Bone Abstracts

Osteoclasts are produced by fusion of pre-osteoclasts derived from stem cells of the monocyte/macrophage lineage in the presence of receptor activator of NF-κB ligand (RANKL) produced by osteoblasts. The phytoestrogens; genistein and daidzein, which are isoflavones found in Leguminosae such as soybeans, are currently being investigated for prevention of postmenopausal osteoporosis. Some polyunsaturated fatty acids (PUFAs) have been shown to exert a bone protective effect in certain communities. The aim of this study was to determine the in vitro effects of genistein and daidzein together with the PUFAs, arachidonic acid (AA) and docosahexanoic acid (DHA) on osteoclastogenesis and bone resorption.

For osteoclast formation, RAW 264.7 murine macrophages were grown in the presence of RANKL (15 μg/ml), 0.02% ethanol (vehicle control), PUFAs (20 μM–80 μM) and phytoestrogens (10⁻⁹ M–10⁻⁵ M) for 5–7 days, stained for tartrate resistant acid phosphatase and the number of multinucleated osteoclasts quantified. Bone resorption assays were conducted on osteoassay plates coated with an inorganic synthetic bone surface. After 7 days of incubation, cells were washed off and after performing a von-Kossa stain, resorption was observed with a microscope. Three experiments were conducted in quadruplicate.

Results show that the formation of multinucleated osteoclasts was dose-dependently inhibited by exposure to either PUFAs or phytoestrogens. When the cells were exposed to these compounds, resorption pits on the bone analogue plates were smaller compared to the vehicle control. This observation may be attributed to a decrease in the number or activity of mature resorbing osteoclasts. Combination of PUFAs and phytoestrogens resulted in major inhibition of osteoclastogenesis and resorption pit formation suggesting an additive effect of these compounds in this model. PUFAs and phytoestrogens may have a protective effect on bone in vitro and combining these agents may exacerbate the inhibition. More work is required to confirm these findings and to clarify the possible mechanisms involved.