臺灣博碩士論文加值系統

牙科黏著劑廣泛運用於現今的牙科治療中，但臨床上仍常出現術後敏感、或牙髓神經發炎壞死的現象。追究其原因，包括未去除完全的受感染齒質、未聚合樹脂單體的細胞毒性、樹脂聚合過程中的收縮以及咬合干擾。牙科使用的單體是否會造成臨床組織壞死，各方論述不一；但多數研究認為：單體濃度決定對細胞的毒性影響程度。
目前有許多研究，為了測量透過牙本質釋放到牙髓腔的單體濃度，設計屏障實驗，但實驗結果往往受制於屏障材料的條件。因為目前的實驗多以人類或動物牙本質為屏障材料，其通透度會隨生物體本身的年齡、飲食習慣、齲齒等因素而改變，進而影響實驗結果。本實驗研究動機即在尋找一種可被認可的材料，以取代牙本質，做為標準化屏障實驗材料。
目前已知樹脂單體對細胞的毒性影響取決於單體濃度，各種細胞對單體的毒性反應也有所不同，此次實驗即測試人類牙髓細胞及NIH-3T3細胞株的細胞毒性，比較兩者對樹脂單體的毒性反應。
• 研究目的
本實驗目的在於比較牙本質與聚氨酯泡棉（Polyurethane foam, 40-pcf, PS, Sawbones®, Washington, DC, USA）的HEMA單體滲透濃度；以及HEMA濃度與Bis-GMA濃度對人類牙髓細胞和NIH-3T3細胞株的影響。
• 研究方法
一、 單體滲透測試：利用0.5mm及1.5mm厚度的牙本質以及Sawbones，在牙科黏著劑3M ESPE Single Bond Universal Adhesive（SBU, 3M）、 ESPE AdperTM Single Bond 2（ASB, 3M）依廠商指示塗抹並聚合後，收集第1、4、8、12、24、48、72小時，共七個時間點的滲透液，並以高效能層析（HPLC）分析滲透液濃度。
二、 在第一（短期）、四（中期）、七天（長期）共三個時間點，利用MTT assay、Alamar blue assay觀察兩種細胞的存活率；測量Caspase-3活性及LDH濃度，以觀察細胞死亡的狀況；利用免疫螢光染色，觀察短期細胞在測試液中的變化。
• 研究結果
一、 在0.5mm的試片厚度，且使用相同牙科黏著劑時，牙本質與Sawbones的HEMA單體濃度無顯著差異。
二、 細胞毒性測試顯示：細胞存活率與單體濃度有關；隨著單體濃度增加，細胞存活率則下降。
三、 短期觀察顯示：在高濃度單體測試液中(HEMA 25mM, HEMA 25mM+Bis-GMA 2.5mM)，細胞會出現破裂情況；在低濃度單體測試液中(HEMA 1.25mM, HEMA 1.25mM+Bis-GMA 0.125mM)，細胞則展現高Caspase-3活性，代表早期凋亡的現象較控制組明顯。
四、 免疫螢光染色顯示：細胞密度在24小時後會因為測試液中的單體濃度不同而改變；單體濃度越高，細胞密度越低。高濃度單體測試液(HEMA 25mM, HEMA 25mM+Bis-GMA 2.5mM)中亦發現細胞破損情形。
• 結論
一、 Sawbones®為穩定、可預期的材料，可取代人類牙本質，用於屏障實驗。
二、 相較之下，HDPC比NIH-3T3易受HEMA影響。
三、 HPLC所得的HEMA濃度，對人類牙髓細胞和NIH-3T3細胞均有毒性影響。

Introduction:
Dental adhesives are widely used in today''s dental treatment, but with clinically undesirable postoperative phenomena, such as sensitivity to heat, dental pulp inflammation and necrosis. These problems may arise from chemical irritation from bacteria by-products of caries lesion, residual monomer, polymerized shrinkage and occlusal interfernce; they can also be caused by the leached-out monomer from dental resin materials. Whether the cytotoxicity of resin monomer is responsible for cell damage, it still remains a controversial issue, with most of the current studies claiming that the concentration level of the leached-out monomer determines the extent of its impacts on the cells.
Many studies have designed barrier tests involving different concentration levels of monomer released through dentin to the pulp chamber. The barrier materials used in the experiment are mostly human dentin or animal dentin. Due to the large variation of dentin structure and difficulty in collecting human teeth, efforts have been made to find substitutes for human dentin. And this study was motivated by such an attempt.
Cytotoxic effects on cells depended on the monomer concentration and also depends on the kind of cells. In this study, two kinds of cells (human dental pulp cell, NIH-3T3 cell line) were used for cytotoxic tests to evaluate the reaction from two kinds of cells.
Objective:
This study aimed to compare the leached-out concentration of HEMA monomer through dentin with that through polyurethane foam disc(Polyurethane foam, 40-pfc, PS, Sawbones®, Washington, DC, USA). It also aimed to evaluate the effects of cytotoxicity of HEMA and Bis-GMA on human dental pulp cells (HDPC) and NIH-3T3 cells.
Methods:
(1) Extracted human molars were collected. Human dentin and Sawbones® discs (Solid Rigid Polyurethane Foam 40 pcf Density) were prepared in two thicknesses, 0.5 mm and 1.5 mm. Two kinds of dentin bonding agent (DBA), Single Bond Universal Adhesive (SBU, 3M ESPE) as self-etch system and Adper™ Single Bond 2 (ASB, 3M ESPE) as total-etch system, were used and photo-cured according to the manufacturer instructions. The leached-out concentration of HEMA through the dentin and Sawbone® (after 1hr, 4hr, 8hr, 12hr, 24hr, 48hr, 72hr) was quantified by high performance liquid chromatography (HPLC).
(2) The effect of cytotoxicity of the monomer on two kinds of cells was evaluated by MTT assay and Alamar blue assay. The cell death rate was evaluated by activity assay of Caspase-3 and LDH assay(1st, 4th, 7th day). Immunofluorescence stain was used to observe cell morphological changes during the first day.

Results:
(1) With the same DBA, there was no significant difference between 0.5mm dentin disc and 0.5mm PS disc in the concentration of HEMA monomer.
(2) The results of cytotoxicity tests showed that the cell viability was related with monomer concentration. As monomer concentration rose, the cell viability declined.
(3) The results of cytotoxicity tests during the first day showed that high monomer concentration (HEMA 25mM, HEMA 25mM+Bis-GMA 2.5mM) induced high LDH released into the medium. On the other hand, low monomer concentration (HEMA 1.25mM, HEMA 1.25mM+Bis-GMA 0.125mM) induced high performance of Caspase-3 activity, which was related to early apoptosis.
(4) Immunofluorescence stain showed that there was negative correlation between cell density and monomer concentration. Cellular breakdown was also found in the high monomer concentration (HEMA 25 mM, HEMA 25 mM + Bis-GMA 2.5 mM).

Conclusions:
(1) Sawbones® is a stable and predictable substitute for human dentin barrier tests.
(2) Compared with NIH-3t3 cell, HDPC was more susceptible to HEMA.
(3) The leached-out concentration of HEMA from the result of HPLC had cytotoxic impact on both human dental pulp cells (HDPC) and NIH-3T3 cells.

目錄
口試委員審定書 I
誌謝 II
摘要 III
Abstract V
圖目錄 XI
表目錄 XIV
第一章 緒論 1
1.1緒論 1
1.2樹脂填補治療術後敏感問題 1
1.3剩餘牙本質厚度之屏障 1
1.4研究動機 2
1.5研究目的 3
第二章 文獻回顧 4
2.1牙科黏著劑的現行狀況 4
2.2牙科黏著劑的介紹 5
2.3常見的牙科樹脂單體 7
2.3.1甲基丙烯酸甲酯 (Methyl Methacrylate resins, MMA ) 9
2.3.2雙酚醇基丙烯酸甲酯(Bis-GMA resin) 9
2.3.3 Triethylene glycol dimethacrylate, TEGDMA 10
2.3.4甲基丙烯酸羥乙酯（Hydroxyethylmethacrylate, HEMA） 11
2.3.5功能性酸性單體（functional acid monomer） 12
2.4樹脂單體的生物影響 13
2.4.1 樹脂基材生物相容性 13
2.4.2樹脂單體的生物影響 14
2.5生物相容性的檢測 19
2.6牙本質屏障測試 22
2.7 聚氨酯泡棉（Polyurethane Foam, Sawbones®） 24
2.8 總結 26
第三章 實驗材料與實驗方法 27
3.1 實驗材料 27
3.2試片備置 28
3.2.1 牙本質試片備置 28
3.2.2 Polyurethane foam試片備置 28
3.2.3試片實驗分組 29
3.4 實驗細胞及培養環境 29
3.4.1人類牙髓細胞(human dental pulp cell, HDPC)： 29
3.4.2 NIH-3T3 cell(ATCC® CRL-1658™)： 30
3.4.3 細胞培養環境 30
3.4.4細胞實驗分組 31
3.5黏著劑滲透測試 32
3.6 黏著劑溶劑比例測試 32
3.7 MTT assay（細胞存活率分析）： 33
3.8 Alamar blue assay 36
3.9 Caspase-3活性測定 39
3.10 LDH Assay 41
3.11 流式細胞分析儀 43
3.12免疫螢光染色 46
3.13統計分析 47
第四章 實驗結果 48
4.1黏著劑滲透測試 48
4.2牙科黏著劑溶劑比例測試 49
4.3 MTT 50
4.3.1人類牙髓細胞 50
4.3.2 NIH-3T3細胞 53
4.4 Alamar blue 56
4.4.1人類牙髓細胞 56
4.4.2 NIH-3T3 59
4.5 Caspase-3 活性測定 62
4.5.1 人類牙髓細胞 62
4.5.2 NIH-3T3 64
4.6 LDH Assay 67
4.6.1人類牙髓細胞 67
4.6.2 NIH-3T3 70
4.7流式細胞分析 73
4.7.1人類牙髓細胞 73
4.7.2 NIH-3T3 76
4.8免疫螢光染色 79
4.8.1人類牙髓細胞 79
4.8.2 NIH-3T3 80
第五章 實驗討論 82
5.1 滲透單體濃度的測量 82
5.2牙本質與聚氨酯泡棉片 82
5.3 單體滲透過牙本質的濃度 85
5.4 樹脂單體的細胞毒性 86
5.4.1 細胞毒性實驗 87
5.4.2 細胞型態改變 89
5.5未來研究方向 90
第六章 結論 91
6.1黏著劑滲透實驗 91
6.2 細胞實驗 91
參考資料： 92
圖目錄
圖2-1、牙科年度健保申報件數 4
圖2-2、民國104年門診合計醫療費用（點數）統計 4
圖2-3、Methyl Methacrylate resins 9
圖2-4、Bis-GMA 9
圖2-5、TEGDMA 10
圖2-6、HEMA 11
圖2-7、Phosphonic group 12
圖2-8、Phosphate group 12
圖2-9、Carboxylic group 12
圖2-10、一名40歲男性出現接觸性過敏皮膚炎，經測試後證實對MMA過敏(35) 15
圖2-11、膠原蛋白沈積於牙本質小管 16
圖2-12、牙本質小管再礦化 16
圖2-13、牙本質橋(37) 17
圖2-14、缺損的牙本質母細胞層(37) 18
圖2-15、階段性生物相容性檢測(35) 20
圖2-16、牙本質小管孔徑在靠近牙髓腔處（a）較寬；遠離處（b）較窄 22
圖2-17、電子顯微鏡下的人類牙本質（Ａ）以及牛的牙本質（Ｂ）（bar:20µm）(52) 23
圖2-18、電子顯微鏡下，40-pcf 聚氨酯泡綿片的影像(58) 25
(50X, 500Ｘ) 25
圖3-1、 為Sawbone® Biomechanical test materials, 40 PCF block 27
圖3-2、ATCC官方網站所提供的NIH-3T3細胞圖片 30
圖3-3、MTT反應示意圖 33
圖3-4、Alamar blue assay 反應示意圖 36
圖3-5、Human Caspase-3 Instant ELISA™ Kit 原理 39
圖3-6、Cytotoxicity Detection Kit (LDH)原理 41
圖3-7、流式細胞分析儀BD LSRFortessa™ （官網圖片） 45
圖4-1、HPLC測得的HEMA濃度 48
圖4-2、SBU(上)、ASB（下）在空氣中的重量改變(g) 49
圖4-3、人類牙髓細胞MTT assay 結果（第一天） 50
圖4-4、人類牙髓細胞MTT assay 結果（第四天） 51
圖4-5、人類牙髓細胞MTT assay 結果（第七天） 52
圖4-6、NIH-3T3 細胞MTT assay 結果（第一天） 53
圖4-7、NIH-3T3 細胞MTT assay 結果（第四天） 54
圖4-8、NIH-3T3 細胞MTT assay 結果（第七天） 55
圖4-9、人類牙髓細胞細胞Alamar blue assay 結果（第一天） 56
圖4-10、人類牙髓細胞細胞Alamar blue assay 結果（第四天） 57
圖4-11、人類牙髓細胞細胞Alamar blue assay 結果（第七天） 58
圖4-12、NIH-3T3 細胞Alamar blue assay 結果（第一天） 59
圖4-13、NIH-3T3 細胞Alamar blue assay 結果（第四天） 60
圖4-14、NIH-3T3 細胞Alamar blue assay 結果（第七天） 61
圖4-15、人類牙髓細胞Caspase-3 assay 結果（第一天） 62
圖4-16、人類牙髓細胞Caspase-3 assay 結果（第四天） 63
圖4-17、人類牙髓細胞Caspase-3 assay 結果（第七天） 63
圖4-18、NIH-3T3細胞Caspase-3 assay 結果（第一天） 64
圖4-19、NIH-3T3細胞Caspase-3 assay 結果（第四天） 65
圖4-20、NIH-3T3細胞Caspase-3 assay 結果（第七天） 66
圖4-21、人類牙髓細胞LDH assay 結果（第一天） 67
圖4-22、人類牙髓細胞LDH assay 結果（第四天） 68
圖4-23、人類牙髓細胞LDH assay 結果（第七天） 69
圖4-24、NIH-3T3細胞LDH assay 結果（第一天） 70
圖4-25、NIH-3T3細胞LDH assay 結果（第四天） 71
圖4-26、NIH-3T3細胞LDH assay 結果（第七天） 72
圖4-27、人類牙髓細胞流式細胞分析儀結果（第一天） 73
圖4-28、人類牙髓細胞流式細胞分析儀結果（第四天） 74
圖4-29、人類牙髓細胞流式細胞分析儀結果（第七天） 75
圖4-30、NIH-3T3流式細胞分析儀結果（第一天） 76
圖4-31、NIH-3T3流式細胞分析儀結果（第四天） 77
圖4-32、NIH-3T3流式細胞分析儀結果（第七天） 78
圖4-33、螢光顯微鏡下的人類牙髓細胞（200X, 30min） 79
圖4-34、螢光顯微鏡下的人類牙髓細胞（200X, 4H） 79
圖4-35、螢光顯微鏡下的人類牙髓細胞（200X, 24H） 79
圖4-36、螢光顯微鏡下的NIH-3T3（200X, 30min） 80
圖4-37、螢光顯微鏡下的NIH-3T3（200X, 4H） 80
圖4-38、螢光顯微鏡下的NIH-3T3（200X, 24H） 81
圖5-1、Bis-GMA與HEMA標準濃度線性關係 82
圖5-2、兩種試片的滲透濃度相對關係 84
圖5-3、析出液中的單體濃度 86

表目錄
表2-1、常見的生物相容性檢測方式 (35) 19
表2-2、Rigid polyurethane foam的型態分類。 24
表2-3、40-pcf聚氨酯泡綿物理性質 25

1.Öztürk B, Üşümez A, Öztürk AN, Ozer F. In vitro assessment of temperature change in the pulp chamber during cavity preparation. The Journal of prosthetic dentistry. 2004;91(5):436-40.
2.Murray PE, About I, Lumley PJ, Franquin JC, Remusat M, Smith AJ. Cavity remaining dentin thickness and pulpal activity. American journal of dentistry. 2002;15(1):41-6.
3.Swift EJ, Trope M, Ritter AV. Vital pulp therapy for the mature tooth – can it work? Endodontic Topics. 2003;5(1):49-56.
4.衛福部中央健康保險署 醫療給付統計資料.
5.衛生福利部統計處 民國104年門診合計(西、牙、中醫門診且包括急診)醫療費用(點數)統計------按性別及年齡別分.
6.Jacobsen T, Söderholm K-J. Some effects of water on dentin bonding. Dental Materials. 1995;11(2):132-6.
7.Kanca J, 3rd. Resin bonding to wet substrate. 1. Bonding to dentin. Quintessence international (Berlin, Germany : 1985). 1992;23(1):39-41.
8.Malacarne J, Carvalho RM, de Goes MF, Svizero N, Pashley DH, Tay FR, et al. Water sorption/solubility of dental adhesive resins. Dental Materials. 2006;22(10):973-80.
9.Liu Y, Tjaderhane L, Breschi L, Mazzoni A, Li N, Mao J, et al. Limitations in bonding to dentin and experimental strategies to prevent bond degradation. Journal of dental research. 2011;90(8):953-68.
10.Peutzfeldt A. Review: Resin Composite in Dentistry : the Monomer Systems. European Journal of Oral Science. 1997;105:97-116.
11.Bowen RL. Dental filling material comprising vinyl silane treated fused silica and a binder consisting of the reaction product of bis phenol and glycidyl acrylate. US Patent. 1962:3,066,112.
12.Sideridou I, Tserki V, Papanastasiou G. Study of water sorption, solubility and modulus of elasticity of light-cured dimethacrylate-based dental resins. Biomaterials. 2003;24(4):655-65.
13.Prakki A. Water sorption of CH(3)- and CF(3)-Bis-GMA based resins. 2012;20(4):472-7.
14.Asmussen E, Peutzfeldt A. Influence of UEDMA BisGMA and TEGDMA on selected mechanical properties of experimental resin composites. Dental materials : official publication of the Academy of Dental Materials. 1998;14(1):51-6.
15.Geurtsen W. Biocompatibility of resin-modified filling materials. Critical reviews in oral biology and medicine : an official publication of the American Association of Oral Biologists. 2000;11(3):333-55.
16.Chirila TV. An overview of the development of artificial corneas with porous skirts and the use of PHEMA for such an application. Biomaterials. 2001;22(24):3311-7.
17.Pashley EL, Zhang Y, Lockwood PE, Rueggeberg FA, Pashley DH. Effects of HEMA on water evaporation from water-HEMA mixtures. Dental materials : official publication of the Academy of Dental Materials. 1998;14(1):6-10.
18.Kazantsev OA, Shirshin KV, Sivokhin AP, Tel''nov SV, Zhiganov IV, Kuznetsov AE, et al. Hydrolysis of 2-Hydroxyethyl Methacrylate in Concentrated Aqueous Solutions. Russian Journal of Applied Chemistry. 2003;76(8):1296-8.
19.Nakabayashi N, Takarada K. Effect of HEMA on bonding to dentin. Dental materials : official publication of the Academy of Dental Materials. 1992;8(2):125-30.
20.Nakaoki Y, Nikaido T, Pereira PN, Inokoshi S, Tagami J. Dimensional changes of demineralized dentin treated with HEMA primers. Dental materials : official publication of the Academy of Dental Materials. 2000;16(6):441-6.
21.Burrow MF, Inokoshi S, Tagami J. Water sorption of several bonding resins. American journal of dentistry. 1999;12(6):295-8.
22.Van Landuyt KL, De Munck J, Snauwaert J, Coutinho E, Poitevin A, Yoshida Y, et al. Monomer-solvent phase separation in one-step self-etch adhesives. Journal of dental research. 2005;84(2):183-8.
23.Feitosa VP, Sauro S, Ogliari FA, Stansbury JW, Carpenter GH, Watson TF, et al. The role of spacer carbon chain in acidic functional monomers on the physicochemical properties of self-etch dental adhesives. J Dent. 2014;42(5):565-74.
24.Chang JC. Amalgam repair with a 4-META resin. The Journal of prosthetic dentistry. 2004;92(5):506-7.
25.Hume WR, Gerzina TM. Bioavailability of Components of Resin-Based Materials Which Are Applied To Teeth. Critical Reviews in Oral Biology & Medicine. 1996;7(2):172-9.
26.Geurtsen W. Substances released from dental resin composites and glass ionomer cements. European Journal of Oral Sciences. 1998;106(2p2):687-95.
27.Gopferich A. Mechanisms of polymer degradation and erosion. Biomaterials. 1996;17(2):103-14.
28.C. Kerezoudia, C. Gogosb, V. Samanidouc, D. Tziafasb, G. Palaghiasa. Evaluation of monomer leaching from a resin cement through dentin by a novel model. dental materials. 2016;32:e297-e305.
29.Peumans M, Van Meerbeek B, Lambrechts P, Vanherle G, Quirynen M. The influence of direct composite additions for the correction of tooth form and/or position on periodontal health. A retrospective study. Journal of periodontology. 1998;69(4):422-7.
30.Kanerva L, Estlander T, Jolanki R. Allergic contact dermatitis from dental composite resins due to aromatic epoxy acrylates and aliphatic acrylates. Contact dermatitis. 1989;20(3):201-11.
31.Kanerva L, Henriks-Eckerman M-L, Estlander T, Jolanki R, Tarvainen K. Occupational allergic contact dermatitis and composition of acrylates in dentin bonding systems. Journal of the European Academy of Dermatology and Venereology. 1994;3(2):157-68.
32.Lyapina MG, Dencheva M, Krasteva-Panova A, Tzekova-Yaneva M, Deliverska M, Kisselova-Yaneva A. Concomitant sensitization to glutaraldehyde and methacrylic monomers among dentists and their patients. Medycyna Pracy. 2016;67(3):311-20.
33.Hensten-Pettersen A, Jacobsen N. Perceived side effects of biomaterials in prosthetic dentistry. The Journal of prosthetic dentistry. 1991;65(1):138-44.
34.Lui JL. Hypersensitivity to a temporary crown and bridge material. J Dent. 1979;7(1):22-4.
35.Gottfried Schmalz DA-B. Biocompatibilty of Dental Materials. 2009.
36.Ten Cate’s Oral Histology Development, Structure, and Function (Sixth Edition).
37.Alves GSLLNL-JnAMJ, . Direct Pulp Capping with a Dentin Bonding System
in Human Teeth: A Clinical and Histological Evaluation. Operative Dentistry. 2006;31(3):297-307.
38.Accorinte MdLR, Loguercio AD, Reis A, Muench A, de Araújo VC. Adverse effects of human pulps after direct pulp capping with the different components from a total-etch, three-step adhesive system. Dental Materials. 2005;21(7):599-607.
39.Eun-Cheol Kim1 HP, Sang-Im Lee3 and Sun-Young Kim2. Effect of the Acidic Dental Resin Monomer 10-methacryloyloxydecyl Dihydrogen Phosphate on Odontoblastic Differentiation of Human Dental Pulp Cells. Basic & Clinical Pharmacology & Toxicology. 2015;117:340-50.
40.C.A. de Souza Costaa, M.A. Vaertenb, C.A. Edwardsc, C.T. Hanksb,. Cytotoxic effects of current dental adhesive systems on immortalized odontoblast cell line MDPC-23. Dental Materials 1999;15:434-41.
41.Inamitsu H, Okamoto K, Sakai E, Nishishita K, Murata H, Tsukuba T. The dental resin monomers HEMA and TEGDMA have inhibitory effects on osteoclast differentiation with low cytotoxicity. Journal of applied toxicology : JAT. 2017.
42.Biological evaluation of medical devices —
Part 5:
Tests for in vitro cytotoxicity. internation standard ISO 10993-5. 2009.
43.Schmalz G. Concepts in biocompatibility testing of dental restorative materials. Clinical oral investigations. 1997;1(4):154-62.
44.Y. YASUDA* HI, H. MAEDA†, A. AKAMINE†, J. E. NO ̈ R‡ & T. SAITO*. Cytotoxicity of one-step dentin-bonding agents toward dental pulp and odontoblast-like cells. Journal of Oral Rehabilitation. 2008;35:940-6.
45.Lee JH, Jun SK, Moon HJ, Lee HH. Cytotoxicity and proinflammatory cytokine expression induced by interim resin materials in primary cultured human dental pulp cells. The Journal of prosthetic dentistry. 2017.
46.Ninghua Konga TJ, ∗, Zongcan Zhouc, Juanling Fuc. Cytotoxicity of polymerized resin cements on human dental pulp cells in vitro
. dental materials. 2009;25:1371-5.
47.Ruey-Song Chena b, Ching-Cheng Liua, Wan-Yu Tsenga, Jiiang-Huei Jenga, Chun-Pin Lina,*. Cytotoxicity of three dentin bonding agents on human dental pulp cells. Journal of Dentistry. 2003;31:223–9.
48.Issa Y, Watts DC, Brunton PA, Waters CM, Duxbury AJ. Resin composite monomers alter MTT and LDH activity of human gingival fibroblasts in vitro. Dental materials : official publication of the Academy of Dental Materials. 2004;20(1):12-20.
49.Garberoglio R, Brännström M. Scanning electron microscopic investigation of human dentinal tubules. Archives of Oral Biology. 1976;21(6):355-62.
50.Schmalz G. Agar overlay method. International endodontic journal. 1988;21(2):59-66.
51.Schmalz G, Arenholt-Bindslev D, Hiller K-A, Schweikl H. Epithelium-fibroblast co-culture for assessing mucosal irritancy of metals used in dentistry. European Journal of Oral Sciences. 1997;105(1):86-91.
52.Schilke R, Lisson JA, Bauss O, Geurtsen W. Comparison of the number and diameter of dentinal tubules in human and bovine dentine by scanning electron microscopic investigation. Arch Oral Biol. 2000;45(5):355-61.
53.ASTM F 1839 Standard - Standard Specification for Rigid Polyurethane Foam for Use as a Standard Material for Testing Orthopaedic Devices and Instruments.
54.Influence of Drilling Speed on Stability of Tapered Dental Implants: An Ex Vivo Experimental Study. The International Journal of Oral & Maxillofacial Implants.31:795-8.
55.Dae-Seung Kima W-JL, Soon-Chul Choib, Sam-Sun Leeb, Min-Suk Heob, Kyung-Hoe Huhb, Tae-Il Kimc,∗∗, Won-Jin Yib,∗. Comparison of dental implant stabilities by impact response and resonance frequencies using artificial bone. 2014;36:715-20.
56.Duck-Rae Kim D, MSD,a Young-Jun Lim, DDS, MSD, PhD,b Myung-Joo Kim, DDS, MS, PhD,c Ho-Beom Kwon, DDS, MSD, PhD,c and, Sung-Hun Kim D, PhD,b Self-cutting blades and their influence on primary stability of tapered dental implants in a simulated low-density bone model: a laboratory study. 2011.112:573-80.
57.Bryan W. Cunningham mS, 1 nianBin hu, m.D.,1 CanDaCe m. Zorn, B.S. eng.,1 anD Paul C. mCafee, m.D.2. Comparative fixation methods of cervical disc arthroplasty versus conventional methods of anterior cervical arthrodesis: serration, teeth, keels, or screws? J Neurosurg Spine 2010;12:214-20.
58.Mi-Joo Kim K-NKaK-MK. Cytotoxicity Test of One-Step Self-Etching Bonding Agents by Standardized Dentin Barrier Test Using Polyurethane Discs. Materials. 2014;7:85-96.
59.https://www.atcc.org/products/all/CRL-1658.aspx.
60.al LRPAe. Identification and Quantification of Monomers Released from Dental Composites Using HPLC. BRAZILIAN ARCHIVES OF BIOLOGY AND TECHNOLOGY. 2009;52(4):855-62.
61.Gerlier D, Thomasset N. Use of MTT colorimetric assay to measure cell activation. Journal of Immunological Methods. 1986;94(1):57-63.
62.Mie-leng Tang1 J-FoN, Michaël Sadoun1 and N Dorin Ruse2*. HPLC Analysis of Monomer Release from Conventionally and High
Temperature High-Pressure Polymerised Urethane Dimethacrylate
Intended for Biomedical Applications. Chromatograph Separat Techniq. 2014:http://dx.doi.org/10.4172/2157-7064.1000227.
63.Bor-Shiunn Lee a Y-DJb, Guo-Shiang Huang c, Chien-Hsun Huang d, Han-Yi Chou a, Juo-Song Wang b, Wan-Yu Tseng b,*. Effect of dentin bonding agent diffusing through dentin slices on the reactive oxygen species production and apoptosis of pulpal cells. Journal of the Formosan Medical Association 2015;114:339-46.
64.Paranjpe A, Bordador LC, Wang MY, Hume WR, Jewett A. Resin monomer 2-hydroxyethyl methacrylate (HEMA) is a potent inducer of apoptotic cell death in human and mouse cells. Journal of dental research. 2005;84(2):172-7.
65.Williams DW, Wu H, Oh JE, Fakhar C, Kang MK, Shin KH, et al. HEMA inhibits migration of dental pulp stem cells. Journal of endodontics. 2013;39(9):1156-60.
66.Hebling J, Giro EM, Costa CA. Biocompatibility of an adhesive system applied to exposed human dental pulp. Journal of endodontics. 1999;25(10):676-82.
67.Rakich DR, Wataha JC, Lefebvre CA, Weller RN. Effects of dentin bonding agents on macrophage mitochondrial activity. Journal of endodontics. 1998;24(8):528-33.
68.Yano J. Apoptosis and survivability of human dental pulp cells under exposure to. 2011;19(3):218-22.
69.Mantellini MG, Botero TM, Yaman P, Dennison JB, Hanks CT, Nor JE. Adhesive resin induces apoptosis and cell-cycle arrest of pulp cells. Journal of dental research. 2003;82(8):592-6.
70.Jun SK, Mahapatra C, Lee HH, Kim HW, Lee JH. Biological Effects of Provisional Resin Materials on Human Dental Pulp Stem Cells. Oper Dent. 2017;42(2):E81-e92.
71.Villa PG, Henzel WJ, Sensenbrenner M, Henderson CE, Pettmann B. Calpain inhibitors, but not caspase inhibitors, prevent actin proteolysis and DNA fragmentation during apoptosis. Journal of cell science. 1998;111 ( Pt 6):713-22.
72.Mills JC, Stone NL, Pittman RN. Extranuclear Apoptosis: The Role of the Cytoplasm in the Execution Phase. The Journal of Cell Biology. 1999;146(4):703-8.
73.Chen Q, Lu G, Wang Y, Xu Y, Zheng Y, Yan L, et al. Cytoskeleton disorganization during apoptosis induced by curcumin in A549 lung adenocarcinoma cells. Planta medica. 2009;75(8):808-13.