SECRETION OF A BONE-INDUCING AGENT (BIA) BY CULTURED SAOS-2 HUMAN OSTEOSARCOMA CELLS

An extractable bone-inducing agent has recently been identified in freeze-dried preparations of Saos-2 cultured human osteosarcoma cells. Although not all osteoinductive components of Saos-2 cell extracts have been identified, we have shown that Saos-2 cells express high levels of mRNA for bone morphogenetic proteins (BMPs)-1,2,3,4 and 6. Any or all of these BMPs (plus possible unknown factors) may be involved in ectopic bone induction, and may act as paracrine agents, conveying morphogenetic information to juxtaposed osteoprogenitor cells. Our objectives in this study were: 1) to determine whether Saos-2 cells secrete BIA into their culture medium; and 2) if secreted, to determine whether released bone-inducing agent is soluble and/or particulate and contains BMPs. Saos-2 cells were grown to confluence, and then overlaid with serum-free DMEM culture medium for 48 hours. The serum-free conditioned medium was then decanted and filtered through 0.45μ pore-size filters to retain any vesicles or other particulates released by the cells. Particulate protein retained on the 0.45μ filter (designated "retentate") was extracted into 6M urea and bioassayed for bone-inducing activity in Nu/Nu mice, along with soluble media protein that had passed through the 0.45μ filter (designated "filtrate") plus freeze-dried Saos-2 cells from which conditioned the culture medium was obtained. Results indicate that the bone-inducing agent of Saos-2 cells is not only retained by the cells, but is also secreted in both soluble and particulate forms into serum-free conditioned medium. Bone-inducing activity (per mg protein) is more concentrated in the particulate fraction, which is shown by electron microscopy to contain a mixture of vesicles (similar to matrix vesicles) plus electron dense granules (resembling ribosomes) and 10 mM microfilaments (of possible collagenous or cytoskeletal origin). BMP-1,2,3,4,6 and 7 were detected by western blots in both the soluble and particulate fractions of conditioned medium. Thus, it is indicated that Saos-2 cells secrete an osteoinductive factor which may function in vivo as a paracrine morphogenetic agent.