Methods: Osteoblasts were isolated from calvariae of newborn rats. These cells express the typical osteoblastic phenotypes such as increased ALP activity and potential to form mineralised tissue after addition of ascorbic acid and β-glycerophosphate to medium. After treatment with ALN, proliferation (cell count or MTT assay) and differentiation (ALP activity) of osteoblasts were analysed.
Results: Treatment with ALN from 10-8 to 10-5 M for 48 h or 72 h did not affect proliferation of osteoblasts, but that from 10-5 to 10-4 M inhibited proliferation in a concentration-dependent manner. This inhibition was partially reversed by addition of a metabolite of the mevalonate pathway (geranylgeraniol) or a pancaspase inhibitor (z-VADfmk). Number of osteoblasts treated with 10-4 M ALN for 72 h during a middle phase of culture period was greater at day 26 than that of those treated during an early phase. However, osteoblasts treated in the middle phase expressed a higher ALP activity at day 26 than did those treated during the early phase.
Conclusions: We confirmed that, as shown in other cells, ALN at higher concentrations decreases viability of osteoblasts through inhibition of the mevalonate pathway and acceleration of apoptosis. Marked effects on ALP activity were observed when treated during the middle phase of culture period rather than during the early phase, suggesting that ALN targets more matured osteoblasts.