郭宗甫臺灣大學：獸醫學研究所黃安婷Huang, An-TingAn-TingHuang2007-11-282018-07-092007-11-282018-07-092006http://ntur.lib.ntu.edu.tw//handle/246246/59941牙周疾病、齲齒、撞傷而造成牙齒缺損，一直是許多人和動物生活中極大的困擾，若能發展出生物性的牙齒再生取代缺損的牙齒將是臨床治療的一種新方法。本研究之目的乃是利用組織工程的方法達成牙齒的再生。取1.5 個月齡迷你豬，將未萌發之臼齒以外科手術取下，其後分離牙胚組織切碎成＜1 mm3的小片段，於培養液中以37℃，5 ％ CO2培養。將培養的牙胚細胞 (Dental bud cells; DBCs) 種入明膠/軟骨素-6-硫酸鹽/透明質酸三重聚合物支架 (Gelatin/chondoitin-6-sulfate /hyaluronan tri-copolymer scaffold; GCHT scaffold) 內培養一週。實驗組將含有DBCs/GCHT scaffold植入同源豬原齒槽骨內及下顎骨骨質中，並取部份DBCs/GCHT scaffold以10 ％中性福馬林及2.5 ％戊二醛固定分別製作組織切片、進行環境掃描式電子顯微鏡掃描，可見細胞於支架中貼附生長並形成細胞外基質。植入24及36週後以X光放射線攝影評估牙齒生長情形，並於36週犧牲豬隻以組織切片評估；對照組為取其臼齒後不種入DBCs/GCHT scaffold者3頭，及取臼齒組織後僅種入不含細胞的支架之豬隻3頭。實驗組6頭豬隻植入DBCs/GCHT scaffold，結果在3頭豬隻原齒槽骨出現放射不透明區域，而另3頭在X光放射線攝影則無任何發現。3頭在原齒槽骨出現放射不透明區域的豬隻，其中2頭各別可見約1×0.5 ㎝2和 0.5×0.5 ㎝2牙齒鈣化形態；另1頭則是出現直徑1 ㎝的放射線不透明區域。再生的牙齒進行組織學的分析，在蘇木紫及伊紅染色 (Haematoxylin and Eosin stain; H&E )，2頭中可觀察到Dentin/pulp-like complex之牙齒結構出現；免疫組織化學染色則可證實牙齒質具有Bone sialoprotein的表現。另1頭圓形的放射線不透明區域則在H&E染色下見到骨組織出現。由上述的結果再次證實，DBCs具有自行更新、分化成不同後裔及選植生殖 (Clonogenic) 的能力。對照組取牙齒組織相對位置都無牙齒生長。本研究證實可由組織工程的方法達成牙齒組織再生，也證實牙齒幹細胞可能存於牙髓組織中。Tooth loss due to periodontal disease, dental caries and trauma affect humans and animals adversely at some time in their lives. A biologically regenerated tooth could replace a lost tooth and the new substituted tooth would provide a vital alternative to currently available clinical treatments. For this purpose, we used a tissue engineering approach to research tooth regeneration. Using surgical operation to remove a molar tooth from a 1.5 month-old miniature pig, before eruption, and then minced dental bud tissues into < 1 mm3 pieces to culture in medium at 37℃ with 5 ％ CO2 . The dental bud cells (DBCs) were isolated, re-suspended, and then the cell suspension was injected into Gelatin/chondoitin-6-sulfate /hyaluronan tri-copolymer scaffold (GCHT scaffold). The DBCs/GCHT scaffold was cultured in a spinner flask bioreactor for 1 week. The DBCs/GCHT scaffold structures were implanted into the pig's original alveolus. In addition, a small piece of DBCs/GCHT scaffold sample was individually fixed with 10 ％ neutral formalin and 2.5 ％ glutaraldehyde. The results showed DBCs growing and attaching on GCHT scaffold, and even secreted extracellular matrix. In experimental groups: 6 pigs were implanted with DBCs/GCHT scaffold into the original alveolus. After 36 weeks, regenerated teeth could be found in 3 pigs by X-ray examination, especially a recognizable 1×0.5 ㎝2 and 0.5×0.5 ㎝2 size tooth formed in 2 different swine mandibular alveolus, and in other swine can see a 1 cm diameter radio-opaque region, the other 3 pigs had no notable results. Which 2 pigs showed tooth regeneration containing dentin/pulp-like complex tooth structures which appeared by H&E staining. We also detected bone sialoprotein (BSP) expressed in dental pulp, odontoblast and dentin by immunohistochemical (IHC) methods. A 1 cm radio-opaque region in a pig stained by H&E staining only presented bone tissue. Above all, we demonstrate the self-renewal capability, multi-lineage differentiation capacity, and clonogenic efficiency of DBCs. In control groups: 3 pigs molar tooth was removed but no implanted DBCs/ GCHT scaffold and 3 pigs receival merely implanted GCHT scaffold. After 36 weeks, no regenerated tooth was found in the original site. Our results demonstrated regeneration of tooth tissue structures could be achieved by applying tissue engineering techniques and suggested that dental stem cells may appear in dental pulp tissues.目錄 誌謝…………………………………………………………………………………..Ⅰ 英文縮寫對照表……………………………………………………………………..Ⅱ 目錄…………………………………………………………………………………..Ⅴ 表次…………………………………………………………………………………..Ⅷ 圖次…………………………………………………………………………………..Ⅸ 中文摘要…………………………………………………………………………..ⅩⅢ 英文摘要…………………………………………………………………………..ⅩⅣ 第一章 文獻回顧 1 第一節 牙齒 (Tooth) 介紹 1 1.1 牙齒的功能 1 1.2 牙齒的種類及功用 1 1.3 牙齒的結構 1 1.4 牙齒相關成份 2 1.5 牙齒的胚胎學發育 4 1.6 牙齒基因調控 7 第二節 組織工程 (Tissue engineering) 8 2.1 組織工程緣由 8 2.2 組織工程三角模式 8 2.2.1 成人前驅/幹細胞 (Adult progenitor/stem cells ) 9 2.2.2 支架 9 2.2.3 形態原 (Inductive morphogenetic signals; Morphogens) 10 2.3 牙齒與組織工程 11 2.4 牙齒組織工程遠景 15 第二章 緒言………………………………………………………………………..17 第三章 材料與方法 19 第一節 培養牙胚細胞之相關藥品配製 19 1.1 牙胚細胞培養液之配製 19 1.2 磷酸鹽緩衝溶液 (Phosphate buffered solution; PBS) 配製 19 1.3 2 mM 乙烯二氨酸 (Ethylene diamietetraacetic acid; EDTA) 之 PBS溶液配製 19 第二節 明膠/軟骨素-6-硫酸鹽/透明質酸三重聚合物支架(Gelatin/chondoitin-6-sulfate /hyaluronan tri-copolymer scaffold; GCHT scaffold) 之製備 19 第三節 實驗動物 20 3.1 實驗動物來源及飼養管理 20 3.2 實驗動物分組 20 3.2.1 實驗豬隻分組 20 3.2.2 實驗兔子分組 21 3.3 實驗動物手術及麻醉 21 3.3.1 豬隻手術及麻醉 21 3.3.2 兔子手術及麻醉 22 第四節 豬隻牙胚細胞分離及培養 23 4.1 從豬隻下顎齒槽骨分離牙齒組織 23 4.2 豬隻牙胚組織之分離 23 4.3 豬隻牙胚細胞的獲得及細胞計數………………………………23 第五節 豬隻牙胚細胞種入明膠/軟骨素-6-硫酸鹽/透明質酸三重聚合物支 架 24 第六節 豬隻牙胚細胞進行細胞流式儀分析步驟 24 第七節 牙齒生長評估 25 7.1 豬隻進行磁振照影 (Magnetic resonance imaging; MRI ) 分析 25 7.2 牙齒組織學分析 25 7.3 免疫組織化學染色 (Immunohistochemical stain； IHC stain) 分析..……………………………….……………………………….25 7.4 PCR分析………………………………………………………..26 7.4.1 石蠟組織DNA萃取 26 7.4.2 進行PCR增幅及進行電泳分析 26 第四章 結果 27 第一節 牙胚細胞培養結果 27 1.1 豬隻牙胚細胞數量及存活率 (Viability) 27 1.2 細胞流式儀結果 27 1.3 豬隻牙胚細胞於明膠/軟骨素-6-硫酸鹽/透明質酸三重聚合物支架上生長情形 27 1.3.1 明膠/軟骨素-6-硫酸鹽/透明質酸三重聚合物支架進行光學顯鏡拍照 (H&E染色) 及以環境掃描式電子顯微鏡 (Environmental scanning electron microscope； ESEM) (FEIQuanta 200， Hitach) 進行掃描之結果 27 1.3.2 豬隻牙胚細胞/明膠/軟骨素-6-硫酸鹽/透明質酸三重聚合物支架進行光學顯微鏡拍照 (H &E染色) 和ESEM掃描之結果 27 第二節 實驗動物之X光放射線攝影 28 2.1 豬隻以外科手術取出牙胚組織，其手術前、後進行X光放射線 攝影 28 2.2 對照組實驗豬隻取牙胚組織後達36週之結果 28 2.2.1 豬隻取出牙胚組織後不再植入細胞或支架 28 2.2.2 豬隻取出牙胚組織後僅植入不含細胞之支架 28 2.3 實驗組實驗動物取出牙胚組織培養後植入牙胚細胞/明膠/軟骨素-6-硫酸鹽/透明質酸三重聚合物支架於原齒槽達36週後之結果 28 2.3.1 豬隻牙齒再生實驗之結果 28 2.3.2 兔子牙齒再生實驗結果 29 第三節 豬隻犧牲後之再生牙齒進行檢查及組織學評估 29 3.1 再生牙齒進行剖檢結果 29 3.2 組織學 (H&E染色) 結果 29 3.3 免疫組織化學染色結果 29 3.4 豬隻植入牙胚細胞/明膠/軟骨素-6-硫酸鹽/透明質酸三重聚合物支架犧牲後再生牙齒進行PCR分析 30 第五章 討論 31 第六章 參考文獻 35 表次 Table 1. Formulas and eruption of deciduous teeth in domestic animals. ………………………………………………………………………..48 家畜乳齒齒式。 Table 2. Formulas and eruption of permanent teeth in domestic animals. 49 家畜恆齒齒式。 Table 3. Genes required for tooth development…………………………...50 牙齒發育相關的基因。 Table 4. Key elements of tissue engineering………………………………52 組織工程的主要元素。 Table 5. Dental bud cell numbers and its viability in each swine. 52 豬隻牙胚細胞培養數目及細胞存活率。 圖次 Fig 1. Dentition of the pig. 53 豬隻齒列。 Fig 2. Tooth structure. 53 牙齒結構。 Fig 3. Formation of the tooth at successive stages of development. 54 牙齒形成發育各階段。 Fig 4. The key elements of tissue engineering and dentin regeneration…. 54 組織工程及牙齒質再生主要元素。 Fig 5. Reciprocal signaling during the early phases of tooth development. 55 早期階段之牙齒發育交互作用訊息調控。 Fig 6. Dental bud tissue in swine mandible……………………………….56 豬隻下顎骨內之牙胚組織。 Fig 7. Morphology of swine dental bud cells are spindle shape (400X) 56 豬隻牙胚細胞形態為紡綞狀 (400X)。 Fig 8. The results of flow cytometry. 58 流式細胞儀結果。 Fig 9. GCHT scaffold stained with H&E and examination by light microscopy. The scaffold stains reddish and shows uniform porosity (100X).. 59 GCHT scaffold進行H&E染色，光學顯微鏡下支架為紫色且呈多孔洞 (100X)。 Fig 10. Environmental scanning electron microscope (ESEM) examination of the GCHT scaffold 59 GCHT scaffold進行環境掃描式電子顯微鏡掃描 (Environmental Scanning Electron Microscope; ESEM)…………………………...59 Fig 11. After 1 week cultivation in spineer flask, results showed swine dental bud cells attached on GCHT scaffold (H&E stain; 400X)....60 豬隻DBCs/GCHT scaffold在spineer flask系統培養1週，細胞貼附於支架上 (H&E stain; 400X)。 Fig 12. After 1 week cultivation in spineer flask, ESEM examination of swine dental bud cells, results showed cells attached on GCHT scaffold (1200X). 60 豬隻DBCs/GCHT scaffold在spineer flask系統培養1週，於環境掃描式電子顯微鏡下可見豬隻牙胚細胞吸附於GCHT scaffold中生長 (1200X)。 Fig 13. ESEM examination of swine dental bud cells attached on the GCHT scaffold and secreted extracellular matrix (5000X). 61 環境掃描式電子顯微鏡下豬隻牙胚細胞吸附於GCHT scaffold且生成細胞外基質 (5000X)。 Fig 14. Dental radiographs of the operation site. Dental radiograph of the swine second molar tooth position………………………………...61 外科手術取豬隻牙胚前進行X光放射線攝影，可見豬隻第二臼 齒相對位置。 Fig 15. During surgery, the swine dental bud tissue was removed. The result showed swine second molar tooth was removed by X-ray. 62 外科手術取出豬隻第二臼齒牙胚後進行X光放射線攝影。 Fig 16. 36 weeks after surgery, the result was evaluated by X-ray. Removing the dental bud, no tooth growth was discovered in the control groups.. 62 對照組，手術後36週以X光放射線攝影，在取豬隻牙胚的相對位置無牙齒生長。 Fig 17. 36 weeks after surgery, the result was evaluated by X-ray. Removing the dental bud, no tooth growth was discovered in the control groups……………………………………………………...63 對照組，手術後36週以X光放射線攝影，取豬隻牙胚的相對位置無牙齒生長。 Fig 18. 36 weeks after surgery, the result was evaluated by X-ray. Removing the dental bud, no tooth growth was discovered in the control groups. 63 對照組，手術後36週以X光放射線攝影，在取豬隻牙胚的相對位置無牙齒生長。 Fig 19. 36 weeks after implanted GCHT scaffold.Results were evaluated by X-ray. No tooth growth in the control groups. 64 植入GCHT scaffold之對照組。手術後36週以X光放射線攝影，在植入GCHT scaffold的相對位置無牙齒生長。 Fig 20. 36 weeks after implanted GCHT scaffold.Results were evaluated by X-ray. No tooth growth in the control group………………………64 植入GCHT scaffold之對照組。手術後36週以X光放射線攝影，在植入GCHT scaffold的相對位置無牙齒生長。 Fig 21. 36 weeks after implanted GCHT scaffold. Results were evaluated by X-ray. No tooth growth in the control groups……………………..65 植入GCHT scaffold之對照組。手術後36週以X光放射線攝影，在植入GCHT scaffold的相對位置無牙齒生長。 Fig 22.36 weeks after implanted DBCs/GCHT scaffold.Results were evaluated by X-ray. Implant location had no tooth growth in the experimental groups………………………………………………65 實驗組 (植入DBCs/GCHT)。手術後36週以X光放射線攝影，在植入DBCs/GCHT scaffold的相對位置無牙齒生長。 Fig 23. 36 weeks after implanted DBCs/GCHT scaffold.Results were evaluated by X-ray. Implant location had no tooth growth in the experimental groups 66 實驗組 (植入DBCs/GCHT)。手術後36週以X光放射線攝影，在植入DBCs/GCHT scaffold的相對位置無牙齒生長。 Fig 24. 36 weeks after implanted DBCs/GCHT scaffold.Results were evaluated by X-ray. Implant location had no tooth growth in the experimental groups.. 66 實驗組 (植入DBCs/GCHT)。手術後36週以X光放射線攝影，在植入DBCs/GCHT scaffold的相對位置無牙齒生長。 Fig 25. 36 weeks after implanted DBCs/GCHT scaffold. Results were evaluated by X-ray. Implant site had radio-opaque region present in the experimental groups………………………………...…………67 實驗組 (植入DBCs/GCHT)。手術後36週以X光放射線攝影，在植入DBCs/GCHT scaffold的相對位置有放射線不透明區域出現。 Fig 26. 36 weeks after implanted DBCs/GCHT scaffold. Results were evaluated by X-ray. Implant site had radio-opaque region present in the experimental groups………………………………………...…67 實驗組 (植入DBCs/GCHT)。手術後36週以X光放射線攝影，在植入DBCs/GCHT scaffold的相對位置有放射線不透明區域出現。 Fig 27. 36 weeks after implanted DBCs/GCHT scaffold. Results were evaluated by X-ray. Implant site had radio-opaque region present in the experimental groups. 68 實驗組 (植入DBCs/GCHT)。手術後36週以X光放射線攝影，在植入DBCs/GCHT的相對位置有放射線不透明區域出現。 Fig 28. The result of Magnetic resonance imaging (MRI) showed regeneration tooth in the implant site.. 69 再生牙齒相對位置進行磁振照影分析。 Fig 29. The result of MRI showed regeneration in the implant site. 69 再生牙齒相對位置進行磁振照影分析。 Fig 30. Results showed 0.5×0.5 ㎝2 size tooth regeneration in alveolus bone.. 70 可見齒槽骨內有0.5×0.5 ㎝2大小的再生牙齒。 Fig 31. Results showed the tooth erupted into the oral cavity. With a 1×0.5×0.5 ㎝3 size regenerated tooth formed in alveolus bone. 70 可見牙齒萌發於口腔中，齒槽骨內有1×0.5×0.5 ㎝3 大小的再生牙齒。 Fig 32. In this H&E stained specimen, results showed bone tissue but no tooth structure present. (H&E stain, 400X). 71 結果顯示具有骨組織出現，但未發現牙齒相關組織出現 (H&E 染色，400X)。 Fig 33. Results show tooth structures including dentin and dental pulp(H&E stain, 100X). 71 結果顯示具有牙齒結構其中包括牙齒質和牙髓 (H&E 染色，100X)。 Fig 34. In another field of view, results show tooth structures-dentin (H&E stain, 100X). 72 在另一個視野，結果顯示具有牙齒結構-牙齒質 (H&E 染色，100X)。 Fig 35. Results show tooth structures including dentin, odontoblast and dental pulp (H&E stain, 100X & 400X). 72 結果顯示具有牙齒結構其中包括牙齒質、牙齒質母細胞和牙髓 (H&E 染色，100X和400X)。 Fig 36. Results show bone sialoprotein expressed in two places (IHC stain, 100X). 73 結果在兩個地方的牙齒質出現Bone sialoprotein (IHC 染色，100X)。 Fig 37. Results show bone sialoprotein expressed in dentin, odontoblast and dental pulp (IHC stain, 400X). 73 結果顯示Bone sialoprotein在牙齒質、牙齒質母細胞和牙髓中表現 (IHC 染色，400X)。 Fig 38. Normal swine tooth expression of bone sialoprotein in dentin (Positive control) (IHC stain, 400X). 74 正常豬隻牙齒質中表現Bone sialoprotein之陽性對照組。 Fig 39. A normal swine tooth that did not express bone sialoprotein in dentin (Negative control) (IHC stain, 400X)….…..……….……..74 正常豬隻牙齒質中表現Bone sialoprotein之陰性對照組。 Fig 40. The result showed normal pig ß-actin and dentin sialophospho- -protein PCR electrophoresis products. 75 正常豬隻之ß-actin和 dentin sialophosphoprotein PCR電泳產物。2488811 bytesapplication/pdfen-US牙胚細胞支架組織工程牙齒再生dental bud cellscaffoldtissue engineeringtooth regeneration[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/59941/1/ntu-95-R93629005-1.pdf