Effects of bisphosphonates APD and HEBP on bone metabolism in vitro

Although the effects of the bisphosphonates on resorption have been well documented, their effects on bone formation are not as clear. Therefore, this investigation was undertaken to elucidate the role played by bisphosphonates in the regulation of bone formation in vitro. To evaluate bisphosphonate-mediated regulation of bone formation in vitro, the effects of two drugs, ethane-1-hydroxy,1-diphosphate (Etidronate) (HEBP), and the second-generation bisphosphonate, disodium-1-hydroxy-1-aminopropylidine-1,1-diphosphate (Pamidronate) (APD), were assessed in the chick periosteal osteogenesis (CPO) model. In this study, drug-induced changes in alkaline phosphatase were assessed at the cellular level by means of quantitative fluorescence histochemistry. Cellular proliferation was quantified by means of autoradiography ([3H]thymidine). Mineralization and matrix production were measured morphometrically, whereas collagen synthesis and degradation were measured biochemically. The data suggest that in addition to their effects on bone resorption, the bisphosphonates have marked and direct effects on bone formation and other parameters of osteogenesis. HEBP may affect cellular proliferation (75–80% reduction, p< 0.05) in zones distant from bone; alkaline phosphatase positive cell numbers were increased in the osteoblastic layer of cells (twofold relative to control, p< 0.05) in 12-day cultures. HEBP, but not APD, prevented mineralization-induced suppression of matrix synthesis in early stages of culture. In 6-day cultures induced to mineralize with β-glycerophosphate, (GP) cotreatment with HEBP induced a 70% increase in collagen synthesis. In addition, degradation of collagen in the CPO cultures was inhibited by HEBP (25%) and to a lesser extent by APD (8%). Although there were no differences in bone-osteoid areas measured in 12-day cultures treated with various regimens of bisphosphonate or GP, a clear increase in bone-osteoid area was detected in 6-day cultures treated with GP and HEBP as compared to GP only. This may suggest that initially, osteoblasts may be induced to synthesize increased volumes of bone matrix when mineralization is inhibited (e.g., with HEBP), but that over time the osteoblasts make the same amount of matrix. Taken together, these findings indicate that whereas the bisphosphonates do have well-documented effects on bone resorption, their effects on bone formation may also be important. The data suggest further that these drugs may affect osteogenesis in vitro, in part through their effects on mineralization and perhaps on differentiation or phenotypic expression in osteoblasts.