Papers by Author: Elena Mavropoulos

Abstract: The knowledge on human tissue is very important to recognize desirable properties of biomaterials. Host cells, extracellular matrix, integrated vessels and interstitial fluids create a complex and dynamic system able to regenerate and respond to environmental stimuli. Myoglobin is a protein with most of α-helices on its secondary structure, and responsible for oxygen binding and release in muscles, by the heme group. This work investigates the Mb adsorption process onto zinc-hydroxyapatite (ZnHA) surface by spectroscopic studies. To do so, ZnHA (0.05 g) was incubated with 4mL of 2mg Mb/mL on phosphate buffer solution pH 6.0 for 24h at 37°C. The FTIR analyses of ZnHA powders before and after protein adsorption provided information concerning the protein content. UV-Vis spectrocopy in the reflectance mode suggested a mixture of MbO₂ and Met Mb on lyophilized solid Mb, and the prevalence of MetMb form when Mb was adsorbed on ZnHA sample. The decrease of UV-Vis secondary bands suggests interactions through the Mb heme group and the ZnHA surfaces. Circular Dichroism (CD) spectroscopy indicated the maintenance of the Mb α-helices secondary structure after the adsorption process on ZnHA powders.

Abstract: In this work, zinc-containing hydroxyapatite (ZnHA) and hydroxyapatite (HA) discs were coated with native bovine serum albumin (BSA) using 100 μg/ml BSA solution at 37°C. Changes on BSA secondary structure from α-helix to β-turn were detected by deconvolution analyses of FTIR amide I band. Fluorescence images of MC3T3-E1 osteoblast cultured on HA and ZnHA discs revealed cells with elongated morphology, typical of 2D experiments, after 24 hours. An intense cytoskeleton organization was observed on samples coated with BSA.

Abstract: The influence of surface chemistry, roughness and hidrophilicity on the protein adhesion onto the biomaterial modulates the cell attachment, proliferation and differentiation. β-TCP is a synthetic bone substitute with bioactive, osteoconductive properties. Insulin is a polypeptide hormone that acts as a growth-stimulating factor for some cells types in culture. Few studies have been discussing this hormone role in bone remodeling and cells metabolism in vitro. The aim of this work was to evaluate the cell responses when insulin is adsorbed on β-TCP commercial discs surfaces. FTIR detected the characteristics bands of insulin adsorbed on sintered powder surface. The insulin adsorption on discs surface increase its wettability and favored growth and differentiation of the pre-osteoblastics cells. Although no addition of differentiation factors was present, it was observed deposits of extracellular matrix and collagen at 7 days in culture. The increase on affinity and cellular activity to the adsorbed insulin β-TCP substract was remarkable. It was quite clear that the insulin presents a role on activate and speed up the bone cells response while adhered to β-TCP substract. However, the insulin application on bone grafts for clinical purposes must be considered after detailed studies on animal models, which are required to fully evaluate the safe use of hormone containing grafts in clinical trials.

Abstract: Synthetic hydroxyapatite (HA) is a widely used ceramic biomaterial due to its well described biocompatibility. Some modifications in HA surface can be made to increase surface porosity. Likewise, HA can be modified by the coating with proteins, which may impact on biocompatibility. In this work, we aimed to evaluate the impact of two surface modifications – coating with albumin, a major serum protein, and augmented porosity - over osteoblast adhesion on stoichiometric HA discs. Dense HA discs were obtained by pressing HA powder at 30 KN and sinterization at 1000°C, while porous HA was molded after the addition of alginate (15:1), followed by thermal treatment. Protein adsorption was attained by incubation on 0.5mg/mL bovine serum albumin (BSA) for 24 h at 37°C. MC3T3 mouse preosteoblasts were seeded over both protein-coated and uncoated dense or porous tablets, and cell viability after 24 h was estimated by XTT and Neutral Red assays. Cell density was quantified by fluorescence microscopy. While both dense and porous discs presented altered surfaces after protein treatment, as observed by scanning electron microscopy, porous HA tablets presented significantly higher levels of adsorbed protein. There was a decrease in the concentration of calcium ions in all samples analyzed. Porous HA treated with protein presented significant higher mitochondrial dehydrogenase activity (XTT) than non treated tablets (p<0.001). Although the BSA adsorption didn`t affect cell adhesion, the results obtained in fluorescence quantification suggests that de dense surface was best for cellular adhesion and spread than the porous one. We conclude that differences in the topography of a biomaterial can directly influence their ability to adsorb proteins, while the dense surface was more favorable for both the adhesion and the spreading of pre-osteoblasts.

Abstract: Hydroxiapatite (HA), one of the most widely employed bioceramic bone substitutes, when applied on its nanostructured form (nHA) may contribute to achieve a crystalline structure which is closer to the size and morphology of biological apatite. Furthermore, HA might also be doped with several different cations with biological effects including Sr2+. Therefore, a biomaterial based on nanostructured HA containing 1% Strontium (nSrHA) could present interesting biological properties, as strontium is described as a modulator of both osteoblast and osteoclast activities, presenting an important regulatory role on bone resorption. However, such modifications may also affect the biocompatibility of this material, which should be accessed initially by in vitro methods. Therefore, the present work aimed to evaluate the in vitro biocompatibility of 1% nSrHA discs with human primary osteoblasts through a multiparametric assay which assesses simultaneously metabolic activity (XTT assay), membrane integrity (NR test) and cell density (CVDE). Extracts of nSrHA, latex fragments (positive control), polystyrene beads (negative control) and nHA (for comparison) were prepared and exposed to 104 cells for 24h at 37°C/5% CO₂ on test plates, according to ISO 10993-5:2009, on quintuplicates. Cells exposed to unconditioned media were used as experimental control. After exposure, cells were tested for viability with a commercial multiparametric kit (In Cytotox, Xenometrix, Germany). The positive and negative controls presented the expected results, validating the assay. Both nHA and SrnHA were considered biocompatible, since the presented a cell viability after exposure statistically similar to the experimental control. In conclusion, the synthesized nSrHA discs are cytocompatible and, consequently, adequate for further in vitro tests on cell adhesion, proliferation and differentiation.

Abstract: 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.

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: Polymeric sponges coated with hydroxyapatite were used in lead and cadmium uptake experiments in order to mimetizes heavy metal incorporation by bone tissue. The hydroxyapatite-coated sponges (S-HA) were obtained by hydrothermal alkali conversion from monetite. The results showed that S-HA were efficient in immobilizing Pb and Cd. The uptake experiments using hydroxyapatite powder (HA) removed almost 100% of Pb2+ and 59% of Cd2+. When polymeric sponges coated with hydroxyapatite were used, the removal of Cd2+ and Pb2+ decreased, respectively, to closely 14 % and 74%, after 24 hours sorption. Before and after sorption experiment MEV analysis indicated precipitation of pyromorphite on hydroxyapatite-coated sponges.

Abstract: 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.