Mineralogical signatures of stone formation mechanisms

Abstract

The mechanisms involved in biomineralization are modulated through interactions with organic matrix. In the case of stone formation, the role of the organic macromolecules in the complex urinary environment is not clear, but the presence of mineralogical ‘signatures’ suggests that some aspects of stone formation may result from a non-classical crystallization process that is induced by acidic proteins. An amorphous precursor has been detected in many biologically-controlled mineralization reactions, which is thought to be regulated by non-specific interactions between soluble acidic proteins and mineral ions. Using in vitro model systems, we find that a liquid-phase amorphous mineral precursor induced by acidic polypeptides can lead to crystal textures that resemble those found in Randall’s plaque and kidney stones. This polymer-induced liquid-precursor (PILP) process leads to agglomerates of coalesced mineral spherules, dense-packed spherulites with concentric laminations, mineral coatings and ‘cements’, and collagen-associated mineralization. Through the use of in vitro model systems, the mechanisms involved in the formation of these crystallographic features may be resolved, enhancing our understanding of the potential role(s) that proteins play in stone formation.

Introduction

Biologically-formed hard tissues, referred to as biominerals, have intrigued the materials engineering community for years because of the high degree of crystallographic control that is exerted during the precipitation of the bioinorganic crystals. While there are precise control mechanisms in place for desirable biominerals, such as in bones and teeth, there is an unfortunate lack of control in pathological deposits, such as mineral plaque and stones associated with nephrolithiasis. There are, however, some commonalities among the processes involved in both biologically-controlled and pathological biomineralization, and one predominant factor is the presence of organic matter. These include the stimulatory or inhibitory influence of biopolymers on crystal nucleation [1–7], growth [8–11], and/or aggregation [12–14]. In the case of stone formation, it is presumed that macromolecules secreted into the urine are ‘designed’ to inhibit one or all of these aspects of crystallization, thus much stone research has focused on the inhibitory potential of various biopolymers. The most potent crystallization inhibitors are typically proteins enriched with acidic residues, such as aspartic or glutamic acid [15], phosphorylated serine or threonine [6, 16, 17], or carboxylate or sulfate containing glycose units [18, 19].

Our research deals with investigating both biologically-controlled and pathological biomineralization because we are interested in the role that acidic biopolymers play in modulating crystal growth [20]. The approach we use is based on biomimetic model systems which allow for the influence of various factors to be examined in a less complex reaction medium than the physiological environment, which is particularly the case for urine. Using this approach, one can identify features that appear relevant to those found in the biological realm, and use the in vitro model system to decipher the underlying crystallochemical mechanism(s). With a better understanding of how these types of crystal aggregates are formed in stones, one is better armed with the knowledge of how to prevent those mechanisms from occurring in stone formers.

Non-classical Crystallization Processes

Kidney stones are polycrystalline aggregates often composed of some combination of calcium oxalate (CaOx) and/or calcium phosphate (CaP), along with a considerable amount of organic matter from the urine that either becomes entrapped, or plays a role, during the formation of the stone [21–25]. Given that crystal growth in pathological deposits is not regulated by highly specific protein-crystal interactions (as is considered the case for biologically-controlled mineralizations), there is not likely just one fundamental mechanism for all the different stone types that are formed. The biopolymers could impact the crystallization process during the nucleation stage, or during crystal growth, and/or during aggregation. Thus, nephrolithiasis is a highly complex problem. Nevertheless, we have found that there are several features of kidney stones that can be reproduced in the beaker, and many of these rely on the transformation of the reaction to a non-classical crystallization process. Rather than the well-recognized conventional crystal nucleation and growth processes, the presence of acidic macromolecules can induce (or stabilize) an amorphous precursor to the mineral [20, 26–30]. According to Ostwald’s rule [31, 32], if the supersaturation is high enough for multiple crystal phases to precipitate, the first occurring phase is often the least stable and closest in free energy to the mother-phase, thus the first phase to form may be an amorphous phase (Fig. 1). The amorphous phase is generally short-lived, but polymers can prolong it transformation, which depending on the relative heights of subsequent energy barriers [32], will eventually transform into one of the more thermodynamically stable phases (e.g., hydroxyapatite (Hap) from amorphous calcium phosphate (ACP)). The final more stable phase will typically be what is observed in the crystal products (such as in a stone), but this multistep crystallization pathway has important consequences with respect to the growth and aggregation behavior of the crystals, as will be discussed below.

Fig. 1.

Multi-step crystallization pathway (a) According to Ostwald’s rule, less stable phases may precipitate out first before transforming to the most thermodynamically stable product. This can include the amorphous phase (e.g. ACP) as well as a PILP phase (b) Schematic representation of how a PILP droplet might be formed, with a polyanionic polypeptide (orange random coil) that has sequestered calcium and phosphate ions along with their associated hydration

Transient amorphous precursors have been identified in the biologically-controlled biominerals of several species, including invertebrates which tend to use calcium carbonate (CaCO₃) in their exoskeletal elements [33–37], as well as vertebrates, which use calcium phosphate (CaP) in their bones and teeth [38, 39]. This paradigm shift towards recognizing the importance of an amorphous precursor has only become well accepted by the biomineralization community in recent years [20, 40, 41].

Our group has discovered that an additional step can occur in the multi-step crystallization pathway induced by acidic polypeptides (e.g. polyaspartic acid), where it seems that an excess of hydration waters become entrapped in the amorphous precursor such that is has fluidic character [20, 42, 43]. We call this a polymer-induced liquid-precursor (PILP) phase (Fig. 1b) [43], to distinguish it from the more traditional, solid amorphous particles. Although the exact molecular mechanism of the PILP process is still being investigated, it appears as though the anionic polymer (polyaspartic acid is deprotonated at neutral pH) sequesters the positively charged calcium ions, which then sequester oppositely charged counterions (e.g., carbonate, phosphate, oxalate), until the solution undergoes liquid-liquid phase separation as nanodroplets of the precursor phase condense within the crystallizing solution [44]. The fluidic character of this newly identified phase leads to interesting features in the mineral products, and some of these features resemble features found in kidney stones. Therefore, our group has proposed the following hypothesis:

Hypothesis: An amorphous mineral precursor may be induced by acidic biopolymers in the urinary environment, thus playing a key role in kidney stone formation through several possible routes:

1. Accumulation of calcium phosphate PILP droplets that solidify can lead to agglomerates of ACP that serves as a nidus for CaOx deposition.

2. Dense-packed spherulites can grow from amorphous globules, and diffusion-limited exclusion of entrapped organic impurities leads to concentrically-laminated spherules.

3. A fluidic mineral precursor can seep into cracks and crevices via capillary action, forming a mineral ‘cement’ throughout the crystals and macromolecular aggregate.

4. When PILP nanodroplets adsorb onto surfaces, the fluidic character enables them to coalesce into smooth and continuous films and coatings; sequential depositions of films could lead to ‘growth ring’ types of laminations.

5. Nanodroplets of PILP phase can promote the mineralization of collagen, such that collagen in the basement membrane and interstitium may serve as a substrate for Randall’s plaque.

Materials and Methods

The data presented here is from a collection of projects dealing with the PILP process, with detailed methods presented in the references indicated. In general, the crystals are grown in aqueous solutions containing micromolar quantities of poly-L-aspartic acid- sodium salt (ranging from 10 – 100 μg/ml). The polyaspartate molecular weight is typically in the range of 5 – 33KDa, depending on the reaction of interest (e.g., lower MW favors CaCO₃ film deposition[43, 45, 46] while higher MW favors collagen mineralization). CaP spherulites and composite ‘stones’ are described in prior reports [47, 48]. In general, the reactions are typically performed at moderately high supersaturation and without stirring, and can take hours to run when at room temperature. The collagen mineralization reaction is done in a tris buffer with stirring at 37°C, and can take up 3 to 16 days, depending on the reaction conditions [49–53].

Results and Discussion

Our group’s initial studies dealt with mimicking biologically-controlled mineralization reactions, but when side products of crystal aggregates kept forming in the reactions, our attention turned to the stone field. In the presence of polyaspartate, aggregates form with CaOx (Fig. 2) [20], CaP (Fig. 3), and CaCO₃ (Fig. 4) [54]. We believe these aggregates result from accumulation of PILP droplets, as evidenced by PILP nanodroplets that grow large enough to observe on an optical microscope before they solidify and crystallize. For example, Fig. 2 shows micron-sized spherules of CaOx which had formed and coalesced into films (Fig. 2a), along with ‘dumbbells’ (Fig. 2b), spherulites (Fig. 2c), and agglomerates (Fig. 2d). Agglomerates of partially coalesced CaP particles are shown in Fig. 3a. Note the similarity between the CaP agglomerates and that seen in the stone shown in Fig. 3b [24]. The central nidus of this stone was CaP, which is shown at higher magnification in Fig. 3c. The agglomerate appears to be formed from partially coalesced amorphous spherules, thus a PILP-like precursor with fluidic character may have been involved.

Fig. 2.

Evidence of CaOx PILP phase. The polarized optical micrographs were taken using a 1^st-order red λ-plate so that amorphous particles can be seen because isotopic materials exhibit a magenta background (including the isotropic glass substrate). (a) Small round crystals are from an amorphous precursor, and the birefringent films (orange and blue patches) are from coalesced precursor droplets (b) Many film patches are seen at lower magnification, along with ‘dumbbell’ partial spherulites (c) Precursor particles appear to have aggregated onto a larger spherulite. The birefringent colors indicates that the spherules are now crystalline, and the Maltese Cross is indicative of a spherulite. (d) SEM shows an agglomerate of CaOx particles cemented to a CaOx film. The darker gray particles are partially embedded in the film. (Reproduced with permission from ref [55]. Copyright 2006 John Wiley & Sons Limited)

Fig. 3.

Evidence of CaP PILP phase and comparison to a stone nidus (a) SEM of an agglomerate of CaP spherules. They have partially coalesced in the top right corner. (b) Composite stone composed of a CaP central core (labeled P) and a CaOx outer layer (labeled O). The CaOx region appears to have a radial spherulitic texture and exhibits some concentric laminations. Bar = 5 μm (d) Higher magnification view of the CaP core, which shows an agglomerate of partially coalesced particles. Bar = 1 μm (Reprinted with permission from ref. [24]. Copyright 1997 Elsevier- American Urological Association Inc.)

Fig. 4.

Concentric laminations in CaCO₃ PILP products (a) Polarized micrograph of a CaCO₃ thin film (~500 nm thick). The film has a spherulitic texture and developed concentric laminations as the amorphous film crystallized. (b) FITC-labeled polyaspartate demonstrates that the laminations are from polymer that became entrapped in diffusion-limited exclusion zones as the polymeric impurities were excluded ahead of the crystallization front. In the case of a radial crystal growth pattern that occurs in spherulite formation, the periodic zones are arranged in a concentric fashion orthogonal to the growth front. (c) Large spherules of CaCO₃ (~10 μm) formed along with the smaller less birefringent solidified PILP droplets (~ 3 μm). (d) These spherules contained a polymer-enriched shell (Reproduced with permission from ref [55]. Copyright 2006 John Wiley & Sons Limited)

Mechanisms of Formation of Polycrystalline Aggregates

It should be noted that there are two pathways that can lead to polycrystalline aggregates that are derived from distinctly different mechanisms (Fig. 5) [55]. The polycrystalline aggregate of a spherulite is formed from radial growth of polycrystals from a centralized nucleation point. The radial growth leads to a ‘Maltese Cross’ pattern in cross-polarized light, which can be seen in the large centralized spherulite of Fig. 2c. As the radial polycrystals are growing, they can form branches which help to fill space, particularly when there is a preference for twinning or splay (Fig. 5b). A partially formed spherulite resembles the shape of a dumbbell (Fig. 5b), which is often the morphology of crystals found in urine [56]. Continued growth will lead to a spherulite, which has a radial texture of the polycrystals (Fig. 5c). If the crystals are growing from within an amorphous globule, the space surrounding the forming crystallites is already filled with mineral precursor, and will eventually crystallize as well, leading to a dense-packed spherulite. On the other hand, if the crystals are growing from solution by the conventional crystallization process, there is likely to be more space between the crystallites as they adopt their needle or platy crystal habits in the solution without being surrounded by amorphous phase to fill in the spaces.

Fig. 5.

Polycrystalline aggregate formation via two different pathways (a) Schematic representation of the radial growth patterns that occur in spherulite formation (b) A partial spherulite (under polarized light), which appears to branch outward due to twinning, has a ‘dumbbell’ morphology (analogous to the top schematic of Fig. (a)) (c) This spherulite has a radial texture, indicating it is polycrystalline aggregate formed by radial growth of the thin CaOx crystals (analogous to the bottom schematic of Fig. (a)) (d) An aggregate of CaOx crystals with the traditional faceted crystal habit represents a true aggregation process consisting of preformed and intergrown crystals (Reproduced with permission from ref [55]. Copyright 2006 John Wiley & Sons Limited)

In contrast to the polycrystalline aggregates formed by the well-defined spherulitic growth process, true aggregation occurs when pre-formed crystals come together (Fig. 5d). Such an aggregate can then be reinforced if the crystals become cemented together, either by an organic ‘glue’ or a mineral ‘cement’ (see for example Figs. 2d and 8c). The PILP droplets have a tendency to aggregate together and with other crystals, as seen in Fig. 2c, where the precursor droplets appear to have been adsorbing onto a larger spherulite. We often observe that these types of aggregates become ‘cemented’ together (Fig. 2d), which presumably arises from the partial liquid-like character of a PILP precursor. Depending on the degree of fluidity of the amorphous precursor, one can imagine that a liquid-like phase could adsorb onto a forming stone and seep into the cracks and crevices via capillary action, thus increasing the contact area and fortifying the mineral cement.

CaP and CaOx Spherulites

In the case of the CaOx crystals in stones, it seems likely that polycrystalline aggregates may form by both processes, where the randomly arranged and more faceted crystals appear to be true aggregates (Fig. 5d), while the spherical deposits with radial texture appear to be dense-packed spherulites (Fig 3b). Spherulites are commonly seen in the in vitro model of CaOx, as shown in Fig.2 and 5c.

In a simple model system for CaP with acidic polypeptides, it was also found that polyaspartate promotes the formation of dense-packed spherulites (Fig. 6a) [20, 47]. As mentioned above, the densely packed structures are thought to arise from radial growth from within a preformed amorphous phase. In the example shown in Fig. 6b–d, it appears that the space filling property arises from polycrystal growth from within an ion enriched gelatinous globule. For example, the early stage spherulites of CaP shown in Fig. 6b are rather faint in birefringence, and they flattened when deposited on a substrate (Fig. 6c). But as they crystallized, the birefringence became brighter until the spherulites had fully densified and could no longer transmit light (when they become 10s of microns in diameter), where they finally appear dark brown (Fig. 6d). Needle-like crystals can also form from the globules (Fig. 6d), and in this case, seem to form from a surface dissolution-recrystallization reaction (since they are not as densely packed).

Fig. 6.

Formation of CaP spherulites from amorphous globules (a) SEM of a dense-packed spherulite with a denser shell and concentric laminations (b) Early stage globules contain a faint birefringence as they are just starting to crystallize (c) Upon drying, some spherulites collapsed, and appeared to be composed of a slightly rigid gelatinous globule (d) Maturing spherulites became highly birefringent, and ultimately became brown as they densified and blocked light transmission. The particle on the right appears to be composed of coalesced globules of spherulites. The middle spherulite is starting to form a brown crust as it undergoes dissolution-recrystallization. The spherulite to the left shows needles that grow from solution, as opposed to the dense-packed smooth-shelled spherulite in image (a). All of the spherulites exhibit pronounced concentric laminations. (Reproduced with permission from ref [55]. Copyright 2006 John Wiley & Sons Limited.)

There is another crystallographic texture observed in these CaP spherulites formed in vitro that may also be relevant to certain features observed in Randall’s plaque, which has taken the spotlight in recent years.

Randall’s Plaque and Concentrically-Laminated Spherules

Recent evidence by Evan and coworkers [57–61] implicates Randall’s plaque as playing an important role in idiopathic calcium oxalate (CaOx) stone formation (Fig. 7) [58]. As originally described by Randall [62], and further examined in greater detail by the Evan team, this pathological mineralization starts with the deposition of calcium phosphate (CaP). The initial deposits appear as small electron-dense multilaminated spherules (roughly 50 nm in diameter), that form first at the basement membrane of the thin loops of Henle (Fig. 7a) [61], and then spread into the nearby interstitial space (Fig. 7b), ultimately extending beyond the urothelial lining into urinary space, where the spherules somehow stimulate attachment and/or nucleation of the CaOx crystals that grow into a large stone.

Fig. 7.

Evan’s work on idiopathic CaOx stone forming patients shows the location and morphology of newly forming precipitates found in Randall’s plaque (a) Histologic optical image (Yasue stained) of a papillary biopsy section with minimal plaque showing spherically shaped, brown-black deposits only in the basement membranes of the thin loops of Henle (arrows) (x 900) (b) A denser region of plaque shows an accumulation of such deposits forming a continuous layer of calcifed matrix (x 500) (c) TEM image of deposits first seen in the basement membrane of the thin loops of Henle (x 25,000) (d) High magnification image of one of the deposits (x 70,000), which are typically multilaminated spherules containing alternating light and dark electron dense rings. (Reprinted with permission from Macmillan Publishers Ltd: [Urological Research] [58], copyright (2006), American Institute of Physics.)

The spherules in Randall’s plaque are quite small, but even full-sized stones often exhibit concentric laminations, and these often occur in the CaOx regions containing a radial sub-texture indicative of spherulites. The concentric layers in these larger multi-laminated structures are typically attributed to daily (or dietary) growth rings, which are thought to form when the urinary environment, which is normally metastable, becomes excessively supersaturated (such as by dehydration, or hyperoxaluria). Our in vitro studies may provide an alternative hypothesis for how such multi-laminated spherical deposits may be formed. For example, multi-laminated spherulites can be generated with an amorphous precursor (Fig. 6), but in this case, the laminations do not occur by sequential deposition of mineral layers. These synthetic spherulites are much larger than the plaque spherules, but commensurate with the laminated spherulitic texture seen in many stones, such as the one shown in Fig. 3b. Our 2-D model system of spherulitic CaCO₃ films (Fig. 3b) shows that the concentric laminations result from diffusion-limited exclusion of the polymeric impurity, which leads to polymer-enriched layers, as seen by the fluorescently labeled polyaspartate [63]. In the case of 3-dimensional spherules (Fig. 4d), the polyaspartate became particularly enriched in an outer shell that had formed. When stained, the spherules of Randall’s plaque show the presence of organic matter in concentric layers (Fig. 7c & 7d), particularly at the outer border. Evan et al. [58] also used immunogold labeling to show an enrichment of osteopontin at the interface between organic and mineral layers in the spherules. In the urinary environment, there are many proteins, lipids, and muco-polysaccharides, all of which could become entrapped in the amorphous precursor, and therefore would be expected to generate pronounced laminations as they are excluded during the crystallization process.

Ryall [64] has put together an interesting collage of multi-laminated spherules found in a wide variety of organisms, ranging from ‘nanobacteria’, to the electron-dense concretions in the midgut of various insects; and calcifying granules in the hepatopancreas of snails; and so on; all of which bear a strikingly resemblance to the multilaminated spherules found in Randall’s plaque. She draws the conclusion that “it is likely that the granules in human kidneys fulfill analogous functions to those in other organisms—particularly in calcium homeostasis”. While the function of the spherules (often times amorphous) in many of these organisms may be for ion storage, their formation does not appear to require a high degree of crystal modulation. Therefore, one could argue that this similarity in morphology and texture is simply a result of how an amorphous mineral precipitates, and particularly with entrapped organics that succumb to diffusion-limited exclusion, leading to concentric laminations. In support of this, the calcium phosphate in plaque has been described as containing amorphous calcium phosphate (ACP), as well as ‘biological’ apatite similar to bone [65]. In the case of CaOx, Ryall found that protease-treated CaOx monohydrate crystals extracted from urine contained sub-crystalline ovoid nanoparticles (50–100 nm in size). Similar features were observed in the CaOx crystals of plant raphides [64]. Nanogranular textures have been observed in numerous CaCO₃ and CaP biominerals as well [66–71], many of which have since been shown to form from an amorphous precursor. AFM and TEM of our PILP-formed films and crystals show a remnant nano-colloidal texture as well [46, 72]. Thus, this nanogranular texture may simply be a ‘mineralogical signature’ of an amorphous precursor in the CaOx biominerals of stones as well.

CaP-CaOx Composite Stones

An important consequence of the Randall’s plaque is that it provides an anchored mineralization site, in which the CaP deposits apparently stimulate CaOx crystal formation into the urinary space [58, 65]. We hypothesize that the CaP minerals that form the nidus of some stones might be generated by an accretion of CaP precursor phase. For example, the spherules of partially coalesced CaP droplets/particles in Fig. 3a appear similar to the partially coalesced CaP spherules seen in the multiphase composite stone of Fig. 3c [24]. It is thought that the differences in pH between the urine and tissue could be responsible for the change in mineral composition because HA forms in a more basic pH, while the lower pH of urine favors CaOx. We have done some in vitro studies on the overgrowth of calcium oxalate on calcium phosphate spherulites, where both minerals were formed in the presence of polyaspartate (Fig. 8) [55]. The CaP precipitates grew from accumulation of PILP droplets that transformed into amorphous globules, which then solidified into dense-packed spherulites, as shown in Fig. 8a. CaOx was then precipitated in the presence of these pre-formed spherulites, and with the polyaspartate additive, it was found that the amorphous CaOx PILP droplets liked to adsorb to the preformed CaP spherulites, and ultimately formed a large spherulitic structure on top of the CaP nidus (Fig. 8b).

Fig. 8.

CaP-CaOx composite particles (a) SEM of a dense-packed spherulite formed from agglomeration of CaP precursor particles (b) CaOx overgrowth on a CaP spherulitic core (marked with an arrow). There is a radial texture, but the CaOx polycrystals have grown large enough to see their facets. (c) A large CaP spherulite as the core with a smooth thin coating of CaOx. Other poorly identified crystals appear to have aggregated and become cemented onto the particle by the mineral coating. (d) A whitlockite stone showing numerous well-defined mineral layers. Notably, the mineral layers in this stone are about the same thickness as the majority of films deposited with the PILP process (about half a micron). It is not clear whether or not this stone has a spherulitic texture, but the relatively separated structure of the laminations suggests they are caused by a sequential deposition of mineral coatings. (Reproduced with permission [55]. Copyright 2006 John Wiley & Sons Limited.)

Depending on the reaction conditions, in some cases the CaOx PILP droplets coalesced into a partial mineral coating (Fig. 8c) [55]. This type of deposition, if performed repeatedly, might be expected to lead to a ‘growth ring’ type of structure, analogous to the kidney stones that appear to have continuous, film-like multilayered coatings (Fig. 8d) [55]. Mineral films and coatings are typical of mineral formed by the PILP process (e.g., Fig. 2 and Fig. 4), which is what led to the initial discovery of this non-classical crystallization process in the CaCO₃ system [43, 45]. From this study of biomimetic composite ‘stones’, one can see that an amorphous precursor can lead to layered textures by two different pathways, 1) diffusion-limited exclusion of organics, or 2) sequential deposition of mineral coatings.

‘Inhibitory’ Proteins

The urinary tract contains many acidic glycoproteins (as well as lipids) which could conceivably induce the amorphous precursor pathway. A more in depth review on this subject is provided in our chapter of the book on Biomineralization- Medical Aspects of Solubility [55]. Many in vitro models of stone formation consider the acidic proteins as having an inhibitory action, which is therefore assumed to provide a protective action against stone formation. However, it should be noted that it is the acidic “inhibitory” polymers (e.g., polyaspartate) that tend to generate the amorphous precursor pathway when a high enough supersaturation is reached. Thus, even though the function of these proteins may well be to protect against urolithiasis, it is quite possible that this inhibitory action could be overwhelmed by the supersaturation or other factors in the reaction environment, and thus lead to the amorphous precursor pathway. In other words, a reaction medium that has inhibitory additives can eventually ‘crash out’ with spherulites and/or droplets of a precursor phase. Spherulitic growth is rapid and can lead to relatively large structures in a short time; likewise, the aggregation tendency of crystals in a solution containing precursor droplets can also generate large structures quickly; so both of these aggregation phenomena could contribute to crystal retention in the renal tubules and augment stone formation.

Osteopontin, which is a prominent protein associated with mineral in the plaque [73] (as well as in bone), is a highly acidic protein which could conceivably induce an amorphous phase in the biological environment. Such acidic proteins are generally considered to be inhibitors of crystal nucleation [15, 74, 75], and indeed, this is true of polyaspartate, our simple model protein, as well. Yet we find that the inhibitory action of polyaspartate promotes the mineralization of collagen [50], which may be relevant to the initial stages of plaque formation in the basement membrane and interstitium In this other project, we are interested the mechanisms involved on bone formation [50]. We have shown that intrafibrillar mineralization of collagen can be achieved using the PILP process [50–52], and have proposed that the precursor phase is drawn into the interstices of collagen fibrils via capillary action [50]. In the control reaction, which did not include polyaspartate, it was found that the collagen fibrils are rather poor at promoting mineralization. This work was done with type-I collagen, thus more studies are needed to determine if this might be the case with the type-IV collagen found in the basement membrane, and if mineralization of such tissues with the PILP process might lead to structures that resemble Randall’s plaque.

It seems reasonable to postulate that osteopontin, which is prevalent in plaque, may be there for the purpose of inhibiting mineralization within the tissue if the cells sense an excessive ion concentration in the papillary tissue. However, if the inhibitory potential of such proteins is overwhelmed (such as by a dehydration rise in supersaturation, or hypercalciluria or hyperoxaluria), a metastable amorphous phase could be formed, which could instigate this unfortunate set of consequences.

Conclusions

In the case of pathological biomineralization, many of the proteins extracted from stones are known to be modulators of crystallization in vitro, which could be active during crystal nucleation, growth, or aggregation. Stones form in a complex urinary environment and are composed of a heterogeneous population of mineral products, so some or all of these stages may be important in the formation or retention of the stone. One might assume that since there are many crystal types and crystal faces present, that highly specific interactions are not involved in the inhibitory and blocking potential of these proteins; instead, more generic crystal binding domains, such as domains of acidic functionality, could be useful for inhibiting both crystal nucleation and growth, as well as aggregation (such as via steric repulsion of a protein of appropriate size). It is possible that these non-specific inhibitory proteins may be secreted by the renal epithelial cells, most often resulting in successful inhibition of the stone; but under certain conditions, the desired inhibitory action of the protein may be overcome by the supersaturation or other environmental factors, leading to amorphous precursors. This seems to be evidenced by some of the mineralogical ‘signatures’ found in stones, such as agglomerates and compact spherulites, core-overgrowth structures, concentric laminations, nanospherical subunits, and generalized aggregation tendencies and cementatious properties. Thus, while an amorphous precursor might be very useful in biologically-controlled mineralization, it could have detrimental effects in contributing to pathological biomineralization.

Abbreviations:

PILP

polymer-induced liquid-precursor

CaCO₃

calcium carbonate

CaP

calcium phosphate

CaOx

Calcium oxalate

SEM

scanning electron microscopy

TEM

transmission electron microscopy

POM

polarized optical microscopy