Effect of caffeine on parameters of osteoblast growth and differentiation of a mineralized extracellular matrix in vitro.

The effects of caffeine exposure on bone formation were examined using a chick osteoblast culture system. Secondary cultures of normal diploid osteoblasts were exposed to chronic doses of 0, 0.1, 0.2, or 0.4 mM caffeine beginning on day 0 through day 28. Neither the rate of cell proliferation nor cell number, as measured by total DNA, was decreased for any of the doses examined. In contrast, osteocalcin levels, alkaline phosphatase activity, and total calcium levels showed a dose-related decrease in cultures treated with caffeine. These parameters were significantly decreased at the highest dose of 0.4 mM. The reduction in total protein levels ranged from 29 to 66% of control values and was independent of dose. In contrast, total collagen levels were more affected by the dose of caffeine used. Inhibition of collagen levels was most apparent on days 17 and 21, time points during the period of active formation of the matrix immediately preceding the deposition of mineral. By day 28 collagen levels in cultures exposed to the lower doses of caffeine had returned to control levels, and only the cultures exposed to the highest dose (0.4 mM) remained significantly inhibited with respect to both collagen and mineral. Histochemically, alkaline phosphatase and mineral staining of day 28 cultures mirrored the biochemical events with the 0.4 mM caffeine exposure. The results indicate that one of the effects of caffeine on bone development is to inhibit the formation of a competent extracellular matrix during the osteoblast differentiation sequence, which results in the inhibition of mineralization analogous to the delayed ossification observed in fetal animals after prenatal caffeine exposure.