The clinical need for bone graft substitutes has led tissue engineering strategies to investigate strategies to create osteoconductive, osteoinductive, and osteogenic constructs for bone repair and regeneration. The strategy in this research work is to use a cell-secreted extracellular matrix (ECM) to influence the osteoblastic differentiation of marrow stromal cells (MSCs). The effects of an in vitro generated ECM on MSC osteoblastic gene expression under static culture indicated that MSCs differentiated down the osteoblastic lineage evidenced by significant increases in expression of the osteoblastic markers, such as collagen type I and osteocalcin. When cultured on an ECM, osteoblastic differentiation was accelerated and enhanced through (1) earlier upregulation of osteopontin and osteocalcin; and (2) maintenance of a high level of expression of these and other osteoblast-specific genes. The upregulation in osteoblastic gene expression leading to significant increases in calcium deposition is likely mediated through the interactions between the growth factors and other matrix molecules found in the in vitro generated ECM since the expression of insulin-like growth factor, vascular endothelial growth factor, fibromodulin, and dentin matrix protein was found to display significant peaks in expression level. To further modulate MSC differentiation, the cellular constructs were cultured in the presence of fluid shear stresses. The effects of fluid shear stress and the bioactivity of the in vitro generated ECM acted synergistically to enhance mineralized matrix deposition. However, this synergy occurred only when the constructs were cultured in the presence of dexamethasone. The spatial distribution of cells and ECM was markedly improved compared to static culture. The osteoinductive and angiogenic potential of ECM constructs generated in a flow perfusion bioreactor for different peroids of time were tested in an ectopic site in a rodent model. No osteoinduction was observed although an increase in the number of blood vessels within the implants with increasing ECM was noted. This study emphasized the challenges in creating ideal conditions for de novo bone formation and underscored the need for optimization of this strategy for in vivo applications. These studies led to the transition from titanium to polymeric fiber scaffolds generated via electrospinning. The system was used to fabricate scaffolds with varying fiber diameters and pore sizes as well as multi-layered scaffolds. It was demonstrated that nanofibers could inhibit the spatial distribution of cells and ECM, despite culture in the bioreactor. These scaffolds hold tremendous potential as tissue engineering scaffolds and will be investigated further as part of the cell-secreted ECM-based scaffold strategy.