三氧礦化物(Mineral trioxide aggregate, MTA)已被廣泛應用於牙齒和骨頭修復之領域。過去本實驗室已成功開發出新型鈣矽根管填充材料(novel Ca-Si root-endfilling material, CS)。CS和MTA組成相似，具有良好的生物相容性與封閉性質，硬化時呈現高鹼性且具有一定之強度，操作上不需特殊儀器，並且可藉由成分添加比率的不同來改變材料之物化性質，缺點則是硬化時間太長。本研究以10%氯化鈣溶液來調拌CS，成功地縮短其硬化時間，以改善操作性質。
首先，探討CS與MTA之水合反應。藉由SEM顯微結構、XRD晶相分析與FT-IR吸收光譜分析結果證實，CS之水合反應和MTA相似，由C3S主導其水合反應，而產生最終產物calcium silicate hydrates (C-S-H) 和Portlandite(氫氧化鈣)。其次，探討CS在模擬體液環境下，SEM觀察到CS材料表面附著類似類氫氧基磷灰石(HAp)結構，經SEM-EDS鑑定Ca/P大於1.67，而XRD分析發現低結晶性之HAp繞射峰出現，FT-IR顯示-PO43-之吸收光譜，同時ICP-AES檢測到環境之磷離子大量被消耗，證明材料具有生物活性(bio-activity)。
最後，使用老鼠前驅骨細胞MC3T3-E1為生物相容性測試模型，評估鈣矽材料之生物相容性與誘導骨細胞礦化表現(mineralization)。細胞毒性分析發現，毒性尚小。SEM顯示，CS和MTA具有很好的細胞貼附行為，經由Alamar Blue測試、細胞鹼性磷酸酶活性分析和礦化染色得知，有助於骨細胞增生、分化與基質生成。因此，綜合總結之實驗結果，CS具有很好的生物相容性、生物活性及骨誘導能力，為一具有潛力應用於牙髓修補之填充材料。

Mineral trioxide aggregate (MTA) has been widely used in the area of dental and orthopedic rehabilitation. Our laboratory has previously reported to successfully develop a novel calcium silicate root canal filling material (Ca-Si root-end filling material, CS). With a similar composition to the conventional commercialized MTA, CS can be easily operated without the need of special equipment. It generates a great amount of alkaline during setting, and shows good mechanical strength and sealing properties, in addition to good biocompatibility after setting. CS can also be easily modified its physical and chemical properties by adding different chemicals. However, its setting time is still too long in clinical practice. In this study, we used 10% calcium chloride solution instead of water in our CS. We successfully reduce its setting time and therefor improve its operability.
The hydration products of CS were calcium silicate hydrates (C-S-H) and Portlandite by the analyses of XRD, FT-IR and SEM evaluations. C3S was the main peak during the reaction of CS hydration. When immersing CS into simulated body fluid (SBF), appetite emerged on the surface of CS observed by SEM and XRD analyses. PO4 was shown in the FT-IR analyses. The phosphate in solution was almost totally consumed confirmed by ICP-AES. The emergence of appetite rendered CS the properties of bioactivity.
We used mouse bone cells MC3T3-E1 to assess the biocompatibility of CS with induced expression of bone cell mineralization. Cytotoxicity of CS was negligent. Bone cell proliferation, differentiation, matrix formation and alkaline phosphatase activity were assessed by Alamar Blue test to know if it can support mineralization. SEM showed that CS has good cell attachment behavior superior to MTA. Therefore, CS has good biocompatibility, bioactivity and osteoconduction, as a potential material for restorative endodontic used.

誌謝 .I
中文摘要 II
英文摘要 III
目錄 IV
圖目錄 VIII
表目錄 XIII
英文縮寫 XIV
前言 1
1.根管治療與牙根穿孔修補材料之重要性 2
2.研究目的 2
3.研究假說 3
4.論文架構 3
第一章 鈣矽材料水合產物之物化性分析 4
1.1 研究背景 4
1.1.1 根管填充材料................................................................................ 4
1.1.2 Mineral trioxide aggregate................................................................5
1.1.3 研究目的.........................................................................................6
1.2研究材料與方法 7
1.2.1 實驗材料.........................................................................................7
1.2.2 實驗儀器.........................................................................................7
1.2.3 新型鈣矽填充材料之組成與製備...............................................7
1.2.4 X光繞射分析儀..............................................................................8
1.2.5 傅立葉紅外線吸收光譜分析儀.....................................................8
1.2.6 硬化時間測試................................................................................8
1.2.7 對徑壓縮強度測試........................................................................ 9
1.2.8 pH變化......................................................................................... 9
1.2.9感應耦合電漿原子發射光譜分析儀...........................................9
1.2.10掃描式電子顯微鏡/能量光譜分析儀............................................9
1.3實驗結果 11
1.3.1 CS和MTA材料粉末性質分析.....................................................11
1.3.2 CS和MTA水合後之X光繞射分析.............................................11
1.3.3 CS和MTA水合後之FT-IR吸收光譜分析.................................11
1.3.4 CS和MTA水合後之離子釋放與pH值變化分析.......................12
1.3.5 CS和MTA硬化時間測試分析.....................................................12
1.3.6 CS以不同溶液調拌之對徑壓縮強度測試...................................13
1.3.7 CS和MTA粉末之顯微結構形態觀察.........................................13
1.3.8 CS和MTA水合後之顯微結構形態觀察....................................13
1.4討論 15
1.4.1 CS和MTA水合後之產物晶相分析.............................................15
1.4.2 CS和MTA水合後之離子釋放與pH值變化分析......................15
1.4.3 CS和MTA硬化時間測試分析.....................................................16
1.4.4 CS和MTA水合後之顯微結構形態觀察.....................................16
1.5結論 18
第二章 鈣矽材料表面活性鈣磷化合物形成 32
2.1研究背景 32
2.1.1 研究目的....................................................................................33
2.2研究材料與方法 34
2.2.1實驗材料............................................................................... 34
2.2.2實驗儀器....................................................................................... 34
2.2.3模擬體液之製備......................................................................... 34
2.2.4測試樣本之製備.......................................................................... 35
2.2.5 X光繞射分析儀............................................................................ 36
2.2.6傅立葉轉換紅外線吸收光譜分析............................................... 36
2.2.7感應耦合電漿原子發射光譜分析儀............................................ 36
2.3實驗結果.............................................................................................................. 37
2.3.1 CS浸泡SBF後材料表面之顯微形態觀察................................. 37
2.3.2 CS浸泡SBF後材料表面元素半定量分析................................. 37
2.3.3 CS浸泡SBF後材料X光繞射分析儀.........................................37
2.3.4 CS浸泡SBF後材料紅外線吸收光譜分析................................. 38
2.3.5 CS泡SBF後離子釋放定量分析...................................................38
2.4討論 39
2.4.1 CS浸泡SBF後材料表面之顯微形態觀察...................................39
2.4.2 CS浸泡SBF後材料表面元素分析與鈣、矽、磷離子之釋
放行為............................................................................................ 39
2.4.3 CS浸泡SBF後材料X光繞射分析儀.........................................39
2.4.4 CS材料之生物活性作用機轉.......................................................40
2.5結論 41
第三章 鈣矽材料對成骨細胞之生物相容性評估與礦化表現 52
3.1研究背景 52
3.1.1生物相容性................................................................................52
3.1.2 體內測試...............................................................................53
3.1.3體外測試...............................................................................53
3.1.4 研究目的.................................................................................54
3.2研究材料與方法 55
3.2.1 實驗儀器.......................................................................................55
3.2.2 實驗材料.......................................................................................55
3.2.3 所使用之細胞株與培養條件.......................................................56
3.2.4 細胞貼附材料表面之觀察 .........................................................57
3.2.5 細胞毒性測試...............................................................................58
3.2.6 細胞增生能力分析.....................................................................59
3.2.7 細胞鹼性磷酸酶活性分析...........................................................60
3.2.8 成骨細胞礦化之定量分析與觀察...............................................60
3.2.9 成骨細胞礦化之定性分析與觀察...............................................62
3.2.10統計分析.......................................................................................64
3.3實驗結果 65
3.3.1細胞貼附於材料表面之形態觀察.................................................65
3.3.2細胞毒性分析.................................................................................65
3.3.3細胞增生能力分析.........................................................................70
3.3.4細胞鹼性磷酸酶活性分析.............................................................74 3.3.5成骨細胞礦化之定量分析與觀察.................................................77
3.3.6成骨細胞礦化之定性分析與觀察................................................ 78
3.4討論 79
3.4.1 細胞貼附於材料表面之形態分析...............................................79
3.4.2 細胞毒性分析 ...........................................................................79
3.4.3細胞增生能力分析........................................................................80
3.4.4 成骨細胞礦化表現分析...............................................................80
3.5 結論......................................................................................................................82
參考文獻...................................................................................................................133

1.Walton RE, Torabinejad M. Principles and practice of endodontics. 1989 Philadelphia: Saunders.
2.Cohen S, Burns RC 2002. Pathways of the pulp. Louis: Mosby.
3.Arens DE, Adams WR, De Castro RA. Endodontic surgery. Philadelphia: Harper & Row1981,154-68.
4.Torabinejad M, Watson TF, Pitt Ford TR. Sealing ability of a mineral trioxide aggregate when used as a root end filling material. Journal of Endodontics 1993;19(12):591-595.
5.Dorn SO, Gartner AH. Retrograde filling materials: a retrospective success-failure study of amalgam, EBA, and IRM. Journal of Endodontics 1990; 16(8):391-393.
6.Alhadainy HA. Root perforations. A review of literature. Oral Surgery, Oral Medicine, Oral Pathology1994; 78(3):368-374.
7.Shabahang S, Torabinejad M, Boyne PP, Abedi H, Mc Millan P. A comparative study of root-end induction using osteogenic protein-1, calcium hydroxide, and mineral trioxide aggregate in dogs. J Endod 1999; 25: 1-5.
8.Asgary S, Parirokh M, Eghbal MJ, Brink F. Chemical differences between white and gray mineral trioxide aggregate. Journal of Endodontics 2005; 31(2):101-103.
9.Asgary S,Parirokh MJ, Brink F. Chemical difference between white and gray mineral trioxide aggregate. J Endod. 2005; 31(2):101-10310.
10.Lee Y-L, Lee B-S, Lin F-H, Lin YA, Lan W-H, Lin C-P. Effects of physiological environments on the hydration behavior of mineral trioxide aggregate. Biomaterials 2004; 25(5):787-793.
11.Torabinejad M, Higa RK, McKendry DJ, Pitt Ford TR . Dye leakage of four root end filling materials: effects of blood contamination. Journal of Endodontics1994; 20(4):159-163.
12.Holland R, de S, V, NeryMJ, Bernabe FE, Filho JA, Junior ED et al. Calcium salts deposition in rat connective tissue after the implantation of Calcium hydroxide-containing sealers. Journal of Endodontics 2002 ; 28(3):173-176.
13.J. Camilleri, Characterization of hydration products of mineral trioxide
Aggregate. International Endodontic Journal 2008; 41(5):408-417.
14.T. Dammaschke, H U. Gerth, H. Züchner, and E. Schäfer. Chemical and physical surface and bulk material characterization of white ProRoot MTA and two Portland cements. Dental Materials2005; 21(8): 731–738.
15.M. Torabinejad, C. U. Hong, F. McDonald, and T. R. Pitt Ford. Physical and chemical properties of a new root-end filling material. Journal of Endodontics 1995 ; 21(7): 349–353.
16.P. Kogan, J. He, G. N. Glickman, and I. Watanabe. The effects of various additives on setting properties of MTA. Journal of Endodontics 2006;(32). 6:569–572.
17.B. S. Ber, J. F. Hatton, and G. P. Stewart. Chemical modification of ProRoot MTA to improve handling characteristics and decrease setting time. Journal of Endodontics 2007; (33) 10:1231-1234.
18.K. B. Wiltbank, S. A. Schwartz, and W. G. Schindler. Effect of selected accelerants on the physical properties of mineral trioxide aggregate and Portland cement. Journal of Endodontics 2007; (33) 10: 1235–1238.
19.E. A. Bortoluzzi, N. J. Broon, C. M. Bramante, W. T. Felippe, M. Tanomaru Filho, and R. M. Esberard. The influence of calcium chloride on the setting time, solubility, disintegration, and pH of mineral trioxide aggregate and white Portland cement with a radiopacifier. Journal of Endodontics 2009; (35) 4: 550-554.
20.E. A. Bortoluzzi, N. J. Broon, C. M. Bramante, R. B. Garcia, I. G. de Moraes, and N. Bernardineli. Sealing ability of MTA and radiopaque Portland cement with or without calcium chloride for root-end filling. Journal of Endodontics 2006; (32) 10: 897-900.
21.E. A. Bortoluzzi, N. J. Broon, C. M. Bramante, A. Consolaro, R. B. Garcia, I. G. de Moraes, and N. Bernadineli. Mineral trioxide aggregate with or without calcium chloride in pulpotomy. Journal of Endodontics 2008; (34) 2: 172-175.
22.B. S. Ber, J. F. Hatton, and G. P. Stewart. Chemical modification of ProRoot MTA to improve handling characteristics and decrease setting time. Journal of Endodontics 2007; (33) 10:1231-1234.
23.T. H. Huang, M. Y. Shie, C. T. Kao, and S. J. Ding. The effect of setting accelerator on properties of mineral trioxide aggregate. Journal of Endodontics 2008; (34) 5:590-593.
24.S. J. Ding, C. T. Kao, M. Y. Shie, C. J. Hung, and T. H. Huang. The physical and cytological properties of white MTA mixed with Na2HPO4 as an accelerant. Journal of Endodontics 2008; (34) 6: 748-751.
25.Kosmatka S, Panarese W. Design and control of concrete mixtures.13th
ed. Skokie, IL: Portland Cement Association, 1988.
26.Abdullah D, Pitt Ford TR, Papaioannous S, Nicholson J, McDonald F. An evaluation of acceleratd Portland cement as a restorative material. Biomaterials 2002; 19:4001-4010.
27.K. Okada, Y. Kameshima, A. Yasumori. Chemical shifts of silicon
X-ray photoelectron spectra by polymerization structures of silicates. J.
Am. Ceram. Soc. 1998; 81 (7):1970-1972.
28.Y. L Lee, B. S. Lee,C. P. Lin. Effects of physiological environments on the hydration behavior of mineral trioxide aggregate. Biomaterials 2004; 25 :787–793.
29. Hence LL, West JK. Biological applications of bioactive glasses.
Life Chem Rep 1996; 13: 187–241.
30.N. K. Sarkar, R. Caidedo, P. Tirwik, R. Moiseyeva, and I. Kawashima,
Physicochemical basis of the biologic properties of mineral trioxide aggregate. Journal of Endodontics 2005; (31)2: 97-100.
31.Yatsushiro JD,Baumgartner JC, Tinkle JS. Longitudinal study of the
microleakage of two root-end filling materials using a fluid conductive system. Journal of Endodontics 1998; ( 24)11: 716-719.
32. Portera AE, Patela N, Skepperb JN, Besta SM, Bonfielda W.
Comparison of in vivo dissolution processes in hydroxyapatite and
silicon-substituted hydroxyapatite bioceramics. Biomaterials 2003; 24:4609–20.
33.Cao W, Hench LL. Bioactive Materials. Ceram Int 1996; 22:493–507.
34.Kotani S, Fujita Y, Kitsugi T, Nakamura T, Yamamuro T. Bone
bonding mechanism of -tricalcium phosphate. J Biomed Mater Res 1991; 25: 1303–15.
35.Fujita Y, Yamamuro T, Nakamura T, Kotani S . The bonding
behaviour of calcite to bone. J Biomed Mater Res 1991; 25: 991–1003.
36.Kokubo T, Takadama H . How to useful is SBF in predicting in vivo
bone bioactivity. Biomaterials 2006; 27: 2907–2915.
37.Mitchell PJ, Pitt Ford TRY Torabinejad My McDonald F. Osteoblast
biocompatibility of mineral trioxide aggregate. Biomaterials 1999 Jan;20(2): 167-173.
38.Nakayama A, Ogiso By Tanabe N, Takeichi , Matsuzaka K, Inoue T.
Behaviour of bone marrow osteoblast-like cells on mineral trioxide aggregate: morphology and expression of type I collagen and bone-related protein mRNAs. Int. Endod J 2005; 38(4):203-210.
39.Torabinejad M, Hong CU, Pitt Ford TR, Kettering JD. Cytotoxicity of four root end filling materials. Journal of Endodontics1995;21:489-492.
40.Trorabinejad M, Pitt Ford TR, McKendry DJ, Abedi HR, Miller DA,
Kariyawasam SP.Histology assessment of mineral trioxide aggregate as a root-end filling in monkeys. J Endod.1997; 23(4):225-228.
41.Torabinejad M, Ford TR, Abedi HR, Kariyawasam SP, Tang HM. Tissue reaction to implanted root-end filling materials in the tibia and mandible of guinea pigs. J Endod. 1998; 24(7):468-471.
42.Koh ET, Torabinejad M, Pitt Ford TR, Brady K, McDonald F. Mineral
trioxide aggregate stimulates a biological response in human osteoblasts.
Journal of Biomedical Matedrials Research1997;37:432-439.
43.Huang TH, Ding SJ, Hsu TC, Kao CT. Effect of mineral trioxide
aggregate (MTA) extracts on mitogen-activated protein kinase activity
in human osteosarcoma cell line (U2OS). Biomaterials 2003; 24:3909-3913.
44.Koh ET, McDonald F, Pitt Ford TR, Torabinejad M. Cellular response to Mineral Trioxide Aggregate. Journal of Endodontic1998; 24:543-547.
45.Pitt Ford TR, T orabinejad M, Abedi HR,Bakland LK, Kariyawasam
SP.Using mineral trioxide aggregate as a pulp-capping materials. J Am Dent Assoc 1996; 127:1491-1494
46.Torabinejad M, Hong CU, Lee SJ, Monsef M, Pitt Ford TR. Investigation of mineral trioxide aggregate for root-end filling in dogs. J Endod. 1995; 21(12):603-608.
47.Bemabe PF, Gomes-Filho JE, Rocha WC, Nery MJ, Otoboni-Filho JAY
Dezan-Jhior E. Histologica1 evaluation of MTA as a root-end filling material. Int Endod J. 2007;40(10):758-765.
48.Yildirim T, Genqoglu N, Firat I, Perk C, Guzel 0. Histologic study of
furcation perforations treated with MTA or Super EBA in dogs'' teeth.
Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology. 2005; 100(1): 120-124.
49.Zhu Q, Haglund R, Safavi KE, Spangberg LS. Adhesion of human osteoblasts on root-end filling materials. J Endod. 2000; 26(7):404-406.
50.Tani-Ishii N, Hamada N, Watanabe K, Tujimoto Y, Teranaka T, Umemoto T.Expression of bone extracellular matrix proteins on
osteoblast cells in the presence of mineral trioxide.J Endod. 2007 ; 33(7):836-839.
51.Al-Rabeah E, Perinpanayagam H, MacFarland D. Human alveolar bone cells interact with ProRoot and tooth-colored MTA. J Endod. 2006 ; 32(9):872-875.
52.Camilleri J, Montesin FE, Papaioannou S, McDonald F, Pitt Ford TR.Biocompatibi1ity of two commercial forms of mineral trioxide aggregate. Int Endod J. 2004;37(10):699-704.
53.Perez AL, Spears R, Gutmann JL, Opperman LA. Osteoblast and MG63 osteosarcoma cells behaver differently when in contact witj ProRoot MTA and White MTA. Int Endod J 2003; 36(8):564-570.
54.Agamy HA, Bakry NS, Mounir MM, Avery DR. Comparison of mineral
trioxide aggregate and formocresol as pulp-capping agents in pulpotomized primary teeth. Pediatr Dent2004; 26(4):302-309.
55.Nobuyuki Tani-Ishii, Nobushiro Hamada, Kiyoko Watanabe, Yasuhisa Tujimoto, Toshio Teranaka and Toshio Umemoto. Expression of Bone Extracellular Matrix Proteins on Osteoblast Cells in the Presence of Mineral Trioxide, Journal of Endodontics 2007; (33) 7: 836-839.
56.Yoshiyuki Yasuda, Masafumi Ogawa, Toshiya Arakawa, Tomoko Kadowaki and Takashi Saito-The Effect of Mineral Trioxide Aggregate on the Mineralization Ability of Rat Dental Pulp Cells: An In Vitro Study , Journal of Endodontics. 2008; 34(9): 1057-1060.
57.J. Camilleri & T. R. Pitt Ford Mineral trioxide aggregate: a review of the constituents and biological properties of the material.-International Endodontic Journal 2006; 39: 747–754.
58.Keiser K, Johnson CC, Tipton DA. Cytotoxicity of mineral trioxide aggregate using human periodontal ligament fibroblasts. J Endod. 2000; 26:288–291.
59.Mitchell PJ, Pitt Ford TR, Torabinejad M, McDonald F. Osteoblast
biocompatibility of mineral trioxide aggregate. Biomaterials1999;
20:167-173
60.Moghaddame-Jafari S, Mantellini MG, Botero TM, McDonald NJ, Nör JE. Effect of ProRoot MTA on pulp cell apoptosis and proliferation in vitro. J Endod. 2005; 31: 387-391.
61.Sema S. Hakki, S. Buket Bozkurt, Erdogan E. Hakki, and Sema Bell-Effects of Mineral Trioxide Aggregate on Cell Survival,Gene Expression Associated with Mineralized Tissues,and Biomineralization of Cementoblasts. Journal of Endodontics.2009; 35(4): 513-519.
62.Nobuyuke TI, Nobushiro H, Kiyoko W, Yasuhisa T, Toshio T, Toshio U. Expression of bone extracellular matrix proteins on osteoblast cells in the presence of mineral trioxide. J Endod. 2007; 33: 836-839.
63.Hakki s, Bozkurt By Hakki E. Effects of miniral trioxide aggregate on cell survival, gene expression associated with mineralized tissues, and biomineralization of cementoblasts. J Endod. 2009; 35(4):513-519.
64.Komori T. , Kishimoto T. Cbfa1 in bone development. Current Opinion in Genetics & Development. 1998; 8(4):494-9.
65.Hunter GK. , Poitras MS., Underhill TM. , Grynpas MD., Goldberg HA.
Induction of collagen mineralization by a bone sialoprotein—decorin
chimeric protein. Journal of Biomedical Materials Research. 2001; 55(4):496-502.
66.Roy ME. , Nishimoto SK., Rho JY. , Bhattacharya SK. , Lin JS., Pharr GM. Correlations between osteocalcin content, degree of mineralization, and mechanical properties of C. carpio rib bone. Journal of Biomedical Materials Research. 2001; 54(4):547-53.