JP2014039517A - Method for controlling size and shape of crown, cusp, and root of tooth, regenerated tooth, and regenerated tooth germ using growth factors including igf - Google Patents
Method for controlling size and shape of crown, cusp, and root of tooth, regenerated tooth, and regenerated tooth germ using growth factors including igf Download PDFInfo
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Abstract
Description
本発明は、臓器、器官および組織を再生させる方法。 The present invention is a method for regenerating organs, organs and tissues.
歯は、エナメル質と象牙質という硬組織を有し、その更に中心部に歯髄を有する器官である。また、歯は齲蝕や歯周病等によって失われることが多く、顎顔面領域の機能性が低下するため、健康維持や質の高い生活を維持するという観点から歯の再生技術が期待されている。 A tooth is an organ having hard tissues of enamel and dentin and further having a pulp in the center. In addition, teeth are often lost due to caries, periodontal disease, etc., and the functionality of the maxillofacial region decreases, so tooth regeneration technology is expected from the viewpoint of maintaining health and maintaining a high quality of life. .
例えば、非特許文献１および特許文献１には、器官原基法によって再構築した再生歯が成体顎骨内に移植することにより機能的な歯として萌出することを報告した。また、再生歯は、移植した再構成歯胚由来の歯根膜を有し、周囲歯槽骨とシャーピー線維様組織によって連結していることが示唆された。さらに、作製した再生歯胚を歯根膜と歯槽骨の伴う再生歯ユニットに作製して成体マウスに移植するという技術の開発が記載されている。しかしながら、再生歯が咀嚼するなどの機能を有するために最も重要な歯冠のサイズを大きくする形態制御の方法は開示されていない。また、歯が咀嚼するために最も重要な咬頭の形態制御の方法も開示されていない。 For example, Non-Patent Document 1 and Patent Document 1 reported that regenerated teeth reconstructed by the organ primordium method erupt as functional teeth when transplanted into the adult jawbone. In addition, it was suggested that the regenerated tooth has a periodontal ligament derived from the transplanted reconstructed tooth germ and is connected to the surrounding alveolar bone by a sharpy fibrous tissue. Furthermore, the development of a technique in which the produced regenerated tooth germ is produced in a regenerative tooth unit with periodontal ligament and alveolar bone and transplanted into an adult mouse is described. However, there is no disclosure of a form control method for increasing the crown size, which is most important for the function of the regenerated tooth to chew. Also, the most important method for controlling the shape of the cusp for the teeth to chew is not disclosed.
例えば、特許文献２には、生理活性物質として線維芽細胞増殖因子（ＦＧＦ）、形質転換細胞増殖因子（ＴＧＦ）などの存在下で歯胚細胞を培養することが開示されている。しかしながら、形態の制御についての技術は開示されていない。また、歯などの臓器の機能において、正常な形態が極めて重要であり、特に歯の場合、歯冠のサイズを大きくする形態制御、および、咬頭の形態制御ができなければ咀嚼機能が欠落し、臨床への応用性および有効性がないといえる。しかしながら、現状では、歯の発生および再生の過程において、形態を制御して再生させる方法は不可能であるというのが現状であった。 For example, Patent Document 2 discloses culturing tooth germ cells in the presence of fibroblast growth factor (FGF), transformed cell growth factor (TGF), and the like as physiologically active substances. However, a technique for controlling the form is not disclosed. Also, in the function of organs such as teeth, the normal form is extremely important, especially in the case of teeth, the form control that increases the size of the crown, and the masticatory function is missing if the form control of the cusp is not possible, It can be said that there is no clinical applicability and effectiveness. However, at present, in the process of tooth generation and regeneration, it has been impossible to control and regenerate the form.
例えば、特許文献３には、担体を使用して歯胚を再生させ、特有の形状の歯を形成することが開示されているが、成長因子を用いた方法は開示されていない。また、担体を用いた形状作製の技術では、歯の器官そのものの全体的な組織学的構造の再現は報告されていない。 For example, Patent Document 3 discloses that a tooth germ is regenerated using a carrier to form a tooth having a specific shape, but a method using a growth factor is not disclosed. In addition, in the technique of shape production using a carrier, the reproduction of the entire histological structure of the tooth organ itself has not been reported.
例えば、特許文献５には、IGF/IGFBP複合体によって骨形成および骨再造形を刺激する方法が記載されているが、形態の制御についての技術は開示されていない。本発明におけるIGF刺激の技術は、歯に対する技術であり、さらに歯の機能性に極めて重要な歯冠および咬頭ならびに歯根の形態制御を実現させ、歯科再生技術に最も必要な技術と考えられる。 For example, Patent Document 5 describes a method of stimulating bone formation and bone remodeling with an IGF / IGFBP complex, but does not disclose a technique for controlling the morphology. The IGF stimulation technique in the present invention is a technique for teeth, and further realizes shape control of crowns and cusps and roots, which are extremely important for the functionality of the teeth, and is considered to be the most necessary technique for the dental regeneration technique.
歯は歯種特異的な形態を有することで、正常な咀嚼機能を維持できる。そのため、人工の歯による補綴処置においても、咬合調整などの精密な咬頭形態の調整が極めて重要となる。正常な歯と同じ機能を有するには、形態の制御が必要であるが、非特許文献1および特許文献１に記載の技術では再生歯は形態の矮小が認められ、再生歯および歯根の咬頭形態も、正常歯と比較すると不十分である。これらの問題を解決するための形態制御について記載していない。本発明技術では、歯冠および咬頭ならびに歯根の形態を制御し、サイズの増大および縮小が可能となる。そのため、上顎骨および下顎骨と再生歯の大きさのバランスの不調和によって起こる咀嚼機能障害を解決し、歯科再生医療の実現化に必須の技術となる。 A tooth can maintain a normal mastication function by having a tooth type specific form. Therefore, in the prosthetic treatment using artificial teeth, precise adjustment of the cusp shape such as occlusion adjustment is extremely important. In order to have the same function as a normal tooth, it is necessary to control the form. However, in the techniques described in Non-Patent Document 1 and Patent Document 1, the regenerated tooth has a reduced form, and the regenerated tooth and root cusp shape However, it is insufficient compared to normal teeth. It does not describe the form control for solving these problems. The technology of the present invention controls the shape of the crown and cusp as well as the root of the tooth, allowing the size to increase and decrease. Therefore, it solves the masticatory dysfunction caused by the unbalance of the size of the maxilla and mandible and the regenerative teeth, and is an indispensable technique for realizing dental regenerative medicine.
日本人の集団の中で９０％以上に叢生が認められ、近年、増加していることも報告されている。叢生は顎骨と歯の大きさの不調和によってもたらされることから、歯の大きさを制御できれば、叢生の発現を抑制することが可能となる。さらに、叢生が誘因となる齲蝕、歯周病や顎関節症もその発現が抑制される。これらは、歯冠のサイズを大きくする形態制御、および歯冠のサイズを小さくする形態制御によって解決される。 More than 90% of the Japanese population has been crowded and has been reported to increase in recent years. Since crowding is caused by a mismatch between the size of the jawbone and the teeth, if the size of the teeth can be controlled, the occurrence of crowding can be suppressed. Furthermore, the occurrence of caries, periodontal disease, and temporomandibular disorders that are caused by crowding are also suppressed. These are solved by a form control for increasing the crown size and a form control for reducing the crown size.
組織、器官、臓器の形態形成は、細胞の外部環境によって制御されている。歯は上皮-間葉相互作用により発生し形態が形成されるが、歯の形態形成にかかわるメカニズムは、ほとんど報告されていない。そのため、歯は歯冠の特異的形態によって、その機能を発揮する器官であるにもかかわらず、歯及び再生歯の形態制御は不可能とされてきた。本発明では、IGFを含む成長因子による刺激を歯の上皮-間葉相互作用の発生メカニズムに組み込んで、形態制御する方法を開発した。この技術によって、歯だけではなく、上皮-間葉相互作用によって発生する肺、腎臓、肝臓、腺組織、四肢、指などの形成における形態制御も可能となる。 The morphogenesis of tissues, organs and organs is controlled by the external environment of the cells. Teeth are generated and formed by epithelial-mesenchymal interactions, but the mechanisms involved in tooth morphogenesis have hardly been reported. For this reason, it has been impossible to control the morphology of teeth and regenerated teeth, despite the fact that the teeth are organs that perform their functions due to the specific morphology of the crown. In the present invention, a method for controlling morphology by incorporating stimulation by growth factors including IGF into the generation mechanism of tooth epithelial-mesenchymal interaction has been developed. This technique enables morphological control not only in teeth but also in the formation of lungs, kidneys, liver, glandular tissues, limbs, fingers, etc., generated by epithelial-mesenchymal interactions.
臓器、器官、組織における移植医療および再生医療がすすめられるなかで、それらが機能性を有するためには、適切な大きさや形態が必須である。そのため、活発化する移植治療および再生医療においても、適切な形態制御技術と組み合わせてすすめられなければ、移植臓器、移植組織、再生臓器、再生組織の機能性は不十分となる。しかしながら、発生過程および再生過程において、形態制御のメカニズムはほとんど解明されておらず、論文報告も認められないのが現状である。また、移植医療や再生医療の技術基盤を向上させるために、その発生過程および再生過程と協調するメカニズムを用いた形態制御技術の開発は臨床成績を上げるうえでも必要とされているものの、その技術開発は不可能とされている。本発明では、まさに、歯の発生過程および再生過程にIGFを含む成長因子による刺激を組み合わせて、形態制御した歯が機能することを実現している。さらに、本発明は歯冠のサイズの制御だけでなく、咬頭形成および歯根形成をもコントロールできるため、機能性を再現できる技術といえる。 Appropriate size and form are indispensable for transplantation and regenerative medicine in organs, organs, and tissues to be functional. Therefore, even in the transplantation therapy and regenerative medicine that are activated, the functionality of the transplanted organ, the transplanted tissue, the regenerated organ, and the regenerated tissue becomes insufficient unless it is recommended in combination with an appropriate form control technique. However, in the developmental process and regeneration process, the mechanism of morphological control is hardly elucidated, and there are no paper reports. In addition, in order to improve the technological base of transplantation medicine and regenerative medicine, the development of morphological control technology using a mechanism that cooperates with the development process and regeneration process is also necessary to improve clinical results. Development is considered impossible. In the present invention, a tooth whose form is controlled is realized by combining stimulation with growth factors including IGF in the tooth generation process and regeneration process. Furthermore, since the present invention can control not only the crown size but also the cusp formation and root formation, it can be said that the technique can reproduce the functionality.
現在の歯科治療では、ブリッジやインプラント治療における人工物における機能代替が進められているが、歯根膜機能の再現が不可能であるため、機能性が不十分である。また、歯胚由来の上皮細胞と間葉細胞を再構築することによって、歯が再生し萌出することが明らかとなった。しかしながら、その大きさが矮小であった。 In current dental treatment, functional replacement of an artificial product in bridge and implant treatment is being promoted, but the functionality is insufficient because the periodontal ligament function cannot be reproduced. It was also clarified that teeth were regenerated and erupted by reconstructing epithelial cells and mesenchymal cells derived from tooth germ. However, its size was small.
従って本発明の目的は、正常な歯と同等の大きさを有し、正常に咬合しうる咬頭に咬頭形態を調整し、また、咀嚼力を支持する十分な歯根形成も調節し、正常な歯と同等の機能性を有するように歯欠損部を修復する修復方法を提供することである。 Therefore, an object of the present invention is to adjust the cusp shape to a cusp having a size equivalent to that of a normal tooth and capable of normal occlusion, and also to adjust sufficient root formation to support mastication, It is to provide a restoration method for repairing a tooth defect portion so as to have a function equivalent to that of the first embodiment.
現在の再生医療では、細胞を移入することで、欠損した組織を再生する方法が進められている。しかしながら、その再生する組織が歯の場合、形態と大きさの制御に限界がある。 In current regenerative medicine, a method of regenerating a deficient tissue by transferring cells is being promoted. However, when the tissue to be regenerated is a tooth, there is a limit to the control of form and size.
従って本発明の目的は、細胞移入療法における、歯という器官を再生させる技術を提供することである。 Accordingly, an object of the present invention is to provide a technique for regenerating an organ called a tooth in cell transfer therapy.
本発明は、以下のとおりである：
項１．インスリン様増殖因子（ＩＧＦ）を含む成長因子の存在下で培養する工程を含む、歯又は歯胚由来の細胞の培養方法であって、
当該培養工程において、歯又は歯胚由来の細胞は支持担体中に配置されており、当該支持担体中にＩＧＦが０．５〜８μｇ／ｍｌ配合されている、方法。
The present invention is as follows:
Item 1. A method for culturing cells derived from teeth or tooth germs, comprising culturing in the presence of a growth factor comprising insulin-like growth factor (IGF),
In the culturing step, cells derived from teeth or tooth embryos are arranged in a support carrier, and IGF is mixed in an amount of 0.5 to 8 μg / ml in the support carrier.
項２．ＩＧＦを含む成長因子の存在下で歯又は歯胚由来の細胞を培養する工程を含む、歯の組織再生能を有する細胞の製造方法であって、
当該培養工程において、歯又は歯胚由来の細胞は支持担体中に配置されており、当該支持担体中にＩＧＦが０．５〜８μｇ／ｍｌ配合されている、方法。
Item 2. A method for producing a cell having a tooth tissue regeneration ability, comprising a step of culturing a tooth or tooth germ-derived cell in the presence of a growth factor containing IGF,
In the culturing step, cells derived from teeth or tooth embryos are arranged in a support carrier, and IGF is mixed in an amount of 0.5 to 8 μg / ml in the support carrier.
項３．ＩＧＦを含む成長因子を含有する支持担体に歯又は歯胚由来の細胞を混合するか、又は当該支持体のなかで歯又は歯胚由来の細胞の凝集体を構築する工程を含む、再生歯胚の製造方法であって、
当該支持担体中にＩＧＦが０．５〜８μｇ／ｍｌ配合されている、方法。
Item 3. Regenerative tooth germ comprising a step of mixing a tooth or tooth germ-derived cell with a support carrier containing a growth factor containing IGF or constructing an aggregate of teeth or tooth germ-derived cells in the support A manufacturing method of
A method in which 0.5 to 8 μg / ml of IGF is blended in the support carrier.
項４．ＩＧＦを含む成長因子を含有する支持担体に歯又は歯胚由来の細胞を混合するか、又は当該支持体のなかで歯又は歯胚由来の細胞の凝集体を構築する工程、及び
上記工程で当該成長因子含有支持担体と混合されるか又は凝集体を構築した歯胚由来の細胞をＩＧＦの存在下で培養する工程を含む、再生歯胚又は再生歯の製造方法であって、
当該支持担体中にＩＧＦが０．５〜８μｇ／ｍｌ配合されている、方法。
Item 4. Mixing a cell derived from a tooth or tooth germ into a support carrier containing a growth factor containing IGF, or constructing an aggregate of cells derived from a tooth or tooth germ in the support, and the above step A method for producing a regenerated tooth germ or a regenerated tooth comprising a step of culturing a cell derived from a tooth germ mixed with a growth factor-containing support carrier or an aggregate in the presence of IGF,
A method in which 0.5 to 8 μg / ml of IGF is blended in the support carrier.
項５．ＩＧＦを含む成長因子を含有する支持担体に歯又は歯胚由来の細胞を混合するか、又は当該支持体のなかで歯又は歯胚由来の細胞の凝集体を構築する工程、及び
上記工程で当該成長因子含有支持担体と混合されるか又は凝集体を構築した歯胚由来の細胞をＩＧＦの存在下で培養する工程を含む、再生歯又は再生歯胚における歯冠、咬頭及び歯根のサイズを大きくし、その形態を制御する方法であって、
当該支持担体中にＩＧＦが０．５〜８μｇ／ｍｌ配合されている、方法。
Item 5. Mixing a cell derived from a tooth or tooth germ into a support carrier containing a growth factor containing IGF, or constructing an aggregate of cells derived from a tooth or tooth germ in the support, and the above step Increasing the size of crowns, cusps and roots in regenerated teeth or regenerated tooth germs, including the step of culturing cells derived from tooth germs mixed with growth factor-containing support carriers or constructed aggregates in the presence of IGF And a method for controlling its form,
A method in which 0.5 to 8 μg / ml of IGF is blended in the support carrier.
項６．ＩＧＦを含む成長因子を含有する支持担体中に再生歯胚を配置する工程、及び
当該再生歯胚を培養する工程を含む、再生歯又は再生歯胚における歯冠、咬頭及び歯根のサイズを大きくし、その形態を制御する方法であって、
当該培養工程において、当該支持担体中にＩＧＦが０．５〜８μｇ／ｍｌ配合されている、方法。
Item 6. Increasing the size of the crown, cusp, and root of the regenerated tooth or regenerated tooth germ, including the steps of placing the regenerated tooth germ in a support carrier containing a growth factor containing IGF and culturing the regenerated tooth germ A method of controlling its form,
In the culturing step, IGF is mixed in the support carrier in an amount of 0.5 to 8 μg / ml.
項７．支持担体が、コラーゲン、フィブリン、ラミニン、細胞外マトリクス混合物、ポリグリコール酸（ＰＧＡ）、ポリ乳酸（ＰＬＡ）、乳酸／グリコール酸共重合体（ＰＬＧＡ）、ハイドロゲル、プロテオグリカン、エンタクチン、多糖類、ハイドロキシアパタイト、β-ＴＣＰ、α-ＴＣＰ、アルミナセラミックス、乾燥骨、乾燥歯、乾燥歯根及び多糖類からなる群より選択される少なくとも一種を含む、項１〜６のいずれか一項に記載の方法。 Item 7. Support carrier is collagen, fibrin, laminin, extracellular matrix mixture, polyglycolic acid (PGA), polylactic acid (PLA), lactic acid / glycolic acid copolymer (PLGA), hydrogel, proteoglycan, entactin, polysaccharide, hydroxy Item 7. The method according to any one of Items 1 to 6, comprising at least one selected from the group consisting of apatite, β-TCP, α-TCP, alumina ceramics, dry bone, dry teeth, dry roots, and polysaccharides.
項８．ＩＧＦを含む成長因子を含有する、歯の再生用組成物であって、歯の再生用支持担体又は培地に最終濃度として0.5〜8μｇ／ｍｌとなるよう添加される量のＩＧＦを含む、組成物。 Item 8. A composition for tooth regeneration comprising a growth factor comprising IGF, the composition comprising IGF in an amount added to a support carrier or medium for tooth regeneration to a final concentration of 0.5 to 8 μg / ml .
項９．間葉系細胞及び／又は上皮系細胞をさらに含む、項８に記載の歯の再生用組成物。 Item 9. Item 9. The tooth regeneration composition according to Item 8, further comprising mesenchymal cells and / or epithelial cells.
項１０．支持担体が、コラーゲン、フィブリン、ラミニン、細胞外マトリクス混合物、ポリグリコール酸（ＰＧＡ）、ポリ乳酸（ＰＬＡ）、乳酸／グリコール酸共重合体（ＰＬＧＡ）、ハイドロゲル、プロテオグリカン、エンタクチン、多糖類、ハイドロキシアパタイト、β-ＴＣＰ、α-ＴＣＰ、アルミナセラミックス、乾燥骨、乾燥歯、乾燥歯根及び多糖類からなる群より選択される少なくとも一種を含む、項８又は９に記載の組成物。 Item 10. Support carrier is collagen, fibrin, laminin, extracellular matrix mixture, polyglycolic acid (PGA), polylactic acid (PLA), lactic acid / glycolic acid copolymer (PLGA), hydrogel, proteoglycan, entactin, polysaccharide, hydroxy Item 10. The composition according to Item 8 or 9, comprising at least one selected from the group consisting of apatite, β-TCP, α-TCP, alumina ceramics, dry bone, dry tooth, dry root, and polysaccharide.
項１１．
歯又は歯胚の間葉系組織から分離した間葉系細胞の細胞懸濁液と、歯又は歯胚の上皮組織から分離した上皮細胞の細胞懸濁液とを、ＩＧＦが０．５〜８μｇ／ｍｌ配合されたコラーゲンゲルに注入することにより得られる、該コラーゲンゲル内に該間葉系細胞が存在する区画と該上皮細胞が存在する区画とを有する、歯組織再生用組成物。
Item 11.
A cell suspension of mesenchymal cells isolated from the mesenchymal tissue of the tooth or tooth germ, and a cell suspension of epithelial cells isolated from the epithelial tissue of the tooth or tooth germ have an IGF of 0.5 to 8 μg. A composition for regenerating tooth tissue having a compartment in which the mesenchymal cells are present and a compartment in which the epithelial cells are present in the collagen gel obtained by injecting into a collagen gel formulated with / ml.
項１２．前記成長因子がＥＧＦ，ＴＧＦ、ＮＧＦ、ＢＤＮＦ、ＶＥＧＦ、Ｇ−ＣＳＦ、ＰＤＧＦ、ＥＰＯ、ＴＰＯ、ＦＧＦ、ＨＧＦ及びＢＭＰからなる群より選択される少なくとも一種をさらに含む、項１〜１１のいずれか一項に記載の方法又は組成物。 Item 12. Any one of Items 1 to 11, wherein the growth factor further comprises at least one selected from the group consisting of EGF, TGF, NGF, BDNF, VEGF, G-CSF, PDGF, EPO, TPO, FGF, HGF, and BMP. The method or composition according to Item.
項１３．項１〜４のいずれか一項に記載の方法により製造された細胞、再生歯胚又は再生歯を歯の欠損部分に移植する工程を含む、歯の欠損部の修復方法。 Item 13. Item 5. A method for repairing a tooth defect part, comprising a step of transplanting a cell, a regenerated tooth embryo or a regenerated tooth produced by the method according to any one of Items 1 to 4 to a tooth defect part.
項１４．項１〜４のいずれか一項に記載の方法により製造された細胞、再生歯胚又は再生歯を患者の歯の欠損部分に移植する工程を含む、欠損部を有する歯の治療方法。 Item 14. Item 5. A method for treating a tooth having a defective part, comprising a step of transplanting a cell, a regenerated tooth embryo or a regenerated tooth produced by the method according to any one of Items 1 to 4 to a defective part of a patient's tooth.
本発明によれば、歯冠と咬頭および歯根を形態制御することで、正常に咬合し、正常な歯と同等の硬度を有し、正常な歯と同等の機能性を有するように歯の再生方法を提供することができる。 According to the present invention, by controlling the form of the crown, the cusp, and the root, the teeth are regenerated so that they normally occlude, have the same hardness as normal teeth, and have the same functionality as normal teeth. A method can be provided.
本発明の再生歯の再構築の方法は、歯胚由来の上皮細胞および間葉細胞であることが望ましい。第１の細胞塊と第２の細胞塊を混合することなく密着させて支持担体の内部に配置し培養して再構成歯胚又は、歯が得られる。 The method for reconstructing a regenerated tooth according to the present invention is preferably an epithelial cell and a mesenchymal cell derived from a tooth germ. The first cell mass and the second cell mass are brought into close contact with each other without being mixed, placed inside the support carrier and cultured to obtain a reconstructed tooth germ or tooth.
以下、本発明について説明する。 The present invention will be described below.
本発明において、「歯」とは、硬組織として内側に象牙質及び外側にエナメル質の層を連続して備え、中心に歯髄が配置し、歯冠や歯根を有する方向性を備えた組織を意味する。歯冠とは、エナメル質と象牙質の層構造の部分をいい、歯根にはエナメル質の層は存在せず、セメント質が存在し、歯根膜によって歯槽骨と連結し歯冠部を支持する。 In the present invention, the term “tooth” refers to a tissue having a directivity having a dentin on the inside and a layer of enamel on the outside as a hard tissue, a pulp disposed in the center, and a crown or root. means. The dental crown is the part of the layer structure of enamel and dentin, and there is no enamel layer in the root of the tooth, cementum is present, and it is connected to the alveolar bone by the periodontal ligament and supports the crown. .
象牙質及びエナメル質は、免疫染色などによって形態的に容易に特定することができる。また、エナメル質は、エナメル芽細胞により産生される。エナメル芽細胞の存在は、アメロブラスチンおよびアメロジェニンの有無によって確認することができる。一方、象牙質は、象牙芽細胞の存在によって特定することができ、象牙芽細胞の存在は、デンチンシアロプロテインの有無によって確認することができる。 Dentin and enamel can be easily identified morphologically by immunostaining or the like. Enamel is also produced by enamel blasts. The presence of enamel blasts can be confirmed by the presence or absence of ameloblastin and amelogenin. On the other hand, dentin can be identified by the presence of odontoblasts, and the presence of odontoblasts can be confirmed by the presence or absence of dentin sialoprotein.
また、歯の方向性は、歯冠や歯根の配置によって特定することができる。歯冠や歯根は、組織構造や組織染色などに基づいて認識することができる。 In addition, the directionality of teeth can be specified by the arrangement of crowns and roots. The crown and root can be recognized based on the tissue structure and tissue staining.
本発明において「歯胚」及び「歯」は、発生段階に基づいて区別されたものに特に言及する場合に用いられる表現である。この場合の「歯胚」とは、将来歯になることが決定付けられた歯の原基であり、歯の発生ステージで一般的に用いられる蕾状期（Bud stage）から鐘状期（Bell stage）までの段階のものを指している。歯胚は最初、口腔粘膜上皮と間葉組織由来の細胞が増殖したものであり、歯胚は、エナメル器、歯乳頭、歯小嚢に分化する。エナメル器はエナメル質、歯乳頭は歯髄と象牙質、歯小嚢はセメント質、歯根膜、歯槽骨になって、歯が完成する。尚、本明細書において、「再生歯」及び「再生歯胚」は、歯又は歯胚由来の細胞を用いて再生した歯及び歯胚をそれぞれ示す。また、本明細書中においては、「再生歯胚」という場合には、細胞培養の結果、蕾状期から鐘状期までの段階に達したものだけでなく、歯又は歯胚由来の細胞を支持担体に混合したものも包含することとする。 In the present invention, “dental germ” and “tooth” are expressions used when specifically referring to those distinguished based on the stage of development. The “tooth germ” in this case is a tooth primordium that has been determined to become a future tooth, and is generally used in the tooth development stage from the Bud stage to the Bell stage (Bell stage). stage). Tooth germs are initially grown from cells derived from the oral mucosal epithelium and mesenchymal tissue, and the tooth germs differentiate into enamel organs, tooth papilla, and dental follicles. The enamel is enamel, the tooth papilla is the pulp and dentin, the dental follicle is cementum, the periodontal ligament, and the alveolar bone, completing the tooth. In the present specification, “regenerated tooth” and “regenerated tooth germ” refer to a tooth and a tooth germ regenerated using a tooth or a tooth germ-derived cell, respectively. In the present specification, the term “regenerated tooth germ” refers to not only cells that have reached the stage from the rod-shaped stage to the bell-shaped stage as a result of cell culture, but also cells derived from teeth or tooth germs. Those mixed with a support carrier are also included.
本発明の方法においては、培養工程に供する「歯又は歯胚由来の細胞」には、上記の「歯」又は「歯胚」から取り出した細胞、当該細胞を適宜酵素処理したもの等だけでなく、これらの細胞と同様に歯又は歯胚の再生能を有するものも含まれる。歯又は歯胚の再生能を有する細胞としては、例えば、歯又は歯胚への分化能を有する幹細胞、ｉＰＳ細胞等も含まれる。従って、本発明の方法には、当該幹細胞及びｉＰＳ細胞を培養して、歯又は歯胚を再生する方法も含まれる。幹細胞、ｉＰＳ細胞を用いて歯又は歯胚を再生するための条件としては自体公知の方法を適宜用いることができる。 In the method of the present invention, “the tooth or tooth germ-derived cell” to be subjected to the culturing step includes not only the above-described “tooth” or “tooth germ” but also the cells appropriately enzyme-treated, etc. Those having the ability to regenerate teeth or tooth germs as well as these cells are also included. Examples of cells having the ability to regenerate teeth or tooth germs include stem cells and iPS cells having the ability to differentiate into teeth or tooth germs. Therefore, the method of the present invention also includes a method of regenerating teeth or tooth germs by culturing the stem cells and iPS cells. As conditions for regenerating a tooth or a tooth germ using stem cells and iPS cells, a method known per se can be appropriately used.
なお本発明において「間葉系細胞」とは、間葉組織由来の細胞を意味し、「上皮系細胞」とは上皮組織由来の細胞を意味する。 In the present invention, “mesenchymal cell” means a cell derived from mesenchymal tissue, and “epithelial cell” means a cell derived from epithelial tissue.
本発明は、ＩＧＦを含む成長因子の存在下で培養することを特徴とする、歯又は歯胚由来の細胞の培養方法を提供する。当該方法により、歯の組織再生能を有する細胞を製造することができる。具体的な培養方法としては、例えば、歯又は歯胚由来の細胞を用いて再生歯胚を再構築し、これを器官培養する方法等が挙げられる。 The present invention provides a method for culturing cells derived from teeth or tooth germs, which comprises culturing in the presence of a growth factor containing IGF. By this method, cells having the ability to regenerate dental tissues can be produced. Specific examples of the culture method include a method of reconstructing a regenerated tooth germ using a tooth or tooth germ-derived cell and organ-culturing the reconstructed tooth germ.
ＩＧＦとしては、ＩＧＦ−１及びＩＧＦ−２が挙げられ、好ましくはＩＧＦ−１が用いられる。 Examples of IGF include IGF-1 and IGF-2, and preferably IGF-1 is used.
ＩＧＦ以外の成長因子としては、ＥＧＦ，ＴＧＦ、ＮＧＦ、ＢＤＮＦ、ＶＥＧＦ、Ｇ−ＣＳＦ、ＰＤＧＦ、ＥＰＯ、ＴＰＯ、ＦＧＦ、ＨＧＦ及びＢＭＰを用いることができる。 As growth factors other than IGF, EGF, TGF, NGF, BDNF, VEGF, G-CSF, PDGF, EPO, TPO, FGF, HGF and BMP can be used.
IGF-1を用いた形態制御した再生歯胚の再構築方法
本発明の方法においては、まずＩＧＦを含む成長因子を含有する支持担体に歯胚由来の細胞を混合する。これにより、再生歯胚が再構築される。例えば、以下の方法が挙げられる。歯胚上皮組織ならびに歯胚間葉組織から採取した単一化細胞を、それぞれシリコンコートした1.5 mlマイクロ遠心管 (Watson, Parsippany, NJ, USA)に入れ、2500rpmで3分間遠心し、その上清をGELoader Tip 0.5-20μl (Eppendorf, Hamburg, Germany)で完全に除去し、上皮細胞と間葉細胞の高密度細胞懸濁液を調製する。次にシリコンコートした35mmペトリディッシュの上に30μlのタイプIコラーゲンゲルをドロップ状に形成し、このコラーゲンゲルにはIGF-1を混合する。さらに、ハミルトンシリンジ(Osaka chemical，Osaka，Japan)を用いて0.05μlの間葉細胞の高密度細胞懸濁液をゲル内に注入する。続いて0.05μlの上皮細胞懸濁液を間葉細胞の凝集体に接合させるようにして注入し、上皮細胞と間葉細胞が高密度で区画化された再生歯胚を作製する。上記のように、本発明の方法において、ＩＧＦを含む成長因子を含有する支持担体に歯又は歯胚由来の細胞を混合する方法には、例えば、当該支持担体に歯又は歯胚由来の細胞を注入する方法も包含される。
Method for Reconstructing Morphologically Controlled Regenerated Tooth Germ Using IGF-1 In the method of the present invention, first, tooth germ-derived cells are mixed with a support carrier containing a growth factor containing IGF. Thereby, the regenerated tooth germ is reconstructed. For example, the following method is mentioned. Single cells collected from tooth germ epithelial tissue and tooth germ mesenchymal tissue are placed in a silicon-coated 1.5 ml microcentrifuge tube (Watson, Parsippany, NJ, USA) and centrifuged at 2500 rpm for 3 minutes. Is completely removed with GELoader Tip 0.5-20 μl (Eppendorf, Hamburg, Germany) to prepare a high-density cell suspension of epithelial and mesenchymal cells. Next, 30 μl of type I collagen gel is formed in a drop shape on a silicon-coated 35 mm Petri dish, and IGF-1 is mixed into the collagen gel. Furthermore, 0.05 μl of a high-density cell suspension of mesenchymal cells is injected into the gel using a Hamilton syringe (Osaka chemical, Osaka, Japan). Subsequently, 0.05 μl of an epithelial cell suspension is injected so as to be joined to an aggregate of mesenchymal cells to produce a regenerated tooth germ in which epithelial cells and mesenchymal cells are compartmentalized at high density. As described above, in the method of the present invention, a method of mixing a tooth or tooth germ-derived cell with a support carrier containing a growth factor containing IGF includes, for example, a tooth or tooth germ-derived cell on the support carrier. Injecting methods are also encompassed.
ここで細胞集合体１個あたりの細胞数を、一般に１０１〜１０８個、好ましくは１０３〜１０８個とすることができる。 Here, the number of cells per cell aggregate can be generally 10 1 to 10 8 , preferably 10 3 to 10 8 .
本発明において、IGFを含む成長因子を支持担体に添加するにあたり、例えば、その濃度は０．５〜２５μg/ｍｌ等の濃度が例示される。歯冠、咬頭及び歯根のサイズを大きくする形態制御の観点から、ＩＧＦを０．５〜８μｇ／ｍｌとすることを特徴とし、０．５〜４μｇ／ｍｌとすることがより好ましい。ただし、再生させる臓器、組織、細胞によってその濃度は変更される。また、添加する支持担体の種類によっても、その濃度は変更される。 In the present invention, when the growth factor containing IGF is added to the support carrier, for example, the concentration is 0.5 to 25 μg / ml. From the viewpoint of morphological control to increase the size of the crown, cusp and root, IGF is 0.5 to 8 μg / ml, and more preferably 0.5 to 4 μg / ml. However, the concentration varies depending on the organs, tissues, and cells to be regenerated. The concentration is also changed depending on the type of support carrier to be added.
歯胚以外に由来する間葉系細胞としては、生体内の他の間葉系組織に由来する細胞である。好ましくは、骨髄細胞や間葉系幹細胞、さらに好ましくは歯髄細胞や歯根膜細胞などの口腔内間葉系細胞や顎骨の内部の骨髄細胞、間葉系前駆細胞やその幹細胞等を挙げることができるが、これに限定されるものではない。 The mesenchymal cells derived from other than the tooth germ are cells derived from other mesenchymal tissues in the living body. Preferably, bone marrow cells and mesenchymal stem cells, more preferably oral mesenchymal cells such as dental pulp cells and periodontal ligament cells, bone marrow cells in the jawbone, mesenchymal progenitor cells and stem cells thereof, etc. However, the present invention is not limited to this.
また、歯胚以外に由来する上皮系細胞としては、生体内の他の上皮系組織に由来する細胞である。好ましくは、皮膚や口腔内の粘膜や歯肉の上皮系細胞、エナメル芽細胞、さらに好ましくは皮膚や粘膜などの分化した、例えば角化した、あるいは錯角化した上皮系細胞とその上皮系前駆細胞、たとえば非角化上皮系細胞やその幹細胞等を挙げることができるが、これに限定されるものではない。 In addition, epithelial cells derived from other than tooth germs are cells derived from other epithelial tissues in the living body. Preferably, epithelial cells of skin and oral mucosa and gingiva, enamel blasts, more preferably differentiated, eg keratinized or keratinized epithelial cells and their epithelial progenitor cells such as skin and mucous membranes, Examples include non-keratinized epithelial cells and stem cells thereof, but are not limited thereto.
歯胚及び他の組織は、哺乳動物の霊長類、例えばヒト、サル、マーモセットなど、有蹄類、例えば豚、牛、馬など、食肉類のイヌなど、小型哺乳類の齧歯類、例えばマウス、ラット、ウサギなどの種々の動物の顎骨等から採取することができる。なお、ヒトの歯胚としては、第３大臼歯いわゆる親知らずの歯胚の他、胎児歯胚を挙げることができるが、自家組織の利用との観点から、親知らず歯胚を用いることが好ましい。また、抜歯した永久歯及び乳歯の歯髄組織、歯根膜組織を用いることも好ましい。また、マウスの場合、胎日齢１０日から１６日の歯胚を用いることが好ましい。 Tooth germs and other tissues include mammalian primates such as humans, monkeys, marmosets, ungulates such as pigs, cows, horses, carnivorous dogs, small mammalian rodents such as mice, It can be collected from jawbones of various animals such as rats and rabbits. Examples of human tooth germs include fetal tooth germs in addition to third molars, so-called wisdom tooth germs, but it is preferable to use wisdom tooth germs from the viewpoint of use of autologous tissue. In addition, it is also preferable to use the dental pulp tissue and periodontal ligament tissue of extracted permanent teeth and deciduous teeth. In the case of a mouse, it is preferable to use a tooth germ of 10 to 16 days of gestation.
この歯胚からの間葉系細胞及び上皮系細胞の調製は、まず周囲の組織から単離された歯胚を、形状に従って歯胚間葉組織及び歯胚上皮組織に外科的に分けることによって行われる。この際、分離を容易に行うため酵素を用いてもよい。このような用途に用いられる酵素としては、ディスパーゼ、コラーゲナーゼ、トリプシン、ヒアルロニダーゼ、エラスターゼ等を挙げることができる。 Preparation of mesenchymal and epithelial cells from the tooth germ is performed by first surgically dividing the tooth germ isolated from the surrounding tissue into tooth germ mesenchymal tissue and tooth germ epithelial tissue according to the shape. Is called. At this time, an enzyme may be used for easy separation. Examples of the enzyme used for such applications include dispase, collagenase, trypsin, hyaluronidase, and elastase.
間葉系細胞及び上皮系細胞は、それぞれ間葉組織及び上皮組織から単一細胞の状態に調製してもよい。単一の細胞に容易に分散可能とするために、ディスパーゼ、コラーゲナーゼ、トリプシン等の酵素を用いてもよい。 Mesenchymal cells and epithelial cells may be prepared in a single cell state from mesenchymal tissue and epithelial tissue, respectively. Enzymes such as dispase, collagenase, and trypsin may be used so that they can be easily dispersed in a single cell.
なお、間葉系細胞及び上皮系細胞は、それぞれ充分な細胞数を得るために、予備的な培養を経たものであってもよい。望ましくは、２〜４継代のものが望ましい。また、凍結保存して、融解したものであっても構わない。 In addition, the mesenchymal cell and the epithelial cell may have undergone preliminary culture in order to obtain a sufficient number of cells. Desirably, those having 2 to 4 passages are desirable. Further, it may be frozen and thawed.
本発明で用いられる、臓器および器官を再構成させる支持担体としては、好ましくは、上記培地との混合物である。このような支持担体としては、コラーゲン、アガロースゲル、カルボキシメチルセルロース、ゼラチン、寒天、ハイドロゲル、プロテオグリカン、エンタクチン、エラスチン、フィブリン、ラミニン、細胞外マトリクス混合物、ポリグリコール酸（ＰＧＡ）、ポリ乳酸（ＰＬＡ）、乳酸／グリコール酸共重合体（ＰＬＧＡ）、多糖類、等を挙げることができる。通常、三次元培養として適用される硬さであることが好ましい。 The support used in the present invention for reconstituting organs and organs is preferably a mixture with the above medium. Such support carriers include collagen, agarose gel, carboxymethylcellulose, gelatin, agar, hydrogel, proteoglycan, entactin, elastin, fibrin, laminin, extracellular matrix mixture, polyglycolic acid (PGA), polylactic acid (PLA) , Lactic acid / glycolic acid copolymer (PLGA), polysaccharides, and the like. Usually, the hardness applied as three-dimensional culture is preferable.
また、組織を再生させるために用いる支持担体は、コラーゲン、フィブリン、ラミニン、細胞外マトリクス混合物、ポリグリコール酸（ＰＧＡ）、ポリ乳酸（ＰＬＡ）、乳酸／グリコール酸共重合体（ＰＬＧＡ）、ハイドロゲル、プロテオグリカン、エンタクチン、ハイドロキシアパタイト、β-ＴＣＰ、α-ＴＣＰ、アルミナセラミックス、乾燥骨、乾燥歯、乾燥歯根及び多糖類からなる群より選択された少なくとも１種であることが好ましい。臓器および器官の再構成又は組織再生のための支持担体の市販品としては、セルマトリクス（コラーゲン）、メビオールゲル（（ポリマー素材を使用した、温度に応答する熱可逆性ハイドロゲル）、マトリゲル（主成分：ラミニン、コラーゲンIV、ヘパラン硫酸プロテオグリカン、およびエンタクチン/ニドジェンの混合物）等が挙げられる。 Supporting carriers used for tissue regeneration are collagen, fibrin, laminin, extracellular matrix mixture, polyglycolic acid (PGA), polylactic acid (PLA), lactic acid / glycolic acid copolymer (PLGA), hydrogel It is preferably at least one selected from the group consisting of proteoglycan, entactin, hydroxyapatite, β-TCP, α-TCP, alumina ceramics, dry bone, dry teeth, dry roots and polysaccharides. Commercially available support carriers for organ and organ reconstruction or tissue regeneration include cell matrix (collagen), meviol gel ((thermoreversible hydrogel that responds to temperature using polymer material), matrigel (main component) : Laminin, collagen IV, heparan sulfate proteoglycan, and a mixture of entactin / nidogen).
培養方法
培養方法としては、器官培養法として自体公知の方法を適宜用いることができる。例えば、細胞培養プレート上で、上記で再構築された再生歯胚を、適切な培地を用いて培養する。
Culturing method As a culturing method, a method known per se as an organ culturing method can be appropriately used. For example, the regenerated tooth germ reconstructed above is cultured on a cell culture plate using an appropriate medium.
培養培地としては、例えば、ダルベッコ改変イーグル(DMEM)培地、αMEM培地、DMEM/F12培地、RPMI培地等が挙げられる。また、ダルベッコ改変イーグル培地と成分系が同等の培地であれば使用可能である。当該培養培地にさらにＩＧＦを添加してもよい。その場合の配合量は、例えば、培地中の最終濃度として、50〜200ｎｇ／ｍｌ、好ましくは100ｎｇ／ｍｌの範囲で適宜設定できる。また、当該培養培地には、前述したＩＧＦ以外の成長因子を添加してもよい。その場合、ＩＧＦ以外の成長因子の配合量は、例えば、培地中の最終濃度として、50〜200ｎｇ／ｍｌ、好ましくは100ｎｇ／ｍｌの範囲で適宜設定できる。培養温度は特に限定されないが、例えば、37℃で適宜設定される。また、培養時間も特に限定されないが、例えば、14日間、より好ましくは10〜14日の範囲で適宜設定できる。動物に移植する場合は、7日間以内の培養期間が好ましい。 Examples of the culture medium include Dulbecco's modified Eagle (DMEM) medium, αMEM medium, DMEM / F12 medium, RPMI medium, and the like. Further, Dulbecco's modified Eagle medium can be used as long as the component system is the same. IGF may be further added to the culture medium. In this case, the blending amount can be appropriately set, for example, in the range of 50 to 200 ng / ml, preferably 100 ng / ml, as the final concentration in the medium. Moreover, you may add growth factors other than IGF mentioned above to the said culture medium. In that case, the compounding quantity of growth factors other than IGF can be suitably set in the range of 50 to 200 ng / ml, preferably 100 ng / ml, for example, as the final concentration in the medium. The culture temperature is not particularly limited, but is appropriately set at 37 ° C., for example. In addition, the culture time is not particularly limited, but can be appropriately set, for example, in the range of 14 days, more preferably in the range of 10 to 14 days. When transplanted to animals, a culture period of 7 days or less is preferred.
歯の再生用組成物
本発明は、ＩＧＦを含む成長因子を含有する、歯の再生用組成物を提供する。有効成分であるＩＧＦ、その他の成長因子等については前述のものを用いることができる。
Tooth regeneration composition The present invention provides a tooth regeneration composition containing a growth factor comprising IGF. As the active ingredient IGF and other growth factors, those described above can be used.
また、本発明の歯の再生用組成物には、担体および添加剤を配合することができる。担体及び添加剤としては、溶剤、賦形剤、コーティング剤、基剤、結合剤、滑沢剤、崩壊剤、溶解補助剤、懸濁化剤、粘稠剤、乳化剤、安定剤、緩衝剤、等張化剤、保存剤、着色剤が挙げられる。 Moreover, a carrier and an additive can be mix | blended with the composition for tooth | gear reproduction | regeneration of this invention. Carriers and additives include solvents, excipients, coating agents, bases, binders, lubricants, disintegrants, solubilizers, suspending agents, thickeners, emulsifiers, stabilizers, buffers, Examples include isotonic agents, preservatives, and coloring agents.
また、本発明においては、歯又は歯胚の間葉系組織から分離した間葉系細胞の細胞懸濁液と、歯又は歯胚の上皮組織から分離した上皮細胞の細胞懸濁液とを、ＩＧＦが０．５〜８μｇ／ｍｌ配合されたコラーゲンゲルに注入することにより得られる、該コラーゲンゲル内に該間葉系細胞が存在する区画と該上皮細胞が存在する区画とを有する、歯組織再生用組成物を提供する。 Further, in the present invention, a cell suspension of mesenchymal cells separated from the mesenchymal tissue of the tooth or tooth germ, and a cell suspension of epithelial cells separated from the epithelial tissue of the tooth or tooth germ, Dental tissue having a section where the mesenchymal cells are present and a section where the epithelial cells are present in the collagen gel obtained by injecting into a collagen gel containing 0.5-8 μg / ml of IGF A regenerating composition is provided.
ここで、細胞懸濁液の細胞密度としては、特に限定されないが、例えば、１×１０８〜１×１０９ｃｅｌｌ／ｍｌの範囲で適宜設定できる。 Here, the cell density of the cell suspension is not particularly limited, but can be appropriately set within a range of 1 × 10 8 to 1 × 10 9 cells / ml, for example.
本発明の歯の再生用組成物は、前述した再生歯胚及び再生歯の製造方法、形態形成方法に用いることができる。 The tooth regeneration composition of the present invention can be used in the above-described method for producing a regenerated tooth embryo and regenerated tooth, and a method for forming a form thereof.
歯の欠損部の修復方法及び治療方法
本発明は、前述の方法により製造された細胞、再生歯胚又は再生歯を歯の欠損部分に移植する工程を含む、歯の欠損部の修復方法、及び前述の方法により製造された細胞、再生歯胚又は再生歯を患者の歯の欠損部分に移植する工程を含む、欠損部を有する歯の治療方法を提供する。
The present invention relates to a method for repairing a tooth defect, comprising the step of transplanting a cell, a regenerated tooth germ or a regenerated tooth produced by the above method into a tooth defect, and There is provided a method for treating a tooth having a defect, which comprises the step of transplanting a cell, a regenerated tooth embryo or a regenerated tooth produced by the above-described method into a tooth defect of a patient.
細胞、再生歯胚又は再生歯の移植方法は、自体公知の方法を用いるか、これに準じて行うことができる。 The method of transplanting cells, regenerated tooth embryos or regenerated teeth can be performed using a method known per se or in accordance with this method.
本発明では、マウスの歯胚の間葉細胞と上皮細胞によって作製した再生歯の歯冠と咬頭の形態制御が実施例によって明らかになっている。マウスはヒトと相同な遺伝子と器官、組織を有している。また、マウスは個体レベルで特定の遺伝子を欠損させることができるため、マウスはヒト遺伝子の組織形成における役割を個体レベルで解明するために最も優れたモデル動物とされている。歯科領域では、ヒトとマウスと両方で歯由来の幹細胞の解析が進んでいる。例えば、文献PLoS ONE. 2006; 1(1): e79.及び 文献Lancet. 2004 Jul 10-16;364(9429):149-55では、歯髄幹細胞がヒトで報告されて, 非特許文献４では、マウスでも報告されている。さらに、歯由来の幹細胞は、文献PLoS ONE. 2006; 1(1): e79.、文献 Lancet. 2004 Jul 10-16;364(9429):149-55、文献Proceeding of the National Academy of Sciences of the United States of America(PNAS) 2003 May 13;100(10):5807-12. Epub 2003 Apr 25、文献J Dent Res. 2003 Dec;82(12):976-81で報告されるとおりに、培養法が確立されているため、本発明技術はヒトへの応用が期待される。 In the present invention, the control of the morphology of the dental crown and cusp of a regenerated tooth produced by mesenchymal cells and epithelial cells of a mouse tooth germ has been clarified by Examples. Mice have genes, organs, and tissues that are homologous to humans. In addition, since a mouse can be deficient in a specific gene at the individual level, the mouse is regarded as the best model animal for elucidating the role of human genes in tissue formation at the individual level. In the dental field, analysis of stem cells derived from teeth is progressing in both humans and mice. For example, in the document PLoS ONE. 2006; 1 (1): e79. And the document Lancet. 2004 Jul 10-16; 364 (9429): 149-55, dental pulp stem cells have been reported in humans. It has also been reported in mice. Furthermore, stem cells derived from teeth are described in literature PLoS ONE. 2006; 1 (1): e79., Literature Lancet. 2004 Jul 10-16; 364 (9429): 149-55, literature Proceeding of the National Academy of Sciences of the. Culture methods as reported in United States of America (PNAS) 2003 May 13; 100 (10): 5807-12.Epub 2003 Apr 25, J J Dent Res. 2003 Dec; 82 (12): 976-81 Therefore, the technique of the present invention is expected to be applied to humans.
本発明では、IGF-1添加によって、咬頭形成が制御できる技術が開示されている。歯は発生過程のなかで、エナメルノットというシグナルセンターが発生し、咬頭形成が調節されている(Mech Dev. 1996 Jan;54(1):39-43.)。また、文献J Cell Physio.2012 April 227(4):1455-1464では、Sonic HedgehogがIGF-1と相互作用して、筋細胞の増殖と分化に影響することが報告されている。同様のメカニズムによってIGF-1がSonic Headgehogと協調し、本発明における歯の咬頭の形態制御に影響していると推察される。 In the present invention, a technique capable of controlling cusp formation by adding IGF-1 is disclosed. During tooth development, a signal center called an enamel knot is generated, and cusp formation is regulated (Mech Dev. 1996 Jan; 54 (1): 39-43.). Further, in the document J Cell Physio. 2012 April 227 (4): 1455-1464, it is reported that Sonic Hedgehog interacts with IGF-1 and affects myocyte proliferation and differentiation. It is surmised that IGF-1 cooperates with Sonic Headgehog by the same mechanism and influences the shape control of the tooth cusp in the present invention.
以下に、実施例及び比較例を挙げて本発明をさらに詳細に説明するが、本発明はこれらの実施例に限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited to these Examples.
実施例１
［１−１］歯胚由来上皮系細胞と間葉系細胞の単一化細胞の調整
Ｃ５７ＢＬ／６Ｊマウスの胎齢14.5日の胎仔下顎臼歯歯胚を50 U/ml Dispase (BD, Franklin Lakes, NJ, USA)及び35U/ml Deoxyribonuclease I (DNase I, Sigma, St. Louis, MO, USA)を含むphosphate buffer saline (PBS)で室温4分間の酵素処理を行った後に、上皮組織及び間葉組織を25G針を用いて分離した。
歯胚上皮組織は、100U/ml Collagenase I (Worthington, Lakewood, NJ, USA) と35U/ml DNase Iを含むPBSを用いて、37℃ 15分間の酵素処理を2回行い、その後0.25% Trypsin (Sigma)と35U/ml DNase Iを含むPBSを用いて、37℃で5分間の酵素処理を行い、単一化細胞にした。歯胚間葉細胞は0.25% Trypsin, 50U/ml Collagenase Iと35U/ml DNase Iを含むPBSを用いて、37℃で15分間の酵素処理を行い、単一化細胞にした。
Example 1
[1-1] Preparation of single cells of tooth germ-derived epithelial cells and mesenchymal cells C57BL / 6J mice were treated with 50 U / ml Dispase (BD, Franklin Lakes, NJ) , USA) and 35 U / ml Deoxyribonuclease I (DNase I, Sigma, St. Louis, MO, USA) with phosphate buffer saline (PBS) for 4 minutes at room temperature. Separated using a 25G needle.
Tooth germ epithelial tissue was subjected to enzyme treatment twice at 37 ° C. for 15 minutes using PBS containing 100 U / ml Collagenase I (Worthington, Lakewood, NJ, USA) and 35 U / ml DNase I, and then 0.25% Trypsin ( (Sigma) and PBS containing 35 U / ml DNase I were subjected to enzyme treatment at 37 ° C. for 5 minutes to form single cells. Tooth germ mesenchymal cells were treated with PBS containing 0.25% Trypsin, 50 U / ml Collagenase I and 35 U / ml DNase I at 37 ° C. for 15 minutes to form single cells.
［１−２］再生歯胚作製とIGF-1 添加
歯胚上皮組織ならびに歯胚間葉組織から採取した単一化細胞を、それぞれシリコンコートした1.5 mlマイクロ遠心管 (Watson, Parsippany, NJ, USA)に入れ、2500rpmで3分間遠心し、その上清をGELoader Tip 0.5-20μl (Eppendorf, Hamburg, Germany)で完全に除去し、上皮細胞及び間葉細胞のそれぞれの高密度細胞懸濁液を調製した（上皮細胞懸濁液及び間葉細胞懸濁液の細胞密度は、それぞれ、５×１０８ cells/ml）。次にシリコンコートした35mmペトリディッシュの上に30μlのタイプIコラーゲンゲルをドロップ状に形成し、ハミルトンシリンジ(Osaka chemical，Osaka，Japan)を用いて0.05μlの間葉細胞の高密度細胞懸濁液をゲル内に注入することにより、間葉細胞の凝集体を作製した。続いて0.05μlの上皮細胞懸濁液を間葉細胞の凝集体に接合させるようにして注入し、上皮細胞と間葉細胞が高密度で区画化された再生歯胚を作製した。IGF-1添加群には4μg/mlとなるようIGF-1が添加されたゲルを用いて再生歯胚を作製した（実施例１−１）。IGF-1を添加していないゲルを用いる以外、上記と同様の操作を行い、再生歯胚を作製した（比較例１−１）。当該再生歯胚の作製方法の概略図を図１３に示す。
[1-2] Preparation of regenerated tooth germ and IGF-1-added tooth germ epithelial tissue and singulated cells collected from tooth germ mesenchymal tissue were each coated with a silicon-coated 1.5 ml microcentrifuge tube (Watson, Parsippany, NJ, USA) ), Centrifuge at 2500 rpm for 3 minutes, and completely remove the supernatant with GELoader Tip 0.5-20 μl (Eppendorf, Hamburg, Germany) to prepare high-density cell suspensions of epithelial cells and mesenchymal cells. (The cell densities of the epithelial cell suspension and the mesenchymal cell suspension were 5 × 10 8 cells / ml, respectively). Next, 30 μl of type I collagen gel is formed in a drop shape on a silicon-coated 35 mm Petri dish, and 0.05 μl of high-density cell suspension of mesenchymal cells using a Hamilton syringe (Osaka chemical, Osaka, Japan) Was injected into the gel to produce mesenchymal cell aggregates. Subsequently, 0.05 μl of an epithelial cell suspension was injected so as to be joined to an aggregate of mesenchymal cells, and a regenerated tooth germ in which epithelial cells and mesenchymal cells were partitioned at high density was produced. In the IGF-1 addition group, regenerated tooth germs were prepared using a gel to which IGF-1 was added so as to be 4 μg / ml (Example 1-1). A regenerated tooth germ was prepared by performing the same operation as described above except that a gel to which IGF-1 was not added was used (Comparative Example 1-1). A schematic diagram of the method for producing the regenerated tooth germ is shown in FIG.
［１−３］IGF添加による形態を増大させた再生歯胚の調整
上記［１−２］で得られた再生歯胚を24-well cell culture plates (BD)に設置した0.4μm pore diameter cell culture inserts (BD)上で、10% FBS (MP Biomedicals, Solon, OH, USA), 100μg/ml ascorbic acid (Sigma), 2mM L-glutamine (Sigma), Dulbecco’s modified eagle medium (DMEM, Sigma)を用いて器官培養を行った。さらにIGF-1添加群には、100ng/ml となるようIGF-1を添加した上記培地を用いて培養した。
培養1日目で上皮細胞と間葉細胞の境界面に上皮・間葉相互作用の指標であるTranslucent zoneが形成され、培養14日目には鐘状期に相当する再生歯胚へと発生した。また、位相差顕微鏡像で、図１(Ｂ)に示すようにIGF-1添加群（実施例１−１）においてサイズの増大が観察された。
[1-3] Preparation of regenerated tooth germ with increased morphology by addition of IGF 0.4 μm pore diameter cell culture in which the regenerated tooth germ obtained in [1-2] above was placed in 24-well cell culture plates (BD) on inserts (BD) using 10% FBS (MP Biomedicals, Solon, OH, USA), 100 μg / ml ascorbic acid (Sigma), 2 mM L-glutamine (Sigma), Dulbecco's modified eagle medium (DMEM, Sigma) Organ culture was performed. Furthermore, in the IGF-1 addition group, it culture | cultivated using the said culture medium which added IGF-1 so that it might become 100 ng / ml.
On the first day of culture, a translucent zone, an index of epithelial-mesenchymal interaction, was formed at the interface between epithelial cells and mesenchymal cells, and on day 14 of culture, it developed into a regenerated tooth germ corresponding to the bell-shaped phase . In addition, as shown in FIG. 1B, an increase in size was observed in the IGF-1 added group (Example 1-1) in the phase contrast microscope image.
［１−４］IGF添加による形態を増大させた再生歯胚とIGF非添加の再生歯胚との比較
上記［１−３］の器官培養を行い、位相差顕微鏡像で再生歯胚のサービカルループを結んだ線と直交する歯胚の最大径を長径、長径と直交する歯冠部の最大径を幅径とし、14日目の時点の長径と幅径を計測した。図２（Ａ）に示すように長径は培養14日目においてIGF-1添加群で944μm、対照群で857μmであった。また図2（Ｂ）に示すように、幅径は培養14日目においてIGF-1添加群で969μm、対照群で814μmであった。これらの結果から、再生歯胚の作製過程でIGF-1を添加することによって再生歯胚の長径および幅径の増大が起こることが示唆された。
[1-4] Comparison of Regenerated Tooth Germ Increased in Morphology with Addition of IGF and Regenerated Tooth Germ without Addition of IGF Organ culture of [1-3] above is performed, and the regenerative tooth germ's cervical loop is observed with a phase contrast microscope The maximum diameter of the tooth germ perpendicular to the line connecting the two was taken as the long diameter, the maximum diameter of the crown part perpendicular to the long diameter was taken as the width diameter, and the long diameter and the width diameter at the 14th day were measured. As shown in FIG. 2 (A), the major axis was 944 μm in the IGF-1 added group and 857 μm in the control group on the 14th day of culture. Further, as shown in FIG. 2 (B), the width was 969 μm in the IGF-1 added group and 814 μm in the control group on the 14th day of culture. From these results, it was suggested that the addition of IGF-1 during the production process of the regenerated tooth germ increases the long diameter and width diameter of the regenerated tooth germ.
［１−５］IGF添加による形態を増大させた再生歯胚とIGF非添加の再生歯胚の組織学的標本の作製
上記［１−３］の培養14日目に歯胚を4％パラフォルムアルデヒド(PFA)にて固定し、10％エチレンジアミン四酢酸(EDTA)にて脱灰し、パラフィン包埋した後に、厚さ8μmの連続切片を作製し、ヘマトキシリン・エオジン(HE)染色にて組織学的評価を行った。HE染色において対照群、IGF-1添加群共にエナメル質、象牙質の形成がみとめられ、鐘状期後期まで発生していることが観察された。
[1-5] Preparation of histological specimens of regenerative tooth germs whose morphology was increased by addition of IGF and regenerative tooth germs without addition of IGF 4% paraform of tooth germ on the 14th day of culture in [1-3] above After fixation with aldehyde (PFA), decalcification with 10% ethylenediaminetetraacetic acid (EDTA) and embedding in paraffin, 8 μm-thick serial sections were prepared and histologically stained with hematoxylin and eosin (HE) Evaluation. In the HE staining, formation of enamel and dentin was observed in both the control group and the IGF-1 addition group, and it was observed that they occurred until the late bell stage.
［１−６］IGF添加による形態を増大させた発生歯胚の調整
Ｃ５７ＢＬ／６Ｊマウスの胎齢14.5日の胎仔下顎臼歯歯胚を外科的に摘出した。シリコンコートした35mmペトリディッシュの上に30μlのタイプIコラーゲンゲルをドロップ状に形成し、その中に歯胚を位置づけた。IGF-1添加群には4μg/ml IGF-1を添加したゲルを用いて再生歯胚を作製した（実施例１−２）。再生歯胚を24-well cell culture plates (BD)に設置した0.4μm pore diameter cell culture inserts (BD)上で、10% FBS (MP Biomedicals, Solon, OH, USA), 100μg/ml ascorbic acid (Sigma), 2mM L-glutamine (Sigma), Dulbecco’s modified eagle medium (DMEM, Sigma)を用いて器官培養を行った。さらにIGF-1添加群には，100ng/ml IGF-1を添加して培養した。位相差顕微鏡像で、図３(Ｂ)に示すように14日目にはIGF-1添加群においてサイズの増大が観察された。
[1-6] Preparation of developmental tooth germ with increased morphology by addition of IGF Fetal mandibular molar tooth embryos of C57BL / 6J mice at 14.5 gestational age were surgically removed. A 30 μl type I collagen gel was formed in a drop shape on a silicon-coated 35 mm Petri dish, and a tooth germ was positioned therein. In the IGF-1 added group, regenerated tooth germs were prepared using a gel added with 4 μg / ml IGF-1 (Example 1-2). 10% FBS (MP Biomedicals, Solon, OH, USA), 100 μg / ml ascorbic acid (Sigma) on 0.4 μm pore diameter cell culture inserts (BD) placed on 24-well cell culture plates (BD). ), 2 mM L-glutamine (Sigma) and Dulbecco's modified eagle medium (DMEM, Sigma). Furthermore, 100ng / ml IGF-1 was added to the IGF-1 addition group and cultured. In the phase contrast microscope image, as shown in FIG. 3 (B), on the 14th day, an increase in size was observed in the IGF-1 added group.
［１−７］IGF添加による形態を増大させた発生歯胚とIGF非添加の発生歯胚との比較
上記［１−６］の器官培養を行い、位相差顕微鏡像で歯胚のサービカルループを結んだ線と直交する歯胚の最大径を長径、長径と直交する歯冠部の最大径を幅径、幅径と直交する歯冠部の最大径を歯冠長径とし14日目の時点の長径、幅径、および歯冠長径を計測した。図４（Ａ）に示すように、長径は培養14日目においてIGF-1添加群で937μm、対照群で857μmであった。また図４（Ｂ）に示すように、幅径は培養14日目においてIGF-1添加群で1084μm、対照群で947μmであった。さらに図４（Ｃ）に示すように、歯冠長径は培養14日目においてIGF-1添加群で588μm、対照群で459μmであった。これらの結果から、発生歯胚はIGF-1を添加することで長径、幅径および歯冠長径が増大することが示唆された。
[1-7] Comparison of developmental tooth germ with increased morphology by addition of IGF and developmental tooth germ without addition of IGF Organ culture of the above [1-6] was performed, and the dental loop of the tooth germ was observed with a phase-contrast microscope image. The maximum diameter of the tooth germ orthogonal to the connected line is the long diameter, the maximum diameter of the crown part orthogonal to the long diameter is the width diameter, and the maximum diameter of the crown part orthogonal to the width diameter is the crown long diameter. The long diameter, the width diameter, and the crown long diameter were measured. As shown in FIG. 4 (A), the major axis was 937 μm in the IGF-1 added group and 857 μm in the control group on the 14th day of culture. As shown in FIG. 4B, the width was 1084 μm in the IGF-1 added group and 947 μm in the control group on the 14th day of culture. Further, as shown in FIG. 4C, the crown diameter was 588 μm in the IGF-1 added group and 459 μm in the control group on the 14th day of the culture. From these results, it was suggested that the long diameter, the width diameter, and the crown long diameter of the developing tooth germ increase by adding IGF-1.
［１−８］IGF添加による形態を増大させた発生歯胚とIGF非添加の発生歯胚の組織学的標本の作製
上記［１−６］の培養14日目に歯胚を4％パラフォルムアルデヒド(PFA)にて固定し、10％エチレンジアミン四酢酸(EDTA)にて脱灰し、パラフィン包埋した後に、厚さ8μmの連続切片を作製し、ヘマトキシリン・エオジン(HE)染色にて組織学的評価を行った。図５のようにHE染色において対照群、IGF-1添加群共にエナメル質、象牙質の形成がみとめられ、鐘状期後期まで発生していることが観察された
実施例２
［２−１］IGF添加による形態を増大させた再生歯胚の腎皮膜下移植
腎皮膜の圧力の影響を回避する目的で、再生歯胚移植のためのスペーサーを用いて移植を行った。OneTouch tips 200 (Sorenson BioScience，Salt Lake City, UT, USA)を内径1.3mm、高さ2.3mmになるようリング状に切断してスペーサーを作製し、内部にコラーゲンゲルを満たして使用した。コラーゲンゲルはIGF-1添加群（実施例２−１）と対照群（比較例２−１）で再生歯胚作製時と同様のものをそれぞれ使用した。上記［１−２］で得られた再生歯胚は、歯根方向への伸長を促すためスペーサーの壁面に歯冠側を近接させるよう配置した。5mg/mlペントバルビタールを腹腔内注射した全身麻酔下で、7週齢C57BL/6マウスの両側腎皮膜下に再生歯胚を含むスペーサーを移植した。
[1-8] Preparation of Histological Specimens of Developmental Tooth Germ Increased in Morphology by Addition of IGF and Developmental Tooth Germ without Addition of IGF On the 14th day of culture in [1-6] After fixation with aldehyde (PFA), decalcification with 10% ethylenediaminetetraacetic acid (EDTA) and embedding in paraffin, 8 μm-thick serial sections were prepared and histologically stained with hematoxylin and eosin (HE) Evaluation. As shown in FIG. 5, in the HE staining, enamel and dentin formation was observed in both the control group and the IGF-1 addition group, and it was observed that they occurred until the late bell-like period.
[2-1] Regenerated tooth germ transplanted under renal capsule with increased morphology by addition of IGF For the purpose of avoiding the influence of the pressure of renal capsule, transplantation was performed using a spacer for regenerated tooth germ transplantation. OneTouch tips 200 (Sorenson BioScience, Salt Lake City, UT, USA) was cut into a ring shape so as to have an inner diameter of 1.3 mm and a height of 2.3 mm to prepare a spacer, and the collagen gel was filled inside and used. Collagen gels used in the IGF-1 added group (Example 2-1) and the control group (Comparative Example 2-1) were the same as those used in the production of regenerated tooth germs. The regenerated tooth germ obtained in the above [1-2] was arranged so that the crown side was brought close to the wall surface of the spacer in order to promote extension in the root direction. Under general anesthesia injected intraperitoneally with 5 mg / ml pentobarbital, a spacer containing regenerated tooth germ was implanted under the bilateral renal capsule of 7-week-old C57BL / 6 mice.
［２−２］腎皮膜下移植における、IGF添加による形態を増大させた再生歯胚とIGF非添加の再生歯胚とのマイクロCT解析
上記［２−１］で移植した移植サンプルは、移植30日目の時点で摘出し、マイクロCT (In vivo Micro X-ray CT System; R_mCT, Rigaku, Tokyo, Japan)にて撮影を行った。画像データは、統合画像処理ソフト (i-VIEW-3DX, Morita, Osaka，Japan)を用いた。
図６に示すように移植した再生歯胚は石灰化が起こり、歯槽骨が形成され、歯根膜腔も認められた。図７に示すように、IGF-1添加した再生歯胚は、腎皮膜下移植によって、非添加の再生歯胚と比較して、サイズの増大(B)が認められた。
[2-2] Micro-CT analysis of regenerative tooth germ with increased morphology due to addition of IGF and regenerative tooth germ without addition of IGF in subrenal capsule transplantation The transplant sample transplanted in [2-1] above is transplanted 30 It was extracted at the time of the day and photographed with a micro CT (In vivo Micro X-ray CT System; R_mCT, Rigaku, Tokyo, Japan). For the image data, integrated image processing software (i-VIEW-3DX, Morita, Osaka, Japan) was used.
As shown in FIG. 6, the transplanted regenerated tooth germ was calcified, an alveolar bone was formed, and a periodontal ligament cavity was also observed. As shown in FIG. 7, the regenerative tooth germ added with IGF-1 showed an increase in size (B) by subrenal transplantation compared to the regenerated tooth germ without addition.
［２−３］腎皮膜下移植における、IGF添加による形態を増大させた再生歯とIGF非添加の再生歯とのマイクロCT解析による大きさの比較
移植30日目の再生歯の最大径を長径、長径と直行する歯冠部の最大径を幅径とし、両者を計測した。図７（Ａ）に示すように、長径はIGF-1添加群で1.37mm、対照群で0.95mmであった。図７（Ｂ）に示すように、幅径はIGF-1添加群で0.49mm、対照群で0.41mmであった。IGF-1を添加して培養した再生歯胚は腎皮膜下移植後に長径および幅径が増大することが示唆された。
[2-3] Comparison of size by micro-CT analysis between regenerative teeth with increased morphology due to IGF addition and non-IGF-added regenerative teeth in subrenal capsule transplantation The longest diameter and the maximum diameter of the orthogonal crown part were taken as the width diameter, and both were measured. As shown in FIG. 7A, the major axis was 1.37 mm in the IGF-1 added group and 0.95 mm in the control group. As shown in FIG. 7B, the width was 0.49 mm in the IGF-1 added group and 0.41 mm in the control group. It was suggested that regenerated tooth germs cultured with IGF-1 increased in length and width after subrenal transplantation.
［２−４］腎皮膜下移植における、IGF添加による形態を増大させた再生歯胚とIGF非添加の再生歯胚と正常歯のヌープ硬度
ダイヤモンド圧子 (19BAA061; Mitutoyo, Tokyo, Japan)と微小硬さ試験機 (HM-102; Mitutoyo, Tokyo, Japan)にて上記［２−２］の移植後30日目の再生歯のエナメル質および象牙質のヌープ硬度を測定した。測定は3週齢マウスの上顎第一臼歯(天然歯)、腎皮膜下移植30日の再生歯の対照群、IGF-1添加群それぞれの歯に対し5点行った。測定条件は、エナメル質に対して25gの圧力10秒間、象牙質に対して10gの圧力10秒間で行った。図８に示すようにエナメル質(A)、象牙質(B)のヌープ硬度はともにIGF-1添加群と対照群では、同等の硬度が認められた。
[2-4] Regenerative tooth germ with increased morphology by IGF addition, regenerative tooth germ without addition of IGF, and Knoop hardness of normal teeth in subrenal transplantation Diamond indenter (19BAA061; Mitutoyo, Tokyo, Japan) and microhardness The Knoop hardness of the enamel and dentin of the regenerated teeth 30 days after the transplantation of the above [2-2] was measured with a length testing machine (HM-102; Mitutoyo, Tokyo, Japan). The measurement was performed on the teeth of each of the control teeth of the upper first molar (natural tooth), regenerated teeth 30 days after subrenal transplantation, and the IGF-1 added group of 3-week-old mice. The measurement conditions were a 25 g pressure for 10 seconds for enamel and a 10 g pressure for 10 seconds for dentin. As shown in FIG. 8, both the enamel (A) and dentin (B) Knoop hardness were found to be equivalent in the IGF-1 added group and the control group.
実施例３
［３−１］口腔内移植における動物モデルの作製
3週齢C57BL/6マウスの上顎第一臼歯を抜歯し、3週間の治癒期間を設けた。その後、同部の歯肉切開・剥離を行い、歯科用マイクロモーター(PAL; Morita)を用いて歯槽骨を切削し、近遠心径0.8mm、頬舌径0.8mmの移植窩を形成した。上記［１−２］で得られた再生歯胚を7日間器官培養したものを顎骨移植窩に埋入し、8-0ナイロン縫合糸 (BEAR Medic, Chiba, Japan)で歯肉縫合した。
Example 3
[3-1] Preparation of animal model for intraoral transplantation
The maxillary first molar was extracted from 3-week-old C57BL / 6 mice, and a 3-week healing period was provided. Thereafter, gingival incision and detachment of the same part were performed, and the alveolar bone was cut using a dental micromotor (PAL; Morita) to form a graft fovea having a near-centrifuge diameter of 0.8 mm and a buccal tongue diameter of 0.8 mm. The regenerated tooth germ obtained in [1-2] above, which had been cultured for 7 days, was embedded in the jaw bone graft fossa and gingival sutured with 8-0 nylon suture thread (BEAR Medic, Chiba, Japan).
［３−２］口腔内移植における、IGF添加による形態を増大させた再生歯胚とIGF非添加の再生歯胚とのマイクロCT解析。 [3-2] Micro-CT analysis of regenerated tooth germs with increased morphology due to the addition of IGF and regenerated tooth germs without addition of IGF in intraoral transplantation.
上記［３−１］で移植した再生歯の移植サンプルは、移植90日目の時点でマイクロCT (In vivo Micro X-ray CT System; R_mCT, Rigaku, Tokyo, Japan)にて撮影を行った。画像データは、統合画像処理ソフト (i-VIEW-3DX, Morita, Osaka，Japan)を用いた。
図9（Ａ）に示すように、IGF-1を添加して培養した再生歯胚は顎骨内移植後に萌出し咬合平面まで到達することが示唆された。
The transplanted sample of the regenerated tooth transplanted in [3-1] above was photographed with a micro CT (In vivo Micro X-ray CT System; R_mCT, Rigaku, Tokyo, Japan) at the 90th day of transplantation. For the image data, integrated image processing software (i-VIEW-3DX, Morita, Osaka, Japan) was used.
As shown in FIG. 9 (A), it was suggested that regenerated tooth embryos cultured with the addition of IGF-1 erupt and reach the occlusal plane after intramaxillary transplantation.
さらに再生歯の最大径を長径、長径と直行する歯冠部の最大径を幅径とし、両者を計測した。図10（Ａ）に示すように、長径はIGF-1添加群で1.49mm、対照群で1.21mmであった。図10（Ｂ）に示すように、幅径はIGF-1添加群で0.64mm、対照群で0.48mmであった。IGF-1を添加して培養した再生歯胚は幅径が増大することが示唆された。IGF-Iを添加した再生歯は咬頭形態が明瞭で、大きさも増加していた。 Furthermore, the maximum diameter of the regenerative tooth was taken as the long diameter, and the maximum diameter of the crown portion perpendicular to the long diameter was taken as the width diameter, and both were measured. As shown in FIG. 10 (A), the major axis was 1.49 mm in the IGF-1 added group and 1.21 mm in the control group. As shown in FIG. 10 (B), the width was 0.64 mm in the IGF-1 addition group and 0.48 mm in the control group. It was suggested that regenerated tooth germs cultured with IGF-1 increased in width. Regenerated teeth with IGF-I added had clear cusp morphology and increased in size.
［３−３］口腔内移植における、IGF添加による形態を増大させた再生歯胚とIGF非添加の再生歯胚と正常歯のヌープ硬度
ダイヤモンド圧子 (19BAA061; Mitutoyo, Tokyo, Japan)と微小硬さ試験機 (HM-102; Mitutoyo, Tokyo, Japan)にてエナメル質および象牙質のヌープ硬度を測定した。測定は3週齢マウスの上顎第一臼歯(天然歯)、顎骨内移植後90日の再生歯の対照群、IGF-1添加群それぞれの歯に対し5点行った。測定条件は、エナメル質に対して25gの圧力10秒間、象牙質に対して10gの圧力10秒間で行った。図11に示すようにエナメル質、象牙質のヌープ硬度はともに天然歯と比較してIGF-1添加群および対照群ともに有意差は認められなかったものの、やや高値である傾向が示された。
[3-3] Regenerative tooth germ with increased morphology due to addition of IGF, regenerative tooth germ without addition of IGF, and Knoop hardness of normal teeth in oral transplantation Diamond indenter (19BAA061; Mitutoyo, Tokyo, Japan) and microhardness The Knoop hardness of enamel and dentin was measured with a testing machine (HM-102; Mitutoyo, Tokyo, Japan). Measurements were made on the teeth of each of the upper 1st molar (natural teeth) of 3-week-old mice, the regenerated tooth control group 90 days after intra-maxillary transplantation, and the IGF-1 added group. The measurement conditions were a 25 g pressure for 10 seconds for enamel and a 10 g pressure for 10 seconds for dentin. As shown in FIG. 11, the Knoop hardness of enamel and dentin were not significantly different in the IGF-1 added group and the control group compared to natural teeth, but showed a tendency to be slightly higher.
実施例４
［４−１］IGF添加による増殖能を上昇させた歯胚由来の間葉系細胞
胎齢14.5日のマウス胎仔の下顎臼歯歯胚の間葉組織から、0.25% Trypsin, 50U/ml Collagenase Iと35U/ml DNase Iを含むPBSを用いて15分間の酵素処理し、歯胚間葉細胞を単離し10% FBSを添加した DMEM培地にて培養し、第一継代目の細胞について増殖能を測定した。測定はIGF-1添加群、対照群、LY294002 (Promega, Madison, WI, USA)処理してIGF-1添加を行った群(阻害剤処理・IGF添加群)について行った。阻害剤処理は細胞懸濁液に10μM LY294002を添加し、37℃で30分放置する方法を用いた。96 well plate (BD)に臼歯歯胚間葉細胞を1000cells/wellで播種し、10% FBSを添加した DMEMを用いて培養を行った。さらにIGF-1添加群には，100ng/ml IGF-1を添加して培養した（実施例４−１）。IFG-1を添加しないこと以外、上記と同様にして対象群の培養を行った（比較例４−１）培養10日目に増殖能を測定した。増殖能の測定はCell counting kit-8 (WST8; DOJINDO, Kumamoto, Japan)を用いた。WST8 10μl/well添加後、37℃で3時間放置し、LS-PLATE manager (Wako, Osaka, Japan)を用いて測定波長450nm、参照波長620nmで吸光度測定した。
Example 4
[4-1] Tooth germ-derived mesenchymal cells whose proliferation ability was increased by the addition of IGF From the mesenchymal tissue of the mandibular molar tooth embryo of a 14.5-day-old mouse embryo, 0.25% Trypsin, 50 U / ml Collagenase I and 35 U / ml DNase I was used for 15 minutes of enzyme treatment, tooth germ mesenchymal cells were isolated and cultured in DMEM medium supplemented with 10% FBS, and the proliferative capacity of the first passage cells was measured . The measurement was performed on the IGF-1 added group, the control group, and the group treated with LY294002 (Promega, Madison, WI, USA) and added with IGF-1 (inhibitor-treated / IGF added group). Inhibitor treatment was performed by adding 10 μM LY294002 to the cell suspension and allowing to stand at 37 ° C. for 30 minutes. Molar embryonic mesenchymal cells were seeded at 1000 cells / well on a 96 well plate (BD) and cultured using DMEM supplemented with 10% FBS. Furthermore, 100ng / ml IGF-1 was added to the IGF-1 addition group and cultured (Example 4-1). The subject group was cultured in the same manner as described above except that IFG-1 was not added (Comparative Example 4-1). Proliferation ability was measured using Cell counting kit-8 (WST8; DOJINDO, Kumamoto, Japan). After adding 10 μl / well of WST8, the mixture was allowed to stand at 37 ° C. for 3 hours, and absorbance was measured at a measurement wavelength of 450 nm and a reference wavelength of 620 nm using LS-PLATE manager (Wako, Osaka, Japan).
［４−２］IGF添加による増殖能を上昇させた歯胚由来の間葉系細胞のWST8による増殖能の解析
図１２に示すように、IGF-1添加群が対照群と比較して増殖能が上昇していることが認められた 。このことからIGF-1添加により歯胚間葉細胞の増殖が促進することが示唆された。さらにLY294002処理・IGF-1添加群でIGF-1添加群にみられた歯胚間葉細胞の増殖の促進は認められず、対照群との間に有意差が認められなかった。このことから、PI3キナーゼ経路を阻害することで歯胚間葉細胞におけるIGF-1の増殖促進効果が阻害されていることが示唆された。
[4-2] Analysis of proliferative ability of tooth germ-derived mesenchymal cells whose proliferation ability was increased by addition of IGF by WST8 As shown in FIG. 12, the IGF-1 addition group had a proliferation ability compared to the control group. Was found to be rising. This suggests that the addition of IGF-1 promotes the proliferation of tooth germ mesenchymal cells. Furthermore, in the LY294002-treated / IGF-1 addition group, the promotion of the proliferation of tooth germ mesenchymal cells observed in the IGF-1 addition group was not observed, and no significant difference was found between the control group and the control group. This suggests that inhibition of the PI3 kinase pathway inhibits the growth promoting effect of IGF-1 in tooth germ mesenchymal cells.
実施例５
ゲル中のIGFの濃度を、それぞれ、0.5μg/ml, 1μg/ml, 2μg/ml, 4μg/ml及び8μg/mlとする以外、上記［１−１］〜［１〜３］に記載の方法に従い、歯胚由来上皮系細胞と間葉系細胞の単一化細胞の調整、再生歯胚作製及び器官培養を行った。0.5μg/ml, 1μg/ml, 2μg/ml, 4μg/ml, 8μg/mlで大きさの増大が認められ、歯冠と咬頭の形態を制御することができた。また、これらの濃度のなかでも、0.5〜4μg/mlの濃度のゲルを用いて作製した再生歯胚で、特に大幅な大きさの増大が認められた。
Example 5
The method according to [1-1] to [1 to 3] above, except that the concentration of IGF in the gel is 0.5 μg / ml, 1 μg / ml, 2 μg / ml, 4 μg / ml and 8 μg / ml, respectively. According to the procedure, preparation of a single cell of tooth germ-derived epithelial cells and mesenchymal cells, preparation of regenerated tooth germ, and organ culture were performed. The increase in size was observed at 0.5 μg / ml, 1 μg / ml, 2 μg / ml, 4 μg / ml, and 8 μg / ml, and the morphology of the crown and cusp could be controlled. Among these concentrations, regenerative tooth germs prepared using gels having a concentration of 0.5 to 4 μg / ml were particularly remarkably increased in size.
Claims (12)
当該培養工程において、歯又は歯胚由来の細胞は支持担体中に配置されており、当該支持担体中にＩＧＦが０．５〜８μｇ／ｍｌ配合されている、方法。 A method for culturing cells derived from teeth or tooth germs, comprising culturing in the presence of a growth factor comprising insulin-like growth factor (IGF),
In the culturing step, cells derived from teeth or tooth embryos are arranged in a support carrier, and IGF is mixed in an amount of 0.5 to 8 μg / ml in the support carrier.
当該培養工程において、歯又は歯胚由来の細胞は支持担体中に配置されており、当該支持担体中にＩＧＦが０．５〜８μｇ／ｍｌ配合されている、方法。 A method for producing a cell having a tooth tissue regeneration ability, comprising a step of culturing a tooth or tooth germ-derived cell in the presence of a growth factor containing IGF,
In the culturing step, cells derived from teeth or tooth embryos are arranged in a support carrier, and IGF is mixed in an amount of 0.5 to 8 μg / ml in the support carrier.
当該支持担体中にＩＧＦが０．５〜８μｇ／ｍｌ配合されている、方法。 Regenerative tooth germ comprising a step of mixing a tooth or tooth germ-derived cell with a support carrier containing a growth factor containing IGF or constructing an aggregate of teeth or tooth germ-derived cells in the support A manufacturing method of
A method in which 0.5 to 8 μg / ml of IGF is blended in the support carrier.
上記工程で当該成長因子含有支持担体と混合されるか又は凝集体を構築した歯胚由来の細胞をＩＧＦの存在下で培養する工程を含む、再生歯胚又は再生歯の製造方法であって、
当該支持担体中にＩＧＦが０．５〜８μｇ／ｍｌ配合されている、方法。 Mixing a cell derived from a tooth or tooth germ into a support carrier containing a growth factor containing IGF, or constructing an aggregate of cells derived from a tooth or tooth germ in the support, and the above step A method for producing a regenerated tooth germ or a regenerated tooth comprising a step of culturing a cell derived from a tooth germ mixed with a growth factor-containing support carrier or an aggregate in the presence of IGF,
A method in which 0.5 to 8 μg / ml of IGF is blended in the support carrier.
上記工程で当該成長因子含有支持担体と混合されるか又は凝集体を構築した歯胚由来の細胞をＩＧＦの存在下で培養する工程を含む、再生歯又は再生歯胚における歯冠、咬頭及び歯根のサイズを大きくし、その形態を制御する方法であって、
当該支持担体中にＩＧＦが０．５〜８μｇ／ｍｌ配合されている、方法。 Mixing a cell derived from a tooth or tooth germ into a support carrier containing a growth factor containing IGF, or constructing an aggregate of cells derived from a tooth or tooth germ in the support, and the above step Increasing the size of crowns, cusps and roots in regenerated teeth or regenerated tooth germs, including the step of culturing cells derived from tooth germs mixed with growth factor-containing support carriers or constructed aggregates in the presence of IGF And a method for controlling its form,
A method in which 0.5 to 8 μg / ml of IGF is blended in the support carrier.
当該再生歯胚を培養する工程を含む、再生歯又は再生歯胚における歯冠、咬頭及び歯根のサイズを大きくし、その形態を制御する方法であって、
当該培養工程において、当該支持担体中にＩＧＦが０．５〜８μｇ／ｍｌ配合されている、方法。 Increasing the size of the crown, cusp, and root of the regenerated tooth or regenerated tooth germ, including the steps of placing the regenerated tooth germ in a support carrier containing a growth factor containing IGF and culturing the regenerated tooth germ A method of controlling its form,
In the culturing step, IGF is mixed in the support carrier in an amount of 0.5 to 8 μg / ml.
The growth factor according to any one of claims 1 to 11, further comprising at least one selected from the group consisting of EGF, TGF, NGF, BDNF, VEGF, G-CSF, PDGF, EPO, TPO, FGF, HGF, and BMP. The method or composition of claim 1.
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