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

Elanagai Rathinam,Dirk Elewaut,Sivaprakash Rajasekharan,Luc Martens,Heidi Declercq,Luc Leybaert,Peter De Coster,Srinath Govindarajan

doi:10.1038/s41598-020-80096-5

Abstract

Calcium (Ca2+) signalling plays an indispensable role in dental pulp and dentin regeneration, but the Ca2+ 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 Ca2+ concentration, intracellular Ca2+ 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 Ca2+ and pH, and intracellular Ca2+ 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 Ca2+ in the α-MEM medium than ProRoot WMTA but this had no cytotoxic impact on hDPSCs. The larger Biodentine-linked Ca2+ release resulted in altered intracellular Ca2+ dynamics, which attained a higher maximum amplitude, faster rise time and increased area under the curve of the Ca2+ changes compared to ProRoot WMTA. Experiments with intracellular Ca2+ chelation, demonstrated that the biomaterial-triggered Ca2+ dynamics affected stem cell-related gene expression, cellular differentiation and mineralization potential. In conclusion, biomaterial-specific Ca2+ dynamics in hDPSCs determine differentiation and mineralization outcomes, with increased Ca2+ dynamics enhancing mineralization.

Highlights

Calcium (Ca2+) signalling plays an indispensable role in dental pulp and dentin regeneration, but the Ca2+ responses of human dental pulp stem cells stimulated with tricalcium silicate-based (TCS-based) dental biomaterials remains largely unexplored
Ca2+ signalling plays an indispensable role in dental pulp and dentin regeneration, there is a lack of information on the C a2+ dynamics of human dental pulp stem cells
In the absence of cells, Biodentine released significantly more Ca2+ in the α-MEM medium than ProRoot White MTA (WMTA) (p < 0.001) (Fig. 1); no significant difference in pH was found between the groups (Supplementary Fig. 1)

Summary

Introduction

Calcium (Ca2+) signalling plays an indispensable role in dental pulp and dentin regeneration, but the Ca2+ 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 Ca2+ concentration, intracellular Ca2+ 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. Experiments with intracellular Ca2+ chelation, demonstrated that the biomaterialtriggered Ca2+ dynamics affected stem cell-related gene expression, cellular differentiation and mineralization potential. The aim of the present study was to identify and correlate extracellular Ca2+ concentration, intracellular Ca2+ dynamics, pH, cytotoxicity, gene expression and mineralization ability of human dental pulp stem cells (hDPSCs) stimulated with two different TCS-based biomaterials; ProRoot WMTA and Biodentine. Our work shows significant differences in extracellular and intracellular Ca2+ changes that link to distinct patterns of hDPSCs gene expression, cellular differentiation and mineralization potential

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Conclusion