George H. Nancollas

Molecular modulation of calcium oxalate crystallization by osteopontin and citrate

Calcium oxalate monohydrate (COM), which plays a functional role in plant physiology, is a source of chronic human disease, forming the major inorganic component of kidney stones. Understanding molecular mechanisms of biological control over COM crystallization is central to development of effective stone disease therapies and can help define general strategies for synthesizing biologically inspired materials. To date, research on COM modification by proteins and small molecules has not resolved the molecular-scale control mechanisms. Moreover, because proteins directing COM inhibition have been identified and sequenced, they provide a basis for general physiochemical investigations of biomineralization. Here, we report molecular-scale views of COM modulation by two urinary constituents, the protein osteopontin and citrate, a common therapeutic agent. Combining force microscopy with molecular modeling, we show that each controls growth habit and kinetics by pinning step motion on different faces through specific interactions in which both size and structure determine the effectiveness. Moreover, the results suggest potential for additive effects of simultaneous action by both modifiers to inhibit the overall growth of the crystal and demonstrate the utility of combining molecular imaging and modeling tools for understanding events underlying aberrant crystallization in disease.

Fluorescent risedronate analogues reveal bisphosphonate uptake by bone marrow monocytes and localization around osteocytes in vivo.

Bisphosphonates are effective antiresorptive agents owing to their bone‐targeting property and ability to inhibit osteoclasts. It remains unclear, however, whether any non‐osteoclast cells are directly affected by these drugs in vivo. Two fluorescent risedronate analogues, carboxyfluorescein‐labeled risedronate (FAM‐RIS) and Alexa Fluor 647–labeled risedronate (AF647‐RIS), were used to address this question. Twenty‐four hours after injection into 3‐month‐old mice, fluorescent risedronate analogues were bound to bone surfaces. More detailed analysis revealed labeling of vascular channel walls within cortical bone. Furthermore, fluorescent risedronate analogues were present in osteocytic lacunae in close proximity to vascular channels and localized to the lacunae of newly embedded osteocytes close to the bone surface. Following injection into newborn rabbits, intracellular uptake of fluorescently labeled risedronate was detected in osteoclasts, and the active analogue FAM‐RIS caused accumulation of unprenylated Rap1A in these cells. In addition, CD14high bone marrow monocytes showed relatively high levels of uptake of fluorescently labeled risedronate, which correlated with selective accumulation of unprenylated Rap1A in CD14+ cells, as well as osteoclasts, following treatment with risedronate in vivo. Similar results were obtained when either rabbit or human bone marrow cells were treated with fluorescent risedronate analogues in vitro. These findings suggest that the capacity of different cell types to endocytose bisphosphonate is a major determinant for the degree of cellular drug uptake in vitro as well as in vivo. In conclusion, this study shows that in addition to bone‐resorbing osteoclasts, bisphosphonates may exert direct effects on bone marrow monocytes in vivo.

Enamel Demineralization in Primary and Permanent Teeth

Although enamel demineralization is important for our understanding of caries formation, no consensus has been reached regarding the possible differences in susceptibility of primary and permanent enamel. We used the constant composition (CC) technique to investigate the acid-induced demineralization of these tissues at a relative undersaturation with respect to hydroxyapatite (HAP) of 0.902, pH = 4.5, and ionic strength = 0.15 mol L−1. The demineralization rates showed significant differences, primary enamel having the greater susceptibility to dissolution during an initial linear stage: 1.5 ± 0.5 x 10−10 mol mm−2 min−1 compared with 2.6 ± 0.5 x 10−11 mol mm−2 min−1 for permanent enamel. During the reactions, we observed nanosized crystallites which attached to the enamel surfaces or escaped into the bulk solution. These nanosized crystallites were kinetically protected against further dissolution, even though the solutions remained undersaturated. It is hypothesized that they may contribute to the remarkable mechanical and dynamic characteristics of enamel.

The dual role of polyelectrolytes and proteins as mineralization promoters and inhibitors of calcium oxalate monohydrate

SummaryPolyelectrolytes and protein molecules appear to be able to act not only as crystallization inhibitors when present in solution, but also as promoters of crystal growth when immobilized onto surfaces. Because this is especially relevant for systems in which heterogeneous nucleation can occur, the influence of poly-L-glutamic (PGlu) acid, poly-L-aspartic (PAsp) acid, and human serum albumin (HSA) on the nucleation and growth inhibition of calcium oxalate monohydrate (COM) was studied using the Constant Composition (CC) kinetics technique. The overgrowth of COM on hydroxyapatite (HAP) seed crystals pretreated with HSA was also investigated. Pronounced differences in inhibiting and nucleating potential were found for the various additives. HSA, a relatively poor growth inhibitor when present in solution, was found to nucleate very regular, hexagonal COM crystals when immobilized on a surface and to enhance the overgrowth of COM when adsorbed on HAP surfaces.

The influence of phytate and phosphonate on the crystal growth of fluorapatite and hydroxyapatite

Abstract Diphosphonates and polyphosphates containing, respectively, P-C-P and P-O-P bonds in their molecules are well known as calcification inhibitors both in vitro and in vivo . The direct precipitation of hydroxyapatite (HAP) on HAP and fluorapatite (FAP) on FAP seed crystals has been studied in the presence of hydroxyethylidene-1,1-diphosphonic acid (EHDP), and phytic acid, from calcium phosphate solutions at low sustained supersaturations. The marked inhibitory influence of the additives on the crystallization reactions can be interpreted in terms of a Langmuirtype adsorption model, assuming that the active growth sites are blocked by adsorption on the surfaces of the inoculating seed crystals. EHDP is almost 25 times more effective than phytic acid in reducing the rate of crystallization of HAP.

Constant composition kinetics study of carbonated apatite dissolution

Abstract The carbonated apatites (CAP) may be more suitable models for biominerals such as bone and dental hard tissues than is pure hydroxyapatite (HAP) since they have similar chemical compositions. Although they contain only a relatively small amount of carbonate, the solubility and dissolution properties are different. The solubility product of the CAP particles used in this dissolution study, 2.88×10 −112 xa0mol 18 xa0l −18 , was significantly greater than that of HAP, 5.52×10 −118 xa0mol 18 xa0l −18 . The kinetics of dissolution of CAP has been studied using the constant composition (CC) method. At low undersaturations, the dissolution reaction appeared to be controlled mainly by surface diffusion with an effective reaction order of 1.9±0.1 with respect to the relative undersaturation. These results together with those obtained by scanning electron microscopy (SEM) suggest a dissolution model. Based on the surface diffusion theory of Burton, Cabrera and Frank (BCF). The interfacial tension between CAP and the aqueous phase calculated from this dissolution model, 9.0xa0mxa0Jxa0m −2 , was consistent with its relatively low solubility. An abnormal but interesting dissolution behavior is that the CAP dissolution rate was relatively insensitive to changes in calcium and phosphate concentrations at higher undersaturations, suggesting the importance of the carbonate component under these conditions.

Constant composition dissolution of mixed phases: II. Selective dissolution of calcium phosphates

Characterization of the dissolution kinetics of individual synthetic and biological calcium phosphates is of considerable importance since these phases often coexist in biological minerals. The constant composition method has been used to study the dissolution kinetics of a series of synthetic calcium phosphates, brushite (DCPD), beta-tricalcium phosphate (TCP), octacalcium phosphate (OCP), hydroxyapatite (HAP), and carbonated apatite (CAP) in the presence and absence of citric acid, as a function of pH and thermodynamic driving force. While citric acid markedly accelerates the dissolution of TCP, HAP dissolution is significantly inhibited. Moreover, this additive has almost no influence on the dissolution of DCPD, OCP, and CAP. Dual constant composition dissolution studies of mixed calcium phosphates in the presence of citric acid have also been made. Another factor, pH, also plays an important role in the dissolution of these calcium phosphates. In suspensions of calcium phosphate mixtures, specific phases can be selectively dissolved by changing experimental parameters such as pH and the presence of rate modifiers. This result has important applications for the dissolution control of dental hard tissues such as dentin, enamel, and calculus.

The mechanism of biological mineralization

Abstract The control of supersaturation and the nucleation and growth of crystals in calcium phosphate systems are important in relation to the physiological deposition of bone and tooth. Other calcium salts such as the carbonate and oxalate hydrates are significant components of pathological mineral deposits. The use of a highly reproducible seeded growth technique has enabled kinetic studies to be made of the crystal growth of these minerals. Under conditions of relatively high supersaturation, secondary nucleation may be induced upon the surface of the seed crystals. In the case of the calcium phosphates, temperature, supersaturation, surface concentration, pH, ionic strength and presence of foreign ions are very important in determining the nature of the phase which grows upon the added seed crystals. Kinetic considerations are of overriding importance in determining the course of the reactions. It is not possible to predict the phase which forms purely on the basis of thermodynamic solubility data. Thus, in solutions appreciably supersaturated with respect to both dicalcium phosphate dihydrate (DCPD) and hydroxyapatite (HAP), the addition of low concentrations of HAP seed results in the exclusive formation of DCPD whereas this phase is absent when the seed concentration is increased. The kinetic results for calcium oxalate and phosphates are discussed in terms of the important problems relating to tooth mineralization and the origin and growth of renal calculi.

Size-effects in the dissolution of hydroxyapatite: an understanding of biological demineralization

A comprehensive dissolution theory that integrates size effects and surface energy control into the overall reaction rate has been developed. This model is successfully applied to the dissolution of hydroxyapatite (HAP) at different undersaturations, using the highly reproducible Constant Composition (CC) method. It is found that the commonly used rate law is not suitable for the dissolution of nanoscale particles where the sizes of the crystallites must be taken into account. Dissolution is induced by the formation of pits and continues with the spreading of their stepwaves. However, only the larger pits (of size greater than a critical value, r*) are active, with stepwaves contributing to dissolution and the spreading velocities are also dependent on the pit sizes, decreasing with decreasing pit size. Size effects dominate the reaction when the crystallite sizes are of the same order as r*, resulting in self-inhibition of dissolution and even reaction suppression. A metastable zone of undersaturation manifests itself when particle and pit critical sizes approach each other. These findings are also applicable for in vivo demineralization of biological materials such as tooth enamel. As the biomaterials select nanosized particles as their basic building blocks, the size-effects are magnified and the materials are insensitive to demineralization. These effects confer on biominerals the ability to be stabilized against dissolution in the undersaturated biological milieux.

Nanosized particles in bone and dissolution insensitivity of bone mineral.

Most of the mineral crystals in bone are platelets of carbonated apatite with thicknesses of a few nanometers embedded in a collagen matrix.We report that spherical to cylindrical shaped nanosized particles are also an integral part of bone structure observed by high resolution scanning electron microscopy. High resolution back scattered electron imaging reveals that the spherical particles have a contrast similar to the crystal platelets, suggesting that they are thus likely to have similar mineral properties. By means of constant composition (CC) dissolution of bone, similar sized nanoparticles are shown to be insensitive to demineralization and are thought to be dynamically stabilized due to the absence of active pits/defects on the crystallite surfaces. Similar reproducible self-inhibited dissolution was observed with these nanoparticles during CC dissolution of synthetic carbonated apatite. This result rules out the possible influence of complicating biological factors such as the possible presence of organic matrix components and other impurities. This phenomenon can be explained by a unique dissolution model involving size considerations at the nanoscale. The unexpected presence of nanoparticles in mature bone may also be due to the stabilization of some nanosized particles during the formation process in a fluctuating biological milieux.