OSTEOCLAST DIFFERENTIATION REQUIRES ASCORBIC-ACID

Osteoclast differentiation assays are usually conducted in alpha minim al essential medium (alpha-MEM). We reasoned that determining which co mponents of this media are critical for osteoclast differentiation mig ht provide insight into the mechanisms that regulate osteoclast differ entiation. This study demonstrates that ascorbic acid is the crucial c omponent of alpha-MEM that stimulates differentiation of murine osteoc lasts in cocultures with murine mesenchymal support cells. Thus, suppl ementation with ascorbic acid allows osteoclast differentiation to occ ur in basal MEM media as well as in RPMI-1640 and basal media Eagle (B ME) media. The conclusion that osteoclast differentiation is stimulate d by ascorbic acid was obtained whether osteoclast differentiation was induced by 1,25-dihydroxyvitamin D-3 or parathyroid hormone, whether Sn or CIMC-2 cells were used as mesenchymal support cells, and whether osteoclast precursors were obtained from spleen or bone marrow, Time course studies revealed that although ascorbic acid only modestly incr eases the rate at which osteoclast precursors begin to express tartrat e-resistant acid phosphatase, it strongly increases the rate at which precursors fuse into mature, multinucleated cells. Moreover, ascorbic acid strongly increases the life span of both osteoclasts and their pr ecursors, The increases in precursor formation, fusion, and life span induced by ascorbic acid are together responsible for the stimulation of osteoclast differentiation by ascorbic acid. Given the known effect s of ascorbic acid on differentiation of mesenchymal cells, it may sti mulate osteoclast differentiation indirectly by regulating the differe ntiation state of the mesenchymal cells that support osteoclast differ entiation.