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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 ([ 3 H]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 p

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, p0.05) in zones distant from bone; alkaline phosphatase positive cell numbers were increased in the osteoblastic layer of cells (twofold relative to control, p0.05) 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 beta-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.(ABSTRACT TRUNCATED AT 400 WORDS)

The bisphosphonates, which are chemically related to pyrophosphate, have been studied extensively both in vivo and in vitro to elucidate their effects on bone tissues and cells. However, because these agents have important effects on bone resorption, the majority of investigations have focused on this area. Few studies regarding direct bisphosphonate effects on bone formation have been carried out in the past and, thus, we chose to use the chick periosteal osteogenesis (CPO) in vitro model system to test the direct effects of pyrophosphate and the bisphosphonates ethane-1-hydroxy-1,1-diphosphate (HEBP) and disodium-1-hydroxy-1-amino-propylidine (APD) on various parameters of osteogenesis in vitro. The data show that the bisphosphonate HEBP inhibits bone mineralization reversibly while APD, at low doses, may actually enhance mineralization of bone. Similarly, pyrophosphate (PPi) will prevent mineralization in CPO cultures. However, CPO cultures can circumvent PPi-mediated blockage of mineralization with longer-term, continuous (10-day) incubation, whereas this does not occur if cultures are incubated continuously with bisphosphonates. Both drugs appear to be able to reverse beta-glycerophosphate-induced changes in alkaline phosphatase activity, but do not appear on their own to regulate the activity of this enzyme. The findings show that in addition to their well-known effects on resorption, bisphosphonates have significant and direct effects on mineralization in bone-forming cultures. Their direct effects on osteoblastic activity and differentiation remain to be determined.

Mineralized bone formation in vitro can be induced by the alkaline phosphatase substrate beta-glycerophosphate (GP). GP may not only be essential for mineralization in vitro, but could also modulate other metabolic activities of bone cells, particularly if GP is presented to these cells during different phases of development. To assess GP modulation of bone cell metabolism, biochemical and autoradiographic analyses of chick periosteal cultures treated with GP were performed. About 50% less (p less than 0.05) Type I collagen was produced in periosteal cultures treated with GP. If the fibrous portion of the periostem was microdissected from the osteogenic layer prior to culture, GP inhibition of Type I collagen synthesis was even more marked (60%: p less than 0.05). To define organic phosphate-sensitive phases of osteogenesis, cultures were exposed to GP for various time periods. Mineralization occurred reproducibly when periosteal cultures were treated with GP from the outset of the incubation period (positive control). However, if GP was added after the third day of incubation, phosphate content was the same as in positive control cultures, whereas calcium content was significantly (20%: p less than 0.05) lower. Moreover, if GP was added on day 6, there was virtually no calcium accumulation by day 12, while massive amounts of phosphate had accumulated. Taken together, these findings indicate that organic phosphates may modulate phenotypic expression of osteogenic cells, and that osteogenic cells traverse an organic phosphate-sensitive phase, after which they may be incapable of normal mineralization.