WO2011004936A1 - Procédé de production de membrane de cellulose bioactive à partir de la couche de peau cornée des ascidiacées, et membrane de cellulose bioactive ainsi obtenue - Google Patents

Procédé de production de membrane de cellulose bioactive à partir de la couche de peau cornée des ascidiacées, et membrane de cellulose bioactive ainsi obtenue Download PDF

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WO2011004936A1
WO2011004936A1 PCT/KR2009/005303 KR2009005303W WO2011004936A1 WO 2011004936 A1 WO2011004936 A1 WO 2011004936A1 KR 2009005303 W KR2009005303 W KR 2009005303W WO 2011004936 A1 WO2011004936 A1 WO 2011004936A1
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cellulose membrane
bioactive
membrane
present
regeneration
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PCT/KR2009/005303
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Korean (ko)
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이석근
김연숙
이상신
진덕희
이종호
김성민
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강릉원주대학교산학협력단
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Publication of WO2011004936A1 publication Critical patent/WO2011004936A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/717—Celluloses
    • 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
    • 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
    • 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
    • A61L27/3683—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
    • 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
    • A61L27/3804—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 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem 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
    • A61L27/50—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

Definitions

  • the present invention relates to a method for producing a bioactive cellulose membrane from the skin keratin of the sea squirt and a bioactive cellulose membrane obtained thereby.
  • Sea urchins belong to the lateral seagrasses of the seaweed rivers, which are native to the world's oceans. Among them, the sea urchins are not toxic, edible and mass-produced through aquaculture. However, in the sea squirt and midder, the meat of the inside is used for food, but the shell is usually thrown away. At this time, since the shell is hard and cannot be easily broken down, it is a cause of environmental pollution.
  • the ecology of the sea squirt is so unusual that the vertebrae have vertebrae and can swim and move, but adults do not move because they adhere to rocks or are buried in the soil at the bottom of the seabed, making structures that resemble the roots of plants and attaching them firmly.
  • In the body there are water inlets and water inlets, which feed seawater and filter out various organic matter and plankton.
  • the flesh has a vegetable flavor, and in particular, the keratin is made of beta-cellulose, similar to land plants.
  • cellulose which is the skin exfoliation of sea squirts and midges, has a very delicate fiber structure, which is a big difference from cellulose derived from general plants.
  • the cellulose membranes of sea squirts and meanders are joined in a form in which fine fibers of about 10 to 100 nm thickness are tightly woven together, and these fine fibers are tightly connected to each other by cellulose binding proteins. Therefore, the cellulose membrane which remove
  • the skin wound protection film and bone formation inducing membrane including the bruise or midderm-derived cellulose membranes proposed in Korean Patent Registration No. 10-0605382 are cellulose membranes obtained by repeated treatment of strong acid and strong alkali and simple protease treatment. Since the part of the cellulose adhesion protein that connects the fine fibers therein is not removed, the water permeability of the cellulose membrane is low, thereby weakening its function as a bioactive membrane in vivo, and in the case of inserting the cellulose membrane in vivo for a long time, The remaining proteins inside are free and can cause inflammatory reactions in the body. In addition, the cellulose membrane does not have affinity with substrate proteins or minerals in vivo, and does not have an activating function of cells or substrates in vivo, resulting in low cell adhesion and thus delayed biological tissue reactions. The function is poor.
  • the present invention improves the conventional method of manufacturing the sea bream and midderm-derived cellulose membrane to more effectively remove the proteins attached to the fine cellulose fibers present in the skin keratin of the sea squirt, and widen the gap of the fine cellulose fibers to permeate the water. It strongly increases the fine cellulose fibers to completely remove attached impurities, and has affinity with substrate proteins or minerals, and activates cells or substrates, and coats calcium hydroxide on the surface of fine cellulose to be used in vivo. It provides a method of producing a functional cellulose membrane that can help regeneration and activate the metabolism of cells and substrates in surrounding tissues.
  • bioactive cellulose membrane that is variously applied to the human body as a pure bioactive cellulose membrane separated from sea squirts, mainly antibacterial, wound protection, blood coagulation or wound healing promoting, fractures for wounds caused by wounds or burns, fractures Or bone grafts that promote healing of bone defects, or bone grafts, and nerve regeneration that repairs neurological disorders such as trauma or amputation, induces regeneration of damaged serosa, induces membrane regeneration, bone marrow cells, and membranes Bioactive cellulose membranes which can be effectively used for culturing and / or transplanting cells, embryonic stem cells or adult stem cells.
  • the permeability of water and the substrate is more effectively achieved by completely removing the proteins attached to the fine cellulose fibers present in the skin keratin of the sea squirt, and widening the gap between the fine cellulose fibers. It strongly increases the oxidation of fine cellulose fibers to completely remove attached impurities, and has affinity with substrate proteins or minerals, and activates cells or substrates. Bioactive cellulose membranes with affinity and activating function of cells or substrates can be prepared.
  • the surface of the fine cellulose is treated with calcium hydroxide after the oxidation treatment, so that the calcium hydroxide apatite nanoparticle microcrystals are coated on the surface to help bone regeneration when the coating material is used in vivo.
  • Free calcium ions can produce functional bioactive cellulose membranes that activate the metabolism of cells and substrates in surrounding tissue.
  • the bioactive cellulose membrane obtained by the method for producing the bioactive cellulose membrane of the present invention has an amino acid content of 0% by weight and a glucose content of 95% by weight or more, so that the protein is completely removed and the purity of glucose is very high. Safer and more versatile.
  • the cellulose of the sea bream is very dense and very low in water permeability, and the cellulose membrane prepared according to the conventional method using such cellulose is also poorly permeable to the substrate liquid in the substrate (according to the conventional method).
  • the bioactive cellulose membrane of the present invention had a problem of low bioactivity.
  • the water permeability is remarkably improved by continuously elongating (at least 0.3 ml of the viable cellulose membrane prepared according to the manufacturing method of the present invention), and most of the water exits the viable cellulose membrane within about 1 minute.
  • the permeability of the substrate material is also significantly improved, Adaptable in the living biological tissue cellulose film well without closing the cells and in use between the cell and the higher the bioactive by it can be applied more secure, in various human body.
  • bioactive cellulose membrane of the present invention can be used in cell culture because it can facilitate the nutrition and oxygen supply due to this significantly improved water and matrix material permeability.
  • the bioactive cellulose membrane of the present invention specifically, antibacterial to wounds caused by wounds or burns, wound protection, promoting blood coagulation or wound healing, bone formation induction or bone graft to promote healing of fractures or bone defects, and It can be effectively used for inducing nerve regeneration that restores nerve disorders caused by trauma or amputation, promoting regeneration of damaged membranes, inducing regeneration of membranes, culturing and / or transplanting bone marrow cells, membrane cells, embryonic stem cells or adult stem cells. .
  • 1 is a view showing the structure of the elastic stretching device of the present invention.
  • FIG. 2 is a view showing a method of using the elastic elongated autonomous and drying process for the cellulose membrane of the present invention.
  • Figure 3 shows the results of High Performane Anion-Exchange Chromatography analysis for the bioactive cellulose membrane of the present invention.
  • 4 to 7 is a view showing the results of observation of the healing effect when the marginal suture after suture of the dorsal skin of the white paper and the case of covering and sutured the bioactive cellulose membrane of the present invention.
  • FIGS. 8 and 9 are diagrams showing the degree of bone regeneration in the case of implanting the bioactive cellulose membrane of the present invention in the cranial permeability damage section of the white paper and the GORETEX membrane instead of the bioactive cellulose membrane of the present invention.
  • FIG. 10 is a view showing the results of observing the effect of transplanting each of the inner and outer surfaces of the bioactive cellulose membrane of the present invention toward the bone surface in the skull cranial permeable injury.
  • Fig. 11 shows the results of observing the bone formation action made by the bioactive cellulose of the present invention implanted into the white frontal cranial permeable bone defect site by electron microscopy.
  • FIG. 12 is a view showing the expression of BMP-2 after transplantation of the cellulose membrane of the present invention to the skull injury site.
  • Fig. 13 shows the structure of a cylindrical (cylindrical) cellulose membrane cell culture device of the present invention.
  • FIG. 14 is a view showing a stem cell culture method using the cylindrical cell culture apparatus of the present invention.
  • Fig. 15 is a view showing the results of transplantation-connection experiments of bioactive cellulose membranes of the present invention after sciatic nerve cutting of white paper.
  • Fig. 16 shows the results of transplanting the bioactive cellulose membrane of the present invention into the hepatic mesentery.
  • Figure 17 shows the experimental results for cell adhesion of the bioactive cellulose membrane of the present invention.
  • This invention is a manufacturing method of the bioactive cellulose membrane which repeats a strong acid and a strong base to the keratin of skin,
  • the present invention is the method according to the above 1, after the step (d).
  • an antibacterial substance on the inner surface of the cellulose membrane obtained in the step Agarose or silicone; Whiplin monomer and thrombin powder; Wound healing substances; Bone formation promoting substances; Nerve tissue forming substances; Curtain regeneration material; And a material selected from the group consisting of mixtures thereof, the antibacterial function; Wound protection; Blood coagulation; Promoting wound healing; Promoting bone formation; Induction of nerve regeneration; And forming a layer having at least one function selected from the group consisting of induction of regeneration of the membrane.
  • the present invention is the above 2,
  • the antimicrobial agent is selected from the group consisting of beta-defensin, lysozyme, lactoferrin, LL-37, histatin, mucocydine, ampicillin, kanamycin, tetracycline, gentamycin, teracin, sulfases and mixtures thereof.
  • the wound healing agents are ampicillin, kanamycin, teramycin, gentamycin, tetracycline, sulfaze, beta-defensin, histatin, lysozyme, lactoferrin, LL-37, mucocydine, recombinant vascular angiogenesis growth factor (VEGF), Collagen, elastin, laminin and mixtures thereof,
  • VEGF vascular angiogenesis growth factor
  • the bone formation promoting substance is a group consisting of recombinant BMPs (bone morphoganetic proteins), collagen, laminin, demineralized bone matrix proteins, osteonectin, osteocalcin, osteopontin, tricalcium phosphate, calcium hydroxide and mixtures thereof Is selected from,
  • the neural tissue-forming substance is selected from the group consisting of recombinant NGFs (nerve growth factors), collagen, laminin, neurotrophin and mixtures thereof,
  • the membrane regeneration material is selected from the group consisting of recombinant VEGF, collagen, laminin, growth hormone, fibroblast growth factors (FGFs), transforming growth factors (TGFs), estrogen, insulin and mixtures thereof.
  • the present invention is one of the above 1 to 3, wherein the bruises are cultured in a closed space to have enlarged cysts, and the cellulose membrane has a thickness of 50 to 100 ⁇ m and a size of 30 mm ⁇ 30
  • a method for producing a bioactive cellulose membrane which is characterized by a mm to 50 mm ⁇ 50 mm.
  • the present invention is to cultivate by attaching a cell selected from the group consisting of bone marrow cells, membranes, embryonic stem cells, adult stem cells to the inner surface of the bioactive cellulose membrane prepared by the method of 1 Provided is a method for culturing cells using a cellulose membrane.
  • the present invention provides a bioactive cellulose membrane prepared by the method of 1, wherein the amino acid content of the cellulose membrane is 0% by weight and the glucose content is 95% by weight or more. do.
  • the present invention provides a bioactive cellulose membrane according to the above 6, wherein the bioactive cellulose membrane is used as a membrane having a function selected from the group consisting of shielding bone regeneration induction, nerve regeneration induction, and shielding membrane regeneration induction. .
  • the present invention is characterized in that the antimicrobial substance on the inner surface of the bioactive cellulose membrane; Agarose or silicone; Whiplin monomer and thrombin powder; Wound healing substances; Bone formation promoting substances; Nerve tissue forming substances; Curtain regeneration material; And an antibacterial function formed by coating a material selected from the group consisting of a mixture thereof; Wound protection; Blood coagulation; Promoting wound healing; Promoting bone formation; Promoting nerve regeneration; And a layer having at least one function selected from the group consisting of promoting membrane regeneration.
  • the antimicrobial agent is selected from the group consisting of beta-defensin, lysozyme, lactoferrin, LL-37, histatin, mucocydine, ampicillin, kanamycin, tetracycline, gentamycin, teracin, sulfases and mixtures thereof.
  • the wound healing agents are ampicillin, kanamycin, teramycin, gentamycin, tetracycline, sulfaze, beta-defensin, histatin, lysozyme, lactoferrin, LL-37, mucocydine, recombinant vascular angiogenesis growth factor (VEGF), Collagen, elastin, laminin and mixtures thereof,
  • VEGF vascular angiogenesis growth factor
  • the bone formation promoting substance is a group consisting of recombinant BMPs (bone morphoganetic proteins), collagen, laminin, demineralized bone matrix proteins, osteonectin, osteocalcin, osteopontin, tricalcium phosphate, calcium hydroxide and mixtures thereof Is selected from,
  • the neural tissue-forming substance is selected from the group consisting of recombinant NGFs (nerve growth factors), collagen, laminin, neurotrophin and mixtures thereof,
  • the membrane regeneration material is selected from the group consisting of recombinant VEGF, collagen, laminin, growth hormone, fibroblast growth factors (FGFs), transforming growth factors (TGFs), estrogen, insulin and mixtures thereof.
  • bioactive cellulose membrane characterized in that.
  • bioactive cellulose membrane is both cultured, transplanted, cultured and transplanted of cells selected from the group consisting of bone marrow cells, membrane cells, bone marrow stem cells, embryonic stem cells, adult stem cells It provides a bioactive cellulose membrane, characterized in that used for.
  • the present invention relates to a method for producing a bioactive cellulose membrane which is repeatedly treated with strong acid and strong base to the skin keratin of sea urchin,
  • the sea urchin is mainly selected
  • a thin cellulose membrane is required, such as a bone regeneration inducing membrane, a nerve regeneration inducing membrane, and a retinal induction membrane
  • a midder is mainly selected.
  • sea squirt avoid the thick, coarse root areas of the skin, and choose a lateral area of the skin that is relatively soft and flat.
  • mid-deck it was advantageous to select a thin cyst area, which was large and sufficiently swollen.
  • the skin keratin of each sea urchin and mesothelioma should choose to have a clear and shiny color, except in some cases discolored or deformed by skin infection. Since the skin keratin of sea squirt and midder is sufficiently strong and firm in water, it does not cause deformation even if it accumulates and stores a large amount, but it is often contaminated by a large number of decaying bacteria.
  • the separation process is selected by selecting the smooth and flat areas of the skin of the sea urchins.
  • Waste skin impurities and various bacteria may be present in the skin keratin of the sea squirt, and sufficient washing is necessary first. It is first washed in running water to remove soluble impurities adhering to the surface, followed by washing for at least 1 hour in a detergent solution such as 5% oxyclean detergent solution.
  • keratin skin keratin Since the inner surface of keratin skin keratin is firmly attached to the muscles and the curtain, it is placed in a strong base such as 0.1M potassium hydroxide solution for about 1 day to remove most of the endothelial soft tissues including meat.
  • Skin keratin treated in paragraph 3) is treated with strong acid at 40-50 ° C., such as 1M hydrochloric acid solution, for about 6 hours with stirring, and after removing the hydrochloric acid solution, fresh acid at 40-50 ° C., for example 1M Treat in hydrochloric acid solution for about 6 hours.
  • strong hydrochloric acid treatment is repeated 5-10 times.
  • Toxic gases are generated during the treatment of strong hydrochloric acid, so they must be carried out in a chemical exhaust chamber, and the exchange of strong hydrochloric acid uses automatic suction and piping.
  • the protein and cationic impurities remaining in the keratin skin are removed.
  • the cellulosic membranes treated in 4) above were treated with a strong base such as 40-50 ° C. for about 6 hours in order to remove the agitated proteins and cationic impurities which are difficult to decompose in the remaining organic substances attached to the skin keratin of the sea urchin. Place in 1M sodium hydroxide solution and stir, remove the sodium hydroxide solution and treat again in fresh 40-50 ° C. strong base such as 1M sodium hydroxide solution for about 6 hours. This strong sodium hydroxide treatment is repeated 2-3 times. Toxic gases are also generated during the treatment of strong sodium hydroxide, so they must be carried out in a chemical exhaust chamber and the use of automatic suction and plumbing equipment for strong sodium hydroxide exchange.
  • the white cellulose membrane becomes whiter and the transparency of the cellulose membrane is slightly reduced, increasing the reflection of light on the surface of the cellulose membrane.
  • Cellulose membranes thinner by strong sodium hydroxide treatment can be damaged by agitation, thus reducing the agitation rate so that the cellulose membranes do not clump or bump together upon stirring.
  • Treatment with strong sodium hydroxide removes most of the hydrophobic proteins and anionic impurities that remain in the skin's keratin.
  • proteolytic enzymes are treated to remove proteins and impurities remaining in the cellulose membrane treated in 5).
  • the proteolytic enzyme for example, trypsin, pepsin or a mixture thereof and the like can be used, and an elastase, collagenase or a mixture thereof and the like can be further added thereto.
  • Specific examples of proteolytic enzyme treatment are as follows.
  • Cellulose membrane treated in the above 6) can be easily stretched as the cellulose membrane is easily removed as the adhesion protein for tightly connecting the fine cellulose membrane to each other, if excessively stretched by mistake, as shown in Figure 1 stator ( 4) and an elastic elongation apparatus capable of stretching the cellulose membrane 1 continuously and evenly by using a rubber band or a spring 2 which fixes the cellulose membrane 1 with the tongs 3.
  • the cellulose membrane treated in the above 7) is easily exposed to the inside of the cellulose by elongation, and again subjected to strong hydrochloric acid treatment to completely remove the remaining adherent protein inside the cellulose. That is, the cellulose membrane treated in 6) is treated for about 6 hours with stirring in 1M hydrochloric acid solution at 40-50 ° C., and after removing the hydrochloric acid solution, it is again treated with fresh 40-40 ° C. 1M hydrochloric acid solution for about 6 hours.
  • This strong hydrochloric acid treatment is repeated 2-3 times. Toxic gases are generated during the treatment of strong hydrochloric acid, so they must be carried out in a chemical exhaust chamber, and the exchange of strong hydrochloric acid uses automatic suction and piping.
  • the hydrochloric acid treated cellulose membrane is washed 2-3 times with distilled water and neutralized again with neutral PBS buffer solution.
  • the cellulose membrane treated in paragraph 8) was treated with sodium hypochlorite (NaClO), potassium persulfate (K 2 S 2 O 8 ), hydrogen peroxide (H 2 O 2 ), oxygen (O 2 ), dichlorine monoxide (Cl 2 O), It is oxidized by treatment with an oxidizing agent selected from the group consisting of ozone (O 3 ) and chlorine (Cl 2 ).
  • an oxidizing agent selected from the group consisting of ozone (O 3 ) and chlorine (Cl 2 ).
  • the cellulose membrane treated in the above 8) is treated with 0.1 M NaOCl solution for 12 hours to oxidize the surface of the cellulose membrane.
  • the membrane surface becomes in a negative charge state, and the reaction entropy of the membrane is increased, thereby facilitating the coating of calcium hydroxide and antimicrobial substances which will be performed later.
  • treatment of the surface of the cellulose membrane with the oxidizing agent according to the method of the present invention increases the reaction entropy of the surface, thereby facilitating their coating because chemically attaching calcium hydroxide and antibacterial substances to the surface of the cellulose.
  • nanoparticle microcrystals refers to crystals having a diameter of 1 to 100 nm.
  • Ca (OH) 2 calcium hydroxide (Ca (OH) 2 ) apatite nanoparticle microcrystals coated on the surface of cellulose help bone regeneration and especially the free calcium ions activate the metabolism of cells and substrates in surrounding tissues.
  • the surface of the cellulose membrane becomes a strong negative charge state due to the above-described process of 9), and the strong negative charge state of the oxide film has a problem that can be toxic in living tissues.
  • Treatment of the membrane with calcium hydroxide by the process of this paragraph neutralizes the strong negative charge state to some extent, resulting in a weak negative cellulose membrane, thereby eliminating the toxicity due to the strong negative charge state.
  • Toxicity removal of such an oxide film is also achieved by coating an oxidized treatment-treated cellulose film with an antimicrobial substance or the like described later.
  • the cellulose membrane treated in step 10) is washed 2-3 times with distilled water and then neutralized in neutral PBS buffer solution. At this time, the neutral PBS buffer solution was exchanged three to five times to prepare a bioactive cellulose membrane in which the cellulose membrane was sufficiently neutral.
  • the bioactive cellulose membrane made in 11 was re-mounted in the elastic stretching device of FIG. 1 used in item 7), and then lightly stretched, and then stretched in the stretching device for 3 to 6 hours immediately in the dryer state. To dry. Care should be taken because the dried bioactive cellulose is very thin and can easily fracture.
  • the dried cellulose membrane (cellulose membrane obtained through all the above processes) is pure cellulose saccharide and has a positive charge easily electrostatically, other impurities can be easily attached, so the inside of the dryer should be clean and the cellulose membrane should be dried in the dryer. Once stored, they should be stored in closed containers immediately. At this time, it is preferable to store in a vacuum dryer etc. if possible.
  • one of two methods can be used for disinfecting the bioactive cellulose membrane.
  • the bioactive cellulose membrane obtained by performing the above steps 1) to 12) is placed in a sealed container with the elastic stretching device in a closed container and sterilized in an autoclave, or the dried bioactive cellulose membrane is removed from the elastic stretching device. After cutting according to the intended use, it is a method of completely sterilizing and packaging by performing gas sterilization using ethylene oxide.
  • the bioactive cellulose membrane of the largest size can be obtained in a pure state.
  • a very wide variety of bioactive cellulose membranes can be prepared immediately. Since a small amount of ethylene oxide may remain in the bioactive cellulose membrane, it is necessary to allow sufficient time for the ethylene oxide to vaporize on its own when opening the closed container.
  • the second sterilization method uses a non-toxic carbon ink on the outer surface of the bioactive cellulose membrane before disinfection, so that the outer and inner surfaces of the cellulose membrane can be easily distinguished. And coating a biological material according to properties such as skin regeneration, bone regeneration, nerve regeneration, and regeneration of the membrane.
  • the packaging material during the disinfection process can easily determine the state of the contents using a material such as transparent vinyl on one side.
  • the soft skin keratin of the sea squirt and the upper part of the sea squirt are selected, first washed in running water, and then washed in a 5% oxyclean detergent solution for at least 1 hour, and then about 1 day. In 0.1M potassium hydroxide solution.
  • the skin keratin was treated in a 1M hydrochloric acid solution at 40-50 ° C. for about 6 hours with stirring, and the process of removing the hydrochloric acid solution was repeated five times, and placed in 1M sodium hydroxide solution at 40-50 ° C. for about 6 hours. The process of stirring and removing the sodium hydroxide solution was repeated 5 times to obtain a cellulose membrane.
  • the cellulose membrane was incubated with pepsin (100 units / mL) in 0.1 M sodium acetate solution (pH 2.0) at 50 ° C for 12 hours, washed with distilled water, neutralized with neutral buffer PBS solution, and then trypsin After 12 hours of incubation with 100 BAEE [N-benzoyl-L-arginine ethyl ester] units / mL), the cellulose membrane was stretched using the elastic stretching device of FIG.
  • the cellulose membrane was treated in a 1M hydrochloric acid solution at 40-50 ° C. for about 6 hours with stirring and the hydrochloric acid solution was removed three times, and the hydrochloric acid treated cellulose membrane was washed three times with distilled water and then neutral PBS buffer solution. Neutralized.
  • the cellulose membrane was treated with 0.1M NaOCl solution for 12 hours to oxidize the surface of the cellulose membrane, washed three times with distilled water, and then immediately precipitated in saturated calcium hydroxide solution for 24 hours to coat calcium hydroxide on the surface of the cellulose microfibers, followed by 3 After three washes, the cellulose membrane was sufficiently neutralized with three exchanges of neutral PBS buffer solution.
  • the cellulose membrane was re-mounted in the elastic stretching apparatus of FIG. 1 and lightly stretched, and immediately dried in the dryer in the stretched state in the stretching apparatus to obtain the cellulose membrane of the present invention.
  • the cellulose membrane obtained by the above-mentioned manufacturing method of the present invention has a large property of inducing biological activity in vivo, the cellulose membrane is called a bioactive cellulose membrane in the present invention.
  • the bioactive cellulose membrane of the present invention has a thickness of 30 to 150 ⁇ m and a size of 10 mm ⁇ 10 mm to 50 mm ⁇ 50 mm.
  • the bioactive cellulose membrane of the present invention has a thickness of 50 to 100 m and a size of 30 mm x 30 mm to 50 mm x 50 mm.
  • Elemental analysis was performed using Field Emission Scanning Electronmicscope (FESEM) to confirm the purity of the oxidized bioactive cellulose membrane of the present invention. As a result, only carbon and oxygen elements corresponding to sugar molecules were detected.
  • FESEM Field Emission Scanning Electronmicscope
  • HPAEC high performance anion-exchange chromatography
  • the mobile solution was maintained at 1.0 mL / min flow rate with 18 mM NaOH and searched using a pulsed amperometric detector (Electrochemical Detector, ED50, Dionex Co., USA).
  • a pulsed amperometric detector Electrochemical Detector, ED50, Dionex Co., USA.
  • the bioactive cellulose membrane obtained by the production method of the present invention was identified as pure ⁇ -1,4-glucose component (95 wt% or more) from which the substrate protein component of sea urchin was completely removed. .
  • cytotoxicity test was performed to confirm the in vivo stability of the bioactive cellulose membrane of the present invention obtained by sufficiently neutralizing with neutral PBS buffer solution.
  • NCTC clone 929 ATCC CLC-1 cells were cultured in a 5% CO 2 , 37 ° C. incubator for at least 24 hours and 80% monolayer formation was confirmed.
  • agar solution (1.5%) was layered, and the positive control groups 1 to 3, the negative control groups 1 to 3 and the experimental groups 1 to 3 were placed in the culture vessel.
  • Agar plates were incubated for 24 hours in a 5% CO 2 , 37 ° C. incubator, and then stained with 0.01% neutral red and observed under an optical microscope.
  • Reverse mutation assay was performed using Salmonella typhimurium according to the OECD guideline to confirm the genotoxicity of cells of the bioactive cellulose membrane of the present invention obtained by sufficiently neutralizing with neutral PBS buffer solution as described in section 0).
  • Experimental medium was prepared using an aqueous solution of the bioactive cellulose membrane eluted at 37 ⁇ 2 ° C. for 72 hours, and the negative control group containing no cellulose eluate in the test tube, the positive control group containing the cellulose eluent, and 0.5 to the experimental group, respectively.
  • S9 mixed solution 0.5 mL of freshly prepared S9 mixed solution was added thereto, and 0.1 mL of the appropriately cultured strain was added.
  • S9 mixed solution addition group only 0.1 mL of the strain except for the S9 mixed solution was added.
  • Each test tube was mixed well, poured into minimal glucose agar (plate) medium, shaken from side to side to solidify, incubated for 48 hours at 37 ° C, and counted colonies formed by reverse variation.
  • the bioactive cellulose membrane of the present invention can be used as a wound protective film for promoting antimicrobial disinfection, protection, and healing of wounds caused by wounds and burns, and inducing bone regeneration to promote damaged bone tissue, promoting bone formation, and bone graft.
  • the bioactive cellulose membrane of the present invention has the effect of promoting the healing in skin wounds of the living body, and after ulceration by forming a ulcer by removing 1 ⁇ 1 cm of skin from the dorsal skin of the white paper, As a result of comparing the treatment with the hemostatic method and the treatment with the bioactive cellulose membrane suture of the present invention, a very rapid healing effect was observed in the case of suture of the skin wound using the bioactive cellulose membrane of the present invention. (FIGS. 4-7).
  • bioactive cellulose membrane of the present invention when used as a protective film for skin wounds, it is possible to promote skin wound healing and to prevent scar generation.
  • bioactive cellulose membrane of the present invention When the bioactive cellulose membrane of the present invention is used for the purpose of protecting and healing wounds on the outer surface of the human body, antimicrobial substances, wound protecting agents such as agarose, blood coagulants such as whiplin and thrombin, and wound healing accelerators are as follows.
  • wound protecting agents such as agarose
  • blood coagulants such as whiplin and thrombin
  • wound healing accelerators are as follows.
  • biotransfer materials such as and the like may be coated on the inner surface of the cellulose film to form a layer containing the above materials.
  • Antimicrobial disinfectants required for wound healing include common chemical fungicides, representative examples of which are anionic or free-radical radicals, including alcohol, chlorhexidine and iodine solutions, most of which are toxic to wounds. It is advantageous to avoid the use of negative ionic agents.
  • bioantibacterial disinfectants which can be used instead of the above-mentioned general chemical disinfectants, consist of positively charged chemicals or short proteins that are easy to attach to negatively charged bacteria. Therefore, a special procedure is required to attach these amphoteric substances to the bioactive cellulose membrane.
  • bioactive cellulose membranes have a positive charge, and thus, bioactive antimicrobial substances having a positive charge are difficult to adhere to. Therefore, it is necessary to make the bioactive cellulose membrane in a negative charge state.
  • the cellulose membrane is subjected to a process of oxidizing the cellulose membrane so that the biomicrobial substance can be easily attached to the cellulose membrane.
  • the drying step of the method 12) of the method for producing the bioactive cellulose membrane from the sea urchin to explain the manufacturing method of the bioactive cellulose membrane of the present invention is as follows. Is deformed.
  • the bioactive cellulose membrane made in 11) is re-attached to the elastic stretching device of FIG. 1 used in 7) so that its inner surface faces upward, and then lightly stretched. 3 to 5 of the bioactive cellulose membrane evenly coated on the inner surface of the thin film, and dried on the dryer for at least 1 hour while attached to the stretcher, and then the same antimicrobial material is applied to the inner surface of the viable cellulose in the same manner and then dried in the dryer. The drying process is repeated about 2-3 times. In this way a large amount of antimicrobial material can be firmly coated on the bioactive cellulose membrane of the present invention.
  • the antimicrobial substance described above a wide variety of medicines can be used.
  • the main antimicrobial substances include beta-defensin, lysozyme, lactoferrin, LL-37, histotin, mucocydine, and human antimicrobial proteins such as ampicillin, kanamycin, Antibiotics such as tetracycline, gentamicin, teramycin and sulfa.
  • a soft aggression material In order to impart a wound-protection function, a soft aggression material, an agarose layer, silicone, or the like is formed on the inner surface of the bioactive cellulose membrane of the present invention and dried to produce a bioactive cellulose membrane having a wound protection function layer.
  • Agarose is non-toxic and flexible, so it can be applied directly to the wound, and it absorbs water even after drying and reduces it to gel agarose.
  • the bioactive cellulose membrane for wound protection of the present invention is prepared that can be coated and then dried for long term storage.
  • drying process of item 12) of the method for producing the bioactive cellulose membrane from the sea urchin which explains the method for producing the bioactive cellulose membrane of the present invention, is modified as follows.
  • the bioactive cellulose membrane made in 11 is re-mounted in the elastic stretching device of FIG. After cooling to 3-5 evenly dropped on the inner surface of the bioactive cellulose membrane and thinly coated, it is cooled to make a gel state, wherein when the thickness of the agarose gel on the bioactive cellulose membrane exceeds about 2 mm dried Since the agarose gel may drop off from the bioactive cellulose membrane, it is desirable to coat the thickness so that it does not exceed about 2 mm.
  • the bioactive cellulose membrane is dried in a 50 ° C. drier while installed in an elastic stretching device.
  • Cellulose membranes made by coating agarose and then drying are wetted by artificially sterilized water or saline, or bleeding, resulting in the original soft, elastic, invasive protective film as the dried agarose absorbs moisture.
  • these agaroses primarily compress the wound to prevent bleeding, reduce pain due to contact, and secondly to allow the bioactive cellulose membrane to be easily removed from the wound, thereby removing the wound protection from the wound. Prevent further damage to the site and facilitate later wound healing.
  • bioactive cellulose membranes are placed on a thin silicone membrane of about 0.5-1 mm.Since the air and water do not pass through, the silicone adheres to the skin wound and lives after 2-3 hours. It is desirable for the cellulose film to adhere to the wound and then to remove the outer silicone film. After removing the silicone membrane, the bioactive cellulose membrane dries easily, so it is better to cover the wound with gauze soaked with saline solution.
  • a layer having a blood coagulation function is formed on the inner surface of the cellulosic cellulose membrane of the present invention, thereby producing a bioactive cellulosic membrane having a blood coagulation function layer.
  • the blood coagulation mechanism in vivo is rapidly progressed by a complex chain reaction.
  • the Hageman factor (Factor XII) is activated by collagen or cell membranes with negative charges to initiate the blood coagulation mechanism.
  • Thrombin (Factor IIa) thus produced polymerizes fibronogen to form a cross-linking substance called fibrin, resulting in blood coagulation.
  • a thrombin powder membrane is formed thereon and then applied to the bleeding wound site, and the fibrin monomer and thrombin coated on the inner surface of the bioactive cellulose membrane
  • the powder causes a blood coagulation polymerization reaction, whereby blood can be coagulated early to obtain an effect of hemostasis.
  • drying process of item 12) of the method for producing the bioactive cellulose membrane from the sea urchin which explains the method for producing the bioactive cellulose membrane of the present invention, is modified as follows.
  • the bioactive cellulose membrane made in 11 is re-mounted to the elastic stretching device of FIG. After application, it is completely dried in a sterile dryer and then sprinkled with thrombin powder evenly. At this time, the diameter of thrombin powder particles is about 50-100 in size and the particles are separated by 200-300 by spraying.
  • the cellulose membrane evenly coated with thrombin powder on the dried whiblin monomer is vacuum packed immediately in a dry place to prevent moisture in the air.
  • bioactive cellulose membrane of this invention which coat
  • membrane in turn can be used extensively in a bleeding wound site
  • Bioactive cellulose membrane inner surface of the present invention for promoting wound healing by applying biomaterials such as antibiotics, antibacterial proteins, vascular or connective tissue growth factors to prevent secondary infection of wounds and promoting healing.
  • biomaterials such as antibiotics, antibacterial proteins, vascular or connective tissue growth factors to prevent secondary infection of wounds and promoting healing.
  • the biological substances include antibiotics such as ampicillin, kanamycin, theramycin, gentamycin, tetracycline, sulfa agent, antibacterial proteins such as beta-defensin, histatin, lysozyme, lactoferrin, LL-37, and mucocydine, And connective tissue growth factors such as recombinant vascular angiogenesis growth factor (VEGF), collagen, elastin, and laminin.
  • antibiotics such as ampicillin, kanamycin, theramycin, gentamycin, tetracycline, sulfa agent, antibacterial proteins such as beta-defensin, histatin, lysozyme, lactoferrin, LL-37, and mucocydine
  • connective tissue growth factors such as recombinant vascular angiogenesis growth factor (VEGF), collagen, elastin, and laminin.
  • VEGF vascular angiogenesis growth factor
  • the method for producing a bioactive cellulose membrane having a wound healing promoting function coated with such a biomaterial is the drying of item 12) in the method for producing the bioactive cellulose membrane from sea urchin, which describes the method for producing the bioactive cellulose membrane of the present invention.
  • the process consists of modifying as follows.
  • the bioactive cellulose membrane made in paragraph 11) is re-mounted in the elastic stretching device of FIG. 1 used in paragraph 7) so that its inner surface faces upward, and then lightly stretched, followed by ampicillin, kanamycin, teramycin, gentamycin, tetracycline, and sulfase.
  • Antibiotics such as beta-defensin, histatin, lysozyme, lactoferrin, antimicrobial proteins such as LL-37 and mucocydine, and connective tissues such as recombinant vascular angiogenesis growth factor (VEGF), collagen, elastin, and laminin.
  • VEGF vascular angiogenesis growth factor
  • An aqueous solution of at least one biomaterial selected from the growth factors (approximately 1% by weight) is evenly deposited on the inner surface of the bioactive cellulose membrane and thinly coated, followed by drying in a drier at about 50-70 ° C. with an elastic stretching device. Let's do it.
  • the bioactive cellulose membrane of the present invention purely separated from the sea urchin, has a bone regeneration-promoting effect.
  • the bioactive cellulose membrane of the present invention was formed after forming a permeable circular bone defect of 8 mm in diameter in the frontal skull of white paper. The bone regeneration effect was observed.
  • the circular permeable bone defect of 8 mm diameter in the frontal skull of white paper is suitable for observing the regeneration effect of the new bone by the bioactive cellulose membrane of the present invention since the damaged bone defect is a bone defect of a size that cannot be completely filled by natural healing. .
  • fibroblasts and collagen fibers were found on the living cellulose membrane of the present invention. They were directly and firmly attached, and the bone tissue was deposited immediately upon contact with the bioactive cellulose membrane of the present invention.
  • the bone regeneration phenomenon was observed through immunohistochemical staining using an antibody against BMP-2, a bone formation factor.
  • a bioactive cellulose membrane was implanted into the permeable bone defect of the frontal skull of the white paper to search for a BMP-2 immune response in the formation of new bone by the bioactive cellulose membrane of the present invention.
  • the positively bioactive cellulose membrane of the present invention rapidly absorbs and deposits the BMP-2 protein distributed in the bone matrix into the bioactive cellulose membrane of the present invention.
  • the bioactive cellulose membrane of the present invention can differentiate into adjacent fibroblasts into osteoblasts and promote bone regeneration.
  • the reason why the bone is deposited directly on the bioactive cellulose membrane of the present invention is probably due to the preferential deposition of bone forming factors or bone matrix proteins such as BMP-2. It is assumed to be.
  • the bioactive cellulose membrane obtained in the present invention is thought to be the cause of absorption and deposition of substrate proteins in the strong positive of ⁇ -1,4-glucose, showing the characteristics of the bioactive induction membrane in various biological reactions.
  • the bone regeneration induction membrane using the bioactive cellulose membrane of the present invention which is primarily useful as a shielding bone regeneration induction membrane.
  • This is a unique function of the inner surface of the bioactive cellulose membrane of the present invention, obtained from purely purified sea urchins, which in theory prevents the proliferation of fibroblasts from surrounding tissues and is inherent in bone defect sites. It is to use the property to increase bone proliferation.
  • the bioactive cellulose membrane of the present invention having a function as such a bone regeneration inducing membrane, it is essential that the pure cellulose membrane is completely purified and also maintain the tissue affinity or biomass transfer function of the cellulose membrane.
  • the bioactive cellulose membrane of the present invention can be made of a bioactive cellulose membrane formed by coating a bone formation promoting material on its inner surface and a bone formation promoting layer, and also on the inner surface of bone marrow cells (autologous bone marrow cells obtained from magnetic bone marrow)
  • bone marrow stem cells can be attached and used for culturing, transplanting cells, and for both culture and transplantation.
  • the bioactive cellulose membrane produced by the method of producing the bioactive cellulose membrane from sea squirts is pure cellulose derived from sea urchin (animal) keratin, and treated with oxide film and calcium Since it is coated, it can be used as a shielding bone regeneration induction membrane.
  • bioactive cellulose membrane of the present invention is cut into a suitable size for application to a bone defect site, then packaged and sterilized by polyethylene oxide gas sterilization, and then used in a living body.
  • the bioactive cellulose membrane of the present invention is cut into a size of about 15 ⁇ 15 mm, and at this time, since the bioactive cellulose membrane of the present invention is very thin, it flows or folds at the periosteal site. In order to prevent this, it is preferable to seal the periphery of the cellulose membrane to the peripheral periosteum. In such a case, it is preferable to cut and use the bioactive cellulose membrane of the present invention more sufficiently in consideration of sealing.
  • various kinds of bone formation promoting substances may be coated on the inner surface of the bioactive cellulose membrane of the present invention.
  • bone formation promoters include, for example, recombinant BMPs (bone morphoganetic proteins), collagen, laminin, demineralized bone matrix proteins, osteonectin, osteocalcin, osteopontin, tricalcium phosphate and calcium hydroxide. Etc.
  • the method for producing a bioactive cellulose membrane having a bone formation promoting function which is obtained by coating such a bone formation promoting substance, is one of the following methods for producing a bioactive cellulose membrane of the present invention.
  • the drying process of item) is made as follows.
  • the bioactive cellulose membrane prepared in paragraph 11) is reattached to the elastic stretching device of FIG. 1 used in paragraph 7) so that its inner surface faces upward, and then lightly stretched, followed by recombinant BMPs (bone morphoganetic proteins), collagen, laminin, and deliming.
  • BMPs bone morphoganetic proteins
  • An aqueous solution of at least one bone formation promoting substance selected from demineralized bone matrix proteins, tricalcium phosphate, and the like (approximately 1% by weight) is evenly dropped on the inner surface of the viable cellulose membrane with a dropper or evenly applied with a brush. After thin coating it is dried in a 50-70 ° C. drier, fitted in an elastic stretching device.
  • bone formation promoting substances In order to coat two or more bone formation promoting substances, they are mixed and coated. In addition, in order to coat the bone formation promoting substance sufficiently thick, for example, to coat it with a thickness of about 10 ⁇ m to 50 ⁇ m, preferably about 20 to 30, it is repeated 2-3 times to coat the bone formation promoting material. However, care should be taken when excessively thick coating of the bone formation promoting substance, for example, when the coating thickness is over 100 ⁇ m or more, the flexibility of the cellulose membrane may be reduced.
  • autologous bone marrow cells obtained from bone marrow, etc. By culturing autologous bone marrow cells obtained from bone marrow, etc., and attaching them to the inner surface of the bioactive cellulose membrane, they can be attached to the periosteum of the bone defect site, thereby obtaining more effective bone formation induction. Since the autologous bone marrow cells are easily adhered to the inner surface of the viable cellulose membrane, the above effects can be obtained.
  • Cell culture on the inner surface of the bioactive cellulose membrane of the present invention requires a cylindrical (or cylindrical) cellulose membrane cell culture apparatus as described in FIG. 13.
  • the cell culture device is composed of a circular (or square) cylinder having a diameter of about 10-50 mm, more specifically, an upper cylinder and a lower cylinder, depending on the size of the cellulose membrane. It is about 5-15 mm depending on the height of the culture petri dish, and its walls and bottom face are made with fine holes (e.g., about 0.5 mm in diameter) to facilitate the circulation of the culture solution, and are sufficient to prevent the culture cells from falling out of the container. Create a culture space.
  • the bioactive cellulose membrane of the present invention is positioned and attached to the bottom surface of the lower cylinder of the cylindrical cellulose membrane culture apparatus so that the inner surface thereof faces the inside of the cylindrical cell culture apparatus, whereby the cellulose membrane is compressed to facilitate de-adhesion of the cellulose membrane.
  • the cylindrical cellulose membrane cell culture apparatus uses a plastic material such as hard non-toxic polystyrene that does not cause deformation during autoclaving. In some cases, reuse a previously used one.
  • the cylindrical cellulose membrane cell culture apparatus is placed inside the petri dish, and the cells to be cultured are attached to the inner surface of the cellulose membrane attached to the cylindrical cell culture apparatus, wherein the cells to be cultured A sufficient amount of the culture solution is filled into a petri dish in which a cylindrical cellulose membrane cell culture apparatus is located. And it cultures using a normal incubator.
  • Cylindrical cellulose membrane cell culture apparatus having a diameter of about 10-50 mm is capable of culturing cells of about 1 ⁇ 10 6-1 ⁇ 10 8 , and the viable cellulose of the present invention in the cultured cells after the cell culture is finished. Cells attached to the inner surface of the membrane are optionally used.
  • the cylindrical (or cylindrical) cellulose membrane cell culture apparatus as shown in FIG. 13, not only a large amount of bone marrow cells can be attached to the viable cellulose membrane of the present invention but also a small amount of bone marrow stem cells (1 ⁇ 10 3 ⁇ ). 1 ⁇ 10 5 ) can be attached and cultured to the bioactive cellulose membrane to increase the number of cells.
  • the bioactive cellulose membrane of the present invention In order to attach a large amount of bone marrow cells to the bioactive cellulose membrane of the present invention, incubation in a 5% CO 2 incubator at 37 ° C. for 1-2 days results in sufficient cell attachment. In the cellulosic membrane cell culture apparatus, the bioactive cellulose membrane of the present invention, to which a large amount of bone marrow cells are sufficiently attached, is removed and used.
  • the cell culture surface When the bioactive cellulose membrane of the present invention is attached to the bone defect site, the cell culture surface must be positioned to face the bone surface.
  • the cells when a small amount of bone marrow stem cells are attached to the viable cellulose membrane of the present invention, the cells must be cultured for a relatively long period of time for two weeks or more. in a 37 ° C, 5% CO 2 incubator using growth media containing nutrients such as osteonectin, osteocalcin, osteoprotegerin, FGF, CTGF, and alkaline phosphatase. It is used after confirming the proper amount of bone marrow stem cell proliferation through microscopic observation while culturing at.
  • the bioactive cellulose membrane of the present invention having sufficient cell culture on the inner side is removed from the cylindrical cellulose membrane cell culture apparatus, and then implanted as it is, with the inner surface of the viable cellulose membrane to which cells are attached facing the bone surface.
  • the bioactive cellulose membrane of the present invention shows an adhesion proliferation effect by easily acting not only on the periosteal tissue but also on the surrounding tissue of the nerve bundle. Bioactive cellulose membrane can be performed.
  • the sciatic nerve of the white paper was sufficiently peeled, the sciatic nerve of about 1 cm was cut and made of a living cellulosic membrane band of a size similar to the length of the sciatic nerve cut and stitched to both ends of the cut sciatic nerve.
  • the state conducted an experiment.
  • the whole sciatic nerve was prepared at 8 weeks after implantation of the bioactive cellulose membrane of the present invention, and the microscopic specimen was observed.
  • the nerve tissue adhered to the inner surface of the bioactive cellulose membrane of the present invention. Phenomenon was observed.
  • These nerve tissues contained several bundles of nerve sheaths, and well-arranged nerve sheaths were clearly observed in Masson trichrome staining and aniline blue staining.
  • the neural tissues were thickly attached to the bioactive cellulose membrane of the present invention and proliferated. Similarly, the neural tissues were well differentiated in Masson trichrome staining and aniline blue staining (FIG. 15).
  • the neural tissues that are regenerated by acting friendly to the cleaved neural tissues rapidly adhere to the inner surface of the bioactive cellulose membranes of the present invention and proliferate along the bioactive cellulose membranes of the present invention. It is believed to induce the direction of regeneration and to promote the maturation of nerve bundles.
  • the bioactive cellulose membrane of the present invention is formed in the form of a band or a tube according to the degree of nerve damage. If the nerve damage is delayed and the nerve regeneration is delayed in the form of a narrow band to induce the growth of nerve sheaths. Each size and length is appropriately adjusted according to the type and location of the damaged nerve.
  • the inner surface of the bioactive cellulose membrane of the present invention serves to quickly attach the nerve membrane and guide the growth direction of the nerve sheath while helping the nerve bundle to stably grow.
  • the purely purified bioactive cellulose membrane of the present invention prepared by the method of producing the bioactive cellulose membrane in the sea urchin should be used.
  • the bioactive cellulose membrane of the present invention should have a form suitable for the anatomical structure as well as the nerve tissue regeneration mechanism.
  • the band-shaped cellulose membrane is about twice the thickness of the damaged nerve and is made 20-30% longer than the length of the damaged nerve.
  • the band-shaped cellulose membrane is mainly suitable for the regeneration of nerve bundles at the distal end.
  • the band-shaped cellulose membrane can be arbitrarily easily adjusted in length and size, and in some cases, even in the case of branched nerves, the band-shaped cellulose membrane can be easily manufactured according to its shape, and thus it is easy to use.
  • the band-shaped cellulose membrane is difficult to maintain its shape in the tissue during the procedure, the band-shaped cellulose membrane should be tightly sealed to the adjacent tissue of the nerve tissue.
  • the cellulose membrane in the form of a band is sufficiently durable and clearly distinguished from the biological tissue, the cellulose band can be accurately implanted in the damaged nerve part during the procedure, and the implanted cellulose band is flexible so that physical discomfort and discomfort do not occur. This is advantageous because the cellulose membrane does not have to be removed after the neural tissue is regenerated.
  • the cellulose membrane in the form of a tube is usually twice as large as the diameter of the damaged nerve bundle and is 20-30% longer than the length of the damaged nerve bundle.
  • the cellulose membrane is folded into a tube and sutured with a surgical thread (e.g. 5-0 silk). I can make it.
  • the cellulose membrane has a hole in the surface of the cellulose membrane with a diameter of 0.5-1 mm to allow the vascular tissue to enter the tube.
  • the cellulose membrane in the form of a tube is mainly used when a large bundle of nerves is damaged and both ends of the cellulose tube must be tightly sealed to the adjacent tissue of the bundle.
  • various types of nerve tissue formation promoting substances may be coated on the inner surface of the bioactive cellulose membrane of the present invention.
  • Neural tissue formation promoting substances include, for example, recombinant NGFs (nerve growth factors), collagen, laminin, neurotrophin and the like.
  • the method for producing a bioactive cellulose membrane having a neuronal regeneration-promoting function obtained by coating such a neural tissue formation promoting substance is one of the methods for producing the bioactive cellulose membrane from 1. It consists of modifying the drying process of 12) as follows.
  • the bioactive cellulose membrane made in paragraph 11) is re-mounted to the elastic extension device of FIG. 1 used in section 7) so that its inner surface faces upward, and then lightly stretched, followed by recombinant NGFs (nerve growth factors), collagen, laminin, and nerves.
  • NGFs nerve growth factors
  • An aqueous solution of at least one neurostimulation promoting substance selected from growth factors or the like is evenly dropped on the inner surface of the bioactive cellulose membrane with a dropper or evenly coated with a brush, and then placed in an elastic stretching device. Dry thoroughly in a 50-70 ° C dryer.
  • nerve regeneration-promoting substances In order to coat two or more nerve regeneration promoting substances, they are mixed and coated. In addition, in order to coat the nerve regeneration-promoting substance sufficiently thick, for example, about 10 to 50 ⁇ m, preferably about 20 to 30, the nerve regeneration-promoting substance is repeatedly coated 2-3 times. However, care should be taken when the nerve regeneration-promoting substance is excessively coated over 100 because the flexibility of the cellulose membrane may be reduced.
  • the purely purified bioactive cellulose membrane of the present invention was cut to a suitable size to sufficiently cover the site of liver resection and pressurized hemostasis for about 5-10 minutes until blood coagulated at the site of liver cutting. It was. After confirming that the bleeding had stopped, the abdominal cavity was closed and sutured, and the animals were stably bred after the treatment of shock and infection.
  • the liver of the white paper was isolated.
  • the bioactive cellulose membrane of the present invention was firmly attached to the surface of the liver, and the parenchyma of the liver was normally enlarged.
  • Microscopic sections were prepared from the liver of the extracted white paper, and microscopic observation showed that the bioactive cellulose membrane of the present invention was attached to the intestinal membrane of the liver tissue, and a new membrane between the bioactive cellulose membrane and the liver tissue of the present invention was detected at the liver cutting site. It was regenerated (FIG. 16).
  • bioactive cellulose membrane of the present invention acts kindly on damaged liver tissues and promotes the regeneration of the membranes of liver tissues.
  • the bioactive cellulose membrane produced by the method of producing bioactive cellulose membrane from sea squirt is a pure cellulose membrane derived from sea urchin (animal) skin keratin, and thus damage to the intestinal membrane. It is a membrane shield to fill the area, which can temporarily stabilize the organ and maintain the shape of the organ until a new one is regenerated.
  • bioactive cellulose membranes In the case of using a wide bioactive cellulose membrane according to the size of the organ damage, several bioactive cellulose membranes may be sealed and used.
  • the bioactive cellulose membrane of the present invention may be coated with a blood coagulant, whiplin and / or thrombin, or with an antimicrobial agent and / or an antibiotic.
  • the bioactive cellulose membrane of the present invention is slowly absorbed and removed in vivo, and it is not necessary to remove the bioactive cellulose membrane by secondary surgery mainly because it is extinguished by the phagocytosis of giant cells with a weak inflammatory response. However, when the pain occurs due to excessive foreign matter or when side effects are feared due to secondary infection or the like, it is preferable to remove the bioactive cellulose membrane.
  • bioactive cellulose membranes into the vein to provide a relatively long-term supply of biomaterials that promote the intestinal regeneration such as recombinant VEGF, collagen, laminin, growth hormone, fibroblast growth factors (FGFs), transforming growth factors (TGFs), estrogens, and insulin.
  • VEGF vascular endothelial growth factor
  • FGFs fibroblast growth factors
  • TGFs transforming growth factors
  • estrogens and insulin.
  • the coating of the biomaterial on the bioactive cellulose membrane of the present invention should be carried out in combination.
  • a general bioactive cellulose membrane is sufficient for a single coating, but in the case of producing a bioactive cellulose membrane having a membrane regeneration promoting function for the purpose of supplying a biological material having the above-mentioned membrane regeneration promoting function for a relatively long time, in order to perform several layers of coatings. It is good to repeat the process of first coating the adhesive material such as collagen or whiplin and then coating the biological material.
  • the method for producing a bioactive cellulose membrane having a membrane regeneration-promoting function which is obtained by coating a membrane regeneration-promoting substance, is described in 1.
  • Method of producing a bioactive cellulose membrane from sea urchins which describes the method for producing a bioactive cellulose membrane of the present invention.
  • the drying process of the term consists of modifying as follows.
  • the bioactive cellulose membrane made in paragraph 11) is re-mounted to the elastic stretching device of FIG. 1 used in paragraph 7) so that its inner surface faces upward, and then lightly stretched, and then coated with an adhesive substance such as collagen or hebrin first. Thereafter, the process of applying a thin film of dropping or evenly applying a thin drop of an aqueous solution of one or more membrane regeneration promoting substances (about 1% by weight) selected from recombinant VEGF, collagen, laminin, growth hormone, estrogen, insulin, etc. By repeating, drying in a 50-70 ° C. drier with the elastic stretching device installed thereon, a bioactive cellulose membrane having a membrane regeneration promoting function obtained by coating the membrane regeneration promoting substance is prepared.
  • the bioactive cellulose membrane of the present invention has a semipermeable membrane when a large amount of biomaterial is supplied for a long time, so that the two bioactive cellulose membranes face each other and are tightly sealed to make a bag, and the biomaterial is placed inside the bag. It is possible by implanting it into the vein after it is placed.
  • the cells to be implanted in the inner surface of the cellulosic cellulose membrane of the present invention, the liver stem cells or neural stem cells in the above example Incubate for 3 to 5 days in a sterile incubator at 37 ° C. 5% carbon dioxide for 3 to 5 days to sufficiently adhere to the bioactive cellulose membrane of the present invention, and then remove the bioactive cellulose membrane of the present invention to which the cells are attached and intact into each organ membrane. Transplant.
  • the cultured cells have excellent adhesion to each organ and loss of cells, so that most of the cells are transferred into the organs, proliferated and differentiated, and the inflammatory response following transplantation is observed to a slight extent.
  • the viable cellulose membrane of the present invention is moderately transparent for microscopic observation for cell culture, it can be confirmed by microscopic observation that the amount of cells to be transplanted to the organs during cell culture is attached to the viable cellulose membrane of the present invention.
  • the bioactive cellulose membrane of the present invention can be easily removed from the cylindrical cellulose membrane cell culture apparatus, it is possible to perform membrane transplantation without loss of cultured cells.
  • a binder such as fibrin glue may be used for preventing bleeding and for coagulation adhesion to prevent cell loss and to prevent excessive bleeding at the implantation site.
  • a cylindrical cellulose membrane cell culture apparatus is inserted into a culture medium of a culture equipment such as a bioreacter for continuous cell culture, or a cell culture of bioreacta is used. After replacing the bottom of the container with the bioactive cellulose membrane, the cell culture may be performed in the same manner, followed by removal of the bioactive cellulose membrane to perform membrane transplantation.
  • the bioactive cellulose membrane of the present invention has affinity with most cells, and in particular, experiments on stem cell adhesion were carried out using a cylindrical cellulose membrane cell culture apparatus to confirm affinity for undifferentiated cells such as stem cells. .
  • Stem cells of bone marrow were removed and stem cells were extracted and cultured using conventional petri dishes for cell culture (control) and cylindrical cellulose membrane cell culture device using the viable cellulose membrane of the present invention.
  • the cultures used in the experiment (test group) were compared with each other.
  • stem cells approximately 1 x 10 7
  • experimental and experimental groups were placed in the same culture area (approximately 3.14 cm 2 ) and observed after incubation in the same manner as described above in a sterile incubator at 37 ° C. 5% carbon dioxide. It was.
  • the number of stem cells attached to the plastic side of conventional petri dishes within 24 hours after incubation was about (1.25 ⁇ 0.187) ⁇ 10 4 / mm 2
  • the cylindrical cellulose membrane cells using the bioactive cellulose membrane of the present invention The number of stem cells attached to the viable cellulose membrane of the culture apparatus was (3.16 ⁇ 0.432) x 10 5 / mm 2, and the number of stem cells attached to the viable cellulose membrane of the present invention was much higher (FIG. 17).
  • bioactive cellulose membrane of the present invention acts kindly on undifferentiated stem cells and promotes stem cell proliferation and cell differentiation by increasing adhesion of stem cells.
  • stem cells obtained from adults are relatively well differentiated and have good biocompatibility, and thus, the living cellulose membrane of the present invention can be easily used.
  • stem cells obtained from embryos and umbilical cord blood are undifferentiated and therefore have less intercellular adhesion because the intercellular signal transduction system is not mature. It must be attached and properly metabolized to allow cell proliferation and differentiation. Therefore, in order to obtain stable proliferation and cell differentiation of these stem cells, it is preferable to attach the stem cells to the extracellular matrix and use them.
  • the use of a bioreactor for stem cell culture is appropriate.
  • the concentration, temperature, CO 2 concentration, pH, etc. of the culture medium should be automatically adjusted, and after one type of growth factor is administered for a certain period of time, it is rapidly changed to a new culture medium. If necessary, an automated device capable of administering other growth factors should be included.
  • the cylindrical cellulosic membrane cell culture apparatus of the present invention can be inserted into the internal cell culture apparatus of such a bioreactor, or the bioactive cellulose membrane of the present invention can be attached to the bottom of the cell incubator of the bioreactor.

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Abstract

La présente invention des procédés améliorant les procédés conventionnels utilisés pour la production d'une membrane cellulosique tirée des ascidies et des ascidies plissées. L'invention permet ainsi de supprimer complètement les protéines de liaison des fines fibres de cellulose sont présentes dans la couche cornée des ascidiacées. L'invention permet aussi d'augmenter plus efficacement la pénétrabilité par l'eau en élargissant les espaces entre les fines fibres de cellulose, d'oxyder fortement les fines fibres de cellulose, ce qui permet finalement de produire une membrane cellulosique bioactive pure. L'invention concerne également une membrane cellulosique bioactive extraite des ascidiacées et qui est utilisable de diverses façons pour le corps humain. L'invention concerne ainsi une membrane cellulosique bioactive qui s'utilisera efficacement lorsqu'on recherchera une action antibactérienne sur une blessure ou une cicatrice provoquée par brûlure ou analogue, la protection d'une lésion, la stimulation de la coagulation sanguine ou de la guérison d'une blessure, l'induction de la formulation osseuse ou de la transplantation osseuse qui stimule la guérison d'une fracture ou de défauts osseux, l'induction d'une régénération nerveuse permettant de récupérer de troubles nerveux provoqués par des lésions externes ou une coupure, l'induction de la régénération de la membrane séreuse qui stimule la régénération de membrane séreuse lésée, et la culture et/ou la transplantation de cellules de moelle osseuse, de cellules de membrane séreuse, de cellules souches embryonnaires, ou de cellules souches adultes.
PCT/KR2009/005303 2009-07-06 2009-09-17 Procédé de production de membrane de cellulose bioactive à partir de la couche de peau cornée des ascidiacées, et membrane de cellulose bioactive ainsi obtenue WO2011004936A1 (fr)

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KR1020090061248A KR101133644B1 (ko) 2009-07-06 2009-07-06 해양 멍게류 피부 각질로부터의 생활성 셀룰로오스 막의 제조 방법 및 이에 의하여 얻어지는 생활성 셀룰로오스 막
KR10-2009-0061248 2009-07-06

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CN115737911A (zh) * 2022-12-16 2023-03-07 华南理工大学 一种高强韧的骨修复复合材料及其制备方法
CN115844753A (zh) * 2022-12-15 2023-03-28 福建工程学院 一种海洋源全生物质面膜基材及其制备方法
CN116036362A (zh) * 2023-02-23 2023-05-02 山东大学 海鞘纳米纤维素/硫酸钙骨支撑材料及其制备方法
WO2023118966A1 (fr) * 2021-12-23 2023-06-29 New York University In Abu Dhabi Corporation Biomatériaux optimisés de diverses cellules et tissus à partir de sources durables

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KR101653141B1 (ko) * 2014-05-28 2016-09-01 주식회사 베네팜 비단멍게 유래 항균 펩타이드의 신규한 유사체 및 그 용도
KR101877703B1 (ko) * 2018-01-24 2018-07-12 충남대학교산학협력단 멍게 껍질 단백질 및 폴리락트산이 적층된 친환경 식품 포장재 제조 방법

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US20050115890A1 (en) * 2003-09-26 2005-06-02 Sartorius Ag Adsorption membranes, method of producing same and equipment, including the adsorption membranes
KR20050105660A (ko) * 2004-05-03 2005-11-08 이석근 멍게 또는 미더덕-유래 셀룰로오스 막을 포함하는 피부상처 보호막 및 골형성 유도막
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Cited By (6)

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Publication number Priority date Publication date Assignee Title
WO2023118966A1 (fr) * 2021-12-23 2023-06-29 New York University In Abu Dhabi Corporation Biomatériaux optimisés de diverses cellules et tissus à partir de sources durables
CN115844753A (zh) * 2022-12-15 2023-03-28 福建工程学院 一种海洋源全生物质面膜基材及其制备方法
CN115844753B (zh) * 2022-12-15 2024-04-30 福建工程学院 一种海洋源全生物质面膜基材及其制备方法
CN115737911A (zh) * 2022-12-16 2023-03-07 华南理工大学 一种高强韧的骨修复复合材料及其制备方法
CN116036362A (zh) * 2023-02-23 2023-05-02 山东大学 海鞘纳米纤维素/硫酸钙骨支撑材料及其制备方法
CN116036362B (zh) * 2023-02-23 2024-05-24 山东大学 海鞘纳米纤维素/硫酸钙骨支撑材料及其制备方法

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