JPWO2011155243A1 - Bone and tissue regeneration guidance membrane - Google Patents

Abstract

Provided is a bone / tissue regeneration-inducing membrane that helps bone formation and is easily extracted after bone formation. A bone / polyester comprising a polyester or polyamide hydrophilized 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 covered on the collagen layer Membrane for inducing tissue regeneration and its manufacturing method.

Description

  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 method for manufacturing the same.

  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.

  In the field of dentistry, there are periodontal tissue regeneration treatments that restore 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 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.

  As such a membrane, two types, a non-absorbent membrane and an absorbent membrane, are known. 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, removal surgery is not necessary, and it is difficult to control and confirm the status of tissue regeneration. 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.

  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.

JP 2007-160011 A

Fumio Watari et al., Functionally Graded Materials, Vol. 20 (2006), 35-40 “Prototype of functionally graded GTR membrane for bone tissue regeneration”

  An object of the present invention is to provide a bone / tissue regeneration-inducing membrane that assists in 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 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.

  Polyester generally refers to a polycondensate of a polyvalent carboxylic acid (dicarboxylic acid) and a 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.

  Examples of collagen or heat-denatured collagen (such as gelatin) that can be used here include type I, type II, or type III atelocollagen derived from bovine, porcine 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 bones are 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.

  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.

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.

  One embodiment of the present invention is 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.

  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. 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.

Next, a crosslinking agent such as carbodiimide is dissolved in an aqueous solution in which collagen or heat-denatured collagen (gelatin or the like) is dispersed, and the 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 peptide bonds (—CONH—), thereby immobilizing the collagen on the polyester surface.

  Next, the collagen-fixed film is placed in an aqueous solution in which urease and carbodiimide are dissolved, and 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.

  Finally, calcium phosphate is deposited on the surface of collagen on which the 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 film immobilized with urease 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.

  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.

  The resulting membrane generally has a collagen layer thickness of about 1-100 μm, preferably about 5-20 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.

  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.

JP 2005-112716 A JP 2005-111255 A

  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.

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 an aqueous CaCl ₂ solution. The immersion time is usually 1 second to 100 minutes, preferably 10 to 60 seconds. The pulling rate of the substrate is usually 1 to 100 cm / min, preferably 15 to 60 cm / min. Although the density | concentration of a calcium ion is not specifically limited, Usually, it is 1-1000 mM, Preferably it is 100-500 mM, More preferably, it is 200-250 mM.

  The step (b) is intended to remove from the surface the calcium solution adhering to the collagen surface in the step (a). 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 rate 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. is there. The immersion time is usually 1 second to 100 minutes, preferably 10 to 60 seconds. The film pulling rate is usually 1 to 100 cm / min, preferably 15 to 60 cm / min. Although the density | concentration of a phosphate ion is not specifically limited, Usually, it is 1-1000 mM, Preferably it is 100-500 mM, More preferably, it is 200-250 mM.

  Step (d) is usually performed by immersing a film with a 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 rate 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.

  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.

  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 carried out by (i) → (b) → (c) → (2) → (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.

[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 a sodium hydroxide aqueous solution 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, and 200 mmol / L of HEPES) to 2 mL of this collagen solution, and hold at 37 ° C. for 4 hours for 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 pH 5.8 phosphate buffer solution, and 0.150 g of carbodiimide is added to an aqueous solution having a collagen fibril concentration of 10 mg / 50 mL. 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 in which 0.030 g of urease and 0.150 g of carbodiimide were dissolved in 50 mL of a phosphate buffer solution having a pH of 5.8, and the mixture was held 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.

  A bone defect with a diameter of 5 mm was formed on the top of the head of an 8-week-old male Wister rat so as not to damage 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.

  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, periosteum was firmly attached to the third group, but periosteum was not attached to the fourth group, and it 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 because collagen has good affinity with bone and epithelial tissue.

  The appearance of this 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.

  In the second group (FIG. 2 (A)) using an untreated PET membrane, new bone formation is not observed, and traces of inflammation like D are 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)

  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.   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.   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.   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.

Images