WO2012081944A2 - Membrane dentaire et procédé de fabrication associé - Google Patents

Membrane dentaire et procédé de fabrication associé Download PDF

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Publication number
WO2012081944A2
WO2012081944A2 PCT/KR2011/009753 KR2011009753W WO2012081944A2 WO 2012081944 A2 WO2012081944 A2 WO 2012081944A2 KR 2011009753 W KR2011009753 W KR 2011009753W WO 2012081944 A2 WO2012081944 A2 WO 2012081944A2
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WIPO (PCT)
Prior art keywords
silk fibroin
membrane
aqueous solution
hexylresorcinol
silk
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PCT/KR2011/009753
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English (en)
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WO2012081944A3 (fr
Inventor
Hae Yong Kweon
You Young Jo
Kang Sun Ryu
Kwang-Gill LEE
Seok Woo Kang
Seong Gon Kim
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Republic Of Korea(Management : Rural Development Administration)
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Priority claimed from KR1020100129445A external-priority patent/KR101183961B1/ko
Priority claimed from KR1020110097751A external-priority patent/KR101403473B1/ko
Application filed by Republic Of Korea(Management : Rural Development Administration) filed Critical Republic Of Korea(Management : Rural Development Administration)
Publication of WO2012081944A2 publication Critical patent/WO2012081944A2/fr
Publication of WO2012081944A3 publication Critical patent/WO2012081944A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61C—DENTISTRY; APPARATUS OR METHODS FOR ORAL OR DENTAL HYGIENE
    • A61C8/00—Means to be fixed to the jaw-bone for consolidating natural teeth or for fixing dental prostheses thereon; Dental implants; Implanting tools
    • A61C8/0003—Not used, see subgroups
    • A61C8/0004—Consolidating natural teeth
    • A61C8/0006—Periodontal tissue or bone regeneration
    • 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
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols

Definitions

  • the present invention relates to a dental membrane and a method of manufacturing the same.
  • membranes made of a variety of materials have been developed.
  • membranes are not a bone graft material, but are a supplementary material that prevents the infiltration of fibroblasts or epithelial cells proliferating in the early stage upon while the bone defects are being treated. Such membranes prevent the defects from regenerating into soft tissue and aid the regeneration into bones.
  • Examples of materials usable for dental membranes include synthetic resins or collagens derived from a variety of animals.
  • the material for dental membranes which is currently prevalent and easily available, is collagen which is a material that is expensive but which decomposes in vivo and thus disappears. Hence, the collagen material is very advantageous because it does not require an additional operation to remove it.
  • Other natural materials for membranes include chitosan, and silk which is a protein derived from insects; however, the membranes of such materials are problematic because they have no antibacterial activities despite being used in the mouth having a variety of many commensal bacteria.
  • the membrane is very important in terms of blocking the migration of cells, which may happen given an appropriate biodegradation rate and a proper pore size. If the membrane contains components able to stimulate the activity of osteoblast in addition to blocking the migration of cells, it enables the formation of a larger amount of bone at a much faster rate.
  • the implant Only when the implant is applied in a state in which the alveolar bone has not been damaged, is it securely fixed. In the case of the alveolar bone having been damaged, it is covered with the membrane to induce the regeneration thereof and then operation may be carried out.
  • Korean Patent No. 10-0845002 discloses a biopolymer composed mainly of glucosamine and acetylglucosamine by chitin, chitosan or its derivative.
  • This prior art provides a biodegradable porous body having a dual pore structure including irregular isotropic pores on the surface thereof and regular anisotropic pores on the inner surface or back surface thereof.
  • Korean Patent No. 10-762928 discloses a membrane for guided bone tissue regeneration of osteoblasts, wherein a silk fibroin solution obtained by removing sericin from silk fibers is dialyzed, quickly frozen and then dried, and the dried silk fibroin is dissolved in an electrospinning solvent and then electrospun.
  • Korean Unexamined Patent Publication No. 2003-2224 discloses a membrane for guided tissue regeneration wherein a porous biodegradable polymer film having micropores is sandwiched between chitosan non-woven fabrics.
  • Korean Patent No. 10-889593 discloses a biodegradable hybrid composite having regular anisotropic pores, which comprises a biopolymer composed mainly of glucosamine and acetylglucosamine by chitin, chitosan or its derivative, silk fibroin derived from bombyx mori or wild silkworm, and a bioceramic having physiological activity.
  • Patent Literature 1 Korean Patent No. 10-0845002 entitled Biodegradable Porous Composite of Dual Porosity and Method for Preparing the Same
  • Patent Literature 2 Korean Patent No. 10-762928 entitled Nonwoven Nanofibrous Membranes of Silk Fibroin for Guided Bone Tissue Regeneration and Their Preparation Method
  • Patent Literature 3 Korean Unexamined Patent Publication No. 2003-2224 entitled Barrier Membrane for Guided Tissue Regeneration and the Preparation thereof
  • Patent Literature 4 Korean Patent No. 10-889593 entitled Hybrid Composite of Biopolymers and Bioceramics, and Method for Preparing the Same
  • an object of the present invention is to provide a dental membrane, which may have increased antibacterial activity and alkaline phosphatase activity in osteoblasts and may significantly improve the formation of bone at a bone defect and at a bone defect around an implant, and a method of manufacturing the same.
  • Another object of the present invention is to provide a dental membrane, which may adhere strongly to the damaged portion and superior bone tissue regeneration ability, with biodegradability in vivo, and a method of manufacturing the same.
  • An aspect of the present invention provides a dental membrane composition, comprising 0.01 ⁇ 10 wt% of 4-hexylresorcinol and 90 ⁇ 99.99 wt% of a silk protein.
  • Another aspect of the present invention provides a dental membrane, comprising 4-hexylresorcinol and a silk protein.
  • 4-hexylresorcinol may be contained in an amount of 0.01 ⁇ 10 wt% based on a total weight of the dental membrane.
  • the dental membrane may comprise 0.01 ⁇ 10 wt% of the 4-hexylresorcinol and 90 ⁇ 99.99 wt% of the silk protein.
  • the silk protein may be silk fibroin.
  • the dental membrane may further comprise, based on the total weight of the dental membrane, 30 wt% or less of one or more bioapplicable materials selected from among collagen, gelatin, chitin, chitosan, keratin, cellulose, fibronectin, elastin, fibrinogen, fibromodulin, laminin, tenascin, vitronectin, alginate, hyaluronic acid, silk protein and derivatives thereof, agarose, polylactic acid (PLA), polyglycolic acid (PGA), polylactic acid-polyglycolic acid copolymer (PLGA), polycaprolactone (PCL), poly ⁇ poly(ethyleneoxide)terephthalate-co-butyleneterephthalate ⁇ (PEOT/PBT), polyphosphoester (PPE), polyphosphazene (PPA), polyanhydride (PA), polyorthoester (POE ⁇ , pluronic 127, glycerin, poly(propylene fumarate)
  • a further aspect of the present invention provides a method of manufacturing a dental membrane, comprising (1) removing sericin from a silk protein, thus obtaining silk fibroin; (2) dissolving the silk fibroin in a salt-containing ethanol aqueous solution, thus preparing a silk fibroin salt ethanol aqueous solution; (3) removing the salt and the ethanol from the silk fibroin salt ethanol aqueous solution, thus preparing a silk fibroin aqueous solution; and (4) pouring the silk fibroin aqueous solution into a flat vessel and drying it.
  • a 4-hexylresorcinol solution may be further added to the silk fibroin aqueous solution.
  • the 4-hexylresorcinol solution may be prepared by dissolving 4-hexylresorcinol in ethanol.
  • the silk fibroin aqueous solution may contain 0.5 ⁇ 20 wt% of silk fibroin based on a total weight thereof.
  • the salt may be any one selected from among lithium bromide, lithium chloride, zinc chloride, calcium chloride, zinc nitrate, and calcium nitrate.
  • the silk fibroin aqueous solution may further comprise a bioapplicable material.
  • the silk fibroin aqueous solution may further comprise one or more selected from among a plasticizer, a softener, an antibiotic agent, an antiviral agent, an antibacterial agent, nucleic acid, a peptide and a protein.
  • the dental membrane can have excellent antibacterial activity and can exhibit superior alkaline phosphatase activity in osteoblasts.
  • the dental membrane can significantly improve the formation of bone at a bone defect.
  • the dental membrane can strongly adhere to the damaged portion and is convenient to use in operations.
  • the dental membrane has high biocompatibility and can regenerate bone tissue without causing an inflammatory reaction.
  • the dental membrane having biocompatibility in vivo obviates the need to remove it.
  • FIG. 1 shows a structure of chemical formula of 4-hexylresorcinol
  • FIG. 2 is a graph showing the alkaline phosphatase activity upon adding 4-hexylresorcinol to MG-63 cells;
  • FIG. 3 is a graph showing the expression level of osteocalcin upon adding 4-hexylresorcinol to MG-63 cells;
  • FIG. 4 shows the antibacterial activity of the dental membrane composition according to the present invention
  • FIG. 5 shows the degree of formation of new bone upon a lapse of 8 weeks at the bone defect around the implant
  • FIG. 6 is of micro-computed tomographic images showing the state of each group upon a lapse of 2 weeks in the cranial bone defect model of a rabbit;
  • FIG. 7 is a graph showing the bone volume based on FIG. 6, wherein control is a group using no membrane, commercials is a group using a commercially available membrane, and silk fibroin is a test group using the membrane including 4-hexylresorcinol and silk fibroin according to the present invention;
  • FIG. 8 shows the transparency of the membrane for guided bone tissue regeneration according to the present invention
  • FIG. 9 is a graph showing FT-IR spectroscopic results
  • FIG. 10A shows the dimensions of the membranes after a lapse of 24 hours following dissolving them in distilled water
  • FIG. 10B is a graph showing the concentration of protein dissolving into distilled water by measuring the solubility after 1 hour;
  • FIG. 10C is a graph showing the concentration of protein dissolving into distilled water by measuring the solubility after 24 hours;
  • FIG. 11A is a micro tomographic image showing the guided bone tissue regeneration ability at the 4 th week of animal testing
  • FIG. 11B is a micro tomographic image showing the guided bone regeneration ability at the 8 th week of animal testing
  • FIG. 12C is a histological image at a high magnification (magnification x 200) upon using the membrane for guided bone tissue regeneration at the 4 th week of in vivo testing to evaluate histological bone regeneration effects, wherein * indicates that the silk thin film may remain but is decomposed from the surface;
  • FIG. 13C is a histological image at a high magnification (magnification x 100) upon using the membrane for guided bone tissue regeneration at the 8 th week of in vivo testing to evaluate histological bone regeneration effects.
  • dental membrane means a membrane that promotes the recovery from bone defects formed by a variety of dental diseases.
  • the dental membrane includes 4-hexylresorcinol and a silk protein.
  • the IUPAC name of 4-hexylresorcinol is 4-hexylbenzene-1,3-diol, having a chemical structure as shown in FIG. 1.
  • the 4-hexylresorcinol may be internally applied in the form of having been added to olive oil in order to treat enteritis, or is recently commonly used to prevent the decolorization of brine shrimps.
  • the dental membrane according to the present invention may include 0.01 ⁇ 10 wt% of 4-hexylresorcinol based on the total weight of the dental membrane. If the amount of 4-hexylresorcinol is less than 0.01 wt% based on the total weight of the dental membrane, it is difficult to exhibit antibacterial activity. In contrast, if the amount of 4-hexylresorcinol exceeds 10 wt% based on the total weight of the dental membrane, effects resulting from increasing the amount thereof are insignificant, and dimensional stability of an agent is not ensured, making it impossible to maintain the dimensional stability of the dental membrane.
  • the dental membrane according to the present invention may include 0.01 ⁇ 10 wt% of 4-hexylresorcinol and 90 ⁇ 99.99 wt% of a silk protein.
  • the silk protein is a protein having the highest purity (97% or more) among many kinds of naturally existing proteins.
  • the silk protein is composed mainly of 75% of fibroin and 25% of sericin, and fibroin and sericin are kinds of polypeptides having a peptide bond resulting from dehydration condensation of amino acid.
  • the amount of the silk protein is less than 90 wt%, the formation of bone may be suppressed instead, and it is difficult to form a membrane. In contrast, if the amount of the silk protein exceeds 99.99 wt%, it is difficult to expect antibacterial effects and the flexibility of the membrane may decrease.
  • silk fibroin includes 18 kinds of amino acids which are present in almost the same amounts, wherein the amounts of glycine and alanine are much higher compared to the keratin protein of wool fiber.
  • Glycine and alanine of the fibroin protein are the components that constitute the greatest amounts among amino acids of collagen proteins which are the main proteins of the skin of the human body.
  • the fibroin protein having the amino acid structure similar to the skin of the human body has superior biocompatibility and no inflammatory reaction.
  • silk fibroin may be used as the silk protein in the present invention.
  • the dental membrane according to the present invention may include 4-hexylresorcinol and silk protein, it may further include a bioapplicable material which is a biocompatible tissue engineering material within the range that does not deteriorate the transparency and properties of the membrane.
  • the bioapplicable material may be added in an amount of 30 wt% or less based on the total weight of the dental membrane. If the amount of the biocompatible material exceeds 30 wt%, the transparency of the membrane may decrease undesirably.
  • the dental membrane according to the present invention may consist of silk fibroin.
  • the amino acid structure of silk fibroin is similar to that of the skin of the human body, thus exhibiting high biocompatibility and no inflammatory reaction.
  • This dental membrane may be manufactured from a procedure of obtaining silk fibroin by removing sericin from the silk protein.
  • the silk fibroin thus obtained is dissolved in an ethanol aqueous solution containing a salt, thus preparing a silk fibroin salt ethanol aqueous solution.
  • the silk fibroin salt ethanol aqueous solution may contain 0.5 ⁇ 20 wt% of silk fibroin based on the total weight of the aqueous solution. If the amount of silk fibroin is less than 0.5 wt%, it is difficult to form the membrane. In contrast, if the amount thereof exceeds 20 wt%, the flexibility of the membrane may decrease.
  • the salt may include any salt material, which includes any one selected from among lithium bromide, lithium chloride, zinc chloride, calcium chloride, zinc nitrate, and calcium nitrate.
  • the salt and ethanol are removed from the silk fibroin salt ethanol aqueous solution, thus preparing a silk fibroin aqueous solution.
  • the salt and ethanol are removed using a dialysis membrane.
  • the silk fibroin aqueous solution thus prepared is poured into a flat vessel and then dried, and thereby formed into the dental membrane according to the present invention.
  • the dental membrane thus manufactured may be composed exclusively of silk fibroin, but may further include a bioapplicable material that is a biocompatible tissue engineering material within the range that does not deteriorate the transparency and properties of the membrane.
  • the bioapplicable material may be added in an amount of 30 wt% based on the total weight of the dental membrane. In contrast, if the amount thereof exceeds 30 wt%, the transparency of the membrane may decrease.
  • transparent and transparency means not only the state of complete transparency but also the state of being transparent enough that the inside of the membrane can be observed.
  • the bioapplicable material may include any bioapplicable material, which may include one or more selected from among collagen, gelatin, chitin, chitosan, keratin, cellulose, fibronectin, elastin, fibrinogen, fibromodulin, laminin, tenascin, vitronectin, alginate, hyaluronic acid, silk protein and derivatives thereof, agarose, polylactic acid (PLA), polyglycolic acid (PGA), polylactic acid-polyglycolic acid copolymer (PLGA), polycaprolactone (PCL), poly ⁇ poly(ethyleneoxide)terephthalate-co-butyleneterephthalate ⁇ (PEOT/PBT), polyphosphoester (PPE), polyphosphazene (PPA), polyanhydride (PA), polyorthoester (POE), pluronic 127, glycerin, poly(propylene fumarate)-diacrylate (PPF-DA ⁇ and polyethylene glycol di
  • the dental membrane according to the present invention may further include one or more additives selected from among an antibiotic agent, antiviral agent, antibacterial agent, nucleic acid, a peptide, and a protein, within the range that does not negatively affect the structure and functions of the dental membrane.
  • the antibiotic, antiviral and antibacterial agents play a role in preventing infections of the membrane.
  • the protein may be any one selected from among a hormone, a cytokine, an enzyme, an antibody, a growth factor, a transfer control factor, vaccine, a structural protein, a ligand protein, an acceptor, a cell surface antigen and a receptor antagonist.
  • the dental membrane according to the present invention is applied to the surface of a bone defect and then an implant is applied thereto.
  • the implant means a replacement for a natural tooth that has been lost.
  • Such a dental membrane may exhibit excellent antibacterial effects and superior alkaline phosphatase activity in osteoblasts.
  • the dental membrane according to the present invention may significantly improve the formation of bone at a bone defect.
  • the dental membrane according to the present invention may adhere strongly to the damaged portion and may be convenient to use during an operation.
  • the dental membrane according to the present invention has high biocompatibility and thus enables bone tissue regeneration to take place without producing any inflammatory reaction.
  • the membrane which is biocompatible in vivo obviates the need to remove it.
  • Cocoons were used as a sample and, in order to remove sericin, the sample was scored with marseilles soap and a sodium carbonate solution at 100°C for 1 hour.
  • the silk fibroin produced by the scoring was dissolved at 80°C for 20 min using a calcium chloride-alcohol aqueous solution comprising calcium chloride, ethanol and water at a molar ratio of 1:2:8, placed in a dialysis membrane, and dialyzed with distilled water for 4 days to remove the salt and ethanol, thus preparing a silk fibroin aqueous solution.
  • 4-Hexylresorcinol was dissolved in ethanol, thus preparing a 4-hexylresorcinol-ethanol solution.
  • the 4-hexylresorcinol-ethanol solution was added to the silk fibroin aqueous solution and mixed together, thus preparing a dental membrane composition comprising 3 wt% of the 4-hexylresorcinol ethanol solution and a remainder of the silk fibroin aqueous solution based on the total weight thereof.
  • the dental membrane composition was poured into a flat polystyrene vessel and then dried at 60°C, thus manufacturing a dental membrane 1.
  • a dental membrane 2 was manufactured in the same manner as in Example 1, with the exception that the dental membrane composition comprising 5 wt% of the 4-hexylresorcinol ethanol solution and the remainder of the silk fibroin aqueous solution based on the total weight thereof was prepared, poured into a flat polystyrene vessel and then dried at 60°C.
  • Cocoons were used as a sample and, in order to remove sericin, the sample was scored with marseilles soap and a sodium carbonate solution at 100°C for 1 hour.
  • the silk fibroin produced by the scoring was dissolved at 80°C for 20 min using a calcium chloride-alcohol aqueous solution comprising calcium chloride, ethanol and water at a molar ratio of 1:2:8, placed in a dialysis membrane, and dialyzed with distilled water for 4 days to remove the salt and ethanol, thus preparing a silk fibroin aqueous solution.
  • 4-Hexylresorcinol was dissolved in ethanol, thus preparing a 4-hexylresorcinol-ethanol solution.
  • the 4-hexylresorcinol-ethanol solution was added to the silk fibroin aqueous solution and mixed together, thus preparing a dental membrane composition comprising 3 wt% of the 4-hexylresorcinol ethanol solution and the remainder of the silk fibroin aqueous solution based on the total weight thereof.
  • Cocoons were used as a sample and, in order to remove sericin, the sample was scored with marseilles soap and a sodium carbonate solution at 100°C for 1 hour.
  • the silk fibroin produced by the scoring was dissolved at 80°C for 20 min using a calcium chloride-alcohol aqueous solution comprising calcium chloride, ethanol and water at a molar ratio of 1:2:8, thus preparing a silk fibroin salt ethanol aqueous solution containing silk fibroin.
  • the silk fibroin salt ethanol aqueous solution was placed in a dialysis membrane and dialyzed with distilled water for four days to remove the salt and ethanol, thus preparing a silk fibroin aqueous solution.
  • the silk fibroin aqueous solution thus prepared was concentrated with PEG 20,000 (polyethylene glycol) as a bioapplicable material.
  • the concentrated silk fibroin aqueous solution containing 1.7 wt% of silk fibroin was poured into a flat polystyrene vessel and then dried at 60°C, thus manufacturing a dental membrane 4 (SM1).
  • a dental membrane 5 (SM2) was manufactured in the same manner as in Example 4, with the exception that the silk fibroin aqueous solution containing 3.5 wt% of silk fibroin was poured into a flat polystyrene vessel and dried at 37°C.
  • a dental membrane 6 (SM3) was manufactured in the same manner as in Example 4, with the exception that the silk fibroin aqueous solution containing 1.7 wt% of silk fibroin was poured into a flat polystyrene vessel and dried at 37°C.
  • MG63 cells ATCC, USA; osteoblast like cell
  • DMEM Dulbecco Modified Eagles Medium
  • PAA fetal bovine serum
  • 100X penicillin/streptomycin
  • the alkaline phosphatase activity was evaluated using a measurement kit by measuring the absorbance showing the degree of conversion of p-nitrophenylphosphate into p-nitrophenol depending on the activity of enzyme at 37°C and pH 10.2.
  • the expression level of osteocalcin was measured according to the protocol of the manufacturer at 72°C using an ELISA kit.
  • the alkaline phosphatase activity was measured for different concentrations of 4-hexylresorcinol on the MG-63-cultured dish.
  • the alkaline phosphatase activity could be seen to increase in proportion to the increase in the amount of added 4-hexylresorcinol up to 10 ⁇ g/ml.
  • the expression level of osteocalcin was measured for different concentrations of 4-hexylresorcinol on the MG-63-cultured dish.
  • the expression level of osteocalcin could be seen to increase in proportion to the increase in the amount of added 4-hexylresorcinol up to 10 ⁇ g/ml.
  • Staphylococcus aureus (ATCC 25923) was purchased from ATCC (American Type Culture Collection).
  • Porphyromonas gingivalis and Prevotella intermedia were cultured in an anaerobic brain heart infusion broth containing 1.0 g/ml Vit K1 and 5 g/ml Hemin under anaerobic conditions.
  • Staphylococcus aureus was cultured on an aerobic nutrient agar medium under aerobic conditions.
  • Respective bacteria were cultured and diluted with a sterile medium at 0.5 absorbance at 450 nm.
  • Staphylococcus aureus (ATCC25923) was spread on a muller hinton agar medium in the same manner as above.
  • a typical membrane and membrane compositions containing 4-hexylresorcinol at 0.1, 1, 10 ⁇ g/ml, 0.1, 1, 10 mg/ml were respectively placed on the surface of each plate per strain.
  • FIG. 4 shows the results of antibacterial test discs impregnated with testing bacteria being applied to the bacterial medium.
  • the use of 0.1 ⁇ 10 wt% of 4-hexylresorcinol based on the total weight of the dental membrane can result in antibacterial activity.
  • Test example 4 Test of bone regeneration at bone defect around implant
  • the tibia area was shaved and disinfected with povidine-iodine. Then, the periosteum was incised. The tibia area was exposed via subperiosteal dissection.
  • Two bone defects having a width of 3.0 mm and a length of 5.0 mm were formed at the unilateral cortical bone of the tibia area. The distance between the bone defects was 10.0 mm. Implants (diameter: 3.0 mm, length: 10.0 mm: IS3010WB, Neobiotech) were implanted at respective bone defects. The periosteum and the skin were ligated with 3-0 silk.
  • gentamicin Kookje Inc.
  • Respective rabbits were penned, and feed and water were supplied thereto. At the 8 th week of the test, ten rabbits were sacrificed and used in this test, and were dyed with a villanueva bone dyeing solution for 7 days.
  • the sections were ground to a thickness of 30 ⁇ m.
  • the digital image of the sections was taken using a digital camera.
  • the cranial bone was shaved and disinfected with povidine-iodine.
  • the periosteum was then incised.
  • the subperiosteal dissection was carried out to expose the parietal bone portion.
  • Two bone defects (diameter: 8.0 mm) were formed per rabbit. Twelve bone defects were obtained in six rabbits, and divided into three groups, comprising the first group as a control to which any material was not applied, the second group to which a commercially available product was applied, and the third group to which the membrane comprising 5% of 4-hexylresorninol and the remainder of silk fibroin (Example 2) according to the present invention was applied.
  • gentamicin Kookje Inc.
  • Respective rabbits were penned, and feed and water were supplied thereto.
  • these rabbits were sacrificed and analyzed using micro-computed tomography.
  • the membrane comprising 5% of 4-hexylresorninol and the remainder of silk fibroin (Example 2) according to the present invention exhibited faster bone regeneration than both the control that did not use any membrane and the group that used the commercially available membrane.
  • the micro-computed tomographic results showed that the volume of new bone was 8.90 ⁇ 1.51 mm3 in the control that did not use any membrane, and 23.27 ⁇ 5.80 mm3 in the membrane comprising 5% of 4-hexylresorninol and the remainder of silk fibroin (Example 2) according to the present invention.
  • the volume of new bone was 11.43 ⁇ 2.46 mm3, which was not statistically significantly different from the control (p>0.05).
  • the dental membrane according to the present invention can exhibit excellent antibacterial activity, superior alkaline phosphatase activity in osteoblasts and a significant improvement in bone regeneration at a bone defect.
  • Test Example 6 Evaluation of appearance of the dental membrane according to the invention using GRB
  • a typical GRB membrane composed of bovine collagen was opaque.
  • the dental membrane according to the present invention was transparent, which is clinically advantageous.
  • Test Example 7 FT-IR Measurement of the dental membrane according to the invention
  • FT-IR Fast Fourier transform infrared absorption spectrum was measured using a microscope equipped with a germanium ATR (attenuated total reflectance) object lens (ATR ⁇ 20) and an MCT detector mounted to a Fourier transform analyzer having a Ge-on-KBr beamsplitter.
  • the spectrum was recorded in the range of spectrum range of 600 ⁇ 3700 cm -1 at a resolution of 2 cm -1 , and each spectrum was repetitively scanned 32 times on average.
  • the spectrum observed for the dental membrane according to the present invention included a specific absorption band for an amide in the range of 600 ⁇ 1,700 cm -1 .
  • the bands of 1630 (amide I), 1530 (amide II), and 1265 cm -1 (amideIII) were in the form of ⁇ sheet.
  • the SF films (SM2 and SM3) represented by 1650 (amide I), 1530 (amide II) and 1235 cm -1 (amideIII) were observed to have the structure of a random coil and a ⁇ -sheet.
  • the morphology of the dental membrane according to the present invention can be estimated to affect the solubility of the membrane.
  • the solubility of the dental membrane according to the present invention was measured via Test Example 7.
  • a 5 mm ⁇ 5 mm sized dental membrane according to the present invention was used.
  • SM1, SM2, SM3 Three kinds of dental membranes (SM1, SM2, SM3) prepared in Examples 4, 5 and 6 according to the present invention were respectively immersed in distilled water.
  • SM3 As shown in FIG. 10A, almost all of SM3 was dissolved within 24 hours, SM1 was slightly dissolved and maintained its original tissue even after being immersed in distilled water for 1 hour.
  • SM1 was used in this test.
  • a typical anesthetic (a combination of 0.5 ml of xylazine and zolazepam and 0.5 ml of tiletamine) was intramuscularly injected to induce anesthesia.
  • the cranial bone area from the nasal bone to the occipital protuberance was shaved, and then disinfected with povidone-iodine.
  • the cranial bone was longitudinally incised, and the medium incision was carried out at the periosteum.
  • the top bone of the head was exposed from the outer table of the cranial bone.
  • the silk fibroin membrane was applied to the right defect of the cranial bone.
  • the left defect was allowed to be naturally treated without the use of a membrane.
  • the head skin and the skin were ligated with 3-0 silk.
  • the rabbits were administered three times with 1 mg/kg gentamicin and 0.5 ml/kg purine per day for 3 days.
  • Respective rabbits were penned, and feed and water were supplied thereto.
  • the rabbits were sacrificed in a humane manner, and samples were then obtained.
  • the prepared samples were subjected to micro tomography using Skyscan 1076.
  • the images were corrected, and the samples were scanned to a thickness of 0.035 mm.
  • the scanned images were reconstructed with CT-AN 1.10 software, and bone volume or BMD (bone mineral density) were measured.
  • the bone samples were dehydrated with ethanol and treated with 5% nitric acid for one week so that calcium was removed from the bone.
  • the right and left cranial bone samples were separated from each other from the symphysis.
  • paraffin blocks were manufactured, cut to a thickness of 5 ⁇ m and dyed with hematoxylin and eosin.
  • the dyed samples were taken using the digital camera of the microscope, and the degree of formation of new bone in the taken images was measured using an image analyzing program (Sigma Scan Pro).
  • the histological results were similar to the micro tomographic results.
  • FIG. 12A shows the histological results of the control at the 4 th week of in vivo testing, wherein the bone defects were still wide, and a large number of fibrous tissue was observed.
  • FIG. 12B shows the results for the group using the silk membrane, which had superior bone regeneration compared to FIG. 12A.
  • FIG. 12C shows the histological image of another test group at a high magnification wherein the formation of new bone was observed underneath the silk membrane and almost no inflammatory reaction was observed. This test group is confirmed to be very biocompatible.
  • FIG. 13A shows the histological results of the control at the 8 th week of in vivo testing, wherein the bone defects were remarkably decreased compared to FIG. 12A but were still wide, and a large number of fibrous tissue was observed.
  • the dental membrane according to the present invention can be applied to membranes for guided alveolar bone regeneration.

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  • Pharmacology & Pharmacy (AREA)
  • Dental Preparations (AREA)
  • Materials For Medical Uses (AREA)

Abstract

La présente invention concerne une membrane dentaire, qui comprend du 4-hexylrésorcinol et une protéine de soie et qui présente une excellente activité antibactérienne et une activité phosphatase alcaline supérieure dans les ostéoblastes, et qui peut considérablement améliorer la formation d'os au niveau d'un défaut osseux.
PCT/KR2011/009753 2010-12-16 2011-12-16 Membrane dentaire et procédé de fabrication associé WO2012081944A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR1020100129445A KR101183961B1 (ko) 2010-12-16 2010-12-16 실크단백질을 이용한 치주 골조직유도 재생용 차폐막 및 그 제조방법
KR10-2010-0129445 2010-12-16
KR10-2011-0097751 2011-09-27
KR1020110097751A KR101403473B1 (ko) 2011-09-27 2011-09-27 치과용 차폐막

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WO2012081944A2 true WO2012081944A2 (fr) 2012-06-21
WO2012081944A3 WO2012081944A3 (fr) 2012-10-11

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CN106421800A (zh) * 2016-09-28 2017-02-22 天津医科大学口腔医院 丝素蛋白改性凹坑结构乳酸基聚合物载药微球及制备方法
CN106535950A (zh) * 2014-08-04 2017-03-22 大韩民国（农村振兴厅长） 利用蚕茧的牙科再生膜及其制造方法
CN106794282A (zh) * 2014-11-05 2017-05-31 大韩民国（农村振兴厅长） 利用蚕茧的血管补片及其制造方法
CN111870740A (zh) * 2020-08-06 2020-11-03 苏州大学 一种复合蛋白膜及其制备方法

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CN106535950A (zh) * 2014-08-04 2017-03-22 大韩民国（农村振兴厅长） 利用蚕茧的牙科再生膜及其制造方法
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CN106794282A (zh) * 2014-11-05 2017-05-31 大韩民国（农村振兴厅长） 利用蚕茧的血管补片及其制造方法
CN106421800A (zh) * 2016-09-28 2017-02-22 天津医科大学口腔医院 丝素蛋白改性凹坑结构乳酸基聚合物载药微球及制备方法
CN111870740A (zh) * 2020-08-06 2020-11-03 苏州大学 一种复合蛋白膜及其制备方法
CN111870740B (zh) * 2020-08-06 2021-12-24 苏州大学 一种复合蛋白膜及其制备方法

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