Electron Microscopy
For scanning electron microscopy (SEM), washed particles, calcium granules, and NB specimens were resuspended in double-distilled water. A small aliquot of the sample was deposited on formvar carbon-coated grids (Electron Microscopy Sciences, Fort Washington, PA, USA). The excess liquid was removed with an absorbent paper and the grids were dried overnight under a laminar flow hood. Prior to observation, the specimens were coated with gold for 90 sec. SEM observations were conducted using a SEM S-5000 field-emission scanning electron microscope (Hitachi Science Systems, Tokyo, Japan).
For transmission electron microscopy (TEM), washed particles and NB samples were deposited on formvar carbon-coated grids and were dried overnight as described above. All TEM observations were performed without staining. Commercially available calcium carbonate (CaCO3, A.C.S. grade reagent, purity 99.6%, Mallinckrodt Baker, Inc., Phillipsburg, NJ, USA), calcium phosphate tribasic (Ca3(PO4)2, Kanto Chemical Co., Tokyo, Japan) and HAP (buffered aqueous suspension, 25% solid, Sigma) were diluted into DMEM or double-distilled water and processed like the other samples as controls.
For thin-sections, washed particles and NB samples were dehydrated with two washes of 100% ethanol. The samples were incubated with Epon 812 resin (Electron Microscopy Sciences) with gentle end-to-end agitation overnight at room temperature. The samples were then centrifuged at 16,000× g for 15 min and incubated at 72°C for 2 days to allow resin polymerization. Thin-sections were prepared using a Leica Ultracut UCT microtome (Leica Microsystems GmbH, Wetzlar, Germany). Thin-sections were deposited on formvar carbon-coated grids. TEM observations and electron diffraction patterns were performed with a JEOL JEM-1230 transmission electron microscope (JEOL, Tokyo, Japan) operated at 120 keV.