Contents lists available at ScienceDirect Archives of Oral Biology journal homepage: www.elsevier.com/locate/archoralbio Live cell imaging reveals different modes of cytotoxic action of extracts derived from commonly used luting cements Rita Trumpaitė-Vanagienė a,1 , Alina Čebatariūnienė b, ⁎ ,1 , Virginijus Tunaitis b , Alina Pūrienė a , Augustas Pivoriūnas b a Institute of Odontology, Faculty of Medicine, Vilnius University, LT-03101, Vilnius, Lithuania b Department of Stem Cell Biology, State Research Institute Centre for Innovative Medicine, LT-08406, Vilnius, Lithuania ARTICLE INFO Keywords: Luting cements Gingival fibroblasts Cytotoxicity Live-cell imaging ABSTRACT Objective: To compare cytotoxicity of extracts derived from commonly used luting cements: Hoffmann’s Zinc Phosphate (ZPC), GC Fuji Plus Resin Modified Glass Ionomer (RMGIC) and 3 M ESPE RelyX Unicem Resin Cement (RC) on primary human gingival fibroblasts (HGFs). Design: HGFs were exposed to different concentrations of the ZPC, RMGIC and RC extracts. The cytotoxicity was assessed with the PrestoBlue Cell Viability Reagent and viable cells were counted by a haemocytometer using the trypan blue exclusion test. In order to determine the primary mechanism of the cell death induced by extracts from different luting cements, the real-time monitoring of caspase-3/-7 activity and membrane integrity of cells was employed. Results: The extracts from the RMGIC and ZPC decreased the metabolic activity and numbers of viable cells. Unexpectedly, the extracts from the RC evoked only small effects on the metabolic activity of HGFs with a decreasing number of viable cells in a dose-and time-dependent manner. The live cell imaging revealed that the apoptosis was the primary mechanism of a cell death induced by the extracts derived from the RMGIC, whereas the extracts from the RC and ZPC induced a cell death through a necrotic and caspase-independent pathway. Conclusions: The apoptosis was the primary mechanism of the cell death induced by the extracts derived from the RMGIC, whereas the extracts from the RC and ZPC induced a cell death via a necrotic pathway. We suggest that metabolic assays commonly used to assess the cytotoxicity of luting cements should be validated by al- ternative methods. 1. Introduction Glass ionomer cements (GIC) and resin-modified GICs (RMGIC) are widely used in the modern clinical dentistry as luting agents. Despite the recent improvements of resin-based restorative materials, the bio- compatibility still represents an important problem (Lad, Kamath, Tarale, & Kusugal, 2014). Unreacted components of resin-based mate- rials are released into an oral cavity due to the incomplete poly- merization and therefore, they are cytotoxic and genotoxic (Schweikl, Spagnuolo, & Schmalz, 2006). These elutable substances represent re- sidual monomers such as 2-hydroxyethylmethacrylate (HEMA), trie- thyleneglycoldimethacrylate (TEGDMA), bisphenol A-glycidyl metha- crylate (Bis-GMA), as well as other components, such as initiating substances (Schweikl et al., 2006). The cytotoxic properties of different resin monomers depend on the chemical structure and concentration (Issa, Watts, Brunton, Waters, & Duxbury, 2004). Thus, more lipophilic monomers are usually more cytotoxic. Lower concentrations of mono- mers suppress the metabolic activity of cells by blocking the activity of mitochondrial dehydrogenases, while at higher concentrations these monomers interact with phospholipid bilayers by altering an integrity and increasing the permeability of cellular membranes (Schuster, Caughman, Rueggeberg, Lefebvre, & Cibirka, 1999). Several studies demonstrated that resin monomers induced an oxidative stress pri- marily via a depletion of intracellular levels of glutathione (Engelmann et al., 2004)(Volk, Engelmann, Leyhausen, & Geurtsen, 2006). Fur- thermore, the cytotoxic effects were inhibited in a presence of reactive oxygen species (ROS) scavengers like N-acetylcysteine, ascorbate, or vitamin E (Spagnuolo et al., 2006). However, the exact mechanism by which resin monomers induce the generation of ROS in cells is not yet clear. Another study demonstrated, that HEMA/Bis-GMA induced DNA double strand breaks through, at least in part, oxidative mechanisms (Blasiak et al., 2012). Furthermore, methacrylic acid, a product of https://doi.org/10.1016/j.archoralbio.2017.11.011 Received 1 January 2017; Received in revised form 19 November 2017; Accepted 26 November 2017 ⁎ Corresponding author at: Department of Stem Cell Biology, State Research Institute Centre for Innovative Medicine, Santariškių Str. 5, LT-08406 Vilnius, Lithuania. 1 These authors contributed equally to this work. E-mail address: alinacebatariuniene@gmail.com (A. Čebatariūnienė). Archives of Oral Biology 86 (2018) 108–115 0003-9969/ © 2017 Elsevier Ltd. All rights reserved. T