The Sequential Production Profiles of Growth Factors and their Relations to Bone Volume in Ossifying Bone Marrow Explants

Osteogenesis is a complex process that involves the synergistic contribution of multiple cell types and numerous growth factors (GFs). To develop effective bone tissue engineering strategies employing GFs, it is essential to delineate the complex and interconnected role of GFs in osteogenesis. The studies investigating the temporal involvement of GFs in osteogenesis are limited to in vitro studies with single cell types or complex in vivo studies. There is a need for platforms that embody the physiological characteristics and the multicellular environment of natural osteogenesis. Marrow tissue houses various cell types that are known to be involved in osteogenesis, and in vitro cultures of marrow inherently undergo osteogenesis process. Self-inductive ossification of marrow explants in vitro can be employed as a representative multicellular and three-dimensional model of osteogenesis. Therefore, the aims of this study were to employ the rat bone marrow explant ossification model to determine (1) the temporal production profiles of key GFs involved in osteogenesis, (2) the relation between GF production and ossification, and (3) the relations between the GF levels throughout ossification. Temporal production profiles of transforming GF beta-1 (TGF-beta 1), bone morphogenetic protein-2 (BMP-2), vascular endothelial GF (VEGF), and insulin-like GF-1 (IGF-1) and the bone-related proteins alkaline phosphatase and osteocalcin were obtained by enzyme-linked immunosorbent assays conducted at days 2, 7, 12, 14, 19, and 21. The final amount of ossification (ossified volume [OV]) was measured by microcomputed tomography at day 21. TGF-beta 1, BMP-2, VEGF, IGF-1, alkaline phosphatase, and osteocalcin were produced by the ossifying marrow explants differentially over time. The early production of IGF-1 (day 2) correlated positively (r -0.868) with OV; however, latent production of IGF-1 correlated negatively (day 14: r =-0.813; day 19: r =-0.865) with OV. OV also correlated with VEGF levels at day 12 (r =0.988) and at day 14 (r=0.970). Production of GFs also correlated to each other across time points, which indicates the complex and interconnected contribution of various GFs in osteogenesis. Therefore, tissue engineering strategies toward bone regeneration should consider the richness of GFs involved in osteogenesis and their dynamically varying participation over time.