Elena Mavropoulos

Dissolution of calcium-deficient hydroxyapatite synthesized at different conditions

Abstract The dissolution characteristics of several calcium-deficient hydroxyapatites (HA) have been investigated. Eleven samples were produced by varying synthesis parameters like temperature, pH, digestion time, reagent concentration and velocity of addition. Powder characterization was conducted using X-ray diffraction. Sample crystallinity was variable and samples examined by transmission electron microscopy showed acicular or plate-like morphology. After sample dissolution in Milli-Q water for 168 h, the calcium and phosphate contents in solution were measured by induced coupled plasma optical emission spectrometry (OES). The dissolution behavior of calcium-deficient HA was highly dependent on the sample calcium/phosphorus (Ca/P) molar ratio of the original HA. Dissolution in water was enhanced with decreasing HA sample molar ratio. However, the dissolution process equilibrium was not achieved at the end of 168 h (7 days).

Adsorption and bioactivity studies of albumin onto hydroxyapatite surface.

Bovine serum albumin (BSA) may have an inhibitory or promoter effect on hydroxyapatite (HA) nucleation when apatite is precipitated in a medium containing the protein. In this study we evaluated the influence of BSA on the precipitation of calcium phosphate phases (CP) from simulated body fluid (SBF) when the protein was previously bounded to HA surface. The kinetics of BSA immobilization onto hydroxyapatite surface was performed in different buffers and protein concentrations in order to adjust experimental conditions in which BSA was tightly linked to HA surface for long periods in SBF solution. It was shown that for BSA concentration higher than 0.1mg/mL the adsorption to HA surface followed Langmuir-Freundlich mechanisms, which confirmed the existence of cooperative protein-protein interactions on HA surface. Fourier Transformed Infrared Attenuated Total Reflectance Microscopy (FTIRM-ATR) evidenced changes in BSA conformational state in favor of less-ordered structure. Analyses from high resolution grazing incident X-ray diffraction using synchrotron radiation (GIXRD), Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) showed that a poorly crystalline calcium phosphate was precipitated on the surface of HA discs coated with BSA, after the immersion in SBF for 4 days. The new bioactive layer had morphological characteristics similar to the one formed on the HA surface without protein. It was identified as a carbonated apatite with preferential crystal growth along apatite 002 direction. The GIXRD results also revealed that BSA layer bound to the surface inhibited the HA dissolution leading to a reduction on the formation of new calcium phosphate phase.

Pb 2+, Cu 2+and Cd 2+ ions uptake by Brazilian phosphate rocks

Neste trabalho, rochas fosfatadas brasileiras (PRs) foram usadas na imobilizacao de Pb2+, Cu2+ e Cd2+ de solucoes aquosas. Concluimos que estes metais sao principalmente imobilizados por fluorapatita, mas outros minerais tais com carbonatos de calcio contribuem para esta imobilizacao. No caso do chumbo, dois mecanismos sao propostos: a dissolucao da Ca10(PO4)6F2 seguida pela precipitacao de (Ca,Pb)10(PO4,CO3) 6(OH,F,Cl)2.56.1.5H2O e a dissolucao de CaCO3 com formacao de PbCO3. Este segundo mecanismo aumenta a eficiencia do processo de remocao, mas contribui para tornar o chumbo mais biodisponivel porque o carbonato de chumbo e mais soluvel que a fluorpiromorfita. Este fato pode limitar a utilizacao das rochas fosfatadas na regeneracao de solos contaminados. A imobilizacao de Cu2+ e do Cd2+ pelas PRs e controlada por mecanismos de adsorcao mas mecanismos de dissolucao/precipitacao tambem contribuem. No caso do Cu2+ a dissolucao da fluorapatita e seguida pela precipitacao da libetenita, Cu2(PO4)(OH).

In vitro and in vivo evaluation of strontium-containing nanostructured carbonated hydroxyapatite/sodium alginate for sinus lift in rabbits

Various synthetic bone substitutes have been developed to reconstruct bone defects. One of the most prevalent ceramics in bone treatment is hydroxyapatite (HA) that is a useful material as bone substitute, however, with a low rate of biodegradation. Its structure allows isomorphic cationic and anionic substitutions to be easily introduced, which can alter the crystallinity, morphology, biocompatibility, and osteoconductivity. The objective of this study was to investigate the in vitro and in vivo biological responses to strontium-containing nanostructured carbonated HA/sodium alginate (SrCHA) spheres (425<ϕ <600 μm) that were used for sinus lifts in rabbits using nanostructured carbonated HA/sodium alginate (CHA) as a reference. Cytocompatibility was determined using a multiparametric assay after exposing murine preosteoblasts to the extracts of these materials. Twelve male and female rabbits underwent bilateral sinus lift procedures and were divided into two groups (CHA or SrCHA) and in two experimental periods (4 and 12 weeks), for microscopic and histomorphometric analyses. The in vitro test revealed the overall viability of the cells exposed to the CHA and SrCHA extracts; thus, these extracts were considered cytocompatible, which was confirmed by three different parameters in the in vitro tests. The histological analysis showed chronic inflammation with a prevalence of macrophages around the CHA spheres after 4 weeks, and this inflammation decreased after 12 weeks. Bone formation was observed in both groups, and smaller quantities of SrCHA spheres were observed after 12 weeks, indicating greater bioresorption of SrCHA than CHA. SrCHA spheres are biocompatible and osteoconductive and undergo bioresorption earlier than CHA spheres.

Short-term in vivo evaluation of zinc-containing calcium phosphate using a normalized procedure

The effect of zinc-substituted calcium phosphate (CaP) on bone osteogenesis was evaluated using an in vivo normalized ISO 10993-6 protocol. Zinc-containing hydroxyapatite (ZnHA) powder with 0.3% by wt zinc (experimental group) and stoichiometric hydroxyapatite (control group) were shaped into cylindrical implants (2×6 mm) and were sintered at 1000 °C. Thermal treatment transformed the ZnHA cylinder into a biphasic implant that was composed of Zn-substituted HA and Zn-substituted β-tricalcium phosphate (ZnHA/βZnTCP); the hydroxyapatite cylinder was a highly crystalline and poorly soluble HA implant. In vivo tests were performed in New Zealand White rabbits by implanting two cylinders of ZnHA/βZnTCP in the left tibia and two cylinders of HA in the right tibia for 7, 14 and 28 days. Incorporation of 0.3% by wt zinc into CaP increased the rate of Zn release to the biological medium. Microfluorescence analyses (μXRF-SR) using synchrotron radiation suggested that some of the Zn released from the biomaterial was incorporated into new bone near the implanted region. In contrast with previous studies, histomorphometric analysis did not show significant differences between the newly formed bone around ZnHA/βZnTCP and HA due to the dissolution profile of Zn-doped CaP. Despite the great potential of Zn-containing CaP matrices for future use in bone regeneration, additional in vivo studies must be conducted to explain the mobility of zinc at the CaP surface and its interactions with a biological medium.

The impact of the RGD peptide on osteoblast adhesion and spreading on zinc-substituted hydroxyapatite surface.

The incorporation of zinc into the hydroxyapatite structure (ZnHA) has been proposed to stimulate osteoblast proliferation and differentiation. Another approach to improve cell adhesion and hydroxyapatite (HA) performance is coating HA with adhesive proteins or peptides such as RGD (arginine–glycine–aspartic acid). The present study investigated the adhesion of murine osteoblastic cells to non-sintered zinc-substituted HA disks before and after the adsorption of RGD. The incorporation of zinc into the HA structure simultaneously changed the topography of disk’s surface on the nanoscale and the disk’s surface chemistry. Fluorescence microscopy analyses using RGD conjugated to a fluorescein derivative demonstrated that ZnHA adsorbed higher amounts of RGD than non-substituted HA. Zinc incorporation into HA promoted cell adhesion and spreading, but no differences in the cell density, adhesion and spreading were detected when RGD was adsorbed onto ZnHA. The pre-treatment of disks with fetal bovine serum (FBS) greatly increased the cell density and cell surface area for all RGD-free groups, overcoming the positive contribution of zinc to cell adhesion. The presence of RGD on the ZnHA surface impaired the effects of FBS pre-treatment possibly due to competition between FBS proteins and RGD for surface binding sites.

Nanometer coatings of hydroxyapatite characterized by glancing-incidence X-ray diffraction

Hydroxyapatite (HAP) crystalline thin-coatings have been grown using a right angle RF magnetron sputtering approach at room temperature. The surface structural information of these biocompatible coatings at nanometer scales was obtained by glancing-incidence X-ray diffraction (GIXRD) with synchrotron radiation. The GIXRD spectra were obtained by fixed incidence theta angles at 0.5 and 1 degree. Structural profile analyses were performed over these nano-coating layers with reduced substrate interference. The coating thickness was calibrated by specular X-ray reflectivity (XRR) curves. Experiments have been performed on thin-coatings of HAP sputtered on silicon wafers and acid etched titanium discs at room temperature. GIXRD analysis has shown that all the principal peaks are attributed to a crystalline HAP. Previous tests of biocompatibility with osteoblasts cells have been encouraging studies on the surface of hydroxyapatite thin coatings prepared by opposing RF magnetron sputtering approach, as a promising candidate for bioimplant materials.

Hydroxyapatite-alginate composite for lead removal in artificial gastric fluid

Millimetric spherical beads of a biocompatible composite were produced from sodium alginate, a natural polysaccharide, and nanostructured hydroxyapatite (HA). It was shown that the composite was effective in the removal of lead ions and lead phosphate nanoparticles from high-contaminated simulated gastric fluid. X-ray diffraction spectroscopy and scanning electron microscopy analyses performed on HA–alginate beads after the Pb 2+ uptake showed that nanocrystals of a lead phosphate, [Pb 10– x Ca x (PO 4 ) 6 Cl 2 ], were precipitated on the bead surface. The cross-linked polymer chain had a double role: (i) keep Pb 2+ ions and lead phosphate nanoparticles bounded to the bead surface, preventing their bioavailability in stomach fluid; and (ii) delay HA dissolution in the acidic conditions of the stomach, assuring that an excess of Ca 2+ will not be released to simulated gastric fluid. Desorption experiments in simulated enteric fluid revealed that lead remained immobilized in the calcium phosphate phase in the intestinal tract. These results indicate HA–alginate composite as a potential system for heavy metals removal from contaminated gastric and enteric human fluids, minimizing its adsorption by the human body.

Effects on insulin adsorption due to zinc and strontium substitution in hydroxyapatite

Insulin-loaded calcium phosphate nanoparticles have been proposed as a potential drug delivery system for the oral treatment of diabetes and to stimulate bone cell proliferation and bone mineralization. The kinetics of insulin incorporation onto hydroxyapatite (HA) and Sr (SrHA)- and Zn (ZnHA)-substituted hydroxyapatite nanoparticles was investigated using X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FTIR) spectroscopy, zeta potential measurements and circular dichroism (CD) spectroscopy. The increase in insulin concentration on HA, SrHA and ZnHA was a typical physical adsorption process controlled by electrostatic forces and followed a Freundlich isotherm model. Zn substitution enhanced the capacity of the apatite surface to adsorb insulin, whereas Sr substitution inhibited insulin uptake. The surface stoichiometry and mesopore specific area induced by Zn and Sr substitution are proposed as the main causes of the difference in insulin adsorption. Despite the weak interaction between insulin and the apatite surface, the CD spectra revealed a decrease in the insulin ellipticity when the protein was adsorbed on the HA, SrHA and ZnHA nanoparticles. A reduction in alpha-helical structures and an increase in beta sheets were observed when insulin interacted with the HA surface. A less pronounced effect was found for ZnHA, for which a subtle decrease in alpha-helical structures was followed by an increase in turn structures. Interaction with the SrHA surface did not change the native insulin conformation. In vitro cell culture experiments lasting 24h using F-OST stromal cells showed that the insulin loaded on HA and ZnHA did not affect cell proliferation but the insulin loaded on SrHA improved cell proliferation. These results suggest that the stability of the native protein conformation is an important factor to consider when cells interact with insulin adsorbed on metal-substituted HA surfaces.

Studies on Cadmium Uptake by Hydroxyapatite

Cadmium uptake by hydroxyapatite from aqueous solution was studied in time intervals between 2 minutes and 96 hours, using different Cd2+ concentrations (21 to 768 mg L-1). The solution pH after Cd2+ uptake was assessed. Dissolution experiments at fixed pH were performed in order to evaluate the ion exchange process. Cd2+sorption by hydroxyapatite is fast and it happens simultaneously by different mechanisms. Ion exchange is the main mechanism, where mechanism Cd2+ present in aqueous solution replaces Ca2+ from hydroxyapatite structure.