JP3668048B2 - Periodontal disease treatment material - Google Patents

Description

【００３７】
その結果は表１に示す様に、実施例品の歯周病治療材を適用した上顎左側ではセメント質の再生率が８５％以上、歯槽骨の再生率が６０％以上であるのに対し、セメント質からの溶出物を含まない比較例の架橋ゼラチン膜を適用した上顎右側での再生率は何れも１０％未満であった。
【００３８】
【発明の効果】
本発明の歯周病治療材は、生体性吸収材料からなる膜状体に、哺乳類の歯のセメント質から溶出させたヒドロキシプロリン残基含有タンパク質を含浸せしめており、上記ヒドロキシプロリン残基含有タンパク質が、繊維芽細胞、骨芽細胞やセメント芽細胞に活性を有することから、土台としての歯槽骨ならびに歯を支持し且つ細菌に対するシーリングを司る歯根膜を再生させ、これにより歯肉の上方成長も促進し、さらに歯と歯根膜とが結合組織性付着するべくセメント質を新生させる。以上のような作用でもって、歯周組織の再生を達成する。
【００３９】
したがって、従来では不可能に近いと考えられていた重度の歯周病に対しても歯を保存し、さらに健全歯周組織と同様の咬合機能を回復することができる。また、セメント質の新生が豊富であることにより、細菌に対するシーリングが達成されるので歯周病再発の危険性がほとんどない。
【００４０】
加えて、本発明の歯周病治療材は、以下のような効果を奏する。
・ 本歯周病治療材の作製は容易で、且つ安価である。
・ 均質な歯周病治療材の作製が可能である。
・ 治療方法が簡単で、早期に治癒するため、歯科医師に技術の差による効果のバラツキが少なく、また手術および手術後に受ける患者の苦痛が少ない。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a therapeutic agent for periodontal disease used in periodontal surgery for the purpose of treating periodontal disease, in particular, regeneration of periodontal tissue and recovery of occlusal function.
[0002]
[Prior art]
Periodontal disease is an inflammatory disease in which plaque (bacterial mass) destroys the cementum that covers the tooth root surface in addition to the tooth supporting tissue (gingiva, periodontal ligament, alveolar bone). It is said to be two major diseases along with tooth decay. This disease destroys the connective tissue between the teeth and gums (periodontal ligament = fibrous ligament), resulting in a pocket. As the disease progresses, this pocket deepens and the alveolar bone is destroyed and absorbed. If left untreated in this situation, teeth may fall off naturally. With the progress of treatment and prevention for dental caries, improvement of periodontal disease treatment technology has become an important issue in recent years as the aging society progresses.
[0003]
Severe periodontal diseases require treatment by surgical techniques. A typical conventional surgical method is gingival ablation curettage. This method is a technique of curettage of periodontal infected tissue, and its main purpose is to stop the progression of periodontal disease. On the other hand, consideration for tissue renewal and regeneration is not sufficient, and cementum renewal is limited to the apex, and regeneration of alveolar bone and periodontal ligament is negligible. For this reason, most of the bonds between teeth and periodontal fibrous tissue are epithelial attachment, and the sealing against bacteria is insufficient, so the risk of recurrence of periodontal disease was also high.
[0004]
Against this background, various treatment methods aiming at ideal regeneration of periodontal tissues have been proposed in recent years. One of them is the GTR method that has begun to be applied to general clinical practice in Japan. The GTR method involves completely scraping the infected tissue by gingival exfoliation curettage, inserting a sheet such as a Teflon sheet between the teeth and the gingiva, and placing the gingiva on the sheet and suturing in this state. Features. This is because the gingiva and the root surface are prevented from adhering to each other through the sheet, so that the remaining periodontal ligament cells and osteoblasts on the apical side grow and regenerate to the crown side (upward), and on the root surface. It was intended to promote the renewal of the cementum and rebuild the periodontal tissue.
[0005]
On the other hand, it has been proposed to use a composite composed of crosslinked gelatin and cementum instead of the sheet in the GTR method (Japanese Patent Laid-Open No. 07-330531, USP 5,792,508). This composite is characterized in that it is a composite film in which bovine cementitious particles are dispersed and supported on a crosslinked gelatin film as a carrier, and the composite is made by dispersing and stirring the ground cementitious particles in an aqueous gelatin solution. A suspension is prepared, impregnated and absorbed in a crosslinked gelatin membrane, and freeze-dried. When this is applied to the root surface, it is advocated that it is useful for forming a new cementum.
[0006]
[Problems to be solved by the invention]
However, the GTR method is difficult to handle and manipulate the sheet, requires a long period of time (2 to 3 months) after the operation, and regenerates the cementum and regenerates the alveolar bone and periodontal ligament. There are problems such as less adhesion, alveolar bone and root adhesion (bone adhesion), and gingival necrosis due to insufficient supply of blood from the alveolar bone side to the periodontal ligament. In some cases, the dentist's trust is not fully obtained.
[0007]
Next, in the complex composed of the crosslinked gelatin and cementum, since the pulverized cementitious particles are attacked by macrophages in the living body, the inflammation is prolonged, or the pulverized cementitious particles are fixed to the crosslinked gelatin film by a body fluid. There is a problem that the gelatin that has started to dissolve immediately and the pulverized cementitious particles are peeled off from the crosslinked gelatin film or settled in the film and the desired effect cannot be obtained.
[0008]
The present invention has been made in view of such circumstances, and its purpose is to treat periodontal disease treatment materials, in particular, severe regeneration requiring periodontal tissue regeneration and occlusal function recovery by periodontal surgery. It is an object of the present invention to provide a stable and high-performance periodontal disease treatment material suitable for use in the treatment of periodontal diseases in an industrial and inexpensive manner.
[0009]
[Means for Solving the Problems]
As a result of intensive studies, the present inventors have found that a membrane-like material obtained by impregnating a membrane made of a bioabsorbable material with a hydroxyproline residue-containing protein eluted from the cementum of mammalian teeth is suitable for the treatment of periodontal disease. The present invention was made by finding that it can be used. The present invention is described in detail below.
[0010]
The hydroxyproline residue-containing protein has activity in fibroblasts, osteoblasts, and cementoblasts. Therefore, in the form directly impregnated with the bioabsorbable material as described later, the alveolar bone as a base and the periodontal ligament supporting the teeth and controlling the bacteria are regenerated, thereby promoting the upward growth of the gingiva, The cementum is renewed so that the teeth and periodontal ligament adhere to connective tissue. The regeneration of periodontal tissue is achieved by the above-described action.
[0011]
As the hydroxyproline residue-containing protein, a protein obtained by immersing the cementum of mammalian teeth in an aqueous collagenase solution and eluting from the cementum can be used. Collagenase, which is a protein enzyme, was confirmed to suitably elute the hydroxyproline residue-containing protein by immersing the cementum in the aqueous solution. Since the dental cementum contains many protein components, it is very difficult to completely identify the protein components. However, in the present invention, it is essential to contain a hydroxyproline residue. To do.
[0012]
The collagenase concentration of the collagenase-containing aqueous solution is preferably 0.1% to 15%, and more effective if it is 0.1% to 5%. In addition, since too much or too little elution of hydroxyproline residues results in a decrease in periodontal tissue regeneration, 0.01 to 10% with respect to all amino acid residues in the elution component is preferable from that viewpoint. 0.1 to 3.0% is more preferable. Centrifugation, filtration and the like may be performed to remove insoluble matters in the eluate, but it is preferable that the eluate is not purified so much. The reason is that the more refined, the lower the periodontal tissue regeneration effect. Therefore, avoid purification to a single protein component, such as by molecular weight fractionation and chromatography.
[0013]
The properties of the cementum are not particularly limited, but it is preferable that the cementum collected from mammals, particularly bovine teeth, is pulverized to be fine powder. This is because protein components can be efficiently extracted from cementum. Tooth cementum is a hard tissue that resembles the bone (alveolar bone) of the periodontal tissue that covers the root surface, and the hydroxyproline residue-containing protein contained therein serves as a growth factor to regenerate periodontal tissue. It is thought that it contributes greatly to The advantage of using dental cementum is that there is a large amount of cementum that can be collected from a single tooth, and that large numbers of mammals such as cattle are bred for food and can be obtained easily and inexpensively.
[0014]
Next, the bioabsorbable material is a material that is decomposed and absorbed after a certain period of time after being implanted in a living body, and includes known natural products and synthetic products, that is, gelatin, collagen, fibrin, chitin, chitosan, and the like. . In addition, examples of synthetic products include polyglycolic acid, polylactic acid, and copolymers thereof. In the present invention, these bioabsorbable materials are formed into a film by means such as crosslinking. As the bioabsorbable membrane, a membrane having a thickness of 2 mm or less and a membrane thickness of 0.1 to 1.0 mm may be used in order to facilitate the operability of the membrane and the entry of cells into the root surface.
[0015]
As such a bioabsorbable film, a crosslinked gelatin film can be preferably used. The cross-linked gelatin film can be easily produced by, for example, forming a mixture of gelatin and a cross-linking agent into a film shape and performing a known cross-linking treatment, for example, heating.
[0016]
As the gelatin, for example, ordinary commercially available gelatin obtained industrially from cow bone, cow skin or pig skin by the alkali method or acidic method, or gelatin prepared by heat denaturation of collagen can be used. Of these, gelatin or purified gelatin as defined in the Japanese Pharmacopoeia 12th Amendment, or gelatin that satisfies the equivalent standards can be preferably used.
[0017]
As the crosslinking agent, for example, a water-soluble epoxy compound, a water-soluble aldehyde, or a water-soluble carbodiimide can be used. Examples of water-soluble epoxy compounds include glycerol polyglycidyl ether, sulfitol polyglycidyl ether, polyglycerol polyglycidyl ether, ethylene glycol polyglycidyl ether, polyethylene glycol diglycidyl ether, propylene glycol diglycidyl ether, propylene glycol diglycidyl ether Polypropylene glycol diglycidyl ether can be exemplified. Examples of water-soluble aldehydes include glutaraldehyde, formaldehyde, and glyoxal. Examples of the water-soluble carbodiimide include 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride. Among these, water-soluble epoxy compounds are preferable because they are easy to handle and have low toxicity, and glycerol polyglycidyl ether is particularly preferable.
[0018]
The amount of the crosslinking agent used varies depending on the type of crosslinking agent, the type of gelatin, and the like, but is usually 1 to 20% by weight based on gelatin.
[0019]
Any known treatment method may be used for crosslinking, but crosslinking by heat treatment is preferable because residual unreacted crosslinking agent can be reduced. For example, when glycerol polyglycidyl ether is used as the crosslinking agent, heat treatment at 80 to 150 ° C. for 1 to 10 hours is preferable.
[0020]
The structure of the bioabsorbable membrane is preferably a porous structure having open holes.
[0021]
A porous structure membrane having an open pore provides a circulation of biological fluid that contributes to regeneration of periodontal tissue in the pore when applied to an affected area, and thus has an advantage in promoting tissue regeneration. Furthermore, the structure of the bioabsorbable membrane is more preferably one surface is a dense surface and the other surface is a porous structure. The reason is that, in addition to the above-mentioned advantages, one surface is a dense surface, so that a stable adhesion between this surface and the root surface can be obtained, and treatment for periodontal disease is facilitated.
[0022]
A porous bioabsorbable membrane having open pores can be obtained by forming a gel-like mixture of a raw material and a crosslinking agent into a membrane and carrying out the above-mentioned crosslinking treatment after lyophilization. In particular, a bioabsorbable membrane in which one surface is a dense surface and the other surface has a porous structure, the gel-like mixture is cast on a metal or plastic plate, cooled to form a membrane gel, The gel can be frozen by cooling from the lower surface of the plate, followed by lyophilization, and can be easily adjusted by performing a crosslinking treatment as described above.
[0023]
Impregnation of the bioprosorbable membrane with the hydroxyproline residue-containing protein is performed, for example, as follows. The collagenase-containing aqueous solution in which the hydroxyproline residue-containing protein is eluted from the cementum is freeze-dried to obtain a hydroxyproline residue-containing protein solid. This solid material is dissolved in distilled water, and the bioabsorbable membrane is uniformly impregnated, followed by lyophilization. At this time, the periodontal tissue regeneration ability decreases if the amount of impregnation of the hydroxyproline residue-containing protein into the bioabsorbable membrane is too large or too small. From this viewpoint, the amount of impregnation of the hydroxyproline residue-containing protein is preferably 0.00001 mg to 10 mg / square centimeter, and more preferably 0.0001 mg to 3 mg / square centimeter. The periodontal disease treatment material of this invention is completed through the above processes.
[0024]
Such a periodontal disease treatment material of the present invention can be suitably used for periodontal surgery, particularly gingival exfoliation curettage. That is, basically after the gingival exfoliation curettage, the treatment is completed simply by inserting the periodontal disease treatment material of the present invention cut into an appropriate size into the exposed root surface, covering it with gingiva, and suturing it. . In recent years, aesthetic requirements for periodontal disease patients have increased, but the periodontal disease treatment material of the present invention is preferably used for gingival alveolar mucoplasty such as free gingival grafting and pedicled gingival flap lateral transplantation. In any case, it is possible to restore healthy gingiva to the exposed root surface as periodontal disease progresses.
[0025]
【Example】
Hereinafter, the present invention will be described in more detail with reference to Examples, Comparative Examples, and Test Examples.
[0026]
Example ( 1) Adjustment of components eluted from cementum Teeth were extracted from bovine jawbone provided by the edible meat slaughterhouse with extraction forceps and stored frozen at -4 ° C. After thawing, the immediately attached gingival tissue was scraped and removed. After these treatments, a surgical scaler is also used to scrape the cementum with 100 to 200 strokes per tooth (the number of strokes of bovine teeth is 0.4 to 0.5 mm because the thickness of the cementum is 0.4 to 0.5 mm). However, the lower dentin was not reached), and cementitious cuttings were obtained from 200 bovine teeth. The excised piece of cementum is freeze-dried using a freeze-drying apparatus (DURA-STOP, FTSSYSTEMS, INK.), Then put into a mortar, crushed with a pestle, further sieved, and a particle size of 0.075 mm or less 9.3 g of cementitious particles were obtained. Further, the obtained cementitious particles were put into 30 ml of 1% collagenase-containing physiological saline at pH 7, and stirred with a stirrer for 1 hour. Insoluble matter was removed by centrifugation at 3000 rpm for 10 minutes to obtain a supernatant. This supernatant was freeze-dried using a freeze-drying apparatus (DURA-STOP, manufactured by FTSSYSTEMS, INK.) To give 0.73 g of a solid (elution component 0.16 g, collagenase 0.3 g, sodium chloride 0.27) Got.
[0027]
Hydroxyproline residues were contained in this eluted component at a ratio of 0.5% with respect to all amino acid residues.
[0028]
(2) Preparation of cross-linked gelatin film Commercially available gelatin produced by an alkali method [manufactured by Nippi Co., Ltd., glycerol as a cross-linking agent in a 5 wt% aqueous solution of a viscosity of 28 mp and a jelly strength of 96 g] Polyglycidyl ether [manufactured by Nagase Kasei Kogyo Co., Ltd.] was added and dissolved in 3% by weight of the gelatin weight to prepare a mixed solution of gelatin and glycerol polyglycidyl ether. One side of a polymethyl methacrylate plate (size 10 x 10 cm, thickness 2 mm) is trimmed with a silicon tape 1 cm wide and 1.0 mm thick, and 3 g of the above mixture is cast inside (8 x 8 cm). did. This was freeze-dried using a freeze-drying apparatus (DURA-STOP, FTSSYSTEMS, INK.) To obtain a gelatin film (film size 8 × 8 cm, film thickness 0.3 mm). The gelatin film was crosslinked by heat treatment at 110 ° C. for 2 hours to obtain a crosslinked gelatin film.
[0029]
(3) Preparation of composite membrane 0.684 g of the solid material prepared in (1) (elution component 0.15 g) was dissolved in 1000 ml of distilled water, 2 g of the solution was collected, and a polymethyl methacrylate plate (size 10) was prepared. The film was trimmed with a silicon tape having a width of 1 cm and a thickness of 1.0 mm on one side of × 10 cm and a thickness of 2 mm, and cast on the inside (8 × 8 cm). Furthermore, the crosslinked gelatin film prepared in (2) is placed on the crosslinked gelatin film, and the dissolved solution is absorbed into the crosslinked gelatin film. Then, using a freeze-drying apparatus (DURA-STOP, FTSSYSTEMS, INK.). Freeze drying was performed to obtain a crosslinked gelatin film (composite film) in which the eluate from the cementum was combined. Moreover, the compounding amount of the elution component from cementum is 0.0156 mg / cubic centimeter. The composite membrane was cut to a size of 1 × 1 cm and used as a periodontal disease treatment material.
[0030]
Comparative Example ( 1) Preparation of 1% collagenase-containing pH 7 physiological saline 30 ml of 1% collagenase-containing physiological saline is prepared, and freeze-dried using a freeze-drying apparatus (DURA-STOP, manufactured by FTSSYSTEMS, INK.). In this way, 0.57 g of solid matter (elution component 0, collagenase 0.3 g, sodium chloride 0.27) was obtained.
[0031]
(2) Preparation of bioabsorbable material A commercially available gelatin manufactured by the alkali method [manufactured by Nippi Co., Ltd., with a viscosity of 28 mp and a jelly strength of 96 g]. Glycerol polyglycidyl ether [manufactured by Nagase Kasei Kogyo Co., Ltd.] was added and dissolved in 3% by weight of the gelatin weight to prepare a mixture of gelatin and glycerol polyglycidyl ether. One side of a polymethylmethacrylate plate (size 10 x 10 cm, thickness 2 mm) is trimmed with a 1 cm wide and 1.0 mm thick silicon tape, and 3 g of the above mixture is cast on the inside (8 x 8 cm). did. This was freeze-dried using a freeze-drying apparatus (DURA-STOP, manufactured by FTSSYSTEMS, INK.), So that a freeze-dried gelatin film (film size 8 × 8 cm, film thickness 0.3 mm) was obtained. Obtained. The gelatin film was crosslinked by heat treatment at 110 ° C. for 2 hours to obtain a crosslinked gelatin film.
[0032]
(3) Preparation of composite membrane 0.534 g of the solid material prepared in (1) (elution component 0 g) was dissolved in 1000 ml of distilled water, 2 g of the solution was collected, and a polymethyl methacrylate plate (size: 10 × 10 cm) , A thickness of 2 mm) was trimmed with a silicon tape having a width of 1 cm and a thickness of 1.0 mm, and cast on the inner side (8 × 8 cm). Furthermore, the crosslinked gelatin film prepared in (2) is placed on the crosslinked gelatin film, and the dissolved solution is absorbed into the crosslinked gelatin film. Then, using a freeze-drying apparatus (DURA-STOP, FTSSYSTEMS, INK.). Freeze-drying was performed to obtain a crosslinked gelatin film containing no eluate from cementum. The composite membrane was cut to a size of 1 × 1 cm.
[0033]
Test example (periodontal tissue regeneration experiment)
After removing the maxillary left and right incisors of the Japanese monkey and the buccal mucosal periosteal flap of the second premolar, 4 mm of the buccal alveolar bone was removed in the apical direction. At this time, the interdental alveolar bone was completely deleted to make the bone defect form a dehiscence model. Thereafter, the periodontal disease treatment material prepared in Example 1 was inserted into the left side incisor of the upper jaw and the exposed root surface portion of the second premolar, while the side incisor and the second premolar of the upper right side were comparative examples. After inserting the crosslinked gelatin film containing no elution components from the cementum prepared in 1, the gingival flap was returned to its original position and sutured.
[0034]
The Japanese macaques were sacrificed 3 weeks after the operation and fixed for perfusion, and then tissue blocks were collected. Then, according to the usual method, a paraffin section is prepared, subjected to HE staining, histological observation is performed, the newly formed and attached cementum length and the newly formed alveolar bone length are measured, The regeneration rate of cementum and alveolar bone was calculated.
[0035]
-Cement regeneration rate (%): Length of new adhesion (mm) ÷ 4 (mm) x 100
・ Alveolar bone regeneration rate (%): Length of new bone (mm) ÷ 4 (mm) x 100
[0036]
[Table 1]

[0037]
As shown in Table 1, the results show that the cementum regeneration rate is 85% or more and the alveolar bone regeneration rate is 60% or more on the left side of the upper jaw where the periodontal disease treatment material of the example product is applied. The regeneration rate on the right side of the upper jaw to which the cross-linked gelatin film of the comparative example containing no eluate from cementum was applied was less than 10%.
[0038]
【The invention's effect】
Periodontal disease treatment material of the present invention, the film-like member made of a bio-absorbing material has impregnated hydroxyproline residues containing protein was eluted from the cementum of a tooth of a mammal, the hydroxyproline residues containing proteins However, because it has activity on fibroblasts, osteoblasts and cementoblasts, it regenerates the alveolar bone as a foundation and the periodontal ligament that supports the teeth and controls sealing against bacteria, thereby promoting the upward growth of the gingiva Further, the cementum is renewed so that the teeth and periodontal ligament adhere to connective tissue. The regeneration of periodontal tissue is achieved by the above-described action.
[0039]
Therefore, the teeth can be preserved even for severe periodontal diseases that have been thought to be almost impossible in the past, and the occlusal function similar to that of healthy periodontal tissues can be recovered. In addition, because of the abundance of new cementum, sealing against bacteria is achieved, so there is almost no risk of periodontal disease recurrence.
[0040]
In addition, the periodontal disease treatment material of the present invention has the following effects.
・ The preparation of this periodontal disease treatment material is easy and inexpensive.
・ A homogeneous periodontal disease treatment material can be produced.
-Because the treatment method is simple and heals early, there is little variation in the effects due to differences in technology among dentists, and there is less pain for patients who undergo surgery and after surgery.

Claims (3)

A periodontal disease treatment material obtained by impregnating a membrane-like body composed of a bioabsorbable material with a hydroxyproline residue-containing protein eluted from a cementum of a mammalian tooth . The periodontal disease treatment material according to claim 1, wherein the amount of the hydroxyproline residue-containing protein impregnated is 0.0001 mg to 3 mg / square centimeter. 3. The tooth according to claim 1, wherein the hydroxyproline residue content in the hydroxyproline residue-containing protein is 0.1 to 3.0% based on the total amino acid residue content. Peri-disease treatment material.