The Leached-Out Concentration of Monomer through the Polyurethane Foam and the Cytotoxicity Tests of HEMA and Bis-GMA

• Main Authors: Ting-San Liang, 梁庭珊
• Other Authors: 曾琬瑜
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/v88p83

碩士 === 國立臺灣大學 === 臨床牙醫學研究所 === 105 === 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.