Setting and incubation environments affect the physicochemical evaluation of two hydraulic calcium silicate-based endodontic materials

Abstract This study evaluated how commonly used setting and incubation conditions influence the properties of hydraulic calcium silicate-based materials, and whether changes observed during prolonged laboratory incubation reflect intrinsic material behaviour or protocol-dependent surface interactions. Setting times of BioRoot RCS and Biodentine were measured using Gillmore needles; crystalline phase changes during setting were assessed by X-ray diffraction (XRD) under dry and humid conditions. Solubility (mass changes), porosity (micro-CT), ion release (inductively coupled plasma optical emission spectrometry), pH, surface morphology (scanning electron microscopy), and chemical composition (XRD, Fourier transform infrared spectroscopy, Raman spectroscopy) changes were measured at 24 h, 7 days, and 28 days. BioRoot RCS was incubated in 0.73 mL (surface area-to-volume ratio of 3 cm 2 /mL) or 15.22 mL (surface area-to-volume ratio of 0.145 cm 2 /mL) of Hank’s balanced salt solution (HBSS). Biodentine was incubated in 0.73 mL HBSS or 0.73 mL human plasma-like medium, both at a surface area-to-volume ratio of 3 cm 2 /mL. Results showed that changes in crystalline phases continued beyond the Gillmore setting endpoints. Under dry conditions, both materials reached the Gillmore setting endpoints but no changes in the tricalcium silicate peaks were observed during the first 24 h. BioRoot RCS showed greater mass loss in the larger HBSS volume; Biodentine showed progressive mass gain in HPLM and significant mass gain only at 28 days in HBSS. Carbonation of the material surfaces increased over time, whereas internal porosity remained relatively stable. These findings highlight the strong influence of moisture availability, immersion volume, medium composition, and incubation time on physicochemical evaluation, with prolonged humid incubation promoting surface-localized dissolution and precipitation.