Papers by Author: Antonella M. Rossi

Abstract: In this present work, we characterize HAp thin films deposited by dual magnetron sputtering device DMS on silicon (Si/HAp). The sputtering RF power was varied from 90 watts to 120 watts and deposition times from 60 to 180 minutes. The argon and oxygen pressure were fixed at 5.0 mTorr and 1.0 mTorr, respectively. Grazing incidence X-ray diffraction (GIXRD) from synchrotron radiation, infrared spectroscopy (FTIR) and atomic force microscopy (AFM) were used for the structural characterization. At lower deposition times, a crystalline phase with preferential orientation along apatite (002) and a disordered nanocrystalline phase were identified. The coating crystallinity was improved with the increase of the deposition time besides the sputtering power.

Abstract: Hydroxyapatite (HA) is one of the most employed materials for bone therapy due to its structural similarity with bone, its biocompatibility and physicochemical properties. Additionally, HA performance may be improved by ionic substitution of calcium with divalent bioactive metallic cations such as zinc. In this context, zinc incorporation into HA have been well studied, in spite of conflicting results regarding its biocompatibility: while previous reports on in vitro cytocompatibility have described 5% zinc containing HA (ZnHA) as slightly cytotoxic, this material presented an excellent response on in vivo studies. In order to bring more information on ZnHA biocompatibility, we performed a multiparametric assay evaluating sequentially on the same cells three different viability parameters: mitochondrial activity (XTT), membrane integrity (Neutral Red) and cell density (Crystal Violet Dye Exclusion test). Additionally, we intended to complement the existing data on ZnHA in vivo performance, by assessing its ability to affect the arrangement of collagen fibers on the grafted area, an important indicative of bone maturation. MC3T3-E1 cells were exposed to 24-hours extracts of ZnHA or stoichiometric HA on culture medium (DMEM) and cell viability was assayed. ZnHA was very cytocompatible, since the levels of viable cells on all 3 tests were similar to the HA and polystyrene (negative control) extracts, but significantly higher than cells treated with 4% phenol (positive control). For the in vivo studies, critical size defects in rats calvaria were filled with HA or ZnHA granules. The histological evaluation after 30 and 180 days revealed an increase along time. Event tough ZnHA is cytocompatible the presence of Zn was unable to alter the interaction between collagen fibers and the mineral bone phase, as compared to stoichiometric HA.

Abstract: The objective of this study was to investigate the bone repair of carbonate apatite (cHA) in comparison to hydroxyapatite (HA, control group), on osseous repair of non-critical size defect in rat calvaria. Spheres (400<ø>500μm) of both materials were synthesized under 37°C (cHA) and 90°C (HA) and were not sintered. Fifteen rats Wistar were submitted to general anesthesia and two perforations (4mm each) were made, one in each parietal bone, for implantation of cHA (left side) and HA spheres (right side). After 1, 3 and 6 weeks, five animals of each group were killed and the two fragments with the biomaterial were collected from the calvaria. The bone blocks with biomaterial were demineralized and 5μm thick semi-serial sections were done for histological analysis. The experimental group of 6 weeks did not show the presence of spheres of both biomaterials and few spheres were observed after 1 and 3 weeks. Histological analysis showed the connective tissue repairing the surgical defect after 1 week and newly formed bone after 3 weeks of surgery. Thus, we concluded both materials are biocompatible, promote osteoconduction and in all studied periods the biomaterials showed to be resorbable.

Abstract: The objective of this study was to investigate the in vitro and in vivo biological responses to carbonate apatite (cHA) in comparison to hydroxyapatite (HA). Spheres (400<ø>500 μm) of both materials were synthesized under 5°C (cHA) and 90°C (HA) and not sintered. The in vitro cytocompatibility was determined by the XTT assay, according to ISO 10993-5:2009, after exposure of MC3T3-E1 cells to the materials extracts. Ethics Commission on Teaching and Research in Animals approved this project (CEPA/NAL 193/10) and, subsequently, the biomaterials were grafted in the subcutaneous tissues of mice (n=15). After 1 and 3 weeks, five animals of each group were killed for samples removal containing biomaterials and surrounding tissues for histological examination. Semi-serial (5-μm thick) sections were cut and stained with Hematoxylin and Eosin (HE) and the presence of inflammatory infiltrates and biomaterials resorption were evaluated. The experimental group of 3 weeks didn’t show the presence of spheres of both biomaterials and few spheres were observed after 1 week. Histological analysis showed the granulation tissue around the biomaterials with the presence of multinucleated giant cells. After 3 weeks it was observed the presence of fibrous tissue around biomaterials and few inflammatory cells. No signals of tissue necrosis were observed in both groups in all experimental studied periods. Nanostructured carbonate apatite spheres are cytocompatible, biocompatible and present initial biosorption on the subcutaneous comparable to stoichiometric HA, indicating its suitability for further studies on regenerative medicine.

Abstract: This study investigated the osteoinductive potential of granules of stoichiometric hydroxyapatite (HA) and 0.5% zinc containing hydroxyapatite (ZnHA) in intramuscular (IM) site of rabbit’s abdomen. The biomaterials were both used in granular form, with 75% porosity and particle diameter between 450 and 500μm, sintered at 1100°C. Both materials performed adequately on a multiparametric in vitro cytocompatibility assay, indicating their suitability for in vivo testing. After approval by the Ethics Commission on Teaching and Research in Animals, fifteen rabbits were submitted to general anesthesia, incision and tissue dilatation, and a small site was created for HA (right incision) and ZnHA (left incision) intramuscular implantation. The animals were killed after 2, 4 and 12 weeks for biomaterials and surrounding tissues removal. Histological analysis after 2 weeks revealed the presence of granulation tissue surrounding biomaterials with multinucleated giant cells and no newly formed bone for both materials. After 4 weeks there was fibrous tissue involving the material and few inflammatory cells. Following 12 weeks it was observed the presence of connective tissue surrounding the biomaterial, cellularized enough for the two experimental groups, but it was not observed the presence of bone matrix associated with the biomaterials. We conclude that both biomaterials are cytocompatible and did not present the property of osseoinduction after 12 weeks of implantation.

Abstract: The objective of this study was to investigate the bone repair of carbonate apatite (cHA) in comparison to hydroxyapatite (HA, control group) on osseous repair of rabbit’s tibia. Spheres (400-500 µm) of both materials were synthesized under 37°C (cHA) and 90°C (HA) and were not sinterized. Ethics Commission on Teaching and Research in Animals approved this project (CEPA/NAL 208/10). Six White New Zealand rabbits were submitted to general anesthesia and one perforation (2mm) was made in each tibia for implantation of cHA (left tibia) and HA spheres (right tibia). After 4 weeks all animals were killed and one fragment of each tibia with the biomaterial was collected with a total of 6 bone blocks for each group. Five bone blocks of each group were demineralized and 5-µm thick semi-serial sections were stain with Hematoxillin and Eosin and Trichromic of Masson for histological analysis and two fragments were collected and embedded in a methacrylate-based resin and cut into slices with ~30 µm and were analyzed by light microscopy (bright field and polarized microscopy). Both groups didn’t show the spheres after 4 weeks, new-formed bone was observed from the periphery toward to the center of the surgical defect, which was even filled with connective tissue. Both materials are biocompatible, promote osteoconduction and showed to be resorbable.

Abstract: Hydroxyapatite (HA, Ca₁₀(PO₄)₆(OH)₂) is one of the most important biomaterials used in bone regeneration therapies due to their chemical properties are very similar to the inorganic phase found in bone tissues. The direct observation of the ultrastructure of HA is very important in the comprehension of their nucleation and interactions with the molecules involved in bone formation. High-resolution Transmission Electron Microscopy (HRTEM) is a currently technique used for this task. However, the interpretation of the images is not straightforward and needs the use of softwares dedicated to high-resolution images simulations. This work presents the applicability of MEGACELL software in the analysis of HRTEM images of HA nanoparticles. MEGACELL is the most newly software, developed to construct nanocrystals models for HRTEM multislice simulations. The output files generated by MEGACELL are raw data format (.xyz), containing all the atomic positions, as well as input files compatible with JEMS (Java Electron Microscopy Software) format files. High-resolution images were acquired using a JEM 3010 URP microscope, with a LaB₆ thermionic electron gun operating at 300 kV, with a point-to-point resolution of 0.17 nm and a CCD Gatan 794SC multiscan digital camera, attached to the DigitalMicrographTM software for recording and image processing. Electron microscopy samples were prepared by dropping HA powder on copper TEM grids. HRTEM experimental images of HA particles, orientated along different zone axes, were interpreted applying the MEGACELL software to construct HA nanocrystal models and the multislice method to simulated them. MEGACELL improves the extraction of the ultrastructural features and facilitates a better interpretation of the phase-contrast images.

Abstract: In the present work, apatite powders were synthesized at pH 10, pH 11 and pH 12 in order to give rise to biphasic and triphasic bioceramics after sintering. A modified gelcasting process, including polyethylene wax spheres addition to the suspension, is proposed in comparison to the original gelcasting method. The aim of the addition is the creation of uniform, open and interconnected pores in the body of samples.

Abstract: B-type carbonate apatite samples were synthesized by wet chemical method and characterized by X-ray Fluorescence Spectrometry, X-ray Diffraction, Fourier Transformed Infrared Spectroscopy and High Resolution Transmission Electron Microscopy. The XRD and FTIR analysis confirmed the presence of one B-type carbonate apatite phase and the HRTEM images revealed the coexistence of amorphous and polycrystalline regions in the order of 2nm with the carbonate apatite structure. Second phases or precursors were not discovered.

Abstract: In the present work, mechanical properties of a stoichiometric hydroxyapatite (HA), synthesized by hydrothermal method, with 1.66 Ca/P molar ratio are investigated as a function of the processing parameters. Cylindrical samples were processed by uniaxial compacting, followed by sintering, aiming to obtain high density HA samples. Density values were obtained by the geometric method and SEM images were taken from HA samples in order to characterize their topography and to determine the grain size for each set of samples. Vickers micro-hardness was measured for each set of samples. Compressive strength of cylindrical samples with 2.0 mean diameter/height ratio was measured reporting load to failure divided by the cross-sectional area of the samples. Vickers micro-hardness and compaction strength values of the samples were found to be in agreement with the relative density and grain size values.