This study aimed to compare the biological performance of a hydraulic calcium silicate cement (EndocemMTA Premixed) containing dimethyl sulphoxide (DMSO), developed to improve handling properties, in comparison with ProRoot mineral trioxide aggregate and Biodentine. Human dental pulp cells were used for in vitro evaluation of cytocompatibility, cell migration and cell–material interactions. Odontogenic differentiation was assessed using a 3D culture model designed to simulate the clinical environment. Pulp capping was performed in vivo on rat maxillary molars, and reparative dentin formation and pulpal inflammatory responses were evaluated using micro-computed tomography (μCT) and histological analyses. In vitro, all investigated materials exhibited comparabl e cytocompatibility, cell migratory behaviour and odontogenic marker expression, with no significant differences among study groups. The μCT analysis demonstrated significantly greater reparative dentin formation in all experimental groups compared with the control, with Biodentine producing a higher dentin volume than EndocemMTA Premixed. Histological evaluation revealed no significant differences among the experimental groups with respect to pulpal inflammation or dentin bridge continuity. These findings suggest that the incorporation of DMSO into premixed formulations would not adversely affect cytocompatibility or pulp healing. Moreover, the 3D model used in this study might serve as a clinically releva
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