Mechanical stimulation effects on functional end effectors in osteoblastic MG-63 cells

Receptor activator of Nf-κB ligand (RANKL) and osteoprotegerin (OPG) have been implicated in bone metabolism. Specifically, the balance of these factors in conjunction with receptor activator of Nf-κB (RANK) is believed to be key in determining the rate of osteoclastogenesis and the net outcome of bone formation/resorption. While it is well accepted that mechanical loading in vivo affects bone formation/resorption and that alterations in the responsiveness of bone cells to mechanical loading have been implicated in metabolic bone diseases, the effect of in vitro mechanical loading on osteoblastic production of OPG and RANKL has not been extensively studied. Thus, in the current study, we developed an in vitro model to load human osteoblasts and studied levels of OPG, RANKL, PGE2 and macrophage colony stimulating factor (M-CSF). We hypothesized that stimulating osteoblastic cells would increase the release of soluble OPG relative to RANKL favoring a bone-forming (and resorption-inhibiting) event. To accomplish this, we developed a small-scale loading machine that imparts via bending, well-defined substrate deformation to bone cells cultured on artificial substrates. Following 2 h of loading and a 1 h incubation period, media was collected and levels of soluble OPG, RANKL, PGE2 and M-CSF were quantified using ELISA and western blotting. We found that mechanical loading significantly increased soluble OPG levels relative to RANKL at this 3 h time point. Levels of soluble and cellular RANKL detected were not significantly affected by mechanical stimulation. The relative shift in abundance of OPG over RANKL associated with applied mechanical stimulation suggests the soluble OPG:RANKL ratio may be important in load-induced coupling mechanisms of bone cells.