Optimal supplementation of dexamethasone for clinical purposed expansion of mesenchymal stem cells for bone repair

Although mesenchymal stem cells (MSCs) are generally considered to represent a very promising tool for bone repair, no optimal protocol has yet been developed for the isolation and expansion of these cells for large-scale clinical applications. Mesenchymal stem cells were supplemented with four different concentrations of dexamethasone: 0 M (Con), 0.2 x 10(-8) M (D0.2), 1.0 x 10(-8) M (D1.0) and 5.0 x 10(-8) M (D5.0); and analyzed every week for 5 weeks (P1-P5). Cells were analyzed via an alkaline phosphatase assay, DNA quantification, Oil Red stain, and flow cytometry for CD105 and CD90. Additionally, P3 and P5 cells were subcutaneously transplanted into nude mice after seeding in ceramic cubes. Proliferation of the cells was significantly higher in the D0.2 group. Alkaline phosphatase activities remained at low levels in the Con and D0.2 groups, but increased to high levels in the D1.0 and D5.0 groups as time elapsed. CD105 expression at P5 was lower than at P1, P2 and P3. Adipocyte differentiation was highest at P3. At the 8th week, in vivo bone formation was enhanced by the MSCs in a dexamethasone-supplemented culture for 3 or 5 weeks, and D0.2 was also higher than Con. The supplementation of MSCs under low-level rather than physiological concentrations (2 x 10(-9) M) of dexamethasone facilitates the culture expansion of these cells for osteogenic purposes by enhancing cell proliferation without diverse differentiation, and also promotes bone formation after in vivo transplantation.