Expression of osteogenic marker genes in both mesenchymal stem cell spheroids and monolayer rat mesenchymal stem cells. Quantitative reverse transcription-polymerase chain reaction results for runt-related transcription factor-2, osterix, osteopontin, and bone sialoprotein, and alkaline phosphatase mRNA expression in mesenchymal stem cell (MSC) spheroids (black column) and monolayer rat MSCs (rMSCs; gray column) treated with an osteoblast-inducer reagent (OIR). Results are expressed as fold-increases in mRNA expression (normalized to that of glyceraldehyde 3-phosphate dehydrogenase mRNA) and compared with results for monolayer rMSCs treated with an OIR on Day 7. mRNA expression levels of osteogenic genes, other than alkaline phosphatase (ALP) (E), in MSC spheroids were increased, compared with those of monolayer MSCs (A-D). Vertical lines represent the mean ± standard deviation of three independent experiments, each performed in triplicate. *Significantly different at p < 0.05 compared with monolayer rMSCs. OSX, osterix; RUNX-2, runt-related transcription factor-2; OPN, osteopontin; BSP, bone sialoprotein; ALP, alkaline phosphatase.