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Mineral trioxide aggregate (MTA) is widely used in various dental endodontic applications such as root-end filling, furcal perforation repair, and vital pulp therapy. In spite of many attempts to improve handling properties and reduce the discoloration of MTA, the ideal root canal filling material has yet to be fully developed. The objective of this study was to investigate the setting time, mechanical properties, and biocompatibility of MTA set by a silk fibroin solution. A 5 wt% silk fibroin (SF) solution (a novel hydration accelerant) was used to set SavDen® MTA and ProRoot® white MTA (WMTA). Changes in setting time, diametral tensile strength (DTS), material crystallization, in vitro cell viability, and cell morphology were assessed by Vicat needle measurement, a universal testing machine, scanning electron microscopy (SEM), and WST-1 assay, respectively. The initial setting time of ProRoot® MTA and SavDen® MTA experienced a drastic decrease of 83.9% and 42.1% when deionized water was replaced by 5 wt% SF solution as the liquid phase. The DTS of SavDen® MTA showed a significant increase after set by the SF solution in 24 h. A human osteoblast-like cell (MG-63)-based WST-1 assay revealed that both ProRoot® MTA and SavDen® MTA hydrated using SF solution did not significantly differ (p > 0.05) in cell viability. MG-63 cells with pseudopodia attachments and nuclear protrusions represent a healthier and more adherent status on the surface of MTA when set with SF solution. The results suggest that the 5 wt% SF solution may be used as an alternative hydration accelerant for MTA in endodontic applications. [ABSTRACT FROM AUTHOR]