- 學位論文
牙科黏著劑於聚氨酯泡棉之單體通透濃度及細胞毒性測試
The Leached-Out Concentration of Monomer through the Polyurethane Foam and the Cytotoxicity Tests of HEMA and Bis-GMA
摘要
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。
並列摘要
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.