JP4836318B2 - Artificial dental root with periodontal ligament formation ability - Google Patents

Description

【００８５】
上記表１に示すように、本発明品のＨＡ−ＴおよびＴｉ−Ｔは、いずれも人工歯根上にセメント質の沈着が見られ、また血管を伴った線維性結合組織が形成されるので、歯根膜が形成されていく途上にあったのに対し、比較例品のＨＡ−Ｂ、Ｔｉ−Ｂはいずれも人工歯根が周囲の組織と骨性癒着していた。
【００８６】
【発明の効果】
以上のように本発明の人工歯根によれば、ヒドロキシプロリン残基含有タンパク質を含むタンパク質成分は線維芽細胞、骨芽細胞、セメント芽細胞に活性を有しているので、表面のヒドロキシプロリン残基含有タンパク質を含むタンパク質成分が基材表面にセメント質を新生させ、さらに骨との間に歯根膜を再生させ、その際、セメント質と歯槽骨がコラーゲン線維束で結びつけられるので、人工歯根周囲に天然歯と同様の機能を有する歯根膜を安全に形成する。
【００８７】
また、本発明の人工歯根は、ヒドロキシプロリン残基含有タンパク質を含むタンパク質成分が哺乳類の歯などから容易かつ安価に入手でき、さらに、その含有濃度が小さくても十分な効果を奏するため、工業的に安定した品質で、かつ安価に供給することができる。
【００８８】
また、本発明において、ヒドロキシプロリン残基含有タンパク質を含むタンパク質成分を含む生体吸収性膜を表面に被着した場合、人工歯根の表面に上記タンパク質成分を容易に固定することができ、タンパク質成分の除放速度を制御ができる。さらに生体吸収性材料が人工歯根周囲にセメント質及び歯根膜が形成されるされるまでの期間では細胞の足場として働き、歯根膜形成後には吸収分解される。以上の理由から人工歯根の周囲にセメント質及び歯根膜を形成する機能が良好なものとなる。
【００８９】
次に、本発明において、上記ヒドロキシプロリン残基含有タンパク質を含むタンパク質成分の担持量を０．００００１mg〜１５ｍｇ／平方センチメートルであるようにすること、及び／又は、上記ヒドロキシプロリン残基含有タンパク質を含むタンパク質成分に含まれるヒドロキシプロリン残基の含有量を全アミノ酸残量に対して０．０１〜１０％であるようにすることで、人工歯根の周囲にセメント質及び歯根膜を形成する機能が良好なものとすることができる。
【００９０】
また、本発明において、上記ヒドロキシプロリン残基含有タンパク質を含むタンパク質成分を哺乳類の歯のセメント質から抽出することで、ヒトは起源が哺乳類と近いため、哺乳類の歯のセメント質に含まれるタンパク質の構造がヒトのセメント質に含まれているタンパク質の構造が他の類よりも類似しているため、人工歯根の周囲にセメント質及び歯根膜を形成する機能が良好なものとなる。
【００９１】
さらに、牛または豚の歯のセメント質を用いるようにすると、食用に牛や豚などが屠殺されるので、それらの歯から容易にまた安価にセメント質を入手することができる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an artificial tooth root, particularly an artificial tooth root intended to restore a healthy occlusal function by forming a periodontal ligament such as a connection structure between a natural tooth root and the surrounding tissue. It is.
[0002]
[Prior art]
Conventionally, a prosthetic treatment mainly using a bed denture or a bridge denture has been performed as a treatment method for a case in which a tooth is lost. However, there are many cases in which mastication, pronunciation, and restoration of aesthetics are not sufficient and patients cannot be satisfied. Therefore, in recent years, an implant method has been developed for such cases and is widely used clinically.
[0003]
This implant method is a method in which an artificial dental root made of a biocompatible material such as titanium or hydroxyapatite is embedded in an alveolar bone or a jawbone, and an upper structure such as an artificial tooth or a denture is held therein. However, the joint mode of the artificial root and surrounding tissue by this treatment is an osteogenic adhesion in which the artificial root directly contacts the alveolar bone.
[0004]
In such an artificial tooth root, the occlusal pressure is directly transmitted to the jawbone due to this bony adhesion. Therefore, the bone gradually disappears (bone resorption) as a response of the living body to these mechanical stresses, and the artificial root is shaken or subsided. Plaques are deposited in the gap caused by the artificial root being shaken or subsidized. It has been regarded as a problem that infection and inflammation are caused by the deposition of selenium, and that the infection and inflammation lead to severe symptoms extending to the alveolar bone and jawbone.
[0005]
These problems are thought to be due to the difference in the joint structure between the artificial root-alveolar bone and the natural root-alveolar bone.
[0006]
In natural tooth roots, cementum (calcified connective tissue covering the root dentin surface, thickness is usually 0.02-0.15 mm) between the root and alveolar bone, periodontal ligament (between cementum and alveolar bone) Is a dense fibrous connective tissue, usually 0.2 to 0.25 mm thick, and a collagen fiber bundle runs from the root to the alveolar bone. This collagen fiber bundle strongly connects the root to the alveolar bone.
[0007]
The periodontal ligament adjusts the occlusal pressure by buffering the occlusal pressure, sensing the occlusal pressure via the baroreceptor (the organ that senses the occlusal pressure), and the neuromodulation effect based on the occlusal pressure detected by the baroreceptor Action). In addition, the periodontal ligament acts as a barrier against infection and has the effect of suppressing the induction of osteoclasts that cause bone resorption.
[0008]
On the other hand, the cementum not only works to connect the root and periodontal ligament, but also plays a role in maintaining the periodontal ligament and maintaining the shape of the alveolar bone. ing.
[0009]
Such a connection structure between the natural tooth root and the surrounding tissue is desirably reproduced also between the planted artificial tooth root and the surrounding tissue. In particular, it is preferable that a periodontal ligament exists.
[0010]
For this purpose, artificial tooth root planting methods using cultured periodontal ligament cells have been proposed in recent years.
[0011]
Japanese Patent Laid-Open No. 6-7381 shows such a technique. In this conventional technique, periodontal ligament cells collected from the extracted tooth are cultured, and a pseudo periodontal ligament formed by further culturing the cultured periodontal ligament cells is interposed between the artificial tooth root and the alveolar bone, thereby artificial tooth root. It has been proposed to regenerate periodontal ligaments around.
[0012]
In addition, Byung-Ho Choi has conducted experiments in which canine periodontal ligament cells are cultured on the surface of a pure titanium artificial tooth root and embedded in the same canine jawbone, and the results have been reported (Int). J Oral Maxilofac Implants 2000; 15: 193-196).
[0013]
According to Choi, in such an experiment, a cement tissue accompanied by the encapsulation of collagen fibers has been observed in a part of the surface of the artificial tooth root. Here, the encapsulation of collagen fibers refers to a state in which collagen fibers are confined and integrated in cementum. Collagen fibers confined in this cement are bound to collagen fibers in the periodontal ligament. In addition, a periodontal ligament-like tissue is observed between the cement-like tissue and the alveolar bone.
[0014]
On the other hand, the use of cementitious particles has been proposed as another type of technology with the above-mentioned purpose of reproducing the joint structure between a natural tooth root and surrounding tissue between the planted artificial tooth root and surrounding tissue. Yes.
[0015]
JP-A-8-266559 and USP 5,772,439 show such a technique.
[0016]
This technique is characterized by using a hybrid artificial tooth root in which cementitious particles are deposited on the surface of the artificial tooth root. The cement particles can be collected from human or animal teeth. In addition, the above-described prior art document describes that a bioabsorbable material or an inorganic compound is used as a medium in order to adhere cement particles to an artificial tooth root.
[0017]
[Problems to be solved by the invention]
However, in the above-described method using cultured periodontal ligament cells, it is necessary to obtain periodontal ligament cells surgically from the same person who implants the artificial tooth root or from another person who does not cause rejection. Furthermore, since the complicated operation of culturing the obtained periodontal ligament cells is also required, the versatility is poor. In addition, the results of the Byung-Ho Choi animal experiment show that the formation of cementitious tissue and periodontal ligament-like tissue is not sufficient. Therefore, at present, a great effect cannot be expected clinically.
[0018]
Next, in a hybrid artificial tooth root in which cementitious particles are applied to the surface of the artificial tooth root, the cementitious particles are recognized as foreign substances in the living body and are likely to be attacked by macrophages. In addition, since the protein component that contributes to the formation of periodontal ligament etc. from cementum is very slow (poorly water-soluble state), the action of forming periodontal ligament etc. is insufficient. And this conventional technique has a prolonged inflammation, it is actually difficult to disperse and fix the cementitious particles uniformly on the artificial tooth root, and the cementitious particles are removed from the hybrid artificial tooth root embedded in the alveolar bone. Since there is a risk of dropping out, a clinically stable effect cannot be expected.
[0019]
The present invention has been made in view of such circumstances, and its object is to industrially produce a safe artificial tooth root that can regenerate a periodontal ligament around the same as in the case of natural teeth. The goal is to supply products with stable quality and at low cost.
[0020]
[Means for Solving the Problems]
As a result of intensive studies, the present inventor has a periodontal ligament forming ability in which a protein component containing a hydroxyproline residue-containing protein is supported in a readily water-soluble state on the surface of a bone-embedded portion of a base material made of titanium. It is an artificial tooth root, and the cross-linked carboxymethyl chitin is deposited on the surface of the bone-implanted portion,TheCross-linked carboxymethyl chitinIs impregnated with a solution of the protein component and dried,The protein component is supported.
[0021]
Protein components, including hydroxyproline residue-containing proteins, promote the growth of undifferentiated mesenchymal cells in the gingiva and jawbone in the environment of the gingiva and jawbone, and further induce differentiation into cementoblasts to produce cementum. Formation and further proliferation of undifferentiated mesenchymal cells into fibroblasts and osteoblasts to induce collagen fiber production and intrinsic alveolar bone (alveolar bone encapsulating collagen fibers on the outermost surface of the alveolar bone) Promote the formation of On the other hand, the newly formed cementum has an effect that the alveolar bone cannot be brought within a certain distance (a distance corresponding to the width of the periodontal ligament within about 0.2 to 0.25 mm). Periodontal ligament is formed during the period.
[0022]
And since the protein component containing the said hydroxyproline residue containing protein exists in a water-soluble state, the effect | action of the said activity expresses from an early stage.
[0023]
As a result, the protein component including the hydroxyproline residue-containing protein on the surface regenerates cementum on the surface of the base material, and further regenerates the periodontal ligament between the bone and the cementum and alveolar bone. They are tied together in bundles. A periodontal ligament having the same function as that of a natural tooth is formed around the artificial tooth root with the above action.
[0024]
In addition, the artificial dental root of the present invention can be obtained easily and inexpensively from protein teeth containing a hydroxyproline residue-containing protein, such as mammalian teeth. Can be supplied at low cost with stable quality.
[0025]
Also,The present inventionThe artificial tooth roots of the skin are coated with a bioabsorbable membrane containing protein components including hydroxyproline residue-containing proteins on the surface.Preferably.
[0026]
According to such a configuration, the protein component can be easily fixed on the surface of the artificial tooth root, and the controlled release rate of the protein component can be controlled. Furthermore, the bioabsorbable material acts as a scaffold for cells until cementum and periodontal ligament are formed around the artificial tooth root, and is absorbed and decomposed after periodontal ligament formation. For the above reasons, the function of forming cementum and periodontal membrane around the artificial tooth root is good.
[0027]
next,The present inventionThe artificial tooth root ofin frontThe carrying amount of the protein component including the hydroxyproline residue-containing protein is 0.00001 mg to 15 mg / square centimeter.Is preferred.
[0028]
According to such a configuration, the function of forming cementum and periodontal membrane around the artificial tooth root is good.
[0029]
The present inventionThe artificial tooth root ofin frontThe hydroxyproline residue contained in the protein component including the hydroxyproline residue-containing protein is 0.01 to 10% with respect to the total amino acid residue content.Is preferred.
[0030]
According to such a configuration, the function of forming cementum and periodontal membrane around the artificial tooth root is good.
[0031]
The present inventionThe artificial tooth root ofin frontProtein components extracted from the cementum of mammalian teethMinBeIs preferred.
[0032]
According to such a configuration, since human origin is close to mammals, the structure of the protein contained in the cementum of mammal teeth is similar to that of other reptiles etc. Yes. For this reason, the protein extracted from the cementum of mammalian teeth has a higher activity against human tissues, and the function of forming cementum and periodontal membrane around the artificial tooth root is improved.
[0033]
The present inventionThe artificial tooth root ofin frontThe mammal is a cow or pigIs preferred.
[0034]
According to such a configuration, since cattle, pigs, and the like are slaughtered for food, cementum can be easily and inexpensively obtained from their teeth.
[0035]
DETAILED DESCRIPTION OF THE INVENTION
The present invention is described in detail below.
[0036]
(About artificial tooth root materials)
The material of the artificial tooth root in the present invention is not particularly limited as long as it has little harmful effect on the living body. Titanium, hydroxyapatite, alumina, zirconia and the like excellent in durability can be suitably used.
[0037]
Of these, hydroxyapatite and titanium can be particularly preferably used. The reason for this is that hydroxyapatite has osteoconductivity, so it promotes the formation of cementum and can be firmly bonded to cementum. This is because titanium has a high affinity with cementum.
[0038]
(Surface treatment of artificial tooth roots)
In the present invention, a known treatment for further enhancing biocompatibility can be applied to the surface of the artificial tooth root.
[0039]
As a treatment for further enhancing the biocompatibility of the surface of the artificial tooth root, for example, when the surface of the artificial tooth root is made of titanium, the surface is immersed in an alkaline aqueous solution to form an alkali titanate phase on the surface. Subsequent heat treatment of the surface can improve the biocompatibility of the surface.
[0040]
By the way, when carrying the protein which contains a hydroxyproline residue on the surface of an artificial tooth root using the bioabsorbable material mentioned later, the well-known surface treatment for fixing a bioabsorbable material more firmly can be given. .
[0041]
For example, when the surface of the artificial tooth root is made of titanium, an ethylene-vinyl alcohol copolymer is welded or applied to the surface as such a treatment method. Subsequently, this is dried to adhere the ethylene-vinyl alcohol copolymer to the surface of the artificial tooth root. Further, the surface is treated with ozone to oxidize the hydroxyl group and introduce a carboxy group.
[0042]
The surface of the artificial tooth root that has been subjected to such treatment can more firmly fix the bioabsorbable material by ion adsorption.
[0043]
For example, collagen can be firmly fixed by immersing the artificial tooth root subjected to the above treatment in an aqueous collagen solution.
[0044]
(About protein components including proteins containing hydroxyproline residues)
Hydroxyproline is C_FiveH₉NO_ThreeIt is represented by the chemical formula (molecular weight 131.13). Proline is hydroxylated at position 3 or 4. The hydroxyproline residue is a group obtained by leaving H from the imino group NH of hydroxyproline and OH from the carboxyl group COOH, that is, one molecule of water.
[0045]
Examples of the protein containing a hydroxyproline residue include tropcollagen alpha chain.
[0046]
The protein component containing the hydroxyproline residue-containing protein can be obtained from dental cementum such as mammals. Dental cementum is a hard tissue that resembles the bone of the periodontal tissue (alveolar bone) that covers the surface of the root, and as mentioned above, the protein component including the hydroxyproline residue-containing protein is contained therein. It has activity on fibroblasts, osteoblasts, and cementoblasts. The advantage of using dental cementum is that it is rich in protein components including hydroxyproline residue-containing proteins involved in the regeneration of cementum and periodontal ligament. Among mammals, when cattle and pig teeth are used, a large amount of mammals such as cattle and pigs are bred for food, so that homogeneous cementum can be obtained easily and inexpensively.
[0047]
As a method for obtaining a protein component containing a hydroxyproline residue-containing protein from the above teeth, a powdered tooth cementum is immersed in an aqueous collagenase solution. Protein components including hydroxyproline residue-containing protein are eluted in this aqueous solution. The protein component containing the hydroxyproline residue-containing protein thus eluted is naturally water-soluble.
[0048]
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, if the content of hydroxyproline residues contained in the elution component (protein component containing hydroxyproline residues) is too much or too little, the function of forming cementum and periodontal ligament around the artificial tooth root is reduced. Come on. In this respect, the content of the hydroxyproline residue is preferably 0.01% to 10%, more preferably 0.1% to 3.0%, based on all amino acid residues contained in the component.
[0049]
In the present invention, a plurality of proteins forming the periodontal ligament are contained in the water-soluble protein component in the dental cementum. Periodontal tissue is regenerated when all or more of this protein is present at a certain concentration, but there is no such activity in a single protein state where all proteins are separated and purified. The protein forming the periodontal ligament has a hydroxyproline residue protein as an essential protein. Therefore, it is essential to contain a hydroxyproline residue.
[0050]
Centrifugation, filtration and the like may be performed in order to remove unnecessary substances 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 function of forming cementum and periodontal membrane around the artificial tooth root. Therefore, avoid purification to a single protein component, such as by molecular weight fractionation and chromatography.
[0051]
The presence or absence of a protein containing a hydroxyproline residue can be confirmed by hydrolyzing the protein component to the amino acid level and examining whether the hydroxyproline residue is contained using a mass spectrometer. it can.
[0052]
Further, the content of the hydroxyproline residue contained in the elution component is determined by hydrolyzing the protein component to the amino acid level in the same manner as above, and fully automated amino acid analyzer, such as mnoAminotac DIC-500 / V manufactured by JEOL. Can be measured by quantifying each amino acid residue. However, when collagenase is used for elution, collagenase is contained in the eluate. Therefore, after measuring the amount of amino acid residues contained in the eluate by the method described above, Calculate the content of hydroxyproline residues by correcting the amount of amino acid residues, or by calculating the protein components in the eluate by removing the collagenase using electrophoresis or the like. It is necessary to measure.
[0053]
(Regarding a method for loading a protein component containing a hydroxyproline residue-containing protein on the surface of an artificial tooth)
The method for supporting the protein component containing the hydroxyproline residue-containing protein of the present invention on the surface of the artificial tooth root is not particularly limited, and various methods can be used. For example, as a method of using a natural polymer or a synthetic polymer, a method of impregnating, adhering or adsorbing the protein component to the surface of an artificial tooth using a bioabsorbable material as a binder, impregnating, adhering or adsorbing the protein component Examples thereof include a method of preparing a polymer sheet and sticking it on the surface of an artificial tooth root. Moreover, the method etc. which form the porous body of a hydroxyapatite on the artificial tooth root surface, and adsorb | suck the said protein component in this can be mentioned. Among these, a method of supporting a protein component containing a hydroxyproline residue-containing protein on the surface of an artificial tooth using a bioabsorbable material is preferable.
[0054]
The bioabsorbable material is a material that is decomposed and absorbed after a certain period of time after being implanted in a living body. The rate of sustained release of protein components including hydroxyproline residue-containing protein in the body can be controlled using the degradation rate of the bioabsorbable material in vivo. Bioabsorbable materials provide a scaffold for cell growth and differentiation.
[0055]
Examples of such bioabsorbable materials include aliphatic polyesters such as polylactic acid, polyglycolic acid, and poly (ε-caprolactone), proteins and polysaccharides such as collagen, gelatin, albumin, dextran, chitin, and chitosan, and these Aliphatic polyesters, protein and polysaccharide derivatives, composites, and cross-linked products can be used. Among these, atelocollagen, cross-linked atelocollagen, cross-linked gelatin, carboxymethyl chitin, and cross-linked carboxymethyl chitin can be more preferably used.
[0056]
(When using bioabsorbable materials-1)
As a method of supporting a protein component containing a hydroxyproline residue-containing protein on the surface of the artificial tooth root using a bioabsorbable material, for example, a bioabsorbable membrane carrying a protein component containing a hydroxyproline residue-containing protein is prepared, This can be applied to the surface of the artificial tooth root. Here, the method for depositing the bioabsorbable membrane on the surface of the artificial tooth root is not particularly limited. Any known method may be used.
[0057]
Specifically, for example, a gelatin aqueous solution in which glyceral polyglycidyl ether is added and dissolved as a crosslinking agent is prepared. This aqueous solution is cast on a plate made of polymethyl methacrylate and then cooled to prepare a film-like gelatin gel. Further, this membrane-like gelatin gel is lyophilized to remove water and then subjected to heat treatment to obtain a porous crosslinked gelatin membrane.
[0058]
On the other hand, the cementum is mechanically scraped from the roots of cattle and dried. A product pulverized with a mortar or the like is put into a physiological saline containing an appropriate amount of collagenase, stirred and dissolved. Thereafter, the solution from which insolubles have been removed by centrifugation is freeze-dried to obtain a solid protein component containing a hydroxyproline-containing protein.
[0059]
Next, the cross-linked gelatin film is impregnated with an aqueous solution in which an appropriate amount of a protein component-containing protein containing a hydroxyproline residue-containing protein is dissolved, and freeze-dried. Thereby, a bioabsorbable membrane carrying a protein component containing a hydroxyproline residue-containing protein can be obtained.
[0060]
Next, a gelatin aqueous solution in which glycerol polyglycidyl ether is added and dissolved is applied to the surface of the artificial tooth root, and a bioabsorbable membrane carrying a protein component containing the hydroxyproline residue-containing protein is attached thereon. And the artificial tooth root of this invention can be obtained by drying this.
[0061]
In addition, when a bioabsorbable material is water-soluble, a crosslinking process etc. can be performed and the solubility to water can be reduced.
[0062]
(When using bioabsorbable materials-2)
In addition, as another method of carrying a protein component containing a hydroxyproline residue-containing protein on the surface of the artificial tooth root using a bioabsorbable material, after attaching the bioabsorbable material to the surface of the artificial tooth root, Protein components including hydroxyproline residue-containing proteins can be introduced on the surface.
[0063]
Specifically, glycerol polyglycidyl ether is added as a crosslinking agent to prepare a dissolved carboxymethylchitin aqueous solution. This aqueous solution is applied to the surface of the base material of the artificial tooth root using a brush or the like. This is freeze-dried and then subjected to heat treatment to adhere cross-linked carboxymethyl chitin to the surface of the artificial tooth root.
[0064]
Next, the surface of the artificial tooth root is impregnated with an aqueous solution in which a protein component containing an appropriate amount of a hydroxyproline residue-containing protein is dissolved. The moisture can be dried to obtain the artificial tooth root of the present invention.
[0065]
It should be noted that if the amount of the protein component containing the hydroxyproline residue-containing protein is too much or too little, the ability to form cementum and periodontal ligament between the artificial tooth root and the alveolar bone is lowered. From this viewpoint, the loading amount of the protein component including the hydroxyproline residue-containing protein in the bone-embedded portion is preferably 0.00001 mg to 15 mg / square centimeter, and more preferably 0.0001 mg to 6 mg / square centimeter.
[0066]
The carrying amount per unit area of the protein component containing the hydroxyproline residue-containing protein can be measured as follows. By immersing and stirring the artificial tooth root in water, the protein component including the hydroxyproline residue-containing protein carried on the surface of the artificial tooth root is eluted. After performing a desalting treatment using a dialysis membrane, the weight of a protein component including a hydroxyproline residue-containing protein from which collagenase has been removed is measured using electrophoresis. It can be determined by dividing the weight of the protein component containing the hydroxyproline residue-containing protein by the surface area of the artificial tooth root surface on which the protein component is supported.
[0067]
【Example】
Hereinafter, the present invention will be described in more detail with reference to Examples, Comparative Examples, and Test Examples.
Example 1
Artificial tooth root (HA-T)
Artificial tooth root material used: Hydroxyapatite
Bioabsorbable material used: Cross-linked gelatin
Extraction of protein components including hydroxyproline residue-containing protein from cementum
Teeth were extracted from the bovine jawbone provided by the edible meat slaughterhouse with extraction forceps and stored frozen at -4 ° C. Immediately after thawing, the gingival tissue that did not adhere to the tooth root was scraped and removed. After these treatments, using a surgical scaler, the cementum was scraped with 100 to 200 strokes per tooth, and excised pieces of cementum were obtained from 100 bovine teeth. 3. The cementitious excised piece is freeze-dried with a freeze-drying device (FREEZE DRYER FD-5N, manufactured by Tokyo Rika Kikai Co., Ltd.), then placed in a mortar, further sieved, and cementum having a particle size of 0.088 mm or less. 9 g was obtained.
[0068]
Further, the obtained cementitious particles were put into 15 ml of physiological saline containing pH 7 containing 1% collagenase (manufactured by Nitta Gelatin Co., Ltd., COLAGGENASE N-2) and stirred at 37 ° C. for 1 hour using a stirrer. Insoluble matter was removed by centrifugation at 3000 rpm for 10 minutes to obtain a supernatant.
[0069]
The supernatant was freeze-dried using a freeze dryer (FREEZE DRYER FD-5N, manufactured by Tokyo Rika Kikai Co., Ltd.) to obtain a solid 0.39 (0.10 g eluted component from cementum, 0.15 g collagenase) Sodium chloride 0.14 g) was obtained.
[0070]
During the elution from the cementum, hydroxyproline residues were contained at a ratio of 0.5% with respect to all amino acid residues.
Preparation of cross-linked gelatin film
Glycerol polyglycidyl ether [Nagase Kasei Kogyo Co., Ltd.] as a cross-linking agent in a commercially available gelatin manufactured by an alkaline method [manufactured by Nitta Gelatin Co., Ltd., with a viscosity of 31 mp and a jelly strength of 94 g when made into a 6.66% aqueous solution] 3% by weight with respect to the weight of gelatin was added and dissolved to prepare a mixed aqueous solution of gelatin and glycerol polyglycidyl ether. One side of a polymethylmethacrylate plate (size: 10 × 10 cm, thickness: 2 mm) is trimmed with a silicon tape having a width of 1 cm and a thickness of 1.0 mm, and 2 g of the above mixed aqueous solution is poured inside (8 × 8 cm). Extended. This was freeze-dried with a freeze dryer (FREEZE DRYER FD-5N manufactured by Tokyo Rika Kikai Co., Ltd.) to obtain a gelatin film (film size 8 × 8 cm, film thickness 0.2 mm). The gelatin film was cross-linked by heat treatment at 110 ° C. for 2 hours to obtain a cross-linked gelatin film.
Crosslinked gelatin membrane carrying a protein component containing a hydroxyproline residue-containing protein
0.0585 g (0.015 g of an elution component from cementum) of the solid prepared in (1) was dissolved in 15 ml of distilled water. A polymethylmethacrylate plate (size: 10 × 10 cm, thickness: 2 mm) is trimmed with a silicon tape having a width of 1 cm and a thickness of 1.0 mm on one side, and 1.5 g of the solution is added on the inside (8 × 8 cm). Was collected and cast. Further, the cross-linked gelatin film prepared in (2) is placed thereon, and the solution is absorbed into the cross-linked gelatin film, followed by freeze-drying machine (FREEZE DRYER FD-5N manufactured by Tokyo Rika Equipment Co., Ltd.) Was freeze-dried to obtain a crosslinked gelatin film carrying an eluate from cementum. Moreover, the carrying amount of the protein component containing the hydroxyproline residue-containing protein was 0.023 mg / square centimeter.
Adjustment of artificial root HA-T
Commercially available artificial tooth root [apaceram, length 9 mm, diameter 4.5 mm, surface material is hydroxyapatite, manufactured by Asahi Optical Co., Ltd.] surface, commercially available gelatin [Nitta Gelatin Co., Ltd., 6.66% aqueous solution The viscosity was 31 mp, and the jelly strength was 94 g]. A 2% by weight aqueous solution was prepared, and this was thinly applied with a small brush. The cross-linked gelatin film carrying the eluate from the cementum prepared in (3) was cut to an appropriate size and pasted so as not to open a single gap on the surface of the artificial tooth root. This is freeze-dried using a freeze-drying apparatus (FREEZE DRYER FD-5N manufactured by Tokyo Rika Kikai Co., Ltd.) to obtain an artificial tooth root (HA-T) carrying a protein component containing a hydroxyproline residue-containing protein. It was. This HA-T is sealed in a polyethylene bag, placed 30 cm below the Toshiba ultraviolet sterilization lamp (15 watts), and subjected to sterilization by irradiating with ultraviolet rays for 30 minutes each on the front and back. Stored in a refrigerator at 5 ° C.
[0071]
(Example 2)
Artificial tooth root (Ti-T)
Artificial tooth root material used: Titanium alloy
Bioabsorbable material used: Cross-linked carboxymethyl chitin
Preparation of artificial roots coated with cross-linked carboxymethyl chitin
Glycerol polyglycidyl ether [Nagase Kasei Co., Ltd.] in a 5% by weight aqueous solution of commercially available carboxymethyl chitin [manufactured by Koyo Chemical Co., Ltd., carboxymethylation substitution degree 75%, deacetylation degree 27%, weight average molecular weight 200,000] Kogyo Kogyo Co., Ltd.] was added to and dissolved in 5% of the weight of carboxymethyl chitin to prepare a mixed aqueous solution of carboxymethyl and glycerol polyglycidyl ether.
[0072]
Apply a mixed aqueous solution of carboxymethyl and glycerol polyglycidyl ether with a brush on the surface of a commercially available artificial tooth [POI system FINAFIX 3Piece Fixture, length 8 mm, diameter 3.7 mm, surface material is titanium alloy, manufactured by Kyocera Corporation]. All the groove portions of the threaded surface of the artificial tooth root surface were filled with the mixed aqueous solution.
[0073]
This was freeze-dried with a freeze dryer (FREEZE DRYER FD-5N manufactured by Tokyo Rika Kikai Co., Ltd.) to obtain an artificial tooth root coated with carboxymethyl chitin. Furthermore, this artificial tooth root was crosslinked by heat treatment at 110 ° C. for 4 hours to obtain a crosslinked carboxymethyl chitin-coated artificial tooth root.
(2) Adjustment of artificial tooth root Ti-T
0.0585 g (elution component 0.015 g) prepared in Example 1 (1) was dissolved in 7.5 ml of distilled water, 0.1 g of the solution was collected, and the crosslinked carboxy prepared in (1). The surface of the methylchitin-coated artificial tooth was uniformly soaked. This was freeze-dried with a freeze dryer (FREEZE DRYER FD-5N manufactured by Tokyo Rika Kikai Co., Ltd.) to obtain an artificial tooth root (Ti-T) carrying a protein component containing a hydroxyproline residue-containing protein. This HA-T is sealed in a polyethylene bag, placed 30 cm below the Toshiba ultraviolet sterilization lamp (15 watts), and subjected to sterilization by irradiating with ultraviolet rays for 30 minutes each on the front and back. Stored in a refrigerator at 5 ° C.
[0074]
Moreover, the average carrying amount of the protein component containing the hydroxyproline residue-containing protein on the surface of the artificial tooth root was 0.12 mg / square centimeter.
[0075]
(Comparative Example 1)
Adjustment of artificial root (HA-B)
Artificial tooth root material used: Hydroxyapatite
Bioabsorbable material used: Cross-linked gelatin
(1) Preparation of physiological saline with pH 7 containing 1% collagenase
Prepare 15 ml of 1% collagenase-containing physiological saline and freeze-dry using a freeze-drying device (FREEZE DRYER FD-5N manufactured by Tokyo Rika Kikai Co., Ltd.) to obtain 0.29 g of solid matter (elution from cementum) Component 0g, collagenase 0.15g, sodium chloride 0.14g).
(2) Preparation of cross-linked gelatin film
A crosslinked gelatin film was obtained in the same manner as in (2) of Example 1.
(3) Preparation of cross-linked gelatin film that does not carry components eluted from cementum
Dissolve 0.044 g of the solid material prepared in (1) (0 g of the elution component from the cementum) in 15 ml of distilled water, 1 cm wide on one side of a polymethyl methacrylate plate (size 10 × 10 cm, thickness 2 mm), A silicon tape having a thickness of 1.0 mm was trimmed, and 1.5 g of the solution was collected on the inside (8 × 8 cm) and cast. Further, the cross-linked gelatin film prepared in (2) is placed thereon, and the solution is absorbed into the cross-linked gelatin film, followed by freeze-drying machine (FREEZE DRYER FD-5N manufactured by Tokyo Rika Equipment Co., Ltd.) Was lyophilized to obtain a membrane.
(4) HA-B adjustment
Commercially available artificial tooth root [apaceram, length 9 mm, diameter 4.5 mm, surface material is hydroxyapatite, manufactured by Asahi Optical Co., Ltd.] surface, commercially available gelatin [Nitta Gelatin Co., Ltd., 6.66% aqueous solution The viscosity was 31 mp, and the jelly strength was 94 g]. A 2% by weight aqueous solution was prepared, and this was thinly applied with a small brush. The crosslinked gelatin film prepared in (3) was cut into an appropriate size and pasted so that a single gap was not opened on the surface of the artificial tooth root. This was freeze-dried with a freeze-drying apparatus (FREEZE DRYER FD-5N manufactured by Tokyo Rika Kikai Co., Ltd.) to obtain a comparative artificial tooth root (HA-B).
[0076]
HA-B is sealed in a polyethylene bag, placed 30 cm below the Toshiba ultraviolet sterilization lamp (15 watts), and subjected to sterilization by irradiating with ultraviolet rays for 30 minutes on each side. Stored in a refrigerator at 0C.
[0077]
(Comparative Example 2)
Adjustment of artificial tooth root (Ti-B)
Artificial tooth root material used: Titanium alloy
Bioabsorbable material used: Cross-linked carboxymethyl chitin
(1) Preparation of cross-linked carboxymethyl chitin-coated artificial tooth root
Adjustment was performed in the same manner as in (1) of Example 2.
(2) Adjustment of artificial tooth root Ti-B
0.044 g (elution component 0 g) prepared in Comparative Example 1 (1) was dissolved in 7.5 ml of distilled water, 0.1 g of the solution was collected, and the crosslinked carboxymethyl chitin prepared in (2) was used. The surface of the coated artificial tooth was soaked uniformly. This was freeze-dried with a freeze dryer (FREEZE DRYER FD-5N manufactured by Tokyo Rika Kikai Co., Ltd.) to obtain a comparative artificial tooth root (Ti-B). Ti-B is sealed in a polyethylene bag, placed 30 cm below the Toshiba ultraviolet sterilization lamp (15 watts), and sterilized by irradiation with ultraviolet rays for 30 minutes each on the front and back sides. Stored in a refrigerator at 0C.
[0078]
(Test example)
Two rhesus monkeys were used as experimental animals. As a preoperative procedure, the first and second premolars and the first premolars on the left and right sides of the lower jaw were removed in order to secure an artificial root planting site. Three months later, one rhesus monkey was planted with HA-T adjusted in Example 1 in the lower right edentulous portion and HA-B adjusted in Comparative Example 1 in the lower left edentulous portion. In another rhesus monkey, the artificial tooth root Ti-T adjusted in Example 2 was planted in the lower right edentulous jaw, and Ti-B was planted in the lower left edentulous jaw.
[0079]
In the operation, first, a vertical incision was performed by centrifuging the edentulous part and a lateral incision on the buccal side 1 mm from the gingival mucosa boundary, and then the gingival flap was peeled off to expose the bone surface. Subsequently, using an artificial tooth rooting kit, an artificial tooth rooting hole was made in the jawbone, and an artificial tooth root was implanted. Thereafter, the gingival flap was sutured in the original position. In HA-T and HA-B, an artificial tooth rooting hole having a diameter of 5 mm was formed, and in Ti-T and TI-B, an artificial tooth rooting hole having a diameter of 4 mm was formed and embedded.
[0080]
The animals were sacrificed 12 weeks after placement of the artificial dental root. Immediately after decapitation, perfusion fixation was performed with 2.5% glutaraldehyde-2.0% paraformaldehyde (pH 7.2, 4 ° C.) from the left and right common carotid arteries.
[0081]
After that, artificial tooth roots, bones, and gums were removed as one lump from the operation part of the monkey jaw. The collected tissue block was fixed with the above fixative solution for 7 days. After fixation, the experimental area was cut cheekly with a diamond saw and embedded in Technobit 7200 resin.
[0082]
Each polymerized embedded sample was cut into a thickness of about 300 μm using a diamond saw, and then a polished piece having a thickness of 100 μm was prepared with a polishing machine. The sections were stained with hematoxin / eosin and observed with an optical microscope.
[0083]
The observation results are shown in the following table.
[0084]
[Table 1]

[0085]
As shown in Table 1 above, since both HA-T and Ti-T of the present invention show cementitious deposits on the artificial tooth root, and fibrous connective tissue with blood vessels is formed, While the periodontal ligament was in the process of being formed, both the HA-B and Ti-B of the comparative examples had an artificial tooth root that was bone-bonded to the surrounding tissue.
[0086]
【The invention's effect】
As described above, according to the artificial tooth root of the present invention, since the protein component containing the hydroxyproline residue-containing protein has activity in fibroblasts, osteoblasts, and cementoblasts, the surface hydroxyproline residue The protein component, including the contained protein, regenerates cementum on the surface of the base material, and further regenerates the periodontal ligament between the bones. At that time, the cementum and the alveolar bone are bound together by collagen fiber bundles. Periodontal ligament having the same function as natural teeth is formed safely.
[0087]
In addition, the artificial dental root of the present invention can be obtained easily and inexpensively from protein teeth containing a hydroxyproline residue-containing protein, such as mammalian teeth. Can be supplied at low cost with stable quality.
[0088]
Further, in the present invention, when a bioabsorbable membrane containing a protein component containing a hydroxyproline residue-containing protein is deposited on the surface, the protein component can be easily fixed on the surface of the artificial tooth root, The release rate can be controlled. Furthermore, the bioabsorbable material acts as a scaffold for cells until cementum and periodontal ligament are formed around the artificial tooth root, and is absorbed and decomposed after periodontal ligament formation. For the above reasons, the function of forming cementum and periodontal membrane around the artificial tooth root is good.
[0089]
Next, in the present invention, the carrying amount of the protein component containing the hydroxyproline residue-containing protein is 0.00001 mg to 15 mg / square centimeter, and / or the protein containing the hydroxyproline residue-containing protein. The function of forming cementum and periodontal membrane around the artificial tooth root is good by making the content of the hydroxyproline residue contained in the component 0.01 to 10% with respect to the remaining amount of all amino acids. Can be.
[0090]
Further, in the present invention, by extracting the protein component containing the hydroxyproline residue-containing protein from mammalian dental cementum, since the origin of humans is close to that of mammals, the protein contained in mammalian dental cementum Since the structure of the protein contained in human cementum is similar to that of the other types, the function of forming cementum and periodontal membrane around the artificial dental root is improved.
[0091]
Furthermore, if the cementum of the teeth of cows or pigs is used, cows and pigs are slaughtered for food, so that the cementum can be easily and inexpensively obtained from those teeth.

Claims (3)

An artificial tooth root having a periodontal ligament forming ability in which a protein component containing a hydroxyproline residue-containing protein is supported on a surface of a bone-incorporated portion of a base material made of titanium in an easily water-soluble state,
With crosslinked carboxymethyl chitin on the surface of the bone in the implantation portion is deposited, the said crosslinking carboxymethyl chitin, by drying was impregnated solution of the protein component, the protein component is supported An artificial tooth root characterized by that .   The artificial tooth root according to claim 1, wherein the protein component is a protein component extracted from cementum of mammalian teeth.   The artificial tooth root according to claim 2, wherein the mammal is a cow or a pig.