Cytotoxicity determination of nano-zinc oxide eugenol on human gingival fibroblast cells

Zinc oxide eugenol (ZOE) is composed of approximately 70% ZnO and approximately 30% eugenol. ZOE has been used in dentistry as a temporary filler, cavity liner, and base for tooth cavities. It is used to form a thin layer of cement to protect the pulp from any influx of bacteria or irritants present in the tooth cavity. Such bacteria and irritants can lead to the formation of an abscess or pus in the gums or periapical tissues. As ZnO has antibacterial effects, it is appropriate to use ZOE as a temporary filler, liner, or base. On the other hand, eugenol alone is acidic and may leach out to have adverse effects on the periapical tissue. Therefore, the toxicity level of ZOE is important to avoid adverse effects or unnecessary dosing. Thus, in the present study, we investigated the potential toxicity of ZOE leaching toward human gingival fibroblast tissue to verify its safety and efficacy by using ZnO particles with different morphologies and sizes. Three types of ZnO powder were used in this study: ZnO-A and ZnO-B (which were produced using the French process) and ZnO-K, which are commercial particles used for ZOE temporary fillers. All three types of ZnO were mixed with eugenol to form ZOE. Then, all three types were characterised to determine their morphology, particle size, and specific surface area. Each type of ZnO was then mixed with resin-bonded eugenol to obtain ZOE pellets. Subsequently, the toxicology of the ZOE leachings was tested on human gingival fibroblast cells using a calorimetric assay for 24 and 48 h. The results show that ZnO-A, ZnO-B, and ZnO-K have particle sizes of 22.3, 48.33, and 1196 nm, respectively. The specific surface areas were 4.44, 3.89, and 0.74 m2/g, respectively. Moreover, the toxicology of the ZOE leachings revealed a lower percentage of viable cells with an increase in the leaching concentration. After 24 and 48 h of cytotoxicity tests, the percentage of viable cells decreased slightly with leaching concentrations of 10%-40% and decreased considerably with leaching concentrations of 50%-100%. This shows that the viable cell percentage depends on the leaching concentration and duration of the exposure. Moreover, the results show that smaller particles have larger specific surface areas and higher percentages of viable cells. This means that the smaller particles released smaller amounts of eugenol and Zn2+ during the toxicity evaluation, which led to less reactive oxygen species (ROS) production and influenced the number of cells that survived after 24 and 48 h.