郭宗甫Kuo, Tzong-Fu臺灣大學:獸醫學研究所王純浩Wang, Chun-HaoChun-HaoWang2010-05-042018-07-092010-05-042018-07-092009U0001-2606200914082500http://ntur.lib.ntu.edu.tw//handle/246246/178951牙齒有咬碎及研磨食物的主要功能，更有助人類發音與支撐臉部輪廓的作用。但牙齒會因牙周疾病、齲齒、撞傷或癌症而造成缺損，如果能利用再生醫學所產生組織工程的牙齒以取代缺損的牙齒將是臨床治療的一大進步。本研究之目的乃是利用組織工程的方法達成整顆牙齒的再生。牙齒發育主要是有許多形態原與生長因子參與上皮及間葉組織交互作用的模式形成。新型態的血小板聚合物-富血小板纖維素(Platelet-rich fibrin；PRF)能夠提供生長因子 (Growth factor) 和形態原 (Morphogens)，包括了Platelet-derived growth factor（PDGF）、Bone morphogenetic protein (BMP)、Epidermal growth factor（EGF）、Transforming growth factor-beta (TGF-β)、及Vascular endothelial growth factor (VEGF)等，這些生長因子及形態原和齒胚胎發育及生長之所需部份是相同的，故本實驗試著將PRF導入生牙作用以期可協同牙胚細胞和支架促進牙齒的再生。分離1.5月齡迷你豬的未萌發牙胚組織，細胞培養後使細胞量Loss of teeth has many causes including physical trauma, gum disease and tooth decay. A biological tooth substitute that could replace lost teeth would provide a vital alternative to currently available clinical treatments. The purpose of this project is to generate a complete tooth crown by tissue engineering. Platelet-rich fibrin(PRP) is a new type of platelet concentrate which can apply a lot of growth factors and morphogens in osteogenesis, including platelet-derived growth factor（PDGF）、bone morphogenetic protein (BMP)、epidermal growth factor（EGF）、transforming growth factor-beta (TGF-β)、and vascular endothelial growth factor (VEGF). Partial of those growth factors and morphogens which involve in tooth development are match with odontogenesis. The aim of this project is to combined the dental bud cells (DBC)、fibrinogen gel and PRF to regenerate a complete tooth. The dental bud cells of molar tooth were removed from 1.5 month-old miniature pigs before eruption. When the DBC yield reached 1×106 cell/ml, the DBC were isolated, re-suspended, and then mixed the cell with fibrinogen gel and PRF. The DBC/fibrinogen gel/PRF constructs were implanted in xenograft into under dermis of nude mice’s pelvic dorsum. After xenogreat implantation for 16 weeks, there were evaluated by macrography and microsections stained with hematoxylin and eosin. We could see mineralized nodule, dentinogenic-like cells and vascular. In the swine experimental groups, one of the 9 pigs generated root, pulp, enamel, ameloblast, dentin, odontoblast, dentin tubular, cementum, blood vessels, periodontal ligament, and the other one pig erupted the regeneration tooth, and generated whole crown, root, pulp, enamel, dentin, odontoblast, dentin tubular, cementum, blood vessels, periodontal ligament. The immunohistochemical analysis results showed ameloblast expressed CK14, the dentin, dentin tubular and odontoblast expressed DMP-1, cementum and alveolar bone expressed OPN and the vascular endothelial cell of pulp expressed VEGF, too. We demonstrated that the porcine dental bud cells combined with fibrinogen gel and PRF which can grow regenerated tooth by tissue engineering when porcine model with autogenic cell transplantation.目錄謝..............................................................................................................................Ⅰ文縮寫對照表..........................................................................................................Ⅱ錄..............................................................................................................................Ⅴ文摘要......................................................................................................................Ⅶ文摘要......................................................................................................................Ⅷ一章:文獻回顧...........................................................................................................1一節:牙齒介紹...................................................................................................1 1.1牙齒的功能......................................................................................1 1.2牙齒的種類......................................................................................1 1.3牙齒的結構與相關成分..................................................................1 1.4牙齒的胚胎學發育..........................................................................5 1.5牙齒的特徵蛋白質表現..................................................................9二節:再生醫學.................................................................................................10 2.1再生醫學與組織工程三角模式....................................................10 2.2組織工程與牙齒再生....................................................................12 2.3牙齒再生醫學遠景........................................................................13三節:富血小板纖維素.....................................................................................13 3.1血小板............................................................................................13 3.2富血小板纖維素............................................................................13二章:緒言.................................................................................................................15三章:材料與方法.....................................................................................................17第一節:培養牙胚細胞之相關藥品配製.............................................................17 1.1牙胚細胞培養液之配製................................................................17 1.2磷酸鹽緩衝溶液配製....................................................................17 1.3 一倍之0.25 % 胰蛋白酵素- 0.02 %乙烯二氨酸配製...............17第二節:纖維素原膠支架之製備.........................................................................18第三節:富血小板纖維素之製備.........................................................................18第四節:實驗動物.................................................................................................19.1實驗動物來源及飼養管理............................................................19.2實驗動物分組................................................................................19.3豬隻手術及麻醉............................................................................20.4裸鼠手術及麻醉............................................................................21五節 豬牙胚組織分離及細胞培養................................................................22.1 豬下顎齒槽骨分離牙齒組織.......................................................22.2 豬牙胚組織之分離.......................................................................22.3 豬之牙胚細胞之獲得及細胞計數...............................................23六節 豬牙胚細胞混合纖維素原膠支架與富血小板纖維素........................23七節 牙齒生長評估........................................................................................23 7.1豬隻進行磁振照影分析................................................................23 7.2 X光放射線攝影分析.....................................................................24 7.3 牙齒組織學分析...........................................................................24 7.4 免疫組織化學染色分析...............................................................24四章 結果................................................................................................................27第一節 牙胚組織分離手術過程........................................................................27.1 豬牙胚組織分離前之X 光放射線攝影......................................27.2 豬牙胚組織分離手術過程...........................................................27.3 豬牙胚組織分離後之X 光放射線攝影......................................27二節 豬牙胚細胞培養結果............................................................................27.1 豬牙胚細胞生長情形...................................................................27.2 豬牙胚細胞數量及存活率...........................................................27第三節 實驗動物移植手術過程........................................................................28 3.1裸鼠異體移植過程........................................................................28 3.2豬隻自體移植過程........................................................................28第四節 裸鼠牙齒再生實驗結果........................................................................28 4.1裸鼠進行剖檢結果........................................................................28 4.2裸鼠進行組織學結果....................................................................28第五節 豬隻牙齒再生實驗結果........................................................................28 5.1對照組-X光放射線攝影結果........................................................28.2實驗組-取出豬牙胚組織後將豬牙胚細胞/纖維素原膠支架/ 富血小板纖維素植入豬原齒槽骨application/pdf5815213 bytesapplication/pdfen-US牙胚細胞富血小板纖維素組織工程牙齒再生裸鼠迷你豬dental bud cellplatelet-rich fibrintissue engineeringtooth regenerationnude micemini pigthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/178951/1/ntu-98-R95629017-1.pdf