0099-2399/97/2309-0572503.00/0 JOURNALOF ENDODONTICS Copyright © 1997 by The American Association of Endodontists Prmted in U.S.A, VOL. 23, No. 9, SEPTE~eER1997 In Vitro Evaluation of the Cytotoxicity of Two Glass-lonomer Root Canal Sealers Panagiotis Beltes, DDS, PhD, Elisabeth Koulaouzidou, DDS, Ioannis Kolokuris, DDS, PhD, and Alexander H. Kortsaris, PhD The cytotoxicity of two glass-ionomer root canal sealers (Ketac-Endo and Endion) was tested by using an established cell line, BHK21/C13. Under aseptic conditions, the sealers were prepared ac- cording to the manufacturers' directions, and 0.1 ml of each material was placed in petri dishes. After setting for 6 h, the sealers were covered with 20 x 104 cells per dish. The cultures were incu- bated at 37°C for 24 h, 48 h, and 72 h. Cytotoxicity was assessed by a quantitative technique at three observation periods. Endion was highly cytotoxic, causing a significant decrease in cell density. Ketac-Endo proved to be a very biocompatible ma- terial. The final stage of endodontic treatment is to fill the entire root canal system and all its complex anatomic pathways completely and densely with a nonirritating hermetic sealing material (1). Various methods have been proposed for root canal obturation. The most frequent methods use semisolid materials such as gutta- percha in combination with a root canal sealer or paste. Since these materials come into direct contact with periradicular tissues for a long period of time, their biocompatibility is important. The glass-ionomer cements were introduced to dentistry by Wilson and Kent in 1972 (2). They are based on the hardening reaction that occurs between particular species of ion-leachable glasses and the aqueous solutions of polyacrylic acids. Glass- ionomer cements bond chemically to dentin and hydroxyapatite of enamel (3, 4) and release fluoride ions (5, 6). Furthermore, they have improved biological tolerance and excellent tissue compati- bility (7). Based on these properties, Pitt Ford proposed the use of glass-ionomer cements as root canal sealers used in conjunction with gutta-percha points (8). Ketac-Endo has longer working time and higher radiopacity than the typical glass-ionomer cements, and due to its thixotropic consistency it can flow into the root canal. According to Ray and Seltzer (9) Ketac-Endo displays equal or superior physical properties to Grossman type sealer. Endion is a very newly proposed glass ionomer root canal sealer, which contains a radiopaquer, polyacrylic acid, and tartaric acid. Its precise formula was not available. The purpose of this study was to assess the cytotoxicity of Ketac-Endo and Endion by using an established cell line. 572 MATERIALS AND METHODS Two glass-ionomer sealers were tested: (a) Ketac-Endo (ESPE, Seefeld, Germany) and (b) Endion (VOCO, Germany). BHK21/C13 baby hamster kidney fibroblasts were cultured in Eagle's minimum essential medium (MEM) supplemented with 10% fetal calf serum (FCS) and antibiotics (100 IU/mt penicillin and 50 ~g/mI streptomycin). The cultures were incubated at 37°C in a humidity atmosphere with 5% CO2. The sealers were prepared under aseptic conditions according to the manufacturers' instruc- tions, and 0.1 ml of each sealer was placed with a micropipette in the center of petri dishes each of an inner diameter of 5 cm. The sealers were allowed to set for 6 h under ultraviolet light to prevent bacteria contamination. Each dish was covered with a 5-ml sus- pension of fibroblasts at a concentration of 40,000 cells/ml. Five ml of the same cell suspension were dispensed in dishes without any sealer to serve as controls. For each sealer and each observa- tion period, four samples and respective controls were prepared. All dishes were incubated at 37°C, and the incubation was con- cluded after 24, 48, and 72 h. At the end of the incubation period, the culture medium was removed and the cells were detached by trypsin (0.25% wt/vol). The viable cells were stained with trypan blue and counted with an hemocytometer chamber under light microscope. The number of cells counted in the experiment dishes was calculated as a percentage using the following formula: percentage of viable cells = (A/B) × 100 where A is viable cells in the experimental dish and B is viable cells in the control dish. This procedure was repeated twice for each sealer and each incubation period. One sample from each group was stained with Giemsa stain and examined under a mi- croscope for morphological observations. RESULTS In control dishes cells appeared viable and morphologically normal (Fig. 1). In the experimental cultures with Ketac-Endo the cells were normal in appearance (Fig. 2). In the experimental cultures with Endion the cells were apparently dead or dying;