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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 楊台鴻(Tai-Horng Young) | |
dc.contributor.author | Rung-Shu Chen | en |
dc.contributor.author | 陳蓉書 | zh_TW |
dc.date.accessioned | 2021-06-15T00:33:18Z | - |
dc.date.available | 2012-02-03 | |
dc.date.copyright | 2009-02-03 | |
dc.date.issued | 2009 | |
dc.date.submitted | 2009-01-13 | |
dc.identifier.citation | 1. Modino SAC, Sharpe PT. Tissue engineering of teeth using adult stem cells. Arch Oral Biol 2005;50:255-258.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/41826 | - |
dc.description.abstract | 牙齒是很獨特的器官,而身體結構內最為堅硬的組織就是牙齒,牙齒具有撕裂、研磨及壓碎食物、輔助發音以及協調臉形等重要功能。牙齒因齲齒、牙周病、外力撞擊以及腫瘤等因素所造成的牙冠缺損會造成生活不便影響生活品質並引發其他疾病。一旦牙齒有缺失,目前在臨床上的治療方式是採用牙冠、牙橋活動義齒或植入生物相容性之金屬材料做為支架然後放置人工牙冠或牙橋即所謂植牙的方式來取代缺損的牙齒。雖然,植牙可以滿足病患的需求,然而植牙費用較為昂貴、且需要進行手術,因此,較不易廣泛使用此方法來修補損失的牙齒。基於牙齒損失會造成生活不便及目前臨床上處理方法的限制加上牙齒是一個器官,探討牙齒再生即有助於調解器官再生,因此引發此研究進入探討牙齒再生工程的動力。生醫材料在組織或器官再生工程中扮演重要的角色,適當的生醫材料可節有某些訊息傳遞路徑而影響細胞的生長與分化,因此,本研究將探討牙齒再生工程中,生醫材料對於牙胚細胞生長分化的影響。
牙胚細胞,是形成牙齒的主要細胞來源,常用於牙齒再生的應用,包含有牙釉質母細胞、牙本質母細胞、間葉細胞及幹細胞。此研究以牙齒再生工程為主軸,探討牙胚細胞與生醫材料的關係,包括四部分:探討不同年齡之Wistar老鼠牙胚細胞之生長潛力;尋找合適與牙胚細胞生長的生醫材料,觀察牙胚細胞之行為表現;探討生醫材料如何提供類似與體內環境的生長條件以促使牙胚細胞增生、礦化及分化;藉由訊息傳遞路徑得知生醫材料與牙胚細胞間之關係。 牙齒隨成長年齡不同而有不同分子、結構等性質。因此,在進行牙齒再生的前提下,尋找適當的牙胚細胞來源是相當重要的。第一部份的研究目的即是藉由不同年齡大小的老鼠取得牙胚細胞,以作為之後應用於牙齒再生的細胞來源。有趣的是,此研究發現介於分化中期之牙胚細胞有良好的增生及分化能力。因此本研究之牙胚細胞將取於4天大的老鼠。 生醫材料已被廣泛的應用於組織工程,然而,牙胚細胞與生醫材料間之關係卻是不明的。在第二部分中,為觀察生醫材料對於牙胚細胞的影響,針對不同親疏水性的醫用高分子進行比較,如較為親水的聚乙烯醇、親疏水性相似於細胞培養皿的共聚乙烯乙烯醇、聚乳酸甘醇酸及較為疏水的聚偏二氟乙烯。結果顯示親疏水性相似於細胞培養皿的生醫材料可以促進牙胚細胞的貼附與生長,然而,較為疏水的生醫材料卻無法提供牙胚細胞良好的貼附與生長。令人驚訝的是,較為親水的聚乙烯醇導致牙胚細胞表現低生長行為且呈現懸浮聚集成球,而這些懸浮成球的牙胚細胞再重新培養回細胞培養皿是可以再次貼附於培養皿的。因次,第二部分的研究顯示出生醫材料是可以用來調控牙胚細胞生長行為。 牙胚細胞培養於聚乙烯醇材料上顯現出較低的增生行為,過去文獻指出當細胞增生行為較為緩慢時,將有利於細胞誘導分化。由於細胞增生行為較為緩慢時,係指細胞與細胞間之接觸較為緊密,因此,延續第二部分的研究,在第三部分中,我們將牙胚細胞培養於聚乙烯醇材料上,藉由不同的細胞密度以得到細胞與細胞間之接觸緊密程度不同,觀察聚乙烯醇材料對於牙胚細胞誘導分化的影響。結果顯示,相較於組織培養皿,不論培養之細胞密度高低,聚乙烯醇材料可有效促進牙胚細胞之鹼性磷酸酶(ALP)活性並提高礦化與分化的表現,特別是此材料亦可促使牙胚細胞表現特定之基因,即便在沒有誘導培養基的條件下。再者,此研究也顯示牙胚細胞的礦化與分化並非單靠懸浮聚集即可,且經由添加短片段之RGD胜肽,顯示聚乙烯醇材料可促使牙胚細胞分泌特定之細胞外基質,進而達到誘導牙胚細胞礦化與分化。 再者,已有許多文獻指出有絲裂原活化蛋白激酶(MAPK)對於骨系列母細胞誘導分化佔相當重要之角色,特別是經由細胞外訊息調控激酶(ERK)路徑,然而,對於此路徑在骨細胞誘導分化中所扮演的角色之說法卻是迥然不同的。過去文獻指出活化細胞外訊息調控激酶路徑可促使細胞趨向分化途徑,然而亦有文獻指出抑制細胞外訊息調控激酶路徑可促使細胞分化。因此,在第四部分中,主要探討聚乙烯醇材料促使牙胚細胞礦化與分化的過程中,是藉由活化或抑制細胞外訊息調控激酶路徑。延續第三部分的研究,在第四部分研究中發現,即便在沒有誘導培養基的條件下聚乙烯醇材料仍可促使牙胚細胞礦化與分化。此外,經由添加細胞外訊息調控激酶抑制劑後,顯示出抑制細胞外訊息調控激酶路徑可促進牙胚細胞的分化。令人驚訝的是,在沒有誘導培養基的條件下,聚乙烯醇材料會促使牙胚細胞抑制細胞外訊息調控激酶路徑。因此,可視聚乙烯醇材料對牙胚細胞之礦化與分化乃經由細胞外訊息調控激酶路徑,且扮演負調控因子之角色。綜合上述之結果,本研究的分析顯示出不同的生醫材料對於牙胚細胞增生,礦化及分化的影響,對於日後將生醫材料應用於牙齒再生將有相當大的幫助。 | zh_TW |
dc.description.abstract | Tooth is a special organ, the hardest tissue in body is tooth, which is used to tear, scrape, milk, and chew food. Tooth losses may affect most adults adversely at some time in their lives due to dental caries, periodontal disease, trauma and/or a variety of genetic disorders. So far, the therapeutic options to treat missing teeth are by using crown-bridge restoration, removable dental, and dental implant. Dental implant can provide an adequate solution for tooth regeneration, however; the cost of dental implant is expensive and the surgical is needed. Therefore, consideration of the quality of live and the limitations of current treatment options have driven the research into tooth regeneration as a new option. In addition, tooth is an organ, to understand the tooth regeneration is leading to understate the organ regeneration. So Far, the hypothesis of this study is as following biomaterials play important roles in tissue or organ regeneration and proper biomaterials may affect the growth and differentiation of cells through signal transduction pathways. Therefore, the purpose of this study is to investigate the effects of biomaterials on tooth germ (TG) cells growth and differentiation in tooth regeneration.
Tooth germ cells, the formation of teeth, constantly using in tooth regeneration, involving ameloblasts, odontoblasts, mesenchyme cells, and stem cells. This study precede the tooth regeneration for investigating relationship of TG cells with biomaterials and including four parts: the first part is to investigate the growth and potential of TG cells isolated from different age of Wistar rats. The second part is to find the best biomaterials for the growth of TG cells and observed the interaction of TG cells. The third part is to investigate the substrate similar to the biological environment for improving proliferation, mineralization, and differentiation of TG cells on biomaterials. The forth part is to investigate the act of biomaterials on mineralization and differentiation of TG cells through cell signaling pathway. The structures and characters of tooth are different in different age during the development of tooth. Therefore, it is important to find the best age source of TG cells for the study on tooth regeneration. In part I, the purpose of this study is to find the adequate TG cells from different age rats for tooth regeneration. Interestingly, the best proliferation and differentiation potential of TG cells might be from cells isolated from four-day-old rats. Hence, TG cells from four-day-old rats are used in this study. Although, biomaterials are generally used in tissue engineering, however; the effects of biomaterials on TG cells are still unknown. In part II, the aim is to observe the interaction of TG cells and biomaterials of varying surface hydrophilicities. The biomaterials including pol (yvinyl alcohol; PVA), poly (lactic-co-glycolic acid; PLGA), poly (ethylene-co-vinyl alcohol; EVAL), and poly (vinylidene fluoride; PVDF) are used. Our results indicate that adhesion of tooth germ cells to biomaterials with moderate hydrophilicity/hydrophobicity was superior compared to extreme analogs. Cellular adhesion and proliferation was evident on all tested biomaterials except PVA. Surprisingly, the spheroidal cellular aggregation on PVA appeared not to be proliferative, cells remained vitally and is able to reattach to tissue culture plates as determined. It is suggested that the behaviors of TG cells can be medicated by biomaterials. Many papers have been reported that low cellular proliferation is conducive to cellular differentiation. Low cellular proliferation may indicate the cell-cell interactions are compacted. In part III, TG cells cultured on PVA with different cell densities to obtain the compacted cell-cell interactions are described. TG cell spheroids on PVA obviously increased the alkaline phosphatase (ALP) activity, the degree of mineralization, and upregulate osteopontin, osteonectin, dentin matrix protein 1, and enamelin genes, regardless of the low or high seeding density. Surprisingly, it seemed that PVA appears to activate the ALP activity and mineralization effects on TG cell spheroids in the absence of differentiation medium. Furthermore, the present study indicates that ECM may play an important role in mineralization on TG cell spheroids by adding Arg-Gly-Asp (RGD) peptides. In addition, many papers have been reported that mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in tightly related to the regulation of cell proliferation, differentiation, motility and death of osteoblast, especially through extracellular signal-regulated kinase1/2 (ERK1/2), however; there were contradictory results about the relationship between ERK1/2 phosphorylation and osteoblast differentiation. In part IV, in order to determine the role of ERK 1/2 pathway in enhancing ALP activity and mineralization of TG cells, the specific inhibitor for blocking the pathway are used. The results reveal that PVA itself can stimulate TG cells with the differentiation and mineralization ability. By showing the direct suppression of extracellular signaling-regulated kinase (ERK1/2) of TG cells treated with inhibitor, known to suppress the activation of ERK1/2, and significant synergistic effects between PVA and inhibitor, we demonstrated the suppression of ERK1/2 pathway is one of the effects of PVA-promoted TG cell differentiation and mineralization. Importantly, analysis of the data of the proliferation, mineralization, and differentiation of TG cells on biomaterials demonstrated the feasibility of utilizing biomaterials in clinical applications for tooth regenerating. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T00:33:18Z (GMT). No. of bitstreams: 1 ntu-98-D92548016-1.pdf: 5709370 bytes, checksum: 61892499b5abb7a25ba7291a32c6e67c (MD5) Previous issue date: 2009 | en |
dc.description.tableofcontents | 摘 要 I
Abstract IV List of contents VII List of Tables XI List of Figures XII Abbreviations XVIII Chapter 1 Background 1 1.1 Motivation and Objectives 1 1.2 The Tissue Engineering Triad 2 Chapter 2 Literature Review 3 2.1 Overview of dental tissues 3 2.1.1 Tooth development 3 2.1.2 Bud, cap, and bell stages 4 2.1.3 Role of the epithelium and mesenchyme in tooth development 4 2.1.4 Molecular control of tooth development 5 2.2 Dental mineralization 7 2.2.1 Matrix vesicles and cell debris in dental mineralization 7 2.2.2 Dentinogenesis: odontoblast differentiation 8 2.2.3 Amelogenesis: ameloblast differentiation 10 2.3 Tooth regeneration 11 2.4 Biomaterials 13 2.4.1 PVA 13 2.4.2 PLGA 14 2.4.3 EVAL 14 2.4.4 PVDF 15 2.5 Polymer membranes 16 2.5.1 The preparation of polymer membrane 16 2.5.2 The structure of polymer membrane 18 Chapter 3 Materials and Methods 19 3.1. Flowchart of Experiment Design 19 3.2. Materials 22 3.3. Instruments 25 3.4. Preparation of solution 26 3.4.1. MTT reagent 26 3.4.2. 2% paraformaldehyde/0.1% Triton X-100 26 3.4.3. Western Blot reagent 26 3.4.3.1. 5X running buffer pH 8.3 distilled water to 1 liter 26 3.4.3.2. 1.5M Tirs-HCl pH 8.8, distilled water to 250 ml, store up to 6 months at 4°C or RT 26 3.4.3.3. 0.5M Tris-HCl pH 6.8, distilled water to 250 ml, store up to 6 months at 4°C or RT 27 3.4.3.4. Semidry transfer buffer distilled water to 1 liter 27 3.4.3.5. Stripping buffer, store at 4°C 27 3.4.3.6. SDS-PAGE 28 3.5. Preparation of membranes 29 3.6. Isolation and identification of TG cells. 30 3.6.1. Isolation of TG cells 30 3.6.2. Identification of TG cells 30 3.7. Evaluation of cell viability (MTT) 32 3.8. Evaluation of cell cytotoxicity (LDH and caspase-3 activity) 33 3.8.1. LDH 33 3.8.2. Caspase-3 activity 33 3.9. Alkaline phosphatase (ALP) activity 35 3.10. Mineralization assay by measurement of Alizarin red S staining 36 3.11. Reverse transcription-polymerase chain reaction (RT-PCR) 37 3.12. Western blot analysis 39 3.13. Statistical analysis 41 Chapter 4 Results 42 4.1. Part I: Tooth germ cells from rats with different ages 42 4.1.1. Cells migration from the tooth germ 42 4.1.2. Cell number and cell proliferation 42 4.1.3. Cell differentiation 43 4.1.4. Radiological observation and histological analysis 44 4.1.5. Identification of TG cells 44 4.2. Part II: Cell-surface interactions of rat tooth germ cells on various biomaterials. 45 4.2.1. Hydrophobic property of the membranes 45 4.2.2. Effect of hydrophobic property on tooth germ cell culture 45 4.3. Part III: The behavior of rat tooth germ cells on poly (vinyl alcohol) 48 4.3.1. Formation of TG cell spheroids 48 4.3.2. Cell proliferation 48 4.3.3. Cell death and apoptosis 49 4.3.4. ALP activity 50 4.3.5. ARS staining 50 4.3.6. Gene expression of mineralization- and differentiation-related markers 51 4.3.7. The effect of PVA on the ALP activity and ARS staining of TG cells in the absence of differentiation medium 52 4.3.8. The effect of PVA on the TG cells in the presence of RGD 53 4.4. Part IV: Induction of differentiation and mineralization in rat tooth germ cells on PVA through inhibition of ERK1/2 54 4.4.1. ALP activity and mineralization of TG cells on PVA and TCPS 54 4.4.2. Effect of U0126 on TG cells differentiation and mineralization 55 4.4.3. Synergism of PVA and U0126 on TG cell differentiation and mineralization 55 4.4.4. Gene expression of TG cells on PVA and TCPS 56 4.4.5. Effect of PVA on inhibiting ERK1/2 phosphorylation 57 Chapter 5 Discussions 59 5.1 Part I: Tooth germ cells from rats with different ages 59 5.2 Part II: Cell-surface interactions of rat tooth germ cells on various biomaterials. 61 5.3 Part III: The behavior of rat tooth germ cells on poly (vinyl alcohol) 63 5.4 Part IV: Induction of differentiation and mineralization in rat tooth germ cells on PVA through inhibition of ERK1/2 67 Chapter 6 Conclusion and Feature works 70 References: 72 Aappendle I 123 List of publication 148 List of Conference 149 Introduction to author 150 | |
dc.language.iso | en | |
dc.title | 不同生醫材料對牙胚細胞增生、礦化及分化的影響 | zh_TW |
dc.title | Proliferation, mineralization, and differentiation of tooth germ cells on different biomaterials. | en |
dc.type | Thesis | |
dc.date.schoolyear | 97-1 | |
dc.description.degree | 博士 | |
dc.contributor.coadvisor | 陳敏慧(Min-Huey Chen) | |
dc.contributor.oralexamcommittee | 林俊彬,姚宗珍,王盈錦,陳志平,邱紫文,廖俊仁 | |
dc.subject.keyword | 牙胚細胞,聚乙烯醇,礦化,分化,成球,細胞外訊息調控激酶, | zh_TW |
dc.subject.keyword | tooth germ cells,polyvinyl alcohol,mineralization,differentiation,spheroid,extracellular signaling-regulated kinase1/2 (ERK1/2), | en |
dc.relation.page | 150 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2009-01-13 | |
dc.contributor.author-college | 工學院 | zh_TW |
dc.contributor.author-dept | 醫學工程學研究所 | zh_TW |
顯示於系所單位: | 醫學工程學研究所 |
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