Nucleation Of Phosphate Research Articles

Insight by Cryo-TEM into the growth and crystallization processes of calcium phosphate nanoparticles in aqueous medium

Hydroxyapatite (HA) is the main constituent of bone mineral in vertebrates and is widely used as a biomaterial in biomedical studies and medical clinics. The synthesis of this calcium phosphate phase is a crucial factor in its potential use in various applications as well as in understanding the nucleation and growth processes in biological mineralization (biomineralization). In this work, a sequential analysis of the different steps of HA synthesis in aqueous media was performed by using cryo-transmission electron microscopy (Cryo-TEM), which allows preservation of the specimens at different stages of the synthesis process, preventing their modification during drying. X-ray energy dispersive spectroscopy and electron diffraction, in addition to high-resolution imaging, were performed on vitrified samples. We showed that drying a specimen from the synthesis solution induces the crystallization of nanoparticles into an HA phase after 30 min of reaction, while when analyzed by Cryo-TEM, crystalline nanoparticles were detected only after 15 h. Calcium phosphate nanoparticles presented three different morphologies: irregular shapes characterized by low contrast in TEM (5 min), isotropic shapes with well-defined contrast (10 min) and needle-like shapes (after 30 min). The long axis of the HA needle-like nanoparticles can be associated with the c-axis direction of the hexagonal unit cell, which can thus be assigned to the preferential crystallization direction. However, a diffraction signal of the nanoparticles in vitreous ice that “mimics” the original solution was detected by Cryo-TEM only after 15 h of synthesis. These apparently controversial results are discussed in this manuscript considering various aspects of the use of Cryo-TEM in relation to the current concepts of calcium phosphate nucleation and growth.

Synthesis of polythiophene nanoparticles by surfactant-free chemical oxidative polymerization method: Characterization, in vitro biomineralization, and cytotoxicity evaluation

Herein, we report the synthesis of polythiophene nanoparticles (PTh-NPs) by surfactant-free chemical oxidative polymerization of thiophene at 37 °C using ammonium persulphate as oxidant. PTh-NPs synthesized without surfactant were compared to those with surfactant in terms of surface morphology, crystallinity, cytotoxicity and some other aspects. Thermogravimetric analysis showed a good thermal stability of as-synthesized PTh-NPs. In vitro biomineralization revealed the nucleation of calcium and phosphate onto the NPs. Cytotoxicity of PTh-NPs was evaluated by measuring cell viability of pre-osteoblast MC3T3-E1 and PC12 cell lines. PTh-NPs synthesized without using surfactant exhibited better cell viability compared to those with surfactant.

A Novel Class of Injectable Bioceramics that Glue Tissues and Biomaterials.

Calcium phosphate cements (CPCs) are clinically effective void fillers that are capable of bridging calcified tissue defects and facilitating regeneration. However, CPCs are completely synthetic/inorganic, unlike the calcium phosphate that is found in calcified tissues, and they lack an architectural organization, controlled assembly mechanisms, and have moderate biomechanical strength, which limits their clinical effectiveness. Herein, we describe a new class of bioinspired CPCs that can glue tissues together and bond tissues to metallic and polymeric biomaterials. Surprisingly, alpha tricalcium phosphate cements that are modified with simple phosphorylated amino acid monomers of phosphoserine (PM-CPCs) bond tissues up to 40-fold stronger (2.5–4 MPa) than commercial cyanoacrylates (0.1 MPa), and 100-fold stronger than surgical fibrin glue (0.04 MPa), when cured in wet-field conditions. In addition to adhesion, phosphoserine creates other novel properties in bioceramics, including a nanoscale organic/inorganic composite microstructure, and templating of nanoscale amorphous calcium phosphate nucleation. PM-CPCs are made of the biocompatible precursors calcium, phosphate, and amino acid, and these represent the first amorphous nano-ceramic composites that are stable in liquids.

Biomineralization of calcium phosphate revealed by in situ liquid-phase electron microscopy

Calcium phosphate biomineralization is essential to the formation of bones and teeth, and other pathological calcifications. Unravelling the mechanism of calcium phosphate nucleation and growth contributes significantly to understanding diseases caused by pathological mineralization, and also to designing biomimetic materials with suitable properties. Recently, calcium phosphate was proposed to mineralize following a non-classical crystal growth pathway of pre-nucleation cluster aggregation. Liquid-phase transmission electron microscopy allows dynamic processes to be recorded continuously inside liquid. Here we present direct evidence, based on continuous monitoring in liquid, to confirm that calcium phosphate mineralization from simulated body fluid occurs by particle attachment, shown with nanoscale spatial resolution and sufficient temporal resolution. This work may lay the foundation for future investigation of mineralization in other relevant biological systems in humans and vertebrates.

Control of Calcium Phosphate Nucleation and Transformation through Interactions of Enamelin and Amelogenin Exhibits the "Goldilocks Effect".

Although amelogenin comprises the vast majority of the matrix that templates calcium phosphate nucleation during enamel formation, other proteins, particularly enamelin, are also known to play an important role in the formation of enamel's intricate architecture. However, there is little understanding of the interplay between amelogenin and enamelin in controlling processes of mineral nucleation and growth. Here, we used an in vitro model to investigate the impact of enamelin interaction with amelogenin on calcium phosphate nucleation for a range of enamelin-to-amelogenin ratios. We found that amelogenin alone is a weak promoter of nucleation, but addition of enamelin enhanced nucleation rates in a highly nonlinear, nonmonotonic manner reaching a sharp maximum at a ratio of 1:50 enamelin/amelogenin. We provide a phenomenological model to explain this effect that assumes only isolated enamelin proteins can act as sites of enhanced nucleation, while enamelin oligomers cannot. Even when interaction is random, the model reproduces the observed behavior, suggesting a simple means to tightly control the timing and extent of nucleation and phase transformation by amelogenin and enamelin.

The apatite forming ability of micro- and nanocomposites of α-Tricalcium phosphate/poly (D,L-lactide-co-glycolide)

ABSTRACTBioresorbable composites consist of micro- and nano-sized α-tricalcium phosphate (α-TCP) particles in a poly(D,L-lactic-co-glycolic acid) (PLGA) matrix were compared for their ability to form bone-like apatite in simulated body fluid (SBF). Due to the uniform distribution of α-TCP nanoparticles over the nanocomposite surface, enhanced apatite formation was observed. This enhanced apatite formation was achieved through faster and more uniform apatite nucleation. The presence of abundant high energy boundaries between α-TCP nanoparticles and PLGA matrix in nanocomposite provided a large number of suitable sites for calcium phosphate (CaP) nucleation. A homogeneous distribution of CaP nuclei formed after 5 days. The resulting apatite crystals grew to form a flake-like apatite layer. In contrast, CaP nucleation was only observed on the micrometre-size α-TCP particles in the microcomposite. After 14 days, a dense flake-like apatite was visible covering the surface of nanocomposite, whilst this surface layer was formed only on α-TCP particles in microcomposite.

Aqueous extract of Sedum oxypetalum induces mineralization and osteogenic differentiation by human Periodontal Ligament-Derived cells

Ethnopharmacological relevanceThe medicinal plant Sedum oxypetalum Kunth (Crassulaceae), locally known as Jiote or in general Siempreviva (always alive) has been traditionally used by people of the Mexican community of Tenango del Valle as a home remedy to treat periodontal diseases, inducing teeth strengthening. Consequently, the aim of this work was to investigate its capacity directed to mineralized tissues regeneration. Materials and methodsThe aerial parts of the plant were processed and its aqueous extract (AE) was chemically characterized. The AE and its components sedoheptulose and syngenite were tested for either osteogenic differentiation or mineral-nucleation induction respectively. ResultsThe AE and one of its components (sedoheptulose) were shown to promote the proliferation and/or osteogenic differentiation by Human Periodontal Ligament-Derived Cells (hPDLs), while inducing the mineralization process. The AE also promoted the nucleation of octacalcium phosphate and its component syngenite, the hydroxyapatite crystals formation in vitro. ConclusionThe findings reported herein support the traditional use of S. oxypetalum due to its potential capacity to promote the regeneration of mineralized tissues.

Heterogeneous Lead Phosphate Nucleation at Organic–Water Interfaces: Implications for Lead Immobilization

Phosphate is added to Pb-contaminated soils to induce lead immobilization through lead phosphate precipitation. Organic coatings on soils, which may affect heterogeneous lead phosphate nucleation, can impact the effectiveness of lead immobilization. Here, SiO2 surfaces were coated with silanol self-assembled thin films terminated with −COOH and −OH functional groups to act as model organic coatings on soil particles. Using grazing incidence small-angle X-ray scattering (GISAXS), heterogeneous lead phosphate nucleation on coatings was measured from mixed Pb(NO3)2 and Na2HPO4/NaH2PO4 solutions at pH 7 with varied ionic strengths (IS = 0.58, 4, and 11 mM). Raman spectroscopy identified the homogeneous precipitates in solution as hydroxylpyromorphite (Pb5(PO4)3OH). The smallest lead phosphate nuclei (4.5 ± 0.5 nm) were observed on −COOH coatings, which resulted from the highest level of lead and phosphate ion adsorption on −COOH coatings. The IS of the solution also affected the sizes of the heterogeneous pre...

Physicochemical properties of calcium silicate-based formulations MTA Repair HP and MTA Vitalcem.

ObjectiveThis study aimed to analyze the following physicochemical properties: radiopacity, final setting time, calcium release, pH change, solubility, water sorption, porosity, surface morphology, and apatite-forming ability of two calcium silicate-based materials.Material and methodsWe tested MTA Repair HP and MTA Vitalcem in comparison with conventional MTA, analyzing radiopacity and final setting time. Water absorption, interconnected pores and apparent porosity were measured after 24-h immersion in deionized water at 37°C. Calcium and pH were tested up to 28 d in deionized water. We analyzed data using two-way ANOVA with Student-Newman-Keuls tests (p<0.05). We performed morphological and chemical analyses of the material surfaces using ESEM/EDX after 28 d in HBSS.ResultsMTA Repair HP showed similar radiopacity to that of conventional MTA. All materials showed a marked alkalinizing activity within 3 h, which continued for 28 d. MTA Repair HP showed the highest calcium release at 28 d (p<0.05). MTA Vitalcem showed statistically higher water sorption and solubility values (p<0.05). All materials showed the ability to nucleate calcium phosphate on their surface after 28 d in HBSS.ConclusionsMTA Repair HP and MTA Vitalcem had extended alkalinizing activity and calcium release that favored calcium phosphate nucleation. The presence of the plasticizer in MTA HP might increase its solubility and porosity. The radiopacifier calcium tungstate can be used to replace bismuth oxide.

The role of confined collagen geometry in decreasing nucleation energy barriers to intrafibrillar mineralization

Mineralization of collagen is critical for the mechanical functions of bones and teeth. Calcium phosphate nucleation in collagenous structures follows distinctly different patterns in highly confined gap regions (nanoscale confinement) than in less confined extrafibrillar spaces (microscale confinement). Although the mechanism(s) driving these differences are still largely unknown, differences in the free energy for nucleation may explain these two mineralization behaviors. Here, we report on experimentally obtained nucleation energy barriers to intra- and extrafibrillar mineralization, using in situ X-ray scattering observations and classical nucleation theory. Polyaspartic acid, an extrafibrillar nucleation inhibitor, increases interfacial energies between nuclei and mineralization fluids. In contrast, the confined gap spaces inside collagen fibrils lower the energy barrier by reducing the reactive surface area of nuclei, decreasing the surface energy penalty. The confined gap geometry, therefore, guides the two-dimensional morphology and structure of bioapatite and changes the nucleation pathway by reducing the total energy barrier.

Phosphating of Mn modified Zn for low alloy steels SA335P22

Zinc phosphating is a well-known chemical conversion coating process for the surface of a metal to increase paint adhesion. The SA335P22 graded low alloy steel is widely used in many aspects due to its hardenability, high fatigue strength and toughness. However, Cr and C contents in SA335P22 steels are found to be obstacles in forming phosphate coating by the traditional process, such as a very few nucleation of Zn phosphate or uncontrollability of crystal size. In this present research, the phosphating of Mn modified Zn was studied to make an efficient phosphating surface for improvement of corrosion resistance as well as good paint adhesion. The surface conditioning solution was applied to assist the nucleation of Zn phosphate at the surface. Moreover, the phosphating temperature was adjusted in the range of 50 to 60 °C. According to the microstructure, it was revealed that the solution having more concentration of phosphoric acid effectively reacted with the steel surface, subsequently uniformed phosphating layers were obtained. After phosphating, paint layers were applied on and the evaluation was performed on adhesion by tape testing and salt spray testing.

Cholesteryl Hemisuccinate Monolayers Efficiently Control Calcium Phosphate Nucleation and Growth

The article describes the phase behavior of cholesteryl hemisuccinate at the air–liquid interface and its effect on calcium phosphate (CP) mineralization. The amphiphile forms stable monolayers with phase transitions at the air–liquid interface from a gas to a tilted liquid-condensed (TLC) and finally to an untilted liquid-condensed (ULC) phase. CP mineralization beneath these monolayers leads to crumpled CP layers made from individual plates. The main crystal phase is octacalcium phosphate (OCP) along with a minor fraction of hydroxyapatite (HAP), as confirmed by X-ray photoelectron spectroscopy, energy dispersive X-ray spectroscopy, bright field transmission electron microscopy, and electron diffraction.

Activation of zinc phosphate coating by silver nitrate pretreatment

ABSTRACTThe effects of a new activation pretreatment based on silver nitrate solution on the formation and protection characteristics of zinc phosphate coating on mild steel surface were studied. The surface morphology of coated samples and microstructural evolution were analysed by the scanning electron microscopy (SEM) and the energy-dispersive spectroscopy. The protection characteristics were evaluated using potentiodynamic polarisation curves and the electrochemical impedance spectroscopy in a 3.5% NaCl solution. Experimental results revealed that the activation pretreatment with silver nitrate solution promotes the nucleation of zinc phosphate coating on steel surface which produced compact coatings with full surface coverage. Introducing the pretreatment with silver nitrate solution prior to the phosphating step strongly influences the surface morphology and corrosion resistance of the phosphate layer. The corrosion resistance of activated phosphate coatings was improved, where the corrosion potential shifted positively, and the corrosion current density was markedly reduced.

Physicochemical properties and cytotoxicity of an experimental resin-based pulp capping material containing the quaternary ammonium salt and Portland cement.

To evaluate invitro the physicochemical properties, cytotoxicity and calcium phosphate nucleation of an experimental light-curable pulp capping material composed of a resin with antibacterial monomer (MAE-DB) and Portland cement (PC). The experimental material was prepared by mixing PC with a resin containing MAE-DB at a 2:1 ratio. Cured pure resin containing MAE-DB served as control resin. ProRoot MTA and Dycal served as commercial controls. The depth of cure, degree of monomer conversion, water absorption and solubility of dry samples, calcium release, alkalinizing activity, calcium phosphate nucleation and the cytotoxicity of materials were evaluated. Statistical analysis was carried out using anova followed by Tukey's HSD test (equal variance assumed) or Tamhane test (equal variance not assumed) and independent-samples t-tests. The experimental material had a cure depth of 1.19mm, and the mean degree of monomer conversion was 70.93% immediately post-cure and 88.75% at 24h post-cure. The water absorption of the experimental material was between those of MTA and Dycal, and its solubility was significantly less (P<0.05) than that of Dycal and higher than that of MTA. The experimental material exhibited continuous calcium release and an alkalinizing power between those of MTA and Dycal throughout the test period. Freshly set experimental material, control resin and all 24-h set materials had acceptable cytotoxicity. The experimental material, MTA and Dycal all exhibited the formation of apatite precipitates after immersion in phosphate-buffered saline. The experimental material possessed adequate physicochemical properties, low cytotoxicity and good calcium phosphate nucleation.

A poly(2-hydroxyethyl methacrylate)-based resin improves the dentin remineralizing ability of calcium silicates

Bioactive polymeric composites have received great attention for their capability to remineralize the dentin tissue.This study was aimed at evaluating if a poly(HEMA-co-TEGDMA) resin (HEMA: 2-hydroxyethyl methacrylate; TEGDMA: triethyleneglycol dimethacrylate) may increase the in vitro apatite forming ability of a calcium silicate cement (CaSi), in view of developing a hydrophilic light-curable composite bio-remineralizing restorative material (R-CaSi). To this purpose, the following experiments were carried out: (1) In vitro apatite forming ability of R-CaSi and CaSi was comparatively assessed by micro-Raman spectroscopy after immersion of the cement disks in Dulbecco's Phosphate Buffered Saline (DPBS) at 37°C for 1–28days; (2) Previously demineralized human dentin slices were soaked for 7days in close contact with the CaSi and R-CaSi cements as well as poly(HEMA), poly(TEGDMA) and poly(HEMA-co-TEGDMA), and then were comparatively analyzed by IR spectroscopy.Micro-Raman spectroscopy showed that in calcium phosphate nucleation tests, the B-type carbonated apatite deposit formed on R-CaSi was thicker than that on CaSi; therefore, the poly(HEMA-co-TEGDMA) resin proved able to increase the in vitro apatite forming ability of the calcium silicate-based cement. Both cements were found to induce dentin remineralization, R-CaSi to a higher extent, in agreement with the calcium phosphate nucleation tests. This result may be ascribed to the positive role played by the polymeric component, which was found to interact with collagen and to chelate calcium ions. Upon remineralization, collagen underwent conformational rearrangements and the formed apatite phase, rather than a simple deposit, was intimately bound to the collagen matrix, thanks to the calcium ions chelated by it.

Detection of Posner's clusters during calcium phosphate nucleation: a molecular dynamics study.

Hydroxyapatite (HA), the main mineral phase of mammalian tooth enamel and bone, originates in body fluids from amorphous calcium phosphate (ACP). ACP presents short-range order in the form of small domains with size of 0.9 nm and chemical formula Ca9(PO4)6, known as Posner's clusters. In this study, the aggregation and clustering of calcium and phosphate ions in water has been investigated by means of shell-model molecular dynamics simulations. Calcium phosphate aggregates form in solution with compositions and Ca coordination that are similar to those found in Posner's cluster, but the stoichiometry of these species is dependent on the ionic composition of the solution: calcium-deficient clusters in solutions with low Ca : P ratio; cluster containing protonated phosphate groups in neutral solutions; sodium ions partially substituting calcium in solutions containing a mixture of sodium and calcium ions. These Posner-like clusters can be connected by phosphate groups, which act as a bridge between their central calcium ions. The simulations of the aggregation in solution of calcium phosphate clusters are an unbiased and unequivocal validation of Posner's model, and reveal for the first time the structure and composition of the species that form during the early stages of ACP nucleation at a scale still inaccessible to experiment.

Counting crystal clusters – a neutron reflectometry study of calcium phosphate nano-cluster adsorption at the air–liquid Interface

An 11 Å mineral film above a dense prenucleation cluster subphase is shown to be the structure of the early stage of calcium phosphate nucleation from a simulated body fluid.

In Situ Evaluation of Calcium Phosphate Nucleation Kinetics and Pathways during Intra- and Extrafibrillar Mineralization of Collagen Matrices.

We revealed that nucleation sites within collagen fibrils determined pathways for calcium phosphate (CaP) nucleation and its transformation, from amorphous species to crystalline plates, during the biomineralization process. Using in situ small-angle X-ray scattering (SAXS), we examined the nucleation and growth of CaP within collagen matrices and elucidated how a nucleation inhibitor, polyaspartic acid (pAsp), governs mineralization kinetics and pathways at multiple length scales. Mineralization without pAsp led initially to spherical aggregates of CaP in the entire extrafibrillar spaces. With time, the spherical aggregates transformed into plates at the outermost surface of the collagen matrix, preventing intrafibrillar mineralization inside. However, mineralization with pAsp led directly to the formation of intrafibrillar CaP plates with a spatial distribution gradient through the depth of the matrix. The results illuminate mineral nucleation kinetics and real-time nanoparticle distributions within organic matrices in solutions containing body fluid components. Because the macroscale mechanical properties of collagen matrices depend on their mineral content, phase, and arrangement at the nanoscale, this study contributes to better design and fabrication of biomaterials for regenerative medicine.

Preservation of Archaeal Surface Layer Structure During Mineralization.

Proteinaceous surface layers (S-layers) are highly ordered, crystalline structures commonly found in prokaryotic cell envelopes that augment their structural stability and modify interactions with metals in the environment. While mineral formation associated with S-layers has previously been noted, the mechanisms were unconstrained. Using Sulfolobus acidocaldarius a hyperthermophilic archaeon native to metal-enriched environments and possessing a cell envelope composed only of a S-layer and a lipid cell membrane, we describe a passive process of iron phosphate nucleation and growth within the S-layer of cells and cell-free S-layer “ghosts” during incubation in a Fe-rich medium, independently of metabolic activity. This process followed five steps: (1) initial formation of mineral patches associated with S-layer; (2) patch expansion; (3) patch connection; (4) formation of a continuous mineral encrusted layer at the cell surface; (5) early stages of S-layer fossilization via growth of the extracellular mineralized layer and the mineralization of cytosolic face of the cell membrane. At more advanced stages of encrustation, encrusted outer membrane vesicles are formed, likely in an attempt to remove damaged S-layer proteins. The S-layer structure remains strikingly well preserved even upon the final step of encrustation, offering potential biosignatures to be looked for in the fossil record.

Apatite layer growth on glassy Zr48Cu36Al8Ag8 sputtered titanium for potential biomedical applications

The bioactivity of magnetron sputtered thin film metallic glasses (TFMGs) of Zr48Cu36Al8Ag8 (at.%) on titanium substrates was tested for bio implant applications. The structural and elemental compositions of TFMGs were analyzed by XRD, XPS and EDAX. X-ray diffraction analysis displayed a broad hump around the incident angle of 30–50°, suggesting that the coatings possess a glassy structure. An in situ crystal growth of hydroxyapatite was observed by soaking the sputtered specimen in simulated body fluid (SBF). The nucleation and growth of a calcium phosphate (Ca–P) bone-like hydroxyapatite on Zr48Cu36Al8Ag8 (at.%) TFMG from SBF was investigated by using XRD, AFM and SEM. The presence of calcium and phosphorus elements was confirmed by EDAX and XPS. In vitro electrochemical corrosion studies indicated that the Zr-based TFMG coating sustain in the stimulated body-fluid (SBF), exhibiting superior corrosion resistance with a lower corrosion penetration rate and electrochemical stability than the bare crystalline titanium substrate.