Papers by Author: J.M. Granjeiro

Abstract: The hydroxyapatite (HA) is a biocompatible and bioactive biomaterial used as bone substitute, however, the high crystallinity of HA and consequently its low solubility may be a limitation for its clinical use. In order to improve the biosorption of HA, the partial substitutions in the chemical structure and doping with small amounts of impurities have been study. The objective of this study was to evaluate the biocompatibility of 3% Zinc-containing nanostructured carbonated hydroxyapatite (ZncHA) compared with the carbonated hydroxyapatite (cHA), both synthesized at 37°C and non-sintered, using as control the stoichiometric HA microspheres in subcutaneous of mice. The X-ray Diffraction (XRD) and Vibrational Spectroscopy in Infra Red Fourier Transform (FTIR) were used to characterize the biomaterials. In vivo test was performed in BALB/c mice by implanting of HA, cHA and ZncHA spheres in the subcutaneous tissue for 1, and 9 weeks (n=5). The negative control consisted in incision without material implantation (Sham group). The samples were histological processed to descriptive analysis of biological effect. The microscopic analysis showed a similar granulation reaction between groups at the first experimental period. In 9 weeks there was a time dependent biosorption of cHA compared with other groups. In conclusion, the biomaterials tested were biocompatible and cHA group showed a significant biosorption in comparison with HA and ZncHA groups. The doping of zinc did not influence the biocompatibility of biomaterial, however, change the biosorption response

Abstract: Bone tissue is a composite material that has hydroxyapatite (HA) as its main inorganic phase component. The biological apatites have low crystallinity and contain cationic and anionic substitutions in their structure, which differ from the available synthetic ceramics. The purpose of this study is to evaluate the biocompatibility of nanostructured carbonated hydroxyapatite microspheres containing 5 wt% strontium (SrcHA) compared with the biocompatibility of carbonated hydroxyapatite (cHA), both synthesized at 37°C and non-sintered, used to control stoichiometric HA microspheres in subcutaneous tissue of mice. The biomaterials (BM) were characterized using X-ray Diffraction (XRD), Vibrational Spectroscopy in an Infrared Fourier Transform (VSIRFT) and Scanning Electron Microscopy (SEM). Forty five balb-C mice were randomly divided into four groups of 15 animals each: SrcHA, cHA, HA, and without material implantation (Sham group). All samples were histologically processed for descriptive evaluation of the biological effect. At each experimental period (1, 3 and 9 weeks), there was a higher biosorption of the tested biomaterials observed in contrast with the HA. The cHA group was the only group completely phagocytosed by macrophages and giant cells after 9 weeks. All biomaterials proved to be biocompatible, and the cHA and SrcHA 3% groups exhibited a faster bioabsorption in comparison with the control group. The doping of strontium did not cause a greater biological response after the 3 experimental periods.

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: Carbonated apatite (CHA) is commonly considered a promising synthetic material for biomedical applications in orthopedic and dental surgery due to its biocompatibility, bioresorption and bioactivity. CHA5, CHA37 and CHA90 powders were synthesized from wet method and the DRX patterns showed that the crystallinity and particle size of CHA samples increased proportionally with the synthesis temperature. Powder extracts medium were obtained from each sample to interact with MC3T3-E1 osteoblastics cells. It was evaluated morphology, citotoxicity, pH and Ca²⁺ concentration. Citotoxicity assays showed high metabolic activity on all samples when compared to control. The polygonal shaped and the confluent monolayer observed in control cells progressively changed according to the crystallinity increase of samples. Cells under mitosis and spindle-like shaped where the main alterations observed. In addition the cell viability could be sensitive to the acid reactivity and crystallinity of carbonated apatite samples.

Abstract: Cell adhesion, proliferation and differentiation are important specific parameters to be evaluated on biocompatibility studies of candidate biomaterials for clinical applications. Several different methodologies have been employed to study, both qualitative and quantitatively, the direct interactions of ceramic materials with cultured mammal and human cells. However, while quantitatively evaluating cell density, viability and metabolic responses to test materials, several methodological challenges may arise, either by impairing the use of some widely applied techniques, or by generating false or conflicting results. In this work, we tested the inherent interference of different representative calcium phosphate ceramic surfaces (stoichiometric dense and porous hydroxyapatite (HA) and cation-substituted apatite tablets) on different tests for quantitative evaluation of osteoblast adhesion and metabolism, either based on direct cell counting after trypsinization, colorimetric assays (XTT, Neutral Red and Crystal Violet) and fluorescence microscopy. Cell adhesion estimation after trypsinization was highly dependent on the time of treatment, and the group with the highest level of estimated adhesion was inverted from 5 to 20 minutes of exposition to trypsin. Both dense and porous HA samples presented high levels of background adsorption of the Crystal Violet dye, impairing cell detection. HA surfaces also were able to adsorb high levels of fluorescent dyes (DAPI and phalloidin-TRITC), generating backgrounds which, in the case of porous HA, impaired cell detection and counting by image processing software (Image Pro Plus 6.0). We conclude that the choice for the most suitable method for cell detection and estimation is highly dependent on very specific characteristics of the studied material, and methodological adaptations on well established protocols must always be carefully taken on consideration.

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.