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

## TL;DR

This study examines how different endodontic sealers affect bone cell development and signaling in pre-osteoblast cells.

## Contribution

The study reveals distinct molecular responses and osteogenic effects of contemporary endodontic sealers on pre-osteoblast cells.

## Key findings

- Calcium-silicate-based BC sealer promoted greater cell viability and osteogenic gene expression compared to ZOE.
- MAPK signaling was activated by BC and ZOE, but BC's effects were more favorable for osteogenesis.
- ZOE impaired early cell attachment and viability despite activating MAPK pathways.

## Abstract

Background/Objectives: 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. Methods: 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. Results: 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 Runx2 and Osx 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. Conclusions: 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.