Formation of Cement Lines, But Not Laminae Limitantes, Requires Contact of Differentiating Osteogenic Cells to Solid Surfaces

We designed in vitro experiments to address the hypothesis that cement lines and laminae limitantes are structurally distinct as a result of cell contact, or absence of cell contact, during their elaboration. We subcultured young adult primary rat bone marrow cells in well inserts with polycarbonate membranes of 0.45 JLm pore size, which allowed protein but not cell passage. The cells, extracellular matrix, lower side of the membrane and the surface of the polystyrene culture well were examined by scanning and transmission electron microscopy. The cells elaborated a bone-like matrix on top of the membrane. In such cultures the cell/substrate interface, beneath developing bone nodules, is occupied by a collagen-free mineralized cement line-like matrix about 0.5JLm thick:. Matrix was also observed on the cell-free surfaces on the lower side of the membrane, and the well-surface. This matrix morphologically resembled laminae limitantes and did not occur in cell-free control cultures. On the lower side of the membrane the matrix varied in thickness from 20-100 nm, while on the polystyrene well bottoms it was of a more uniform thickness (about 40 nm). Calcium phosphate mineralization, in the presence of osteopontin and bone sialoprotein, was detected at both cell-free surfaces by X-ray photoelectron spectroscopy (XPS) and immunofluorescence. These data suggest that the key factor which results in the structurally divergent features of these bony extracellular architectures, lamina limitans and cement line, is direct cell contact to osteogenic cells at solid surfaces.