研發新型含幾丁聚醣材料應用於乳牙根管治療之封填─細胞毒性與抗菌分析

Abstract

此研究乃利用UV光接枝技術，合成以幾丁聚醣為基底的高分子聚合材料，應用於乳牙根管治療中，形成一種具有抗菌與良好生物相容性的乳牙根管治療封填材料。 本研究包括四個部分，第一個部分為使用兩種低分子量的幾丁聚醣粉末(Chitosan, Mw=15kDa, 100kDa)，經不同時間研磨後製備出次微米化的幾丁聚醣樣品，並進行表面形貌與粒徑分析，發現粒徑大小於研磨60分鐘開始有大幅的下降。第二部分為根據不同分子量及研磨時間之幾丁聚醣樣品，依重量比例分別與甲基丙烯酸羥乙酯(2-Hydroxyethyl methacrylate, HEMA)、N-異丙基丙烯醯胺(N-isopropylacrylamide, NIPAAm)進行UV光接枝聚合，製成含幾丁聚醣之根管封填材料，並分析樣品接枝聚合後的物化特性。於SEM觀察下發現，幾丁聚醣研磨時間越長，與HEMA單體聚合後樣品會呈現沒有什麼孔隙的片狀；而研磨後粒徑較小的幾丁聚醣較容易被包覆在NIPAAm的結構網絡之內。在膨潤率測試的部分，發現樣品皆在測試1小時後為最飽和狀態。於黏度測試部分，發現研磨10分鐘的低分子量(Mw=15kDa)幾丁聚醣與HEMA聚合之樣品組別，無論是25℃、37℃溫度下，皆可以得到較高的黏度；高分子量(Mw=100kDa)幾丁聚醣，於研磨時間達30分鐘以上的樣品組別，在25℃、37℃溫度下，皆顯示較高的黏度。在第三部分為進行各組樣品之體外細胞毒性，培養老鼠纖維母細胞(NIH-3T3)，觀察不同組別之樣品與市售材料的生物相容性，以細胞存活率(cell viability)來表示試驗結果，由觀察結果發現，本實驗合成之乳牙根管封填材料基質，皆沒有細胞毒性的產生。第四部分為培養糞腸球菌(Enterococcus faecalis, ATCC 29212)，以Kirby-Bauer試驗進行抑菌效果的觀察，測試不同樣品以及市售材料的抗菌能力，結果顯示chitosan-pHEMA，隨著幾丁聚醣研磨時間越長其抗菌效果越好；但chitosan-pNIPAAm，只有在研磨時間0、10、20、30分鐘，有看到抑菌圈而研磨60分鐘後就沒辦法測得，推測有可能是因為經研磨後粒徑變小，幾丁聚醣與NIPAAm聚合程度較佳，以致於具有抗菌能力的幾丁聚醣被包覆在網絡結構之內，而無法達成抗菌的效果。本實驗測試市售材料Vitapex®的抗菌結果則顯示無明顯的抑菌圈出現。 藉由以上的研究結果發現，經改質後的幾丁聚醣樣品，觀察到有抗菌能力、生物相容性、良好的流動性質，具有成為根管治療封填材料的潛力。

The purpose of this study was to develop materials containing chitosan as root canal filling material for pediatric patients. Using UV-radiation grafting techniques to synthesize polymer materials based on chitosan for root canal treatment of deciduous teeth, forming root canal filling materials that are both antibacterial and biocompatible. The study had four parts. First, using two low-molecular-weight chitosan powders (Chitosan, Mw=15kDa, 100kDa) that were ground for different periods to prepare submicron-sized chitosan samples and analyzed for structure and particle size. Second, we used chitosan samples with different molecular weights and grinding times to graft 2-Hydroxyethyl methacrylate (HEMA) and N-isopropylacrylamide (NIPAAm) via UV-graft polymerization to create root canal filling materials, and we analyzed the physical and chemical properties of the samples. The swelling rate test showed that the samples reached their saturation point after one hour of testing. The viscosity test showed that chitosan with a molecular weight of 15kDa ground for 10 minutes and polymerized with HEMA had higher viscosity at both 25℃ and 37℃. Chitosan with a molecular-weight of 100kDa ground for over 30 minutes and polymerized with HEMA showed higher viscosity at both temperatures. In the third part, in vitro cell cytotoxicity was conducted on cultured mouse fibroblast cells (NIH-3T3) to assess the biocompatibility of different groups of samples and commercially available material. The cell viability was used to represent the test results. From the observations, it was found that the synthesized matrix of the dental root canal filling material in this experiment did not exhibit any cytotoxicity. The fourth part, we used Enterococcus faecalis (ATCC 29212) and conducted Kirby-Bauer tests to observe the antibacterial ability of the different samples and commercially available materials. The chitosan-pHEMA filling material had better antibacterial effects with longer chitosan grinding times. However, the chitosan-pNIPAAm filling material only showed antibacterial effects with grinding times of 0, 10, 20, and 30 minutes, but not with above 60 minutes of grinding times. It is speculated that the antibacterial properties of chitosan may be limited to the network-like chitosan-pNIPAAm material, and also due to its smaller particle size after grinding, lead to better polymerization with NIPAAm, making it unable to achieve antibacterial effects. The antibacterial test of the commercially available material Vitapex® in this experiment showed no significant inhibition zone. Based on the above results, it was found that the synthesized materials of chitosan have antibacterial ability, biocompatibility, and good flow properties, which have the potential to become a root canal filling material.