Papers by Author: Gloria Dulce de Almeida Soares

Abstract: Hybrid composites with chitosan (CHI), silk fibroin (SF) and hydroxyapatite (HA) are biocompatible and attractive for bone engineering applications. The objective of this work was to evaluate in vitro cells behavior in contact with CHI-SF-HA scaffolds. The scaffolds were produced from a chitosan solution (2%wt) with SF (1%wt) and HA powders (1%wt) in acetic acid. This solution was molded, frozen and the scaffolds were freeze-dried, crosslinked, and then, submitted to in vitro tests with STRO+1A, MC3T3-E1 and SaOS₂ cells under static conditions for 7, 14 and 21 days. The scaffolds were characterized through X-ray diffraction (XRD), infrared spectroscopy (FTIR) and scanning electron microscopy with energy dispersive spectroscopy (SEM/EDS). Cell viability and activity was assessed by MTT reduction and alkaline phosphate (ALP) activity detection. XRD patterns showed characteristic peaks at 8.8º, 20.3º and 24.6º (corresponding to SF) and peaks at 31.8º, 32.2º and 32.9º (corresponding to HA). The FTIR presented characteristic bands of the amide groups (SF) and of the phosphate and carbonate groups (HA). The EDS showed the presence of the C, O, N, P and Ca elements. SEM analyses showed the scaffold morphology, and indicated cell growth across the surface of the sample. The STRO+1A and MC3T3-E1 cells presented the best cell adhesion. MTT assay showed an increase of the cell number with time and the 21 days analyses showed the best proliferation for SaOS₂ cells (p<0.01), STRO+1A cells (p<0.04) and MC3T3-E1 cells, respectively. The ALP activity was higher at 21 days for all cells types and the SaOS2 cells presented the best results in all analyses (p<0.01). Molecular studies are under progress to evaluate more deeply the biocompatibility of these scaffolds. Moreover, dynamic studies in bioreactor are under progress to improve cell colonization inside scaffolds.

Abstract: Porous granules were obtained through a route using a mixture of calcium phosphate powder with sodium alginate solution. The sintered β-TCP and β-TCMP granules with diameters ranging from 250 µm to 500 µm were implanted into dental alveoli of 30 rats (Rattus norvegicus). The animals were divided in three groups: group I (control, no treatment), group II (β-TCP) and group III (β-TCMP); the sacrifices occurred at 7 and 21 days (n=5/group/period). Histological and histomorphometric analysis were performed to observe and measure connective tissue, bone neoformation and biomaterial areas. Most animals showed acute inflammatory response with many neutrophil granulocytes and foreign body giant multinucleated cells associated to both biomaterials, at 7 and 21 days. The utilization of sodium alginate as additive in the porous granules might explain these results. Fourier-transformed infrared spectroscopy (FTIR) pointed out residue in the granules surface that could exacerbate the inflammatory response. Additional studies are in course to confirm such hypothesis.

Abstract: The current trends in bioactive ceramics point out the ionic substitution in hydroxyapatite (HA) as a concrete way to create new active ceramics with a high developed biomimetic character. Accordingly, our objective in this work was investigating the effects of the simultaneous replacement of Ca²⁺ ions for Mg²⁺, Sr²⁺ and Mn²⁺ into the crystalline structure of HA.

Abstract: The knowledge of the degradation process is an important role to understand and predict in vitro results. Granules of strontium-hydroxyapatite (Sr-HA) were submitted to a degradation process under dynamic conditions using a bioreactor system and using a solution with pH=5. The degradation process under dynamic conditions can be represented by a curve in which the calcium content firstly increases (dissolution part) followed by calcium decreasing (reprecipitation). The use of a low pH solution does not affect the curve’s shape but reduces the precipitation step.

Abstract: Natural bone constitutes of an inorganic phase (a biological nanoapatite) and an organic phase (mostly type I collagen). The challenge is to develop a material that can regenerate lost bone tissue with degradation and resorption kinetics compatible with the new bone formation. The aim of this study was to prepare self-organized magnesium and carbonate substituted apatite/collagen scaffolds, cross-linked with glutaraldehyde (GA). Bovine tendon was submitted to alkaline treatment resulting in a negatively charged collagen surface. The scaffolds were prepared by precipitation: simultaneous dropwise addition of solution containing calcium (Ca) and magnesium (Mg) ions and collagen into a buffered solution containing carbonate and phosphate ions in reaction vessel maintained at 37 °C, pH=8. The reaction products were cross-linked with 0.125 and 0.25% (v/v) glutaraldehyde (GA) solution and freeze-dried. The samples were characterized by Fourier-transformed infrared spectroscopy (FTIR). In vitro cytotoxicity (based on three parameters assays) and scaffolds degradation in culture medium and osteoblastic cells culture were performed in the cross-linked materials. No cytotoxic effects were observed. The cross-linked samples with the lower GA concentration showed a lower stability when placed in contact with culture medium. Human osteoblasts attached on the scaffolds surface cross-linked with 0.25% GA, forming a continuous layer after 14 days of incubation. These results showed potential application of the designed scaffolds for bone tissue engineering.

Abstract: In this study, commercially pure titanium (Ti-cp) discs were used as substrates. Octacalcium phosphate (OCP) layers were deposited by immersion of Ti-cp discs up to 7 days into Solution for Bioactivity Evaluation (SBE) and characterized by SEM-EDS and XRD. The OCP coatings were doped with cephazolin or cefalexin by individual immersion in 300 ppm of each solution for 24 h at room temperature. The non-existence of mathematical models to explain drug release from these matrixes made the choosing of correct model, a complex process. Five non-linear mathematical methods were employed in order to identify the possible drug release mechanism using Akaike and Bayesian Information Criterion (AIC and BIC respectively) and its derivatives. The best model was Peppas & Sahlin that consider two stages in release: pure diffusion in first stage, and drug dissolution and migration through the porous matrix at second stage.

Abstract: Composites based on hydroxyapatite (HA) and collagen are being used for bone engineering applications. Synthetic composites were produced by precipitating hydroxyapatite in a reaction medium containing collagen fibers. The effect of saline treatment during the procedure of fibrils extraction on the composite morphology, phases presented and in vitro stability was evaluated. The FTIR analysis showed collagen bands well preserved. By Rietveld analysis, additional peaks of sodium chloride were identified on sample made from collagen submitted to saline precipitation. Besides that, the mineral formed phase is a carbonated apatite. This step during fibrils extraction results in larger areas of collagen uncoated and higher stability in culture medium. Despite of this, a cross-linking agent will be necessary to maintain the composite in culture for longer times.

Abstract: In this study, commercially pure titanium (Ti-cp) sheets grade 2 were used as substrates. To investigate the role of composition and characteristics of titanium surface oxides in cellular behavior of osteoblasts, the surface of titanium were modified by anodic oxidation with sulphuric acid. Subsequently were treated part of anodized samples with FN and cultured with osteoblast cells for 30, 120 and 240 minutes. The chemical composition and topography have influenced the interaction between fibronectin and substrate. Cells were found more associated to the surfaces that have been submitted to anodization and FN coating than to the related controls.

Abstract: In this study we report on the microstructure and its mechanical behavior of a Ca-P coating produced on bioactive titanium by immersion in a simplified simulated body fluid (S-SBF). The coating was probed by nanoindentation in several point times up to the formation of octacalcium phosphate (OCP). Amorphous calcium phosphate, formed after 1h of immersion in SSBF, presented the highest values of hardness (H) and elastic modulus (E). Nucleation of OCP was observed after 2-2,5h of immersion in S-SBF. From this stage on, lower values of H and E were obtained, probably due to the low dense structure of the coating.

Abstract: The aim of this paper was to evaluate the usefulness of coupling digital image analysis with immunohistochemistry and histomorphometry data to the study of tissue response to hydroxyapatite in a model of critical size bone defect in calvaria of rats. A transosseous defect measuring 8 mm in diameter was performed with a surgical trephine in the parietal bone of 40 rats and divided into two experimental groups according to the treatment: group I (blood clot, control), group II (HA) and killed 1, 3, 6 and 9 months after implantation (n=5/group/period). The skullcaps with overlaying skin were collected and processed for paraffin embedding. The specimens were cut in the laterolateral direction into 5-µm thick semi-serial sections and stained with hematoxylin-eosin for identification and counting of polymorphonuclears cells, mastocytes, and multinucleated giant cells, MNG, or immunolabeled with anti- lysozyme, -factor VIII and –PCNA. Digital images were obtained and analyzed with the ImagePro-Plus® software for cell couting (polymorphonuclears cells, mastocytes, macrophages and MNG) and microvessel density. Image segmentation of anti-PCNA immunostaining was used for cell proliferation analysis. The digital images obtained allowed clear identification of cells of interest by through morphological aspects or immunostaining. Data recording and analysis was facilitated by the use of specific software for image processing and graphical and statistical analysis. It can be concluded that the techniques applied were usefull to identify and count cells, structures and process of interest making easier the effectiveness of hydroxyapatite in the critical size defect in rat calvaria model.