WO2003101502A1 - Method of regenerating tooth germ and regenerated tooth germ - Google Patents

Method of regenerating tooth germ and regenerated tooth germ Download PDF

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Publication number
WO2003101502A1
WO2003101502A1 PCT/JP2003/006842 JP0306842W WO03101502A1 WO 2003101502 A1 WO2003101502 A1 WO 2003101502A1 JP 0306842 W JP0306842 W JP 0306842W WO 03101502 A1 WO03101502 A1 WO 03101502A1
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Prior art keywords
tooth germ
cells
tooth
carrier
regenerating
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PCT/JP2003/006842
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French (fr)
Japanese (ja)
Inventor
Minoru Ueda
Masaki Honda
Hideaki Kagami
Hiromichi Shimizu
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Hitachi Medical Corporation
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Priority to JP2004508855A priority Critical patent/JPWO2003101502A1/en
Publication of WO2003101502A1 publication Critical patent/WO2003101502A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A61L27/3843—Connective tissue
    • A61L27/3865—Dental/periodontal tissues
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3895—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis

Definitions

  • the present invention relates to a method for regenerating tooth germ. More specifically, the present invention relates to a method for regenerating tooth germ by culturing tooth germ cells on a carrier such as collagen. The present invention further relates to a method of treating a dental patient using the tooth germ regenerated by the above method.
  • Background art
  • the GTR method is a method of suppressing the invasion of epithelial cells and the like to the root surface by using a membrane such as MILLIPORE FILTER (trade name of Millipore) to form a space necessary for proliferation of periodontal ligament cells. (Nyman et al., J. Clin. Periodontol., 9, 290 (1982)) 0
  • the GTR method aims to regenerate the alveolar bone and periodontal ligament around the tooth affected by periodontal disease. In light cases, great results have been achieved. In recent years, proteins that enable periodontal ligament regeneration have been developed and put into practical use. However, GTR cannot be applied to the resorption of high alveolar bone that causes tooth loss, nor can it repair tooth decay due to dental caries.
  • An object of the present invention is to solve the above-mentioned problems of the conventional technology. That is, the present invention provides a method for regenerating tooth germ, and more specifically, it is possible to treat a patient whose teeth have been lost or damaged due to a dental disease such as alveolar leakage or dental caries. An object of the present invention is to provide a method for regenerating a tooth germ that can be activated. Furthermore, an object of the present invention is to provide a method for treating a patient having a missing or damaged tooth using a regenerated tooth germ.
  • the present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, by culturing tooth germ cells on a carrier consisting of collagen, a comparison was made with the case of using a carrier consisting of polyglycolic acid (PGA).
  • PGA polyglycolic acid
  • the present inventors have found that high cell adhesion and low cytotoxicity are exhibited, and that tooth germ can be efficiently formed from tooth germ cells.
  • the present invention has been completed based on these findings.
  • the present invention provides a method for regenerating tooth germ by culturing at least one of tooth germ cells and cells that can be differentiated into these cells on a carrier.
  • a carrier Preferably, at least one type of cells cultured on the carrier is transplanted into an animal body and regenerated into a tooth germ in the animal body.
  • the animal is a mammal.
  • the at least one type of cells is transplanted into the body of the animal at a site where blood flow is abundant.
  • the carrier is capable of adhesion to the at least one cell without toxicity.
  • the material has an affinity for the living body to be transplanted and a bioabsorbing property.
  • a carrier having a target shape of a tooth germ to be regenerated and having a blood circulation introducing portion is used.
  • the carrier comprises at least one of the following forms: mesh, sponge, and gel.
  • the carrier is made of collagen.
  • the at least one cell is an odontoblast, an ameloblast, a dental pulp or papillary cell, a dental sac cell, or a precursor cell thereof.
  • the at least one cell is obtained by fragmenting tissue collected from a living body. Separated and recovered after enzymatic treatment.
  • the regenerated tooth germ has at least one of dentin, tooth papilla, and enamel pulp.
  • a tooth germ regenerated by any one of the above methods is transplanted into a jaw bone of a patient having a tooth germ defect or damage, thereby giving the patient a regenerated tooth germ.
  • a method of treatment is provided.
  • FIG. 1 is a diagram showing the appearance of cells 15 weeks after transplantation.
  • FIG. 1A calcified matter that forms tooth germ-like hard tissue and is considered to correspond to dentin was observed. Calcification was also promoted by mechanical stimulation.
  • Figure 2 is a diagram comparing the size of tooth germ-like tissue formation with (+) and without (1) mechanical stimuli.
  • FIG. 3 shows the results of comparing the affinity and adhesion of tooth germ-derived cultured cells to the PGA carrier or collagen carrier.
  • FIG. 4 shows a histological image (hematoxylin and eosin staining) of a sample extracted 25 weeks after transplantation (microscope magnification: 10 ⁇ 10 ⁇ ).
  • FIG. 4A shows a specimen using a collagen sponge carrier
  • FIG. 4B shows a specimen using a PGA mesh carrier.
  • the method for regenerating tooth germ according to the present invention is characterized in that at least one of tooth germ cells and at least one of these cells that can be differentiated into these cells is cultured on a carrier.
  • a carrier One of the factors that determines the success or failure of tissue regeneration by tissue engineering is a carrier.
  • the number of cells remaining after transplantation into a living body is an important factor, including the cell affinity of the carrier and ⁇
  • the cell growth promoting effect, differentiation induction effect or toxicity of the carrier itself has an effect. Therefore, selection of a carrier needs to be determined by considering these factors.
  • Factors that determine the number of remaining cells in the early stage of transplantation are considered to be the ability of cells to adhere to the carrier and the cytotoxicity of the carrier. Therefore, in the present invention, a change in the number of cells after seeding of cultured tooth germ cells was compared and studied using a PGA mesh and collagen sponge. As a result, it was found that a larger number of cells were maintained on the carrier when the collagen sponge was used than when the PGA mesh was used.
  • a carrier that can withstand the time required for the formation of the tooth germ and that is rapidly absorbed thereafter is preferable. That is, it is preferable to use a carrier having a suitable absorption rate and characteristics in a living body such as a gastric omentum or a jawbone and a material having high affinity with cells.
  • a carrier made of collagen satisfies these conditions.
  • a mesh form, a sponge form, a gel form, and the like can be used as a form of the collagen carrier.
  • a gel-shaped carrier is useful as a carrier for culturing tooth germ cells because cells can easily contact each other as compared with a mesh-shaped or sponge-shaped carrier.
  • the carrier is preferably processed into a shape that allows cells to be easily transplanted, and is preferably a plate, a sphere, or a hollow, one end of which is open, and preferably has a site where blood circulation can be easily introduced from the periphery.
  • the carrier is preferably prepared in a form suitable for the purpose.
  • a mold is obtained using an impression material after the desired form is made of resin. After that, the resin form is taken out and the synthetic material that makes up the carrier is poured in to reproduce the desired form.
  • the type of tooth germ cells used in the present invention is not particularly limited as long as it is a cell constituting tooth germ or a cell capable of differentiating into these cells. Examples thereof include odontoblasts, ameloblasts, dental pulp, and the like. Tooth papillary cells, tooth sac cells, or precursor cells thereof can be used. These cells are a single type of cell The cells may be cultured as cells or as a cell mixture composed of two or more types of cells.
  • Tooth germ cells can be obtained from the mandible of mammals (eg, humans, pigs, etc.). Aseptically remove the impacted teeth and store them in an appropriate storage solution such as Hanks balanced salt solution (HBSS) solution. Remove the calcified part of the tooth, scalpel the tissue with a scalpel, and wash the tissue with an HBSS solution. Subsequently, it is preferable to subject the tissue to enzyme treatment using collagenase and dispase. After the enzyme treatment, the cells can be collected by pitting and centrifugation.
  • HBSS Hanks balanced salt solution
  • the tooth germ regenerated according to the method of the present invention is used for treatment of a dental patient (ie, a patient having a tooth defect or damage) by transplanting the tooth germ to the patient.
  • a dental patient ie, a patient having a tooth defect or damage
  • Cells that constitute tooth germ or cells that differentiate into tooth germ can also be collected from wisdom teeth (wisdom teeth).
  • teeth are formed through five stages from development to maturity.
  • the first phase called the onset phase, induces epithelial and mesenchymal tissue in the basement membrane.
  • the second stage is called the bud stage and enamel is made.
  • the third stage forms the papillae and the tooth germ.
  • the fourth stage called the bell-shaped stage, begins the differentiation of tooth germ into cells that form enamel and the differentiation of tooth papillae into cells that form dentin and pulp.
  • the fifth stage called the maturation period, differentiates into tissues that make up the teeth, such as enamel, dentin, and pulp.
  • cells at a suitable time among these can be collected and used.
  • the pulp and periodontal ligament can be removed from the root and the cells can be separated and collected.
  • the extraction of dental pulp from tooth strength can be performed according to the method described in About I. and other Experimental cell research. 258. 33-41, 2000.
  • regeneration of tooth germ refers to regeneration of tooth germ in the second and subsequent stages of the above five steps.
  • tooth germ cells are cultured on a carrier made of collagen.
  • the culturing can be performed while applying a mechanical stimulus.
  • the mechanical stimulus can be given by, for example, shaking culture of the cell, sonication of the cell, extension stimulation or culture under pressure.
  • the cells are cultured with shaking.
  • Shaking culture can be performed by placing a container containing cells and a medium on a shaker, and culturing the cells while shaking at an appropriate speed (for example, about 100 to 100 times z minutes). it can.
  • Cultured cells the normal used to culture animal cells containing serum with a medium, culture conditions typical animal cells (e.g., 3 7 temperature ° C to room temperature; and 5% C_ ⁇ 2 incubator within terpolymers) of Can be done below.
  • the separated tooth germ cells be cultured and proliferated, and then seeded and cultured on a carrier made of collagen.
  • a carrier made of collagen By performing the culture before seeding on the carrier, it becomes possible to obtain a sufficient number of cells to grow on the carrier.
  • the culture of the separated tooth germ cells can be performed according to the usual animal cell culture conditions, as described above.
  • the cultured cells after culturing tooth germ cells on a carrier composed of collagen, the cultured cells may be transplanted together with the carrier into a transplanted animal, and the tooth germ may be regenerated in the transplanted animal.
  • the cultured cells may be directly transplanted into the patient's jawbone together with the carrier.
  • the isolated tooth germ cells are cultured and proliferated, and then seeded on a carrier made of collagen, or cultured after seeding, and then the cultured cells are transplanted together with the carrier into a transplanted animal. Then, the tooth germ can be regenerated in the body of the transplanted animal.
  • the type of the transplanted animal is not particularly limited, but is preferably a mammal, and for example, a rodent such as a rat (eg, a nude rat), a rabbit, or a mouse can be used.
  • a site for transplantation a site that easily supplies factors necessary for the formation of tooth germ is preferable, and specifically, a site with abundant blood flow is preferable. For example, a gastric omentum below the abdominal cavity is particularly preferable. By transplanting to such a site, the growth of tooth germ cells can be promoted, and the formation of tooth germ can be accelerated.
  • Tooth germ (tooth germ cells mechanically regenerated by the method for regenerating tooth germ Tooth germ tissue obtained by culturing while applying a stimulus, or tooth germ tissue obtained by transplanting this tooth germ tissue into a transplanted animal and regenerating it further in the body of the transplanted animal) may be a defective or damaged tooth
  • the dental patient can be treated by implanting it in the jaw bone of a patient having the above. That is, a method for treating a dental patient using a tooth germ obtained by the method for regenerating a tooth germ according to the present invention is also within the scope of the present invention.
  • the tooth can be formed by continuing the growth of the tooth germ even after being implanted into the jaw bone of a dental patient.
  • the tooth root may be formed outside the patient's body, and then implanted into the patient's jawbone to form a crown using conventional dental techniques.
  • HBSS Hanks balanced salt solution
  • the washed tissue was treated with an enzyme solution containing 2 mg / ml collagenase and dispase 11A dissolved in 35 ml of HBSS solution for 30 minutes.
  • Pipetting was performed for 10 minutes using a 25 ml pipette.
  • the cells were collected by centrifuging 25 ml of the supernatant (1500 rpm, 10 minutes) (1500 rpm, 10 minutes) (collected cells 1).
  • the remaining tissue in the 10 ml collagenase solution was pitted with 10 ml pits for 10 minutes (collected cells 2).
  • the recovered cells 1 were mixed with the recovered cells 2 and centrifuged again to recover the cells. After washing three times with a medium containing 10% serum, the cells were collected by centrifugation.
  • the isolated cells were adjusted to l ⁇ 10 6 cells / ml and 3 ⁇ 10 6 cells were transferred to one carrier (collagen).
  • the seeds were seeded on a sponge carrier (porosity: 99%, Nipro, Japan).
  • the carrier in which the cells were seeded was shaken at 50 times / min with a shaker for 12 hours before transplantation.
  • stationary culture was performed for 12 hours.
  • the culture medium for the cells used was Dulbeco's Modified Eagle Medium supplemented with 10% fetal bovine serum, glutamic acid and antibiotics. Further, culturing of cells was carried out at 37 ° C, C0 2 5% and Rere U conditions.
  • F344 nude rats were used as transplant animals. After laparotomy of the F344 nude rat, the cultured cell was transplanted into the omentum. The cells after the culture were transplanted for 15 weeks and then excised.
  • PGA mesh carrier volume density 50 ° /. To 60 ° thickness 2 bulge, Albany International Research, MA, USA
  • collagen sponge carrier 99% porosity, Professional, Japan. The density of the cell suspension was adjusted to 2 ⁇ 10 5 / ml, and 1 ml was inoculated on one carrier.
  • a culture medium for the cells after seeding a medium obtained by adding 10% fetal bovine serum to Dulbeco's Modified Eagle Medium and containing glutamic acid and an antibiotic were used. Cell culture is also 37. KoTsuta under the condition that the C, C0 2 5%. After culture for 48 hours after seeding, the number of adherent cells was measured. The results are shown in Figure 3. - From the results shown in FIG. 3, it can be seen that the number of cells on the collagen sponge is larger in the initial stage after culture for 48 hours than in the PGA mesh group. This result is considered to indicate that collagen has higher cell adhesion to tooth germ cells and lower cytotoxicity. These results indicate that collagen is more advantageous than PGA as a carrier for cultured and isolated tooth germ cells in tooth germ regeneration.
  • Example 3 Comparison of in vivo transplant specimens
  • the differentiation is induced to form the tooth germ structures.
  • tooth germ structures such as dentin, tooth papillae, and enamel pulp in the body of the transplanted animal or on the medium.
  • the root or the tooth can be regenerated.
  • QOL quality of life
  • the period required for regeneration is reduced by using a physiologically active substance such as a growth factor and a transcription factor, Alternatively, differentiation induction can be promoted.

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Abstract

It is intended to provide a method of regenerating a tooth germ, more specifically, a method of regenerating a tooth germ whereby a patient suffering from the a defect or injury in a tooth germ due to a dental disease such as marginal periodontitis (pyorrhea alveolaris) or tooth decay can be treated. Namely, a method of regenerating a tooth germ by culturing at least one type of cells selected from among tooth germ cells and cells capable of differentiating into the same on a support and a regenerated tooth germ are provided.

Description

明細書  Specification
歯胚の再生方法および再生歯胚 技術分野  Method of regenerating tooth germ and regenerated tooth germ
本発明は、 歯胚の再生方法に関する。 より詳細には、 本発明は、 歯胚細胞をコ ラーゲンなどの担体上で培養することにより歯胚を再生する方法に関する。 本発 明はさらに、 上記方法により再生された歯胚を用いて歯科患者を治療する方法に 関する。 背景技術  The present invention relates to a method for regenerating tooth germ. More specifically, the present invention relates to a method for regenerating tooth germ by culturing tooth germ cells on a carrier such as collagen. The present invention further relates to a method of treating a dental patient using the tooth germ regenerated by the above method. Background art
現代社会は高齢化社会であり、 数年後には日本国民人口の約 2 0 %が 6 5歳以 上の高齢者になることが予想されている。 これら高齢者の大多数は、 何らかの理 由により一部又は全部の歯牙を喪失しており、 多くの人は可綴式義歯 （いわゆる 入れ歯） を使用している。 従来の義歯は、 着脱が必要で装着感もよくないなどの 実際的問題のみならず、 心理的にも老化の象徴といった印象があり、 できれば義 歯を使用したくないというのが患者の一般的認識である。 さらに、 全ての歯牙を 喪失した場合に、 総義歯を装着すると、 その咀嚼能力は通常の天然歯牙の約 5分 の 1となることが知られている。 多くの高齢者にとって楽しみの一つである食事 が歯の喪失のため苦痛となる場合も少なくない。 さらに、 脳に対する咀嚼刺激は 痴呆防止の効果があり、 咀嚼力の低下は痴呆の促進になることが明らかになって きている。  The modern society is an aging society, and it is expected that about 20% of the Japanese population will be older than 65 years old in a few years. The majority of these elderly people have lost some or all of their teeth for some reason, and many use mandible dentures (so-called dentures). Conventional dentures not only have practical problems such as the need to attach and remove and have a poor feeling of wearing, but also have the impression that they are a symbol of aging in terms of psychology, and it is common for patients not to use dentures if possible. It is recognition. In addition, it is known that if all the teeth are lost and a complete denture is worn, the chewing ability will be about one-fifth that of ordinary natural teeth. Diet, which is one of the pleasures of many elderly people, is often painful due to tooth loss. Furthermore, it has become clear that mastication stimulation of the brain is effective in preventing dementia, and that reduction in masticatory power promotes dementia.
これに対して近年、 人工歯根が開発され臨床に応用されている。 人工歯根の応 用により義歯が固定され、 維持がよくなり、 咀嚼力も改善される。 し力 し、 審美 性、 装着感に関しては未だ満足のいくものではない。 また、 手術が必要であるこ と、 一定量の骨が必要であり、 全身状態によっても制限されること、 さらに多額 の費用がかかり、 信頼できる医療機関も限られることなどの理由から、 未だイン プラント義歯は広く普及しているとは言えない。 その結果、 義歯に不満を感じて いる患者が少なくないにもかかわらず、 インプラント義歯の使用者は義歯使用者 のうちの極わずかである。 On the other hand, in recent years, artificial dental roots have been developed and applied to clinical practice. The application of artificial roots fixes the dentures, improves their maintenance and improves their mastication. However, their aesthetics and comfort are still unsatisfactory. In addition, implants are still needed because of the need for surgery, a certain amount of bone, limited by general condition, and the high cost and limited number of reliable medical institutions. Dentures are not widely used. As a result, I feel dissatisfied with the denture Despite the fact that many patients have implanted dentures, very few of them use dentures.
—方、 他家移植による歯牙移植の報告はあるが、 移植できる健康な抜去歯牙を 確保することは困難であるのみならず、 感染症の危険もあり、 一般的な治療とは なっていない。 義歯に不満を感じながらインプラントに踏み込めないか、 あるい は条件的にインプラント治療が困難な多数の患者が存在している。  —On the other hand, there are reports of tooth transplantation by allogeneic transplantation, but it is difficult to secure healthy extracted teeth that can be transplanted, and there is a risk of infectious disease. There are a large number of patients who are dissatisfied with dentures and unable to step into the implant, or who have difficulty condition the implant.
現在までに、 歯科に関する再生の研究は、 歯周組織の再生に注目が置かれ、 骨 の再生、 歯根膜の再生を中心に研究されてきた。 これらの研究の成果として、 G T R法（Guided Tissue Regeneration法）が開発された。 G T R法とは、 例えばミ リポアフィルター（MILLIPORE FILTER,ミリポア社商品名）などの膜によって、歯根 面への上皮細胞等の侵入を抑制し、 歯根膜細胞の増殖に必要な空間を形成させる 方法である （Nymanら， J. Clin. Periodontol. , 9, 290 (1982) ) 0 G T R法は、 歯周 病に罹患した歯牙周囲に歯槽骨と歯根膜を再生させることを目的とするものであ り、 軽症例では大きな成果を挙げている。 また、 近年、 歯根膜再生を可能にする タンパク質が開発され、 実用化されている。 しかしながら、 G T R法は、 歯牙喪 失の原因となる高度の歯槽骨の吸収には応用できず、 またう蝕による歯牙の崩壊 を修復することはできない。 To date, research on dental regeneration has focused on periodontal tissue regeneration, and has focused on bone regeneration and periodontal regeneration. As a result of these studies, the GTR method (Guided Tissue Regeneration method) was developed. The GTR method is a method of suppressing the invasion of epithelial cells and the like to the root surface by using a membrane such as MILLIPORE FILTER (trade name of Millipore) to form a space necessary for proliferation of periodontal ligament cells. (Nyman et al., J. Clin. Periodontol., 9, 290 (1982)) 0 The GTR method aims to regenerate the alveolar bone and periodontal ligament around the tooth affected by periodontal disease. In light cases, great results have been achieved. In recent years, proteins that enable periodontal ligament regeneration have been developed and put into practical use. However, GTR cannot be applied to the resorption of high alveolar bone that causes tooth loss, nor can it repair tooth decay due to dental caries.
上記した問題点を根本的に解決する方策として、 歯胚そのものを再生する方法 が提案され、 検討が行われているが （C. S. Young et al J. Dent. Res. 81 (10) , 695-2000， 2002；および Tissue Engineering , Vol. 7， Number 5, 624 , October 2001)、未だ小さな組織が形成できたのみで、実際の治療に供することが可能な程 の大きさの組織形成はできていなかった。 発明の開示  As a method to fundamentally solve the above problems, a method of regenerating tooth germ itself has been proposed and studied (CS Young et al J. Dent. Res. 81 (10), 695-2000) , 2002; and Tissue Engineering, Vol. 7, Number 5, 624, October 2001), only small tissue was formed, but not large enough to be used for actual treatment. Was. Disclosure of the invention
本発明は上記した従来技術の問題点を解消することを解決すべき課題とした。 即ち、 本発明は、 歯胚を再生する方法を提供すること、 より具体的には、 歯槽膿 漏やう蝕などの歯科疾患により歯牙を欠損又は損傷した患者を治療することを可 能にする歯胚の再生方法を提供することを解決すべき課題とした。 さらに本発明 は、 再生した歯胚を用いて歯牙を欠損又は損傷した患者を治療する方法を提供す ることを解決すべき課題とした。 An object of the present invention is to solve the above-mentioned problems of the conventional technology. That is, the present invention provides a method for regenerating tooth germ, and more specifically, it is possible to treat a patient whose teeth have been lost or damaged due to a dental disease such as alveolar leakage or dental caries. An object of the present invention is to provide a method for regenerating a tooth germ that can be activated. Furthermore, an object of the present invention is to provide a method for treating a patient having a missing or damaged tooth using a regenerated tooth germ.
本発明者らは、 上記課題を解決するために鋭意検討した結果、 歯胚細胞をコラ 一ゲンから成る担体上で培養することにより、 ポリグリコール酸 （P G A) から 成る担体を使用した場合と比較して高い細胞接着能および低い細胞毒性が示され ること見出し、 これにより歯胚細胞から歯胚を効率よく形成できることを見出し た。 本発明はこれらの知見に基づいて完成したものである。  The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, by culturing tooth germ cells on a carrier consisting of collagen, a comparison was made with the case of using a carrier consisting of polyglycolic acid (PGA). In addition, the present inventors have found that high cell adhesion and low cytotoxicity are exhibited, and that tooth germ can be efficiently formed from tooth germ cells. The present invention has been completed based on these findings.
即ち、 本発明によれば、 歯胚細胞およびこれらの細胞に分化可能な細胞のうち 少なくとも 1種類を担体上で培養することで歯胚を再生する方法が提供される。 好ましくは、 上記担体上で培養した少なくとも 1種類の細胞を動物の体内に移 植して該動物の体内で歯胚に再生させる。  That is, the present invention provides a method for regenerating tooth germ by culturing at least one of tooth germ cells and cells that can be differentiated into these cells on a carrier. Preferably, at least one type of cells cultured on the carrier is transplanted into an animal body and regenerated into a tooth germ in the animal body.
好ましくは、 上記動物は哺乳動物である。  Preferably, the animal is a mammal.
好ましくは、 上記動物の体内の血流の豊富な部位に上記少なくとも 1種類の細 胞を移植する。  Preferably, the at least one type of cells is transplanted into the body of the animal at a site where blood flow is abundant.
好ましくは、 上記担体は上記少なくとも 1種類の細胞への毒性がなく接着が可 能である。  Preferably, the carrier is capable of adhesion to the at least one cell without toxicity.
好ましくは、 上記担体について、 材質は移植先の生体に対する親和性と生体吸 収性を有する。  Preferably, regarding the carrier, the material has an affinity for the living body to be transplanted and a bioabsorbing property.
好ましくは、上記担体について、形状は再生する歯胚の目的形状でありかつ血行 の導入部分を有する担体を用レヽる。  Preferably, for the carrier, a carrier having a target shape of a tooth germ to be regenerated and having a blood circulation introducing portion is used.
好ましくは、上記担体には、 メッシュ、スポンジ、およびゲルの形態のうち少な くとも 1つの形態部分が含まれる。  Preferably, the carrier comprises at least one of the following forms: mesh, sponge, and gel.
好ましくは、 上記担体はコラーゲンからなる。  Preferably, the carrier is made of collagen.
好ましくは、 上記少なくとも 1種類の細胞は、 象牙芽細胞、 エナメル芽細胞、 歯髄あるいは歯乳頭細胞、 歯嚢細胞、 またはこれらの前駆細胞である。  Preferably, the at least one cell is an odontoblast, an ameloblast, a dental pulp or papillary cell, a dental sac cell, or a precursor cell thereof.
好ましくは、 上記少なくとも 1種類の細胞は生体から採取した組織を細片化し 酵素処理してから分離回収して得る。 Preferably, the at least one cell is obtained by fragmenting tissue collected from a living body. Separated and recovered after enzymatic treatment.
本発明によればさらに、 上記のいずれかの方法によって再生された歯胚が提供 される。  According to the present invention, there is further provided a tooth germ regenerated by any of the above methods.
好ましくは、上記再生された歯胚は、象牙質、歯乳頭、エナメル髄のうち少なく とも 1つを有する。  Preferably, the regenerated tooth germ has at least one of dentin, tooth papilla, and enamel pulp.
本発明によればさらに、 上記のいずれかの方法によって再生された歯胚を歯胚 欠損または損傷を有する患者の顎骨に移植することで該患者に再生歯胚を付与す ることを特徴とする治療方法が提供される。 図面の簡単な説明  According to the present invention, further, a tooth germ regenerated by any one of the above methods is transplanted into a jaw bone of a patient having a tooth germ defect or damage, thereby giving the patient a regenerated tooth germ. A method of treatment is provided. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 移植後 1 5週間後の細胞の様子を示す図である。 図 1 Aでは、 歯胚様 の硬組織を形成し、 象牙質に相当すると考えられる石灰化物を認めた。 また、 機 械的刺激により石灰化が促進された。  FIG. 1 is a diagram showing the appearance of cells 15 weeks after transplantation. In FIG. 1A, calcified matter that forms tooth germ-like hard tissue and is considered to correspond to dentin was observed. Calcification was also promoted by mechanical stimulation.
図 2は、 機械的 （Mechanical) 刺激を加えた （+ ) 場合と加えない （一） 場合 で、 歯胚様組織が形成の大きさを比較した図である。  Figure 2 is a diagram comparing the size of tooth germ-like tissue formation with (+) and without (1) mechanical stimuli.
図 3は、 歯胚由来培養細胞の P G A担体又はコラーゲン担体に対する親和性と 接着能を比較した結果を示す。  FIG. 3 shows the results of comparing the affinity and adhesion of tooth germ-derived cultured cells to the PGA carrier or collagen carrier.
図 4は、 移植 2 5週後に摘出した試料の組織像 （へマトキシリン 'ェォジン染 色） を示す （顕微鏡倍率： 1 0 X 1 0倍)。 図 4 Aはコラーゲンスポンジ担体、 図 4 Bは PGAメッシュ担体を使用した標本を示す。  FIG. 4 shows a histological image (hematoxylin and eosin staining) of a sample extracted 25 weeks after transplantation (microscope magnification: 10 × 10 ×). FIG. 4A shows a specimen using a collagen sponge carrier, and FIG. 4B shows a specimen using a PGA mesh carrier.
発明を実施するための最良の形態 BEST MODE FOR CARRYING OUT THE INVENTION
以下、 本発明の実施の形態について詳細に説明する。  Hereinafter, embodiments of the present invention will be described in detail.
本発明による歯胚の再生方法は、 歯胚細胞おょぴこれらの細胞に分化可能な細 胞のうち少なくとも 1種類を担体上で培養することを特徴とするものである。 組織工学による組織再生の成否を決める要因の一つに担体が挙げられる。 特に 生体に移植後の残存細胞数は重要な要素であり、 これには担体の細胞親和性およ ぴ担体自身の持つ細胞增殖促進効果、分化誘導効果あるいは毒性が影響を与える。 従って、 担体の選択は、 これらの要素を検討の上決定する必要がある。 The method for regenerating tooth germ according to the present invention is characterized in that at least one of tooth germ cells and at least one of these cells that can be differentiated into these cells is cultured on a carrier. One of the factors that determines the success or failure of tissue regeneration by tissue engineering is a carrier. In particular, the number of cells remaining after transplantation into a living body is an important factor, including the cell affinity of the carrier and 增 The cell growth promoting effect, differentiation induction effect or toxicity of the carrier itself has an effect. Therefore, selection of a carrier needs to be determined by considering these factors.
移植初期における残存細胞数を決定する要因としては、 細胞の担体への接着能 と、 担体の細胞毒性とが考えられる。 そこで、 本発明では、 PGA メッシュとコラ 一ゲンスポンジを用いて， 培養歯胚細胞播種後の細胞数の変化を比較検討した。 その結果、 PGA メッシュを用いた場合と比較してコラーゲンスポンジを用いた場 合の方が、 多数の細胞が担体上に維持されることが判明した。  Factors that determine the number of remaining cells in the early stage of transplantation are considered to be the ability of cells to adhere to the carrier and the cytotoxicity of the carrier. Therefore, in the present invention, a change in the number of cells after seeding of cultured tooth germ cells was compared and studied using a PGA mesh and collagen sponge. As a result, it was found that a larger number of cells were maintained on the carrier when the collagen sponge was used than when the PGA mesh was used.
歯胚の再生に用いる担体としては、 歯胚の形成に必要とされる時間を耐久する ことができ、 かつその後、 速やかに吸収されるものが好ましい。 即ち、 胃大網又 は顎骨内などの生体内において適切な吸収速度と特性を有し、 かつ細胞と高い親 和性を有する材料から成る担体を使用することが好ましい。 コラーゲンから成る 担体はこれらの条件を満たすものである。  As the carrier used for the regeneration of the tooth germ, a carrier that can withstand the time required for the formation of the tooth germ and that is rapidly absorbed thereafter is preferable. That is, it is preferable to use a carrier having a suitable absorption rate and characteristics in a living body such as a gastric omentum or a jawbone and a material having high affinity with cells. A carrier made of collagen satisfies these conditions.
コラーゲン担体の形態としては、 メッシュ形態、 スポンジ形態、 ゲル形態など が可能である。 中でも、 ゲル形態の担体は、 メッシュ形態やスポンジ形態の担体 と比較して、 細胞同士の接触が容易になるため歯胚細胞の培養のための担体とし て有用である。  As a form of the collagen carrier, a mesh form, a sponge form, a gel form, and the like can be used. Among them, a gel-shaped carrier is useful as a carrier for culturing tooth germ cells because cells can easily contact each other as compared with a mesh-shaped or sponge-shaped carrier.
担体は細胞を移植しやすい形状に加工したものが好ましく、 板状、 球状あるい は中空で一端が開放されており、 周囲から血行が導入されやすい部位を有するこ とが好ましい。  The carrier is preferably processed into a shape that allows cells to be easily transplanted, and is preferably a plate, a sphere, or a hollow, one end of which is open, and preferably has a site where blood circulation can be easily introduced from the periphery.
担体は、目的に適合した形態のものを作製することが好ましい。このためには、 目的とする形態をレジンで作製した後に印象材を用いて型を取得する。 その後、 レジンの型を取り出し、 担体を構成する合成材料を流しこむことによって目的の 形態を再現することができる。  The carrier is preferably prepared in a form suitable for the purpose. For this purpose, a mold is obtained using an impression material after the desired form is made of resin. After that, the resin form is taken out and the synthetic material that makes up the carrier is poured in to reproduce the desired form.
本発明で用いる歯胚細胞としては、 歯胚を構成する細胞あるいはこれらの細胞 に分化することができる細胞であれば特にその種類は限定されず、 例えば、 象牙 芽細胞、 エナメル芽細胞、 歯髄あるいは歯乳頭細胞、 歯嚢細胞、 又はこれらの前 駆細胞を使用することができる。 これらの細胞は、 1種類の細胞から成る単一の 細胞として培養してもよいし、 2種類以上の細胞から成る細胞混合物として培養 してもよい。 The type of tooth germ cells used in the present invention is not particularly limited as long as it is a cell constituting tooth germ or a cell capable of differentiating into these cells. Examples thereof include odontoblasts, ameloblasts, dental pulp, and the like. Tooth papillary cells, tooth sac cells, or precursor cells thereof can be used. These cells are a single type of cell The cells may be cultured as cells or as a cell mixture composed of two or more types of cells.
歯胚細胞は、 哺乳動物 （例えば、 ヒト、 豚など） の下顎骨から採取することが できる。 埋伏歯を無菌的に取り出し、 Hanks balanced salt solution (HBSS) 溶 液などの適当な保存液で保存する。歯牙の中の石灰化した部分を取り除き，メスに て組織を小片にして、 HBSS溶液などを用いて組織を洗浄する。 次いで、 コラゲナ ーゼとディスパーゼを用いて組織を酵素処理することが好ましい。 酵素処理後、 ピぺッティング操作と遠心操作により細胞を回収することができる。  Tooth germ cells can be obtained from the mandible of mammals (eg, humans, pigs, etc.). Aseptically remove the impacted teeth and store them in an appropriate storage solution such as Hanks balanced salt solution (HBSS) solution. Remove the calcified part of the tooth, scalpel the tissue with a scalpel, and wash the tissue with an HBSS solution. Subsequently, it is preferable to subject the tissue to enzyme treatment using collagenase and dispase. After the enzyme treatment, the cells can be collected by pitting and centrifugation.
本発明の方法に従って再生した歯胚は、 歯科患者 （即ち、 歯牙の欠損又は損傷 を有する患者） に移植することにより、 該患者の治療のために用いられる。 この 場合、 移植に伴う生体適合性などの観点から、 再生に用いる歯胚細胞は、 該患者 に由来する自分の歯胚細胞を用いることが好ましい。 歯胚を構成する細胞あるい は歯胚に分化する細胞は、 親知らず （智歯） からも採取することができる。  The tooth germ regenerated according to the method of the present invention is used for treatment of a dental patient (ie, a patient having a tooth defect or damage) by transplanting the tooth germ to the patient. In this case, from the viewpoint of the biocompatibility associated with the transplantation, it is preferable to use the own tooth germ cells derived from the patient as the tooth germ cells used for regeneration. Cells that constitute tooth germ or cells that differentiate into tooth germ can also be collected from wisdom teeth (wisdom teeth).
また、 歯牙は、 発生から成熟するまでに 5つの段階を経て形成されることが知 られている。 第一期は、 開始期と呼ばれ、 基底膜に上皮組織と間葉組織が誘導さ れる。 第二期は、 蕾状期と呼ばれエナメル器が作られる。 第三期は帽状期と呼ば れ、 歯乳頭が形成され、 歯胚が形成される。 第四期は鐘状期と呼ばれ、 歯胚から エナメル質を形成する細胞への分化と歯乳頭から象牙質と歯髄を形成する細胞へ の分化が開始される。 第五期は成熟期と呼ばれ、 エナメル質と象牙質と歯髄など の歯牙を構成する組織へと分化する。 本発明においては、 これらのうちの好適な 時期の細胞を採取して用いることができる。 また、 歯胚が存在していない症例で は、 歯根より歯髄や歯根膜を摘出して細胞を分離採取することができる。 なお、 歯牙力 らの歯髄の摘出は、 About I.，他 Experimental cell research. 258. 33-41, 2000に記載の方法に従って行うことができる。  In addition, it is known that teeth are formed through five stages from development to maturity. The first phase, called the onset phase, induces epithelial and mesenchymal tissue in the basement membrane. The second stage is called the bud stage and enamel is made. The third stage, called the cap stage, forms the papillae and the tooth germ. The fourth stage, called the bell-shaped stage, begins the differentiation of tooth germ into cells that form enamel and the differentiation of tooth papillae into cells that form dentin and pulp. The fifth stage, called the maturation period, differentiates into tissues that make up the teeth, such as enamel, dentin, and pulp. In the present invention, cells at a suitable time among these can be collected and used. In cases where tooth germ is not present, the pulp and periodontal ligament can be removed from the root and the cells can be separated and collected. The extraction of dental pulp from tooth strength can be performed according to the method described in About I. and other Experimental cell research. 258. 33-41, 2000.
また、 本発明で言う 「歯胚の再生」 とは、 上記 5段階のうちの第二期以降の歯 胚を再生することを言う。  The term “regeneration of tooth germ” as used in the present invention refers to regeneration of tooth germ in the second and subsequent stages of the above five steps.
本発明では、 歯胚細胞をコラーゲンから成る担体上で培養する。 本発明の特に 好ましい態様によれば、 培養は機械的刺激を与えながら行うことができる。 機械 的刺激は、 例えば、 細胞を振盪培養したり、 細胞に超音波処理を施したり、 伸展 刺激あるいは加圧下で培養することなどにより与えることができる。好ましくは、 細胞を振盪培養する。 振盪培養は、 細胞と培地を含む容器を振盪器の上に設置し て、 適当な速度 （例えば、 1 0〜1 0 0回 z分程度） で振盪しながら細胞を培養 することで行うことができる。 In the present invention, tooth germ cells are cultured on a carrier made of collagen. Especially of the present invention According to a preferred embodiment, the culturing can be performed while applying a mechanical stimulus. The mechanical stimulus can be given by, for example, shaking culture of the cell, sonication of the cell, extension stimulation or culture under pressure. Preferably, the cells are cultured with shaking. Shaking culture can be performed by placing a container containing cells and a medium on a shaker, and culturing the cells while shaking at an appropriate speed (for example, about 100 to 100 times z minutes). it can.
細胞の培養は、 動物細胞の培養に用いる通常の血清入り培地を用いて、 通常の 動物細胞の培養条件 （例えば、 室温から 3 7 °Cの温度； 5 % C〇2インキュベー ター内など） の下で行なうことができる。 Cultured cells, the normal used to culture animal cells containing serum with a medium, culture conditions typical animal cells (e.g., 3 7 temperature ° C to room temperature; and 5% C_〇 2 incubator within terpolymers) of Can be done below.
本発明の方法では、 分離した歯胚細胞を培養して増殖させた後に、 コラーゲン から成る担体上に播種して培養することが好ましい。 担体上に播種する前に培養 を行うことにより、 担体上で生育するのに十分な数の細胞を取得することが可能 になる。 分離した歯胚細胞の培養は、 上記と同様、 通常の動物細胞の培養条件に 準じて行うことができる。  In the method of the present invention, it is preferable that the separated tooth germ cells be cultured and proliferated, and then seeded and cultured on a carrier made of collagen. By performing the culture before seeding on the carrier, it becomes possible to obtain a sufficient number of cells to grow on the carrier. The culture of the separated tooth germ cells can be performed according to the usual animal cell culture conditions, as described above.
本発明の方法では、 歯胚細胞をコラーゲンから成る担体上で培養した後に、 該 培養細胞を担体と一緒に移植動物に移植し、 該移植動物の体内で歯胚を再生させ てもよいし、 該培養細胞を担体と一緒に直接患者の顎骨に移植してもよい。 ある いはさらに好ましくは、 分離した歯胚細胞を培養して増殖させた後にコラーゲン から成る担体上に播種して、 または播種後培養し、 次いで該培養細胞を担体と一 緒に移植動物に移植し、 該移植動物の体内で歯胚を再生させることができる。 移植動物の種類は特に限定されないが、 好ましくは哺乳動物であり、 例えば、 ラット （ヌードラットなど）、 ゥサギ又はマウスなどのげつ歯類動物を使用するこ とができる。 移植の部位としては、 歯胚の形成に必要な因子を供給しやすい部位 が好ましく、 具体的には、 血流の豊富な部位が好ましく、 例えば、 腹腔下の胃大 網などが特に好ましい。 このような部位に移植することにより、 歯胚細胞の成長 を促進することができ、 歯胚の形成を早めることが可能えだる。  In the method of the present invention, after culturing tooth germ cells on a carrier composed of collagen, the cultured cells may be transplanted together with the carrier into a transplanted animal, and the tooth germ may be regenerated in the transplanted animal. The cultured cells may be directly transplanted into the patient's jawbone together with the carrier. Or more preferably, the isolated tooth germ cells are cultured and proliferated, and then seeded on a carrier made of collagen, or cultured after seeding, and then the cultured cells are transplanted together with the carrier into a transplanted animal. Then, the tooth germ can be regenerated in the body of the transplanted animal. The type of the transplanted animal is not particularly limited, but is preferably a mammal, and for example, a rodent such as a rat (eg, a nude rat), a rabbit, or a mouse can be used. As a site for transplantation, a site that easily supplies factors necessary for the formation of tooth germ is preferable, and specifically, a site with abundant blood flow is preferable. For example, a gastric omentum below the abdominal cavity is particularly preferable. By transplanting to such a site, the growth of tooth germ cells can be promoted, and the formation of tooth germ can be accelerated.
上記した本発明による歯胚の再生方法により再生した歯胚 （歯胚細胞を機械的 刺激を与えながら培養して得られる歯胚組織、 あるいはこの歯胚組織を移植動物 に移植し、 該移植動物の体内でさらに再生させた歯胚組織の何れでもよい） は、 歯牙の欠損又は損傷を有する患者の顎骨に移植することによって、 該歯科患者を 治療することができる。 即ち、 本発明による歯胚の再生方法により得られた歯胚 を用いる歯科患者の治療方法も本発明の範囲内のものである。 歯科患者の顎骨内 に移植された後も歯胚の成長を継続させることにより、 歯牙を形成させることが できる。 あるいは歯根までを患者の体外で形成させた後、 これを患者の顎骨内に 移植し、 従来の歯科的な手法により歯冠を形成してもよい。 Tooth germ (tooth germ cells mechanically regenerated by the method for regenerating tooth germ Tooth germ tissue obtained by culturing while applying a stimulus, or tooth germ tissue obtained by transplanting this tooth germ tissue into a transplanted animal and regenerating it further in the body of the transplanted animal) may be a defective or damaged tooth The dental patient can be treated by implanting it in the jaw bone of a patient having the above. That is, a method for treating a dental patient using a tooth germ obtained by the method for regenerating a tooth germ according to the present invention is also within the scope of the present invention. The tooth can be formed by continuing the growth of the tooth germ even after being implanted into the jaw bone of a dental patient. Alternatively, the tooth root may be formed outside the patient's body, and then implanted into the patient's jawbone to form a crown using conventional dental techniques.
以下の実施例により本発明をさらに具体的に説明するが、 本発明は実施例によ つて限定されるものではない。 実施例  The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples. Example
実施例 1 ：機械的刺激を用いた歯胚再生 Example 1: Tooth germ regeneration using mechanical stimulation
生後数ケ月の新鮮豚から下顎骨を採取した。 実験に使用するまでは 4 °Cの冷蔵 庫にて保存し、 運搬中は氷上にて保存した。 埋伏歯を無菌的に取り出し、 Hanks balanced salt solution (HBSS) 溶液にて保存した。 歯牙の中の石灰化した部分 を取り除き，メスにて組織を約 2讓の小片にした。 HBSS溶液にて小片にした組織 を約 5回洗浄した。  Mandibles were collected from fresh pigs a few months after birth. They were stored in a refrigerator at 4 ° C until used for experiments, and stored on ice during transportation. Impacted teeth were aseptically removed and stored in Hanks balanced salt solution (HBSS) solution. The calcified part of the tooth was removed, and the tissue was cut into small pieces of about 2 rows with a scalpel. The tissue cut into small pieces with the HBSS solution was washed about five times.
2mg/mlコラゲ^ "一ゼとディスパーゼ 1 1 Aを 35mlの HBSS溶液に溶解した酵素 溶液を用いて、 洗浄した組織を 30分間酵素処理した。  The washed tissue was treated with an enzyme solution containing 2 mg / ml collagenase and dispase 11A dissolved in 35 ml of HBSS solution for 30 minutes.
25ml用のピぺットを用いて 10分間ピペッティングした。 25mlの上澄み液を遠 心 （1500rpm, 10分） して細胞を回収した （1500rpm， 10分） （回収細胞 1とする）。 残りの 10mlコラゲナーゼ溶液中の組織を 10mlのピぺットにて 1 0分間ピぺッテ ィングした（回収細胞 2とする）。回収細胞 1を回収細胞 2に混ぜて再度遠心して 細胞を回収した。 1 0 %血清入り培地にて 3回洗浄した後に遠心することによつ て細胞を回収した。  Pipetting was performed for 10 minutes using a 25 ml pipette. The cells were collected by centrifuging 25 ml of the supernatant (1500 rpm, 10 minutes) (1500 rpm, 10 minutes) (collected cells 1). The remaining tissue in the 10 ml collagenase solution was pitted with 10 ml pits for 10 minutes (collected cells 2). The recovered cells 1 were mixed with the recovered cells 2 and centrifuged again to recover the cells. After washing three times with a medium containing 10% serum, the cells were collected by centrifugation.
単離した細胞を l X l06cell/mlに調整し、 3 X 106cellを一個の担体 （コラーゲ ンスポンジ担体（空孔率 9 9 %、 二プロ，日本）） に播種した。 細胞を播種した担体 を実験群では移植前に 1 2時間振盪器にて 5 0回/分振盪させた。対照群において は，静置培養を 1 2時間行った。 細胞の培養培地としては、 Dulbeco's Modified Eagle Medium に 1 0 %牛胎児血清を加えたものとグルタミン酸と抗生剤を含有し たものを用いた。 また、 細胞の培養は、 37°C， C02 5 %とレヽぅ条件下で行った。 移植動物としては F344ヌードラットを用いた。 F344ヌードラットを開腹後、 培養後の細胞を大網に移植した。 培養後の細胞は 15週間移植した後、 摘出した。 The isolated cells were adjusted to l × 10 6 cells / ml and 3 × 10 6 cells were transferred to one carrier (collagen). The seeds were seeded on a sponge carrier (porosity: 99%, Nipro, Japan). In the experimental group, the carrier in which the cells were seeded was shaken at 50 times / min with a shaker for 12 hours before transplantation. In the control group, stationary culture was performed for 12 hours. The culture medium for the cells used was Dulbeco's Modified Eagle Medium supplemented with 10% fetal bovine serum, glutamic acid and antibiotics. Further, culturing of cells was carried out at 37 ° C, C0 2 5% and Rere U conditions. F344 nude rats were used as transplant animals. After laparotomy of the F344 nude rat, the cultured cell was transplanted into the omentum. The cells after the culture were transplanted for 15 weeks and then excised.
1 5週間移植された細胞は、 歯胚様の硬組織を形成し、 象牙質に相当すると考 えられる石灰化物を認めた（図 1 A)。 さらに、 機械的刺激により石灰化が促進さ れ（図 1 B )、肉眼的所見にて対照群の約 2倍の大きさの歯胚様組織が形成された (図 2 )。 これは、 機械的刺激によって、 細胞の分化誘導のみでなく、 細胞増殖が 促進され、 またその生存率が高まったことによると考えられる。 また、 このよう な機械的ストレスに代表される歯胚細胞の增殖促進技術により、 採取した一つの 歯胚細胞を分離、 培養することで複数の歯胚を再生できることが示された。 実施例 2 ：歯胚由来培養細胞の担体に対する親和性と接着能の比較  The cells transplanted for 15 weeks formed tooth germ-like hard tissue and found calcified substances that were considered to correspond to dentin (Fig. 1A). Furthermore, calcification was accelerated by mechanical stimulation (Fig. 1B), and tooth germ-like tissue about twice as large as the control group was formed by macroscopic observation (Fig. 2). This is probably because mechanical stimulation not only induces cell differentiation, but also promotes cell proliferation and increases the survival rate. In addition, it was shown that a plurality of tooth germs can be regenerated by separating and culturing one collected tooth germ cell by using such a technique for promoting the proliferation of tooth germ cells represented by mechanical stress. Example 2: Comparison of affinity and adhesion of tooth germ-derived cultured cells to carriers
実施例 1の方法によりブタ歯胚細胞を分離した後、 1 0 %牛胎児血清を含む DMEM (Dalbeco's Modified Eagle Medium)へグルタミン酸と抗生剤を加えた培地に て培養を行い、 必要な細胞を獲得した。  After isolating porcine tooth germ cells by the method of Example 1, culturing the cells in a medium containing glutamate and antibiotics in DMEM (Dalbeco's Modified Eagle Medium) containing 10% fetal bovine serum to obtain the necessary cells did.
この細胞をプレートから剥離したのち、 PGAメッシュ担体（体積密度 50°/。 〜60 °ん 厚さ 2讓、 Albany International Research, MA, USA)、 又はコラーゲンスポンジ 担体（空孔率 9 9 %、 二プロ，日本） に播種した。 細胞懸濁液の密度は 2 X 105/mlに 調整し、 1mlを 1個の担体に播種した。 After detaching the cells from the plate, a PGA mesh carrier (volume density 50 ° /. To 60 ° thickness 2 bulge, Albany International Research, MA, USA) or a collagen sponge carrier (99% porosity, Professional, Japan). The density of the cell suspension was adjusted to 2 × 10 5 / ml, and 1 ml was inoculated on one carrier.
播種後の細胞の培養培地としては、 Dulbeco's Modified Eagle Mediumに 1 0 % 牛胎児血清を加えたものとグルタミン酸と抗生剤を含有したものを用いた。また、 細胞の培養は、 37。C， C02 5 %という条件下で行つた。 播種後 4 8時間培養後に、 接着細胞数を測定した。 結果を図 3に示す。 - 図 3の結果から、 培養 4 8時間後の初期の段階において、 PGA メッシュ群と比 較して、 コラーゲンスポンジ上での細胞数が多いことが分かる。 この結果は、 コ ラーゲンの歯胚細胞に対するより高い細胞接着能、 低い細胞毒性を示したものと 考えられる。 この結果より、 歯胚再生において培養および分離歯胚細胞の担体と してコラーゲンが PGAより有利であることが判明した。 実施例 3 ： In vivo移植標本の比較 As a culture medium for the cells after seeding, a medium obtained by adding 10% fetal bovine serum to Dulbeco's Modified Eagle Medium and containing glutamic acid and an antibiotic were used. Cell culture is also 37. KoTsuta under the condition that the C, C0 2 5%. After culture for 48 hours after seeding, the number of adherent cells was measured. The results are shown in Figure 3. - From the results shown in FIG. 3, it can be seen that the number of cells on the collagen sponge is larger in the initial stage after culture for 48 hours than in the PGA mesh group. This result is considered to indicate that collagen has higher cell adhesion to tooth germ cells and lower cytotoxicity. These results indicate that collagen is more advantageous than PGA as a carrier for cultured and isolated tooth germ cells in tooth germ regeneration. Example 3: Comparison of in vivo transplant specimens
実施例 1記載と同様の方法により、 コラーゲンスポンジ担体 （空孔率 9 9 %、 二プロ） に細胞を播種した。 対照として、 PGA メッシュ担体 （体積密度 5 0〜6 0 %、 厚さ 2 mm、 Albany International Research, MA, USA) に同様に細胞を播 種した。各々を実施例 1記載と同様の方法により F 3 4 4ヌードラットに移植し、 2 5週後に摘出した。  Cells were seeded on a collagen sponge carrier (porosity: 99%, Nipro) in the same manner as described in Example 1. As a control, cells were similarly seeded on a PGA mesh carrier (volume density: 50 to 60%, thickness: 2 mm, Albany International Research, MA, USA). Each was transplanted into F344 nude rats in the same manner as described in Example 1, and was extracted 25 weeks later.
摘出した各試料を常法に従って固定、 包埋し、 組織切片を作成した。 その後、 切片にへマトキシリン 'ェォジン染色を施し、 組織学的に観察した。 結果を図 4 に示す。  Each extracted sample was fixed and embedded according to a conventional method to prepare a tissue section. Thereafter, the sections were stained with hematoxylin and eosin and observed histologically. Figure 4 shows the results.
コラーゲンスポンジ担体を使用した試料 （図 4 A) は、 PGA メッシュ担体を使 用した試料 （図 4 B ) に比較して、 象牙質様組織及びエナメル質様組織を顕著に 多く形成することが判明した。 産業上の利用の可能性  It was found that the sample using the collagen sponge carrier (Fig. 4A) formed significantly more dentin-like and enamel-like tissues than the sample using the PGA mesh carrier (Fig. 4B). did. Industrial applicability
本発明の方法を利用して、 移植動物の体内または培地上で象牙質、 歯乳頭、 ェ ナメル髄などの歯胚構造を形成してから、 あるいは歯胚構造を形成するように分 化誘導された細胞塊を担体とともに歯科患者の顎骨内に移植することにより、 歯 根または歯牙を再生きせることができる。 この結果、 歯牙を欠損した患者の咀嚼 能力の回復に極めて有効な治療となる。 また、 審美的な回復も見込まれ、 患者の Quality of Life (Q O L ) の向上に大きく貢献する。 また、 本発明の方法では、 増殖、転写因子など生理活性物質を用いることにより再生に要する期間を短縮し、 あるいは分化誘導を促進することができる。 Utilizing the method of the present invention, after the formation of tooth germ structures such as dentin, tooth papillae, and enamel pulp in the body of the transplanted animal or on the medium, the differentiation is induced to form the tooth germ structures. By implanting the cell mass together with the carrier into the jaw bone of a dental patient, the root or the tooth can be regenerated. As a result, it is an extremely effective treatment for restoring the masticatory ability of patients with tooth loss. Aesthetic recovery is also expected and will greatly contribute to improving the quality of life (QOL) of patients. Further, in the method of the present invention, the period required for regeneration is reduced by using a physiologically active substance such as a growth factor and a transcription factor, Alternatively, differentiation induction can be promoted.

Claims

請求の範囲 The scope of the claims
1 . 歯胚細胞おょぴこれらの細胞に分化可能な細胞のうち少なくとも 1種類 を担体上で培養することで歯胚を再生する方法。 1. Tooth germ cells-A method of regenerating tooth germ by culturing at least one of these cells that can be differentiated into these cells on a carrier.
2 . 上記担体上で培養した少なくとも 1種類の細胞を動物の体内に移植して 該動物の体内で歯胚に再生させることを含む請求項 1に記載の歯胚を再生する方 法。  2. The method for regenerating a tooth germ according to claim 1, comprising transplanting at least one kind of cells cultured on the carrier into an animal body and regenerating the tooth germ in the animal body.
3 . 上記動物は哺乳動物であることを特徴とする請求項 2に記載の歯胚を再 生する方法。  3. The method for regenerating tooth germ according to claim 2, wherein the animal is a mammal.
4 . 上記動物の体内の血流の豊富な部位に上記少なくとも 1種類の細胞を移 植することを特徴とする請求項 2または 3のいずれかに記載の歯胚を再生する方 法。  4. The method for regenerating a tooth germ according to any one of claims 2 and 3, wherein the at least one cell is transplanted to a site where the blood flow is abundant in the body of the animal.
5 . 上記担体は上記少なくとも 1種類の細胞への毒性がなく接着が可能であ ることを特徴とする請求項 1から 4のいずれかに記載の歯胚を再生する方法。  5. The method for regenerating tooth germ according to any one of claims 1 to 4, wherein the carrier is capable of adhering to the at least one type of cell without toxicity.
6 . 上記担体について、 材質は移植先の生体に対する親和性と生体吸収性を 有することを特徴とする請求項 1から 5のいずれかに記載の歯胚を再生する方法。  6. The method for regenerating a tooth germ according to any one of claims 1 to 5, wherein the material of the carrier has an affinity for a living body to be transplanted and a bioabsorbability.
7 . 上記担体について、形状は再生する歯胚の目的形状でありかっ血行の導入 部分を有する担体を用いることを特徴とする請求項 1から 7のいずれかに記載の 歯胚を再生する方法。  7. The method for regenerating a tooth germ according to any one of claims 1 to 7, wherein the carrier has a target shape of a tooth germ to be regenerated and a carrier having a portion into which blood circulation is introduced.
8 . 上記担体には、 メッシュ、 スポンジ、およびゲルの形態のうち少なくとも 1つの形態部分が含まれることを特徴とする請求項 1カゝら 7のいずれかに記載の 歯胚を再生する方法。  8. The method for regenerating a tooth germ according to any one of claims 1 to 7, wherein the carrier includes at least one of a mesh, a sponge, and a gel.
9 . 上記担体はコラーゲンからなることを特徴とする請求項 1から 4のいず れかに記載の歯胚を再生する方法。  9. The method for regenerating tooth germ according to any one of claims 1 to 4, wherein the carrier is made of collagen.
1 0 . 上記少なくとも 1種類の細胞は、 象牙芽細胞、 エナメル芽細胞、 歯髄 あるいは歯乳頭細胞、 歯嚢細胞、 またはこれらの前駆細胞であることを特徴とす る請求項 1から 9のいずれかに記載の歯胚を再生する方法。 10. The at least one kind of cell is an odontoblast, an ameloblast, a dental pulp or a papillary cell, a dental sac cell, or a precursor cell thereof, any one of claims 1 to 9. A method for regenerating a tooth germ according to claim 1.
1 1 . 上記少なくとも 1種類の細胞は生体から採取した組織を細片化し酵素 処理してから分離回収して得ることを特徴とする請求項 1から 1 0のいずれかに 記載の歯 Eを再生する方法。 11. The tooth E according to any one of claims 1 to 10, wherein the at least one kind of cells is obtained by fragmenting a tissue collected from a living body, treating it with an enzyme, and separating and collecting the tissue. how to.
1 2 . 請求項 1から 1 1に記載のいずれかの方法によって再生された歯胚。  12. Tooth germ regenerated by the method according to any one of claims 1 to 11.
1 3 . 上記再生された歯胚は、象牙質、歯乳頭、エナメル髄のうち少なくとも1 3. The regenerated tooth germ should be at least one of dentin, tooth papilla, and enamel pulp.
1つを有することを特徴とする請求項 1 2に記載の再生された歯胚。 13. The regenerated tooth germ according to claim 12, comprising one.
1 4 . 請求項 1から 1 1に記載のいずれかの方法によって再生された歯胚を 歯胚欠損または損傷を有する患者の顎骨に移植することで該患者に再生歯胚を付 与することを特徴とする治療方法。  14. A method of providing a regenerated tooth germ to a patient having a tooth germ defect or injury by transplanting the tooth germ regenerated by the method according to any one of claims 1 to 11 into the jaw bone of the patient having the tooth germ defect or damage. A characteristic treatment method.
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