WO2011155243A1 - Membrane for induction of regeneration of bone/tissue - Google Patents

Membrane for induction of regeneration of bone/tissue Download PDF

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Publication number
WO2011155243A1
WO2011155243A1 PCT/JP2011/056638 JP2011056638W WO2011155243A1 WO 2011155243 A1 WO2011155243 A1 WO 2011155243A1 JP 2011056638 W JP2011056638 W JP 2011056638W WO 2011155243 A1 WO2011155243 A1 WO 2011155243A1
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Prior art keywords
collagen
bone
membrane
layer
calcium phosphate
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PCT/JP2011/056638
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French (fr)
Japanese (ja)
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英郎 鵜沼
貴裕 川井
利武 古澤
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国立大学法人山形大学
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Priority to JP2012519294A priority Critical patent/JPWO2011155243A1/en
Publication of WO2011155243A1 publication Critical patent/WO2011155243A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/02—Inorganic materials
    • A61L27/12—Phosphorus-containing materials, e.g. apatite
    • 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
    • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/12—Materials or treatment for tissue regeneration for dental implants or prostheses

Definitions

  • the present invention relates to a bone / tissue regeneration-related material used in the field of dentistry or surgery, and more particularly to a membrane for inducing bone / tissue regeneration and a manufacturing method thereof.
  • Periodontal tissue regenerative treatment restores tissues and bones lost due to moderate or higher periodontal disease.
  • Periodontal disease progresses, bone around the teeth and tissues around the root of the periodontal ligament are destroyed.
  • the GTR method Guided Tissue Regeneration
  • the GBR method Guided Bone Regeneration
  • autologous cancellous or cortical bone, xenogeneic or homogenous bone grafts, hydroxyapatite or tricalcium phosphate granules, coral, natural polymers such as collagen, bone induction such as bone morphogenetic proteins Proteins are used.
  • non-absorbent membranes Two types of such membranes are known: non-absorbent membranes and absorbent membranes.
  • Known non-absorbable membranes include titanium mesh that is familiar with living bodies and easily deformed, and stretched polytetrafluoroethylene (ePTFE). This type of membrane must be removed by surgery after bone formation is complete.
  • ePTFE stretched polytetrafluoroethylene
  • As the absorbent membrane a copolymer of lactic acid and glycolic acid or collagen is used. Since this type of membrane is decomposed and absorbed in the living body after a certain period of time, it is difficult to control and confirm the status of tissue regeneration while eliminating the need for removal surgery.
  • Patent Document 1 two layers of a bioabsorbable collagen membrane and hydroxyapatite (hydroxyapatite) or a layer of concentrated platelet gel for promoting healing are also known, An absorptive three-layer film made of hydroxyapatite (hydroxyapatite), collagen, polylactic acid and the like as described in Non-Patent Document 1 is known.
  • Non-absorbable membranes are required to be removed smoothly from the affected area without causing unnecessary adhesion with surrounding tissues after bone regeneration is complete. It may penetrate and make it difficult to extract the titanium mesh after bone regeneration.
  • Two-layer membrane composed of hydroxyapatite (hydroxyapatite) and collagen described in Patent Document 1 or three-layer composed of hydroxyapatite (hydroxyapatite), collagen and polylactic acid described in Non-patent Document 1
  • the absorption in the body is faster than the bone formation, so that the invasion of the epithelium is allowed, and the alveolar bone cannot be regenerated to the initial membrane implantation position.
  • An object of the present invention is to provide a bone / tissue regeneration-inducing membrane that helps bone formation and is easily extracted after bone formation.
  • the bone / tissue regeneration-inducing membrane of the present invention comprises a polyester or polyamide hydrophilized film substrate, a collagen layer made of collagen or heat-denatured collagen provided on both sides or one side of the film, and a collagen layer on the collagen layer. And a covered calcium phosphate layer.
  • the method for producing a membrane for inducing bone / tissue regeneration includes a first immobilization step of immobilizing collagen or heat-denatured collagen on a hydrophilized film of polyester or polyamide to generate a collagen layer, and calcium phosphate on the collagen layer. Laminating step of placing the layer.
  • Polyester generally refers to a polycondensate of polycarboxylic acid (dicarboxylic acid) and polyalcohol (diol). It is basically made by reacting (dehydrating and condensing) a polyalcohol (a compound having a plurality of alcoholic groups —OH) and a polyvalent carboxylic acid (a compound having a plurality of carboxyl groups —COOH).
  • a polyalcohol a compound having a plurality of alcoholic groups —OH
  • a polyvalent carboxylic acid a compound having a plurality of carboxyl groups —COOH.
  • PET polyethylene terephthalate
  • PET polyethylene naphthalate
  • polybutylene terephthalate polytrimethylene terephthalate
  • polybutylene naphthalate etc.
  • polyamide such as nylon or aramid
  • Polyamide is a general term for polymers having an amide group in a monomer. This is because both polyester and polyamide can provide a carboxyl group necessary for immobilization with collagen by applying the hydrophilic treatment described below.
  • collagen or heat-denatured collagen there are types such as type I, type II or type III atelocollagen derived from bovine, pig or fish.
  • This collagen can be replaced by a polypeptide such as a collagen peptide, and in this application the term collagen is used in a broad sense encompassing such a polypeptide. It is known that this collagen plays a role of adhesion between the calcium phosphate layer and the polyester, and also helps the osteoblast to form an environment for proliferation.
  • Calcium phosphate is a substance composed of calcium ions and phosphate groups (PO 4 3 ⁇ ) or pyrophosphate groups (P 2 O 7 4 ⁇ ). May have hydrogen or hydroxy groups. About 70% of the bone is made of hydroxyapatite (hydroxyapatite) (Ca 5 (PO 4 ) 3 (OH)), which is a kind of calcium phosphate ceramics. The three hydrogen atoms (H) and calcium of phosphoric acid are each replaced, resulting in the following three different phosphates.
  • bone / tissue regeneration-inducing membrane of the present invention smooth growth of bone tissue can be expected and extraction after bone formation is easy.
  • the micro CT photograph of the rat skull defect part which compares the case where the membrane of one embodiment of the present invention and other membranes are used, and the case where a membrane is not used is shown.
  • the tissue section image of the membrane embedding part which compared the case where the membrane of one Embodiment of this invention and the other membrane is used is shown.
  • the membrane of this embodiment comprises (1) hydrophilicity of the surface of a polyester or polyamide film as a base material, (2) fixation of collagen to the film surface, and (3) to collagen immobilized on the film. Of urease and (4) precipitation of calcium phosphate on the surface of this collagen.
  • an alkaline aqueous solution such as sodium hydroxide
  • the surface of the polyester film is hydrolyzed, and hydroxy groups (—OH) and carboxyl groups (—COOH) appear on the surface.
  • the thickness of the polyester or polyamide film should be determined by considering factors such as the rigidity of the film itself, the size of the bone defect, the rigidity required to support stress from external tissues, and ease of handling. is there.
  • the hydrophilic treatment includes electron beam treatment, acid solution treatment, oxidizing agent treatment, hydrophilic functional group grafting treatment, silane coupling treatment, anodizing treatment, and roughening treatment. The method is known. In addition, film materials that have already been hydrophilized are on sale.
  • a cross-linking agent such as carbodiimide is dissolved in an aqueous solution in which collagen or heat-denatured collagen (gelatin or the like) is dispersed, and a hydrophilic film is immersed in the solution and held at a temperature of 20 to 50 ° C. for 6 to 72 hours.
  • the carboxyl group (—COOH group) on the polyester surface and the amino group (—NH 2 ) of collagen are dehydrated and condensed to form a peptide bond (—CONH—), thereby immobilizing the collagen on the polyester surface.
  • the collagen-fixed film is placed in an aqueous solution in which urease and carbodiimide are dissolved, and is held at a temperature of 20 to 50 ° C., preferably 30 to 40 ° C. for 6 to 72 hours.
  • urease and carbodiimide are dissolved in an aqueous solution in which urease and carbodiimide are dissolved, and is held at a temperature of 20 to 50 ° C., preferably 30 to 40 ° C. for 6 to 72 hours.
  • the carboxyl group or amino group on the surface of collagen and the amino group or carboxyl group of urease undergo dehydration condensation, and urease is immobilized on the collagen surface.
  • calcium phosphate is deposited on the surface of collagen to which this urease is fixed. That is, in an aqueous solution containing 1 to 100 mmol / L of calcium ions, 1 to 100 mmol / L of phosphate ions, and 10 to 100 mmol / L of urea.
  • the urease-immobilized film prepared in the previous step is immersed and held at a temperature of 20 to 50 ° C. for 15 minutes to 24 hours.
  • urea is converted into ammonia by the action of urease, and ammonia selectively precipitates calcium phosphate on the surface of the film.
  • a membrane having a three-layer structure of a film substrate and calcium phosphate on the film can be obtained.
  • the resulting membrane generally has a collagen layer thickness of about 1 to 100 ⁇ m, preferably about 5 to 20 ⁇ m.
  • the thickness of the calcium phosphate layer is generally about 1-100 ⁇ m, preferably about 20-70 ⁇ m.
  • the thickness of the collagen layer is less than 1 ⁇ m, there is a problem that the adhesion property of the calcium phosphate layer is inferior, and when the thickness is more than 100 ⁇ m, the collagen layer itself is easily peeled off.
  • the thickness of the calcium phosphate layer is less than 1 ⁇ m, the effect of promoting the growth of bone tissue is poor, and it is not preferable.
  • the thickness is more than 100 ⁇ m, it is not preferable because it easily peels from the collagen layer.
  • the thickness of the collagen layer and the calcium phosphate layer may be wide, and high uniformity is not necessarily required, and there may be some degree of unevenness.
  • the substrate is treated with a solution containing at least calcium or phosphate ions, and the substrate to which the solution is adhered is washed with water.
  • an apatite nucleating agent composed of calcium phosphate is immobilized on the surface of hydrophilic collagen.
  • a method in which the following steps are combined is preferable. (B) a step of treating a film having a collagen layer with a solution containing at least calcium; (b) a step of treating a film to which the calcium solution is adhered with a medium containing water and drying; and (c) water.
  • the step (a) is mainly intended to adsorb calcium ions on the surface of collagen, and is usually performed by immersing the film in a solution containing calcium ions such as a CaCl 2 aqueous solution.
  • the immersion time is usually 1 second to 100 minutes, preferably 10 to 60 seconds.
  • the pulling speed of the substrate is usually 1 to 100 cm / min, preferably 15 to 60 cm / min.
  • the concentration of calcium ions is not particularly limited, but is usually 1 to 1000 mM, preferably 100 to 500 mM, more preferably 200 to 250 mM.
  • the step (b) is mainly intended to remove from the surface the calcium solution adhering to the collagen surface in the step (b). By this step, calcium ions adsorbed by hydrogen bonds or the like selectively remain on the surface. Without this water treatment step, the apatite nucleating agent is formed thick, so that not only the adhesive strength between the apatite layer and the collagen layer provided on it is weakened, but also crystals other than calcium phosphate are precipitated in large quantities. End up.
  • the step (b) is usually performed by immersing a film having a calcium solution attached in a medium containing water.
  • the immersion time is usually 1 to 60 seconds, preferably 1 to 5 seconds.
  • the film pulling speed is usually 1 to 100 cm / min, preferably 15 to 60 cm / min.
  • the drying time is usually 10 seconds to 60 minutes, preferably 1 to 10 minutes.
  • the step (c) is mainly intended to react the calcium ions adsorbed on the surface of the collagen obtained in the step (b) with phosphate ions to obtain an apatite nucleating agent composed of calcium phosphate.
  • the immersion time is usually 1 second to 100 minutes, preferably 10 to 60 seconds.
  • the film pulling speed is usually 1 to 100 cm / min, preferably 15 to 60 cm / min.
  • the concentration of phosphate ions is not particularly limited, but is usually 1 to 1000 mM, preferably 100 to 500 mM, more preferably 200 to 250 mM.
  • the step (d) is usually performed by immersing the film with the phosphorus solution attached in a medium containing water.
  • the immersion time is usually 1 to 60 seconds, preferably 1 to 5 seconds.
  • the film pulling speed is usually 1 to 100 cm / min, preferably 15 to 60 cm / min.
  • the drying time is usually 10 seconds to 60 minutes, preferably 1 to 10 minutes.
  • the order of immersion in the calcium solution and the phosphorus solution is not particularly limited to the above-described embodiment.
  • the order of immersion in the calcium solution and the phosphorus solution is changed as follows to fix the apatite nucleating agent.
  • the following steps may be taken as the converting means.
  • a step of processing with a medium to be dried is not particularly limited to the above-described embodiment.
  • the order of immersion in the calcium solution and the phosphorus solution is changed as follows to fix the apatite nucleating agent.
  • the following steps may be taken as the converting means.
  • (a) ⁇ (b) ⁇ (c) ⁇ (2) is performed in the order of steps, but the introduction amount of the apatite nucleating agent is increased, and the surface of the substrate is not sufficiently high in hydrophilicity.
  • the immersion steps (a) to (2) are performed by (i) ⁇ (b) ⁇ (c) ⁇ (ii) ⁇ (B) ⁇ (b)... May be repeated a predetermined number of times. However, if the number of repetitions is increased, the adhesive strength between the substrate and the apatite layer formed on the surface thereof is lowered. Therefore, the number of repetitions is usually preferably 1 or more and 4 or less.
  • a PET film was used as the polyester film substrate.
  • a product having a thickness of 50 ⁇ m manufactured by Toyobo Co., Ltd. was used (product name A4100).
  • the PET film cut to a size of 25 mm ⁇ 50 mm was immersed in an aqueous solution of sodium hydroxide having a concentration of 3.0 mol / L, and kept hydrophilic at a temperature of about 70 ° C. for about 3 hours.
  • As the collagen bovine dermis-derived atelocollagen solution I-PC 50 (5 mg / mL) manufactured by Koken Co., Ltd. was used.
  • the collagen fibrils after washing are dispersed in 50 mL of a phosphate buffer solution having a pH of 5.8, 0.150 g of carbodiimide is added to an aqueous solution having a collagen fibril concentration of 10 mg / 50 mL, and the hydrophilized polyester is immersed in the solution.
  • the collagen was fixed to the PET film by maintaining at a temperature of 37 ° C. for 18 hours.
  • urease used was a product of Kanto Chemical Co., Ltd. (derived from Tachinata beans, 5000 U / g).
  • a PET film on which collagen was immobilized was placed in an aqueous solution prepared by dissolving 0.030 g of this urease and 0.150 g of carbodiimide in 50 mL of a phosphate buffer solution having a pH of 5.8, and kept at a temperature of about 37 ° C. for 24 hours.
  • the urease prepared in the previous step is fixed in an aqueous solution containing calcium nitrate tetrahydrate 10.0 mmol / L, ammonium dihydrogen phosphate 6.0 mmol / L, urea 10.0 mmol / L.
  • the soaked PET film was immersed and held at a temperature of about 37 ° C. for 1 hour.
  • the thickness of the collagen layer was about 10 ⁇ m, and the thickness of the hydroxyapatite (hydroxyapatite) layer was about 30 ⁇ m.
  • col represents collagen
  • HA represents a kind of hydroxyapatite (hydroxyapatite) of calcium phosphate.
  • a bone defect with a diameter of 5 mm was formed on the top of the head of an 8-week-old Wister rat to avoid damaging the dura mater.
  • No membrane (Group 1), untreated PET film (PET film alone) (Group 2) , PET film coated with collagen (3rd group, PET-col coating), PET film coated with collagen and hydroxyapatite (hydroxyapatite) (4th group, PET-col-HA coated)
  • Three animals were used in the group.
  • the bone defect portion was covered with a membrane, the opening was sutured thereon, and the progress was observed with the membrane completely under the scalp.
  • the bone defect covered with the membrane was filled with blood clots.
  • FIG. 1 shows a micro CT photograph of a rat skull defect.
  • A is the first group
  • B the second group
  • C the third group
  • D the fourth group
  • the wavy line in the figure is the boundary line between the natural bone and the bone defect.
  • the boundary line is clear, and it can be seen that bone formation hardly progresses.
  • the third group and the fourth group FIGS. 1C and 1D
  • it is observed that the new bone is growing toward the defect.
  • the periosteum adhered firmly to the third group, but the fourth group did not adhere to the periosteum and could be removed smoothly.
  • the ease of extracting the membrane is remarkable, and the membrane can be easily pulled out from a small opening by pinching one end or a part of the membrane. Therefore, the size of the incision when the membrane is removed can be limited, and the invasion to the patient's body can be minimized.
  • the ease of extraction of the third group was inferior, but this is thought to be due to the fact that collagen has good affinity with both bone and epithelial tissue.
  • FIG. 2 shows a tissue section image of the membrane embedded portion.
  • A is the second group, (B) the third group, and (C) the fourth group.
  • A indicates the region where the membrane is embedded, B indicates the defect side, C indicates the periosteum side, and D indicates a small point in this region.
  • E is new bone, F is calcium phosphate and osteoblasts dissolved from the membrane, and G is new blood vessel.
  • FIG. 2A In the second group using the untreated PET membrane (FIG. 2A), no new bone was formed, and traces of inflammation like D were observed in the periosteum.
  • FIG. 2B In the third group using the PET-col membrane (FIG. 2B), formation of new bone is observed in the portion E, but inflammation is observed in the vicinity of D.
  • FIG. 2C In the fourth group using the PET-col-HA membrane (FIG. 2C), new bone formation is thick in the region E, and there is no evidence of inflammation.
  • the region F indicates that osteoblasts are actively proliferating with calcium phosphate dissolved from the membrane. Furthermore, formation of new blood vessels is observed in the vicinity of G.
  • the membrane of the present invention has a calcium phosphate layer that does not dissolve in the part sandwiched between the base material and the bone surface in the living body, and improves the peelability of the membrane during the removal of the membrane while In the contact portion, the calcium phosphate layer dissolves, and in combination with the action of collagen, the activity of osteoblasts can be increased. From the above results, the effectiveness of the membrane of the present invention is shown.

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Abstract

Disclosed is a membrane for inducing the regeneration of a bone/tissue, which can assist in the formation of a bone and can be removed easily after the formation of the bone. Specifically disclosed are: a membrane for inducing the regeneration of a bone/tissue, which comprises a hydrophilized polyester or polyamide film that serves as a base, a collagen layer that is formed on both surfaces or one surface of the film and comprises a collagen or a thermally modified collagen, and a calcium phosphate layer that is superposed on the collagen layer; and a process for producing the membrane.

Description

骨・組織再生誘導用メンブレンBone and tissue regeneration guidance membrane
　本発明は、歯科あるいは外科分野において用いられる骨・組織再生関連材料に関し、特に、骨・組織再生誘導用メンブレンとその製造方法に関する。 The present invention relates to a bone / tissue regeneration-related material used in the field of dentistry or surgery, and more particularly to a membrane for inducing bone / tissue regeneration and a manufacturing method thereof.
　形成外科分野における外傷や疾病による骨欠損部の再生、歯科における歯周病により失われた組織の再生、歯科インプラント治療におけるインプラント埋入部位の骨の増量は、それぞれの分野において重要な課題である。一般的に、骨再生を望んでも、骨よりもその周囲の上皮組織または粘膜の方が成長のスピードが速いため、骨を再生または増量しようとする部位が肉組織で覆われてしまい、骨再生が難しい。そこで、遮蔽用のメンブレンを利用して骨再生を促す技術が開発されている。 Regeneration of bone defects due to trauma and disease in the field of plastic surgery, regeneration of tissue lost due to periodontal disease in dentistry, and increase of bone at the implant placement site in dental implant treatment are important issues in each field. . Generally, even if bone regeneration is desired, the surrounding epithelial tissue or mucous membrane grows faster than bone, so the site where bone is to be regenerated or increased is covered with meat tissue, and bone regeneration Is difficult. Therefore, a technique for promoting bone regeneration using a shielding membrane has been developed.
　歯科の分野では、中等度以上の歯周病で失われた組織や骨を元通りに戻す歯周組織再生治療がある。歯周病が進行すると、歯の周りの骨と歯根膜という歯根の周りの組織が破壊されるが、この歯根膜組織と骨を再生する治療がある。その方法としては、メンブレンを使用するＧＴＲ法(Guided Tissue Regeneration)や、メンブレンを用いて骨再生のスペースを作り、その中に移植骨、骨代替材等の誘導因子などを封入して、骨を再生させるＧＢＲ法（Guided Bone Regeneration）がある。ＧＢＲ法においては、自己由来の海綿骨もしくは皮質骨、異種または同種の骨移植片、ヒドロキシアパタイト又はリン酸三カルシウムの顆粒、サンゴ、コラーゲンのような天然ポリマー、骨形態形成タンパク質のような骨誘導タンパク質などが用いられる。 In the field of dentistry, there is periodontal tissue regenerative treatment that restores tissues and bones lost due to moderate or higher periodontal disease. When periodontal disease progresses, bone around the teeth and tissues around the root of the periodontal ligament are destroyed. There is a treatment for regenerating the periodontal ligament and bone. As a method for this, the GTR method (Guided Tissue Regeneration) using a membrane, or a bone regeneration space using a membrane, encapsulating inductive factors such as transplanted bone and bone substitute material, etc. are encapsulated in the bone. There is a GBR method (Guided Bone Regeneration) to regenerate. In the GBR method, autologous cancellous or cortical bone, xenogeneic or homogenous bone grafts, hydroxyapatite or tricalcium phosphate granules, coral, natural polymers such as collagen, bone induction such as bone morphogenetic proteins Proteins are used.
　このようなメンブレンとしては、非吸収性メンブレンと吸収性メンブレンの２種類が知られている。非吸収性メンブレンとしては、生体とのなじみがよく変形が容易なチタンのメッシュや、延伸加工したポリテトラフルオロエチレン（ePTFE）が知られている。このタイプのメンブレンは、骨生成が完了したあと、手術により除去する必要がある。そして、吸収性メンブレンとしては、乳酸とグリコール酸の共重合体やコラーゲンなどが使用される。このタイプのメンブレンは、ある程度の時間がたつと生体内で分解されて吸収されるので、除去手術の必要がない一方、組織再生の状況のコントロールと確認が困難である。最近では、特許文献１にあるような、生体吸収性のコラーゲンメンブレンとハイドロキシアパタイト（水酸アパタイト）との２層あるいは、それにさらに治癒促進用の濃縮血小板ゲルを重ねたものも知られており、非特許文献1にあるようなハイドロキシアパタイト（水酸アパタイト）、コラーゲン、ポリ乳酸などからなる吸収性の三層膜が知られている。 Two types of such membranes are known: non-absorbent membranes and absorbent membranes. Known non-absorbable membranes include titanium mesh that is familiar with living bodies and easily deformed, and stretched polytetrafluoroethylene (ePTFE). This type of membrane must be removed by surgery after bone formation is complete. As the absorbent membrane, a copolymer of lactic acid and glycolic acid or collagen is used. Since this type of membrane is decomposed and absorbed in the living body after a certain period of time, it is difficult to control and confirm the status of tissue regeneration while eliminating the need for removal surgery. Recently, as disclosed in Patent Document 1, two layers of a bioabsorbable collagen membrane and hydroxyapatite (hydroxyapatite) or a layer of concentrated platelet gel for promoting healing are also known, An absorptive three-layer film made of hydroxyapatite (hydroxyapatite), collagen, polylactic acid and the like as described in Non-Patent Document 1 is known.
　非吸収性メンブレンは、骨再生完了後に周辺組織と不必要な癒着を起こすことなく、患部からスムーズに摘出できることが求められているが、上記のチタンメッシュでは、メッシュのわずかな間隙にも骨が貫入し、骨再生後のチタンメッシュの摘出が困難になる場合がある。特許文献１に記載されているハイドロキシアパタイト（水酸アパタイト）及びコラーゲンからなる２層のメンブレン、又は非特許文献１に記載されているハイドロキシアパタイト（水酸アパタイト）、コラーゲン及びポリ乳酸からなる３層のメンブレンでは、体内での吸収が骨形成よりも速いために、上皮の侵入を許してしまい、歯槽骨が最初のメンブレンの埋入位置まで再生できないという問題がある。 Non-absorbable membranes are required to be removed smoothly from the affected area without causing unnecessary adhesion with surrounding tissues after bone regeneration is complete. It may penetrate and make it difficult to extract the titanium mesh after bone regeneration. Two-layer membrane composed of hydroxyapatite (hydroxyapatite) and collagen described in Patent Document 1, or three-layer composed of hydroxyapatite (hydroxyapatite), collagen and polylactic acid described in Non-patent Document 1 In this membrane, the absorption in the body is faster than the bone formation, so that the invasion of the epithelium is allowed, and the alveolar bone cannot be regenerated to the initial membrane implantation position.
特開2007-160011号公報JP 2007-160011 A
　本発明は、骨形成を助け、骨形成後の摘出が容易な骨・組織再生誘導用メンブレンを提供することを目的とする。 An object of the present invention is to provide a bone / tissue regeneration-inducing membrane that helps bone formation and is easily extracted after bone formation.
　本発明の骨・組織再生誘導用メンブレンは、ポリエステルまたはポリアミドの親水化フィルムの基材と、該フィルムの両面または片面に設けたコラーゲンまたは熱変性コラーゲンからなるコラーゲン層と、該コラーゲン層の上に被せたリン酸カルシウム層とを含んでなる。 The bone / tissue regeneration-inducing membrane of the present invention comprises a polyester or polyamide hydrophilized film substrate, a collagen layer made of collagen or heat-denatured collagen provided on both sides or one side of the film, and a collagen layer on the collagen layer. And a covered calcium phosphate layer.
　本発明の骨・組織再生誘導用メンブレンの製造方法は、ポリエステルまたはポリアミドの親水化フィルムへコラーゲンまたは熱変性コラーゲンを固定してコラーゲン層を生成する第一固定化工程と、コラーゲン層の上にリン酸カルシウム層を置く積層工程とを含んでなる。 The method for producing a membrane for inducing bone / tissue regeneration according to the present invention includes a first immobilization step of immobilizing collagen or heat-denatured collagen on a hydrophilized film of polyester or polyamide to generate a collagen layer, and calcium phosphate on the collagen layer. Laminating step of placing the layer.
　ポリエステルとは、一般に、多価カルボン酸（ジカルボン酸）とポリアルコール（ジオール）との重縮合体をいう。ポリアルコール（アルコール性の基－ＯＨを複数有する化合物）と、多価カルボン酸（カルボキシル基－ＣＯＯＨを複数有する化合物）を反応（脱水縮合）させて作ることを基本とするものである。ポリエチレンテレフタレート（ＰＥＴ）、ポリエチレンナフタレート、ポリブチレンテレフタレート、ポリトリメチレンテレフタレート、ポリブチレンナフタレートなどがあるが、中でも最も多く生産されているものはテレフタル酸とエチレングリコールから製造されるポリエチレンテレフタレートである。ポリエステル以外の材料としては、ナイロンや、アラミドのようなポリアミドを使用することができる。ポリアミドは、モノマー内にアミド基を持つポリマーの総称である。ポリエステルもポリアミドも、下に記す親水化処理を施すことによって、コラーゲンとの固定化に必要なカルボキシル基を提供することができるからである。 Polyester generally refers to a polycondensate of polycarboxylic acid (dicarboxylic acid) and polyalcohol (diol). It is basically made by reacting (dehydrating and condensing) a polyalcohol (a compound having a plurality of alcoholic groups —OH) and a polyvalent carboxylic acid (a compound having a plurality of carboxyl groups —COOH). Polyethylene terephthalate (PET), polyethylene naphthalate, polybutylene terephthalate, polytrimethylene terephthalate, polybutylene naphthalate, etc., but the most widely produced is polyethylene terephthalate produced from terephthalic acid and ethylene glycol . As materials other than polyester, polyamide such as nylon or aramid can be used. Polyamide is a general term for polymers having an amide group in a monomer. This is because both polyester and polyamide can provide a carboxyl group necessary for immobilization with collagen by applying the hydrophilic treatment described below.
　ここで使用できるコラーゲンまたは熱変性コラーゲン(ゼラチンなど)としては、ウシ、ブタまたは魚類由来のI型、II型またはIII型のアテロコラーゲンといったタイプのものがある。このコラーゲンは、コラーゲンペプチドといったポリペプチドで代替でき、本願においては、コラーゲンという用語は、このようなポリペプチドを包含する広い意味で使用する。このコラーゲンは、リン酸カルシウム層とポリエステルの接着の役割を果たすとともに、骨芽細胞に対しては増殖のための環境形成の助けになることが知られている。 As the collagen or heat-denatured collagen (gelatin etc.) that can be used here, there are types such as type I, type II or type III atelocollagen derived from bovine, pig or fish. This collagen can be replaced by a polypeptide such as a collagen peptide, and in this application the term collagen is used in a broad sense encompassing such a polypeptide. It is known that this collagen plays a role of adhesion between the calcium phosphate layer and the polyester, and also helps the osteoblast to form an environment for proliferation.
　また、リン酸カルシウムは、カルシウムイオンとリン酸基（ＰＯ４ ３－）またはピロリン酸基（Ｐ２Ｏ７ ４－）からなる物質である。水素やヒドロキシ基を持つこともある。骨の約７０％は、リン酸カルシウム系のセラミックスの一種であるハイドロキシアパタイト（水酸アパタイト）（Ｃａ５（ＰＯ４）３（ＯＨ））からできている。リン酸の持つ三個の水素原子(H)とカルシウムがそれぞれ置換されて、次の三種類の異なったリン酸塩となる。水素原子一個がカルシウムと置き換った場合には、第一リン酸カルシウムＣａ（Ｈ２ＰＯ４）２となり、水素原子二個がカルシウムと置き換った場合には、第二リン酸カルシウムＣａＨＰＯ４、水素原子三個がカルシウムと置き換ると第三リン酸カルシウムＣａ３（ＰＯ４）２となる。 Calcium phosphate is a substance composed of calcium ions and phosphate groups (PO 4 3− ) or pyrophosphate groups (P 2 O 7 4− ). May have hydrogen or hydroxy groups. About 70% of the bone is made of hydroxyapatite (hydroxyapatite) (Ca 5 (PO 4 ) 3 (OH)), which is a kind of calcium phosphate ceramics. The three hydrogen atoms (H) and calcium of phosphoric acid are each replaced, resulting in the following three different phosphates. When one hydrogen atom is replaced with calcium, it becomes primary calcium phosphate Ca (H 2 PO 4 ) 2 , and when two hydrogen atoms are replaced with calcium, dicalcium phosphate CaHPO 4 , hydrogen atoms When three of them are replaced with calcium, tricalcium phosphate Ca 3 (PO 4 ) 2 is obtained.
　本発明の骨・組織再生誘導用メンブレンによれば、順調な骨組織の成長が期待できるとともに、骨形成後の摘出が容易である。 According to the bone / tissue regeneration-inducing membrane of the present invention, smooth growth of bone tissue can be expected and extraction after bone formation is easy.
本発明の一実施形態のメンブレンとそれ以外のメンブレンを用いた場合と、メンブレンを用いなかった場合とを比較したラット頭蓋骨欠損部のマイクロＣＴ写真を示す。The micro CT photograph of the rat skull defect part which compares the case where the membrane of one embodiment of the present invention and other membranes are used, and the case where a membrane is not used is shown. 本発明の一実施形態のメンブレンとそれ以外のメンブレンを用いた場合を比較したメンブレン埋入部分の組織切片画像を示す。The tissue section image of the membrane embedding part which compared the case where the membrane of one Embodiment of this invention and the other membrane is used is shown.
　本発明の一つの実施形態を下記に説明する。この実施形態のメンブレンは、（１）基材となるポリエステルまたはポリアミドのフィルムの表面の親水化と、（２）フィルム表面へのコラーゲンの固定化と、（３）フィルム上に固定されたコラーゲンへのウレアーゼの固定化と、（４）このコラーゲンの表面上へのリン酸カルシウムの析出との４工程により製造することができる。 One embodiment of the present invention will be described below. The membrane of this embodiment comprises (1) hydrophilicity of the surface of a polyester or polyamide film as a base material, (2) fixation of collagen to the film surface, and (3) to collagen immobilized on the film. Of urease and (4) precipitation of calcium phosphate on the surface of this collagen.
　まず、１０～３００μｍ程度の厚さ、好ましくは２０～１００μｍの厚さのポリエステルまたはポリアミドのフィルムを０．１～１０ｍｏｌ／Ｌのアルカリ性水溶液（水酸化ナトリウムなど）に浸し、６０～１００℃の温度で１５分～６時間程度保持する。これにより、ポリエステルフィルムの表面が加水分解し、ヒドロキシ基（－ＯＨ）とカルボキシル基（－ＣＯＯＨ）が表面に現れる。なお、ポリエステルまたはポリアミドのフィルムの厚さは、フィルム自体の剛性、骨欠損部の大きさ、外部組織からの応力を支えるために求められる剛性、取り扱いやすさといった要素を検討して決めるべきものである。あまり薄いと外部組織からの応力を支えきれずに骨形成のための空間を維持することができなくなるし、あまり厚いと摘出の際の切開部を大きくしなければならなくなる。親水化には、電子線処理、酸溶液処理、酸化剤処理、親水性官能基のグラフト処理、シランカップリング処理、陽極酸化処理、粗面化処理など、アルカリ水溶液を用いるほかにもいくつかの方法が知られている。また、すでに親水化処理の済んだフィルム素材も販売されている。 First, a polyester or polyamide film having a thickness of about 10 to 300 μm, preferably 20 to 100 μm, is immersed in an alkaline aqueous solution (such as sodium hydroxide) of 0.1 to 10 mol / L, and a temperature of 60 to 100 ° C. Hold for about 15 minutes to 6 hours. As a result, the surface of the polyester film is hydrolyzed, and hydroxy groups (—OH) and carboxyl groups (—COOH) appear on the surface. The thickness of the polyester or polyamide film should be determined by considering factors such as the rigidity of the film itself, the size of the bone defect, the rigidity required to support stress from external tissues, and ease of handling. is there. If it is too thin, it will not be possible to maintain the space for bone formation without being able to support the stress from the external tissue, and if it is too thick, the incision part at the time of extraction will have to be enlarged. In addition to using an alkaline aqueous solution, the hydrophilic treatment includes electron beam treatment, acid solution treatment, oxidizing agent treatment, hydrophilic functional group grafting treatment, silane coupling treatment, anodizing treatment, and roughening treatment. The method is known. In addition, film materials that have already been hydrophilized are on sale.
　次に、コラーゲンまたは熱変性コラーゲン(ゼラチンなど)を分散した水溶液に、カルボジイミドなどの架橋剤を溶かし、これに親水化フィルムを浸して、２０～５０℃の温度で６～７２時間保持する。この過程で、ポリエステル表面のカルボキシル基（－ＣＯＯＨ基）とコラーゲンのアミノ基（－ＮＨ２）が脱水縮合し、ペプチド結合（－ＣＯＮＨ－）することによって、コラーゲンがポリエステル表面に固定化される。 Next, a cross-linking agent such as carbodiimide is dissolved in an aqueous solution in which collagen or heat-denatured collagen (gelatin or the like) is dispersed, and a hydrophilic film is immersed in the solution and held at a temperature of 20 to 50 ° C. for 6 to 72 hours. In this process, the carboxyl group (—COOH group) on the polyester surface and the amino group (—NH 2 ) of collagen are dehydrated and condensed to form a peptide bond (—CONH—), thereby immobilizing the collagen on the polyester surface.
　次いで、このコラーゲンが固定されたフィルムを、ウレアーゼとカルボジイミドを溶かした水溶液に入れ、２０～５０℃の温度、好ましくは３０～４０℃の温度で６～７２時間保持する。この過程で、コラーゲンの表面のカルボキシル基またはアミノ基と、ウレアーゼのアミノ基またはカルボキシル基が脱水縮合し、コラーゲン表面にウレアーゼが固定化される。 Next, the collagen-fixed film is placed in an aqueous solution in which urease and carbodiimide are dissolved, and is held at a temperature of 20 to 50 ° C., preferably 30 to 40 ° C. for 6 to 72 hours. In this process, the carboxyl group or amino group on the surface of collagen and the amino group or carboxyl group of urease undergo dehydration condensation, and urease is immobilized on the collagen surface.
　最後に、このウレアーゼが固定されているコラーゲンの表面に、リン酸カルシウムを析出させる。すなわち、カルシウムイオン１～１００ｍｍｏｌ／Ｌ、リン酸イオン１～１００ｍｍｏｌ／Ｌ、尿素１０～１００ｍｍｏｌ／Ｌを含む水溶液に。先の工程で準備されたウレアーゼを固定化したフィルムを浸し、２０～５０℃の温度で１５分から２４時間保持する。この過程で、ウレアーゼの作用により尿素がアンモニアに変わり，アンモニアはリン酸カルシウムをフィルムの表面に選択的に析出させる。その結果、フィルムの基体と、フィルムの上のリン酸カルシウムとの３層構造を有しているメンブレンを得ることができる。 Finally, calcium phosphate is deposited on the surface of collagen to which this urease is fixed. That is, in an aqueous solution containing 1 to 100 mmol / L of calcium ions, 1 to 100 mmol / L of phosphate ions, and 10 to 100 mmol / L of urea. The urease-immobilized film prepared in the previous step is immersed and held at a temperature of 20 to 50 ° C. for 15 minutes to 24 hours. In this process, urea is converted into ammonia by the action of urease, and ammonia selectively precipitates calcium phosphate on the surface of the film. As a result, a membrane having a three-layer structure of a film substrate and calcium phosphate on the film can be obtained.
　上記の過程では、ポリエステルフィルムの両面にコラーゲンとリン酸カルシウムが付着することになるが、片面だけに付着させたい場合には、予めポリエステルフィルムを２枚重ねておくか、１枚のポリエステルフィルムの１面を別のポリマーフィルムで覆っておくなどして、上記の工程が終了後に重なったフィルムを剥がせばよい。 In the above process, collagen and calcium phosphate adhere to both sides of the polyester film. However, if you want to adhere only to one side, either stack two polyester films in advance or one side of one polyester film. May be covered with another polymer film, and the overlapped film may be peeled off after the above process is completed.
　その結果得られるメンブレンは、コラーゲンの層の厚さが、一般的に、約１～１００μｍであり、約５～２０μｍであることが好ましい。リン酸カルシウムの層の厚さは、一般的に、約１～１００μｍであり、約２０～７０μｍであるのが好ましい。コラーゲン層の厚さが１μｍより薄いと、リン酸カルシウム層の接着性に劣るといった問題があり、１００μｍより厚いとコラーゲン層自体が剥がれやすいという問題が生じる。また、リン酸カルシウム層の厚さは、１μｍより薄いと、骨組織の成長を促す効果に乏しくなり好ましくなく、１００μｍより厚いとコラーゲン層から剥がれやすくなるので好ましくない。このようなコラーゲン層およびリン酸カルシウム層の厚さには幅があってもよく、からならずしも高い均一性が求められるものではなく、ある程度のムラがあってもかまわない。 The resulting membrane generally has a collagen layer thickness of about 1 to 100 μm, preferably about 5 to 20 μm. The thickness of the calcium phosphate layer is generally about 1-100 μm, preferably about 20-70 μm. When the thickness of the collagen layer is less than 1 μm, there is a problem that the adhesion property of the calcium phosphate layer is inferior, and when the thickness is more than 100 μm, the collagen layer itself is easily peeled off. Further, if the thickness of the calcium phosphate layer is less than 1 μm, the effect of promoting the growth of bone tissue is poor, and it is not preferable. If the thickness is more than 100 μm, it is not preferable because it easily peels from the collagen layer. The thickness of the collagen layer and the calcium phosphate layer may be wide, and high uniformity is not necessarily required, and there may be some degree of unevenness.
　また、ポリエステルに固定したコラーゲン上にリン酸カルシウムを析出させる方法としては、ウレアーゼを用いる方法以外にも、少なくともカルシウムまたはリン酸のイオンを含む溶液で基材を処理し、溶液が付着した基材を水を含む媒体で処理し乾燥させ、水処理後のいずれかのイオンを吸着した基材を少なくともリンまたはカルシウムを含む溶液で処理して、水を含む媒体で基材を処理し乾燥させる方法(特許文献２)や、その表面が親水性を有する基材表面にリン酸カルシウムからなるアパタイト核形成剤が固定化されてなる基材とリン酸カルシウム過飽和溶液を接触させるアパタイト複合体の製造方法(特許文献３)などを利用することができる。 Further, as a method for precipitating calcium phosphate on collagen immobilized on polyester, in addition to the method using urease, the substrate is treated with a solution containing at least calcium or phosphate ions, and the substrate to which the solution is adhered is washed with water. A method of treating a substrate that has adsorbed any ions after water treatment with a solution containing at least phosphorus or calcium and treating the substrate with a medium containing water and then drying (patented) Document 2), and a method for producing an apatite complex in which a substrate in which an apatite nucleating agent composed of calcium phosphate is immobilized on a surface of a substrate having hydrophilicity is contacted with a calcium phosphate supersaturated solution (Patent Document 3), etc. Can be used.
特開2005-112716号公報JP 2005-112716 A 特開2005-111255号公報JP 2005-111255 A
　この別法においては、親水性を有するコラーゲンの表面にリン酸カルシウムからなるアパタイト核形成剤を固定化する。コラーゲンの表面にアパタイト核形成剤を固定化するには、次の工程を組み合わせる方法が好ましい。すなわち、（イ）コラーゲン層を有するフィルムを、少なくともカルシウムを含む溶液で処理する工程と、（ロ）カルシウム溶液が付着したフィルムを、水を含む媒体で処理し乾燥する工程と、（ハ）水処理後のカルシウムイオン吸着フィルムを、少なくともリンを含む溶液で処理する工程と、（ニ）リン溶液が付着したフィルムを、水を含む媒体で処理し乾燥する工程とからなる方法である。 In this alternative method, an apatite nucleating agent composed of calcium phosphate is immobilized on the surface of hydrophilic collagen. In order to immobilize the apatite nucleating agent on the surface of collagen, a method in which the following steps are combined is preferable. (B) a step of treating a film having a collagen layer with a solution containing at least calcium; (b) a step of treating a film to which the calcium solution is adhered with a medium containing water and drying; and (c) water. It is a method comprising a step of treating a calcium ion-adsorbed film after treatment with a solution containing at least phosphorus and a step of (d) treating and drying the film to which the phosphorus solution is adhered with a medium containing water.
　（イ）の工程は、まず、コラーゲンの表面にカルシウムイオンを吸着させることを主眼としたものであり、通常、ＣａＣl２水溶液などのカルシウムイオンを含む溶液にフィルムを浸漬することにより行われる。浸漬時間は通常１秒～１００分好ましくは１０～６０秒である。また基材の引き上げ速度は、通常１～１００cm/分、好ましくは１５～６０cm/分である。カルシウムイオンの濃度は特に限定されないが、通常１～１０００mM、好ましくは１００～５００mM、さらに好ましくは２００～２５０mMである。 The step (a) is mainly intended to adsorb calcium ions on the surface of collagen, and is usually performed by immersing the film in a solution containing calcium ions such as a CaCl 2 aqueous solution. The immersion time is usually 1 second to 100 minutes, preferably 10 to 60 seconds. The pulling speed of the substrate is usually 1 to 100 cm / min, preferably 15 to 60 cm / min. The concentration of calcium ions is not particularly limited, but is usually 1 to 1000 mM, preferably 100 to 500 mM, more preferably 200 to 250 mM.
　（ロ）の工程は、（イ）の工程でコラーゲンの表面に付着したカルシウム溶液を表面から除くことを主眼としたものである。この工程により、水素結合等により吸着したカルシウムイオンが、表面に選択的に残存するようになる。この水処理工程を欠くと、アパタイト核形成剤が厚く形成されてしまうので、その上に設けられるアパタイト層とコラーゲン層との接着強度が弱くなるだけでなく、リン酸カルシウム以外の結晶も多量に析出してしまう。（ロ）の工程は通常、水を含む媒体中に、カルシウム溶液が付着したフィルムを浸漬することにより行われる。浸漬時間は通常１～６０秒、好ましくは１～５秒である。またフィルムの引き上げ速度は、通常１～１００cm/分、好ましくは１５～６０cm/分である。乾燥時間は、通常１０秒～６０分、好ましくは１～１０分である。 The step (b) is mainly intended to remove from the surface the calcium solution adhering to the collagen surface in the step (b). By this step, calcium ions adsorbed by hydrogen bonds or the like selectively remain on the surface. Without this water treatment step, the apatite nucleating agent is formed thick, so that not only the adhesive strength between the apatite layer and the collagen layer provided on it is weakened, but also crystals other than calcium phosphate are precipitated in large quantities. End up. The step (b) is usually performed by immersing a film having a calcium solution attached in a medium containing water. The immersion time is usually 1 to 60 seconds, preferably 1 to 5 seconds. The film pulling speed is usually 1 to 100 cm / min, preferably 15 to 60 cm / min. The drying time is usually 10 seconds to 60 minutes, preferably 1 to 10 minutes.
　（ハ）の工程は、前記（ロ）の工程で得られたコラーゲンの表面に吸着したカルシウムイオンとリン酸イオンを反応させて、リン酸カルシウムからなるアパタイト核形成剤を得ることを主眼としたものである。浸漬時間は通常１秒～１００分好ましくは１０～６０秒である。またフィルムの引き上げ速度は、通常１～１００cm/分、好ましくは１５～６０cm/分である。リン酸イオンの濃度は特に限定されないが、通常１～１０００mM、好ましくは１００～５００mM、さらに好ましくは２００～２５０mMである。 The step (c) is mainly intended to react the calcium ions adsorbed on the surface of the collagen obtained in the step (b) with phosphate ions to obtain an apatite nucleating agent composed of calcium phosphate. is there. The immersion time is usually 1 second to 100 minutes, preferably 10 to 60 seconds. The film pulling speed is usually 1 to 100 cm / min, preferably 15 to 60 cm / min. The concentration of phosphate ions is not particularly limited, but is usually 1 to 1000 mM, preferably 100 to 500 mM, more preferably 200 to 250 mM.
　（ニ）の工程は通常、水を含む媒体中に、リン溶液が付着したフィルムを浸漬することにより行われる。浸漬時間は通常１～６０秒、好ましくは１～５秒である。またフィルムの引き上げ速度は、通常１～１００cm/分、好ましくは１５～６０cm/分である。乾燥時間は、通常１０秒～６０分、好ましくは１～１０分である。この工程により、表面にアパタイト核形成剤が固定化される。 (D) The step (d) is usually performed by immersing the film with the phosphorus solution attached in a medium containing water. The immersion time is usually 1 to 60 seconds, preferably 1 to 5 seconds. The film pulling speed is usually 1 to 100 cm / min, preferably 15 to 60 cm / min. The drying time is usually 10 seconds to 60 minutes, preferably 1 to 10 minutes. By this step, the apatite nucleating agent is immobilized on the surface.
　この場合、カルシウム溶液とリン溶液への浸漬順序は上記のような態様に特に限定されるものではなく、カルシウム溶液とリン溶液への浸漬の順番を下記のごとく変更し、アパタイト核形成剤の固定化手段として、次の工程をとっても良い。すなわち、（イ）少なくともリンを含む溶液で処理する工程と、（ロ）水を含む媒体で処理し乾燥する工程と、（ハ）少なくともカルシウムを含む溶液で処理する工程と、（ニ）水を含む媒体で処理し乾燥する工程とからなる方法である。 In this case, the order of immersion in the calcium solution and the phosphorus solution is not particularly limited to the above-described embodiment. The order of immersion in the calcium solution and the phosphorus solution is changed as follows to fix the apatite nucleating agent. The following steps may be taken as the converting means. (B) a step of treating with a solution containing at least phosphorus; (b) a step of treating with a medium containing water and drying; (c) a step of treating with a solution containing at least calcium; And a step of processing with a medium to be dried.
　通常、前記（イ）→（ロ）→（ハ）→（二）の工程順により行われるが、アパタイト核形成剤の導入量を多くし、また親水性の十分高くない基材表面にも確実に、かつ基材の表面全面にアパタイト核形成剤を固定化するためには、工程（イ）～（二）の浸漬工程を、（イ）→（ロ）→（ハ）→（二）→（イ）→（ロ）・・・の如く所定回数繰り返せばよい。ただし、繰り返す回数を多くすると、基材と、その表面に形成されるアパタイト層の間の接着強度が低くなってしまうので、繰り返す回数は通常１回以上、４回以内とするのが好ましい。 Usually, (a) → (b) → (c) → (2) is performed in the order of steps, but the introduction amount of the apatite nucleating agent is increased, and the surface of the substrate is not sufficiently high in hydrophilicity. In addition, in order to immobilize the apatite nucleating agent on the entire surface of the base material, the immersion steps (a) to (2) are performed by (i) → (b) → (c) → (ii) → (B) → (b)... May be repeated a predetermined number of times. However, if the number of repetitions is increased, the adhesive strength between the substrate and the apatite layer formed on the surface thereof is lowered. Therefore, the number of repetitions is usually preferably 1 or more and 4 or less.
［実験例］
　以下に、本発明のメンブレンを用いて、ラット頭蓋骨の骨誘導再生を行った実験例を記す。ポリエステルフィルム基材にはＰＥＴフィルムを用いた。ＰＥＴフィルムは、東洋紡績株式会社製の厚さ50μｍの製品を用いた（製品名A4100）。このＰＥＴフィルムを25mm×50mmの大きさに切り取ったものを3.0ｍｏｌ／Ｌの濃度の水酸化ナトリウム水溶液に浸し、約70℃の温度で3時間程度保持して親水化を行った。また、コラーゲンは、株式会社高研製のウシ真皮由来アテロコラーゲン溶液I-PC 50（5mg/mL）を用いた。このコラーゲン溶液2mLに純水48mLと再構成用緩衝溶液5mL（炭酸水素ナトリウム50mmol/L、水酸化ナトリウム260mmol/L、HEPES 200mmol/Lを含むもの）を加え、37℃で4時間保持してコラーゲンをゲル化（フィブリル化）させた。このゲルをホモジナイザーでせん断して分散させたものを遠心分離で取り出し、pH5.8のリン酸緩衝溶液で2回洗浄した。洗浄後のコラーゲンフィブリルをpH5.8のリン酸緩衝溶液50mLに入れて分散させ、コラーゲンフィブリル濃度10mg/50mLの濃度にした水溶液に、カルボジイミドを0.150g加え、これに親水化ポリエステルを浸して、約37℃の温度で18時間保持して、コラーゲンをＰＥＴフィルムに固定した。さらに、ウレアーゼは、関東化学株式会社の製品（タチナタ豆由来、5000U/g）を用いた。このウレアーゼ0.030gとカルボジイミド0.150gをpH 5.8のリン酸緩衝溶液50mLに溶かした水溶液に、コラーゲンを固定化したPETフィルムを入れ、約37℃の温度で24時間保持した。そして、リン酸カルシウムを析出させるために、硝酸カルシウム四水和物10.0mmol/L、リン酸二水素アンモニウム6.0mmol/L、尿素10.0mmol/Lを含む水溶液に、先の工程で準備されたウレアーゼを固定化したＰＥＴフィルムを浸し、約37℃の温度で1時間保持した。その結果、コラーゲンの層の厚さは、約10μｍ、ハイドロキシアパタイト（水酸アパタイト）の層の厚さは、約30μｍであった。以下、ｃｏｌはコラーゲン、ＨＡはリン酸カルシウムの一種のハイドロキシアパタイト（水酸アパタイト）を表す。 [Experimental example]
Below, the experimental example which performed bone guidance reproduction | regeneration of the rat skull using the membrane of this invention is described. A PET film was used as the polyester film substrate. As the PET film, a product having a thickness of 50 μm manufactured by Toyobo Co., Ltd. was used (product name A4100). The PET film cut to a size of 25 mm × 50 mm was immersed in an aqueous solution of sodium hydroxide having a concentration of 3.0 mol / L, and kept hydrophilic at a temperature of about 70 ° C. for about 3 hours. As the collagen, bovine dermis-derived atelocollagen solution I-PC 50 (5 mg / mL) manufactured by Koken Co., Ltd. was used. Add 48 mL of pure water and 5 mL of reconstitution buffer solution (containing 50 mmol / L of sodium bicarbonate, 260 mmol / L of sodium hydroxide, 200 mmol / L of HEPES) to 2 mL of this collagen solution, and maintain at 37 ° C for 4 hours to maintain collagen. Was gelled (fibrillated). The gel dispersed by shearing with a homogenizer was removed by centrifugation and washed twice with a phosphate buffer solution having a pH of 5.8. The collagen fibrils after washing are dispersed in 50 mL of a phosphate buffer solution having a pH of 5.8, 0.150 g of carbodiimide is added to an aqueous solution having a collagen fibril concentration of 10 mg / 50 mL, and the hydrophilized polyester is immersed in the solution. The collagen was fixed to the PET film by maintaining at a temperature of 37 ° C. for 18 hours. Further, urease used was a product of Kanto Chemical Co., Ltd. (derived from Tachinata beans, 5000 U / g). A PET film on which collagen was immobilized was placed in an aqueous solution prepared by dissolving 0.030 g of this urease and 0.150 g of carbodiimide in 50 mL of a phosphate buffer solution having a pH of 5.8, and kept at a temperature of about 37 ° C. for 24 hours. In order to precipitate calcium phosphate, the urease prepared in the previous step is fixed in an aqueous solution containing calcium nitrate tetrahydrate 10.0 mmol / L, ammonium dihydrogen phosphate 6.0 mmol / L, urea 10.0 mmol / L. The soaked PET film was immersed and held at a temperature of about 37 ° C. for 1 hour. As a result, the thickness of the collagen layer was about 10 μm, and the thickness of the hydroxyapatite (hydroxyapatite) layer was about 30 μm. Hereinafter, col represents collagen, and HA represents a kind of hydroxyapatite (hydroxyapatite) of calcium phosphate.
　８週齢Wister系ラット雄の頭頂部に、硬膜を傷つけないように直径５ｍｍの骨欠損を形成し、メンブレンなし（第１群）、未処理ＰＥＴフィルム（ＰＥＴフィルム単独）（第２群）、ＰＥＴフィルムをコラーゲンで被覆したもの（第３群、PET-col被覆）、ＰＥＴフィルムをコラーゲンとハイドロキシアパタイト（水酸アパタイト）で被覆したもの（第４群、PET-col-HA被覆）の各群に３匹ずつ使用した。メンブレンで骨欠損部分を覆い、その上で、開口部を縫合し、メンブレンが完全に頭皮の下にある状態で経過を観察した。メンブレンで覆われた骨欠損部は、血餅で満たされていた。メンブレンを埋入してから１週間後に摘出し、マイクロＣＴにより骨欠損部を観察し、組織切片の観察を行った。図１にその結果を示す。なお、いずれの例でも、被覆がある場合は、その被覆はＰＥＴフィルムの両面に行った。 A bone defect with a diameter of 5 mm was formed on the top of the head of an 8-week-old Wister rat to avoid damaging the dura mater. No membrane (Group 1), untreated PET film (PET film alone) (Group 2) , PET film coated with collagen (3rd group, PET-col coating), PET film coated with collagen and hydroxyapatite (hydroxyapatite) (4th group, PET-col-HA coated) Three animals were used in the group. The bone defect portion was covered with a membrane, the opening was sutured thereon, and the progress was observed with the membrane completely under the scalp. The bone defect covered with the membrane was filled with blood clots. One week after embedding the membrane, it was removed, the bone defect was observed by micro CT, and the tissue section was observed. The result is shown in FIG. In any case, when there was a coating, the coating was performed on both sides of the PET film.
　図１には、ラット頭蓋骨欠損部のマイクロＣＴ写真を示す。それぞれ、（Ａ）が第１群、（Ｂ）第２群、（Ｃ）第３群、（Ｄ）第４群である。図中の波線は、天然骨と骨欠損部との境界線である。第１群と第２群（図１（Ａ）、（Ｂ））では、境界線が明確であり、骨形成はほとんど進行していないことがわかる。これに対して第３群と第４群（図１（Ｃ）、（Ｄ））では、欠損部に向かって新生骨が成長している様子が観察される。またメンブレン摘出の際、第３群には骨膜が強固に付着していたが、第４群には骨膜の付着がなく、滑らかに摘出することができた。このメンブレン摘出の容易性は特筆すべきであり、メンブレンの一端あるいは一部をつまんで、小さな開口からメンブレンを容易に引き出すことができる。そのため、メンブレンを摘出する際の切開の大きさを限定することができ、患者の身体への侵襲を最小化することができる。第３群の摘出の容易性は劣っていたが、これは、コラーゲンが骨とも上皮組織などとも親和性がいいことが原因であると考えられる。 FIG. 1 shows a micro CT photograph of a rat skull defect. (A) is the first group, (B) the second group, (C) the third group, and (D) the fourth group, respectively. The wavy line in the figure is the boundary line between the natural bone and the bone defect. In the first group and the second group (FIGS. 1A and 1B), the boundary line is clear, and it can be seen that bone formation hardly progresses. In contrast, in the third group and the fourth group (FIGS. 1C and 1D), it is observed that the new bone is growing toward the defect. At the time of membrane extraction, the periosteum adhered firmly to the third group, but the fourth group did not adhere to the periosteum and could be removed smoothly. The ease of extracting the membrane is remarkable, and the membrane can be easily pulled out from a small opening by pinching one end or a part of the membrane. Therefore, the size of the incision when the membrane is removed can be limited, and the invasion to the patient's body can be minimized. The ease of extraction of the third group was inferior, but this is thought to be due to the fact that collagen has good affinity with both bone and epithelial tissue.
　本メンブレンが新生骨の形成を促進している様子は、メンブレンを摘出後、脱灰した組織の切片観察からも示された。図２にその結果を示す。図２には、メンブレン埋入部分の組織切片画像を示す。（Ａ）は、第２群、（Ｂ）第３群、（Ｃ）第４群である。図中のＡはメンブレンが埋入されていた領域を示し、Ｂは欠損部側、Ｃは骨膜側、Ｄはこの領域の小さな点は炎症を表す。Ｅは新生骨であり、Ｆはメンブレンから溶解したリン酸カルシウムと骨芽細胞、Ｇは新生血管である。 The appearance of the membrane promoting the formation of new bone was also shown by observation of a section of tissue decalcified after the membrane was removed. The results are shown in FIG. FIG. 2 shows a tissue section image of the membrane embedded portion. (A) is the second group, (B) the third group, and (C) the fourth group. In the figure, A indicates the region where the membrane is embedded, B indicates the defect side, C indicates the periosteum side, and D indicates a small point in this region. E is new bone, F is calcium phosphate and osteoblasts dissolved from the membrane, and G is new blood vessel.
　未処理のＰＥＴメンブレンを使用した第２群（図２（Ａ））では、新生骨の生成が見られず、骨膜中にはＤのように炎症の痕跡が観察される。PET-colメンブレンを使用した第３群（図２（Ｂ））では、Ｅの部分に新生骨の形成が観察されるが、Ｄの付近に炎症が見られる。PET-col-HAメンブレンを使用した第４群（図２（Ｃ））では、Ｅの領域に厚く新生骨形成が認められ、炎症の痕跡がない。Ｆの領域は、メンブレンから溶解したリン酸カルシウムによって、骨芽細胞が旺盛に増殖していることを示している。さらにＧ付近には新生血管の形成が認められる。つまり、本発明のメンブレンは、生体内において、基材と骨表面に挟まれている部分においては、リン酸カルシウムの層が溶けず、メンブレン摘出の際のメンブレンの剥離性を高める一方、骨欠損部に接している部分では、リン酸カルシウムの層は溶解して、コラーゲンの作用と相まって、骨芽細胞の活性を高めることができる。以上の結果より、本発明のメンブレンの有効性が示されている。 In the second group using the untreated PET membrane (FIG. 2A), no new bone was formed, and traces of inflammation like D were observed in the periosteum. In the third group using the PET-col membrane (FIG. 2B), formation of new bone is observed in the portion E, but inflammation is observed in the vicinity of D. In the fourth group using the PET-col-HA membrane (FIG. 2C), new bone formation is thick in the region E, and there is no evidence of inflammation. The region F indicates that osteoblasts are actively proliferating with calcium phosphate dissolved from the membrane. Furthermore, formation of new blood vessels is observed in the vicinity of G. In other words, the membrane of the present invention has a calcium phosphate layer that does not dissolve in the part sandwiched between the base material and the bone surface in the living body, and improves the peelability of the membrane during the removal of the membrane while In the contact portion, the calcium phosphate layer dissolves, and in combination with the action of collagen, the activity of osteoblasts can be increased. From the above results, the effectiveness of the membrane of the present invention is shown.

Claims (4)

  1. 　基材となるポリエステルまたはポリアミドの親水化フィルムと、該フィルムの両面または片面に設けたコラーゲンまたは熱変性コラーゲンからなるコラーゲン層と、該コラーゲン層の上に被せたリン酸カルシウム層とを含んでなる骨・組織再生誘導用メンブレン。 A bone / polyester comprising a polyester or polyamide hydrophilic film as a base material, a collagen layer made of collagen or heat-denatured collagen provided on both or one side of the film, and a calcium phosphate layer placed on the collagen layer Tissue regeneration guidance membrane.
  2. 　ポリエステルまたはポリアミドの親水化フィルムへコラーゲンまたは熱変性コラーゲンを固定してコラーゲン層を生成する第一固定化工程と、コラーゲン層の上にリン酸カルシウム層を置く積層工程とを含んでなる骨・組織再生誘導用メンブレンの製造方法。 Bone / tissue regeneration induction comprising a first immobilization step of immobilizing collagen or heat-denatured collagen on a polyester or polyamide hydrophilic film to form a collagen layer, and a laminating step of placing a calcium phosphate layer on the collagen layer Membrane manufacturing method.
  3. 　前記積層工程が、該コラーゲン層へウレアーゼを固定する第２固定化工程と、ウレアーゼを固定化したコラーゲン層の上にリン酸カルシウムを析出させる析出工程とを含んでなる、請求項２に記載の骨・組織再生誘導用メンブレンの製造方法。 The bone and bone according to claim 2, wherein the laminating step includes a second immobilization step of immobilizing urease to the collagen layer, and a precipitation step of precipitating calcium phosphate on the collagen layer immobilizing urease. A method for producing a membrane for inducing tissue regeneration.
  4. 　前記積層工程が、該コラーゲン層の表面にリン酸カルシウムからなるアパタイト核形成剤が固定化されてなる該コラーゲン層とリン酸カルシウム過飽和溶液を接触させることを含む、請求項２に記載の骨・組織再生誘導用メンブレンの製造方法。 The bone / tissue regeneration-inducing guidance according to claim 2, wherein the laminating step includes contacting the collagen layer formed by immobilizing an apatite nucleating agent composed of calcium phosphate on the surface of the collagen layer and a calcium phosphate supersaturated solution. Membrane manufacturing method.
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