Morphological effects of serum-starvation on RANKL-induced osteoclastogenesis in RAW264.7

Objectives: Stable and repetitive induction of osteoclast formation in vitro is important for bone-related researches. There are contraversial arguments about serum starvation before induction of cell differentiation. On one hand, it is generally taken that exit from cell cycle and synchronization into G1/G0 is a frequent prelude to differentiation, and serum starvation is one of the effective ways. On the other hand, recent evidence showed that differentiation is not restricted to cessation of cell proliferation and entry to G0 , and receptor activator of nuclear factor-kappa B ligand (RANKL) could induce osteoclasts from asynchronously proliferating precusors. Moreover, serum starvation may induce cell apoptosis. The aim of this study was to compare the morphological effects of serum starvation on osteoclastogenesis induced by RANKL RAW264.7.

Methods: RAW264.7 cells were seeded in 48-well paltes at density of 5x103 per well, followed by serum starvation (1% fetal bovine serum) for 12 hours or not (control). Then for osteoclastgenesis, the cells were treated with RANKL (50ng/ml) ina-MEM supplemented with 10% serum for 4 days. As additional experimental groups, 24-hour starvation with 1% serum and 12-hour starvation without serum were also included. The morphological change was observed after tartrate-resistant acid phosphatase (TRAP) staining microscopically.

Results: 24-hour starvation with 1% serum and 12-hour starvation without serum both induced large amount of apoptosis. Compared with the control wells (no starvation), 12-hour starvation with 1% serum dramatically increased osteoclast size (especially >600µm), indicating increased osteoclast development, fusion and cytoplasmic extension.

Conclusion: Although serum-starvtion is not necessary to osteoclastgenesis, 12-hour starvation with 1% serum will facilitate osteoclastgenesis induced by RANKL in RAW264.7. Excessive or severe starvation will induce cell apoptosis.