent.
  • the present invention also provides a method for producing a biocompatible small intestinal submucosa sheet capable of controlling the degradation period in vivo.
  • the present invention also provides a support for tissue engineering, drug carrier, wound dressing, and hemostatic agent including the small intestinal submucosa sheet.
  • the small intestinal submucosa sheet according to the present invention is characterized by separating small intestinal submucosa of animals other than humans, preferably mammals except humans, and reacting it with a crosslinking agent to form crosslinks.
  • the crosslinking agent that can be used in the present invention may be at least one selected from the group consisting of aldehyde compounds, water-soluble carbodiimide compounds, organic solvent soluble carbodiimide compounds, epoxy compounds and diisocyanates, more preferably water-soluble carbodiimide 1-ethyl-3- (3-dimethylaminopropylcarbodiimide), 1-ethyl-3- (2-morpholinyl-4-ethyl) carbodiimide as the compound, and dicyclohexyl as the organic solvent soluble carbodiimide compound
  • aldehyde-based compounds include, but are not limited to, compounds shown in FIG. 1 such as formaldehyde, glutaraldehyde, dextrin aldehyde, and the like.
  • the crosslinking agent that can be used in the present invention may be a biocompatible polymer having a carboxyl group at the terminal or side chain
  • the biocompatible polymer is polyethylene glycol (PEG, poly ethylene glycol), polycaprolactone (PCL, polycaprolactone), glycolide (GA, glycolide), lactide (LA, lactide), or one or more selected from the group consisting of copolymers (co-polymers), but is not limited thereto.
  • the biocompatible polymer having a carboxyl group at the terminal or side chain may be a crosslinking agent represented by the following Formula 1 or Formula 2.
  • n is an integer representing a repeating unit of polyethylene glycol, preferably an integer of 1 to 20.
  • n is an integer representing a repeating unit of polyethylene glycol, preferably an integer of 1 to 20
  • m represents an integer constituting the polyester and is an integer of 1 to 10.
  • the animal includes, but is not limited to, pigs, cows, rabbits, mice, and the like.
  • Small intestinal submucosa sheet according to the present invention can be used for various purposes as a sheet that can control the decomposition period by forming crosslinks using various crosslinking agents.
  • the small intestinal submucosa is rich in ECM (extracellular matrix) and cytokines to help the cell's functional aspects such as cell adhesion, growth, migration, differentiation, and wound healing in addition to the tissue engineering support.
  • ECM extracellular matrix
  • cytokines to help the cell's functional aspects such as cell adhesion, growth, migration, differentiation, and wound healing in addition to the tissue engineering support.
  • Including a drug in a dressing, a tissue sheet, or a drug carrier, a wound dressing, or a hemostatic agent may be applied to many fields.
  • Adipose tissue was removed using a pig's ileum within 4 hours after death. After washing the inside and outside of the plant cleanly with water, the plant was cut to a length of about 10 cm and placed in saline. A physical force was applied to the washed factory to remove the dense layer on the outer layer, and then reversed to remove the mucosal muscle layer, thereby separating only the small intestinal submucosa layer. It was washed again with brine and then stored in a -80 ° C low temperature cooler. One side of the small intestinal submucosa stored at ⁇ 80 ° C. was cut and unfolded, and then lyophilized to prepare a sheet-like small intestinal mucosa.
  • FIG. 1 A crosslinking agent capable of causing a crosslinking reaction to the small intestinal submucosa sheet is shown in FIG. 1.
  • EDC Aminopropyl carbodiimide
  • CDI carbonyldiimidazole
  • DCC dicyclohexylcarbodiimide
  • formalin formailn
  • toluene was used as a solvent, 1.3 times glutaric anhydride (Glutaric anhydride) was added to the hydroxyl group (OH) of polyethylene glycol, and the mixture was stirred for 24 hours under acetic acid as a catalyst.
  • Polyethylene glycol, 1-ethyl-3- (3-dimethylaminopropylcarbodiimide) (EDC) and N-hydroxysuccinimide (NHS) having a carboxyl group were dissolved in distilled water in a molar ratio of 1: 2: 2. After stirring for 2 hours, a crosslinking agent having biocompatibility and biodegradability represented by Chemical Formula 1 was prepared.
  • n is an integer representing a repeating unit of polyethylene glycol, preferably an integer of 1 to 20.
  • PCLA polyethylene glycol / poly (caprolactone-co-lactide)
  • toluene is used as a solvent, and 1.3 times glutaric anhydride is added to the hydroxyl group (OH) of polyethylene glycol / poly (caprolactone-co-lactide), and acetic acid is used as a catalyst. Stir for hours.
  • Polyethylene glycol / poly (caprolactone-co-lactide) (PCLA) and dicyclohexylcarbodiimide (DCC) having a carboxyl group were dissolved in distilled water in a molar ratio of 1: 1, followed by stirring for 2 hours.
  • a crosslinking agent having biocompatibility and biodegradation represented by 2 was prepared.
  • n is an integer representing a repeating unit of polyethylene glycol, preferably an integer of 1 to 20
  • m represents an integer constituting the polyester and is an integer of 1 to 10.
  • Example 1-1 To 20 ml of a phosphate buffer solution in which the biocompatible crosslinker prepared in Example 2-1 was dissolved at 50 mM concentration, the small intestinal submucosa sheet prepared in Example 1-1 was added and stirred at room temperature for 24 hours. Crosslinking reaction was performed.
  • N is an integer which represents the repeating unit of polyethylene glycol, Preferably it is an integer of 1-20.
  • n is an integer which shows the repeating unit of polyethyleneglycol, Preferably it is an integer of 1-20, m shows the segment which comprises polyester, and is an integer of 1-10.
  • the small intestinal submucosa prepared in Example 1 was cut into 2 ⁇ 2 cm 2 and placed in a 20 ml vial, followed by 5 ml of phosphate buffer solution added with collagenase (60 ⁇ g / ml), for 60 hours. Decomposition behavior was carried out at 37 °C, 1000 rpm environment for a while to determine the degree of decomposition.
  • FIG. 4 A schematic diagram of the degradation behavior experiment of the biocompatible small intestinal submucosa sheet of the present invention is shown in FIG. 4, and the results of the degradation behavior observation are shown in FIG. 5.
  • the cross-linked small intestinal submucosa sheet is not decomposed than the general small intestinal submucosa sheet was not cross-linked, it was confirmed that the shape is maintained excellently.
  • Crosslinking was performed for 24 hours (1 day) or 72 hours (3 days) using the biocompatible crosslinking agent prepared in Example 2, and then lyophilized to prepare a crosslinked small intestinal submucosa sheet.
  • Subcutaneous intestinal mucosa in sheet form was cut into 2 ⁇ 2 cm 2 and placed in a 20 ml vial. Then, 5 ml of phosphate buffer solution containing collagenase (60 ⁇ g / ml) was added. Decomposition behavior was carried out at to confirm the degree of decomposition morphologically.
  • the small intestinal submucosa sheet crosslinked with a biocompatible crosslinking agent was not degraded compared to the general small intestinal submucosa sheet without crosslinking, and the shape was maintained excellently.
  • the shape retention of the small intestinal submucosa sheet was superior to the case where the crosslinking was carried out for 24 hours compared to the case where the crosslinking was performed for 24 hours. Therefore, it can be seen that the longer the crosslinking reaction time, the better the physical properties of the small intestinal submucosa sheet.
  • the initial weight of the small intestinal submucosa sheet was not significantly different depending on the type of crosslinking agent. However, as shown in 2-1, the sheet was very well preserved.

Abstract

The present invention provides a biocompatible small intestinal submucosa sheet having adjustable in vivo degradation time in which the small intestinal submucosa of an animal reacts with a crosslinking agent to form a crosslinked bond. The present invention also provides a method for preparing the biocompatible small intestinal submucosa sheet. The biocompatible small intestinal submucosa sheet according to the present invention may delay in vivo degradation time by the crosslinked bond of the crosslinking agent, and may adjust the degradation time depending on the degree of the formation of the crosslinked bond based on the type of the crosslinking agent and crosslinking time. Therefore, the biocompatible small intestinal submucosa sheet according to the present invention can be significantly valuably used as a scaffold for tissue engineering, a drug carrier, wound dressing or an antihemorrhagic agent.

Description

생체 내 분해기간 조절이 가능한 생체적합성 소장점막하조직 시트, 및 이의 제조방법Biocompatible small intestinal submucosa sheet capable of controlling the degradation period in vivo, and a preparation method thereof
본 발명은 생체 내 분해기간 조절이 가능한 생체적합성 소장점막하조직 시트, 및 이의 제조방법에 관한 것이다.The present invention relates to a biocompatible small intestinal submucosa sheet capable of controlling the degradation period in vivo, and a method of manufacturing the same.
천연소재는 천연물질, 동물, 인체에서 유래한 물질로서 매우 우수한 생체적합성을 가지고 있다. 대표적인 일례로 세포외기질(extracellular matrix; ECM)을 들 수 있는데 이는 복잡한 인체 및 동물에서 추출된 생체재료로서 세포의 기능을 제어할 수 있는 특징이 있다. 천연소재로 제작된 지지체는 생체에 이식 후 염증반응이 적을 뿐 아니라, 뛰어난 생체 기능성 및 생분해성 등을 제공할 수 있어 이상적인 조직공학용 지지체의 재료로 평가된다.Natural materials are derived from natural materials, animals and the human body and have very good biocompatibility. A representative example is an extracellular matrix (ECM), which is a biomaterial extracted from a complex human body and an animal, and has characteristics that can control the function of cells. The support made of natural material is evaluated as an ideal tissue engineering support material because it can provide excellent bio-functionality and biodegradability as well as less inflammatory response after implantation into a living body.
이러한 천연소재 물질 중 돼지의 소장점막하조직은 세포가 존재하지 않는 조직으로 면역반응이 거의 일어나지 않으며, 구성성분의 90% 이상이 피부에 있는 제Ⅰ형 및 제Ⅱ형 콜라겐으로 구성되어 있고, 그 외에는 소량의 제Ⅴ형 및 제Ⅵ형 콜라겐으로 구성된다. 또한, 돼지의 소장점막하조직은 세포외기질(ECM)로서 글리코오스아미노글리칸 및 피브로넥틴, 콘드로이틴 설페이트, 헤파린, 헤파린 설페이트, 히아루론산과 염기성 섬유아세포 성장인자-2(base fibroblast growth factor - 2; FGF-2), 신경성장인자(nerve growth factor; NGF), 변환성장인자-β(transforming growth factor-β; TGF-β), 상피세포 성장인자(epidermal growth factor; EGF), 혈관 내피세포 성장인자(vascular endothelial growth factor; VEGF) 및 인슐린 성장인자(insulin-like growth factor-1; IGF-1) 등의 다양한 사이토카인을 다량 함유하고 있어 세포의 점착이나 성장, 이동, 분화 등의 세포의 기능적인 면에 도움을 줌으로써 조직 재생 이외에도 많은 분야에 응용될 수 있다.Among these natural materials, the small intestinal submucosa of pigs is a cell-free tissue and almost no immune response. More than 90% of the components are composed of type I and type II collagen in the skin. Consisting of small amounts of type V and VI collagen. In addition, the small intestinal submucosa of pigs is glycoaminoglycan and fibronectin, chondroitin sulfate, heparin, heparin sulfate, hyaluronic acid and basic fibroblast growth factor-2 (FGF-) as extracellular matrix (ECM). 2) nerve growth factor (NGF), transforming growth factor-β (TGF-β), epidermal growth factor (EGF), vascular endothelial growth factor (vascular) It contains a large amount of various cytokines such as endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1), so that the cell functions in terms of cell adhesion, growth, migration and differentiation. By helping, it can be applied to many fields besides tissue regeneration.
각종 질병으로 인한 장기의 손상을 치유하기 위해서는 때때로 골수 이식이나 심장, 신장, 안구 등의 장기 기증 등으로 치료를 받아야 하는 경우가 있지만, 수요에 비해 공급이 상당히 부족하기 때문에 조직공학을 이용한 인공장기의 개발이 필요하다. 이러한 조직공학은 생명과학과 공학의 원리를 이용하여 손실된 기관의 기능을 복원 혹은 보존을 목적으로 생물학적 대체물의 재생에 적용되는 학문간 상호 협조를 필요로 하는 분야로서, 상기 돼지의 소장점막하조직은 이러한 인공장기를 개발하여 손상된 조직을 재생하기 위한 조직공학적 지지체로 응용할 수 있다. In order to cure organ damage caused by various diseases, sometimes it is necessary to be treated by bone marrow transplantation or donation of organs such as heart, kidney, eyeball, etc. Need development Tissue engineering is a field that requires mutual cooperation between disciplines applied to the regeneration of biological substitutes for the purpose of restoring or preserving lost organ function using the principles of bioscience and engineering. Artificial organs can be developed and applied as a tissue engineering support for regenerating damaged tissue.
상기 소장점막하조직에 대한 연구는 미국의 "Purdue University"에 있는 Badylak에 의해 시작되었으며 "COOK"사 등 유수의 수많은 제약회사에서 활발하게 연구가 진행되고 있다. 이러한 연구 결과로 소장점막하조직은 정맥이나 동맥의 혈관 및 진피, 상피, 뼈 등의 이식물, 담즙관의 재생 및 요실금 환자의 방광을 조여 주는 이식물 등으로 현재 상용화되어있다. 따라서, 소장점막하조직은 여러 가지 생체 대체물로써 다양하게 응용가능한 생체적합성 재료로서 많은 연구가 기대되고 있다.The study of the small intestinal submucosa was initiated by Badylak at "Purdue University" in the United States, and is being actively researched by many leading pharmaceutical companies such as "COOK". As a result of this study, small intestinal submucosa is currently commercialized as a vein or artery, and a graft of the dermis, epithelium, bone, and graft regeneration of the bile duct and tightening the bladder of the incontinence patient. Therefore, many studies are expected as a small intestinal submucosa as a biocompatible material that can be variously applied as various bio substitutes.
그러나, 천연소재로서 높은 생체적합성을 지닌 돼지의 소장점막하조직은 짧은 분해기간 등, 물성이 취약하여 적용에 한계가 존재하기 때문에, 이의 물리적 또는 화학적 처리를 통하여 물성을 증진시키는 방법의 개발이 필요하다. 특히, 생체적합성있는 가교제 및 이를 이용하여 물성을 증진시키는 방법에 대해서는 전혀 알려져 있지 않으며, 이에 대한 연구도 전무한 상태이다. However, since the small intestinal submucosa of a pig with high biocompatibility as a natural material has a limitation in its application due to its weak physical properties such as a short decomposition period, it is necessary to develop a method of improving physical properties through physical or chemical treatment thereof. . In particular, there is no known biocompatible crosslinking agent and a method of improving physical properties using the same, and there is no research on this.
따라서, 소장점막하조직을 가교제와 반응시켜 가교결합을 형성함으로써, 생체 내 분해기간 조절이 가능한 생체적합성 소장점막하조직 시트 개발의 필요성이 절실히 요구되고 있다.Therefore, there is an urgent need for the development of a biocompatible small intestinal submucosa sheet capable of controlling the in vivo degradation period by forming a crosslink by reacting the small intestinal submucosa with a crosslinking agent.
본 발명자들은 생체적합성 재료에 관하여 연구하던 중 면역반응이 거의 일어나지 않는 소장점막하조직에 가교제와 반응시켜 가교결합을 형성한 결과, 가교결합의 유무 및 가교반응시간에 비례하여 기계적 물성이 향상되어 분해기간이 지연되는 것을 확인하고 본 발명을 완성하였다.The present inventors formed a crosslink by reacting with a crosslinking agent in the small intestinal submucosa, which hardly caused an immune reaction while studying biocompatible materials, and thus the mechanical properties were improved in proportion to the presence or absence of crosslinking and the crosslinking reaction time. This delay was confirmed and the present invention was completed.
따라서, 본 발명은 인간을 제외한 동물의 소장점막하조직을 가교제와 반응시켜 가교결합이 형성된 생체 내 분해기간이 조절가능한 생체적합성 소장점막하조직 시트를 제공하고자 한다. Accordingly, the present invention is to provide a biocompatible small intestinal submucosa sheet capable of controlling the in vivo degradation period in which crosslinking is formed by reacting the small intestinal submucosa of animals other than humans with a crosslinking agent.
또한, 본 발명은 상기 생체 내 분해기간이 조절가능한 생체적합성 소장점막하조직 시트의 제조방법을 제공하고자 한다.In addition, the present invention is to provide a method for producing a biocompatible small intestinal submucosa sheet capable of controlling the degradation period in vivo.
본 발명은 인간을 제외한 동물의 소장점막하조직을 가교제와 반응시켜 가교결합이 형성된 생체 내 분해기간이 조절가능한 생체적합성 소장점막하조직 시트를 제공한다. The present invention provides a biocompatible small intestinal submucosa sheet capable of controlling in vivo degradation period in which crosslinking is formed by reacting small intestinal submucosa of animals other than humans with a crosslinking agent.
또한, 본 발명은 상기 생체 내 분해기간이 조절가능한 생체적합성 소장점막하조직 시트의 제조방법을 제공한다.The present invention also provides a method for producing a biocompatible small intestinal submucosa sheet capable of controlling the degradation period in vivo.
본 발명에 따른 생체적합성 소장점막하조직 시트는 가교제와 가교결합을 형성함으로써 생체 내 분해기간을 지연시킬 수 있으며, 가교제의 종류 및 가교 시간에 따른 가교결합의 형성 정도에 따라 분해기간을 조절할 수 있으므로, 조직공학용 지지체, 약물전달체, 창상드레싱 또는 지혈제로서 매우 유용하게 사용할 수 있다.The biocompatible small intestinal submucosa sheet according to the present invention can delay the degradation period in vivo by forming a crosslink with a crosslinking agent, and can control the degradation period according to the type of crosslinking agent and the degree of crosslinking formation according to the crosslinking time. It can be used very usefully as a support for tissue engineering, drug carrier, wound dressing or hemostatic agent.
도 1은 본 발명의 소장점막하조직 시트의 가교제로 이용할 수 있는 가교제의 화학적 구조를 나타낸 도이다.1 is a view showing the chemical structure of the crosslinking agent that can be used as a crosslinking agent of the small intestinal submucosal tissue sheet of the present invention.
도 2는 본 발명의 실시예 2-1에 따른 PEG 말단에 카르복실기를 도입한 후 EDC-NHS와 반응시켜 제조한 가교제의 화학적 구조 및 이의 NMR 스펙트럼을 나타낸 도이다. Figure 2 is a diagram showing the chemical structure of the crosslinking agent prepared by reacting with EDC-NHS after introducing a carboxyl group at the PEG terminal according to Example 2-1 of the present invention and its NMR spectrum.
도 3은 본 발명의 실시예 2-2에 따른 PCLA 고분자의 말단 및/또는 측쇄에 카르복실기를 도입한 후 DCC와 반응시켜 제조한 가교제의 화학적 구조 및 이의 NMR 스펙트럼을 나타낸 도이다. Figure 3 is a diagram showing the chemical structure of the crosslinking agent prepared by introducing a carboxyl group in the terminal and / or side chain of the PCLA polymer according to Example 2-2 of the present invention and reacted with DCC and its NMR spectrum thereof.
도 4는 본 발명의 가교결합을 통한 생체적합성 소장점막하조직 시트의 분해거동 실험을 모식적으로 나타낸 도이다.Figure 4 is a diagram schematically showing the degradation behavior of the biocompatible small intestinal submucosa sheet through crosslinking of the present invention.
도 5는 본 발명의 실시예 1에서 제조한 생체적합성 소장점막하조직 시트의 분해거동을 관찰한 도이다.5 is a view illustrating the degradation behavior of the biocompatible small intestinal submucosa sheet prepared in Example 1 of the present invention.
도 6은 본 발명의 실시예 1에서 제조한 생체적합성 소장점막하조직 시트의 시간에 따른 무게의 변화를 가교제의 종류별로 나타낸 도이다. Figure 6 is a diagram showing the change in weight with time of the biocompatible small intestinal submucosa sheet prepared in Example 1 of each cross-linking agent.
도 7은 본 발명의 실시예 2에서 제조한 생체적합성 소장점막하조직 시트의 분해거동을 가교반응 시간에 따라 나타낸 도이다.Figure 7 is a diagram showing the decomposition behavior of the biocompatible small intestinal submucosa sheet prepared in Example 2 according to the crosslinking reaction time.
도 8은 본 발명의 실시예 2에서 제조한 생체적합성 소장점막하조직 시트의 시간에 따른 무게 변화를 가교제의 종류별로 나타낸 도이다.8 is a view showing the weight change with time of the biocompatible small intestinal submucosa sheet prepared in Example 2 according to the type of crosslinking agent.
본 발명은 인간을 제외한 동물의 소장점막하조직을 가교제와 반응시켜 가교결합이 형성된 생체 내 분해기간이 조절가능한 생체적합성 소장점막하조직 시트를 제공한다.  The present invention provides a biocompatible small intestinal submucosa sheet capable of controlling in vivo degradation period in which crosslinking is formed by reacting small intestinal submucosa of animals other than humans with a crosslinking agent.
또한, 본 발명은 상기 생체 내 분해기간이 조절가능한 생체적합성 소장점막하조직 시트의 제조방법을 제공한다.The present invention also provides a method for producing a biocompatible small intestinal submucosa sheet capable of controlling the degradation period in vivo.
또한, 본 발명은 상기 소장점막하조직 시트를 포함하는 조직공학용 지지체, 약물전달체, 창상드레싱, 및 지혈제를 제공한다.The present invention also provides a support for tissue engineering, drug carrier, wound dressing, and hemostatic agent including the small intestinal submucosa sheet.
이하 본 발명에 관하여 더욱 구체적으로 설명한다. Hereinafter, the present invention will be described in more detail.
본 발명에 따른 소장점막하조직 시트는 인간을 제외한 동물, 바람직하게는 인간을 제외한 포유류의 소장점막하조직을 분리하고, 이를 가교제와 반응시켜 가교결합이 형성된 것을 특징으로 한다. The small intestinal submucosa sheet according to the present invention is characterized by separating small intestinal submucosa of animals other than humans, preferably mammals except humans, and reacting it with a crosslinking agent to form crosslinks.
본 발명에서 사용될 수 있는 가교제로는 알데히드계 화합물, 수용성 카보이미드계 화합물, 유기용매 가용성 카보이미드계 화합물, 에폭시화합물 및 디이소시아네이트로 이루어진 군으로부터 선택된 1종 이상일 수 있고, 더욱 바람직하게는 수용성 카보이미드계 화합물로서 1-에틸-3-(3-디메틸 아미노프로필카보이미드), 1-에틸-3-(2-몰포리닐-4-에틸) 카보이미드, 유기용매 가용성 카보이미드계화합물로서 디시클로헥실카보이미드, 알데히드계 화합물로서 포름알데히드, 글루타르알데히드, 덱스트린 알데히드 등 도 1에 나타낸 화합물을 포함하나 이에 한정되지 않는다.The crosslinking agent that can be used in the present invention may be at least one selected from the group consisting of aldehyde compounds, water-soluble carbodiimide compounds, organic solvent soluble carbodiimide compounds, epoxy compounds and diisocyanates, more preferably water-soluble carbodiimide 1-ethyl-3- (3-dimethylaminopropylcarbodiimide), 1-ethyl-3- (2-morpholinyl-4-ethyl) carbodiimide as the compound, and dicyclohexyl as the organic solvent soluble carbodiimide compound Carbodiimide, aldehyde-based compounds include, but are not limited to, compounds shown in FIG. 1 such as formaldehyde, glutaraldehyde, dextrin aldehyde, and the like.
또한, 본 발명에서 사용될 수 있는 가교제는 말단 또는 측쇄에 카르복실기를 갖는 생체적합성 고분자일 수 있으며, 상기 생체적합성 고분자는 폴리에틸렌글리콜(PEG, poly ethylene glycol), 폴리카프로락톤(PCL, polycaprolactone), 글리콜라이드(GA, glycolide), 락타이드(LA, lactide), 또는 이들의 공중합체(co-polymer)들로 이루어진 군으로부터 선택된 1종 이상일 수 있으나, 이에 한정되지 않는다. 본 발명에서 상기 말단 또는 측쇄에 카르복실기를 갖는 생체적합성 고분자는 하기 화학식 1 또는 화학식 2로 표시되는 가교제일 수 있다.In addition, the crosslinking agent that can be used in the present invention may be a biocompatible polymer having a carboxyl group at the terminal or side chain, the biocompatible polymer is polyethylene glycol (PEG, poly ethylene glycol), polycaprolactone (PCL, polycaprolactone), glycolide (GA, glycolide), lactide (LA, lactide), or one or more selected from the group consisting of copolymers (co-polymers), but is not limited thereto. In the present invention, the biocompatible polymer having a carboxyl group at the terminal or side chain may be a crosslinking agent represented by the following Formula 1 or Formula 2.
[화학식 1][Formula 1]
Figure PCTKR2013008067-appb-I000001
Figure PCTKR2013008067-appb-I000001
상기 화학식 1에서, n은 폴리에틸렌글리콜의 반복단위를 나타내는 정수이며, 바람직하게는 1~20의 정수이다.In Formula 1, n is an integer representing a repeating unit of polyethylene glycol, preferably an integer of 1 to 20.
[화학식 2][Formula 2]
Figure PCTKR2013008067-appb-I000002
Figure PCTKR2013008067-appb-I000002
상기 화학식 2에서, n은 폴리에틸렌글리콜의 반복 단위를 나타내는 정수이며, 바람직하게는 1~20의 정수이고, m은 폴리에스터를 구성하는 부분(segment)을 나타내는 것으로 1~10의 정수이다.In the formula (2), n is an integer representing a repeating unit of polyethylene glycol, preferably an integer of 1 to 20, m represents an integer constituting the polyester and is an integer of 1 to 10.
상기 동물은 돼지, 소, 토끼, 마우스 등을 포함하나, 이에 한정되지 않는다. The animal includes, but is not limited to, pigs, cows, rabbits, mice, and the like.
본 발명에 따른 소장점막하조직 시트는 다양한 가교제를 이용하여 가교결합을 형성시켜 분해기간을 조절할 수 있는 시트로서 다양한 용도로 활용될 수 있다. 상기 소장점막하조직은 ECM(extracellular matrix) 및 사이토카인이 풍부하게 존재하여 세포의 점착이나 성장, 이동, 분화 등의 세포의 기능적인 면에 도움을 주며, 조직공학적인 조직재생용 지지체 이외에도 상처 치유를 위한 드레싱, 조직 시트에 약물을 포함시킬 경우 약물의 전달체, 창상용 드레싱, 또는 지혈제 등 많은 분야에 응용될 수 있다. Small intestinal submucosa sheet according to the present invention can be used for various purposes as a sheet that can control the decomposition period by forming crosslinks using various crosslinking agents. The small intestinal submucosa is rich in ECM (extracellular matrix) and cytokines to help the cell's functional aspects such as cell adhesion, growth, migration, differentiation, and wound healing in addition to the tissue engineering support. Including a drug in a dressing, a tissue sheet, or a drug carrier, a wound dressing, or a hemostatic agent may be applied to many fields.
이하 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나, 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred examples are provided to help understanding of the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.
실시예 1. 가교제를 이용한 소장점막하조직 시트의 제조Example 1 Preparation of Small Intestinal Submucosa Sheet Using Crosslinking Agent
1-1. 돼지에서의 소장점막하조직 분리, 보관 및 소장점막하조직 시트의 제조1-1. Isolation, Storage and Preparation of Small Intestinal Submucosa Sheets in Swine
사후 4시간 이내의 돼지의 공장(ileum)을 이용하여 지방조직을 제거하였다. 물로 깨끗이 공장 안과 밖을 세척한 다음, 공장을 대략 10 cm 정도의 길이로 잘라 식염수에 넣고 세척하였다. 세척한 공장에 물리적 힘을 가하여 바깥층에 있는 치밀층을 제거한 후, 다시 뒤집어 점막 근육층을 제거하여 소장점막하조직 층만을 분리하였다. 이를 다시 식염수로 세척한 다음, -80 ℃ 급저온 냉각기에 보관하였다. -80 ℃에서 보관된 소장점막하조직의 한쪽 면을 절단하고 펼쳐 고정시킨 후, 동결건조하여 시트상의 소장점막하조직을 제조하였다.Adipose tissue was removed using a pig's ileum within 4 hours after death. After washing the inside and outside of the plant cleanly with water, the plant was cut to a length of about 10 cm and placed in saline. A physical force was applied to the washed factory to remove the dense layer on the outer layer, and then reversed to remove the mucosal muscle layer, thereby separating only the small intestinal submucosa layer. It was washed again with brine and then stored in a -80 ° C low temperature cooler. One side of the small intestinal submucosa stored at −80 ° C. was cut and unfolded, and then lyophilized to prepare a sheet-like small intestinal mucosa.
1-2. 가교제를 이용한 소장점막하조직 시트의 가교결합 반응1-2. Crosslinking Reaction of Small Intestinal Submucosa Sheet Using Crosslinking Agent
소장점막하조직 시트에 가교결합 반응을 일으킬 수 있는 가교제는 도 1에 나타내었다. 도 1에 나타낸 가교제 중 N-하이드록시석신이미드/1-에틸-3-(3-디메틸아미노프로필카보이미드/디카르복실(NHS/EDC/dicarboxyl), 1-에틸-3-(3-디메틸아미노프로필카보이미드(EDC), 카보닐디이미다졸(CDI), 디시클로헥실카르보디이미드(DCC), 및 포르말린(formailn)을 선택하여 이를 50 mM의 농도로 용해시킨 인산완충용액 20 ml 에 상기 1-1 에서 제조된 소장점막하조직 시트를 넣고 24시간 동안 상온에서 교반하여 가교반응을 수행하였다.A crosslinking agent capable of causing a crosslinking reaction to the small intestinal submucosa sheet is shown in FIG. 1. N-hydroxysuccinimide / 1-ethyl-3- (3-dimethylaminopropylcarbodiimide / dicarboxylic (NHS / EDC / dicarboxyl), 1-ethyl-3- (3-dimethyl) in the crosslinking agent shown in FIG. Aminopropyl carbodiimide (EDC), carbonyldiimidazole (CDI), dicyclohexylcarbodiimide (DCC), and formalin (formailn) were selected and dissolved in a concentration of 50 mM, in 1 ml of 20 ml of phosphate buffer solution. The small intestinal submucosa sheet prepared in -1 was added and stirred at room temperature for 24 hours to carry out the crosslinking reaction.
실시예 2. 시트상의 소장점막하조직과 가교결합을 하는 생체적합성 가교제의 제조Example 2 Preparation of Biocompatible Crosslinking Agents Crosslinking with Sheet-like Small Intestinal Submucosa
2-1. 화학식 1의 생체적합성 가교제의 제조2-1. Preparation of Biocompatible Crosslinkers of Formula 1
폴리에틸렌글리콜(PEG)의 말단 또는 측쇄에 카르복실기를 도입하기 위하여 하기와 같은 실험을 수행하였다. In order to introduce a carboxyl group into the terminal or side chain of polyethylene glycol (PEG), the following experiment was performed.
구체적으로는 톨루엔을 용매로 하여, 폴리에틸렌글리콜의 히드록시기(OH) 대비 1.3배의 글루타르산 무수물(Glutaric anhydride)을 넣고 아세트산을 촉매로 하여 100℃ 하에서 24시간 동안 교반하였다. 제조된 카르복실기를 갖는 폴리에틸렌글리콜, 1-에틸-3-(3-디메틸아미노프로필카보이미드)(EDC) 및 N-하이드록시석신이미드(NHS)를 1:2:2의 몰비로 증류수에 녹인 후, 2시간 동안 교반하여 하기 화학식 1로 표시되는 생체적합성 및 생분해성을 가지는 가교제를 제조하였다. Specifically, toluene was used as a solvent, 1.3 times glutaric anhydride (Glutaric anhydride) was added to the hydroxyl group (OH) of polyethylene glycol, and the mixture was stirred for 24 hours under acetic acid as a catalyst. Polyethylene glycol, 1-ethyl-3- (3-dimethylaminopropylcarbodiimide) (EDC) and N-hydroxysuccinimide (NHS) having a carboxyl group were dissolved in distilled water in a molar ratio of 1: 2: 2. After stirring for 2 hours, a crosslinking agent having biocompatibility and biodegradability represented by Chemical Formula 1 was prepared.
[화학식 1][Formula 1]
Figure PCTKR2013008067-appb-I000003
Figure PCTKR2013008067-appb-I000003
상기 화학식 1에서, n은 폴리에틸렌글리콜의 반복단위를 나타내는 정수이며, 바람직하게는 1~20의 정수이다.In Formula 1, n is an integer representing a repeating unit of polyethylene glycol, preferably an integer of 1 to 20.
상기 제조된 화학식 1의 가교제의 NMR 분석 결과는 도 2에 나타내었다.The NMR analysis results of the prepared crosslinking agent of Formula 1 are shown in FIG. 2.
2-2. 화학식 2의 생체적합성 가교제의 제조2-2. Preparation of Biocompatible Crosslinkers of Formula 2
하이드로겔의 원료이자 생체적합성이 뛰어난 폴리에틸렌글리콜/폴리(카프로락톤-co-락타이드)(PCLA, polyethylene glycol/poly(caprolactone-co-lactide))의 말단 또는 측쇄에 카르복실기를 도입하기 위하여 하기와 같은 실험을 수행하였다.In order to introduce a carboxyl group into the terminal or side chain of a polyethylene glycol / poly (caprolactone-co-lactide) (PCLA), which is a raw material of hydrogel and excellent in biocompatibility, The experiment was performed.
구체적으로는 톨루엔을 용매로 하여, 폴리에틸렌글리콜/폴리(카프로락톤-co-락타이드)의 히드록시기(OH) 대비 1.3배의 글루타르산 무수물(Glutaric anhydride)을 넣고 아세트산을 촉매로 하여 100℃ 하에서 24시간 동안 교반하였다. 제조된 카르복실기를 갖는 폴리에틸렌글리콜/폴리(카프로락톤-co-락타이드)(PCLA) 및 디시클로헥실카르보디이미드(DCC)를 1:1의 몰비로 증류수에 녹인 후, 2시간 동안 교반하여 하기 화학식 2로 표시되는 생체적합성 및 생분해성을 가지는 가교제를 제조하였다. Specifically, toluene is used as a solvent, and 1.3 times glutaric anhydride is added to the hydroxyl group (OH) of polyethylene glycol / poly (caprolactone-co-lactide), and acetic acid is used as a catalyst. Stir for hours. Polyethylene glycol / poly (caprolactone-co-lactide) (PCLA) and dicyclohexylcarbodiimide (DCC) having a carboxyl group were dissolved in distilled water in a molar ratio of 1: 1, followed by stirring for 2 hours. A crosslinking agent having biocompatibility and biodegradation represented by 2 was prepared.
[화학식 2][Formula 2]
Figure PCTKR2013008067-appb-I000004
Figure PCTKR2013008067-appb-I000004
상기 화학식 2에서, n은 폴리에틸렌글리콜의 반복 단위를 나타내는 정수이며, 바람직하게는 1~20의 정수이고, m은 폴리에스터를 구성하는 부분(segment)을 나타내는 것으로 1~10의 정수이다.In the formula (2), n is an integer representing a repeating unit of polyethylene glycol, preferably an integer of 1 to 20, m represents an integer constituting the polyester and is an integer of 1 to 10.
상기 제조된 화학식 2의 가교제의 NMR 분석 결과는 도 3에 나타내었다.The NMR analysis results of the prepared crosslinking agent of Formula 2 are shown in FIG. 3.
실시예 3. 생체적합성 가교제에 의한 소장점막하조직의 가교실험Example 3. Crosslinking test of small intestinal submucosa by biocompatible crosslinking agent
실시예 2-1 에서 제조한 생체적합성 가교제를 50 mM 농도로 용해시킨 인산완충용액 20 ml에, 상기 실시예 1-1에서 제조된 소장점막하조직 시트를 넣고 24시간 동안 상온에서 교반하여 하기와 같은 가교반응을 수행하였다.To 20 ml of a phosphate buffer solution in which the biocompatible crosslinker prepared in Example 2-1 was dissolved at 50 mM concentration, the small intestinal submucosa sheet prepared in Example 1-1 was added and stirred at room temperature for 24 hours. Crosslinking reaction was performed.
Figure PCTKR2013008067-appb-I000005
Figure PCTKR2013008067-appb-I000005
상기 n은 폴리에틸렌글리콜의 반복단위를 나타내는 정수이며, 바람직하게는 1~20의 정수이다.N is an integer which represents the repeating unit of polyethylene glycol, Preferably it is an integer of 1-20.
또한, 상기와 같은 방법으로 실시예 2-2 에서 제조한 생체적합성 가교제를 이용하여 소장점막하조직 시트에 하기와 같은 가교반응을 수행하였다.In addition, the crosslinking reaction was performed on the small intestinal submucosal tissue sheet using the biocompatible crosslinking agent prepared in Example 2-2 in the same manner as described above.
Figure PCTKR2013008067-appb-I000006
Figure PCTKR2013008067-appb-I000006
상기 반응식에서, n은 폴리에틸렌글리콜의 반복 단위를 나타내는 정수이며, 바람직하게는 1~20의 정수이고, m은 폴리에스터를 구성하는 부분(segment)을 나타내는 것으로 1~10의 정수이다.In said reaction formula, n is an integer which shows the repeating unit of polyethyleneglycol, Preferably it is an integer of 1-20, m shows the segment which comprises polyester, and is an integer of 1-10.
실험예 1. 가교제를 이용하여 제조한 소장점막하조직 시트의 분해거동Experimental Example 1. Degradation Behavior of Small Intestinal Submucosa Sheets Prepared Using a Crosslinking Agent
1-1. 가교된 소장점막하조직 시트의 분해실험1-1. Degradation Test of Crosslinked Small Intestinal Submucosa Sheet
상기 실시예 1 에서 제조한 시트상의 소장점막하조직을 2×2 cm2 로 절단하여 20 ml 바이알에 넣은 다음, 콜라겐분해효소(60μg/ml)를 첨가한 인산 완충 용액을 5 ml씩 넣고, 60시간 동안 37 ℃, 1000 rpm 환경에서 분해거동을 실시하여 분해정도를 형태학적으로 확인하였다.The small intestinal submucosa prepared in Example 1 was cut into 2 × 2 cm 2 and placed in a 20 ml vial, followed by 5 ml of phosphate buffer solution added with collagenase (60 μg / ml), for 60 hours. Decomposition behavior was carried out at 37 ℃, 1000 rpm environment for a while to determine the degree of decomposition.
본 발명의 생체 적합성 소장점막하조직 시트의 분해 거동 실험의 모식도는 도 4에 나타내었으며, 분해 거동 관찰 결과는 도 5에 나타내었다.A schematic diagram of the degradation behavior experiment of the biocompatible small intestinal submucosa sheet of the present invention is shown in FIG. 4, and the results of the degradation behavior observation are shown in FIG. 5.
도 5에 나타낸 바와 같이, 가교된 소장점막하조직 시트는 가교가 되지 않은 일반 소장점막하조직 시트보다 분해되지 않고, 형태가 월등히 우수하게 유지됨을 확인하였다.As shown in Figure 5, the cross-linked small intestinal submucosa sheet is not decomposed than the general small intestinal submucosa sheet was not cross-linked, it was confirmed that the shape is maintained excellently.
1-2. 가교된 소장점막하조직 시트의 무게변화 측정1-2. Measurement of Weight Change of Crosslinked Small Intestinal Submucosa Sheets
상기 실험예 1-1의 실시 후 잔존하는 불순물 및 인산완충용액을 제거하고, 소장점막하조직 시트를 간단하게 세척한 다음, 동결건조하여 시간에 따른 무게의 변화로 분해거동을 수치화하였다. After the implementation of Experimental Example 1-1, residual impurities and phosphate buffer solution were removed, the small intestinal submucosa sheet was simply washed, and then lyophilized to quantify the degradation behavior by the change in weight over time.
결과는 도 6에 나타내었다.The results are shown in FIG.
도 6에 나타낸 바와 같이, 가교제의 종류에 따라 차이는 있으나, 가교를 실시한 경우에 소장점막하조직 시트의 형태 및 초기무게가 더 우수하게 유지되는 것을 확인하였다. As shown in Figure 6, there is a difference depending on the type of crosslinking agent, it was confirmed that the shape and initial weight of the small intestinal submucosa sheet is more excellent when crosslinking is carried out.
실험예 2. 생체적합성 가교제를 이용하여 제조한 소장점막하조직 시트의 분해거동Experimental Example 2 Degradation Behavior of Small Intestinal Submucosa Sheets Prepared Using a Biocompatible Crosslinker
2-1. 가교시간에 따른 소장점막하조직 시트의 분해실험2-1. Decomposition of Small Intestinal Submucosa Sheets with Crosslinking Time
상기 실시예 2에서 제조한 생체적합성 가교제를 이용하여 24시간(1 일) 또는 72시간(3 일) 동안 가교를 실시한 후, 동결 건조하여 가교결합된 소장점막하조직 시트를 제조하였다. 시트형태의 소장점막하조직을 2×2 cm2 로 절단하여 20ml 바이알에 넣은 다음, 콜라겐분해효소(60μg/ml)를 첨가한 인산 완충 용액을 5 ml씩 넣고, 60시간 동안 37 ℃, 1000 rpm 환경에서 분해거동을 실시하여 분해정도를 형태학적으로 확인하였다. Crosslinking was performed for 24 hours (1 day) or 72 hours (3 days) using the biocompatible crosslinking agent prepared in Example 2, and then lyophilized to prepare a crosslinked small intestinal submucosa sheet. Subcutaneous intestinal mucosa in sheet form was cut into 2 × 2 cm 2 and placed in a 20 ml vial. Then, 5 ml of phosphate buffer solution containing collagenase (60 μg / ml) was added. Decomposition behavior was carried out at to confirm the degree of decomposition morphologically.
결과는 도 7에 나타내었다.The results are shown in FIG.
도 7에 나타낸 바와 같이, 생체적합성 가교제로 가교된 소장점막하조직 시트는 가교가 되지 않은 일반 소장점막하조직 시트보다 분해되지 않고, 형태가 월등히 우수하게 유지되었다. 또한, 가교를 24시간 실시한 경우보다, 72시간 실시한 경우에서 소장점막하조직 시트의 형태유지가 우수하였다. 따라서, 가교반응시간이 길수록 소장점막하조직 시트의 물성이 우수해짐을 알 수 있다.As shown in FIG. 7, the small intestinal submucosa sheet crosslinked with a biocompatible crosslinking agent was not degraded compared to the general small intestinal submucosa sheet without crosslinking, and the shape was maintained excellently. In addition, the shape retention of the small intestinal submucosa sheet was superior to the case where the crosslinking was carried out for 24 hours compared to the case where the crosslinking was performed for 24 hours. Therefore, it can be seen that the longer the crosslinking reaction time, the better the physical properties of the small intestinal submucosa sheet.
2-2. 가교시간에 따른 소장점막하조직 시트의 무게변화 측정2-2. Measurement of Weight Changes of Small Intestinal Submucosa Sheets with Crosslinking Time
상기 실험예 2-1의 실시 후 잔존하는 불순물 및 인산완충용액을 제거하고, 소장점막하조직 시트를 간단하게 세척한 다음, 동결건조하여 시간에 따른 무게의 변화로 분해거동을 수치화하였다. After the implementation of Experimental Example 2-1, the remaining impurities and phosphate buffer solution were removed, the small intestinal submucosa sheet was simply washed, and then lyophilized to quantify the degradation behavior by the change in weight over time.
결과는 도 8에 나타내었다.The results are shown in FIG.
도 8에 나타낸 바와 같이, 가교제의 종류에 따라 소장점막하조직 시트의 초기무게는 큰 차이는 없었으나, 상기 2-1에서와 같이 시트의 형태는 매우 우수하게 보존되는 것을 확인하였다.As shown in FIG. 8, the initial weight of the small intestinal submucosa sheet was not significantly different depending on the type of crosslinking agent. However, as shown in 2-1, the sheet was very well preserved.

Claims (13)

  1. 인간을 제외한 동물의 소장점막하조직을 가교제와 반응시켜 가교결합이 형성된 생체 내 분해기간이 조절가능한 생체적합성 소장점막하조직 시트를 제조하는 방법. A method for producing a biocompatible small intestinal submucosa sheet capable of controlling the in vivo degradation period in which crosslinking is formed by reacting a small intestinal submucosa of an animal other than a human.
  2. 제1항에 있어서, 상기 가교제는 말단 또는 측쇄에 카르복실기를 갖는 생체적합성 고분자인 것을 특징으로 하는, 생체 내 분해기간이 조절가능한 생체적합성 소장점막하조직 시트를 제조하는 방법.The method of claim 1, wherein the crosslinking agent is a biocompatible polymer having a carboxyl group at the terminal or side chain thereof.
  3. 제2항에 있어서, 상기 생체적합성 고분자는 폴리에틸렌글리콜(PEG, poly ethylene glycol), 폴리카프로락톤(PCL, polycaprolactone), 글리콜라이드(GA, glycolide), 락타이드(LA, lactide), 및 이들의 공중합체(co-polymer)들로 이루어진 군으로부터 선택된 1종 이상인 것을 특징으로 하는, 생체 내 분해기간이 조절가능한 생체적합성 소장점막하조직 시트를 제조하는 방법. The method of claim 2, wherein the biocompatible polymer is polyethylene glycol (PEG, poly ethylene glycol), polycaprolactone (PCL, polycaprolactone), glycolide (GA, glycolide), lactide (LA, lactide), and their air A method for producing a biocompatible small intestinal submucosa sheet having a controlled in vivo degradation period, characterized in that at least one selected from the group consisting of co-polymers.
  4. 제2항에 있어서, 상기 생체적합성 고분자는 하기 화학식 1로 표시되는 가교제인 것을 특징으로 하는, 생체 내 분해기간이 조절가능한 생체적합성 소장점막하조직 시트를 제조하는 방법:The method of claim 2, wherein the biocompatible polymer is a crosslinking agent represented by Formula 1 below.
    [화학식 1][Formula 1]
    Figure PCTKR2013008067-appb-I000007
    Figure PCTKR2013008067-appb-I000007
    상기 n은 1~20의 정수이다.N is an integer of 1 to 20.
  5. 제2항에 있어서, 상기 생체적합성 고분자는 하기 화학식 2로 표시되는 가교제인 것을 특징으로 하는, 생체 내 분해기간이 조절가능한 생체적합성 소장점막하조직 시트를 제조하는 방법:The method of claim 2, wherein the biocompatible polymer is a crosslinking agent represented by Formula 2 below.
    [화학식 2][Formula 2]
    Figure PCTKR2013008067-appb-I000008
    Figure PCTKR2013008067-appb-I000008
    상기 화학식 2에서, n은 폴리에틸렌글리콜의 반복 단위를 나타내는 것으로 1~20의 정수이며, m은 폴리에스터를 구성하는 부분(segment)을 나타내는 것으로 1~10의 정수이다.In Formula 2, n represents a repeating unit of polyethylene glycol, and is an integer of 1 to 20, and m represents an integer constituting a polyester and is an integer of 1 to 10.
  6. 제1항에 있어서, 상기 가교제는 알데히드계 화합물, 수용성 카보이미드계 화합물, 유기용매 가용성 카보이미드계 화합물, 에폭시화합물 및 디이소시아네이트로 이루어진 군으로부터 선택된 1종 이상인 것을 특징으로 하는, 생체 내 분해기간이 조절가능한 생체적합성 소장점막하조직 시트를 제조하는 방법.According to claim 1, wherein the cross-linking agent is at least one member selected from the group consisting of aldehyde-based compound, water-soluble carbodiimide-based compound, organic solvent soluble carbodiimide-based compound, epoxy compound and diisocyanate, A method of making an adjustable biocompatible small intestinal submucosa sheet.
  7. 제6항에 있어서, 상기 가교제는 포름알데히드, 글루타르알데히드, 덱스트린알데히드, 1-에틸-3-(3-디메틸아미노프로필카보이미드), 디시클로헥실카보이미드, 또는 1-에틸-3-(2-몰포리닐-4-에틸)카보이미드인 것을 특징으로 하는, 생체 내 분해기간이 조절가능한 생체적합성 소장점막하조직 시트를 제조하는 방법.The method of claim 6, wherein the crosslinking agent is formaldehyde, glutaraldehyde, dextrinaldehyde, 1-ethyl-3- (3-dimethylaminopropylcarbodiimide), dicyclohexylcarbodiimide, or 1-ethyl-3- (2 A method for producing a biocompatible small intestinal submucosa sheet having a controlled in vivo degradation period, characterized in that it is -morpholinyl-4-ethyl) carbodiimide.
  8. 제1항에 있어서, 상기 동물은 돼지, 소, 토끼, 마우스로 이루어진 군으로부터 선택된 1종 이상인 것을 특징으로 하는, 생체 내 분해기간이 조절가능한 생체적합성 소장점막하조직 시트를 제조하는 방법.The method of claim 1, wherein the animal is one or more selected from the group consisting of pigs, cows, rabbits, and mice.
  9. 제1항 내지 제8항 중 어느 한 항의 방법에 의하여 제조된, 생체 내 분해기간이 조절가능한 생체적합성 소장점막하조직 시트.A biocompatible small intestinal submucosa sheet manufactured by the method of any one of claims 1 to 8, wherein the in vivo degradation period is adjustable.
  10. 제9항의 소장점막하조직 시트를 포함하는 조직공학용 지지체.A support for tissue engineering, comprising the submucosal tissue sheet of claim 9.
  11. 제9항의 소장점막하조직 시트를 포함하는 약물전달체.A drug delivery system comprising the submucosal tissue sheet of claim 9.
  12. 제9항의 소장점막하조직 시트를 포함하는 창상드레싱.A wound dressing comprising the small intestinal submucosa sheet of claim 9.
  13. 제9항의 소장점막하조직 시트를 포함하는 지혈제.A hemostatic agent comprising the submucosal tissue sheet of claim 9.
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