**10. Pertinent question: is the dental filling material toxic to the living tooth? Contemplations on the making of live and artificial teeth**

Monomers from methacrylate based dental materials both prior to and post polymerization have demonstrated adverse effects both *in vitro* and *in vivo* in terms of cytotoxicity [68], mutagenicity/genotoxicity [69–72], negative effects on fertility [73], xenoestrogenicity [74–76], and allergy induction [77]. The degree of cytoxicity will vary from the type assay used, materials tested, time intervals for testing, and cell types tested [78].

It is most pertinent to perform *in vitro* cytotoxicity testing on cells from cell types and tissues relevant to the area of *in vivo* placement of dental materials [79]. Recent studies on elution of monomers available in both dental composites and methacrylate- and epoxy-based root canal sealers looked at reactions in submandibular salivary gland acinar cells for the evaluation of cytotoxicity, cell proliferation, and apoptosis [69, 80]. The findings in such studies are of great interest and importance, but the authors of one of these studies [80] stated that their model would have been more realistic had they utilized human primary cells from direct target tissue. Tissues that often share the closest proximity to dental fillings are certainly not salivary glands but rather gingiva, mucosa, and, in particular, pulp tissues [79].

The pulp is a loose connective tissue within a nonresilient capsule of dentin and enamel. Pulpal inflammation is considered a protective mechanism and can either be of an acute or chronic nature. Acute and chronic responses are related to the "magnitude and duration of the insult [81]." Inflammation will inevitably cause vasodilation, increased vessel permeability which in turn will result in relatively large changes in tissue pressure [82]. Bacterial infection is the most common reason for pulpal inflammation, but any insult or stimuli will most probably result in a response. It is an established fact that many of the constituents in dental adhesive resin are cytotoxic [81], and the difference in cytoxicity varies among commercial materials commonly used by public dentists in Norway [unpublished results in a report to the Norwegian National Directorate of Health].

This project aims at elucidating the cellular effects of "leachables" (residual monomers) from dental filling materials exerted on dental pulp stem cells (DPSCs) *ex vivo*. It is important to ensure that the cells used in the study are, indeed, stem cells. The International Society for Cellular Therapy has released a position statement wherein they list three criteria to define human stem cells: (1) adherence to plastic, (2) specific surface antigen expression, and (3) multipotent differentiation potential [83]. The cells to be used in this project fulfil all three criteria [84], and were isolated in accordance with a published procedure described by Sorrentino et al. [85].
