The effects of simvastatin on cellular viability, stemness and osteogenic differentiation using 3-dimensional cultures of stem cells and osteoblast-like cells

Abstract

Background. Simvastatin has been reported to increase the therapeutic effects of many kinds of stem cells by increasing the number of those cells. However, the effects of simvastatin on the differentiation potential of stem cells have not been clearly determined. Objectives. The aim of the study was to evaluate the effects simvastatin has on cellular viability, stemness and osteogenic differentiation using 3-dimensional cell spheroids of stem cells and osteoblast-like cells. Material and methods. Three-dimensional cell spheroids were fabricated using concave silicon elastomer-based microwells in the presence of simvastatin at concentrations of 1 μM and 10 μM. Qualitative cellular viability was determined with a confocal microscope, and quantitative cellular viability was evaluated using a cell-counting assay kit. The expression of stem cell surface markers was tested. A quantitative real-time polymerase chain reaction (qRT-PCR) was performed to evaluate the expression of collagen I and RUNx2. Alkaline phosphatase activity and alizarin red S staining were used to assess osteogenic differentiation. Results. The spheroids formed well in the concave silicon elastomer-based microwells, and the application of simvastatin caused no significant morphological changes. No significant changes in cellular viability were noted with the addition of simvastatin on days 1, 3 and 5. Secretion of the vascular endothelial growth factor (VEGF) was observed on day 1 and remained stable throughout the culture period. Expression of the CD90 surface marker was seen on day 7. The addition of simvastatin caused a statistically significant increase in the expression of collagen I and RUNX2. It also caused decreases in alkaline phosphatase activity and alizarin red S staining. Conclusions. The study clearly showed that the application of simvastatin enhanced collagen I and RUNX2 expression; however, this did not lead to increases in alkaline phosphatase activity or alizarin red S staining.