Phosphatase actions at the site of appositional mineralization in bisphosphonate-affected bones of the rat

Tissue-nonspecifi c alkaline phosphatase(TNSALP) and Ca-ATPase are known to play rolesin bone mineralization, but how these enzymescontribute to appositional mineralization hasbeen illusive. Here we examined the active sitesof these enzymes in appositional mineralizationusing the bones of young rats being administeredwith 1-hydroxyethylidene-1,1-bisphosphonate(HEBP) for 5 days. The doses of HEBP totallyabolished mineralization of newly formed bonematrix except in matrix vesicles (MVs), and henceallowed precise localization of MVs and phosphatasereactions within non-mineralized extracellularmatrix. Intense TNSALP and ATPase reactionswere confi rmed along the limited portionsof osteoblast membranes where intimate cell-cellcontacts were maintained. Diffuse reactions ofthese enzymes were throughout the osteoid implicatingeffl ux of TNSALP and ATPase moleculesinto extracellular matrix from the osteoblast membranes.Phosphatase reactions associated withMVs varied both in intensity and location amongthe individual vesicles; newly formed MVs werealmost free of reactions but appeared to gain thoseactivities later in the osteoid. These data suggestthat TNSALP and ATPase are released from theosteoblast membrane and later integrated intoMVs within the osteoid. The osteoblasts may thusregulate appositional mineralization of bone from adistance at least in part by providing phosphatasesvia MVs.