Papers by Author: G.V.O. Fernandes

Abstract: The pursuit for an ideal bone substitute remains the main focus of many tissue engineering researchers. Among the myriad types of grafts available, synthetic bone grafts are of special importance, because it is available in large amounts, reduce the surgical trauma and eliminate the risk of diseases’ transmission. In this context, bioactive glasses have received attention mostly due to its described biocompatibility and rapid rate of surface reactivity when compared with other materials, allowing for faster interactions with the local tissue. The addition of niobium to this material has been shown as increasing the chemical resistance of the compound and providing greater stability. However, alterations on the chemical composition of biomaterials may impact on its biocompatibility. Therefore, the aim of this study was to evaluate the in vitro biocompatibility of bioglass-Niobium (BgNb) granules, in comparison with standard commercial bioglass (Biogran®) throughout an interesting multiparametrical approach, employing Phenol 2% and dense polystyrene beads as positive and negative controls, respectively. Extracts from each material were prepared by 24 hours incubation in culture medium (DMEM). Human primary osteoblasts were then exposed for 24 hours to each extract and cell viability was evaluated by three parameters: mitochondrial activity (XTT method), membrane integrity (neutral red dye uptake) and cell density (crystal violet dye exclusion test). BgNb extracts were highly compatible, since the levels of viable cells were similar to the control group (unexposed cells), on all parameters studied. The mean cell density on the Biogran® group was slightly lower than BgNb, even though this material was also non-cytotoxic. The excellent in vitro response for BgNb granules indicates the suitability of this material to future studies on its biological and physical properties when applied in vivo.

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: 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 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.