- 學位論文
單瓶式牙科黏著劑透過人類牙本質及聚氨酯泡棉屏障實驗對單體濃度與生物相容性之探討
The leached-out monomer concentration and biocompatibility of one-bottled adhesives by barrier test using human dentin and polyurethane
摘要
前言 現今的牙科治療中廣泛運用牙科黏著劑，舉凡樹脂填補、嵌體或貼片治療皆有使用。但在臨床上副作用常出現術後敏感的情形，在靠近牙髓腔的位置可能會引發牙髓發炎甚至導致壞死的可能。其原因可能包含未去除完全的受感染齒質、未聚合樹脂單體的細胞的毒性、樹脂聚合過程中收縮的力量以及咬合干擾。牙科使用的樹脂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影響的毒性程度。 為了測量透過牙本質釋放到牙髓腔的單體濃度，目前許多研究設計各種屏障實驗，但實驗結果往往受制於屏障材料的條件，目前實驗多以人類或動物的牙齒牙本質作為屏障材料，但其通透度會隨生物體本身的年齡、飲食習慣、齲齒或個體間的差異等因素而有所不同，進而影響實驗結果。本實驗研究動機即在尋找一種穩定且被認可的材料，以取代牙本質作為標準化的屏障實驗材料。 10-MDP酸性單體在現今臨床應用十分廣泛，尤其常見於自酸蝕牙科黏著系統與自聚合/光聚合樹脂黏合劑；但是針對10-MDP單體對人體牙髓細胞反應的研究結果遠少於其他常用樹脂單體。因此，本次實驗第二部分是利用牙本質屏障實驗測量臨床10-MDP釋放出來的濃度，並培養細胞，量測10-MDP單體對牙髓細胞造成的影響。 研究目的 本實驗目的在於比較人類牙本質與聚氨酯泡棉（Polyurethane foam, 40-pcf, PS, Sawbones®, Washington, DC, USA）的HEMA及10-MDP單體滲透濃度，以及牙科黏著劑透過屏障的單體對人類牙髓細胞的影響；另外配置不同濃度的10-MDP單體溶液培養細胞以觀察細胞的反應及變化。 研究方法 一、 單體滲透測試：屏障材料使用0.5 mm及1 mm厚度的牙本質及聚氨酯泡棉，在材料上依廠商指示塗抹兩種含10-MDP單體的牙科黏著劑（3M Single Bond Universal Adhesive, Ivoclar Tetric-N Bond Universal Adhesive）並照光聚合後，收集第15mins、30mins、1hr、8hr、24hr、48hr共六個時間點的滲透液，並以高效能液相層析儀（HPLC）分析滲透液之單體濃度。 二、 細胞活性測試：利用0.5 mm及1 mm厚度的牙本質及聚氨酯泡棉試片，分別塗抹兩種牙科黏著劑，使其滲透進細胞培養液中，並在第一（短期）、第四（中期）、第七（長期）天共三個時間點，利用Alamar Blue assay觀察牙髓細胞存活率。 三、 針對現今應用較廣泛的10-MDP酸性單體，參考單體滲透測試中透過人類牙本質的濃度配置溶液來培養細胞，利用Alamar Blue assay、LDH assay以及免疫螢光染色觀察此單體對細胞活性的影響。 研究結果 一、 聚氨酯泡棉在與牙本質在相同的厚度下滲出的HEMA濃度無顯著差異。 二、 不論使用1 mm或0.5 mm的聚氨酯泡棉與牙本質，聚合完成的牙科黏著劑滲透過屏障的物質對細胞活性的影響在1、4、7天皆無顯著差異。 三、 在Alamar Blue實驗中觀察濃度400 μM的10-MDP組別第4、7天結果顯示對細胞具有抑制生長的影響；在LDH實驗中則是觀察到濃度大於50 μM的組別，在第1、4天隨著10-MDP濃度提升而造成LDH明顯上升，到第7天LDH則無顯著差異。 結論 以本實驗HEMA滲出濃度結果，以及屏障實驗對細胞影響觀察，推測聚氨酯泡棉可以在屏障實驗中作為牙本質的替代品；觀察本實驗結果10-MDP濃度400 μM到第7天仍會對細胞有顯著的生長影響，LDH數值在第1、4天較控制組顯著，但是到第7天則沒有顯著差異。
並列摘要
Introduction Dental adhesives are widely used in the procedure of dental treatment, such as composite resin filling, inlay/onlay or veneer treatment, etc. The side effect such as postoperative sensitivity are often reported. If the position of cavity/restorations are close to the pulp, it may cause pulpitis even pulp tissue necrosis. The reasons of side effects may include incomplete removal of infected dentin, cellular toxicity of unpolymerized resin monomers, force of shrinkage during resin polymerization, and occlusal disturbance. Whether the cytotoxicity of resin monomer is reasonable for pulp cell damage, numbers of studies approved that the monomer concentration determines the degree of toxicity to cells. In order to measure the concentration of monomers eluted into the pulp tissue through dentin, various barrier designs was investigated in many experiments. But the results are often limited by the variations of the barrier material. Human or animal dentin is usually used as the barrier material in most experiments. Its permeability will vary with factors such as the age of the organism itself, dietary habits, dental caries or variations between individuals, which in turn affects the experimental results. One of the aims of the study was to find a stable and recognized material to replace dentin as a standardized barrier test material. 10-MDP monomers are widely used in clinical practice nowadays, especially in self-etching dental adhesive systems and dual cure resin cements. However, there are few studies on the influence of 10-MDP to human dental pulp cells compared with other resin monomers. Therefore, the second part of this study was to use the exuded concentration of 10-MDP measured from the HPLC results mentioned above. To observe the influence of 10-MDP to dental pulp cells. Research goal 1. Measurement of exuded monomer concentration: Dentin and polyurethane foam with thickness of 0.5 mm and 1 mm as barrier were used. Two dental adhesives (3M Single Bond Universal Adhesive, Ivoclar Tetric-N Bond Universal Adhesive) were applied on the material according to the manufacturer's instructions. Eluted monomers were collected at six time points of 15mins, 30mins, 1hr, 8hr, 24hr, and 48hr, and the concentration was measured by high performance liquid chromatography (HPLC). 2. Cell viability test: The test samples were described above. After sample preparation, it was placed on the insert of 24-well transwell, and then cultured with DPSC. The Alamar Blue assay was used to observe the cell viability at Day 1, Day 4 and Day 7. 3. To investigate the cytotoxicity of 10-MDP, 25 μM, 50 μM, 100 μM, 200 μM, 400 μM of 10-MDP solution were cultured with DPSC. Alamar Blue assay, LDH assay and immunofluorescence staining were utilize to observe the influences of this monomer to dental pulp cells. Result 1. There was no significant difference in the concentration of HEMA exuded from polyurethane foam and dentin at the same thickness. 2. Whether using polyurethane foam or dentin at thickness of 1 mm or 0.5 mm, the effects of the polymerized dental adhesive permeating the barrier to cell viability were no significant difference at 1, 4, and 7 days. 3. In the Alamar Blue experiment, the concentration of 400 μM 10-MDP was cytotoxic on cell growth on the 4th and 7th day. The higher LDH was measured when the 10-MDP concentration was higher than 50 μM on day 1 and day 4. However, there was no significant difference on day 7. Conclusion Based on the results of the HEMA exuded concentration in this experiment and the effect of bonding agents through the barrier test to cells. It is reliable that polyurethane foam can be used as a substitute for dentin in dentin barrier test. It was observed that 10-MDP concentration of 400 μM still had significant effect on the growth of cells on the 7th day. The LDH of 400 μM 10-MDP was significantly higher than control group on the 1st and 4th days, but there was no significant difference on the 7th day.