臺灣博碩士論文加值系統

傳統上而言，介穩水泥(鈣矽水泥)的製程是藉由一般的氧化物粉末經由適當的混合後，再以高溫爐加熱製備而成，並且所有的成分必需分別製備，然而這樣的製程不僅需要極高的溫度及相當長的時間，而且所製備出來產品其反應性也相當的低，因此也限制了介穩水泥作為牙科逆向封填材料的發展性。因此本研究希望能使用溶膠-凝膠法發展出一個較為簡單及經濟的製備方式。
本研究的第一部份著重於材料的製備及材料性質的分析。首先，因為在溶膠-凝膠法中使用的有機金屬化合物及金屬鹽類，其反應性差異相當的大，因此為了避免因反應性差異所造成的自我聚合及相分離現象，本研究使用acetylacetone對aluminum sec-butoxide的表面作修飾，降低其反應性，使其能與其他反應物進行反應。在對反應物作修飾之後，再加入適當的催化劑催化，在這研究中是使用鹼(氨水)作為催化劑，不使用酸(硝酸)作為催化劑的原因是因為鹼在本製程有較好的催化效率。在所得的產物中，籍由XRD, FTIR, 及MAS NMR的結果，不但可以得知在產物中可以鑑定出介穩水泥中的所有成份，而且擁有反應性較高的monoclinic的結晶相，而這個現象也與水合產物及微硬度測試的結果一致，經由溶膠-凝膠法製備出的介穩水泥其起始強度在第24小時的時候就可以在微硬度儀上可被偵測到，比起傳統製程的介穩水泥早了大約72個小時達到可被偵測的強度，而到了第7天時，溶膠-凝膠法製備的介穩水泥其微硬度為27.26HV，而傳統製程的介穩水泥的微硬度才2.05HV。
本研究的第二部份是著重於材料的體外測試，經由crystal violet, MTT,及LDH的測試方法可得知，溶膠-凝膠法製備的介穩水泥並不會影響到細胞的增殖能力、粒腺體活性及細胞毒性，除此之外，根據鹼性磷酸酶及骨鈣素的測試結果可知，由跟材料萃取後的培養基與細胞共養後，細胞比較不容易失去其phenotype，而且根據PI cell cycle及Annexin V-FITC cell apoptosis test的結果，細胞也比較不會朝凋亡的方向進行，而維持較高的活性，而且這些現象的原因都是來自於材料所釋放出的鈣離子及氫氧基離子。鈣離子對於骨母細胞及細胞凋亡扮演相當重要角色，而氫氧基離子的釋放會促進鹼性磷酸酶的作用及減緩因為細胞代謝物所造成的酸化。
由溶膠-凝膠法製備出的介穩水泥其生物相容性及其細胞反應並不會因為製程的改變而有所變化，而且，籍由溶膠-凝膠法製備出的介穩水泥擁有較高反應性的結晶相及較高的水合反應速率，由以上這些結果我們可以知道，對於作為牙科逆向封填材料而言，溶膠-凝膠法製備出的介穩水泥是有相當大的潛力及發展空間。

Conventionally, Partial-Stablized Cement (PSC), which is a kind of calcium silicate cement, is prepared through powder mixing method and have to prepare each component separately. Low reaction efficiency in conventional process leads to a very time- and energy-consuming preparation process. Moreover, low initial strength of conventional PSC limited the application of PSC as a dental root-end filling material. This study provides a one-step sol-gel process for the synthesis of PSC for more simple and economic preparation process.

First part of this study will focus on the preparation and characterization of sol-gel synthesized PSC. Because of inconsistency between alkoxide and metal salts used in the sol-gel process, complexing ligand (acetylacetone) is used for tuning down the activity of aluminum sec-butoxide (ASB) in order to avoid possible self-polymerization and phase separation. After the modification with complex ligand, ammonia water is used instead of nitric acid as catalyst in the process because of better reaction efficiency. Each component of PSC is identified and more active monoclinic phase is formed in the product according to the result XRD, FTIR, and MAS NMR. This fact of more active product is also in agreement with result of hydration product formation and microhardness test. The initial strength of sol-gel-synthesized PSC achieves detectable level 24 hours which is 72 hours earlier than PSC synthesized by conventional process. Moreover, the microhardness value of sol-gel-synthesized PSC at 7th day is 27.26 HV which is much higher than conventional PSC which is 2.05 HV.

Second part is the in vitro evaluation of PSC. PSC synthesized by sol-gel will not alter the proliferation ability, mitochondria activity, and cytoxicity of the target cells according to result of crystal violet, MTT and LDH assays. Cell phenotype is maintained when incubated with medium extract from PSC (both groups) according to the result of alkaline phosphatase and osteocalcin. Moreover, cell apoptosis is also delayed according to the PI cell cycle and Annexin V-FITC cell apoptosis test. This phenomenon is due to the higher Ca2+ ion and OH- group content in the extract medium. Ca2+ ion plays a very important in the regulation of osteoblast maturation and cell apoptosis. Higher content of OH- group in the medium can not only favors the function of alkaline phosphatase but also delays acidized tendency due to the cell metabolites.
Sol-gel synthesized PSC remains its biocompatibility and similar cell response as conventional PSC. Moreover, more active monoclinic phase and much faster hydration rate are observed in PSC synthesized by sol-gel process. The results above suggest that sol-gel prepared PSC is a potential candidate as dental retrograde-filling materials.

口試委員會審定書………………………………………………………….I
謝誌…………….…………………………………………………………II
English abstract……………………………………………………………III
Chinese abstract……………………………………………………………..VI
Chart list…………………………………………………………………VIII
Table list…………………………………………………………………….XII
Table of content…………………………………………………………….XIII
Abbreviation Table………………………………………………………XVIII
Chapter 1 Introduction…………….……………….…………………………1
1-1 Root-End Surgery……………………………...……….....……….1
1-2 Root-End Filling Materials………………………………………….3
1-2-1 Amalgam…………………………………………………..…3
1-2-2 Zinc Oxide Eugenol (ZOE) and Reinforced ZOE cement………………………………………………………………4
1-2-3 Composite Resin……………………………………….……5
1-2-4 Mineral trioxide aggregate (MTA)…………………………5
1-3 Calcium Silicate Cement……………..………………………………7
Chapter 2 Theoretical Basis………………………………….....………..13
2-1 Sol-Gel Process………………………………………………….13
2-2 Precursors……………………………………………………….15
2-2-1 Hydrolysis and condensation reactions of metal salt precursors…………………………………………….…………16

2-2-2 Hydrolysis and condensation of metal alkoxide precursors………………..….................................................19
2-3 Purpose of the Study………………………………………………………………..21
Chapter 3 Materials and Method……………………………………...22
3-1 Materials Preparation…………………....................................23
3-2 Materials Analysis……………………………………………24
3-2-1 XRD…………………………………………….……..24
3-2-2 FTIR……………………………………………………24
3-2-3 Microhardness Test………………………………..…..24
3-2-4 pH Variation in the Solution Incubate with Material…………25
3-2-5 Scanning Electron Microscopy (SEM)…………………………………….26
3-3 In vitro Evaluation………………………………..…………..27
3-3-1 Cell Culture……………………………………………27
3-3-2 Lactate Dehydrogenase Assay…………………………28
3-3-3 Crystal Violet Assay………………………….………..29
3-3-4 Mitochondria Activity Assay…………………………….29
3-3-5 Alkaline Phosphatase Activity Assay…………………..30
3-3-6 Osteocalcin Assay………………………….…………31
3-3-7 PI Cell Cycle………………………………………..…..32
3-3-8 Annexin V-FITC Cell Apoptosis………………………33
3-4 Genotoxicity ……………………………………......……….34
3-4-1 Chromosome Aberration…………………..……………34
3-4-2 Sister Chromatid Exchange……………………………..35
3-5 Morphological Examination of Cell on the Developed Material………………………………………………….…….37
3-6 The Effect of Developed Material in Antibacterial………………………………………………………38
3-7 Statistics Analysis……………………………………………….39
Chapter 4 Results ………………………………………………………40
4-1. Materials Analysis……………………………………………40
4-1-1 Synthesis of PSC by Sol-Gel Process………..……..40
4-1-2 Characterization of PSC Synthesized by Sol-Gel Process……………………………………………………….…….44
4-2. In-vitro Evaluation……………………………………………59
4-2-1 Cell proliferation and Cytotoxicity……………………..59
4-2-2 Protein Secretion in Osteoblast….…………………..….63
4-2-3 Cell Cycle Analysis……………………………………..67
4-2-4 Cell Apoptosis Assay………………….………...……..67
4-2-5 Genotoxicity……………………………………..……..71
4-2-6 The Effect of Developed Material in Antibacterial………………………………...................………73
4-2-7 Morphological Examination of Cell on the Developed Material……………………………………………………..75
Chapter 5 Discussion…………………………………………………..76
Discussion of Synthesis of PSC by Sol-Gel Process………….76
Discussion of Characterization of PSC Synthesized by Sol-Gel Process………………..……………………………………..81
Discussion of In vitro Evaluation……………………………84
Chapter 6 Conclusion………………….…………………………..….90
Reference………………………………………………………….……91
Appendix…………………………………………………………………99
Appendix A Resume…………………………………………………..100
Appendix B Publication List…………………………………………..101

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