Introduction: Tricalcium silicate based materials are recognized as bioactive materials through their capacity to induce hard tissue formation both in the dental pulp and bone. Sealing the apex implies that the root canal filling materials interact with the periapical tissues. This work was designed to study the interactions of newly developed tricalcium silicate cement (BioRoot RCS; Septodont, Saint Maur Des Fosses, France) with apical tissue compared with a standard zinc oxide eugenol sealer (Pulp Canal Sealer [PCS]; SybronEndo, Orange, CA). Methods: Cell viability was assessed by direct contact between human periodontal ligament (PDL) cells and BioRoot RCS or PCS. In addition, an in vitro tooth model was used to study the interactions between these materials and PDL cells. For this purpose, human extracted incisors were sectioned at the enamel-cementum junction; root canals were prepared, sterilized, and filled with lateral condensation with both materials. The root apices were dipped in the culture medium for 24 hours. These conditioned media were used to investigate their effects on human PDL cells. Cell proliferation was investigated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MU) assay, and the secretion of angiogenic and osteogenic growth factors was quantified using an enzyme-linked immunosorbent assay. Results: BioRoot RCS has less toxic effects on PDL cells than PCS and induced a higher secretion of angiogenic and osteogenic growth factors than PCS. Conclusions: Taken together, these preclinical results suggest that the calcium silicate cement (BioRoot RCS) has a higher bioactivity than the zinc oxide eugenol sealer (PCS) on human PDL cells.