JP4714741B2 - 歯の再生方法 - Google Patents
歯の再生方法 Download PDFInfo
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- JP4714741B2 JP4714741B2 JP2007528466A JP2007528466A JP4714741B2 JP 4714741 B2 JP4714741 B2 JP 4714741B2 JP 2007528466 A JP2007528466 A JP 2007528466A JP 2007528466 A JP2007528466 A JP 2007528466A JP 4714741 B2 JP4714741 B2 JP 4714741B2
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Classifications
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Description
1):生体内で歯形成に関与している組織(歯乳頭、歯髄、歯根膜など)を利用する方法。
1)−1、歯胚、歯髄、歯根膜などの組織をそのまま移植する(例えば、非特許文献5参照)。
1)−2、歯胚を上皮系細胞と間葉系細胞とに分離して各々培養し、それぞれからエナメル芽細胞、象牙芽細胞を分化誘導してin vitroで歯の形成を試みる。
1)−3、分離培養した歯胚上皮細胞と間葉系細胞を再度混合し、マウス皮下などの生体組織に移植して、歯の形成を試みる。(例えば、非特許文献6参照。)
2):胚性幹細胞や間葉性幹細胞など、多分化能を持つ未分化細胞を利用する方法。
3):1)と2)の双方を利用する方法。
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morphogenetic protein−4、BMP−4;SIGMA)、インスリン成長因子1(insulin growth factor−1、IGF−1;TOYOBO)、繊維芽細胞成長因子2(fibroblast
growth factor−2、FGF−2;TOYOBO)及びトランスフォーミング増殖因子β1(transforming growth factor−β1、TGF−β1;TOYOBO)を適切に使用することができる。
growthになる前の歯胚間葉系細胞にマウスES細胞を加えて約5日間培養し、当該ES細胞を十分に分化させる。この細胞塊の作製過程において、マウスの歯胚間葉系細胞とマウスES細胞との細胞混合比は、約2:1であり、具体的には、本実施態様において培養した上記マウスの歯胚間葉系細胞1.7x104個に対しマウスES細胞8x103個であった。共存培養1日後に上記の誘導因子を加えたMSCGM培地を用い、37℃、5%CO2下で上述のように計5日間培養を行う。その後、トリプシンとEDTAの処理により細胞を回収してプラスチックフィルターを通してシングル化し、低接着性の96穴プラスチックプレートに播種し、次いで24穴プラスチックプレートに播種して誘導因子の存在下で培養して、マウスES細胞とマウスの歯胚間葉系細胞との細胞塊を作製する。
growthになる前の歯胚間葉系細胞にマウスES細胞を加え5日間培養し、ES細胞を十分に分化させた(図5左)。細胞混合比はMDU1
1.7x104個に対しマウスES細胞8x103個であった(図5中央)。誘導因子非存在下で1日共存培養をした後、培地に種々の誘導因子を加えたMSCGM培地を用い、37℃、5%
CO2下で計5日間培養を行った。使用した誘導因子は、アクチビン(味の素)、骨誘導因子4(bone
morphogenetic protein−4、BMP−4;SIGMA)、インスリン成長因子1(insulin growth factor−1、IGF−1;TOYOBO)、繊維芽細胞成長因子2(fibroblast
growth factor−2、FGF−2;TOYOBO)、トランスフォーミング増殖因子β1(transforming growth factor−β1、TGF−β1;TOYOBO)だった。その後トリプシン−EDTA処理(GIBCO)により細胞を回収してプラスチックフィルターを通してシングル化し、低接着性の96穴プラスチックプレート(Nunc社)に播種して1日培養し、次いで、低接着性の24穴プラスチックプレート(Nunc社)に移して上記の誘導因子の存在下で4日間培養して、細胞塊を作製した。つまり、この過程を説明すると、歯胚間葉系細胞とマウスES細胞との混合細胞を回収してシングル化し、低接着性96穴プラスチックプレートに播種して1日培養した時点で混合細胞は丸くなるが、この混合細胞はまだ小さくて細胞数が十分ではないので、低接着性24穴プラスチックプレートに移して栄養(すなわち、血清培地と誘導因子)を十分に与えて大きくし、ほぼ100〜200micro径の細胞塊が作製された時点(すなわち、4日間培養)で終了する。
Sponge HoneycombTM)上で、上記の誘導因子を除いた血清培地で3日間培養した。コラーゲン担体に接着した細胞塊は、ハニカム構造をしたコラーゲン担体の梁部分に接着し伸展していることが分かった(図5右)。
Claims (4)
- 哺乳類の上皮系細胞への分化制御機能を有する歯胚間葉系細胞を継代培養して細胞系列を作製する工程と、
該細胞系列をフィーダー細胞として前記哺乳類と同種の胚性幹細胞を、誘導因子の存在下で共に培養して混合細胞群を作製する工程と、
該混合細胞群を単一化する工程と、
該単一化された混合細胞群を誘導因子の存在下で浮遊培養する工程とを有し、前記誘導因子は、アクチビン、骨誘導因子4、インスリン成長因子1、繊維芽細胞成長因子2又は、トランスフォーミング増殖因子β1である、象牙芽細胞及びエナメル芽細胞に分化誘導可能であり、歯の形成に使用できる細胞塊の作製方法。 - 請求項1に記載の作製方法で得られた細胞塊を、アクチビン、骨誘導因子4、トランスフォーミング増殖因子β1、インスリン成長因子1及び繊維芽細胞成長因子2のいずれも存在しない血清培地中で接着培養する工程を有する歯の形成方法。
- 請求項1に記載の作製方法で得られた細胞塊を、アクチビン、骨誘導因子4、トランスフォーミング増殖因子β1、インスリン成長因子1又は繊維芽細胞成長因子2の存在下で接着培養する工程と、アクチビン、骨誘導因子4、トランスフォーミング増殖因子β1、インスリン成長因子1及び繊維芽細胞成長因子2のいずれの因子も存在しない血清培地中で接着培養する工程を有する歯の形成方法。
- 請求項1に記載の方法で作製した、象牙芽細胞及びエナメル芽細胞に分化誘導可能であり、歯の形成に使用できる細胞塊。
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JP2008206500A (ja) | 2007-02-28 | 2008-09-11 | Tokyo Univ Of Science | 歯の製造方法及びこれにより得られた歯 |
EP2331153A4 (en) * | 2008-08-25 | 2014-01-15 | Laser Abrasive Technologies Llc | METHOD AND DEVICE FOR REGENERATING ORAL CAVITY TISSUE |
WO2012045097A1 (en) | 2010-10-01 | 2012-04-05 | The Trustees Of Columbia University In The City Of New York | Production of dentin, cementum and enamel by cells |
EP2633870B1 (en) * | 2012-02-29 | 2018-08-01 | Technische Universität Berlin | Method of preparing an artificial tooth primordium in vitro and artificial tooth primordium derived therefrom |
JP6000021B2 (ja) * | 2012-08-23 | 2016-09-28 | 国立大学法人東北大学 | Igfを含む成長因子による歯、再生歯および再生歯胚における歯冠および咬頭ならびに歯根の大きさおよび形の制御の方法 |
JP6874953B2 (ja) * | 2015-04-01 | 2021-05-19 | 学校法人慶應義塾 | ヒトiPS細胞から、ヒト歯原性上皮細胞やヒト歯原性間葉細胞を製造する方法 |
KR101957034B1 (ko) * | 2017-08-24 | 2019-03-11 | 서울대학교산학협력단 | 인간유래 유도만능줄기세포에서 치계상피줄기세포로의 분화방법 및 이의 용도 |
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