Thin-Section Electron Microscopy of Fetuin-A and Albumin-Containing Nanoparticles Reveal Unique Morphologies
Next, thin-sections of the protein-mineral particles were prepared in order to compare their internal structures with those of the NB-like formations and calcium granules characterized earlier. Protein-mineral particles were prepared by using BSF and HSA as well as precipitating ions at concentrations that favored maximal precipitation as described earlier in Figure 6. In this case, calcium and phosphate were added each at 0.3 mM to DMEM containing either BSF at 2.1 µg/ml, HSA at 120 µg/ml, or both BSF and HSA at these same concentrations, followed by incubation in cell culture conditions and preparation of thin-sections as described in the  Materials and Methods .
As shown in Figure 11, large BSF-NLP around 1 µm in diameter tended to show variegated, multi-layered structures with alternating electron dense and pervious laminations resembling at times “bull's eyes” or “tree-age-ring-like” morphology typical of NB [3], [5], [27] as well as calcium granules found in the serum [3] and throughout nature (see the galleries of granule pictures assembled by Ryall [51]). In this case, the dark rings appeared to consist mainly of crystalline material (Fig. 11B and C). In fact, dark, crystalline needle-like projections could also be seen to protrude transversally from either the surface or from the core interior (see a magnified image in Fig. 11C). Like the many calcium granules found in nature [51], the alternating electron dense and pervious layers found here can also be interpreted as representing intercalated layers of mineral and organic matrices, or alternatively, they may represent distinct stages of nucleation and growth. That is, the multi-ring formations may represent the result of several successive cycles of growth-and-inhibition of the apatite crystals formed in the presence of organic material, in this case fetuin-A. Not surprisingly, inhibitors of calcification like osteopontin, as well as other proteins like alpha-anti-trypsin, were shown to be present at the mineral-protein interface within the multi-layer particles found in kidney tissues [119]–[125].
10.1371/journal.pone.0008058.g011 Figure 11  Thin-sections of protein-mineral nanoparticles seeded by fetuin-A or albumin show distinct morphologies.
Protein-mineral nanoparticles were prepared as described in Fig. 6, by diluting either BSF at 2.1 µg/ml (A–C), HSA at 120 µg/ml (D–F), or both proteins at these same concentrations (G–I) into DMEM and then adding 0.3 mM each of CaCl2 and NaH2PO4, followed by incubation in cell culture conditions for 1 month. Thin-sections were prepared without fixation or staining, as described in the  Materials and Methods . BSF-NLP (A–C) resembled multi-layered laminations with alternate electron densities. HSA-NLP (D–F) appeared mostly as incompletely sealed, single-layered formations. The BSF-HSA-NLP particles had rough surfaces covered with elongated crystal projections (G), with some structures appearing either as multi-layered (H) or single-layered (I). Control, commercial grades of CaCO3 (J), Ca3(PO4)2 (K), and HAP (L) were incubated in DMEM as in Fig. 10. These controls showed mainly monolithic platelets or aggregates of crystalline formations that contrasted with the round nanoparticles shown above. Scale bars: 100 nm (H, I, L); 200 nm (C, K); 250 nm (F); 500 nm (B, D, E, G, J); 1 µm (A).   When albumin was used instead to seed mineral particles in supersaturated solutions, the particles showed morphologies that were significantly different from those of the fetuin-A-NLP seen earlier. Noticeably, HSA-NLP were less likely to form multi-layered structures. Instead, they tended to adopt morphologies resembling incomplete, single-layered rings consisting of electron-dense spindles or needle-like projections surrounding a core material that appeared more electron-pervious (Fig. 11D–F). Assuming that the outer, dark, electron-dense layer represents a mixture of organic and mineral interphases, as interpreted from the vast literature on calcium granules [51], [119]–[126], it is possible that the incomplete rings seen associated with HSA-NLP may represent incomplete enclosure or inhibition of nascent apatite! That is, albumin, being a much less potent inhibitor of mineralization than fetuin-A, may not be capable of inhibiting fully the nucleation of nascent apatite crystals, and this in turn can be translated further into the predominance of incomplete rings seen in Figure 11D–F. Assuming that this reasoning is correct, it can be further inferred that, during the process of nanoparticle assembly, the presence of organic inhibitors actually helps sustain the rounded shapes of nanoparticles, thereby delaying or repressing their conversion into larger aggregates and films.
In comparison, nanoparticles that had been assembled in the presence of both fetuin-A and albumin (Fig. 11G–I) assumed a variety of shapes, including multi-layered formations (Fig. 11G) and single-walled rings (Fig. 11I). Intriguingly, the incomplete rings seen earlier with HSA-NLP were not found among the BSF-HSA-NLP samples. As before, electron-dense, crystalline, needle-like projections could be seen protruding from the rings (Fig. 11H and I). It should be noted that we used combinations of BSF and HSA here at concentrations that favored maximal mineral precipitation as seen in Figure 6, indicating that these images may correspond in fact to optimal protein-mineral interactions that lead to mineralization.
As controls, commercially available CaCO3 (Fig. 11J), Ca3(PO4)2 (Fig. 11K), and HAP (Fig. 11L), were each diluted and incubated in DMEM for various amounts of time, and processed for thin-section TEM (only data for 1 hour incubation shown here). As can be seen from Figure 11J-L, these same controls appeared as large homogeneous and monolithic crystalline formations, lacking the characteristic round and multi-layered morphologies linked otherwise to the protein-mineral nanoparticles. Longer incubations of these control compounds in DMEM resulted in further homogenization and consolidation of the aggregates (not shown). These results further indicate that the ring-like structures, be them complete and multi-layered, or incomplete and single-walled, may correspond to organic-mineral interphases that are in turn absent when formed in protein-free medium that had been supplemented with precipitating minerals.