Comparison of in vitro and in vivo 44Ca labeling of bone by scanning ion microprobe

To determine whether Ca incorporation from medium into cultured bone represents normal mineralization, we labeled some neonatal mouse calvariae in vitro and others in vivo with the stable isotope 44Ca and compared surface label localization with a scanning ion microprobe utilizing secondary ion mass spectrometry. To label in vitro, we incubated live calvariae in medium containing 40Ca or 44Ca for 3 h. Compared with a 44Ca/40Ca ratio of 0.020 with 1 mM 40Ca, the ratio with 1 mM 44Ca was 0.135 and with 2 mM 44Ca was 0.556. Erosion revealed a marked decrease in 44Ca/40Ca with depth. To label in vivo, we subcutaneously injected 40Ca or 44Ca into mice equal to a percentage of their total body weight and dissected the calvariae 24 h later. Compared with a 44Ca/40Ca ratio of 0.021 with 2% 40Ca, the ratio with 2% 44Ca was 0.120 and with 6% 44Ca was 0.205. Erosion revealed only a slight decrease in 44Ca/40Ca with depth. Elemental distribution maps of in vivo labeled samples show broad deposition of 44Ca, whereas maps of in vitro labeled bones show 44Ca preferentially localized at the surface in contact with the medium. Thus calvariae can be labeled with 44Ca both in vitro and in vivo. However, the differing patterns of isotope localization under the conditions of this study indicate that in vitro Ca deposition differs from normal in vivo bone mineralization.