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Introduction The aim of this article was to analyze biocompatibility and bioactivity of new endodontic materials on the basis of nanosynthesized calcium silicates (ALBO-MPCA₁ and ALBO-MPCA₂) combined with different radiopacifiers in comparison with MTA⁺. Methods Morphology of the samples was studied by scanning electron microscopy, and the pH and ion release analysis were also assessed. Biocompatibility of materials' eluates (24-hour, 7-day, and 21-day) was conducted by using MTT test. Twelve New Zealand white rabbits were used for intraosseous implantation. Four calvarial defects per animal were created and filled with freshly prepared investigated materials. Results Samples mostly consisted of agglomerates built up from nanoparticles, preferably spherical and rod-like. There was no significant difference among pH values of materials' eluates after 24 hours (P > .05). The amount of calcium and aluminum ion release decreased, whereas the amount of magnesium and bismuth (ALBO-MPCA₁, MTA⁺) and barium (ALBO-MPCA₂) increased during 21-day period. The metabolic activity of cells increased after the extraction time, except in case of undiluted elutes of ALBO-MPCA₂ and ALBO-MPCA₁ (21-day). Histologic analysis of the samples revealed newly formed bone tissue with moderate inflammation for all investigated materials, which subsided during 90-day period to mild. Both MTA⁺ and ALBO-MPCA₁ were in direct contact with the newly formed bone tissue. After 90 days, statistically significant difference in hard tissue formation was observed in comparison of MTA⁺ and ALBO-MPCA₁ with control group (P < .05). Conclusions Experimental materials ALBO-MPCA₁ and ALBO-MPCA₂ possess both biocompatibility and bioactivity. Because ALBO-MPCA₁ provokes favorable biological response, it is especially good candidate for further clinical investigations. © 2016 American Association of Endodontists