1 Vol.:(0123456789) Scientifc Reports | (2021) 11:645 | https://doi.org/10.1038/s41598-020-80096-5 www.nature.com/scientificreports The calcium dynamics of human dental pulp stem cells stimulated with tricalcium silicate‑based cements determine their diferentiation and mineralization outcome Elanagai Rathinam 1* , Srinath Govindarajan 2,3 , Sivaprakash Rajasekharan 1 , Heidi Declercq 4,5 , Dirk Elewaut 2,3 , Peter De Coster 6 , Luc Martens 1 & Luc Leybaert 7 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 diferent 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 signifcantly 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 afected stem cell‑related gene expression, cellular diferentiation and mineralization potential. In conclusion, biomaterial‑specifc Ca 2+ dynamics in hDPSCs determine diferentiation and mineralization outcomes, with increased Ca 2+ dynamics enhancing mineralization. Tricalcium silicate-based (TCS-based) cements are hydraulic bioactive materials widely used as endodontic cements in dentistry and as bone substitutes in orthopedics 1 . Several commercial TCS-based cements with subtle modifcations in the manufacturing process and composition are available. ProRoot White MTA (WMTA) (Dent- sply, Tulsa Dental, OK, USA) and Biodentine (Septodont, Saint-Maur-des-Fossés, France) are two representative TCS-based cements with superior clinical success in dentistry. Te widespread clinical indications of TCS-based cements are primarily based on their ability to form calcium hydroxide as a by-product of hydration 2 . Te sub- sequent dissolution of calcium hydroxide to release hydroxide (OH - )and calcium ions (Ca 2+ ) creates a desirable OPEN 1 Department of Paediatric Dentistry and Special Care, PAECOMEDIS Research Cluster, Ghent University, Ghent University Hospital, 9000 Ghent, Belgium. 2 Department of Internal Medicine and Paediatrics, Ghent University, Ghent University Hospital, 9000 Ghent, Belgium. 3 Unit for Molecular Immunology and Infammation, VIB-Center for Infammation Research, Technologiepark 71, 9052 Zwijnaarde, Ghent, Belgium. 4 Tissue Engineering and Biomaterials Group, Department of Human Structure and Repair, Ghent University, Ghent University Hospital, 9000 Ghent, Belgium. 5 Tissue Engineering Lab, Department of Development and Regeneration, KU Leuven, 8500 Kortrijk, Belgium. 6 Department of Reconstructive Dentistry and Oral Biology, Dental School, Ghent University, Ghent University Hospital, 9000 Ghent, Belgium. 7 Department of Basic And Applied Medical Sciences - Physiology Group, Ghent University, Ghent, Belgium. * email: Elanagai.Rathinam@ugent.be