Alexandre Mello

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.

Osteoblast proliferation on hydroxyapatite thin coatings produced by right angle magnetron sputtering

Right angle magnetron sputtering (RAMS) was used to produce hydroxyapatite (HA) film coatings on pure titanium substrates and oriented silicon wafer (Si(0 0 1)) substrates with flat surfaces as well as engineered surfaces having different forms. Analyses using synchrotron XRD, AFM, XPS, FTIR and SEM with EDS showed that as-sputtered thin coatings consist of highly crystalline hydroxyapatite. The HA coatings induced calcium phosphate precipitation when immersed in simulated body fluid, suggesting in vivo bioactive behavior. In vitro experiments, using murine osteoblasts, showed that cells rapidly adhere, spread and proliferate over the thin coating surface, while simultaneously generating strong in-plane stresses, as observed on SEM images. Human osteoblasts were seeded at a density of 2500 cells cm(-2) on silicon and titanium HA coated substrates by RAMS. Uncoated glass was used as a reference substrate for further counting of cells. The highest proliferation of human osteoblasts was achieved on HA RAMS-coated titanium substrates. These experiments demonstrate that RAMS is a promising coating technique for biomedical applications.

Growth of Crystalline Hydroxyapatite Thin Films at Room Temperature by Tuning the Energy of the RF-Magnetron Sputtering Plasma

Right angle radio frequency magnetron sputtering technique (RAMS) was redesigned to favor the production of high-quality hydroxyapatite (HA) thin coatings for biomedical applications. Stoichiometric HA films with controlled crystallinity, thickness varying from 254 to 540 nm, crystallite mean size of 73 nm, and RMS roughness of 1.7 ± 0.9 nm, were obtained at room temperature by tuning the thermodynamic properties of the plasma sheath energy. The plasma energies were adjusted by using a suitable high magnetic field confinement of 143 mT (1430 G) and a substrate floating potential of 2 V at the substrate-to-magnetron distance of Z = 10 mm and by varying the sputtering geometry, substrate-to-magnetron distance from Z = 5 mm to Z = 18 mm, forwarded RF power and reactive gas pressure. Measurements that were taken with a Langmuir probe showed that the adjusted RAMS geometry generated a plasma with an adequate effective temperature of Teff ≈ 11.8 eV and electron density of 2.0 × 10(15) m(-3) to nucleate nanoclusters and to further crystallize the nanodomains of stoichiometric HA. The deposition mechanism in the RAMS geometry was described by the formation of building units of amorphous calcium phosphate clusters (ACP), the conversion into HA nanodomains and the crystallization of the grain domains with a preferential orientation along the HA [002] direction.

Osteoblast proliferation on hydroxyapatite coated substrates prepared by right angle magnetron sputtering

The preparation of hydroxyapatite (HA) coatings via a versatile right-angle magnetron sputtering (RAMS) approach for use as a biomaterial has recently been reported. RAMS coatings show some advantages over conventionally sputtered films in that room temperature deposition yields nanocrystalline and nearly stoichiometric HA coatings under appropriate conditions, thereby avoiding the troublesome post deposition annealing treatment. In this article, we present an exploratory study of the biocompatibility of RAMS HA coatings deposited on metallic substrates. RAMS HA coatings with a thickness around 500nm were prepared on various substrates. X-ray diffraction (XRD) analysis showed that the as-deposited HA coatings were polycrystalline with some strongly preferred orientations. Atomic force microscopy (AFM) results showed that the coatings were rather smooth with surface roughness on the order of 10 nm. X-ray photoelectron spectroscopy (XPS) confirmed that the surface chemistry was nearly stoichiometric. To study the biocompatibility of these coatings, murine pre-osteoblastic MC3T3-E1 cells were seeded onto various substrates. Cell density counts using fluorescence microscopy showed that the best osteoblast proliferation is achieved on an HA RAMS-coated titanium substrate. Additionally, in preliminary studies the influence of Zn, Mg, and Al incorporation in the HA crystal lattice on the in vitro behavior was also evaluated. These experiments demonstrate that RAMS is a promising coating technique for biomedical applications.

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.

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.

Characterization of Crystalline Hydroxyapatite Thin Coatings for Biomedical Applications

Crystalline hydroxyapatite thin coatings have been prepared using a novel opposing RF magnetron sputtering approach at room temperature. X-ray diffraction (XRD) analysis shows that all the principal peaks are attributable to HA, and the as-deposited HA coatings are made up of crystallites in the size range of 50-100nm. Fourier transform infrared spectroscopy (FTIR) studies reveal the existence of phosphate, carbonate and hydroxyl groups, suggesting that HA coatings are carbonated. Finally, in vitro cell culture experiments have demonstrated that murine osteoblast cells attach and grow well on the as-sputtered coatings. These results encourage further studies of hydroxyapatite thin coatings prepared by the opposing RF magnetron sputtering approach as a promising candidate for next-generation bioimplant materials.

CF-LIBS analysis of frozen aqueous solution samples by using a standard internal reference and correcting the self-absorption effect

The aim of this paper is to present an innovative procedure to determine the composition of a liquid sample using Laser Induced Breakdown Spectroscopy without calibration curves, or the so-called CF-LIBS (Calibration Free-Laser Induced Breakdown Spectroscopy). Our results of CF-LIBS for the elemental composition were compared with the sample standard concentration determined by Microwave Plasma-Atomic Emission Spectroscopy (MP-AES). In this work, a controlled amount of an internal standard, an extra chemical element, is placed into a liquid sample that is afterward frozen to produce a solid target. The plasma of the target material is produced at atmospheric pressure by focusing a Nd:YAG laser on the frozen target surface. Time-resolved emission spectra are acquired and used for quantitative analysis. An extraordinary increase in the accuracy of elemental concentration is achieved when the spectroscopic data are corrected through an analytical elimination of the self-absorption effect. The corrected data plotted in a Boltzmann diagram show that CF-LIBS can provide good analytical sensitivity and accurate and reliable elemental concentrations when the local thermodynamic equilibrium conditions are experimentally fulfilled and the self-absorption effect on the line intensities is corrected. In the case analyzed here, the concentration results obtained by the improved CF-LIBS procedure give relative deviations of 3.3% maximum when compared with those of MP-AES.

Spectroscopic Studies of Adsorbed Myoglobin on Hydroxyapatite Surface

In this work myoglobin (Mb) adsorption was carried out in a batch system using hydroxyapatite (HA) powder during 24 hours at 37°C. The HA samples were analyzed after protein adsorption by Fourier Transformed Infrared (FTIR) and UV-Vis Spectroscopy in reflectance mode. UV-Vis analyses showed that Soret and Q bands shifted to lower wavelengths when Mb is associated with HA surface. This result suggests that Mb Heme group is sensitive to the protein adsorption onto HA surface. HA disks coated with myoglobin and cultured with human osteoblastic cells during 7 days showed that cell adhesion and proliferation were not inhibited by the protein coating after 7 days in cell culture.