Simvastatin-enriched Chitosan-scaffold Promotes Odontoblastic Phenotype Expression in Pulp Cells

Objectives: The synergistic potential of chitosan-calcium scaffolds associated with simvastatin was assessed stimulating the odontoblastic phenotype expression of dental pulp stem cells (DPSCs).
Methods: To obtain chitosan-calcium (CH-Ca) scaffold a calcium-rich suspension was incorporated into chitosan solution followed by phase separation technique. Chitosan (CH) scaffold with no calcium were also fabricated. Firstly, cells were seeded in 96-well plates in osteogenic medium with different SV concentrations (0 mM, 0.01 mM, 0.1 mM, or 1 mM) until 21 days. Cell viability (alamar blue), odontoblastic markers expression (ALP pNPP assay and alizarin red) and cell migration (wound healing) were evaluated at several time-points (n=6). Then, DSPP/DMP-1 gene expression (real-time PCR) and active cell migration (0.8 mm pore transwell) were evaluated on DPSCs exposed to 0 mM or 0.1 mM SV (n=4). Thereafter, DPSCs/scaffolds constructs were incubated in osteogenic medium supplemented or not with 0.1 mM SV up to 21 days, and the cell viability (live/dead), proliferation (alamar blue), ALP activity (pNPP assay), DSPP/DMP-1 gene expression (real-time PCR) and Ca deposition (alizarin red) were assessed (n=6) (ANOVA/Tukey; α=5%).
Results: Proliferation decrease was observed at 7 and 14 days for the cells cultivated with all SV concentrations in comparison with negative control. However, 0.1 mM SV enhanced cell migration, ALP activity (14 days), mineralized matrix deposition (21 days) and DSPP/DMP-1 gene expression in comparison with control. The CH/DPSCs or CH-Ca/DPSCs construct incubated in osteogenic medium supplemented with 0.1 mM SV presented significant higher ALP activity and mineralized matrix deposition. Finally, transwell assay demonstrated intense DPSCs migration on CH-Ca scaffold compared with control (CH scaffold). This phenomenon was more intense when the culture medium was supplemented with SV.
Conclusions: Thereby, low SV concentrations increase the bioactive potential of scaffolds and induce odontoblastic phenotypes on DPSCs.