The influence of simvastatin on the osteogenic differentiation induced by calcium silicate–based cements

Objective: The combination of simvastatin with hydraulic materials might offer added benefits for enhancing mineralisation, reducing inflammation, and supporting pulp healing. Therefore, the aim of the present in vitro study was to assess the effect of hydraulic calcium silicate–based cements in addition to simvastatin on cell viability and osteogenic differentiation. Methods: Human osteogenic sarcoma (Saos-2) cells were cultured in presence of 0.1 μM of simvastatin, and with ProRoot MTA (Mineral Trioxide Aggregate) and Biodentine in combination or not with simvastatin. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at 24, 48, and 72 hours and immunofluorescence microscopy analysis was conducted after 72 hours. Osteogenic differentiation was evaluated by Alizarin Red Staining (ARS) after 18 days. Moreover, gene expression (RUNX-2, COL1A1, ALP, OCN) was analysed at 12 and 18 days. Statistical analysis was performed using analysis of variance, Bonferroni’s test, and Student’s tests. Results: Simvastatin at 0.1 μM preserved cell viability comparable to that of the untreated control. Lesser cytotoxicity was reported when simvastatin was combined with Biodentine, rather than ProRoot MTA. Simvastatin alone or in combination with both cements induced the reorganisation of the actin cytoskeleton into lamellipodia. Alizarin Red Staining showed that simvastatin significantly enhanced mineralisation, mainly when combined with Biodentine. Conversely, both cements alone showed moderate effects. Regarding gene expression, analyses revealed that these materials, in addition to simvastatin, influenced differently osteogenic markers, suggesting the involvement of distinct molecular pathways probably due to different chemical composition. Conclusions: Hydraulic calcium silicate–based cements exhibited distinct effects on the expression of differentiation-related genes, while showing a comparable influence on mineralisation nodule deposition. These findings suggest material-specific biological responses when the cements are combined with simvastatin.