Lovastatin increases the proliferation and osteoblastic differentiation of human gingiva-derived stem cells in three-dimensional cultures

Lovastatin is a cholesterol-lowering agent that also has effects of cell proliferation and apoptosis. The present study was performed to evaluate the effects of lovastatin on the proliferation and osteogenic differentiation of three-dimensional cell spheroids formed from human gingiva-derived stem cells (GDSCs) using concave microwells. GDSCs were plated on polydimethylsiloxane-based concave micromolds and grown in the presence of lovastatin at concentrations of 0, 2 and 6 µM. The morphology of the cells was viewed under an inverted microscope, and cell viability was determined with Cell Counting kit-8 on days 2, 7 and 14. Alkaline phosphatase activity assays were performed to evaluate the osteogenic differentiation on days 2 and 8. Alizarin red-S staining was also used to assess the mineralization of the stem cell spheroids at day 14. The results confirmed that GDSCs formed spheroids in concave microwells. No significant changes were noted with longer incubation time, and no significant differences in cell viability were noted between the three lovastatin groups at each time point. Higher osteogenic differentiation was observed in the 2 µM group when compared with the control. Mineralized extracellular deposits were visible after Alizarin red-S staining, and higher mineralization was noted in the 2 and 6 µM lovastatin groups when compared with the 0 µM control. The relative mineralization values of the 0, 2 and 6 µM groups on day 14 were 39.0±9.6, 69.3±6.0 and 60.9±7.5, respectively. This study demonstrated that the application of lovastatin enhanced the osteogenic differentiation of cell spheroids formed from GDSCs. This suggests that combinations of lovastatin and stem cell spheroids may have the potential for use in tissue engineering.