Expansion and trilineage differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). (A) Cell counts, (B) population doublings per day, and (C) cumulative population doublings at each passage during expansion of BMSCs under normoxia or hypoxia. Data points represent mean ± standard error of the mean of cells from six donors, and P values are listed. (D) Osteogenic differentiation of BMSCs verified with Alizarin Red S staining following expansion under hypoxia and monolayer culture within medium containing β-glycerophosphate, dexamethasone, and fetal bovine serum. (E) Adipogenic differentiation of BMSCs verified with Oil Red O staining following expansion under hypoxia and monolayer culture within medium containing isobutyl-1-methylxanthine (IBMX), indomethacin, dexamethasone, and fetal bovine serum. (F) Chondrogenic differentiation of BMSCs verified by safranin O staining following expansion under hypoxia and culture performed in pellets (left) or scaffolds composed of collagen (middle) or hyaluronic acid (right) submersed in a defined serum-free chondrogenic medium containing transforming growth factor-beta 3 and dexamethasone.