Mechanistic Signaling Induced by Calcium Hydroxide for Osteodentin Repair

Objectives: Pulp capping with calcium-hydroxide (CaOH) and Mineral Trioxide Aggregate (MTA) are used routinely in dental clinics today. The prevailing hypothesis is that the extreme initial pH generated by mixing this cement results in a sclerotic pulp-dentin tissue response. However, the precise molecular mechanism remains to be precisely elucidated that motivated this study.
Methods: We used mouse odontoblasts cells, MDPC-23, and treated them with freshly mixed CaOH (Dycal) and assessed mineralization with Alkaline Phosphatase (ALP) activity and Alizarin Red staining. To examine the contributions of specific molecular pathways, we treated the cells with pH 1 to 11 pH in HEPES, Nigericin (intracellular pH), hydrogen peroxide (30 uM), and TGF-b1 (2.5 ng/ml) and assessed ALP. To dissect their roles, we pre-treated these cells with neutralization reagents for pH (Omeprazole), ROS (N-Acetyl cysteine and Catalase), or TGF-b inhibitors (SB431542 or SIS3). Data was organized in Excel, and statistical tests were performed to assess significance. All studies were performed with technical replicates and repeated at least twice.
Results: We observed a robust induction of ALP in odontoblasts following TGF-β1, Hydrogen Peroxide, pH 9, and CaOH treatments. We noted CaOH increased ROS and phospho-Smad 2/3 in odontoblasts. Neutralizing ROS, pH, or TGF-β signaling abolished these responses.
Conclusions: This study outlines the interplay of three discrete signaling pathways involving ROS, TGF-β, and pH that mediate mineralized repair in pulp-dentin following calcium hydroxide pulp capping. This mechanistic understanding could further promote pulp capping as a viable, reliable approach to preserve tooth vitality and eventual tooth loss.