In vitro Comparison of Cytotoxicity and Osteoclastogenesis of Four Bioceramic Sealers in Murine Bone Marrow Cells

Abstract

Introduction and aims

Root canal sealers are indispensable in endodontic therapy, but the diffusion of their chemical components into periapical tissues may trigger osteoclast activation and bone resorption. This study aims to evaluate the pH values of 4 calcium silicate-based bioceramic sealers (C-root SP, iMTA SP, iRoot SP and NeoSEALER Flo) and their effects on the cell viability of osteoclast precursor cells, while further exploring their regulatory roles in receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis in vitro.

Methods

After pH measurement of sealer extracts, the cell viability following sealer treatment was quantitatively assessed using the Cell Counting Kit-8 (CCK-8) assay, while tartrate-resistant acid phosphatase (TRAP) staining and phalloidin staining were employed to evaluate RANKL-induced osteoclast formation and fibrous actin (F-actin) ring formation, respectively. Furthermore, quantitative real-time PCR (qPCR) and Western blot analyses were conducted to examine the expression patterns of osteoclast-related genes and proteins.

Results

All sealer extracts exhibited alkaline properties. Low-concentration sealer extracts exhibited no significant impact on cell viability, whereas high-concentration extracts (50 mg/mL) displayed time-dependent cytotoxic effects. Notably, all tested sealers exhibited inhibitory effects on osteoclast differentiation, with NeoSEALER Flo demonstrating the most potent suppression. This was evidenced by a reduction in the formation of TRAP-positive multinucleated cells, disruption of F-actin ring organisation, and a coordinated downregulation of osteoclast-related gene and protein expression.

Conclusion

All tested sealers showed no obvious cytotoxicity at low concentrations and exhibited varying degrees of inhibitory effects on osteoclast formation and function. Among them, NeoSEALER Flo extracts showed the strongest inhibitory effect on osteoclasts.

Clinical Relevance

NeoSEALER Flo, a calcium silicate-based bioceramic sealer, effectively minimises cytotoxicity while inhibiting osteoclast formation and activity. This suggests that it may effectively prevent periapical tissue destruction and promote bone healing in apical periodontitis, highlighting its promising potential as a root canal obturation material.