Substrate induction of osteogenesis from marrow-derived mesenchymal precursors

Therapeutic modalities aimed at bone regeneration are increasingly employing extracellular matrix (ECM) constituents to control bone marrow progenitor cell (BMPC) commitment, growth, and differentiation. However, the precise role these ECM elements play during stem cell differentiation remains unclear. (See also Salaszynk et al., Stem Cells Dev 14(6): 608-620, 2005; and Schwartz et al., Stem Cells Dev. 14(6), 643-655, 2005, both in this issue.) Because bone formation ultimately begins with the recruitment and commitment of BMPCs into the osteogenic lineage, factors that enhance this process are clearly therapeutic targets. We hypothesized that BMPC attachment, proliferation, and osteogenic differentiation would be potentiated when cultured on ECM proteins normally found in the bone niche. To examine this, we cultured murine BMPCs on laminin-1, fibronectin, and collagen type-1 substrates for up to 14 days and assessed their homogeneity, attachment, proliferation, and expression of the specific bone lineage markers RUNX2, collagen-1, alkaline phosphatase, and osteocalcin. We found that freshly harvested mBMPCs contain a mixed population of progenitor cells and that the mesenchymal pool can be enriched by adherent culture in the presence of leucine methyl ester. Furthermore, mBMPCs attached to laminin, fibronectin, and collagen-1 with varying affinity up to 3 h (fibronectin >= collagen > laminin), after which time no difference could be detected. Despite this, growth was unaffected; cells thereafter proliferated equally well on all substrates up to confluence (7 days). Notably, commitment to the osteoblast lineage (RUNX2) increased up to 14 days for cells cultured on the various substrates, yet no difference was observed at day 14 in the expression of collagen-1, alkaline phosphatase, or osteocalcin. We conclude that mBMPC differentiation down the osteoblastic lineage is time-dependent in osteogenic culture and that attachment to ECM matrices potentiates lineage commitment rather than growth.