Effect of Combined Action of Extracellular ATP and Elevated Calcium on Osteogenic Differentiation of Primary Cultures from Rat Calvaria

Abstract

The in vitro osteogenic differentiation has been intensively studied. However, it is not yet clear precisely how osteogenesis can be optimized.Changes in extracellular Ca2þ concentration ([Ca2þ]e), as well as modulation of purinergic receptors play an important role in the regulation ofosteoblasts differentiation and bone formation. In this study, we investigated the effects of a combined treatment of ATPg-S and high [Ca2þ]e(5.35 mM) on osteogenic differentiation and function of primary cell cultures from rat calvaria. Our results indicate that ATPg-S stimulates celltransition from the G0 to S phase of cell cycle, involving the PI3K signaling pathway. Treatment with 10 or 100 mM ATPg-S and [Ca2þ]e (ATP-[Ca2þ]e) for 48 h increases cell number significantly above the control. ATPg-S treatment in osteogenic medium containing [Ca2þ]e stimulatesthe gene expression of BMP-4, BMP-5, and OPN at 16, 48, and 72 h, respectively, above control. In same conditions, treatment for 6 days with10mMUTP or 100mMUDP significantly increased the ALP activity respect to control. Cells grown in osteogenic medium showed a statisticallysignificant increase in calcium deposits at 15 and 18 days, for 10mMATPg-S treatment, and at 18 and 22 days, for [Ca2þ]e treatment, respect tocontrol but ATP-[Ca2þ]e treatment shown a significant greater mineralization at 15 days respect to ATPg-S, and at 18 days respect to bothagonists. In conclusion, we demonstrated that an osteogenic medium containing 10mM ATPg-S and 5.35mM [Ca2þ]e enhance osteogenesisand mineralization by rat primary calvarial cells cultures.