Osteogenic Differentiation of Mesenchymal Stem Cells on Pregenerated Extracellular Matrix Scaffolds in the Absence of Osteogenic Cell Culture Supplements

Abstract

This study utilized a full-factorial design to investigate the effect of four factors: presence of whole bone marrow cells, presence of in vitro-generated mineralized extracellular matrix (ECM), presence of dexamethasone, and variations in culture duration, on the proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) cultured on a polymer scaffold. Electrospun poly(epsilon-caprolactone) (PCL) fiber mesh scaffolds were seeded with rat MSCs and cultured in complete osteogenic medium for 12 days to generate constructs containing mineralized ECM. MSCs or MSCs and whole bone marrow cells were seeded onto decellularized ECM constructs (PCL/ECM) or plain PCL scaffolds and cultured statically for 4, 8, and 16 days in medium either with or without dexamethasone. After each culture period, the cell number was determined by DNA analysis, and the osteogenic differentiation state of the cells was determined by alkaline phosphatase activity and calcium assays. MSCs seeded onto PCL/ECM constructs and cultured in medium either with or without dexamethasone demonstrated similar amounts of calcium deposition after 16 days. A significant increase in cell number over time compared with all other groups was observed when whole bone marrow cells were cocultured with MSCs on PCL scaffolds in medium without dexamethasone. This study establishes that the osteogenic differentiation of MSCs seeded onto ECM-containing constructs is maintained even in the absence of dexamethasone and that the coculture of MSCs and whole bone marrow cells without dexamethasone and ECM enhances the proliferation of a cell population (or populations) present in the whole bone marrow.