Bone regeneration is a complex process that involves the synergistic contribution of multiple cell types and numerous growth factors (GFs). It is widely accepted that effective and efficient reconstruction of critical size skeletal defects and non-unions can be achieved by tissue engineering approaches employing multi-factor and multi-phase GF delivery strategies. However, the studies investigating the involvement of multiple factors in osteogenesis are limited to simplified 2-dimensional in vitro studies with particular cell types or complex in vivo studies with associated experimental hurdles. There is a need for an in vitro model that embodies the multicellular and 3-dimensional (3D) nature of osteogenesis without the complexities of in vivo animal models. Bone marrow tissue consists of multiple cell types, houses the multipotent mesenchymal and hematopoietic stem cells, and plays a major role in bone regeneration. Marrow has a unique microenvironment and inherently ossifies in vitro under basal conditions (i.e. without addition of excipient osteoinductive factors). Therefore the main objective of this dissertation was to harness the inherent ossification potential of rat bone marrow tissue and develop a representative 3D, multicellular, scaffold-free in vitro model of osteogenesis as a platform to study the temporal and interconnected involvement of multiple GFs. The specific aims of this work were:​ 1) optimizing and characterizing the in vitro ossification of marrow tissue, 2) tracing the sequential production profiles of key GFs in osteogenesis and their relation to ossified volume in marrow ossification model, and 3) assessing the mechanoresponsiveness of marrow ossification process and the effect of mechanical stimulation on the temporal production levels of GFs. Specifically, the osteogenic involvement of bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1) and transforming growth factor beta-1 (TGF-beta1) were studied. The key findings of this dissertation are:​ 1) in vitro ossification of bone marrow can be achieved under serum-free conditions resulting in a 3D tissue structure with characteristic morphological, compositional and cellular properties of newly forming bone, 2) BMP-2, VEGF, IGF-1 and TGF-beta1 are sequentially produced and secreted during in vitro ossification of marrow, 3) The levels of BMP-2, VEGF, IGF-1 and TGF-beta1 at specific time points correlate with the final ossified volume and they are highly interdependent to each other, 4) in vitro ossification model is mechanoresponsive and responds to mechanical stimulus by increased bone volume with enhanced or sustained release of VEGF, IGF-1 and TGF-beta1, but not BMP-2. These outcomes are essential for delineating the temporal and interconnected involvement of multiple growth factors in osteogenesis and the role of mechanical cues in this process