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研究生：	陳昭安 Chen, Chao-An
論文名稱：	牙科填補物對於人類牙髓幹細胞的影響 Effects of Dental Restorative Materials on Human Dental Pulp Stem Cells
指導教授：	莊淑芬 Chuang, Shu-Fen 陳玉玲 Chen, Yuh-Ling
學位類別：	碩士 Master
系所名稱：	醫學院 - 口腔醫學研究所 Institute of Oral Medicine
論文出版年：	2014
畢業學年度：	102
語文別：	英文
論文頁數：	64
中文關鍵詞：	牙髓幹細胞、複合樹脂、玻璃離子黏合劑、氧化锌丁香油酚基底黏合劑、細胞毒性、分化、礦化
外文關鍵詞：	Dental pulp stem cell, Composite resin, Glass isomer cement, Zinc oxide eugenol-based cement, Cytotoxicity, Mineralization, Differentiation
相關次數：	點閱：151 下載：0
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在組織工程發展中，許多文獻已證明牙髓幹細胞是可分化為多種細胞的可靠來源，而且已應用於幹細胞治療。但在具有牙髓幹細胞的牙齒來源中，可能因蛀牙而需要填補。牙科常用的填補材料包括複合樹脂、玻璃離子黏著劑、汞合金、以及氧化锌丁香油酚基底黏合劑等。從體外細胞實驗研究中，對於這些填補材料釋放出的殘留物質所造成的細胞毒性及基因毒性，已有完整的論文發表結果，證實其中某些材料成份，即使在完整的牙本質的阻隔下，依然能穿透牙本質小管到達牙髓腔，造成影響。然而，只有少數體外(in vitro)研究顯示牙科材料能干擾牙髓幹細胞的分化，目前更缺乏相關的體內實驗。因此這些研究對將來組織工程或幹細胞治療等發展具有重要性。本篇研究的目的是利用體內實驗來調查在牙齒填補牙科材料後，對牙髓幹細胞所造成的反應。
本實驗於36顆牙齒上分別填補三種牙科材料(複合樹脂、玻璃離子黏合劑、以及氧化锌丁香油酚基底黏合劑)，經過7日，及30-60日後拔除。首先將牙齒脫鈣後，利用組織學方法來研究牙髓對牙科材料的反應以及牙髓幹細胞的分佈情況。在組織學檢查中可見這些填補物會導致些微的牙髓傷害。再進一步利用組織免疫染色方法去計算幹細胞的數目，在所有組別中都發現於牙髓中心有CD44以及α-SMA陽性免疫反應。比較CD44(+)細胞在所有牙髓細胞的百分比例，相對於填補7日的百分比例，填補30-60日的牙齒幹細胞百分比例逐漸減少，特別在複合樹脂組別的數值最低，但是各組別間並沒有顯著差異。
為了比較牙齒填補後對牙髓幹細胞型態、活性、幹性以及分化特性，於是從各組別培養出的牙髓幹細胞，進行檢查分析。結果發現牙齒經填補後所培養出的牙髓幹細胞，在形態上都呈細小、梭狀的同質性表徵。在細胞增生試驗中，實驗組以及對照組牙髓幹細胞能形成附著性細胞群集，其細胞群落單位以及生長曲線並無明顯差異。代表幹性的Oct4、Nanog以及CD44表達，樹脂填補30-60日組的Oct4表達較高，但各組之間並無明顯差異。各組經過2及4周骨分化後，都有礦物化沈積現象。本結果提示著複合樹脂所釋放出的單體可能會減少牙髓幹細胞的存活數量，但是能提高存活牙髓幹細胞的幹性。此外顯示牙髓幹細胞在牙科材料填補後，雖然對牙髓組織可能造成傷害，但牙髓幹組織密度上只有微量變化，而且保有骨分化的能力。

Numerous studies have approved the significance of DPSC since it may differentiate into various cells to support tissue engineering and stem cell therapy. The source teeth are frequently filled with material including composite resin, glass ionomer, and zinc oxide eugenol-base materials, due to caries. Cytotoxicity and genotoxicity of these restorative materials have been well-documented in in vitro studies. However, only some of these compounds were also found to diffuse through the dentinal tubules and reach the pulp tissue, even in the presence of an intact dentin barrier. However, there is until now fragmentary information available on their biological effects on dental pulp stem cell (DPSC). Only few in vitro studies report that dental materials disturb the differentiation of DPSC. Therefore, it is important to develop further in vivo study in tissue engineering and stem cell therapy. The purpose of this study was to investigate the response of dental pulp stem cell after application of dental restorations in vivo.
In this study, volunteers with scheduled extracted human molar teeth were collected under informed consent. The cavity floor prepared on the occlusal surface of molars was restored with composite resin, glass ionomer and zinc oxide eugenol-base materials. The 36 molars teeth filled with dental restoration were extracted after 7 days and 30-60 days, then were processed for histopathological evaluation in order to examine the pulp response and the distribution of stem-cells. The histopathological finding showed mild pulp damage in teeth with restoration in cavities. The immunoreactivity of CD44 and α-SMA was obviously found in the pulp core of all groups. The stem cell percentage of the groups at day 30-60 decreased comparing with the groups at day 7, with the composite resin group showing the lowest value among groups. There was no significant difference among the tested groups and control group.
In established culture experiment, we investigated the morphology, proliferation, stemness, and differentiation potential of DPSCs. For each group, isolated DPSCs exhibited small, spindle-like, homogeneous morphology. In cell proliferation, all groups of cell formed adherent clonogenic cell cluster; however, there were similar CFU counts between all groups. In WST1 assay, no significant difference was observed among groups. Oct4 expression increased in resin-30days group comparing with control group. Mineralized nodules were formed after 2 and 4 weeks of osteogenic induction. The result suggested that resinous monomers may reduce the viability of DPSC but increased the stemness of the survival DPSCs under inflammatory stimulation. Additionally, the dental restoration caused mild damage of pulp tissue with little alteration in density of DPSCs, and the osteogenic potential was kept after dental restoration.

中文摘要 Ⅰ
ABSTRACT	Ⅲ
誌謝	Ⅴ
Contents Ⅵ	
List of tables IX     
List of figures X	
符號Abbreviation	XII
Chapter 1.Introduction	1
1.1The Stem Cell	1
1.2 Mesenchymal Stem Cells	1
1.2.1 Dental Pulp Stem Cell	2
1.2.2 Tooth Regeneration of DSPC	3
1.2.3 Osteogenic Differentiation of DPSCs in Regenerative Medicine.	4
1.2.4 Neural Differentiation of Dental Stem Cells in Regenerative Medicine	4
1.3 Dental Restoration Material	5
1.3.1 Composite Resin and its Cytotoxicity	5
1.3.2 Glass Inomer Cements and its Cytotoxicity	6
1.3.3 Zinc Oxide-eugenol and its Cytotoxicity	7
1.4 Effects of the Dental Restoration Material on Stem Cell in Vitro	7
1.5 Motivation and Specific Aims	8
Chapter 2. Materials and Methods	9
2.1 Tooth Collection and Preparation	11
2.2 Immunohistochemical Stain	14
2.3 Primary Culture of Human Dental Pulp Stem Cells	15
2.4 Cell Morphology	15
2.5 Colony Forming Unit Fibroblast (CFU-F) Assays	16
2.6 WST-1 Assay	16
2.7 Osteogenic Differentiation Assays	17
2.8 RNA Isolation and Quantitative Real-time PCR (qRT-PCR) Analysis	17
2.9 Alizarin Red S Stain	18
2.10 Statistical Analysis	18
Chapter 3 Results	19
3.1 The inflammatory reaction of dental restorative material on DPSCs	19
3.2. The influence of the density and distribution of DPSCs with dental restorative material	20
3.3 Cell morphology	21
3.4 The proliferation of DPSCs with dental restorative material	21
3.5 mRNA expression of Oct-4, Nanog and CD44 in DPSCs of teeth with dental restorative material	22
3.6 Osteogenic differentiation of DPSCs of teeth with dental restoration material	22
Chapter 4. Discussion	24
Chapter 5. Conclusion	29
Reference       30
Figures 	40
Appendix1. Reagents	62
Appendix2. Equipments	63
Curriculum Vitae 64

List of tables
Table 2.1 Experimental material used in experimental group 9
Table 2.2 Experimental and control groups 10
Table 2.3 Histopathologic event and scores 13
Table 2.4. Antibody	15
Table 2.5 Primers used in qRT-PCR 18
Table 3.1. The tissue response of experimental groups from histologic observations 20

List of Figures
Figure 1. Source of dental stem cells and potential regeneration.	40
Figure 2. Dental stem cell-based tissue engineering.	41
Figure 3. Chemical structures of monomers of dental resin material	42
Figure 4. Eugenol diffuses into the pulp.	43
Figure 5. The experimental scheme.	44
Figure 6. Adhesive system and restoration used in this study.	45
Figure 7. Flow chart 1	46
Figure 8. Flow chart 2	47
Figure 9. Post-operative radiography.	48
Figure 10. Pulp response.	49
Figure 11. Stro-1 expression in pulp tissue.	50
Figure 12.α-SMA expression in odontoblastic layer and pulp core.	51
Figure 13. CD44 expression in odontoblastic layer and pulp core.	52
Figure 14. CD44 (+) cell percentage.	53
Figure 15. Cell morphology.	54
Figure 16. Growth curves analyzed by WST1 assay	55
Figure 17. Colony formation in each group.	56
Figure 18. Colony forming unit fibroblast (CFU-F) assays.	57
Figure 19. mRNA expression of Nanog, Oct-4 and CD44 in DPSCs with dental restorative material	58
Figure 20. Osteogenic induction for 2 weeks in microscope.	59
Figure 21. Osteogenic induction for 2 weeks.	60
Figure 22. Osteogenic induction for 4 weeks.	61
                                    

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