根尖充填劑之生物學效應

The biological effects of the root-end filling materials

黃翠賢(Tsui-Hsien Huang)

指導教授 ： 周明勇

中山醫學大學/醫學院/醫學研究所/博士(2006年)

https://doi.org/10.6834/CSMU.2006.00065

摘要

臨床上常用之根尖充填材料有以氫氧化鈣為基底(Life)、氧化鋅丁香油為基底(Super EBA)和以三氧礦化物(MTA)為基底之充填劑。此等材料於修復根尖組織上無系列性之研究與報告，且無法明顯得知各種不同材料間之差異性，本研究之目的乃對於哺乳類細胞與動物之生物學效應上作探討，以了解ㄧ、於活體外(in vitro)和活體內(in vivo)，不同根尖充填材料是否對於細胞或組織具有生物相容性；二、當根尖充填材料作用於細胞或組織後，細胞或組織是否有凋亡現象；三、其可能於細胞內之訊息傳遞機轉。四、於活體內植入材料後觀察其組織之變化與血液中生化值之變化，以清楚了解根尖充填材料之生物學上特性。
研究方法採用活體外研究與活體內研究，活體外研究則以MTT分析細胞毒性，顯微鏡觀察細胞型態變化，西方墨點分析蛋白訊息傳遞之因子，以ELISA分析細胞素之產生，以流式細胞儀觀測Sub G1 DNA之含量變化。而於活體試驗中則將根尖充填材料植入於Balb/c小鼠體內，以X 光觀察是否有鈣化組織之生成，以切片評估植入周圍組織之變化，以血液之生化分析比較鹼性磷酸酶、乳酸去氫酶，鈣離子與磷離子之含量，並分析IL4、IL10和 interferon garma之含量。
結果顯示，於試管內之實驗研究：MTA 之生物相容性優於氫氧化鈣為基底(Life)、氧化鋅丁香油酚等為基底(Super EBA)；細胞型態變化未見有細胞凋亡特徵；蛋白訊息傳遞之因子只於MTA組出現ERK Kinase，其他如Capcase 3, p53, JNK kinase 則未出現。以ELISA分析細胞素IL2之表現。結果發現三種根尖充填劑之細胞素IL2之表現較控制組高(P<0.01)。MTA組根尖充填劑組之細胞素IL4之表現最高(P<0.01)。MTA組有明顯較高的IL10，而於Life 和 Super EBA 組之IL10則較低。流式細胞儀觀測Sub G1 DNA之含量於浸泡一天或七天的根尖充填劑作用48小時後，只有於Life 組之SubG1期有較控制組高之比值，代表細胞有傷害死亡，而於其他Super EBA和MTA組則SubG1期之DNA之比值與控制組間沒有差異性。
活體之研究結果顯示，經X 光觀察是否有鈣化組織之生成，結果發現只有Life組於6週與8週X光上有放射不透光之區域出現。組織切片發現Life根尖充填材料被膜包覆，無發現有發炎細胞之浸潤。Super EBA組之組織變化，發現Super EBA根尖充填材料6 週與8週之組織切片，組織與Super EBA根尖充填材料剝離，周圍組織生長不良。血液之生化分析，所有實驗組與控制組之ALP、Ca、P均無差異，結果代表植入之根尖充填材料不影響ALP、Ca、P於血液中之表現。MTA和Super EBA組第六週植入後LDH值之表現均較控制組高，而Life組則較控制組低。細胞素之變化則顯示根尖充填材料植入於老鼠後第四週、第八週由採血液分析IL2、IL4和INF－γ之結果顯示，只有IL4表現出統計學上之差異(p=0.000<0.05)，但第四週其平均值均小於控制組，而第八週則只有Super EBA組大於控制組。
結語：根尖充填材料以MTA 對於細胞或是組織均呈現良好之生物相容性，MTA應是一臨床上根管治療時可優先考慮使用之材料。

關鍵字

根尖充填劑

並列摘要

Three types of root end filling materials used in clinical are calcium hydroxide base (Life), zinc oxide eugenol base (Super EBA) and mineral trioxide aggregate (MTA). The past study in comparing root end filling materials does not consistent. The purpose of present study are to compare above root canal filling materials biologic effects in vitro and in vivo.
In vitro biocompatibility study, the Life, Super EBA and MTA root end filling materials were mixed according to the manufacturer’s recommendations and placed into the bottom of 48 well tissue culture plates to achieve a thickness of about 5 mm. The surface area of the test material exposed was 64 mm2. With different time period, the extract were collected and stored at 4℃ for use. In vitro study, MTT assay was used to detect the cytotoxicity of human osteosarcoma cell (U2OS ). The morphology of the cell after interaction with the extracts was recorded. To detect the intracellular protein change, western blot analysis was applied to analyze MAPK kinase signal transduction pathway. By ELISA method, the cytokine was analyze on U2OS cell after adding the extract of root end filling materials. The flow cytometry was to detect the DNA content of U2OS cell.
In vivo study, the root end fillng material was implanted into the Balb/c rat. The implanted area was observed by X ray and biopsy the area to observe the tissue change under microscope. The blood biochemical properties was used to analysis such as ALP, LDH, Ca ion and P ion content.
The result of the present study showed the biocompatibility as MTA>Life >Super EBA. There is no apoptic body found in U2OS cell morphology observation. The ERK kinase was strong singal express in U2OS cell treated with MTA. The Capase 3, p53 and JNK kinase did not appeare in treated cell. The IL2 cytokine showed significant difference in all experiment groups. In MTA group, IL4 and IL10 were more prominent than other root end filling materials. SUbG1 DNA content was expressed strongest in Life group. The Super EBA and MTA did not showed significant difference (p>0.05) from the control group in SUbG1 DNA content.
The in vivo study showed radiopaque was found in Life group after six or eight weeks implantation. The histologic section tissue was separated with the Super EBA material was found after six and eight weeks implantation. Biochemistry analysis of implanted animal blood showed no significant difference in ALP, Ca ion and P ion content was found. Only LDH showed higher level in MTA and Super EBA group than that of the control group. IL4 showed statistical difference in 4th week in all implanted group.
In conlcusion: the MTA root end filling material is a good biocompatible material and can be applied in clinical.

並列關鍵字

mineral trioxide aggregate (MTA)

參考文獻

2. Oguntebi B, Clark A, Wilson J. Pulp capping with Biograss and autologous deminealized dentin inminiature swine. J Dent Res, 72:484-489, 1993.

4. West TL, Brustein DD. Freeze-dried bone and coralline implants compared in the dog. J Periodontol , 56: 348-351, 1985.

5. Hench LL, Ethridge EC, Biomaterials, an interfacial approach. New York: Academic Press,1982.

6. Rawlings CE, Wilkins RH. Hanker JS. Georgiade NG, Harrelson JM. Evaluation in cats of a new material for cranioplasty: A composite of plaster of Paris and hydroxylapatite. J Neurosurg, 69:269-275, 1988.

7. Tamburic SD, Vuleta GM, Ognjanovic JM. In vitro release of calcium and hydroxyl ions from two types of calcium hydroxide preparation. Int Endod J, 26:125-130, 1993.