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 »  Home  »  Endodontic Articles 2  »  Cytotoxicity of resin-, zinc oxide–eugenol-, and calcium hydroxide-based root canal sealers on human periodontal ligament cells and permanent V79 cells
Cytotoxicity of resin-, zinc oxide–eugenol-, and calcium hydroxide-based root canal sealers on human periodontal ligament cells and permanent V79 cells
Discussion - References.

Many cell culture techniques have been applied to assess the cytotoxicity of root canal sealers. These methods are based on cell cultures with established or diploid cell lines and a few tissue explant techniques. Any material used for obturation will come into contact with, or close proximity to the periodontal ligament. Various cell lines (normal diploid or transformed) are commonly used in cytotoxicity evaluations, and since PDL cells are critical to a healthy periodontium, they were used in this study. In addition, the selection of a permanent cell line derived from the Chinese hamster was used because they are easily maintained in culture. Furthermore, donor biopsy variability was eliminated and greater reproducibility was possible.
The MTT assays are colorimetric methods for quantifying viable cell numbers. The methyl-tetrazolium ring is cleared by mitochondrial dehydrogenouses in viable cells to formazan, which has a blue colour and can be measured with a spectrophotometer (Mosmann 1983). The amount of formazan produced is directly proportional to the total viable cell number over a wide range of cell numbers. Whilst in proliferating cells, comparison of bioassays performed with 3 H-thymidine incorporation versus the MTT assay utilized in this investigation display similar results, the MTT assay reflects cell numbers at any stage in their growth cycle. Since dead cells are unable to produce the coloured formazon product, this assay can be distinguished from dead cells (Mosmann 1983). The advantages of this method are its simplicity, rapidity, and precision, in addition, it does not require radioisotopes.
In this study, the freshly prepared root canal sealers were placed immediately into medium. Clearly, canal sealer should be tested immediately after mixing and also after a period of time when it is assumed that they have reached their final chemical structure. Root canal sealers are inserted into the mouth in a freshly mixed, incompletely polymerized stage, and thus it is probable that, during a relatively short period after clinical application of the material, local responses are provoked by unreacted or only partially reacted components. After setting, it is possible that potentially toxic constituents may be released from the materials. The difference in toxicity patterns at the various elution times may be related to the degree of setting. This would be reflected in the rate of component leaching. Thus, the different time extracts might be important to determine long-term cytotoxicity of root canal sealers.
The cytotoxicity of extracts of six root canal sealers was evaluated using MTT assay in human PDL cells and V79 cells. Our results agree with previous reports (Arenholt-Bindslev & Horsted-Bindslev 1989, Matsumoto et al . 1989, Briseno & Willershausen 1991, Gerosa et al . 1995, Beltes et al . 1995, Vajrabhaya et al . 1997, Osorio et al . 1998, Koulaouzidou et al . 1998, Geurtsen et al . 1998, Guigand et al . 1999, Cohen et al . 2000, Leonardo et al . 2000) that all materials tested were cytotoxic. In the longterm extracts of sealers, human PDL cells were more sensitive than V79 cells, except for Endomethansone and N2. However, the final extracts of Sealapex, Canals, AHPlus, and AH26 did not exert cytotoxic effects on PDL cells. The different responses between PDL and V79 cells to various sealers is difficult to explain; it is probably due to differences in the origin of cells.
Both resin-based sealers, AH26 and AHPlus, were cytotoxic. The cytotoxicity of resin-based sealers may be related to the release of formaldehyde (Spångberg et al . 1993, Cohen et al . 1998). In addition, bisphenol A diglycidyl ether was identified as a mutagenic component of resin-based materials, which may also be cytotoxic (Heil et al . 1996). AHPlus exhibited a lower cytotoxic potential compared to AH26 in this study, to confirm previous reports (Koulaouzidou et al . 1998, Cohen et al . 2000). Moreover, the amount of formaldehyde released by AH26 is significantly higher than AHPlus (Spångberg et al . 1993, Cohen et al . 1998, Leonardo et al . 1999).
In the case of zinc oxide–eugenol-based sealers, moderate to severe cytotoxicity was observed. The toxicity decreased in an order of N2 > Endomethansone > Canals. It was suggested by Lindqvist & Otteskog (1981) that the cytotoxicity of zinc oxide–eugenol root canal sealers was attributable to free eugenol liberated from the set material. A previous study has shown that both N2 and Endomethansone sealers can release formaldehyde after setting (Leonardo et al . 1999). The combined effects of eugenol and formaldehyde might explain why N2 and Endomethansone were highly toxic. Apart from formaldehyde and eugenol, N2 also contains a variety of aromatic oils that are cytotoxic (Table 2). These might be the reason why N2 is the most toxic sealer.
The use of so called ‘biological’ sealers based on calcium hydroxide has been proposed for the permanent obturation of the root canal system. Sealapex is primarily made of calcium hydroxide, and it demonstrated only slight toxicity in the fresh state. However, it exhibited increasing toxicity when set to confirm the results of previous studies that reported considerable leakage of cytotoxic substances from the disintegrating sealer (Gerosa et al . 1995, Beltes et al . 1995, Leonardo et al . 2000). This instability in an aqueous environment might enhance the release of substances from set Sealapex.


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