Ion microprobe analysis of mouse calvariae in vitro: evidence for a "bone membrane"

It is not clear whether the bone mineral is in passive physicochemical equilibrium with the extracellular fluid (ECF) or is separated from it by a metabolically active partition, a so-called "bone membrane." We used a sensitive high spatial resolution scanning ion microprobe utilizing secondary ion mass spectrometry to compare the relative concentrations of 23Na, 39K, and 40Ca on the surface, subsurface, and cross section of cultured live bone with the concentrations in similar regions of dead bone. Calvariae from neonatal mice were dissected and either incubated for 24 h (live) or subjected to 3 freeze-thaw cycles to kill the bone cells prior to incubation (dead). The live bone has abundant surface Na and K relative to Ca and the Na/K is approximately unity. With dead bone there is a dramatic fall in the K/Ca and an increase in the Na/K. These findings are most consistent with an egress of bone K after cell death. Flux measurements indicate a net influx of Ca into the dead bone. The marked change in relative ion concentrations with cell death indicates that live bone is not in passive physiochemical equilibrium with the surrounding medium. There appears to be a metabolically active partition, a so-called bone membrane, between the mineral and the culture medium that utilizes bone cells to maintain ion gradients.