Abstract

Statement of the problem: Universal adhesives have gained popularity simplifying dental restorative procedures. However, limited information exists regarding formulation variations among manufacturers and their impact on cytocompatibility using human-derived cell lines. Purpose: This study aimed to investigate the molecular mechanisms of cytotoxicity of three universal adhesives-Ambar Universal (AU), Single Bond Universal (SB), and Prime & Bond Universal (PB)-on human-derived Saos-2 osteoblast-like cells, elucidating the differential induction of apoptosis and necrosis pathways, contributing to a greater understanding of their biological effects. Methods: Saos-2 cells were incubated with 0.1 % v/v AU, SB, or PB during 2, 4, or 6. h. Populations of viable, early apoptotic, late apoptotic, and necrotic cells were estimated by cell sorting after Annexin V/Propidium Iodide staining. Apoptosis indicators, caspase-3 activation or nuclei fragmentation were addressed by immunoblot or immunofluorescence, respectively. Cytotoxicity was evaluated after 24 h incubation, using MTT Cell Proliferation Assay, with or without the apoptosis inhibitor Z-VAD-FMK. Results: SB exhibited a higher percentage of early apoptotic cells, and a significant increase in activated caspase-3 compared to control. AU and PB significantly increased necrosis, reducing cell viability by 100 %, compared to a 52 % reduction with SB. Immunofluorescence showed apoptotic nuclei morphology for SB, and necrotic characteristics for AU and PB. The apoptosis inhibitor Z-VAD-FMK only abolished SB-evoked cytotoxicity. Conclusion: The universal adhesives studied induced cell death in Saos-2 cells through different mechanisms. SB primarily evoked apoptosis, whereas AU and PB predominantly led to necrosis. These varying cytotoxic effects should be considered when selecting safer universal adhesives for clinical use. However, these findings should be interpreted with caution, as the exclusive use of the Saos-2 osteosarcoma cell line may not fully reflect the biological behavior of dental pulp cells or other oral tissues. © 2025 Elsevier Ltd