Protein-mineral nanoparticles containing fetuin-A and albumin resemble NB and calcium granules as revealed by TEM.

<p>Protein-mineral nanoparticles were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008058#pone-0008058-g009" target="_blank">Fig. 9</a>, from solutions containing either BSF (A), HSA (B), or both proteins (C), followed by the addition of 0.3 mM each of CaCl₂ and NaH₂PO₄, and incubation for 1 month in cell culture conditions. The samples were then prepared for TEM without fixation or staining. The various protein-seeded particles showed small, rounded formations that tended to aggregate (A–C). The samples of calcium granules prepared from addition of either CaCl₂ (D), NaH₂PO₄ (E), or a combination of both (F) to FBS (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008058#s3" target="_blank"><i>Materials and Methods</i></a>) showed stacks of crystalline spindles and stacks forming ellipsoid structures (D and E), while other granules appeared mostly as round aggregated particles (F). The morphologies of the protein-mineral nanoparticles and the calcium granules were similar to NB obtained from either 10% HS (G, “HS-NB”) or the two NB strains “Nanons” (H) and “DSM 5820” (I) which were both obtained from 10% FBS. In this case, the NB samples consisted of rounded particles with rough surfaces and variable sizes (G–I). Commercially available CaCO₃ (J), Ca₃(PO₄)₂ (K), and HAP (L) incubated in DMEM for 1 hour showed mainly large, crystalline, monolithic structures. Scale bars: 50 nm (A, F); 100 nm (B, D, E, G–J, L); 200 nm (C, K).