Effects of tricalcium silicate cements on osteogenic differentiation of human bone marrow-derived mesenchymal stem cells in vitro

Tricalcium silicate cements have been successfully employed in the biomedical field as bioactive bone and dentin substitutes, with widely acclaimed osteoactive properties. This research analyzed the effects of different tricalcium silicate cement formulations on the temporal osteoactivity profile of human bone marrow-derived mesenchymal stem cells (hMW-MSCs). These cells were exposed to four commercially available tricalcium silicate cement formulations in osteogenic differentiation medium. After 1, 3, 7 and 10 days, quantitative real-time polymerase chain reaction and Western blotting were performed to detect expression of the target osteogenic markers ALP, RUNX2, OSX, OPN, MSX2 and OCN. After 3, 7, 14 and 21 days, alkaline phosphatase assay was performed to detect changes in intracellular enzyme level. An Alizarin Red S assay was performed after 28 days to detect extracellular matrix mineralization. In the presence of tricalcium silicate cements, target osteogenic markers were downregulated at the mRNA and protein levels at all time points. Intracellular alkaline phosphatase enzyme levels and extracellular mineralization of the experimental groups were not significantly different from the untreated control. Quantitative polymerase chain reaction results showed increases in downregulation of RUNX2, OSX, MSX2 and OCN with increasing time of exposure to the tricalcium silicate cements, while ALP showed peak downregulation at day 7. For Western blotting, OSX, OPN, MSX2 and OCN showed increased downregulation with increased exposure time to the tested cements. Alkaline phosphatase enzyme levels generally declined after day 7. Based on these results, it is concluded that tricalcium silicate cements do not induce osteogenic differentiation of hBM-MSCs in vitro