CALCIUM AND ORTHOPHOSPHATE DEPOSITS INVITRO DO NOT IMPLY OSTEOBLAST-MEDIATED MINERALIZATION - MINERALIZATION BY BETAGLYCEROPHOSPHATE IN THE ABSENCE OF OSTEOBLASTS

It has been shown in several laboratories that addition of beta-glycerophosphate (βGP), a substrate for alkaline phosphatase (AP), to cultured osteoblast-like cells induces deposition of orthophosphate (Pi) and Ca within seven days. Even though this effect is regarded as an in vitro model of bone mineralization, it is not known whether it is specific for osteoblasts. We have, therefore, studied the amounts of Pi and Ca deposited after seven days with 10 mM βGP in culture wells containing confluent cultures of osteoblast-like cells (OB) derived from human trabecular bone expiants, human skin fibroblasts (SF), or culture medium alone (MED). Ox liver AP at an activity considerably greater than the endogenous AP activity of the cells, but comparable with that of other osteoblast models, was added to ensure a similar rate of Pi generation from βGP in all wells. βGP was converted quantitatively to Pi within seven days, leading to a nonphysiological 10-fold increase in the Pi concentration in the culture medium. After thorough rinsing on day seven, the OB and SF wells contained deposits of Pi and Ca, but the amounts were comparable for the two cell types. Smaller, but significant, amounts of Pi and Ca were also detectable even in rinsed MED wells. This suggests that the detection of such deposits in βGP experiments cannot necessarily be interpreted as a specific property of osteoblast cultures in vitro, and may simply reflect the presence of AP. It was also shown that, even though phosphatases in human plasma incubated for seven days in vitro with added 10 mM βGP can hydrolyze a large fraction of the βGP to Pi, endogenous organic phosphates in the plasma, which occur at a similar concentration, are hydrolyzed only slowly. This implies that no naturally occurring analogue of βGP is present at high concentrations in extracellular fluid in humans. © 1990.