Synthesis of nanocrystalline calcium phosphate in microemulsion--effect of nature of surfactants

Nanosized calcium phosphate (CP) powders have been synthesized by an inverse microemulsion system using kerosene as the oil phase, a cationic surfactant Aliquat 336, a non-ionic surfactant Tween 20 and their mixture and aqueous solutions of calcium nitrate tetrahydrate and biammonium hydrogen phosphate as the water phase. It has been found that the nature of surfactants played an important role to regulate the size and morphologies of the calcium phosphate nanoparticles. The cationic surfactant Aliquat 336 has been found to regulate the nucleation and crystal growth. The synthesized powders have been comprehensively characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), powder X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). Our results show that the brushite (DCPD) is the major phase comprising the calcium phosphate nanoparticles. In mixed surfactants mediated system a morphological controlled highly crystalline particles have been synthesized. Further, the role of Aliquat 336 has been established and a plausible synthetic mechanism has been proposed.     

A new model for the rationalization of the thermal behavior of carbonated apatites

A new model for the location and distribution of carbonate ions in carbonated apatite was used to assign the IR spectra of A- and AB-carbonated apatites. The percentage of total carbonate as measured by the mass loss in the TGA of these compounds is in good agreement with the percentage obtained by combustion analysis. The decomposition of pure A-type carbonate appears at temperatures of 985–1123 °C, whereas the decomposition of AB-type carbonated apatites occurs in the range of 600–800 °C. This difference is attributed to changes in the environment of channel carbonate brought about by B-type substitution of carbonate for phosphate. In the presence of sodium ions, the channel is changed by substitution of sodium for calcium in order to accommodate the difference between the charge of the carbonate and phosphate ions. A thermodynamic cycle is introduced to rationalize the differences in decomposition temperatures of A- and B-type carbonate. Preferential loss of B-type carbonate upon heating to 600 °C also suggests the migration of B-type carbonate to A-sites.

     

Amphiphilic phosphoprotein-controlled formation of amorphous calcium carbonate with hierarchical superstructure

Amorphous calcium carbonate (ACC) plays important roles in biomineralization, and the phosphoproteins extracted from biogenic stable ACC can induce and stabilize synthetic ACC in vitro. Here, mineralization of square-shaped ACC plates with micrometer-sized channels has been reported in the presence of the amphiphilic phosphoprotein casein. Casein can be assumed to take a key role during ACC plate formation, where it serves as an effective stabilization agent for ACC and assembles spherical ACC particles into ACC plates. The stabilizing effect of casein arises from the electrostatic attraction between phosphate groups as well as carbonate groups (especially the former) and the calcium ions, preventing the transformation from unstable ACC to the more stable crystalline phase of CaCO(3). The assembling effect of casein mainly comes from the hydrophobic interaction between casein molecules bound on CaCO(3) particle surface. The inclusion of casein in ACC plates revealed by the thermogavimetric analysis confirms the proposed stabilizing and assembling mechanism. The ability to fabricate such novel hierarchical structured ACC holds the promise for creating more complex micro- and nanostructured materials by use of biological proteins with special structure.     

From synthetic to biogenic Mg-containing calcites: a comparative study using FTIR microspectroscopy

The formation mechanism of the thermodynamically unstable calcite phase, very high Mg calcite, in biological organisms such as sea urchin or corallina algae has been an enigma for a very long time. In contrast to conventional methods such as KBr pellet Fourier Transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD), FTIR microspectroscopy (FTIRM) provides additional information about a local disorder such as an amorphous phase or the occlusion of Mg ions in the calcite lattice. In this work, we characterise for the first time systematically synthetic and biogenic Mg-containing calcium carbonate samples (especially sea urchin teeth--SUT) in detail by using two FTIRM instruments and compare these samples with KBr pellet FTIR measurements. Furthermore, we present spectra from geogenic calcite and dolomite minerals, recorded with both FTIRM systems, as well as KBr pellet FTIR spectra as references. We analyse the spectra by applying multi-peak curve fitting on the in-plane-bending (ν(4)) and out-of-plane (ν(2)) bands. Based on the obtained results we attribute the two singlet bands at ～860-865 cm(-1) and ～695-704 cm(-1) observed in the SUT FTIRM spectra to the existence of amorphous calcium carbonate (ACC), and report for the first time the existence of ACC at the mature end of SUT. In the other three studied biominerals, however, we did not find any ACC. Also, based on the FTIRM results, we observe that not only ν(4), but also ν(2) shifts to higher wavenumbers if more calcium ions are replaced by magnesium ions in the calcite lattices.     

Coordination of biologically important alpha-amino acids to calcium(II) at high pH: insights from crystal structures of calcium alpha-aminocarboxylates

A series of calcium alpha-aminocarboxylates was prepared by refluxing aqueous solutions/suspensions of calcium hydroxide and the respective alpha-amino acid. The colorless, crystalline hydrates Ca(gly)2.H2O (1), Ca(ala)2.3H2O (2), Ca(val)2.H2O (3), Ca(leu)2.3H2O (4), Ca(met)2.nH2O (5, n approximately 2), and Ca(pro)2.H2O (6) have been isolated in yields between 29 and 67% (gly- = glycinate, ala- = rac-alaninate, val- = rac-valinate, leu- = rac-leucinate, met- = rac-methioninate, pro- = rac-prolinate). The compounds 1-6 are readily soluble in water. The 0.10 M solutions have ca. pH 10-11 which is consistent with a noticeable degree of dissociation. The 13C NMR spectra of 1-6 in D2O were measured, and their comparison with those of the corresponding tetramethylammonium alpha-aminocarboxylates point to carboxylate coordination in solution, but no indication of nitrogen coordination was found. Infrared spectra of 1-6 gave similar results for the solid state. Complete single-crystal X-ray structure analyses of 1-4 and preliminary ones of 5 and 6, however, revealed that all aminocarboxylate ligands are N,O-chelating. Crystals of 2 consist of mononuclear complexes, while the other five compounds form three different types of one-dimensional coordination polymers. Structural diversity is also observed with the binding modes of the aminocarboxylate ligands and the calcium environment. Besides terminal aminocarboxylate coordination, there are three different types of aminocarboxylate bridges. The calcium ions are seven- or eight-coordinate in N2O5 and N2O6 coordination environments, respectively; one or three water molecules are part of the first ligand sphere of each metal ion. The crystal structures support conjectures about the existence of the yet undetected solution species [Cax(aa)2x(H2O)n] (aa- = alpha-aminocarboxylate). For example, x = 1 is realized in crystalline [Ca(ala)2(H2O)3] (2), and in 4 [Ca2(leu)4(H2O)4] complexes (x = 2) are linked to infinite chains by bridging aqua ligands.     

A novel process for the fabrication of nanoporous apatites from Moroccan phosphate rock

Naturally-abundant Moroccan phosphate (Bengurir ores) was used as a source for calcium and phosphorus precursors for the synthesis of nanoporous apatites, following a dissolution–precipitation method. The influence of temperature, ripening time and solvent nature on the crystal growth of apatite was investigated. For all products, poorly crystalline apatite phases were obtained after drying, exhibiting a mesoporous structure with specific surface area up to 150 m² g⁻¹. These materials could be converted into a pure crystalline hydroxyapatite phase after treatment at 800 °C. This simple, eco-friendly and cheap procedure may be useful for the future development of sorbents for water treatment.     

Giant liposome microreactors for controlled production of calcium phosphate crystals

Calcium phosphates are among the most important biominerals in living organisms, where they play both a mechanical and a calcium storage role. Their growth in vivo is under strong biological control, and this process occurs in closed spaces. Our aim in this paper is to describe a microreactor system able to control the mineralization process within closed spaces. To this aim we produce giant liposomes containing calcium ions as active ions in the mineralization process, spermine as an activator of crystal growth, and alkaline phosphatase as a catalyst to convert phosphate esters into phosphates. These phosphate esters are provided in the form of p-nitrophenyl phosphate outside of the liposomes. It is demonstrated that these amphiphilic molecules are able to diffuse through the lipidic container and to be subsequently hydrolyzed under enzymatic catalysis into active phosphate species which interact with the already available calcium and spermine to produce calcium phosphates only in the interior of the liposomes. This opens the route to control the calcium phosphate particle size in biomimetic systems.     

FT-Raman-Spectroscopic Study on the Luminescence of Synthetic and Mineral Apatites

 Strong luminescence bands can be present in NIR-excited FT-Raman spectra of certain inorganic materials like hydroxyapatite. Since the origin of these emissions exclusively occurring on NIR-excitation is still unclear, a variety of synthetic and mineral apatites and related calcium salts was studied by means of Stokes and anti-Stokes FT-Raman spectroscopy, X-ray diffraction, and geochemical analyses. The results indicate that the occurrence of the luminescence is in some way related to foreign ion contents. However, a direct correlation to a specific ion could not be found on the basis of available data.     

Sorption of phosphates on macroporous synthetic calcium silicates

The sorption of phosphate ions on macroporous synthetic calcium silicates has been studied and it has been evaluated whether the latter are suitable to serve as sorbents of geochemical barriers preventing phosphorus losses from agricultural soils.     

Vibrational spectral fingerprinting for chemical recognition of biominerals

Pathologies associated with calcified tissue, such as osteoporosis, demand in vivo and/or in situ spectroscopic analysis to assess the role of chemical substitutions in the inorganic component. High energy X-ray or NMR spectroscopies are impractical or damaging in biomedical conditions. Low energy spectroscopies, such as IR and Raman techniques, are often the best alternative. In apatite biominerals, the vibrational signatures of the phosphate group are generally used as fingerprint of the materials although they provide only limited information. Here, we have used first principles calculations to unravel the complexity of the complete vibrational spectra of apatites. We determined the spectroscopic features of all the phonon modes of fluoroapatite, hydroxy-apatite, and carbonated fluoroapatite beyond the analysis of the phosphate groups, focusing on the effect of local corrections induced by the crystalline environment and the specific mineral composition. This provides a clear and unique reference to discriminate structural and chemical variations in biominerals, opening the way to a widespread application of non-invasive spectroscopies for in vivo diagnostics, and biomedical analysis.     

Calcification mechanism and bony bonding studies of calcium carbonate and composite aluminosilicate/calcium phosphate applied as biomaterials by using radioactivation methods

Bony grafts are used as a filling biomaterial for defective bone. The introduction of new range of synthetic materials offers to surgeons additional possibilities to avoid virus transmission risks by using natural grafts in bony surgery. In this work, two materials, synthetic calcium carbonate and composite aluminosilicate/calcium phosphate were synthesized by an original method and experimented “in vivo” as biomaterials for bony filling. Extracted biopsies were studied by several physico chemical and biological methods. The aim was to evaluate the kinetic resorption and bioconsolidation of these materials. We focused on the bioconsolidation between implant and bone by realising cartographies from the implant to the bone and on the calcification mechanism by determination of the origin of Ca and Sr responsible of the neo-formed bone. Neutron activation analysis (NAA), radiotracers ⁴⁵Ca* and ⁸⁵Sr* and proton-induced X-ray emission (PIXE) were used. Concerning the synthetic calcium carbonate, results show that twelve months after implantation, the mineral composition of implant becomes similar to that of the mature bone. The neoformed bone is composed with Ca and Sr coming from the organism when the Ca and Sr of the implant were progressively eliminated. Concerning the composite geopolymer/calcium phosphate, PIXE and histological studies reveal the intimate links between the bone and the implant starting with the first month after implantation.     

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.     

Mineralization mechanism of calcium phosphates under three kinds of Langmuir monolayers

Three kinds of Langmuir monolayers formed by dipalmitoylphosphatidylcholine (DPPC), arachidic acid (AA), and octadecylamine (ODA) were used as templates to study the initial stage of nucleation and crystallization of calcium phosphates. It was demonstrated that the combination of calcium ions (or phosphates) to the monolayer/subphase interface is a prerequisite for subsequent nucleation. It was found that calcium phosphate dihydrate (DPCD) formed at 25.0 degrees C for 12 h has a biphasic structure containing both amorphous and crystalline phases. These results showed that calcium phosphates were formed through a multistage assembly process, during which an initial amorphous phase DPCD was followed by a phase transformation into a crystalline phase and then the most stable hydroxyapatite (HAp). This provided new insights into the template-biomineral interaction and a mechanism for biomineralization.     

Annealing dependent synthesis of cyto-compatible nano-silver/calcium hydroxyapatite composite for antimicrobial activities

Crystalline hydroxyapatite were synthesized from synthetic/human urine through precipitation which were further doped with silver nanoparticle for effective biomedical application. The aim were to improve overall biological compatibility of the synthesized bone-graft material even in oncogenesis cases. The thermal calcinated material was characterized by several techniques including UV–vis, Laser Raman, Fourier transmittance infrared spectroscopy, X-ray diffraction analysis, Transmission Eelectron microscopy and X-ray fluorescence spectroscopy. The quantitative and qualitative analysis revealed that the synthesized material was highly crystalline and nanosized with majority of silver and phosphate components. The antibacterial, anticancer and invitro cytotoxicity of the synthesized material was evaluated with Escherichia coli, Hela cells and brine shrimp assay, respectively. The brine shrimp assay revealed that the synthesized material is compatible with biological system, whereas anticancer activity showed the application of the synthesized biomaterial in cancer treatment in which antibacterial activity adds more advantage on preventing the bone-graft from microbial attack.     

Covalent modification of calcium hydroxyapatite surface by grafting phenyl phosphonate moieties

The reaction between phenyl phosphonic dichloride (C₆H₅P(O)Cl₂) and synthetic calcium hydroxy- and fluorapatite has been investigated. The presence of mono- or polymeric (C₆H₅PO) fragment bound to hydroxyapatite was evidenced by IR, and solid-state ³¹P NMR spectroscopy. X-ray powder analysis has shown that the apatitic structure remains unchanged during the reaction. In contrast, no reaction was found using fluorapatite. According to the results found for these two different apatites a mechanism was proposed for the formation of covalent P-O-P bonds as the result of a reaction between the C₆H₅P(O)Cl₂ organic reagent and (HPO₄)⁻ and/or OH⁻ ions of the hydroxyapatite.     

Infrared spectra of calcium-strontium phosphate apatites

The ir spectra of calcium-strontium phosphate apatites were studied and the OH, PO₄, CO₃, and cation-oxygen fundamental frequencies were assigned. Furthermore, X-ray diffraction patterns were obtained and lattice parameters were calculated.     

The mineral phase in the cuticles of two species of Crustacea consists of magnesium calcite, amorphous calcium carbonate, and amorphous calcium phosphate

The cuticules (shells) of the woodlice Porcellio scaber and Armadillidium vulgare were analysed with respect to their content of inorganic material. It was found that the cuticles consist of crystalline magnesium calcite, amorphous calcium carbonate (ACC), and amorphous calcium phosphate (ACP), besides small amounts of water and an organic matrix. It is concluded that the cuticle, which constitutes a mineralized protective organ, is chemically adapted to the biological requirements by this combination of different materials.     

Formate incorporation in the structure of Ca-deficient apatite: Rietveld structure refinement

Two sets of non-stoichiometric apatites (Ca-deficient apatites) were prepared from calcium phosphate solutions by homogeneous precipitation through the hydrolysis of formamide at 95°C. One set of products contained monetite (CaHPO₄) and apatite, whilst the second, with more formamide, contained only apatite. Rietveld whole pattern fitting structure refinements were undertaken on all samples, and chemical analyses, IR and NMR spectroscopy, on the second set of samples. The Ca/P mol ratio was 1.596. Rietveld analysis gave lattice parameters a=9.4729(20) and c=6.8855(9) Å and showed that Ca²⁺ ions were lost exclusively from Ca2 sites, and that the PO₄ tetrahedron volume and P-O bonds were 4.4% and 1.4% smaller, respectively, than in hydroxyapatite (OHAp). Formate, HCO₂⁻, was clearly visible in the IR and NMR spectra, but the diffraction studies showed it was not present as a separate crystalline phase. Chemical analysis gave 5.8 wt % formate. We propose that the enlarged a-axis compared with OHAp (a=9.4243(55) Å) and reduced PO₄ dimensions and P occupancy are, respectively, caused by the partial replacement of OH⁻ and PO₄³⁻ ions in the structure by HCO₂⁻ ions. These substitutions would parallel the similar known substitutions of CO₃²⁻ ions in precipitated carbonate apatites.     

An electrokinetic study on a synthetic adsorbent of crystalline calcium silicate hydrate and its mechanism of endotoxin removal

A synthetic, disposable adsorbent of crystalline calcium silicate hydrate, LRA product by Advanced Minerals Corp., has been found highly effective for endotoxin removal from aqueous solutions. Endotoxin removal by this adsorbent is greatly enhanced by the addition of an electrolyte, such as NaCl or Tris-HCl. The electrophoretic method has been used to study the mechanism of endotoxin adsorption. In many cases, adding the electrolyte increases the magnitude of negative zeta potential of the adsorbent in water, while endotoxin adsorption reduces the magnitude. It is hypothesized that ion-exchange between monovalent cations from the aqueous phase and Ca2+ ions near the surface of the adsorbent shift zeta potential of the adsorbent to the more negative direction. It is further hypothesized that endotoxins form cationic species through binding between its phosphate groups and Ca2+ ions dissolved from the adsorbent. The adsorption of endotoxins in the form of cationic species is enhanced by the increased negative zeta potential of the adsorbent when an electrolyte is added.     

Formation of calcium phosphates in gelatin with a novel diffusion system

The present paper demonstrated a novel and simple diffusion system to precipitate calcium phosphates in gelatin gel. In this system, a gelatin cup was specially used as the membrane separating reservoirs of calcium and phosphate ions. Relative to the conventional diffusion system, the novel one in our experiment decreased the time required for the deposition from 5-7 days to 20 h and increased the amount of the precipitated mineral phases significantly. The influence of pH values and concentrations of calcium and phosphate solutions buffered with Tris-HCl and NaOH, respectively, was investigated. The results showed that precipitation of the mineral phase at low pH values (7 for calcium and 11 for phosphate) and concentrations (200 mM for calcium and 15 mM for phosphate) resulted in the formation of plate-like octacalcium phosphate (OCP) crystals. With increasing the pH values of calcium and phosphate solutions to 8 and 12, respectively, spherical amorphous calcium phosphate (ACP) particles were obtained uniquely. Furthermore, flower-like hydroxyapatite (HAP) aggregates composed of many nano-sized needles were formed from the solutions with high pH values (8 for calcium and 12 for phosphate) and concentrations (500 mM for calcium and 37.5 mM for phosphate). The novel diffusion system is proposed to play an important role in both studying the process of biological mineralization and synthesizing calcium phosphates in different forms.