Background/PurposeRadio-opacity is an essential attribute of ideal root-end filling materials because it is important for clinicians to observe root canal filling and to facilitate the follow-up instructions. The novel root-end filling material (NRFM) has good cytocompatibility and physicochemical properties but low intrinsic radio-opacity value. To improve its radio-opacity value, three novel radio-opaque root-end filling materials (NRRFMs) were developed by adding barium sulphate (NRFM-Ba), bismuth trioxide (NRFM-Bi) and zirconium dioxide (NRFM-Zr) to NRFM, respectively. The purpose of this study was to identify the suitable radio-opacifier for NRFM through evaluating their physicochemical and biological properties, in comparison with NRFM and glass ionomer cement (GIC).MethodsNRRFMs were characterized using X-ray diffraction (XRD) and Fourier transform infrared spectrophotometry (FTIR). Physicochemical properties including setting time, compressive strength, porosity, pH variation, solubility, washout resistance, contact angle and radiopacity were investigated. Cytocompatibility of both freshly mixed and set NRRFMs was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Alkaline phosphatase (ALP) activity assay and alizarin red staining were used to investigate the osteogenic differentiation potential of NRFM-Zr. Data were analyzed using two-way ANOVA (pH variation, solubility and ALP activity) and one-way ANOVA (for the other variables).Results(1) NRRFMs were primarily composed of hydroxyapatite, calcium carboxylate salt and the corresponding radio-opacity agents (barium sulphate, bismuth trioxide or zirconium dioxide). (2) Besides similar physicochemical properties in terms of setting time, pH variation, solubility, washout resistance and contact angle to NRFM, NRFM-Bi and NRFM-Zr exhibited lower porosity and greater compressive strength after being set for 7 days and their radio-opacity were greater than the 3 mm aluminium thickness specified in ISO 6876 (2001). (3) MTT assay revealed that freshly mixed and set NRFM-Zr presented better cell viability than NRFM-Ba and NRFM-Bi at 24 hours and 48 hours (P<0.05). (4) NRFM-Zr significantly enhanced ALP activity and calcium formation of human osteoblast-like Saos-2 cells when compared with negative group and GIC (P<0.05).ConclusionNRFM-Zr presents desirable physicochemical and biological properties, thus zirconium dioxide may be a suitable radio-opacifier for NRFM.