Matrix Vesicles-Induced Mineralization and Phosphatase Actions in Bisphosphonate-Affected Bones

Objective: It is known that matrix vesicles (MVs) play a pivotal role in determining the sites and timing of mineral deposition in bone and that certain phosphatases contribute to the process. This study aims to elucidate the exact site of actions of MVs-associated phosphatases in the osteoid and its functional significance in appositional mineralization of bone. Methods: Five-day-old rats were intraperitoneally injected with bisphosphonate (HEBP) (8mg P/Kg/day) for 5 days and fixed by perfusion at the 6th day. Long bones of the HEBP-affected animals were either embedded in Technovit 7100 or processed for frozen sectioning with or without decalcification, and subjected to histo-cyotochemical localization of Ca-ATPase and tissue-nonspecific ALPase.

Results: HEBP-affected osteoblasts did not show structural abnormality and continued to form osteoid matrix. The doses of HEBP totally abolished mineral deposition in the newly formed bone matrix except in MVs, and hence allowed precise localization of MVs and phosphatase reactions associated with MVs throughout the thick osteoid layers. In the osteoblasts, intense ATPase and ALPase reactions were only seen along the limited portions of plasma membranes where an intimate cell-cell contact was maintained. Cytochemical localization of enzymatic reactions of both phosphatases in MVs varied considerably. Newly formed MVs appeared almost free of phosphatases and gained the activities of these enzymes later in the osteoid along the outer surface. In MVs, the site of actions of ATPase but not of ALPase further shifted to the inner aspect of the limiting membrane. Conclusions: These data implicate that considerable amounts of ATPase and ALPase molecules are liberated from the osteoblast membranes and released into the osteoid matrix, and later incorporated in the limiting membranes of MVs by undefined mechanisms. The osteoblasts thus appear to regulate bone mineralization at least in part by providing phosphatases via MVs to the site of appositional mineralization.