This study addresses the role of bone morphogenetic protein-7 (BMP-7) in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells (BM MSCs) in vitro. BM MSCs were expanded and differentiated in the presence or absence of BMP-7 in monolayer and three-dimensional cultures. After 3 days of stimulation, BMP-7 significantly inhibited MSC growth in expansion cultures. When supplemented in commonly used induction media for 7-21 days, BMP-7 facilitated both chondrogenic and osteogenic differentiation of MSCs. This was evident by specific gene and protein expression analyses using real-time PCR, Western blot, histological, and immunohistochemical staining. BMP-7 supplementation appeared to enhance upregulation of lineage-specific markers, such as type II and type IX collagens (COL2A1, COL9A1) in chondrogenic and secreted phosphoprotein 1 (SPP1), osteocalcin (BGLAP), and osterix (SP7) in osteogenic differentiation. BMP-7 in the presence of TGF-beta3 induced superior chondrocytic proteoglycan accumulation, type II collagen, and SOX9 protein expression in alginate and pellet cultures compared to either factor alone. BMP-7 increased alkaline phosphatase activity and dose-dependently accelerated calcium mineralization of osteogenic differentiated MSCs. The potential of BMP-7 to promote adipogenesis of MSCs was restricted under osteogenic conditions, despite upregulation of adipocyte gene expression. These data suggest that BMP-7 is not a singular lineage determinant, rather it promotes both chondrogenic and osteogenic differentiation of MSCs by co-ordinating with initial lineage-specific signals to accelerate cell fate determination. BMP-7 may be a useful enhancer of in vitro differentiation of BM MSCs for cell-based tissue repair.

This study addresses synergistic effects of bone morphogenetic protein-2 (BMP-2) and transforming growth factor-beta3 (TGF-beta3) in the induction of chondrocytic differentiation of bone marrow multipotent mesenchymal stromal cells (BM MSCs) in vitro for potential use in intervertebral disc (IVD) repair. Human BM MSCs encapsulated in alginate beads were induced to differentiate in serum-free medium containing BMP-2 and TGF-beta3. The expression of chondrocytic genes and proteins was analyzed by real-time PCR, western blot, histological, and immunohistochemical assays. This differentiation system showed a potent induction of chondrocytic phenotypes. The expression of chondrocytic markers, such as aggrecan (ACAN) and type II collagen (COL2A1), was upregulated at higher levels than using TGF-beta3 alone. Blocking BMP-2 by noggin completely suppressed BMP-2-enhanced gene and protein expression, confirming a crucial input of BMP-2 signaling in this differentiation process. Inhibition of extracellular signal-regulated kinases 1 and 2 signaling resulted in an increase in ACAN and COL2A1 gene expression, suggesting a negative regulatory role of this pathway. In conclusion, BMP-2 enhances TGF-beta3-mediated chondrogenesis of MSCs. The combination of BMP-2 and TGF-beta3 in alginate culture is superior to the standard differentiation method using TGF-beta alone. This potent induction system may provide an alternative cell source for IVD and cartilage regeneration in clinical practice.