Osteogenic differentiation was evaluated by detecting calcium depositions with Alizarin red staining after 21 days of differentiation (
Fig. 4a). Staining of MSCs with Alizarin red solution did not indicate any calcium depositions before differentiation (
Fig. 4a). Strong Alizarin red staining was observed in the differentiated ASCs and, to a lesser extent, in BMMSCs, UCSCs, and CSSCs after 21 days of differentiation (
Fig. 4a). To confirm these observations, the expression of four osteoblast-associated genes was analyzed at days 7 and 21 of differentiation: alkaline phosphatase
(ALP), runt-related transcription factor 2 (
RUNX2),
46 osteocalcin (
BGLAP),
47 and osteonectin (
SPARC).
48 ALP expression was upregulated by 3.5-fold (
P < 0.05) in all differentiated MSCs (
Fig. 4b).
RUNX2 mRNA levels were elevated (
P < 0.05) more than 2.1-fold after 7 and 21 days of differentiation of all MSCs (
Fig. 4c).
BGLAP expression was not increased in any of the MSC types after differentiation for 7 or 21 days (
Fig. 4d).
SPARC expression was upregulated 6.5-fold in UCSCs and 1.5-fold in CSSCs (
P < 0.05) on day 7, and
SPARC expression was upregulated 2.7-fold in BMMSCs on day 21 of differentiation (
Fig. 4e).
SPARC expression was downregulated in ASCs after 7 and 21 days of differentiation. These results can be explained by the later expression of
SPARC and
BGLAP during osteogenic differentiation.
47–49 Twenty-one days of differentiation was not long enough to stimulate the increased expression of those genes in the investigated MSCs; this finding has been observed previously in another study of BMMSCs.
46 Overall, ASCs and BMMSCs displayed the most Alizarin red staining, and this staining was correlated with increased levels of
ALP and
RUNX2 after differentiation.