Dry Ultrathin Sectioning Combined With High Pressure Freezing

Cell culture models of bone mineralization: UMR106-01 cells do it faster, are temporally synchronized, and produce more mineral

We have used cultured UMR106-01 osteoblastic cells to investigate the process of bone mineralization. UMR106-01 cells as well as primary calvarial bone cells assemble spherical extracellular supramolecular protein-lipid complexes, termed biomineralization foci (BMF), in which the first crystals of hydroxyapatite mineral are deposited (Midura et al., 2004; Wang et al., 2004). A major difference between these culture models is the speed with which mineralization occurs, ranging from 12–16 days after plating for primary osteoblastic cells to 88 h for UMR106-01 cells.

If mineralization is blocked by omission of phosphate source or by addition of serine protease inhibitor AEBSF, BMF complexes are formed but no mineralization occurs. Interestingly, ultra structural studies have shown that prior to mineralization BMF contain numerous membrane limited vesicles ranging in size from 50 nm to 2 microns in diameter. However, the first mineral crystals are not detected until 78 h after plating of UMR106-01 cells and are localized within spherical sites presumed to be vesicles.

Specifically, confocal Raman spectral analyses have shown that mineralization within BMF is a progressive, multi-step process occurring simultaneously in all BMF within a culture flask (Wang et al., 2009). Importantly, several protein spectral changes are detectable within each BMF prior to the deposition of poorly crystalline hydroxyapatite and when mineralization was blocked, these changes did not occur. Thus, mineralization within BMF is a temporally synchronized process. However, understanding the biochemical mechanism of mineralization requires a detailed appreciation of calcium and phosphorus ion handling prior to crystal nucleation within BMF.

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