In Vitro Assessment of Osteogenic Modulation and Molecular Responses Induced by Contemporary Endodontic Sealers in MC3T3-E1 Pre-Osteoblasts

<b>Background/Objectives</b>: Endodontic sealers can interact with periapical tissues through extrusion, yet the molecular mechanisms underlying their biological effects remain poorly defined. This study investigated how commonly used sealers influence mitogen-activated protein kinase (MAPK) signaling, cell viability, and osteogenic-associated responses in MC3T3-E1 pre-osteoblasts. <b>Methods</b>: Four commercial sealers, Calcium-silicate-based Bioceramic Sealer (EndoSequence^® BC Sealer, BC), Zinc oxide eugenol sealer (Kerr Pulp Canal Sealer, ZOE), Sealapex™, and AH26^®, were applied as standardized pellets, allowed to set, and cultured with MC3T3-E1 cells. Calcium deposition was assessed by Alizarin Red S (ARS) staining, and MAPK activation was evaluated by Western blotting. Due to excessive solubility (Sealapex™) or poor cell survival (AH26^®), mechanistic analyses were performed only for BC and ZOE. Osteogenic-associated gene expression was measured by qRT-PCR, and the functional role of MAPK signaling was assessed using ERK, JNK, and p38 inhibitors. <b>Results</b>: BC and Sealapex™ produced robust ARS staining, while ZOE and AH26^® produced minimal mineral-associated staining. Both BC and ZOE activated ERK, JNK, and p38, with ZOE inducing higher phosphorylation. However, BC maintained greater cell viability and increased <i>Runx2</i> and <i>Osx</i> expression, whereas ZOE impaired early cell attachment and viability. MAPK inhibition in BC-treated cultures reduced osteogenic-associated gene expression and ARS staining, indicating MAPK involvement in BC-mediated responses. <b>Conclusions</b>: BC and ZOE elicit distinct MAPK activation patterns and cellular responses. Under the conditions tested, BC promoted a more favorable osteogenic-associated response, whereas ZOE compromised early cell viability. These mechanistic insights may help explain clinical differences in periapical tissue responses to sealer extrusion.