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and Future Perspectives\nThe results presented in this study further confirm a dual inhibitory-seeding role for serum insofar as the formation of NB-like calcifying particles is concerned, a notion that was first advanced through our earlier studies [2], [3] but that can now be extended to encompass the effects of serum-associated proteinaceous factors and at least those of the two main serum proteins albumin and fetuin-A. That is, calcification-inhibitory factors such as albumin and fetuin-A are seemingly capable of combining with calcium and phosphate to form amorphous nanoparticles, thereby repressing them from progressing further onto crystalline phases that appear in turn as spindles, aggregates, and biofilms. It is only when these same inhibitors are overwhelmed by excess calcium phosphate that mineral seeding will then occur. Since serum and other body fluids are already supersaturated with respect to calcium and phosphate, our results support fully the notion that it is really the de-repression of this same predominantly inhibitory state that results in the seeding of mineral crystals [52]–[58].\nOur focus on serum and serum-derived factors as an experimental platform to study the broader issue of nanoparticle assembly can be seemingly justified by both the physiological relevance of this system to biomineralization as well as the known fact that serum has been used in the past as the body fluid of choice for the culture of the putative NB [4], [5], [7]–[9]. These same earlier studies had shown that when FBS and adult HS were inoculated into DMEM at a final concentration of 10%, followed by incubation in cell culture conditions for several weeks, structures described as mineralized NB could be observed in most samples in the form of a white precipitate adherent to the cell culture flask [4], [5], [7]–[9]. Given that this precipitate was absent in DMEM alone incubated in the same conditions, it was concluded that the serum provided the source of putative NB [4], [5], [7]–[9]. Our studies indicate that, in retrospect, the levels of serum used (generally 10% and at times 5%) for the bulk of NB studies were essentially and paradoxically inhibitory on NB formation. In our own studies, as little as 0.1% serum triggered mineral precipitation while the dose-dependent precipitation produced by serum followed typically a bell-shaped curve, with maximal precipitation achieved at 1–3% serum and with FBS generally giving peak precipitations at lower concentrations as compared to HS. The prominent inhibitory effect on mineral seeding shown by serum can also explain now the slow time-course of mineral precipitation associated with the entire NB phenomenology—generally taking several weeks to months for noticeable precipitation to develop. When these observations are factored in, it is now possible to view NB from an entirely different perspective, as follows. In spite of the fact that NB are lifeless mineralo-organic entities, they are nonetheless real and verifiable, and they may in fact represent a general mechanism of calcium homeostasis used throughout nature [2], [3].\nIt is with these perspectives in mind that we sought to dissect the putative serum factor(s) that might account for the inhibitory-seeding attributes associated with serum. It is not clear for example whether the calcification inhibitors and nucleators found in the serum represent the same exact entities. This latter question is all the more relevant when one takes into account that, presumably, it is the presence of prevailing inhibitory influences that keep supersaturated solutions like serum from calcifying spontaneously [52]–[58]. This is also to say that releasing such inhibitory influences, as accomplished through protease or heat treatment, may be sufficient to trigger calcification in supersaturated solutions. According to Mann, whereas biomineralization can be construed as a result of phase transformation from amorphous to crystalline apatite phases, “‘promotion’ of various phases and polymorphs does not occur in the literal sense, but that the mineralization pathway is controlled through intermittent release of a system under chemical repression” [58].\nIn fact, our studies point to a definitive role for proteinaceous (e.g. protease and heat sensitive) factors in the inhibition of calcification reactions associated with both bovine and human serum. Indeed, once de-repressed from this same inhibition, be it through protease or heat treatment of serum, it appears that this same supersaturated solution, e.g. serum, will naturally nucleate and precipitate minerals. Accordingly, this same chain reaction—de-repression of inhibition, followed by calcification—can be enhanced several fold by the addition of small amounts of calcium and phosphate (0.3–1 mM).\nIn turn, the addition of these small amounts of precipitating ions (calcium and phosphate) to metastable culture solutions like DMEM has helped unravel attributes associated with protein-linked calcification that are seemingly repressed or subdued in metastable solutions. For instance, fetuin-A and/or albumin inoculated into DMEM alone fail to produce any mineral precipitation. On the other hand, mineral deposition is seen when submillimolar amounts of calcium and phosphate are also added concomitantly, and these ions can be effective even at the low ion concentration levels that normally result in negligible or no precipitation on their own. This synergistic interaction seen between the proteins fetuin-A/albumin and precipitating ions, resulting in apatite nucleation and deposition, further confirms the propensity of these proteins to behave as calcification inhibitors. It is only when the calcium-phosphate equilibrium is perturbed and the apatite-binding capacities of these proteins are somehow overwhelmed through calcium and phosphate loading that mineralization is finally triggered.\nThe fact that the small amounts of precipitating ions added do not result in any significant calcification on their own but that they do in the presence of fetuin-A and/or albumin, suggests further that the calcium or apatite-binding sites on these proteins may not only bind to excess calcium or apatite, but that they may somehow anchor and bridge the apatite chains so as to allow them to grow in size, thereby forming in effect the nidi required for crystallization. Thus, fetuin-A, itself a much stronger binder of apatite than albumin ([74]; see also our apatite-binding calculations discussed earlier), displays not only stronger inhibition of calcification as seen in our experiments, but it clearly synergizes with precipitating ions to produce calcification; however, this seeding effect is seen at much lower protein-to-ion concentration ratios as compared with albumin (Figs. 4– 7). In other words, both seeding and inhibition of NB-like particles appear to represent in fact two sides of the same coin, with inhibition seen as the predominant state that nonetheless gives in to seeding when it is somehow de-repressed.\nIn this sense also it would be reasonable to expect that a stronger calcification inhibitor like fetuin-A, by it binding more avidly to apatite, would be much more likely to be tightly associated with nascent apatite nuclei as compared with other more weakly apatite-binding proteins like albumin—a concept that has been confirmed and extended through the elegant work on primary and secondary CPPs done by Heiss and his co-workers [73], [74], [94], [141]. Our own morphological data indicate that the fetuin-A-mediated mineral particles are more likely to look as multi-walled sealed rings whereas albumin-particles are more prone to resemble incomplete single-walled rings. Presumably, fetuin-A, by it being a more avid apatite binder or inhibitor, is more likely to cover an apatite nucleus entirely, thereby blocking its access to further growth, whereas weaker inhibitors (albumin) tend to produce an incomplete seal, which may facilitate the further seeding and growth of apatite crystals. Nonetheless, the results shown here and elsewhere [2], [3], [73], [74], [94], [141] demonstrate that any such inhibition, be it by a potent inhibitor like fetuin-A or a weaker inhibitor like albumin, is only transient at best and, that, eventually, there is a progressive and irreversible transformation of the round particles to spindles and these to films and aggregates. In full agreement with this notion and as shown by our own spectroscopic and ultrastructural data, when fetuin-A and/or albumin particles are fully developed, they end up becoming virtually indistinguishable from the purported NB or calcium granules grown out of whole serum and other more complex body fluids.\nStill in this respect, it should be noted that, to date, all NB-related studies have been done in the presence of body fluids or tissue homogenates inoculated into culture medium (reviewed in refs. [2], [43]). This procedure may very well have introduced small amounts of calcium and phosphate, not to mention inorganic and organic modulators with calcification-inhibitory-and-seeding properties, a situation that may be comparable to the conditions used here with purified proteins, in which addition of exogenous precipitating ions is also required for calcification to develop.\nProteins are present in high amounts in the serum and many of them have been shown to bind to NB-like particles or granules derived from the serum [2], [3]. As far as biomineralization is concerned, proteins are known to modulate the formation, morphology, orientation, and growth of the apatite crystals found in the mineralized tissues of living organisms (see the excellent reviews on this topic in refs. [99], [100], [142]). Given their binding affinities for calcium and apatite, certain proteins may interact with the apatite crystals during the mineralization process and as such they are expected to be enriched in mineralized tissues like bones and teeth. Several questions however remain to be addressed, including whether the same inhibitory-seeding role in calcification seen here with serum and the serum proteins fetuin-A and albumin has been documented elsewhere in the literature. It is also not clear whether the seeding or inhibitory attributes associated with a protein may not in fact be a function of the protein's conformational state, as we presumed to be the case when attempting to unfold fetuin-A and albumin through heat and immobilization treatments described in this study.\nAs it turns out, both inhibitory and seeding roles have been ascribed to a number of mineralization-related proteins. Table 1 lists the many proteins implicated in mineralization in vertebrates along with their tissue distribution and their (seeding and/or inhibitory) effects on mineralization as seen both in vitro and in vivo. To address further the role of a protein's conformational state in effecting either inhibition or seeding, we further segregated the in vitro experiments referenced in Table 1 into proteins that had been adsorbed onto solid substrates or dissolved in solution. Our summary table confirms that, depending on the conditions used for the studies, e.g. whether the proteins are adsorbed onto substrates or are dissolved in solution, several proteins present in the serum like albumin, fibrinogen, and vitronectin have actually been shown to exhibit both seeding and inhibitory roles in vitro. That is, as expected from our own heat treatment studies, these same calcium and apatite binding proteins when adsorbed onto solid substrates express nucleating or seeding tendencies while their counterparts in solution tend to be inhibitory!\n10.1371/journal.pone.0008058.t001 Table 1 Proteins associated with mineralization in vertebrates along with their tissue distribution, their effect on mineralization in vitro, and their potential role in vivo. a The effect of the proteins on mineralization was studied in vitro following their adsorption onto solid substrates such as agarose beads, agarose gels, or collagen fibrils. b The term “Nucleator” refers to a protein which is able to induce mineral formation in a metastable solution where precipitation does not occur spontaneously. An “Inhibitor” consists of a protein which has the ability to delay or prevent mineral formation. c The effect of various proteins on mineralization was also studied following dissolution of each protein into a liquid buffer. d The word “Structure” refers to a protein which does not appear to induce or inhibit mineral formation by itself, but which is known to be important for the disposition and the arrangement of minerals formed in vivo. *The potential role of each protein during mineralization in vivo was proposed based on gene deletion studies in laboratory animals when available; in this case, the proteins were denoted with a single asterisk. A role for the other proteins shown was proposed based on limited functional studies giving sometimes divergent results; the role of these proteins should therefore be considered with strong reservation. **Inhibition of pancreatic stones of calcium carbonate. ***Inhibition of kidney stones of calcium oxalate or calcium phosphate. This Table was adapted and modified from the review by Benesch et al. [100]. We have also noticed that these dual seeding-inhibitory tendencies seen in vitro with some proteins may not in fact correlate with their net mineralizing effect seen in vivo (Table 1). As for the in vivo role of proteins presented in Table 1, a definitive assignment could only be derived from the phenotype of mice that were engineered to lack the gene coding for the protein in question, as already done in the case of dentin matrix protein-1, bone sialoprotein, fetuin-A, matrix gla protein, and osteopontin (see the proteins marked with a single asterisk in Table 1 and the corresponding references therein). However, in the absence of gene knockout data, a potential role for some of the proteins was proposed by the respective authors based on limited functional studies and thus must be viewed with caution. Based on such analyses, for example, albumin has been deemed to be a inhibitor of mineralization in vivo [56], [62], and yet this protein can display either inhibitory or nucleating properties in vitro depending on the conditions used (Table 1). As for fetuin-A, on the other hand, we were not able to find any study documenting the effect of the adsorbed protein; our own experiments reported here, however, demonstrate that fetuin-A can also behave as a nucleator when subjected simultaneously to heat treatment as well as to challenge with submillimolar amounts of calcium and phosphate.\nSurprisingly, few studies to date have addressed the possibility of fetuin-A representing a nucleator of calcium phosphate minerals. In a recent report [141], the early stage of CPP formation was studied based on a dynamic small-angle X-ray scattering analysis. It was observed that fetuin-A at various concentrations (1, 5, or 15 µM) added to 20 mM calcium and 12 mM phosphate ions did not in fact act as a nucleating agent of CPPs, but it essentially inhibited their formation by “shielding” nascent mineral nuclei [141]. Given that relatively high concentrations of calcium and phosphate ions were used in these experiments, it is not clear whether they are in fact representative of the slow and spontaneous formation of NB grown in metastable solutions or of NLP assembled in metastable solutions that had been challenged with submillimolar amounts of precipitating ions, both of which were meticulously addressed here. Other studies have reported that by fetuin-A was effective only in delaying mineral formation temporarily [71], [205]. For instance, when fetuin-A was present at 5 mg/ml in solutions containing 4 mM of calcium and phosphate, precipitation in the form of a fetuin-A-mineral complex appeared only after 4 to 5 days of incubation at room temperature [71], [205]. When calcium and phosphate ions were present at 5 mM in these same conditions, the incubation time was shortened to 20 to 24 hours [71], [205]. These latter observations clearly support our hypothesis of a dual inhibitory-seeding role for fetuin-A.\nThe dichotomy seen between inhibitory and seeding tendencies as a function of protein solubility (conformational state) is not in fact limited to serum proteins. Thus, Table 1 illustrates that several proteins associated with bones or teeth have also been found to possess a dual seeding and inhibitory role on mineralization, including biglycan, bone sialoprotein, decorin, dentin phosphophoryn, osteocalcin, and osteonectin. These proteins also show marked propensity to act as apatite crystal nucleators when adsorbed in vitro, consistent with the assumption that, once bound to collagen fibrils, they may unfold and help nucleate the first crystals required for collagen mineralization in vivo [99], [100]. Given the variabilities in seeding versus inhibition seen with the same proteins under different conformational states, it would appear that designating a protein as inhibitor or nucleator can be rather arbitrary. We contend that, in principle, any proteinaceous inhibitor of mineralization can be induced to nucleate under conditions that result in a conformational change in its structure.\nWith regards then to the differences seen between adsorbed and soluble proteins and still in the context of Table 1, it would appear that apatite-binding proteins tend to act as nucleators of calcification while the same proteins free in solution tend to act as inhibitors, a notion that has also been advanced in several earlier studies [97]–[100]. Presumably, an adsorbed protein may provide binding sites that recruit and bridge ions so as to position them in a specific tridimensional configuration needed to form a mineral nucleus [101]. On the other hand, when free in solution, the protein may still bind to precipitating ions, but it somehow may either prevent them from forming a mineral nucleus or coat the nascent crystal, thereby blocking any growing sites and preventing further growth [99], [101].\nFor our own experiments, we also attempted to immobilize albumin and fetuin-A onto various substrates and monitored their calcification effects in metastable solutions. Despite repeated trials, we were unable to directly seed mineral nanoparticles under the conditions used. While two earlier studies had reported that albumin could promote the formation and growth of apatite crystals when adsorbed to collagen [81], [82] or to apatite itself, using ceramics comprising of commercial apatite and calcium phosphate [83], [84], it is not clear how these results relate to our own observations given the differences in experimental conditions used. For one, the nature of the substrate used here could have played a role in the absence of effect observed. It is also not clear how the effect of albumin can be ascertained from its immobilization onto apatite or calcium phosphate crystals, which are known to promote the nucleation of other identical crystals by secondary nucleation [118]. In addition, the concentrations of calcium and phosphate used could also have played a role in the different responses obtained. Additional experiments are needed to reconcile these differences.\nIt should also be noted that although we did not obtain calcification in a metastable medium (DMEM) using either immobilized fetuin-A or immobilized albumin, we were however able to demonstrate seeding with heat-treated albumin (Fig. 7). Heat-treated fetuin-A, on the other hand, failed to seed under the same conditions. The only seeding seen with fetuin-A (also seen with albumin) was in the presence of submillimolar amounts of calcium and phosphate added simultaneously to DMEM. This property may be attributed to the potent inhibitory effect on apatite formation associated with fetuin-A. Albumin, by comparison, being a much less effective inhibitor of apatite nucleation [74], [94], has shown a greater tendency to mineralize under the conditions studied.\nViewed from a different perspective, the differential display of inhibitory versus seeding tendencies may depend on the stoichiometric relationship between the number of protein molecules versus the number of precipitating ions available in any given environment. Thus, our results indicate that, for any calcium or apatite binding protein and for given amounts of calcium and phosphate present in a medium, the protein concentration should certainly influence its effect on mineralization. This relationship can be illustrated by the significant differences seen with the amounts of fetuin-A versus albumin needed to trigger calcification (Figs. 4– 7). That is, fetuin-A is such an effective binder of apatite that it will seed apatite minerals not only when the protein-to-mineral ratios are low compared to that seen with albumin but also only with the concomitant addition of submillimolar amounts of calcium and phosphate. Based on these considerations, and extrapolating further to body conditions, fetuin-A should behave as a potent inhibitor of calcification at levels found in the body fluids, while it will nucleate at low protein concentrations only in the presence of excess calcium and phosphate. This is not surprising since according to our calculations, each bovine fetuin-A molecule binds to 54–58 apatite units while each human albumin molecule binds to 4 apatite units. In fact, due to these same stoichiometric considerations, low concentrations of calcium or apatite binding proteins have been generally associated with nucleation of mineral formation whereas high concentrations are known to usually delay or inhibit this same process [100]. In fact, according to Boskey, any macromolecule that can bind and coat nascent crystals will inhibit crystal growth when present in high concentrations in solution [206].\nProteins that inhibit mineral formation are thought to play an important role by preventing spontaneous calcification of the serum and the extracellular fluids [52], [54], [56]. This role is critical given the high natural propensity of these supersaturated extracellular fluids in vertebrates to calcify [52]–[58]. Direct evidence for the role of these proteins in preventing calcification in vivo comes from the observations that mice designed to lack any one of the calcification inhibitors like fetuin-A, matrix gla protein, or osteopontin are prone to ectopic calcification and to the debilitating effects associated with this process [67], [68], [186], [196].\nWhile most proteins that can bind to apatite crystals can be seen as inhibitors of mineralization, several proteins listed in Table 1 have been shown to be able to initiate mineral formation as well. Proteins like bone sialoprotein, biglycan, and decorin interact with the collagen fibrils and are thought to initiate the formation of the first mineral nucleus required for collagen mineralization [99], [142]. Collagen fibrils are often described to play a structural role by forming a receptacle which can delineate the main area destined to become mineralized in bones [99], [100]. Proteins can initiate mineralization by lowering the activation energy required for the formation of the first mineral nucleus in metastable solutions which usually does not occur spontaneously [101]. Given that the extracellular fluids are saturated in calcium and phosphate ions [52]–[58], the formation of mineral nuclei is thought to be sufficient to initiate mineralization, which in turn can proceed further on its own by the deposition of calcium and phosphate ions onto the nascent nuclei. It is further assumed that an effective mineral nucleator should provide an array of functional groups, particularly carbonate groups as well as phosphorylated residues, which possess high affinity for calcium and phosphate ions [101], [142]. Accordingly, several studies have suggested that proteins present in the serum may also act as nucleators of calcification. Strong evidence for the presence of a putative nucleator(s) in the serum comes from the observation that demineralized bones incubated in DMEM containing as low as 1.5% serum can remineralize following incubation for a few days [207]–[209]. This remineralization did not proceed if the demineralized bones were incubated in DMEM alone for the same period of time [207]–[209]. The factor responsible for this remineralization, which remains unidentified, was attributed to one or more protein(s) with a molecular weight between 55–150 kDa [207], [209] and which required dephosphorylation by alkaline phosphatase to become activated [210]. At first sight, this suggestion might appear paradoxical given that various systems including proteins are present in the serum to prevent calcification at any given time. Nevertheless, the observation that blood is continually in contact with bones through the so-called basic multicellular unit and bone-remodeling compartment—two anatomical structures that are associated with remodeling of cortical and cancellous bones, respectively [211], [212]—leads to the possibility that a protein secreted by osteoblasts to initiate mineralization in bones might end up circulating in the blood at low concentrations [208], [209]. Were this scenario to occur in vivo, it is likely that spontaneous calcification of blood would be prevented as long as the other major inhibitory systems remain in place [208], [209]. Whether or not this putative nucleator(s) plays a role in the formation of NB in cell culture conditions is still unclear.\nStill with regards to fetuin-A and albumin, it should be noted that we selected these two proteins for our demonstration of a dual inhibitory-seeding model for protein-mediated mineralization since they represent the two main proteins found in association with NB-like particles [1]–[3], [46]. Their effect on mineral formation has been studied extensively in the past mainly because they are present in high amounts in mineralized tissues. These findings have long suggested that the two proteins might play a role in the mineralization process [56], [57], [81], [82]. Alternatively, the binding of these two proteins to the mineral phase of bones may simply reflect their high affinity for both calcium phosphate and apatite [94]. In support of this alternative view and as noted earlier, proteins from blood are in constant contact with bones and this could account for the gradual enrichment of albumin and fetuin-A in mineralized tissues. Both albumin and fetuin-A are also found at high concentrations in the serum used to culture NB. In fact, albumin represents the main protein found in both the fetal bovine and adult human sera while fetuin-A is more abundant in FBS than in HS [92]–[96]. Both proteins can inhibit or delay the precipitation of calcium phosphate in vitro [2], [3], [62]–[64], [66], [110], [111] and both bovine fetuin-A and human albumin have been shown to account for a significant portion of the calcification-inhibitory effect associated with each serum [62], [66]. In fact, earlier studies made on the inhibitory capacity of human serum showed that two-thirds of the inhibitory potential of the serum studied was due to proteins and other macromolecules of high molecular weights while the other one-third of the inhibitory capacity could be attributed to compounds of low molecular weights [62]. Further observations made on albumin-depleted serum indicated that albumin accounted for about half of the inhibitory effect of the high molecular weight fraction present in the serum [62].\nSimilarly, when fetuin-A-depleted FBS was incubated in DMEM in cell culture conditions, it was reported that a precipitate of calcium phosphate would form in the bottom of the flask within 6 days of incubation [205]. It thus appeared that the removal of fetuin-A was sufficient to induce mineral precipitation from DMEM under these conditions. Given that the DMEM used in this study was also supplemented with phosphate to a final concentration of 2 mM [205], further experiments are needed to evaluate whether these observations are relevant to our own findings with NB and protein-minerals described here. While more experiments are required to integrate and compare the various systems of calcification used here and elsewhere, together, these results strongly support the notion that both fetuin-A and albumin represent major repressors of spontaneous calcification associated with the serum.\nThe calcification-inhibitory effects associated with fetuin-A and albumin can be attributed to the presence of several calcium and apatite binding sites on these proteins which may presumably sequester calcium and phosphate ions before they bind to the growing crystals [2], [3], [62]–[64], [66], [110], [111]. In addition, the calcium or apatite binding sites may tentatively mediate inhibition by binding onto the growing sites of the nascent crystals and blocking further mineralization. Albumin has been shown to possess as much as thirty calcium-binding sites with three different binding affinities [60] while fetuin-A has been shown to have six calcium-binding sites per molecule with two different binding affinities [65]. The calcium-binding constants associated with the fetuin-A molecule are generally lower than the ones obtained for albumin, an observation which suggests that the binding sites of fetuin-A have a higher affinity for calcium than those of albumin [65]. In addition to the potential influence of calcium-binding sites, the inhibitory effect of these proteins on mineral formation can also be attributed to the presence of specific protein motifs which promote the interaction of the proteins with growing crystals. Indeed, fetuin-A possesses a motif consisting of a cystatin-like domain with several basic amino acids which has been shown to interact with calcium phosphate and apatite crystals [66], [94]. The removal of this motif on fetuin-A by mutational analysis showed that it was responsible for the relatively strong inhibition effect of this protein [66], [94]. While no specific mineral-binding motif has been described for albumin, the interaction of this protein with apatite crystals has been attributed instead to the multiple calcium-binding sites present on this protein [62]-[64]. The possibility that a protein conformation might be associated with the inhibitory effect of albumin was also suggested by the observation that heat-denatured albumin showed increased inhibitory effect compared to the native protein [62], a result that we could not confirm however through our own experiments. Given that these two proteins are present in relatively high amounts in the serum, it is possible that they would play a predominant role in the inhibition effect seen during the culture of NB or during the assembly of NLP in the body. Taken together, these earlier studies suggest that the high inhibitory effect of albumin may be attributed mainly to its relatively high concentration in the serum [62], [74] while the stronger inhibitory effect of fetuin-A may be attributed in turn to its strong interaction with apatite crystals [74], [94].\nIn the context of biomineralization, perhaps no other protein has given more divergent and even contradictory results in terms of seeding or inhibition than albumin. These discrepancies were more noticeable when studies were done via adsorption of albumin to various solid substrates of well-defined chemical composition. For instance, albumin had earlier been shown to promote the nucleation of calcium phosphate crystals when adsorbed onto a substrate, but to essentially act as an inhibitor of mineral deposition when dissolved in solution [83]. In these experiments, albumin was adsorbed onto ceramics of apatite and calcium phosphate and immersed into a buffer containing high concentrations of calcium and phosphate similar to those contained in the serum [83], [84]. Precipitation of calcium phosphate, verified by SEM and FTIR, was found to be more prominent when albumin was adsorbed on the surface [83], [84]. However, other authors have reported that albumin seemed to exert an inhibitory effect or a lack of nucleating effect irrespective of its being adsorbed to titanium or agarose beads or its being dissolved in solution [102], [105]. The discrepancy seen between these observations has been attributed to the nature of the substrates which may seemingly have produced different protein conformations [83], [84], [149]. As such, the presence of a specific albumin conformation which allows for both firm adsorption to a surface and binding of calcium from the solution is thought to be required for albumin to induce mineralization [83], [149]. In another set of experiments, the presence of albumin in solution has also been described to promote the mineralization of phosphate ceramics [84]. In this case, the presence of a carbonate-buffered solution was found to alter the charge characteristics of albumin and to increase mineralization of phosphate ceramics when compared to solutions where no albumin or carbonate buffer was used [84]. In yet another study, the dual inhibitor-nucleator effect of albumin described here could also be seen in the context of collagen type I-mediated mineralization and was shown to depend on the concentration of albumin used [81]. When albumin was present at low concentrations (below 10 mg/ml) in supersaturated solutions containing 3.6 mM of calcium and 2.7 mM of phosphate ions, it appeared to exert a nucleating effect on collagen mineralization and to lead to increased crystal growth whereas high concentrations (above 10 mg/ml) essentially inhibited mineral formation [81]. Albumin also had an effect on the induction time needed for mineralization to start, extending the period of time for mineral to appear when compared to solutions where albumin was absent [81]. Accordingly, the disparate results may be attributed to several factors which can include the nature of the mineralization assay used, the provenance, purity, and characteristics of the protein used, as well as the different criteria used to describe its mineralizing function [82], [100].\nTogether, these studies illustrate the complexities as well as the subtleties seen in terms of seeding and inhibition functions that can be attributed to a single protein, all of which may depend on both the conformational changes as well as the concentrations of the same protein found in any given compartment. These inherent attributes of calcification-related proteins make it difficult if not altogether impossible to classify the respective roles of the same proteins during the mineralization process without first considering their physiological context (see also the excellent reviews in refs. [82], [100]).\nSince we were not able to produce NB-like minerals using only fetuin-A and/or albumin inoculated into a metastable medium like DMEM, without first heat-inactivating these proteins and adding exogenous calcium and phosphate, it is possible that other serum factors may be involved in the formation of the NB-like particles seen here and in the earlier studies. Lipids had earlier been shown to be associated with NB-like particles derived from the serum (ref. [2] and unpublished observations). Preliminary experiments show that, in addition to proteinaceous factor(s), lipids in the form of membrane or matrix vesicles (MV) may actually be involved. MV are small lipid-bound vesicles released by the vast majority of cells, but in the context of collagen-independent mineralization, they are especially significant in that they are known to be actively released by skeletal cells like osteoblasts, chondrotonblasts, and odontoblasts, and are in turn capable of nucleating the deposition of calcium phosphate in specific areas of the body (see the comprehensive review by Anderson et al. [213]). The release of these vesicles is associated with the mineralization of bones, cartilage, and teeth [213]. In bones, the release of MV is thought to be associated with mineralization away from collagen fibrils in the endochondral plaque [214]. Ectopic calcifications are thought to be associated with the release of MV-like vesicles by vascular smooth muscle cells which experience insults of high concentration of phosphate ions [215], [216]. The mechanism of mineral deposition by matrix vesicles is thought to involve the presence of calcium and phosphate channels in the membrane delineating MV; these channels gradually increase the concentration of calcium and phosphate ions within MV until mineral precipitation ensues [213]. In the context of NB formation, while our earlier studies have failed to provide conclusive evidence for a role of lipids in this process [2], we are now able to extract lipids, in the form of MV, directly from serum samples and use them to seed NLP in vitro (data not shown). These same membrane vesicles have earlier been shown to be found in the blood and in most if not all body compartments [213], [217]. In this context, it should be noted that the earlier study by Cisar et al. [44] had in fact shown that lipids like phosphatidylinositol are indeed capable of seeding apatite particles that are morphologically similar to the putative NB, thereby providing a direct link between lipid moieties and the seeding of apatite nanoparticles in a metastable medium like DMEM.\nA yet another recently proposed mechanism of mineralization that needs to be considered in the context of NLP formation involves the hydrolysis of polyphosphate present in dense calcium phosphate granules seen in tissues experiencing mineralization [218]. Presumably, this hydrolysis can be catalyzed by alkaline phosphatase [218].\nFinally, it is not clear whether the protein-mineral nanoparticles and NLP described here are related in any way to the so-called proteons and proteon-nucleation centers (PNCs) previously shown to represent blood-derived, metallic nanoclusters associated with serum protein fragments such as hemoglobin alpha-chain [219]. It is also not clear whether the more recently described proteon-associated, cell-like nanoforms in the blood that can be produced by hypotonic hemolysis are in any way related to the nanoparticles or their underlying assembly mechanisms described here [220].\nMineralization inside the body is thought to occur through the formation of an amorphous phase of calcium phosphate which gradually converts into the more stable apatite ([221]; see also ref. [116] addressing current controversies surrounding this topic). Our own data indicate that much of this amorphous-crystalline phase transformation can be mimicked through in vitro studies of the kind reported here. Our studies indicate that the protein-mineral particles assembled using fetuin-A or albumin are in many respects similar to the particles described earlier in association with NB and calcium granules. Our results point to an orderly and successive morphological conversion from spherical nanoparticles to spindles, and these to films, that are comparable despite the diverse environments in which these same particles and aggregates are found. The binding of proteins and possibly other organic compounds to nascent apatite is what apparently sustains the particles in their spherical conformations, while the growth of apatite crystals will eventually de-repress this same inhibitory influence resulting in the fusion of the round nanoparticles allowing them to coalesce to form spindle and film-like shapes. Whether this general progression of morphologies is restricted to calcium phosphate or may be conceptually extended to other minerals in general remains to be explored."}

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span":{"begin":39253,"end":39261},"obj":"CHEBI_36080"},{"id":"T4160","span":{"begin":39310,"end":39317},"obj":"CHEBI_52254"},{"id":"T4161","span":{"begin":39414,"end":39421},"obj":"CHEBI_52254"},{"id":"T4162","span":{"begin":39520,"end":39525},"obj":"PATO_0002397"},{"id":"T4163","span":{"begin":39520,"end":39525},"obj":"PATO_0000411"},{"id":"T4164","span":{"begin":39729,"end":39737},"obj":"CHEBI_46662"},{"id":"T4165","span":{"begin":36206,"end":36228},"obj":"PM3723"},{"id":"T4166","span":{"begin":22499,"end":22520},"obj":"PM3722"}],"text":"Conclusion and Future Perspectives\nThe results presented in this study further confirm a dual inhibitory-seeding role for serum insofar as the formation of NB-like calcifying particles is concerned, a notion that was first advanced through our earlier studies [2], [3] but that can now be extended to encompass the effects of serum-associated proteinaceous factors and at least those of the two main serum proteins albumin and fetuin-A. That is, calcification-inhibitory factors such as albumin and fetuin-A are seemingly capable of combining with calcium and phosphate to form amorphous nanoparticles, thereby repressing them from progressing further onto crystalline phases that appear in turn as spindles, aggregates, and biofilms. It is only when these same inhibitors are overwhelmed by excess calcium phosphate that mineral seeding will then occur. Since serum and other body fluids are already supersaturated with respect to calcium and phosphate, our results support fully the notion that it is really the de-repression of this same predominantly inhibitory state that results in the seeding of mineral crystals [52]–[58].\nOur focus on serum and serum-derived factors as an experimental platform to study the broader issue of nanoparticle assembly can be seemingly justified by both the physiological relevance of this system to biomineralization as well as the known fact that serum has been used in the past as the body fluid of choice for the culture of the putative NB [4], [5], [7]–[9]. These same earlier studies had shown that when FBS and adult HS were inoculated into DMEM at a final concentration of 10%, followed by incubation in cell culture conditions for several weeks, structures described as mineralized NB could be observed in most samples in the form of a white precipitate adherent to the cell culture flask [4], [5], [7]–[9]. Given that this precipitate was absent in DMEM alone incubated in the same conditions, it was concluded that the serum provided the source of putative NB [4], [5], [7]–[9]. Our studies indicate that, in retrospect, the levels of serum used (generally 10% and at times 5%) for the bulk of NB studies were essentially and paradoxically inhibitory on NB formation. In our own studies, as little as 0.1% serum triggered mineral precipitation while the dose-dependent precipitation produced by serum followed typically a bell-shaped curve, with maximal precipitation achieved at 1–3% serum and with FBS generally giving peak precipitations at lower concentrations as compared to HS. The prominent inhibitory effect on mineral seeding shown by serum can also explain now the slow time-course of mineral precipitation associated with the entire NB phenomenology—generally taking several weeks to months for noticeable precipitation to develop. When these observations are factored in, it is now possible to view NB from an entirely different perspective, as follows. In spite of the fact that NB are lifeless mineralo-organic entities, they are nonetheless real and verifiable, and they may in fact represent a general mechanism of calcium homeostasis used throughout nature [2], [3].\nIt is with these perspectives in mind that we sought to dissect the putative serum factor(s) that might account for the inhibitory-seeding attributes associated with serum. It is not clear for example whether the calcification inhibitors and nucleators found in the serum represent the same exact entities. This latter question is all the more relevant when one takes into account that, presumably, it is the presence of prevailing inhibitory influences that keep supersaturated solutions like serum from calcifying spontaneously [52]–[58]. This is also to say that releasing such inhibitory influences, as accomplished through protease or heat treatment, may be sufficient to trigger calcification in supersaturated solutions. According to Mann, whereas biomineralization can be construed as a result of phase transformation from amorphous to crystalline apatite phases, “‘promotion’ of various phases and polymorphs does not occur in the literal sense, but that the mineralization pathway is controlled through intermittent release of a system under chemical repression” [58].\nIn fact, our studies point to a definitive role for proteinaceous (e.g. protease and heat sensitive) factors in the inhibition of calcification reactions associated with both bovine and human serum. Indeed, once de-repressed from this same inhibition, be it through protease or heat treatment of serum, it appears that this same supersaturated solution, e.g. serum, will naturally nucleate and precipitate minerals. Accordingly, this same chain reaction—de-repression of inhibition, followed by calcification—can be enhanced several fold by the addition of small amounts of calcium and phosphate (0.3–1 mM).\nIn turn, the addition of these small amounts of precipitating ions (calcium and phosphate) to metastable culture solutions like DMEM has helped unravel attributes associated with protein-linked calcification that are seemingly repressed or subdued in metastable solutions. For instance, fetuin-A and/or albumin inoculated into DMEM alone fail to produce any mineral precipitation. On the other hand, mineral deposition is seen when submillimolar amounts of calcium and phosphate are also added concomitantly, and these ions can be effective even at the low ion concentration levels that normally result in negligible or no precipitation on their own. This synergistic interaction seen between the proteins fetuin-A/albumin and precipitating ions, resulting in apatite nucleation and deposition, further confirms the propensity of these proteins to behave as calcification inhibitors. It is only when the calcium-phosphate equilibrium is perturbed and the apatite-binding capacities of these proteins are somehow overwhelmed through calcium and phosphate loading that mineralization is finally triggered.\nThe fact that the small amounts of precipitating ions added do not result in any significant calcification on their own but that they do in the presence of fetuin-A and/or albumin, suggests further that the calcium or apatite-binding sites on these proteins may not only bind to excess calcium or apatite, but that they may somehow anchor and bridge the apatite chains so as to allow them to grow in size, thereby forming in effect the nidi required for crystallization. Thus, fetuin-A, itself a much stronger binder of apatite than albumin ([74]; see also our apatite-binding calculations discussed earlier), displays not only stronger inhibition of calcification as seen in our experiments, but it clearly synergizes with precipitating ions to produce calcification; however, this seeding effect is seen at much lower protein-to-ion concentration ratios as compared with albumin (Figs. 4– 7). In other words, both seeding and inhibition of NB-like particles appear to represent in fact two sides of the same coin, with inhibition seen as the predominant state that nonetheless gives in to seeding when it is somehow de-repressed.\nIn this sense also it would be reasonable to expect that a stronger calcification inhibitor like fetuin-A, by it binding more avidly to apatite, would be much more likely to be tightly associated with nascent apatite nuclei as compared with other more weakly apatite-binding proteins like albumin—a concept that has been confirmed and extended through the elegant work on primary and secondary CPPs done by Heiss and his co-workers [73], [74], [94], [141]. Our own morphological data indicate that the fetuin-A-mediated mineral particles are more likely to look as multi-walled sealed rings whereas albumin-particles are more prone to resemble incomplete single-walled rings. Presumably, fetuin-A, by it being a more avid apatite binder or inhibitor, is more likely to cover an apatite nucleus entirely, thereby blocking its access to further growth, whereas weaker inhibitors (albumin) tend to produce an incomplete seal, which may facilitate the further seeding and growth of apatite crystals. Nonetheless, the results shown here and elsewhere [2], [3], [73], [74], [94], [141] demonstrate that any such inhibition, be it by a potent inhibitor like fetuin-A or a weaker inhibitor like albumin, is only transient at best and, that, eventually, there is a progressive and irreversible transformation of the round particles to spindles and these to films and aggregates. In full agreement with this notion and as shown by our own spectroscopic and ultrastructural data, when fetuin-A and/or albumin particles are fully developed, they end up becoming virtually indistinguishable from the purported NB or calcium granules grown out of whole serum and other more complex body fluids.\nStill in this respect, it should be noted that, to date, all NB-related studies have been done in the presence of body fluids or tissue homogenates inoculated into culture medium (reviewed in refs. [2], [43]). This procedure may very well have introduced small amounts of calcium and phosphate, not to mention inorganic and organic modulators with calcification-inhibitory-and-seeding properties, a situation that may be comparable to the conditions used here with purified proteins, in which addition of exogenous precipitating ions is also required for calcification to develop.\nProteins are present in high amounts in the serum and many of them have been shown to bind to NB-like particles or granules derived from the serum [2], [3]. As far as biomineralization is concerned, proteins are known to modulate the formation, morphology, orientation, and growth of the apatite crystals found in the mineralized tissues of living organisms (see the excellent reviews on this topic in refs. [99], [100], [142]). Given their binding affinities for calcium and apatite, certain proteins may interact with the apatite crystals during the mineralization process and as such they are expected to be enriched in mineralized tissues like bones and teeth. Several questions however remain to be addressed, including whether the same inhibitory-seeding role in calcification seen here with serum and the serum proteins fetuin-A and albumin has been documented elsewhere in the literature. It is also not clear whether the seeding or inhibitory attributes associated with a protein may not in fact be a function of the protein's conformational state, as we presumed to be the case when attempting to unfold fetuin-A and albumin through heat and immobilization treatments described in this study.\nAs it turns out, both inhibitory and seeding roles have been ascribed to a number of mineralization-related proteins. Table 1 lists the many proteins implicated in mineralization in vertebrates along with their tissue distribution and their (seeding and/or inhibitory) effects on mineralization as seen both in vitro and in vivo. To address further the role of a protein's conformational state in effecting either inhibition or seeding, we further segregated the in vitro experiments referenced in Table 1 into proteins that had been adsorbed onto solid substrates or dissolved in solution. Our summary table confirms that, depending on the conditions used for the studies, e.g. whether the proteins are adsorbed onto substrates or are dissolved in solution, several proteins present in the serum like albumin, fibrinogen, and vitronectin have actually been shown to exhibit both seeding and inhibitory roles in vitro. That is, as expected from our own heat treatment studies, these same calcium and apatite binding proteins when adsorbed onto solid substrates express nucleating or seeding tendencies while their counterparts in solution tend to be inhibitory!\n10.1371/journal.pone.0008058.t001 Table 1 Proteins associated with mineralization in vertebrates along with their tissue distribution, their effect on mineralization in vitro, and their potential role in vivo. a The effect of the proteins on mineralization was studied in vitro following their adsorption onto solid substrates such as agarose beads, agarose gels, or collagen fibrils. b The term “Nucleator” refers to a protein which is able to induce mineral formation in a metastable solution where precipitation does not occur spontaneously. An “Inhibitor” consists of a protein which has the ability to delay or prevent mineral formation. c The effect of various proteins on mineralization was also studied following dissolution of each protein into a liquid buffer. d The word “Structure” refers to a protein which does not appear to induce or inhibit mineral formation by itself, but which is known to be important for the disposition and the arrangement of minerals formed in vivo. *The potential role of each protein during mineralization in vivo was proposed based on gene deletion studies in laboratory animals when available; in this case, the proteins were denoted with a single asterisk. A role for the other proteins shown was proposed based on limited functional studies giving sometimes divergent results; the role of these proteins should therefore be considered with strong reservation. **Inhibition of pancreatic stones of calcium carbonate. ***Inhibition of kidney stones of calcium oxalate or calcium phosphate. This Table was adapted and modified from the review by Benesch et al. [100]. We have also noticed that these dual seeding-inhibitory tendencies seen in vitro with some proteins may not in fact correlate with their net mineralizing effect seen in vivo (Table 1). As for the in vivo role of proteins presented in Table 1, a definitive assignment could only be derived from the phenotype of mice that were engineered to lack the gene coding for the protein in question, as already done in the case of dentin matrix protein-1, bone sialoprotein, fetuin-A, matrix gla protein, and osteopontin (see the proteins marked with a single asterisk in Table 1 and the corresponding references therein). However, in the absence of gene knockout data, a potential role for some of the proteins was proposed by the respective authors based on limited functional studies and thus must be viewed with caution. Based on such analyses, for example, albumin has been deemed to be a inhibitor of mineralization in vivo [56], [62], and yet this protein can display either inhibitory or nucleating properties in vitro depending on the conditions used (Table 1). As for fetuin-A, on the other hand, we were not able to find any study documenting the effect of the adsorbed protein; our own experiments reported here, however, demonstrate that fetuin-A can also behave as a nucleator when subjected simultaneously to heat treatment as well as to challenge with submillimolar amounts of calcium and phosphate.\nSurprisingly, few studies to date have addressed the possibility of fetuin-A representing a nucleator of calcium phosphate minerals. In a recent report [141], the early stage of CPP formation was studied based on a dynamic small-angle X-ray scattering analysis. It was observed that fetuin-A at various concentrations (1, 5, or 15 µM) added to 20 mM calcium and 12 mM phosphate ions did not in fact act as a nucleating agent of CPPs, but it essentially inhibited their formation by “shielding” nascent mineral nuclei [141]. Given that relatively high concentrations of calcium and phosphate ions were used in these experiments, it is not clear whether they are in fact representative of the slow and spontaneous formation of NB grown in metastable solutions or of NLP assembled in metastable solutions that had been challenged with submillimolar amounts of precipitating ions, both of which were meticulously addressed here. Other studies have reported that by fetuin-A was effective only in delaying mineral formation temporarily [71], [205]. For instance, when fetuin-A was present at 5 mg/ml in solutions containing 4 mM of calcium and phosphate, precipitation in the form of a fetuin-A-mineral complex appeared only after 4 to 5 days of incubation at room temperature [71], [205]. When calcium and phosphate ions were present at 5 mM in these same conditions, the incubation time was shortened to 20 to 24 hours [71], [205]. These latter observations clearly support our hypothesis of a dual inhibitory-seeding role for fetuin-A.\nThe dichotomy seen between inhibitory and seeding tendencies as a function of protein solubility (conformational state) is not in fact limited to serum proteins. Thus, Table 1 illustrates that several proteins associated with bones or teeth have also been found to possess a dual seeding and inhibitory role on mineralization, including biglycan, bone sialoprotein, decorin, dentin phosphophoryn, osteocalcin, and osteonectin. These proteins also show marked propensity to act as apatite crystal nucleators when adsorbed in vitro, consistent with the assumption that, once bound to collagen fibrils, they may unfold and help nucleate the first crystals required for collagen mineralization in vivo [99], [100]. Given the variabilities in seeding versus inhibition seen with the same proteins under different conformational states, it would appear that designating a protein as inhibitor or nucleator can be rather arbitrary. We contend that, in principle, any proteinaceous inhibitor of mineralization can be induced to nucleate under conditions that result in a conformational change in its structure.\nWith regards then to the differences seen between adsorbed and soluble proteins and still in the context of Table 1, it would appear that apatite-binding proteins tend to act as nucleators of calcification while the same proteins free in solution tend to act as inhibitors, a notion that has also been advanced in several earlier studies [97]–[100]. Presumably, an adsorbed protein may provide binding sites that recruit and bridge ions so as to position them in a specific tridimensional configuration needed to form a mineral nucleus [101]. On the other hand, when free in solution, the protein may still bind to precipitating ions, but it somehow may either prevent them from forming a mineral nucleus or coat the nascent crystal, thereby blocking any growing sites and preventing further growth [99], [101].\nFor our own experiments, we also attempted to immobilize albumin and fetuin-A onto various substrates and monitored their calcification effects in metastable solutions. Despite repeated trials, we were unable to directly seed mineral nanoparticles under the conditions used. While two earlier studies had reported that albumin could promote the formation and growth of apatite crystals when adsorbed to collagen [81], [82] or to apatite itself, using ceramics comprising of commercial apatite and calcium phosphate [83], [84], it is not clear how these results relate to our own observations given the differences in experimental conditions used. For one, the nature of the substrate used here could have played a role in the absence of effect observed. It is also not clear how the effect of albumin can be ascertained from its immobilization onto apatite or calcium phosphate crystals, which are known to promote the nucleation of other identical crystals by secondary nucleation [118]. In addition, the concentrations of calcium and phosphate used could also have played a role in the different responses obtained. Additional experiments are needed to reconcile these differences.\nIt should also be noted that although we did not obtain calcification in a metastable medium (DMEM) using either immobilized fetuin-A or immobilized albumin, we were however able to demonstrate seeding with heat-treated albumin (Fig. 7). Heat-treated fetuin-A, on the other hand, failed to seed under the same conditions. The only seeding seen with fetuin-A (also seen with albumin) was in the presence of submillimolar amounts of calcium and phosphate added simultaneously to DMEM. This property may be attributed to the potent inhibitory effect on apatite formation associated with fetuin-A. Albumin, by comparison, being a much less effective inhibitor of apatite nucleation [74], [94], has shown a greater tendency to mineralize under the conditions studied.\nViewed from a different perspective, the differential display of inhibitory versus seeding tendencies may depend on the stoichiometric relationship between the number of protein molecules versus the number of precipitating ions available in any given environment. Thus, our results indicate that, for any calcium or apatite binding protein and for given amounts of calcium and phosphate present in a medium, the protein concentration should certainly influence its effect on mineralization. This relationship can be illustrated by the significant differences seen with the amounts of fetuin-A versus albumin needed to trigger calcification (Figs. 4– 7). That is, fetuin-A is such an effective binder of apatite that it will seed apatite minerals not only when the protein-to-mineral ratios are low compared to that seen with albumin but also only with the concomitant addition of submillimolar amounts of calcium and phosphate. Based on these considerations, and extrapolating further to body conditions, fetuin-A should behave as a potent inhibitor of calcification at levels found in the body fluids, while it will nucleate at low protein concentrations only in the presence of excess calcium and phosphate. This is not surprising since according to our calculations, each bovine fetuin-A molecule binds to 54–58 apatite units while each human albumin molecule binds to 4 apatite units. In fact, due to these same stoichiometric considerations, low concentrations of calcium or apatite binding proteins have been generally associated with nucleation of mineral formation whereas high concentrations are known to usually delay or inhibit this same process [100]. In fact, according to Boskey, any macromolecule that can bind and coat nascent crystals will inhibit crystal growth when present in high concentrations in solution [206].\nProteins that inhibit mineral formation are thought to play an important role by preventing spontaneous calcification of the serum and the extracellular fluids [52], [54], [56]. This role is critical given the high natural propensity of these supersaturated extracellular fluids in vertebrates to calcify [52]–[58]. Direct evidence for the role of these proteins in preventing calcification in vivo comes from the observations that mice designed to lack any one of the calcification inhibitors like fetuin-A, matrix gla protein, or osteopontin are prone to ectopic calcification and to the debilitating effects associated with this process [67], [68], [186], [196].\nWhile most proteins that can bind to apatite crystals can be seen as inhibitors of mineralization, several proteins listed in Table 1 have been shown to be able to initiate mineral formation as well. Proteins like bone sialoprotein, biglycan, and decorin interact with the collagen fibrils and are thought to initiate the formation of the first mineral nucleus required for collagen mineralization [99], [142]. Collagen fibrils are often described to play a structural role by forming a receptacle which can delineate the main area destined to become mineralized in bones [99], [100]. Proteins can initiate mineralization by lowering the activation energy required for the formation of the first mineral nucleus in metastable solutions which usually does not occur spontaneously [101]. Given that the extracellular fluids are saturated in calcium and phosphate ions [52]–[58], the formation of mineral nuclei is thought to be sufficient to initiate mineralization, which in turn can proceed further on its own by the deposition of calcium and phosphate ions onto the nascent nuclei. It is further assumed that an effective mineral nucleator should provide an array of functional groups, particularly carbonate groups as well as phosphorylated residues, which possess high affinity for calcium and phosphate ions [101], [142]. Accordingly, several studies have suggested that proteins present in the serum may also act as nucleators of calcification. Strong evidence for the presence of a putative nucleator(s) in the serum comes from the observation that demineralized bones incubated in DMEM containing as low as 1.5% serum can remineralize following incubation for a few days [207]–[209]. This remineralization did not proceed if the demineralized bones were incubated in DMEM alone for the same period of time [207]–[209]. The factor responsible for this remineralization, which remains unidentified, was attributed to one or more protein(s) with a molecular weight between 55–150 kDa [207], [209] and which required dephosphorylation by alkaline phosphatase to become activated [210]. At first sight, this suggestion might appear paradoxical given that various systems including proteins are present in the serum to prevent calcification at any given time. Nevertheless, the observation that blood is continually in contact with bones through the so-called basic multicellular unit and bone-remodeling compartment—two anatomical structures that are associated with remodeling of cortical and cancellous bones, respectively [211], [212]—leads to the possibility that a protein secreted by osteoblasts to initiate mineralization in bones might end up circulating in the blood at low concentrations [208], [209]. Were this scenario to occur in vivo, it is likely that spontaneous calcification of blood would be prevented as long as the other major inhibitory systems remain in place [208], [209]. Whether or not this putative nucleator(s) plays a role in the formation of NB in cell culture conditions is still unclear.\nStill with regards to fetuin-A and albumin, it should be noted that we selected these two proteins for our demonstration of a dual inhibitory-seeding model for protein-mediated mineralization since they represent the two main proteins found in association with NB-like particles [1]–[3], [46]. Their effect on mineral formation has been studied extensively in the past mainly because they are present in high amounts in mineralized tissues. These findings have long suggested that the two proteins might play a role in the mineralization process [56], [57], [81], [82]. Alternatively, the binding of these two proteins to the mineral phase of bones may simply reflect their high affinity for both calcium phosphate and apatite [94]. In support of this alternative view and as noted earlier, proteins from blood are in constant contact with bones and this could account for the gradual enrichment of albumin and fetuin-A in mineralized tissues. Both albumin and fetuin-A are also found at high concentrations in the serum used to culture NB. In fact, albumin represents the main protein found in both the fetal bovine and adult human sera while fetuin-A is more abundant in FBS than in HS [92]–[96]. Both proteins can inhibit or delay the precipitation of calcium phosphate in vitro [2], [3], [62]–[64], [66], [110], [111] and both bovine fetuin-A and human albumin have been shown to account for a significant portion of the calcification-inhibitory effect associated with each serum [62], [66]. In fact, earlier studies made on the inhibitory capacity of human serum showed that two-thirds of the inhibitory potential of the serum studied was due to proteins and other macromolecules of high molecular weights while the other one-third of the inhibitory capacity could be attributed to compounds of low molecular weights [62]. Further observations made on albumin-depleted serum indicated that albumin accounted for about half of the inhibitory effect of the high molecular weight fraction present in the serum [62].\nSimilarly, when fetuin-A-depleted FBS was incubated in DMEM in cell culture conditions, it was reported that a precipitate of calcium phosphate would form in the bottom of the flask within 6 days of incubation [205]. It thus appeared that the removal of fetuin-A was sufficient to induce mineral precipitation from DMEM under these conditions. Given that the DMEM used in this study was also supplemented with phosphate to a final concentration of 2 mM [205], further experiments are needed to evaluate whether these observations are relevant to our own findings with NB and protein-minerals described here. While more experiments are required to integrate and compare the various systems of calcification used here and elsewhere, together, these results strongly support the notion that both fetuin-A and albumin represent major repressors of spontaneous calcification associated with the serum.\nThe calcification-inhibitory effects associated with fetuin-A and albumin can be attributed to the presence of several calcium and apatite binding sites on these proteins which may presumably sequester calcium and phosphate ions before they bind to the growing crystals [2], [3], [62]–[64], [66], [110], [111]. In addition, the calcium or apatite binding sites may tentatively mediate inhibition by binding onto the growing sites of the nascent crystals and blocking further mineralization. Albumin has been shown to possess as much as thirty calcium-binding sites with three different binding affinities [60] while fetuin-A has been shown to have six calcium-binding sites per molecule with two different binding affinities [65]. The calcium-binding constants associated with the fetuin-A molecule are generally lower than the ones obtained for albumin, an observation which suggests that the binding sites of fetuin-A have a higher affinity for calcium than those of albumin [65]. In addition to the potential influence of calcium-binding sites, the inhibitory effect of these proteins on mineral formation can also be attributed to the presence of specific protein motifs which promote the interaction of the proteins with growing crystals. Indeed, fetuin-A possesses a motif consisting of a cystatin-like domain with several basic amino acids which has been shown to interact with calcium phosphate and apatite crystals [66], [94]. The removal of this motif on fetuin-A by mutational analysis showed that it was responsible for the relatively strong inhibition effect of this protein [66], [94]. While no specific mineral-binding motif has been described for albumin, the interaction of this protein with apatite crystals has been attributed instead to the multiple calcium-binding sites present on this protein [62]-[64]. The possibility that a protein conformation might be associated with the inhibitory effect of albumin was also suggested by the observation that heat-denatured albumin showed increased inhibitory effect compared to the native protein [62], a result that we could not confirm however through our own experiments. Given that these two proteins are present in relatively high amounts in the serum, it is possible that they would play a predominant role in the inhibition effect seen during the culture of NB or during the assembly of NLP in the body. Taken together, these earlier studies suggest that the high inhibitory effect of albumin may be attributed mainly to its relatively high concentration in the serum [62], [74] while the stronger inhibitory effect of fetuin-A may be attributed in turn to its strong interaction with apatite crystals [74], [94].\nIn the context of biomineralization, perhaps no other protein has given more divergent and even contradictory results in terms of seeding or inhibition than albumin. These discrepancies were more noticeable when studies were done via adsorption of albumin to various solid substrates of well-defined chemical composition. For instance, albumin had earlier been shown to promote the nucleation of calcium phosphate crystals when adsorbed onto a substrate, but to essentially act as an inhibitor of mineral deposition when dissolved in solution [83]. In these experiments, albumin was adsorbed onto ceramics of apatite and calcium phosphate and immersed into a buffer containing high concentrations of calcium and phosphate similar to those contained in the serum [83], [84]. Precipitation of calcium phosphate, verified by SEM and FTIR, was found to be more prominent when albumin was adsorbed on the surface [83], [84]. However, other authors have reported that albumin seemed to exert an inhibitory effect or a lack of nucleating effect irrespective of its being adsorbed to titanium or agarose beads or its being dissolved in solution [102], [105]. The discrepancy seen between these observations has been attributed to the nature of the substrates which may seemingly have produced different protein conformations [83], [84], [149]. As such, the presence of a specific albumin conformation which allows for both firm adsorption to a surface and binding of calcium from the solution is thought to be required for albumin to induce mineralization [83], [149]. In another set of experiments, the presence of albumin in solution has also been described to promote the mineralization of phosphate ceramics [84]. In this case, the presence of a carbonate-buffered solution was found to alter the charge characteristics of albumin and to increase mineralization of phosphate ceramics when compared to solutions where no albumin or carbonate buffer was used [84]. In yet another study, the dual inhibitor-nucleator effect of albumin described here could also be seen in the context of collagen type I-mediated mineralization and was shown to depend on the concentration of albumin used [81]. When albumin was present at low concentrations (below 10 mg/ml) in supersaturated solutions containing 3.6 mM of calcium and 2.7 mM of phosphate ions, it appeared to exert a nucleating effect on collagen mineralization and to lead to increased crystal growth whereas high concentrations (above 10 mg/ml) essentially inhibited mineral formation [81]. Albumin also had an effect on the induction time needed for mineralization to start, extending the period of time for mineral to appear when compared to solutions where albumin was absent [81]. Accordingly, the disparate results may be attributed to several factors which can include the nature of the mineralization assay used, the provenance, purity, and characteristics of the protein used, as well as the different criteria used to describe its mineralizing function [82], [100].\nTogether, these studies illustrate the complexities as well as the subtleties seen in terms of seeding and inhibition functions that can be attributed to a single protein, all of which may depend on both the conformational changes as well as the concentrations of the same protein found in any given compartment. These inherent attributes of calcification-related proteins make it difficult if not altogether impossible to classify the respective roles of the same proteins during the mineralization process without first considering their physiological context (see also the excellent reviews in refs. [82], [100]).\nSince we were not able to produce NB-like minerals using only fetuin-A and/or albumin inoculated into a metastable medium like DMEM, without first heat-inactivating these proteins and adding exogenous calcium and phosphate, it is possible that other serum factors may be involved in the formation of the NB-like particles seen here and in the earlier studies. Lipids had earlier been shown to be associated with NB-like particles derived from the serum (ref. [2] and unpublished observations). Preliminary experiments show that, in addition to proteinaceous factor(s), lipids in the form of membrane or matrix vesicles (MV) may actually be involved. MV are small lipid-bound vesicles released by the vast majority of cells, but in the context of collagen-independent mineralization, they are especially significant in that they are known to be actively released by skeletal cells like osteoblasts, chondrotonblasts, and odontoblasts, and are in turn capable of nucleating the deposition of calcium phosphate in specific areas of the body (see the comprehensive review by Anderson et al. [213]). The release of these vesicles is associated with the mineralization of bones, cartilage, and teeth [213]. In bones, the release of MV is thought to be associated with mineralization away from collagen fibrils in the endochondral plaque [214]. Ectopic calcifications are thought to be associated with the release of MV-like vesicles by vascular smooth muscle cells which experience insults of high concentration of phosphate ions [215], [216]. The mechanism of mineral deposition by matrix vesicles is thought to involve the presence of calcium and phosphate channels in the membrane delineating MV; these channels gradually increase the concentration of calcium and phosphate ions within MV until mineral precipitation ensues [213]. In the context of NB formation, while our earlier studies have failed to provide conclusive evidence for a role of lipids in this process [2], we are now able to extract lipids, in the form of MV, directly from serum samples and use them to seed NLP in vitro (data not shown). These same membrane vesicles have earlier been shown to be found in the blood and in most if not all body compartments [213], [217]. In this context, it should be noted that the earlier study by Cisar et al. [44] had in fact shown that lipids like phosphatidylinositol are indeed capable of seeding apatite particles that are morphologically similar to the putative NB, thereby providing a direct link between lipid moieties and the seeding of apatite nanoparticles in a metastable medium like DMEM.\nA yet another recently proposed mechanism of mineralization that needs to be considered in the context of NLP formation involves the hydrolysis of polyphosphate present in dense calcium phosphate granules seen in tissues experiencing mineralization [218]. Presumably, this hydrolysis can be catalyzed by alkaline phosphatase [218].\nFinally, it is not clear whether the protein-mineral nanoparticles and NLP described here are related in any way to the so-called proteons and proteon-nucleation centers (PNCs) previously shown to represent blood-derived, metallic nanoclusters associated with serum protein fragments such as hemoglobin alpha-chain [219]. It is also not clear whether the more recently described proteon-associated, cell-like nanoforms in the blood that can be produced by hypotonic hemolysis are in any way related to the nanoparticles or their underlying assembly mechanisms described here [220].\nMineralization inside the body is thought to occur through the formation of an amorphous phase of calcium phosphate which gradually converts into the more stable apatite ([221]; see also ref. [116] addressing current controversies surrounding this topic). Our own data indicate that much of this amorphous-crystalline phase transformation can be mimicked through in vitro studies of the kind reported here. Our studies indicate that the protein-mineral particles assembled using fetuin-A or albumin are in many respects similar to the particles described earlier in association with NB and calcium granules. Our results point to an orderly and successive morphological conversion from spherical nanoparticles to spindles, and these to films, that are comparable despite the diverse environments in which these same particles and aggregates are found. The binding of proteins and possibly other organic compounds to nascent apatite is what apparently sustains the particles in their spherical conformations, while the growth of apatite crystals will eventually de-repress this same inhibitory influence resulting in the fusion of the round nanoparticles allowing them to coalesce to form spindle and film-like shapes. Whether this general progression of morphologies is restricted to calcium phosphate or may be conceptually extended to other minerals in general remains to be explored."}