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- Electrochemical characterization of cobalt-based alloys using the mini-cell systemPublication . Nascimento, M. Lúcia; Mueller, Wolf-Dieter; Carvalho, Ana C.; Tomás, HelenaThe aim of this work was to investigate the electrochemical behavior of cobaltbased alloys of different compositions using the mini-cell system (MCS) and to test the sensitivity of this technique in the detection of variations in the electrochemical behavior caused by the different compositions of cobalt-based alloys. Methods. The electrochemical measurements were performed on two cobalt-based alloys, having a small content of gold (B¨arlight and Gold Core); one cobalt–platinum–ruthenium alloy (Porta Smart) and one classical Co-based alloy (Wironit), in two different electrolytes, 1% NaCl and phosphate buffered solution (PBS). Results. Based on I versus E curves, enhanced pitting corrosion capability was observed for the cobalt-based alloys, especially for those with lower chromium content and with the addition of a small amount of gold. The alloying of Co with Pt and Ru (Porta Smart), and the standard Co-based alloy do not show any critical instability; in contrast, a small addition of Au enhanced the pitting corrosion activity and reduced the corrosion stability. For higher alloy stability, a minimum 30% atomic ratio of chromium in the alloy composition is necessary. Furthermore, it was shown that MCS has sensitivity for the qualitative comparison of the alloys as well as in the electrochemical characterization of each alloy. Significance. Electrochemical measurements are essential to assess the quality of an alloy. MCSmighthelpinunderstandingtheroletheelementsplayintheelectrochemicalbehavior of the alloys and at the same time contribute to the selection of the alloys in terms of their quality,evenbeforemorecomplextestsinvitroorinvivoareapplied,andmightreducethe costs for materials research.
- Electrochemical characterization of titanium biomaterials using the Mini-cell SystemPublication . Nascimento, M. L.; Mueller, W. D.; Carvalho, A. C.; Tomás, H. M.The aim of this work was the electrochemical characterization of modified Titanium surfaces for implants with help of the Mini-cell System (MCS). The materials tested were Cp titanium specimens, designed for animal experiments, with 8 mm length and 4 mm diameter, with different surface conditions: one had machined surface, the other was blasted with a bioceramic (GB14+AP40) and the third was blasted with aluminum oxide. Roughness measurements were also made on the materials. The results showed that MCS has sensitivity to detect differences in the electrochemical point of view due to surface modifications. The differences between the materials are small, but systematic. The aluminum oxide blasted material had the highest current density, however due to the roughness increase. The material with bioceramics blasting had the highest chemical activity. The higher activity of the bioceramics treated surface was explained as (i) an interaction between the modified surface and electrolyte; or (ii) the calcium phosphate dissolves in the electrolyte and leaves the surface of the metal, and then the free metal reacts immediately to form new titanium oxide. These results may help to explain the biological effects observed by modified surfaces, in particular with bioceramics.
- Osteoblastic behavior of human bone marrow cells cultured over adsorbed collagen layer, over surface of collagen gels, and inside collagen gelsPublication . Fernandes, Luís F.; Costa, Maria A.; Fernandes, Maria H.; Tomás, HelenaWhile collagen type I is often used as a substrate for cell culturing and as a coating in biomedical implants, as far as we know a simple systematic study comparing the effects of the different presentations of collagen type I on the osteoblastic behavior of cells is missing. In this work, human bone marrow cells (hBMCs) were cultured under osteoblastic-inducing conditions, for 21 days, over a layer of adsorbed collagen (monomeric) and on the surface and inside collagen gels (fibrillar). Comparison was made based on three classical parameters; cell proliferation/viability, alkaline phosphatase (ALP) activity, and production of mineral deposits. The three types of collagen type I substrates allowed the adhesion, proliferation, and the osteoblastic differentiation of cells. However, hBMCs behavior was influenced by the monomeric/fibrillar and 2-/3-dimensional nature of the collagen substrates, namely: monomeric collagen favored cell attachment; cells on 2D substrates presented higher proliferation rates during the exponential phase of growth with formation of spiral-like multilayered structures; cells seeded inside 3D collagen gels formed a regular dense cellular mesh and had a low proliferating rate; cells cultured over or inside fibrillar collagen differentiated faster, with the 3D cultures presenting higher levels of ALP activity; and the extension of mineralization was greater for the cultures done over or inside fibrillar collagen. Thus, cells cultured over collagen gels showed both the ability for cell proliferation and for earlier differentiation, a fact that can be exploited in the biomaterials field.
- Guanidinoacetate methyltransferase deficiency identified in adults and a child with mental retardationPublication . Araújo, H. Caldeira; Smit, W.; Verhoeven, N.M.; Salomons, G.S.; Silva, S.; Vasconcelos, R.; Tomás, H.; Almeida, I. Tavares de; Jakobs, C.; Duran, M.Our study describes the adult clinical and biochemical spectrum of guanidinoacetate methyltransferase (GAMT) deficiency, a recently discovered inborn error of metabolism. The majority of the previous reports dealt with pediatric patients, in contrast to the present study. A total of 180 institutionalized patients with a severe mental handicap were investigated for urine and plasma uric acid and creatinine. Patients with an increased urinary uric acid/creatinine ratio and/or decreased creatinine were subjected to the analysis of guanidinoacetate (GAA). Four patients (three related and one from an unrelated family) were identified with GAMT-deficiency. A fifth patient had died before a biochemical diagnosis could be made. They all had shown a normal psychomotor development for the first year of life, after which they developed a profound mental retardation. Three out of four had convulsions and all four totally lacked the development of speech. Their GAMT activity in lymphoblasts was impaired and two novel mutations were identified: the 59 G > C and 506 G > A missense mutations. Urinary GAA was increased, but highly variable 347-1,624 mmol/mol creat (Controls <150 mmol/mol creat). In plasma and CSF the GAA levels were fairly constant at 17.3-27.0 mumol/L (Controls 1.33-3.33) and 11.0-12.4 mumol/L, respectively (Controls 0.068-0.114). GAMT deficiency in adults is associated with severe mental retardation and absence or limited speech development. Convulsions may be prominent. The nonspecific nature of the clinical findings as well as the limited availability of GAA assays and/or in vivo magnetic resonance spectroscopy of the brain may mean that many more patients remain undiagnosed in institutions for persons with mental handicaps.
- Osteogenic differentiation of mesenchymal stem cells using PAMAM dendrimers as gene delivery vectorsPublication . Santos, José Luís; Oramas, Elena; Pêgo, Ana Paula; Granja, Pedro Lopes; Tomás, HelenaThis paper reports the use of different generations of polyamidoamine (PAMAM) dendrimers for the in vitro transfection of mesenchymal stem cells (MSCs). A systematic study was carried out on the transfection efficiency achieved by the PAMAM dendrimers using a beta-galactosidase reporter gene system. Transfection results were shown to be dependent upon the generation of dendrimers, the amine to phosphate group ratio and the cell passage number. In all cases, the transfection efficiency was very low. Nevertheless, it was hypothesized that a low transfection level could be sufficient to promote the in vitro differentiation of MSCs towards the osteoblastic lineage. To address this possibility, dendrimers carrying the human bone morphogenetic protein-2 (hBMP-2) gene-containing plasmid were used. All quantitative (alkaline phosphatase activity, osteocalcin secretion and calcium deposition) and qualitative (von Kossa staining) osteogenic markers were significantly stronger in transfected cells when compared to non-transfected ones. This study not only clearly demonstrates that a low transfection level can be sufficient for inducing in vitro differentiation of MSCs to the osteoblast phenotype but also highlights the importance of focusing research on the development of gene delivery vectors in the concrete application.
- Separation of poly(amidoamine) (PAMAM) dendrimer generations by dynamic coating capillary electrophoresisPublication . Sedláková, P.; Svobodová, J.; Mikšík, I.; Tomás, H.The separation of compounds possessing amino groups (peptides, proteins, polyamino compounds) by capillary zone electrophoresis suffers from the interaction (sticking) of these solutes with the capillary wall. This sticking can result in the absence or incomplete separation of compounds or even in their retention in the capillary. Polyamidoamine (PAMAM) dendrimers are a class of spherical polymers with primary amino groups at the surface. These compounds can be separated reasonably well at acidic pH but not at neutral pH. A new method based on the dynamic coating of the capillary was developed for the separation of these compounds at pH 7.4. The method comprises separation in a fused-silica capillary (57 cm total length, 50 cm to the detector, ID 75 microm) and a background electrolyte consisting of a Tris-phosphate buffer (50 mmol/L, pH 7.4) and 0.05% (w/v) polyethyleneimine. This system is suitable for the separation of 7 generations of dendrimers (generations 0-6). The dynamic coating agent (polyethyleneimine) also improves the separation at acid pH.
- Bioinspired superhydrophobic poly(L-lactic acid) surfaces control bone marrow derived cells adhesion and proliferationPublication . Alves, Natália M.; Shi, Jun; Oramas, Elena; Santos, José L.; Tomás, Helena; Mano, João F.The aptitude of a cell to adhere, migrate, and differentiate on a compact substrate or scaffold is important in the field of tissue engineering and biomaterials. It is well known that cell behavior can be controlled and guided through the change in micro- and nano-scale topographic features. In this work, we intend to demonstrate that special topographic features that control wettability may also have an important role in the biological performance of biodegradable substrates. Poly(L-lactic acid) surfaces with superhydrophobic characteristics were produced, based on the so-called Lotus effect, exhibiting dual micro- and nano-scale roughness. The water contact angle could be higher than 150 degrees and a value of that order could be kept even upon immersion in a simulated body fluid solution for more than 20 days. Such water repellent surfaces were found to prevent adhesion and proliferation of bone marrow derived cells previously isolated from the femurs of 6-week-old male Wistar rats, when compared with smoother surfaces prepared by simple solvent casting. Such results demonstrate that these superhydrophobic surfaces may be used to control cell behavior onto biodegradable substrates.