Biomineralization Mechanisms: A New Paradigm for Crystal Nucleation in Organic Matrices

There is substantial practical interest in the mechanism by which the carbonated apatite of bone mineral can be initiated specifically in a matrix. The current literature is replete with studies aimed at mimicking the properties of vertebrate bone, teeth, and other hard tissues by creating organic matrices that can be mineralized in vitro and either functionally substitute for bone on a permanent basis or serve as a temporary structure that can be replaced by normal remodeling processes. A key element in this is mineralization of an implant with the matrix and mineral arranged in the proper orientations and relationships. This review examines the pathway to crystallization from a supersaturated calcium phosphate solution in vitro, focusing on the basic mechanistic questions concerning mineral nucleation and growth. Since bone and dentin mineral forms within collagenous matrices, we consider how the in vitro crystallization mechanisms might or might not be applicable to understanding the in vivo processes of biomineralization in bone and dentin. We propose that the pathway to crystallization from the calcium phosphate–supersaturated tissue fluids involves the formation of a dense liquid phase of first-layer bound-water hydrated calcium and phosphate ions in which the crystallization is nucleated. SIBLING proteins and their in vitro analogs, such as polyaspartic acids, have similar dense liquid first-layer bound-water surfaces which interact with the dense liquid calcium phosphate nucleation clusters and modulate the rate of crystallization within the bone and dentin collagen fibril matrix.     

Polyphosphates inhibit extracellular matrix mineralization in MC3T3-E1 osteoblast cultures

Studies on various compounds of inorganic phosphate, as well as on organic phosphate added by post-translational phosphorylation of proteins, all demonstrate a central role for phosphate in biomineralization processes. Inorganic polyphosphates are chains of orthophosphates linked by phosphoanhydride bonds that can be up to hundreds of orthophosphates in length. The role of polyphosphates in mammalian systems, where they are ubiquitous in cells, tissues and bodily fluids, and are at particularly high levels in osteoblasts, is not well understood. In cell-free systems, polyphosphates inhibit hydroxyapatite nucleation, crystal formation and growth, and solubility. In animal studies, polyphosphate injections inhibit induced ectopic calcification. While recent work has proposed an integrated view of polyphosphate function in bone, little experimental data for bone are available. Here we demonstrate in osteoblast cultures producing an abundant collagenous matrix that normally show robust mineralization, that two polyphosphates (PolyP5 and PolyP65, polyphosphates of 5 and 65 phosphate residues in length) are potent mineralization inhibitors. Twelve-day MC3T3-E1 osteoblast cultures with added ascorbic acid (for collagen matrix assembly) and β-glycerophosphate (a source of phosphate for mineralization) were treated with either PolyP5 or PolyP65. Von Kossa staining and calcium quantification revealed that mineralization was inhibited in a dose-dependent manner by both polyphosphates, with complete mineralization inhibition at 10 μM. Cell proliferation and collagen assembly were unaffected by polyphosphate treatment, indicating that polyphosphate inhibition of mineralization results not from cell and matrix effects but from direct inhibition of mineralization. This was confirmed by showing that PolyP5 and PolyP65 bound to synthetic hydroxyapatite in a concentration-dependent manner. Tissue-nonspecific alkaline phosphatase (TNAP, ALPL) efficiently hydrolyzed the two PolyPs as measured by P_i release. Importantly, at the concentrations of polyphosphates used in this study which inhibited bone cell culture mineralization, the polyphosphates competitively saturated TNAP, thus potentially interfering with its ability to hydrolyze mineralization-inhibiting pyrophosphate (PP_i) and mineralizing-promoting β-glycerophosphate (in cell culture). In the biological setting, polyphosphates may regulate mineralization by shielding the essential inhibitory substrate pyrophosphate from TNAP degradation, and in the same process, delay the release of phosphate from this source. In conclusion, the inhibition of mineralization by polyphosphates is shown to occur via direct binding to apatitic mineral and by mixed inhibition of TNAP.     

Proteomic analysis of a rare urinary stone composed of calcium carbonate and calcium oxalate dihydrate: A case report

The objective of the present study was to investigate the matrix protein of a rare urinary stone that contained calcium carbonate. A urinary stone was extracted from a 34‐year‐old male patient with metabolic alkalosis. After X‐ray diffractometry and infrared analysis of the stone, proteomic analysis was carried out. The resulting mass spectra were evaluated with protein search software, and matrix proteins were identified. X‐ray diffraction and infrared analysis confirmed that the stone contained calcium carbonate and calcium oxalate dihydrate. Of the identified 53 proteins, 24 have not been previously reported from calcium oxalate‐ or calcium phosphate‐containing stones. The protease inhibitors and several proteins related to cell adhesion or the cytoskeleton were identified for the first time. We analyzed in detail a rare urinary stone composed of calcium carbonate and calcium oxalate dihydrate. Considering the formation of a calcium carbonate stone, the new identified proteins should play an important role on the urolithiasis process in alkaline condition.     

Differential expression patterns of the dentin matrix proteins during mineralized tissue formation

Sequential and reciprocal interactions between the oral ectoderm and neural crest-derived mesenchyme are responsible for tooth development. During dentin formation, there are three components that are necessary for proper mineralization, namely, collagen which forms a scaffold, noncollagenous proteins that can specifically bind to the collagen template and function as a mineral nucleator and crystalline calcium phosphate deposited in an ordered manner. It is well established that noncollagenous proteins play an important role during mineralized tissue formation. Here we demonstrate by in situ hybridization techniques that the noncollagenous dentin matrix proteins 1, 2 (DMP1, 2) and dentin sialoprotein (DSP) have characteristic temporal and spatial expression patterns within odontogenic tissues during dentin mineralization. DMP1, DMP2 and DSP mRNA are expressed in the odontoblasts at specific and overlapping time points and are thus presumably used for different functions during dentin formation. In developing rat incisors and molars, high levels of expression of DMP2 mRNA were seen in polarized odontoblasts and preameloblasts, while DSP mRNA was expressed at significantly lower levels and was expressed by highly differentiated odontoblasts. However, their expression was continuously maintained during the mineralization of the organic matrix. In the adult rats, DMP2 and DSP mRNA was also detected in the osteoblasts. The expression of DMP1 mRNA was found to coincide with the start of the mineral nucleation process and gradually decreased during the maturation of the mineralized matrix during odontogenesis. In this study, we have also correlated the expression of these proteins relative to the presence of type I collagen and calcium phosphate crystals. Thus, the temporal and spatial differences between DMP1, DMP2 and DSP might implicate a direct demonstration of the functional difference between these three genes during calcified tissue formation.     

Presence of lipids in urine,crystals and stones: implications for the formation of kidney stones

BACKGROUND: Cell membranes and their lipids play critical roles in calcification. Specific membrane phospholipids promote the formation of calcium phosphate and become a part of the organic matrix of growing calcification. We propose that membrane lipids also promote the formation of calcium oxalate (CaOx) and calcium phosphate (CaP) containing kidney stones, and become a part of their stone matrix. METHODS: Human urine, crystals of CaOx and CaP produced in the urine of healthy individuals, and urinary stones containing struvite, uric acid, CaOx and CaP crystals for the presence of membrane lipids were analyzed. Crystallization of CaOx monohydrate at Langmuir monolayers of dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylserine (DPPS), dioleoylphosphatidylglycerol (DOPG), palmitoyloleoylphosphatidylglycerol (POPG) and dimyristoylphosphatidylglycerol (DMPG) was investigated to directly demonstrate that phospholipid assemblies can catalyze CaOx nucleation. RESULTS: Urine as well as CaOx and CaP crystals made in the urine and various types of urinary stones investigated contained some lipids. Urine of both CaOx and uric acid stone formers contained significantly more cholesterol, cholesterol ester and triglycerides than urine of healthy subjects. However, urine of CaOx stone formers contained more acidic phospholipids. The organic matrix of calcific stones contained significantly more acidic and complexed phospholipids than uric acid and struvite stones. For each Langmuir monolayer precipitation was heterogeneous and selective with respect to the orientation and morphology of the CaOx crystals. Crystals were predominantly monohydrate, and most often grew singly with the calcium rich (10-1) face toward the monolayer. The number of crystals/mm2 decreased in the order DPPG> DPPC and was inversely proportional to surface pressure and mean molecular area/molecule. CONCLUSIONS: Stone forming conditions in the kidneys greatly impact their epithelial cells producing significant differences in the urinary lipids between healthy and stone forming individuals. Altered membrane lipids promote face selective nucleation and retention of calcium oxalate crystals, and in the process become a part of the growing crystals and stones.     

Quantification of proteins extracted from calcium oxalate and calcium phosphate crystals induced in vitro in the urine of healthy controls and stone-forming patients.

We have previously identified proteins extracted from calcium oxalate (CaOx) and calcium phosphate (CaP) crystals generated experimentally in vitro in whole urine of healthy controls and stone formers. No significant differences were detected between protein components in matrices of crystals obtained from both groups. The aim of the present study was to estimate the amounts of six proteins identified earlier in order to investigate the differences, if any, between healthy controls and lithiasis patients. CaOx and CaP crystals were generated in the urine samples by adding an oxalate and phosphate load, respectively. Crystals were harvested, washed, dried, and analyzed. Crystal matrix protein was extracted by demineralizing crystals with EDTA solution, analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and identified immunochemically using Western blot analysis. The quantity of each protein was estimated by densitometric analysis. The predominant proteins found in organic matrices of CaOx crystals induced in the urine of healthy controls were prothrombin-related proteins followed by albumin and osteopontin. In matrices of CaP crystals, the principal proteins were Tamm-Horsfall protein followed by albumin, prothrombin-related proteins and osteopontin. However, when crystals were induced in the urine of stone formers, albumin was the major component of the organic matrix of both CaOx and CaP crystals. We concluded that a subset of urinary proteins is present in crystal matrix. Among them, albumin seems to play a crucial role in kidney stone formation.     

Lipids and Membranes in the Organic Matrix of Urinary Calcific Crystals and Stones

The ultrastructure of the organic matrix of demineralized urinary stones was examined by standard transmission and scanning electron microscopy as well as after malachite green-glutaraldehyde fixation. Crystal ghosts of both calcium oxalate and calcium phosphate were made of amorphous material and were dispersed in a matrix containing amorphous, fibrillar, and membranous substances. Malachite green positive material was seen to be associated with the ghosts, as well as with the membranous and fibrillar components of the organic matrix. Calcium oxalate and calcium-phosphate crystals, induced in human urine in vitro were also found to be associated with an organic matrix containing lipids and proteins. It is suggested that the intimate association between crystals and lipids is a result of the involvement of cellular membranes in the nucleation of these crystals. Received: 28 August 1995 / Accepted: 22 December 1995     

Cultured podocytes establish a size-selective barrier regulated by specific signaling pathways and demonstrate synchronized barrier assembly in a calcium switch model of junction formation

Podocytes form unique cell-cell junctions (slit diaphragms) that are central to glomerular selectivity, although regulation and mechanisms of slit diaphragm assembly are poorly understood. With the use of cultured podocytes, a paracellular permeability flux assay was established to characterize properties of the size-selective barrier. Paracellular flux of differentiated podocytes was measured using anionic fluorescent dextrans of 3, 10, 40, and 70 kD. Podocytes form a highly selective barrier with a 160-fold difference in flux from the 3-kD dextran (11 pmol/min) to the 70-kD dextran (0.06 pmol/min). Barrier development was dependent on podocyte differentiation and not affected by dextran charge. Puromycin, a known podocyte toxin, increased flux 250% in a dose-dependent manner without affecting cell viability. Screening with modulators of specific signaling pathways identified reversible increases in flux with Src tyrosine and Rho kinase inhibition. The calcium switch model of epithelial junction assembly was modified to determine whether podocytes regulate barrier assembly. When cultured in low calcium for 90 min, flux increased by 300% and consistently returned to baseline 24 to 48 h after switching to normal calcium. Similar to classical epithelial junctions, barrier recovery occurred in the presence of cyclohexamide, an inhibitor of protein synthesis. During the calcium switch, there were reversible changes in localization and detergent solubility of the slit diaphragm protein ZO-1 and alpha-actinin-4, whereas nephrin and podocin solubility were unchanged. Taken together, these findings demonstrate that cultured podocytes develop a selective size barrier that is regulated by specific signaling pathways, and similar to classical epithelial junctions, podocytes demonstrate synchronized assembly of the barrier.     

Refinement of collagen–mineral interaction: A possible role for osteocalcin in apatite crystal nucleation,growth and development

Mineralization of vertebrate tissues such as bone, dentin, cementum, and calcifying tendon involves type I collagen, which has been proposed as a template for calcium and phosphate ion binding and subsequent nucleation of apatite crystals. Type I collagen thereby has been suggested to be responsible for the deposition of apatite mineral without the need for non-collagenous proteins or other extracellular matrix molecules. Based on studies in vitro, non-collagenous proteins, including osteocalcin and bone sialoprotein, are thought to mediate vertebrate mineralization associated with type I collagen. These proteins, as possibly related to mineral deposition, have not been definitively localized in vivo. The present study has reexamined their localization in the leg tendons of avian turkeys, a representative model of vertebrate mineralization. Immunocytochemistry of osteocalcin demonstrates its presence at the surface of, outside and within type I collagen while that of bone sialoprotein appears to be localized at the surface of or outside type I collagen. The association between osteocalcin and type I collagen structure is revealed optimally when calcium ions are added to the antibody solution in the methodology. In this manner, osteocalcin is found specifically located along the a_4–1, b₁, c₂ and d bands defining in part the hole and overlap zones within type I collagen. From these data, while type I collagen itself may be considered a stereochemical guide for intrafibrillar mineral nucleation and subsequent deposition, osteocalcin bound to type I collagen may also possibly mediate nucleation, growth and development of platelet-shaped apatite crystals. Bone sialoprotein and osteocalcin as well, each immunolocalized at the surface of or outside type I collagen, may affect mineral deposition in these portions of the avian tendon.     

Ultrastructure and growth of the sea urchin tooth

Individual sea urchin teeth consist of many elements, each secreted by a syncytium formed for the purpose. The numerous syncytia of each tooth take up secondary connection with one another in the vicinity of needles and prisms. The elements of the primary tooth skeleton are surrounded by cytoplasm and are therefore intracellular. Following the origin of a syncytium in the plumula, a new tooth element sheath originates in the form of a vesicle, which develops a unified crystallization cavity in the shape of the future tooth element. During the early growth of the sheath, calcium carbonate crystallization begins within the sheath. An inner coating of the sheath functions as a crystallization matrix, and further growth of calcium carbonate takes place centripetally. Collagen does not take part in mineralization. Neither an axial thread nor other organic material inside the hardened mineral was found.     

Influence of aluminum on mineralization during matrix-induced bone development

A model of de novo mineralization employing matrix-induced endochondral bone formation in rats was used to study the short-term effects of aluminum on the deposition of calcium and phosphate in vivo. In experiments where systemic aluminum concentrations were elevated, the cellular processes associated with bone development appeared to be normal, if somewhat delayed, however precipitation of the mineral phase was prevented. This suggests a primary direct physical chemical effect of aluminum in vivo on calcification, as suggested by in vitro studies which demonstrate that aluminum is a potent inhibitor of calcium phosphate precipitation. Aluminum salts implanted locally with the matrix appeared to be toxic to the cellular processes leading to chondrogenesis and osteogenesis.     

Triamterene and renal stone formation: The influence of triamterene and triamterene stones on calcium oxalate crystallization

Summary A constant composition method has been used to compare the effects of triamterene renal stone material, synthetic triamterene precipitates, and soluble triamterene on the nucleation and crystallization kinetics of calcium oxalate in aqueous solutionin vitro. Crystallization studies have been carried out with the concentrations of calcium and oxalate ions maintained constant by the potentiometrically controlled addition of concentrated reagent solutions containing these ions. Triamterene renal stones were found to be much less effective than synthetic triamterene towards promoting the nucleation and crystallization of calcium oxalate from supersaturated solution. Renal stones composed of triamterene and matrix did not significantly enhance the deposition of calcium oxalate compared to nonseeded controls. The triamterene stones were also found to be ineffective in promoting calcium oxalate crystallization compared to other precipitates thought to be involved in the etiology of stone disease such as calcium hydroxyapatite. For stones of mixed triamterene/calcium oxalate composition, the enhancement of the nucleation and crystallization of calcium oxalate was directly related to the calcium oxalate content of the stone seed material. The presence of soluble triamterene or its metabolites in solution did not influence the crystallization kinetics of pure calcium oxalate seed materials. The results of this study indicate that triamterene in stones does not significantly contribute to further stone development through the enhancement of calcium oxalate crystallization processes.     

Analysis of Proteinaceous Components of the Organic Matrix of Endoskeletal Sclerites from the Alcyonarian Lobophytum crassum

The mesoglea of alcyonarians is occupied by an abundance of minute calcitic sclerites. The sclerites of the alcyonarian Lobophytum crassum contain a water-soluble organic matrix comprising 0.48% of the sclerite weight and a water-insoluble fraction comprising 1.15% of the sclerite weight. Analysis of proteinaceous components in the soluble fraction shows a particularly high content of aspartic acid, followed by alanine, glycine, and glutamate. Aspartic acid, glycine, alanine, and glutamate are the most abundant residues in the insoluble fraction. In both cases, the fractions show the highest concentration of aspartic acid from the total proteins. In an in vitro assay, we show that the matrix proteins extracted from the calcitic sclerites induce the formation of amorphous calcium carbonate prior to its transformation into the calcitic crystalline form. We also show scanning electron micrographs of the rhombohedral calcite crystals used as template, the protein imprinted with these crystals. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of both matrices shows the protein fractions at 67 and 48 kDa. The soluble matrix shows two additional faint bands. Both fractions stain for a carbohydrate at 67 kDa, indicating a glycoprotein at this molecular weight. A newly derived protein sequence was subjected to bioinformatics analysis involving identification of similarities to other acidic proteins. The identification of these proteins in alcyonarian endoskeletal sclerites emphasizes the fundamental importance of such acidic proteins and sheds more light on the functions of these proteins in the processes of biocalcification.     

Properties of osteoconductive biomaterials: calcium phosphates

Bone is formed by a series of complex events involving the mineralization of extracellular matrix proteins rigidly orchestrated by cells with specific functions of maintaining the integrity of the bone. Bone, similar to other calcified tissues, is an intimate composite of the organic (collagen and noncollagenous proteins) and inorganic or mineral phases. The bone mineral idealized as calcium hydroxyapatite, Ca10 (PO4)(6)(OH)2, is a carbonatehydroxyapatite, approximated by the formula: (Ca,X)(10)(PO4,HPO4,CO3)(6)(OH,Y)2, where X are cations (magnesium, sodium, strontium ions) that can substitute for the calcium ions, and Y are anions (chloride or fluoride ions) that can substitute for the hydroxyl group. The current author presents a brief review of CaP biomaterials that now are used as grafts for bone repair, augmentation, or substitution. Commercially-available CaP biomaterials differ in origin (natural or synthetic), composition (hydroxyapatite, beta-tricalcium phosphate, and biphasic CaP), or physical forms (particulates, blocks, cements, coatings on metal implants, composites with polymers), and in physicochemical properties. CaP biomaterials have outstanding properties: similarity in composition to bone mineral; bioactivity (ability to form bone apatitelike material or carbonate hydroxyapatite on their surfaces), ability to promote cellular function and expression leading to formation of a uniquely strong bone-CaP biomaterial interface; and osteoconductivity (ability to provide the appropriate scaffold or template for bone formation). In addition, CaP biomaterials with appropriate three-dimensional geometry are able to bind and concentrate endogenous bone morphogenetic proteins in circulation, and may become osteoinductive (capable of osteogenesis), and can be effective carriers of bone cell seeds. Therefore, CaP biomaterials potentially are useful in tissue engineering for regeneration of hard tissues.     

Physical chemical studies of calcium oxalate crystallization

The physical chemical approach to the investigation of the calcium oxalate (CaOx) crystallization and urolith formation is the systematic examination of the various aspects of mineral precipitation and growth in pure solution, in the presence of individual urinary components, and in whole urine media. Recent experimental studies have indicated that while small urinary ions such as citrate, magnesium, and phosphocitrate retard the mineralization rate of CaOx, urinary macromolecules may act either as inhibitors of growth or promoters of nucleation. Some CaOx mineralization inhibitors have also been found to influence the growth mechanism of the phase and its flocculation properties. Therefore, urinary macromolecules that are adsorbed on the mineralizing crystals and incorporated into the developing stone may play a significant role in urolithiasis.     

Differential involvement of matrix vesicles during the initial and appositional mineralization processes in bone, dentin, and cementum

The distribution of matrix vesicles and its role in biological mineralization were examined in bone and dental hard tissues of the rat after daily administrations of 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP), a potent inhibitor of mineralization, for 7 or 14 days. Newly formed, nonmineralized matrices of the HEBP-affected bone and mesodermal dental hard tissues other than circumpulpal dentin contained numerous mineral-filled matrix vesicles (MV), randomly distributed throughout the collagenous matrix. The distribution density of the mineral-filled MV in the HEBP-affected matrices of calvaria, metaphyseal trabecular bone, alveolar bone, and cellular cementum ranged from 60 to 70 per 100 microm(2), and no statistically significant differences were noted among the values. In the HEBP-affected dentin, however, MV were located only in the nonmineralized matrix of mantle dentin and totally absent in the circumpulpal dentin layers. Instead, the HEBP-affected circumpulpal dentin contained a dense meshwork of noncollagenous matrix enriched with calcium and phosphorus. Comparable meshwork structures were undetectable in nonmineralized matrices of the other hard tissues affected by HEBP. These observations suggest that a certain population of MV (60-70 per 100 microm(2)) is involved in the process of appositional mineralization in most of the mesodermal hard tissues, in addition to their well-known role in initial mineral induction in these tissues. Circumpulpal dentin appears to be an exception, where MV are not required for the appositional mineralization process. Exclusive localization of dentin phosphoproteins in circumpulpal dentin layers must take place to facilitate appositional mineralization at the calcification front, in the absence of MV.     

Lanthanum carbonate: a new phosphate binder

PURPOSE OF REVIEW: Hyperphosphatemia remains an important aspect in the management of end-stage renal disease patients. Consequently, there is a need for new, efficient and well-tolerated phosphate binders. In this review, a new phosphate-binding drug, lanthanum carbonate, with an attractive preclinical efficacy profile compared with existing binders, is discussed. Although the available human efficacy and safety data over 3 years are encouraging, the consequences of low-level tissue deposition continue to be evaluated in longer-term clinical studies. RECENT FINDINGS: Lanthanum carbonate has been shown in clinical studies of up to 3 years to be an effective, well-tolerated phosphate binder. Reported adverse effects are mainly gastrointestinal, and do not differ from those of calcium carbonate. The gastrointestinal absorption of lanthanum is very low. Whereas the element is mainly excreted by the liver, renal excretion of the absorbed fraction is less than 2%. Bone lanthanum levels seen after long-term treatment (up to 4 years) seem not to affect the physicochemical process of mineralization, or osteoblast number/function. Preliminary data on the localization of lanthanum in bone have shown the element to be present at both active and quiescent sites of bone mineralization, independent of the type of renal osteodystrophy, a profile distinct from aluminum, as well as diffusely distributed throughout the mineralized bone matrix especially in rats/humans with an increased bone turnover. A randomized, comparator-controlled, parallel group, open-label study comparing lanthanum carbonate with calcium carbonate in dialysis patients showed no evolution towards low bone turnover in the lanthanum group, and no aluminum-like effect on bone. SUMMARY: Lanthanum carbonate seems to be a potent phosphate-binding drug, minimally absorbed from the gut, with an encouraging safety profile, and no deleterious effects on bone.     

Bone impairment in oxalosis: An ultrastructural bone analysis

Deposition of calcium oxalate crystals in the kidney and bone is a hallmark of systemic oxalosis. Since the bone compartment can store massive amounts of oxalate, patients present with recurrent low-trauma fractures, bone deformations, severe bone pains and specific oxalate osteopathy on plain X-ray. Bone biopsy from the iliac crest displays specific features such as oxalate crystals surrounded by a granulomatous reaction due to an invasion of bone surface by macrophages. We present data obtained in 10 samples from 8 patients with oxalosis (16–68 years) who underwent iliac crest bone biopsy and bone quality analysis using modern methods (microradiography, microindentation, Fourier Transform InfraRed Microspectroscopy, transmission electron microscopy) in addition to histomorphometry. Disseminated calcium oxalate deposits (whewellite) were found in the bone marrow space (with a granulomatous reaction) but not in the bone matrix. Calcium oxalate deposits were totally surrounded by macrophages and multinucleated giant cells, and a phagocytosis activity was sometimes observed. Very few calcium oxalate crystals were directly in close contact with the mineral substance of the bone. Bone mineralization was not modified by the presence of calcium oxalate even in close vicinity. Bone quality analysis also revealed a harder bone than normal, perhaps in relationship with decreased carbonate content in the mineral. This increase in bone hardness could explain a more “brittle” bone. In patients with oxalosis, the formation and growth of calcium oxalate crystals in the bone appeared independent of apatite. The mechanisms leading to nucleation and growth of oxalate deposits are still unclear and deserve further studies.     

A Review of Phosphate Mineral Nucleation in Biology and Geobiology

Relationships between geological phosphorite deposition and biological apatite nucleation have often been overlooked. However, similarities in biological apatite and phosphorite mineralogy suggest that their chemical formation mechanisms may be similar. This review serves to draw parallels between two newly described phosphorite mineralization processes, and proposes a similar novel mechanism for biologically controlled apatite mineral nucleation. This mechanism integrates polyphosphate biochemistry with crystal nucleation theory. Recently, the roles of polyphosphates in the nucleation of marine phosphorites were discovered. Marine bacteria and diatoms have been shown to store and concentrate inorganic phosphate (Pi) as amorphous, polyphosphate granules. Subsequent release of these P reserves into the local marine environment as Pi results in biologically induced phosphorite nucleation. Pi storage and release through an intracellular polyphosphate intermediate may also occur in mineralizing oral bacteria. Polyphosphates may be associated with biologically controlled apatite nucleation within vertebrates and invertebrates. Historically, biological apatite nucleation has been attributed to either a biochemical increase in local Pi concentration or matrix-mediated apatite nucleation control. This review proposes a mechanism that integrates both theories. Intracellular and extracellular amorphous granules, rich in both calcium and phosphorus, have been observed in apatite-biomineralizing vertebrates, protists, and atremate brachiopods. These granules may represent stores of calcium-polyphosphate. Not unlike phosphorite nucleation by bacteria and diatoms, polyphosphate depolymerization to Pi would be controlled by phosphatase activity. Enzymatic polyphosphate depolymerization would increase apatite saturation to the level required for mineral nucleation, while matrix proteins would simultaneously control the progression of new biological apatite formation.