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Calcium (Ca 2+ ) signalling plays an indispensable role in dental pulp and dentin regeneration, but the Ca 2+  responses of human dental pulp stem cells (hDPSCs) stimulated with tricalcium silicate-based (TCS-based) dental biomaterials remains largely unexplored. The objective of the present study was to identify and correlate extracellular Ca 2+  concentration, intracellular Ca 2+  dynamics, pH, cytotoxicity, gene expression and mineralization ability of human dental pulp stem cells (hDPSCs) stimulated with two different TCS-based biomaterials: Biodentine and ProRoot white MTA. The hDPSCs were exposed to the biomaterials, brought in contact with the overlaying medium, with subsequent measurements of extracellular Ca 2+  and pH, and intracellular Ca 2+  changes. Messenger RNA expression (BGLAP, TGF-β, MMP1 and BMP2), cytotoxicity (MTT and TUNEL) and mineralization potential (Alizarin red and Von Kossa staining) were then evaluated. Biodentine released significantly more Ca 2+  in the α-MEM medium than ProRoot WMTA but this had no cytotoxic impact on hDPSCs. The larger Biodentine-linked Ca 2+  release resulted in altered intracellular Ca 2+  dynamics, which attained a higher maximum amplitude, faster rise time and increased area under the curve of the Ca 2+  changes compared to ProRoot WMTA. Experiments with intracellular Ca 2+  chelation, demonstrated that the biomaterial-triggered Ca 2+  dynamics affected stem cell-related gene expression, cellular differentiation and mineralization potential. In conclusion, biomaterial-specific Ca 2+  dynamics in hDPSCs determine differentiation and mineralization outcomes, with increased Ca 2+  dynamics enhancing mineralization.

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The calcium dynamics of human dental pulp stem cells stimulated with tricalcium silicate-based cements determine their differentiation and mineralization outcome