Current protocols for inducing osteogenic differentiation in mesenchymal stem/stromal cells (MSCs) in culture for tissue engineering applications depend on the use of biochemical supplements. However, standard in vitro culture conditions expose cells to ambient oxygen concentrations and high levels of serum (21% O2, 10% FBS) that do not accurately recapitulate the physiological milieu. While we and others have examined MSC behavior under hypoxia, the synergistic effect of low serum levels, such as those present in ischemic injury sites, on osteogenic differentiation has not been clearly examined. We hypothesized that a concomitant reduction of serum and O2 would enhance in vitro osteogenic differentiation of MSCs by more accurately mimicking the fracture microenvironment. We show that serum deprivation, in conjunction with hypoxia, potentiates osteogenic differentiation in MSCs. These data demonstrate the role of serum levels in regulating osteogenesis and its importance in optimizing MSC differentiation strategies.
Highlights: Serum levels, in addition to hypoxia, have a significant effect on MSC osteogenic differentiation. Both naïve and osteogenically induced MSCs exhibit higher osteogenic markers in reduced serum. MSCs deposit the most calcium under 5% O2 in osteogenic media supplemented with 5% FBS. Standard culture conditions (21% O2, 10% FBS) may not be optimal for MSC osteogenic differentiation.