Mário A. Barbosa

Chemical modification of chitosan by phosphorylation: an XPS, FT-IR and SEM study

In the present work, the surface of chitosan membranes was modified using a phosphorylation method carried out at room temperature. Phosphorylation may be of particular interest in materials for orthopaedic applications, due to the cation-exchange properties of phosphate functionalities. Phosphate groups chelate calcium ions, thus inducing the deposition of an apatite-like layer known to improve the osteoconduction of polymer-based implants. Additionally, the negatively charged phosphate functionalities, together with the positively charged amine groups from chitosan, are expected to provide chitosan with an amphoteric character, which may be useful as a combinatorial therapeutic strategy, by simultaneously allowing the immobilization of signalling molecules like growth factors. Phosphorylation was carried out at room temperature using the H3PO4/Et3PO4/P2O5/butanol method. Surface characterization was performed by XPS, ATR–FT-IR, and SEM. Cross-sections were analyzed by SEM fitted with EDS. The phosphate content increased with the reaction time, as shown by XPS and ATR–FT-IR, a P/N atomic ratio of 0.73 being obtained after 48 h of treatment. High-resolution XPS spectra regarding C1s, O1s, N1s and P2p are discussed. The introduction of a neutralization step led to a reduction of P content, which pointed out to the presence of phosphates ionically bound to protonated amines, in addition to phosphate esters. EDS analysis of cross-sections revealed a gradual P reduction up to 50% towards the inner part of the membrane.

Corrosion behaviour of commercially pure titanium shot blasted with different materials and sizes of shot particles for dental implant applications

It is well known that the osseointegration of the commercially pure titanium (c.p. Ti) dental implant is improved when the metal is shot blasted in order to increase its surface roughness. This roughness is colonised by bone, which improves implant fixation. However, shot blasting also changes the chemical composition of the implant surface because some shot particles remain adhered on the metal. The c.p. Ti surfaces shot blasted with different materials and sizes of shot particles were tested in order to determine their topographical features (surface roughness, real surface area and the percentage of surface covered by the adhered shot particles) and electrochemical behaviour (open circuit potential, electrochemical impedance spectroscopy and cyclic polarisation). The results demonstrate that the increased surface area of the material because of the increasing surface roughness is not the only cause for differences found in the electrochemical behaviour and corrosion resistance of the blasted c.p. Ti. Among other possible causes, those differences may be attributed to the compressive residual surface stresses induced by shot blasting. All the materials tested have an adequate corrosion and electrochemical behaviour in terms of its possible use as dental implant material.

The effect of the co-immobilization of human osteoprogenitors and endothelial cells within alginate microspheres on mineralization in a bone defect.

Bone regeneration seems to be dependant on cell communication between osteogenic and endothelial cells arising from surrounding blood vessels. This study aims to determine whether endothelial cells can regulate the osteogenic potential of osteoprogenitor cells in vitro and in vivo, in a long bone defect, when co-immobilized in alginate microspheres. Alginate is a natural polymer widely used as a biomaterial for cell encapsulation. Human osteoprogenitors (HOP) from bone marrow mesenchymal stem cells were immobilized alone or together with human umbilical vein endothelial cells (HUVEC) inside irradiated, oxidized and RGD-grafted alginate microspheres. Immobilized cells were cultured in dynamic conditions and cell metabolic activity increased during three weeks. The gene expression of alkaline phosphatase and osteocalcin, both specific markers of the osteoblastic phenotype, and mineralization deposits were upregulated in co-immobilized HOPs and HUVECs, comparing to the immobilization of monocultures. VEGF secretion was also increased when HOPs were co-immobilized with HUVECs. Microspheres containing co-cultures were further implanted in a bone defect and bone formation was analysed by muCT and histology at 3 and 6 weeks post-implantation. Mineralization was observed inside and around the implanted microspheres containing the immobilized cells. However, when HOPs were co-immobilized with HUVECs, mineralization significantly increased. These findings demonstrate that co-immobilization of osteogenic and endothelial cells within RGD-grafted alginate microspheres provides a promising strategy for bone tissue engineering.

Corrosion behaviour of titanium in biofluids containing H2O2 studied by electrochemical impedance spectroscopy

Abstract This work aims at studying the electrochemical behaviour of titanium in the presence of an artificial biofluid containing H 2 O 2 , mimicing the situation, where the metal is implanted in the human body and hydrogen peroxide is generated by an inflammatory reaction. A phosphate buffered saline (PBS) solution and two PBS/H 2 O 2 solutions containing 50 and 150 mM of H 2 O 2 were used to simulate the body fluids. The behaviour of the metal was monitored as a function of time by electrochemical impedance spectroscopy (EIS) for three weeks. After one week, the PBS/H 2 O 2 solutions were replaced by fresh PBS solutions in order to simulate the end of the inflammatory process and recovery of the system. All the experiments were carried out at a constant temperature of 37°C. From the simulation of the experimental EIS spectra, it was concluded that the corrosion resistance of titanium is strongly affected by the presence of H 2 O 2 and when the peroxide is removed, the metal displays a sharp resistance increase. Furthermore, the oxides formed in H 2 O 2 are rougher and display higher ionic conductivities than the oxides formed in the absence of peroxide. The study was complemented with potentiostatic experiments and scanning electron microscopy observation of the metal surfaces.

The correlation between the adsorption of adhesive proteins and cell behaviour on hydroxyl-methyl mixed self-assembled monolayers

The objective of this study was to compare the biological effects of two key cell-adhesive proteins, fibronectin (FN) and vitronectin (VN), upon adsorption onto molecularly-designed model surfaces. Single-component and mixed self-assembled monolayers (SAMs) of alkanethiols on gold with OH and CH(3) terminal groups were prepared at 100%, 65%, 36% and 0% of OH at the surface, to generate a range of surfaces with a simple chemistry and a wettability gradient. FN and VN were adsorbed under non-competitive (single-protein solutions) and competitive (multi-protein solutions) conditions, and compared at different levels: adsorbed amount (radiolabelling), elution, functional presentation of cell-binding domains (ELISA), and role in mediating cell adhesion (antibody-based assay). The observed trends were related to mesenchymal stem cell response in terms of adhesion and overall cell morphology. Under non-competitive conditions, adsorption of both proteins increased with surface hydrophobicity. The presence of competitive proteins significantly decreased the adsorbed amounts, although both proteins were still detected in all SAMs. Adsorption of FN followed a trend similar to that of non-competitive conditions, while adsorption of VN was higher on 100%OH-SAMs. Concerning elution, retention of adsorbed VN was always higher than that of FN. For both proteins, functional presentation of cell-binding domains was more effective on the more hydrophilic 100%OH-SAMs. This fact, coupled to the ability of this type of SAMs to selectively recruit and retain VN in the presence of competitive serum proteins, seems to correlate with the better cell response observed on these surfaces, as compared with hydrophobic 0%OH(100%CH(3))-SAMs.

Pectin-Based Injectable Biomaterials for Bone Tissue Engineering

A variety of natural polymers and proteins are considered to be 3D cell culture structures able to mimic the extracellular matrix (ECM) to promote bone tissue regeneration. Pectin, a natural polysaccharide extracted from the plant cell walls and having a chemical structure similar to alginate, provides interesting properties as artificial ECM. In this work, for the first time, pectin, modified with an RGD-containing oligopeptide or not, is used as an ECM alternative to immobilize cells for bone tissue regeneration. The viability, metabolic activity, morphology, and osteogenic differentiation of immobilized MC3T3-E1 preosteoblats demonstrate the potential of this polysaccharide to keep immobilized cells viable and differentiating. Preosteoblasts immobilized in both types of pectin microspheres maintained a constant viability up to 29 days and were able to differentiate. The grafting of the RGD peptide on pectin backbone induced improved cell adhesion and proliferation within the microspheres. Furthermore, not only did cells grow inside but also they were able to spread out from the microspheres and to organize themselves in 3D structures producing a mineralized extracellular matrix. These promising results suggest that pectin can be proposed as an injectable cell vehicle for bone tissue regeneration.

Immobilization of Human Mesenchymal Stem Cells within RGD-Grafted Alginate Microspheres and Assessment of Their Angiogenic Potential

In this work, human mesenchymal stem cells (hMSC) immobilized in RGD-coupled alginate microspheres, with a binary composition of high and low molecular weight alginate, were investigated. Cells immobilized within RGD-alginate microspheres (during 21 days) showed metabolic activity, with an overall viability higher than 90%, short cell extensions, and, when induced, they were able to differentiate into the osteogenic lineage. In osteogenic conditions (comparing to basal conditions), immobilized cells presented alkaline phosphatase (ALP) activity and an upregulation of ALP, collagen type I, and Runx 2 expression. Moreover, mineralization was also detected in immobilized cells under osteogenic stimulus. In addition, it was demonstrated for the first time that MSCs immobilized in this 3D matrix were able to enhance the ability of neighboring endothelial cells to form tubelike structures. Overall, these findings represent a step forward in the development of injectable stem cell carriers for bone tissue engineering.

Apatite deposition on titanium surfaces — the role of albumin adsorption

Titanium implant surfaces are known to spontaneously nucleate apatite layers when in contact with simulated body fluids. However, adsorption of proteins may influence the process of apatite layer formation. In this study the role of bovine serum albumin (BSA) adsorption in the process of apatite deposition on titanium substrates is investigated. Deposition of calcium phosphate was induced by immersing titanium substrates in a Hanks balanced salt solution (HBSS) for times ranging from 1 to 23 days. The resulting substrates were studied by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS), wettability measurements and electrochemical impedance determinations. All these methods indicate the presence of a calcium phosphate layer. The same procedure was repeated substituting HBSS with a solution of BSA in HBSS. Although SEM, EDS and electrochemical impedance spectra do not reveal the presence of an apatite layer, XPS analysis strongly indicates that the inhibition of apatite formation by BSA is only partial. The competition between BSA adsorption and apatite deposition seems to lead to a mixed film where the protein co-exists with calcium phosphate. Wettability studies suggest that this surface film is heterogeneous and porous, similar to the thicker films formed in albumin-free HBSS.

Effect of hydroxyapatite thickness on metal ion release from Ti6Al4V substrates

The electrochemical dissolution behaviour of Ti6Al4V alloy coated with hydroxyapatite (HA) by plasma spraying was studied in Hanks balanced salt solution (HBSS) and compared with that of polished and grit-blasted passivated surfaces. Two different nominal thicknesses of HA (50 and 200 micro m) were used. Taking a polished passivated surface as reference, grit blasting of the substrate increased the electrical charge used in the oxidation of Ti6Al4V alloy at constant potential, as a result of increased surface area. However, only HA coatings with a thickness of 200 micro m were capable of reducing the charge to values lower than those measured for polished surfaces. Electrochemical impedance spectroscopy has also shown that only 200 micro m thick coatings are effective in reducing the oxidation rate of the substrate. Furthermore, in potentiostatic experiments the 50 micro m thick coating detached from the substrate, which did not occur with the 200 micro m thick coating. However, after 6 months immersion in HBSS, detachment occurred in some regions of both coatings. No titanium, aluminium or vanadium were detected in solution by electrothermal atomic absorption spectroscopy. These data indicate that HA is an effective barrier to metal ion release, even for the thinner coatings, due to formation of metal phosphates or to incorporation of metal ions in the HA structure.

Improving chitosan-mediated gene transfer by the introduction of intracellular buffering moieties into the chitosan backbone

Chitosan was functionalized with imidazole moieties (CHimi) with the aim of improving its buffering capacity and promoting the endosomal escape ability of chitosan-DNA complexes, ultimately increasing their transfection efficiency. 5.6%, 12.9% and 22.1% of the glucosamine residues of chitosan were substituted. Complexes with different molar ratios of primary amines to DNA phosphate anion (N/P) were prepared by a coacervation method. For an N/P>3, CHimi polymers are able to complex electrostatically with DNA and condense it into positively charged nanostructures (average size 260 nm and zeta potential +16 mV at pH 5.5). In the concentration range 2.5-100 microg ml(-1), the modified polymers had no cytotoxic effect on 293T cells. CHimi polymers with the highest degree of substitution were found to enhance beta-gal expression in 293T and HepG2 cells. Bafilomycin A1 inhibited transfection, indicating that the protonation of the imidazole groups in the endolysosome pathway favors the escape of the complexes from the endosomes, increasing the amount of transgene that can reach the cell nucleus.