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Keridou, I., Cailloux, J., Martínez, J. C., Santana, O., Maspoch, M. L., Puiggalí, J., Franco, L., (2020). Biphasic polylactide/polyamide 6,10 blends: Influence of composition on polyamide structure and polyester crystallization Polymer 202, 122676

Blends with different ratios of polylactide and polyamide 6,10 (PA610) have been prepared by melt-mixing using a Brabender mixer equipment. Previously, a rheologically modified polylactide (PLAREx) was obtained through reactive extrusion using a multifunctional epoxide agent. It was expected that unreacted epoxy groups of PLAREx were able to improve the compatibility between the two polymers. SEM observations revealed a logical dependence of the morphology of immiscible phases with composition, and more interestingly a co-continuity at relatively low PA content (around 50%) was detected. This result contrasts with previous observations performed with non-modified PLA. Confined PA domains increased with the PA content and hardly crystallized at the typical crystallization temperature of the pure PA (195 °C). Synchrotron X-ray diffraction studies indicated that a PA crystallization at a lower temperature close to 120 °C was enhanced and led to a pseudohexagonal γ phase that differs from the characteristic layered structure of PA610. SAXS data revealed also that well differentiated lamellar entities could be assigned at both immiscible polymer phases. Clear differences were observed in the spherulitic morphologies attained under isothermal melt crystallization experiments. Results indicated that the texture of PLAREx spherulites was modified by the presence of PA. Compatibilization of PA molecules on the crystal lamellar boundaries of PLAREx led to an enhancement of the lamellar twisting frequency. Optical microscopy results also indicated that the crystal growth rate of PLAREx increased by the incorporation of PA, but in contrast this had an adverse effect on the nucleation process.

Keywords: Crystal growth rate, Epoxy modified polylactide, Nucleation, Polyamide 6,10, Polyamide crystalline structure, Polyamide/polylactide blend morphology, Thermal properties

Rodríguez-Contreras, A., Torres, D., Guillem-Marti, J., Sereno, P., Ginebra, M. P., Calero, J. A., Manero, J. M., Rupérez, E., (2020). Development of novel dual-action coatings with osteoinductive and antibacterial properties for 3D-printed titanium implants Surface and Coatings Technology 403, 126381

Gallium (Ga) has been recently proposed as a novel therapeutic agent, since it promotes bone formation and exhibits antibacterial properties. This work focuses on the optimization of a thermochemical treatment that incorporates Ga ions by the addition of the body-friendly Ga nitrate approved by the Food and Drug Administration. The objective was to simultaneously provide the inner and the outer surfaces of porous‑titanium surfaces obtained by 3D-printing with bioactivity and antibacterial properties. The apatite-forming ability of the coating, as well as the antibacterial activity and SaOS-2 cell adhesion, proliferation, differentiation and mineralization were evaluated and compared with untreated Ti surfaces. The characterization of the surfaces revealed the presence of a Ga-containing calcium titanate layer, which was non cytotoxic and in simulated body fluid produced a homogeneous apatite coating well adhered to the substrate. The formation of this apatite layer was accelerated with increasing Ga amounts present on the surface, resulting also in an increase in thickness. An initial quick release of Ga ion promoted the antibacterial effect against gram positive strains, especially for Pseudomonas aeruginosa, one of the most frequent resistant pathogens in nosocomial infections. SaOS-2 cells adhered and proliferated on the Ga-doped Ti surfaces, its presence contributed to cell differentiation and to considerably increase the mineralization levels. Thus, the developed multifunctional coatings could provide bioactivity to the porous Ti implants while protecting them from the most frequent gram-negative pathogens.

Keywords: 3D-printing, Antibacterial activity, Biomaterials, Gallium, Porous structures, Titanium implants

Minguela, J., Slawik, S., Mücklich, F., Ginebra, M. P., Llanes, L., Mas-Moruno, C., Roa, J. J., (2020). Evolution of microstructure and residual stresses in gradually ground/polished 3Y-TZP Journal of the European Ceramic Society 40, (4), 1582-1591

A comprehensive study of progressively ground/polished 3Y-TZP was performed with the aim of better understanding the mechanisms driving the microstructural modifications observed after such procedures, and identifying the processing parameters leading to optimal microstructures (i.e. ageing-protective and damage-free). Gradually ground/polished surfaces were produced, yielding four different topographies of increasing roughness (grades 1–4) and two different textures (unidirectionally, U, and multidirectionally, M). Phase transformation, microstructure and residual stresses were investigated by means of advanced characterization techniques. It was found that low-roughness mildly ground/polished specimens (i.e. 2-M/U) presented a nanometric layer with the ageing-related protective features generally associated with coarsely ground specimens. A lower limit for grain refinement in terms of surface abrasion was also found, in which partial recrystallization took place (i.e. 1-M/U). A mathematical relation was established between average surface roughness (Sa), monoclinic volume fraction (Vm) and surface compressive residual stresses, demonstrating that if the processing parameters are controlled, both Vm and residual stresses can be predicted by the measurement of Sa.

Keywords: Grinding, Microstructure, Phase transformation, Residual stresses, Zirconia

Riccobelli, D., Noselli, G., Arroyo, M., DeSimone, A., (2020). Mechanics of axisymmetric sheets of interlocking and slidable rods Journal of the Mechanics and Physics of Solids 141, 103969

In this work, we study the mechanics of metamaterial sheets inspired by the pellicle of Euglenids. They are composed of interlocking elastic rods which can freely slide along their edges. We characterize the kinematics and the mechanics of these structures using the special Cosserat theory of rods and by assuming axisymmetric deformations of the tubular assembly. Through an asymptotic expansion, we investigate both structures that comprise a discrete number of rods and the limit case of a sheet composed by infinitely many rods. We apply our theoretical framework to investigate the stability of these structures in the presence of an axial load. Through a linear analysis, we compute the critical buckling force for both the discrete and the continuous case. For the latter, we also perform a numerical post-buckling analysis, studying the non-linear evolution of the bifurcation through finite elements simulations.

Keywords: Biomimetic structures, Elastic structures, Helical rods, Mechanical instabilities, Metamaterials, Post-buckling analysis

Barba, A., Diez-Escudero, A., Espanol, M., Bonany, M., Sadowska, J. M., Guillem-Marti, J., Öhman-Mägi, C., Persson, C., Manzanares, M. C., Franch, J., Ginebra, M. P., (2019). Impact of biomimicry in the design of osteoinductive bone substitutes: Nanoscale matters ACS Applied Materials and Interfaces 11, (9), 8818-8830

Bone apatite consists of carbonated calcium-deficient hydroxyapatite (CDHA) nanocrystals. Biomimetic routes allow fabricating synthetic bone grafts that mimic biological apatite. In this work, we explored the role of two distinctive features of biomimetic apatites, namely, nanocrystal morphology (plate vs needle-like crystals) and carbonate content, on the bone regeneration potential of CDHA scaffolds in an in vivo canine model. Both ectopic bone formation and scaffold degradation were drastically affected by the nanocrystal morphology after intramuscular implantation. Fine-CDHA foams with needle-like nanocrystals, comparable in size to bone mineral, showed a markedly higher osteoinductive potential and a superior degradation than chemically identical coarse-CDHA foams with larger plate-shaped crystals. These findings correlated well with the superior bone-healing capacity showed by the fine-CDHA scaffolds when implanted intraosseously. Moreover, carbonate doping of CDHA, which resulted in small plate-shaped nanocrystals, accelerated both the intrinsic osteoinduction and the bone healing capacity, and significantly increased the cell-mediated resorption. These results suggest that tuning the chemical composition and the nanostructural features may allow the material to enter the physiological bone remodeling cycle, promoting a tight synchronization between scaffold degradation and bone formation.

Keywords: Biomimetic, Calcium phosphate, Carbonated apatite, Foaming, Nanostructure, Osteogenesis, Osteoinduction

Roki, N., Tsinas, Z., Solomon, M., Bowers, J., Getts, R. C., Muro, S., (2019). Unprecedently high targeting specificity toward lung ICAM-1 using 3DNA nanocarriers Journal of Controlled Release 305, 41-49

DNA nanostructures hold great potential for drug delivery. However, their specific targeting is often compromised by recognition by scavenger receptors involved in clearance. In our previous study in cell culture, we showed targeting specificity of a 180 nm, 4-layer DNA-built nanocarrier called 3DNA coupled with antibodies against intercellular adhesion molecule-1 (ICAM-1), a glycoprotein overexpressed in the lungs in many diseases. Here, we examined the biodistribution of various 3DNA formulations in mice. A formulation consisted of 3DNA whose outer-layer arms were hybridized to secondary antibody-oligonucleotide conjugates. Anchoring IgG on this formulation reduced circulation and kidney accumulation vs. non-anchored IgG, while increasing liver and spleen clearance, as expected for a nanocarrier. Anchoring anti-ICAM changed the biodistribution of this antibody similarly, yet this formulation specifically accumulated in the lungs, the main ICAM-1 target. Since lung targeting was modest (2-fold specificity index over IgG formulation), we pursued a second preparation involving direct hybridization of primary antibody-oligonucleotide conjugates to 3DNA. This formulation had prolonged stability in serum and showed a dramatic increase in lung distribution: the specificity index was 424-fold above a matching IgG formulation, 144-fold more specific than observed for PLGA nanoparticles of similar size, polydispersity, ζ-potential and antibody valency, and its lung accumulation increased with the number of anti-ICAM molecules per particle. Immunohistochemistry showed that anti-ICAM and 3DNA components colocalized in the lungs, specifically associating with endothelial markers, without apparent histological changes. The degree of in vivo targeting for anti-ICAM/3DNA-nanocarriers is unprecedented, for which this platform technology holds great potential to develop future therapeutic applications.

Keywords: 3DNA, DNA nanostructure, Drug nanocarrier, Endothelial and lung targeting, ICAM-1, In vivo biodistribution

Noselli, G., Arroyo, M., DeSimone, A., (2019). Smart helical structures inspired by the pellicle of euglenids Journal of the Mechanics and Physics of Solids 123, 234-246

This paper deals with a concept for a reconfigurable structure bio-inspired by the cell wall architecture of euglenids, a family of unicellular protists, and based on the relative sliding of adjacent strips. Uniform sliding turns a cylinder resulting from the assembly of straight and parallel strips into a cylinder of smaller height and larger radius, in which the strips are deformed into a family of parallel helices. We examine the mechanics of this cylindrical assembly, in which the interlocking strips are allowed to slide freely at their junctions, and compute the external forces (axial force and axial torque at the two ends, or pressure on the lateral surface) necessary to drive and control the shape changes of the composite structure. Despite the simplicity of the structure, we find a remarkably complex mechanical behaviour that can be tuned by the spontaneous curvature or twist of the strips.

Keywords: Bio-inspired structures, Euglenoid pellicle, Helical bundles, Morphing structures, Reconfigurable structures

Blithikioti, C., Miquel, L., Batalla, A., Rubio, B., Maffei, G., Herreros, I., Gual, A., Verschure, P., Balcells-Oliveró, M., (2019). Cerebellar alterations in cannabis users: A systematic review Addiction Biology 24, (6), 1121-1137

Cannabis is the most used illicit substance in the world. As many countries are moving towards decriminalization, it is crucial to determine whether and how cannabis use affects human brain and behavior. The role of the cerebellum in cognition, emotion, learning, and addiction is increasingly recognized. Because of its high density in CB1 receptors, it is expected to be highly affected by cannabis use. The aim of this systematic review is to investigate how cannabis use affects cerebellar structure and function, as well as cerebellar-dependent behavioral tasks. Three databases were searched for peer-reviewed literature published until March 2018. We included studies that focused on cannabis effects on cerebellar structure, function, or cerebellar-dependent behavioral tasks. A total of 348 unique records were screened, and 40 studies were included in the qualitative synthesis. The most consistent findings include (1) increases in cerebellar gray matter volume after chronic cannabis use, (2) alteration of cerebellar resting state activity after acute or chronic use, and (3) deficits in memory, decision making, and associative learning. Age of onset and higher exposure to cannabis use were frequently associated with increased cannabis-induced alterations. Chronic cannabis use is associated with alterations in cerebellar structure and function, as well as with deficits in behavioral paradigms that involve the cerebellum (eg, eyeblink conditioning, memory, and decision making). Future studies should consider tobacco as confounding factor and use standardized methods for assessing cannabis use. Paradigms exploring the functional activity of the cerebellum may prove useful as monitoring tools of cannabis-induced impairment.

Keywords: Behavior, Cannabis use, Cerebellum, Cognitive function, Structure

Samitier, Josep, Correia, A., (2019). Biomimetic Nanotechnology for Biomedical Applications (NanoBio&Med 2018) Biomimetics MDPI

Emerging nanobiotechnologies can offer solutions to the current and future challenges in medicine. By covering topics from regenerative medicine, tissue engineering, drug delivery, bionanofabrication, and molecular biorecognition, this Special Issue aims to provide an update on the trends in nanomedicine and drug delivery using biomimetic approaches, and the development of novel biologically inspired devices for the safe and effective diagnosis, prevention, and treatment of disease.

Keywords: Bioinspired nanotechnologies, Bionanofabrication, Bio-nano measurement and microscopy, Nanomaterials for biological and medical applications, Nanoassemblies, Nanostructured surfaces, Drug delivery, Nanobioelectronics, Integrated systems/nanobiosensors, Nanotoxicology, Graphene-based applications

Sebastian, P., Giannotti, M. I., Gómez, E., Feliu, J. M., (2018). Surface sensitive nickel electrodeposition in deep eutectic solvent ACS Applied Energy Materials , 1, (3), 1016-1028

The first steps of nickel electrodeposition in a deep eutectic solvent (DES) are analyzed in detail. Several substrates from glassy carbon to Pt(111) were investigated pointing out the surface sensitivity of the nucleation and growth mechanism. For that, cyclic voltammetry and chronoamperometry, in combination with scanning electron microscopy (SEM), were employed. X-ray diffraction (XRD) and atomic force microscopy (AFM) were used to more deeply analyze the Ni deposition on Pt substrates. In a 0.1 M NiCl2 + DES solution (at 70 °C), the nickel deposition on glassy carbon takes place within the potential limits of the electrode in the blank solution. Although, the electrochemical window of Pt|DES is considerably shorter than on glassy carbon|DES, it was still sufficient for the nickel deposition. On the Pt electrode, the negative potential limit was enlarged while the nickel deposit grew, likely because of the lower catalytic activity of the nickel toward the reduction of the DES. At lower overpotentials, different hydrogenated Ni structures were favored, most likely because of the DES co-reduction on the Pt substrate. Nanometric metallic nickel grains of rounded shape were obtained on any substrate, as evidenced by the FE-SEM. Passivation phenomena, related to the formation of Ni oxide and Ni hydroxylated species, were observed at high applied overpotentials. At low deposited charge, on Pt(111) the AFM measurements showed the formation of rounded nanometric particles of Ni, which rearranged and formed small triangular arrays at sufficiently low applied overpotential. This particle pattern was induced by the (111) orientation and related to surface sensitivity of the nickel deposition in DES. The present work provides deep insights into the Ni electrodeposition mechanism in the selected deep eutectic solvent.

Keywords: AFM, Deep eutectic solvent, Glassy carbon, Nanostructures, Nickel electrodeposition, Platinum electrode, Pt(111), SEM, Surface sensitive

Burgués, Javier, Marco, Santiago, (2018). Multivariate estimation of the limit of detection by orthogonal partial least squares in temperature-modulated MOX sensors Analytica Chimica Acta 1019, 49-64

Metal oxide semiconductor (MOX) sensors are usually temperature-modulated and calibrated with multivariate models such as Partial Least Squares (PLS) to increase the inherent low selectivity of this technology. The multivariate sensor response patterns exhibit heteroscedastic and correlated noise, which suggests that maximum likelihood methods should outperform PLS. One contribution of this paper is the comparison between PLS and maximum likelihood principal components regression (MLPCR) in MOX sensors. PLS is often criticized by the lack of interpretability when the model complexity increases beyond the chemical rank of the problem. This happens in MOX sensors due to cross-sensitivities to interferences, such as temperature or humidity and non-linearity. Additionally, the estimation of fundamental figures of merit, such as the limit of detection (LOD), is still not standardized in multivariate models. Orthogonalization methods, such as Orthogonal Projection to Latent Structures (O-PLS), have been successfully applied in other fields to reduce the complexity of PLS models. In this work, we propose a LOD estimation method based on applying the well-accepted univariate LOD formulas to the scores of the first component of an orthogonal PLS model. The resulting LOD is compared to the multivariate LOD range derived from error-propagation. The methodology is applied to data extracted from temperature-modulated MOX sensors (FIS SB-500-12 and Figaro TGS 3870-A04), aiming at the detection of low concentrations of carbon monoxide in the presence of uncontrolled humidity (chemical noise). We found that PLS models were simpler and more accurate than MLPCR models. Average LOD values of 0.79 ppm (FIS) and 1.06 ppm (Figaro) were found using the approach described in this paper. These values were contained within the LOD ranges obtained with the error-propagation approach. The mean LOD increased to 1.13 ppm (FIS) and 1.59 ppm (Figaro) when considering validation samples collected two weeks after calibration, which represents a 43% and 46% degradation, respectively. The orthogonal score-plot was a very convenient tool to visualize MOX sensor data and to validate the LOD estimates.

Keywords: Metal oxide sensors, Partial least squares, Orthogonal projection to latent structures, Maximum likelihood principal component regression, Limit of detection, Temperature modulation

Barba, A., Diez-Escudero, A., Maazouz, Y., Rappe, K., Espanol, M., Montufar, E. B., Bonany, M., Sadowska, J. M., Guillem-Marti, J., Öhman-Mägi, C., Persson, C., Manzanares, M. C., Franch, J., Ginebra, M. P., (2017). Osteoinduction by Foamed and 3D-Printed Calcium Phosphate Scaffolds: Effect of Nanostructure and Pore Architecture ACS Applied Materials & Interfaces 9, (48), 41722-41736

Some biomaterials are osteoinductive, that is, they are able to trigger the osteogenic process by inducing the differentiation of mesenchymal stem cells to the osteogenic lineage. Although the underlying mechanism is still unclear, microporosity and specific surface area (SSA) have been identified as critical factors in material-associated osteoinduction. However, only sintered ceramics, which have a limited range of porosities and SSA, have been analyzed so far. In this work, we were able to extend these ranges to the nanoscale, through the foaming and 3D-printing of biomimetic calcium phosphates, thereby obtaining scaffolds with controlled micro- and nanoporosity and with tailored macropore architectures. Calcium-deficient hydroxyapatite (CDHA) scaffolds were evaluated after 6 and 12 weeks in an ectopic-implantation canine model and compared with two sintered ceramics, biphasic calcium phosphate and β-tricalcium phosphate. Only foams with spherical, concave macropores and not 3D-printed scaffolds with convex, prismatic macropores induced significant ectopic bone formation. Among them, biomimetic nanostructured CDHA produced the highest incidence of ectopic bone and accelerated bone formation when compared with conventional microstructured sintered calcium phosphates with the same macropore architecture. Moreover, they exhibited different bone formation patterns; in CDHA foams, the new ectopic bone progressively replaced the scaffold, whereas in sintered biphasic calcium phosphate scaffolds, bone was deposited on the surface of the material, progressively filling the pore space. In conclusion, this study demonstrates that the high reactivity of nanostructured biomimetic CDHA combined with a spherical, concave macroporosity allows the pushing of the osteoinduction potential beyond the limits of microstructured calcium phosphate ceramics.

Keywords: 3D-printing, Calcium phosphate, Foaming, Nanostructure, Osteoinduction

Gumí-Audenis, Berta, Costa, Luca, Carlá, Francesco, Comin, Fabio, Sanz, Fausto, Giannotti, M. I., (2016). Structure and nanomechanics of model membranes by atomic force microscopy and spectroscopy: Insights into the role of cholesterol and sphingolipids Membranes , 6, (4), 58

Biological membranes mediate several biological processes that are directly associated with their physical properties but sometimes difficult to evaluate. Supported lipid bilayers (SLBs) are model systems widely used to characterize the structure of biological membranes. Cholesterol (Chol) plays an essential role in the modulation of membrane physical properties. It directly influences the order and mechanical stability of the lipid bilayers, and it is known to laterally segregate in rafts in the outer leaflet of the membrane together with sphingolipids (SLs). Atomic force microscope (AFM) is a powerful tool as it is capable to sense and apply forces with high accuracy, with distance and force resolution at the nanoscale, and in a controlled environment. AFM-based force spectroscopy (AFM-FS) has become a crucial technique to study the nanomechanical stability of SLBs by controlling the liquid media and the temperature variations. In this contribution, we review recent AFM and AFM-FS studies on the effect of Chol on the morphology and mechanical properties of model SLBs, including complex bilayers containing SLs. We also introduce a promising combination of AFM and X-ray (XR) techniques that allows for in situ characterization of dynamic processes, providing structural, morphological, and nanomechanical information

Keywords: Atomic force microscopy, Force spectroscopy, Lipid membranes, Supported lipid bilayers, Nanomechanics, Cholesterol, Sphingolipids, Membrane structure, XR-AFM combination

A. R. Dalton, J., Lans, I., Rovira, X., Malhaire, F., Gómez-Santacana, X., Pittolo, S., Gorostiza, P., Llebaria, A., Goudet, C., Pin, J-P., Giraldo, J., (2016). Shining light on an mGlu5 photoswitchable NAM: A theoretical perspective Current Neuropharmacology , 14, (5), 441-454

Metabotropic glutamate receptors (mGluRs) are important drug targets because of their involvement in several neurological diseases. Among mGluRs, mGlu5 is a particularly high-profile target because its positive or negative allosteric modulation can potentially treat schizophrenia or anxiety and chronic pain, respectively. Here, we computationally and experimentally probe the functional binding of a novel photoswitchable mGlu5 NAM, termed alloswitch-1, which loses its NAM functionality under violet light. We show alloswitch-1 binds deep in the allosteric pocket in a similar fashion to mavoglurant, the co-crystallized NAM in the mGlu5 transmembrane domain crystal structure. Alloswitch-1, like NAM 2-Methyl-6-(phenylethynyl)pyridine (MPEP), is significantly affected by P655M mutation deep in the allosteric pocket, eradicating its functionality. In MD simulations, we show alloswitch-1 and MPEP stabilize the co-crystallized water molecule located at the bottom of the allosteric site that is seemingly characteristic of the inactive receptor state. Furthermore, both NAMs form H-bonds with S809 on helix 7, which may constitute an important stabilizing interaction for NAM-induced mGlu5 inactivation. Alloswitch-1, through isomerization of its amide group from trans to cis is able to form an additional interaction with N747 on helix 5. This may be an important interaction for amide-containing mGlu5 NAMs, helping to stabilize their binding in a potentially unusual cis-amide state. Simulated conformational switching of alloswitch-1 in silico suggests photoisomerization of its azo group from trans to cis may be possible within the allosteric pocket. However, photoexcited alloswitch-1 binds in an unstable fashion, breaking H-bonds with the protein and destabilizing the co-crystallized water molecule. This suggests photoswitching may have destabilizing effects on mGlu5 binding and functionality.

Keywords: Allosteric modulation, Docking, Metabotropic glutamate receptor, Molecular dynamics, Mutation, Protein structure, Transmembrane domain

Moles, Ernest, Valle-Delgado, Juan José, Urbán, Patricia, Azcárate, Isabel G., Bautista, José M., Selva, Javier, Egea, Gustavo, Ventura, Salvador, Fernàndez-Busquets, Xavier, (2015). Possible roles of amyloids in malaria pathophysiology Future Science OA , 1, (2), FSO43

The main therapeutic and prophylactic tools against malaria have been locked for more than a century in the classical approaches of using drugs targeting metabolic processes of the causing agent, the protist Plasmodium spp., and of designing vaccines against chosen antigens found on the parasite’s surface. Given the extraordinary resources exhibited by Plasmodium to escape these traditional strategies, which have not been able to free humankind from the scourge of malaria despite much effort invested in them, new concepts have to be explored in order to advance toward eradication of the disease. In this context, amyloid-forming proteins and peptides found in the proteome of the pathogen should perhaps cease being regarded as mere anomalous molecules. Their likely functionality in the pathophysiology of Plasmodium calls for attention being paid to them as a possible Achilles’ heel of malaria. Here we will give an overview of Plasmodium-encoded amyloid-forming polypeptides as potential therapeutic targets and toxic elements, particularly in relation to cerebral malaria and the blood–brain barrier function. We will also discuss the recent finding that the genome of the parasite contains an astonishingly high proportion of prionogenic domains.

Keywords: Amyloids, Intrinsically unstructured proteins, Malaria, Prions

Estévez, M., Martínez, Elena, Yarwood, S. J., Dalby, M. J., Samitier, J., (2015). Adhesion and migration of cells responding to microtopography Journal of Biomedical Materials Research - Part A , 103, (5), 1659-1668

It is known that cells respond strongly to microtopography. However, cellular mechanisms of response are unclear. Here, we study wild-type fibroblasts responding to 25 μm2 posts and compare their response to that of FAK-/- fibroblasts and fibroblasts with PMA treatment to stimulate protein kinase C (PKC) and the small g-protein Rac. FAK knockout cells modulated adhesion number and size in a similar way to cells on topography; that is, they used more, smaller adhesions, but migration was almost completely stalled demonstrating the importance of FAK signaling in contact guidance and adhesion turnover. Little similarity, however, was observed to PKC stimulated cells and cells on the topography. Interestingly, with PKC stimulation the cell nuclei became highly deformable bringing focus on these surfaces to the study of metastasis. Surfaces that aid the study of cellular migration are important in developing understanding of mechanisms of wound healing and repair in aligned tissues such as ligament and tendon.

Keywords: Adhesion, Cell migration, Cell morphology, Focal adhesion kinase, Microstructures

Cuervo, A., Dans, P. D., Carrascosa, J. L., Orozco, M., Gomila, G., Fumagalli, L., (2014). Direct measurement of the dielectric polarization properties of DNA Proceedings of the National Academy of Sciences of the United States of America 111, (35), E3624-E3630

The electric polarizability of DNA, represented by the dielectric constant, is a key intrinsic property that modulates DNA interaction with effector proteins. Surprisingly, it has so far remained unknown owing to the lack of experimental tools able to access it. Here, we experimentally resolved it by detecting the ultraweak polarization forces of DNA inside single T7 bacteriophages particles using electrostatic force microscopy. In contrast to the common assumption of low-polarizable behavior like proteins (εr ~ 2–4), we found that the DNA dielectric constant is ~ 8, considerably higher than the value of ~ 3 found for capsid proteins. State-of-the-art molecular dynamic simulations confirm the experimental findings, which result in sensibly decreased DNA interaction free energy than normally predicted by Poisson–Boltzmann methods. Our findings reveal a property at the basis of DNA structure and functions that is needed for realistic theoretical descriptions, and illustrate the synergetic power of scanning probe microscopy and theoretical computation techniques.

Keywords: Atomic force microscopy, Atomistic simulations, DNA packaging, DNA-ligand binding, Poisson-Boltzmann equation, capsid protein, DNA, double stranded DNA, amino acid composition, article, atomic force microscopy, bacteriophage, bacteriophage T7, dielectric constant, dipole, DNA binding, DNA packaging, DNA structure, electron microscopy, ligand binding, nonhuman, polarization, priority journal, protein analysis, protein DNA interaction, scanning probe microscopy, static electricity, virion, virus capsid, virus particle, atomic force microscopy, atomistic simulations, DNA packaging, DNA-ligand binding, Poisson-Boltzmann equation, Bacteriophage T7, Capsid, Cations, Dielectric Spectroscopy, DNA, DNA, Viral, DNA-Binding Proteins, Electrochemical Techniques, Ligands, Microscopy, Atomic Force, Models, Chemical, Nuclear Proteins

Urra, O., Jané, R., (2014). New sleep transition indexes for describing altered sleep in SAHS IFMBE Proceedings XIII Mediterranean Conference on Medical and Biological Engineering and Computing 2013 (ed. Roa Romero, Laura M.), Springer International Publishing (London, UK) 41, 1017-1020

Traditional Sleep Structure Indexes (TSSIs) are insufficient to identify patterns of altered sleep. TSSIs mainly account for absolute time measures, but different levels of state instability may lead to similar absolute time distribution. Therefore, sleep stability remains beyond the scope of TSSIs. However, recent studies suggest that sleep disorders may be rather influenced by a breakdown in the sleep-stage switching mechanisms. In this study, we propose a set of 11 Sleep Transition Indexes (STIs) that characterize sleep fragmentation and account for the state-stability governed by the ultradian, homeostatic and circadian rhythms. We demonstrate that most of the proposed STIs are potential markers of SAHS severity, while TSSIs are not. In addition, we provide a new framework to analyze sleep disorders from the direct perspective of sleep regulatory mechanisms. In particular, our results indicate that SAHS may be influenced by a dysregulation of homeostatic rhythms but not of ultradian or circadian rhythms.

Keywords: SAHS, Sleep Transitions, Sleep Structure, Polysomnography, Hypnogram

Salerno, A., Levato, R., Mateos-Timoneda, M. A., Engel, E., Netti, P. A., Planell, J. A., (2013). Modular polylactic acid microparticle-based scaffolds prepared via microfluidic emulsion/solvent displacement process: Fabrication, characterization, and in vitro mesenchymal stem cells interaction study Journal of Biomedical Materials Research - Part A , 101A, (3), 720-732

The present study reports a novel approach for the design and fabrication of polylactic acid (PLA) microparticle-based scaffolds with microstructural properties suitable for bone and cartilage regeneration. Macroporous PLA scaffolds with controlled shape were fabricated by means of a semicontinuous process involving (1) microfluidic emulsification of a PLA/ethyl lactate solution (5% w/v) in a span 80/paraffin oil solution (3% v/v) followed by (2) particles coagulation/assembly in an acetone/water solution for the development of a continuous matrix. Porous scaffolds prepared from particles with monomodal or bimodal size distribution, overall porosity ranges from 93 to 96%, interparticles porosity from 41 to 54%, and static compression moduli from 0.3 to 1.4 MPa were manufactured by means of flow rate modulation of of the continuous phase during emulsion. The biological response of the scaffolds was assessed in vitro by using bone marrow-derived rat mesenchymal stem cells (MSCs). The results demonstrated the ability of the scaffolds to support the extensive and uniform three-dimensional adhesion, colonization, and proliferation of MSCs within the entire construct.

Keywords: Green solvent, Microfluidic, Microstructure, Stem cells, Scaffold

Valle-Delgado, J. J., Liepina, I., Lapidus, D., Sabaté, R., Ventura, S., Samitier, J., Fernàndez-Busquets, X., (2012). Self-assembly of human amylin-derived peptides studied by atomic force microscopy and single molecule force spectroscopy Soft Matter 8, (4), 1234-1242

The self-assembly of peptides and proteins into amyloid fibrils of nanometric thickness and up to several micrometres in length, a phenomenon widely observed in biological systems, has recently aroused a growing interest in nanotechnology and nanomedicine. Here we have applied atomic force microscopy and single molecule force spectroscopy to study the amyloidogenesis of a peptide derived from human amylin and of its reverse sequence. The spontaneous formation of protofibrils and their orientation along well-defined directions on graphite and DMSO-coated graphite substrates make the studied peptides interesting candidates for nanotechnological applications. The measured binding forces between peptides correlate with the number of hydrogen bonds between individual peptides inside the fibril structure according to molecular dynamics simulations.

Keywords: Amyloid fibril, Amyloidogenesis, Binding forces, Fibril structure, Graphite substrate, Molecular dynamics simulations, Nanometrics, Protofibrils, Single molecule force spectroscopy, Spontaneous formation, Atomic force microscopy, Atomic spectroscopy, Graphite, Hydrogen bonds, Medical nanotechnology, Molecular dynamics, Molecular physics, Self assembly, Thickness measurement, Peptides

Azevedo, S., Diéguez, L., Carvalho, P., Carneiro, J. O., Teixeira, V., Martínez, Elena, Samitier, J., (2012). Deposition of ITO thin films onto PMMA substrates for waveguide based biosensing devices Journal of Nano Research , 17, 75-83

Biosensors' research filed has clearly been changing towards the production of multifunctional and innovative design concepts to address the needs related with sensitivity and selectivity of the devices. More recently, waveguide biosensors, that do not require any label procedure to detect biomolecules adsorbed on its surface, have been pointed out as one of the most promising technologies for the production of biosensing devices with enhanced performance. Moreover the combination of optical and electrochemical measurements through the integration of transparent and conducting oxides in the multilayer structures can greatly enhance the biosensors' sensitivity. Furthermore, the integration of polymeric substrates may bring powerful advantages in comparison with silicon based ones. The biosensors will have a lower production costs being possible to disposable them after use ("one use sensor chip"). This research work represents a preliminary study about the influence of substrate temperature on the overall properties of ITO thin films deposited by DC magnetron sputtering onto 0,5 mm thick PMMA sheets.

Keywords: ITO thin films, PMMA sheets, Waveguide biosensing devices, Biosensing devices, Conducting oxides, Dc magnetron sputtering, Electrochemical measurements, Enhanced performance, Innovative design, ITO thin films, Multilayer structures, Overall properties, PMMA sheets, Polymeric substrate, Production cost, Sensor chips, Silicon-based, Substrate temperature, Biosensors, Deposition, Design, Film preparation, Optical multilayers, Thin films, Vapor deposition, Waveguides, Substrates

Serra, T., Navarro, M., Planell, J. A., (2012). Fabrication and characterization of biodegradable composite scaffolds for tissue engineering Innovative Developments in Virtual and Physical Prototyping 5th International Conference on Advanced Research and Rapid Prototyping (ed. Margarida, T., Ferreira, D.), Taylor & Francis (Leiria, Portugal) VR@P, 67-72

In this study, polylactic acid (PLA) and polyethylene glycol (PEG) were combined with soluble CaP glass particles and processed by rapid prototyping to obtain fully biodegradable structures for Tissue Engineering applications. The obtained 3D biodegradable structures were characterized in terms of their architecture and mechanical properties. The scaffold morphology, internal micro-architecture and mechanical properties were evaluated using Scanning Electron Microscopy (SEM), micro-computed tomography (micro-CT) and mechanical testing, respectively. Well defined structures with pore size of 350-400μm (in the axial view), struts width of approximately 70-80μm, and a porosity ranging between 60-65% were obtained. The combination RP and PLA/PEG/CaP glass turned into promising fully degradable, mechanically stable, bioactive and biocompatible composite scaffolds for TE.

Keywords: Axial view, Biodegradable composites, Composite scaffolds, Glass particles, Mechanically stable, Micro architectures, Micro computed tomography (micro-CT), Poly lactic acid, Scaffold morphology, Tissue engineering applications, Well-defined structures, Bioactive glass, Mechanical properties, Mechanical testing, Polyethylene glycols, Polymer blends, Rapid prototyping, Scaffolds (biology), Scanning electron microscopy, Computerized tomography

Castaño, O., Eltohamy, M., Kim, H. W., (2012). Electrospinning technology in tissue regeneration Nanotechnology in Regenerative Medicine - Methods and Protocols (Methods in Molecular Biology) (ed. Navarro, M., Planell, J. A.), Springer (New York, USA) 811, 127-140

Electrospinning is one of the most versatile and effective tools to produce nanostructured fibers in the biomedical science fields. The nanofibrous structure with diameters from tens to hundreds of nanometers largely mimics the native extracellular matrix (ECM) of many tissues. Thus far, a range of compositions including polymers and ceramics and their composites/hybrids have been successfully applied for generating electrospun nanofibers. Different processing tools in electrospinning set-ups and assemblies are currently developed to tune the morphology and properties of nanofibers. Herein, we demonstrate the electrospinning process and the electrospun biomaterials for specific use in tissue regeneration with some examples, involving different material combinations and fiber morphologies.

Keywords: Ceramic, Composites, Electrospinning, Nanofi bers, Nanostructured fi bers, Polymer, Tissue regeneration

Auffarth, Benjamin, Gutierrez, Agustin, Marco, Santiago, (2011). Statistical analysis of coding for molecular properties in the olfactory bulb Frontiers in Systems Neuroscience 5, (62), 1-8

The relationship between molecular properties of odorants and neural activities is arguably one of the most important issues in olfaction and the rules governing this relationship are still not clear. In the olfactory bulb (OB), glomeruli relay olfactory information to second-order neurons which in turn project to cortical areas. We investigate relevance of odorant properties, spatial localization of glomerular coding sites, and size of coding zones in a dataset of 2-deoxyglucose images of glomeruli over the entire OB of the rat. We relate molecular properties to activation of glomeruli in the OB using a nonparametric statistical test and a support-vector machine classification study. Our method permits to systematically map the topographic representation of various classes of odorants in the OB. Our results suggest many localized coding sites for particular molecular properties and some molecular properties that could form the basis for a spatial map of olfactory information. We found that alkynes, alkanes, alkenes, and amines affect activation maps very strongly as compared to other properties and that amines, sulfur-containing compounds, and alkynes have small zones and high relevance to activation changes, while aromatics, alkanes, and carboxylics acid recruit very big zones in the dataset. Results suggest a local spatial encoding for molecular properties.

Keywords: Molecular-receptive range, Odor, Olfactory bulb, Olfactory coding, Property-activity relationship, Structure-odor relationship

Cordeiro, T. N., Schmidt, H., Madrid, C., Juarez, A., Bernado, P., Griesinger, C., Garcia, J., Pons, M., (2011). Indirect DNA readout by an H-NS related protein: Structure of the DNA complex of the C-terminal domain of Ler PLoS Pathogens Plos Pathogens , 7, (11), 12

Ler, a member of the H-NS protein family, is the master regulator of the LEE pathogenicity island in virulent Escherichia coli strains. Here, we determined the structure of a complex between the DNA-binding domain of Ler (CT-Ler) and a 15-mer DNA duplex. CT-Ler recognizes a preexisting structural pattern in the DNA minor groove formed by two consecutive regions which are narrower and wider, respectively, compared with standard B-DNA. The compressed region, associated with an AT-tract, is sensed by the side chain of Arg90, whose mutation abolishes the capacity of Ler to bind DNA. The expanded groove allows the approach of the loop in which Arg90 is located. This is the first report of an experimental structure of a DNA complex that includes a protein belonging to the H-NS family. The indirect readout mechanism not only explains the capacity of H-NS and other H-NS family members to modulate the expression of a large number of genes but also the origin of the specificity displayed by Ler. Our results point to a general mechanism by which horizontally acquired genes may be specifically recognized by members of the H-NS family.

Keywords: Enteropathogenic escherichia-coli, Nucleoid-associated protein, Nmr structure determination, Encoded regulator ler, Controls expression, Binding domain

Melchels, Ferry P. W., Tonnarelli, Beatrice, Olivares, Andy L., Martin, Ivan, Lacroix, Damien, Feijen, Jan, Wendt, David J., Grijpma, Dirk W., (2011). The influence of the scaffold design on the distribution of adhering cells after perfusion cell seeding Biomaterials 32, (11), 2878-2884

In natural tissues, the extracellular matrix composition, cell density and physiological properties are often non-homogeneous. Here we describe a model system, in which the distribution of cells throughout tissue engineering scaffolds after perfusion seeding can be influenced by the pore architecture of the scaffold. Two scaffold types, both with gyroid pore architectures, were designed and built by stereolithography: one with isotropic pore size (412 ± 13 [mu]m) and porosity (62 ± 1%), and another with a gradient in pore size (250-500 [mu]m) and porosity (35%-85%). Computational fluid flow modelling showed a uniform distribution of flow velocities and wall shear rates (15-24 s-1) for the isotropic architecture, and a gradient in the distribution of flow velocities and wall shear rates (12-38 s-1) for the other architecture. The distribution of cells throughout perfusion-seeded scaffolds was visualised by confocal microscopy. The highest densities of cells correlated with regions of the scaffolds where the pores were larger, and the fluid velocities and wall shear rates were the highest. Under the applied perfusion conditions, cell deposition is mainly determined by local wall shear stress, which, in turn, is strongly influenced by the architecture of the pore network of the scaffold.

Keywords: Scaffolds, Microstructure, Cell adhesion, Confocal microscopy, Image analysis, Computational fluid dynamics

Crona, Mikael, Torrents, Eduard, Rohr, Asmund K., Hofer, Anders, Furrer, Ernst, Tomter, Ane B., Andersson, K. Kristoffer, Sahlin, Margareta, Sjoberg, Britt-Marie, (2011). NrdH-redoxin protein mediates high enzyme activity in manganese-reconstituted ribonucleotide reductase from bacillus anthracis Journal of Biological Chemistry , 286, (38), 33053-33060

Bacillus anthracis is a severe mammalian pathogen encoding a class Ib ribonucleotide reductase (RNR). RNR is a universal enzyme that provides the four essential deoxyribonucleotides needed for DNA replication and repair. Almost all Bacillus spp. encode both class Ib and class III RNR operons, but the B. anthracis class III operon was reported to encode a pseudogene, and conceivably class Ib RNR is necessary for spore germination and proliferation of B. anthracis upon infection. The class Ib RNR operon in B. anthracis encodes genes for the catalytic NrdE protein, the tyrosyl radical metalloprotein NrdF, and the flavodoxin protein NrdI. The tyrosyl radical in NrdF is stabilized by an adjacent Mn(2)(III) site (Mn-NrdF) formed by the action of the NrdI protein or by a Fe(2)(III) site (Fe-NrdF) formed spontaneously from Fe(2+) and O(2). In this study, we show that the properties of B. anthracis Mn-NrdF and Fe-NrdF are in general similar for interaction with NrdE and NrdI. Intriguingly, the enzyme activity of Mn-NrdF was approximately an order of magnitude higher than that of Fe-NrdF in the presence of the class Ib-specific physiological reductant NrdH, strongly suggesting that the Mn-NrdF form is important in the life cycle of B. anthracis. Whether the Fe-NrdF form only exists in vitro or whether the NrdF protein in B. anthracis is a true cambialistic enzyme that can work with either manganese or iron remains to be established.

Keywords: Escherichia-coli, Corynebacterium-ammoniagenes, Crystal-structure, Cofactor, Cubunit, Growth, Genes

Tahirbegi, I. B., Mir, M., (2011). Slit-wave model for band structures in solid state physics Modern Physics Letters B , 25, (3), 151-161

The reason behind the entire development in silicon technology was band models in solid state physics. However, the theories postulated in order to give response to this phenomenon do not explain all kinds of materials. In a bid to overcome this limitation, we approach the problem from another point of view. In this work, the wave properties of the electrons from the external orbitals of the atoms and its diffraction patterns through the lattice structure of the material have been used to explain the band structure of metals, semiconductor and insulators. In order to probe this hypothesis, a simulation has been used and according to the relation between the lattice constant and the atomic diameter, the splitting of the bands have been observed for different kind of materials, showing a strong correlation between the simulation and the experimental results.

Keywords: Electrical band structure, Band gap, Fraunhofer diffraction, Semiconductor, Insulator

Valle-Delgado, J. J., Alfonso-Prieto, M., de Groot, N. S., Ventura, S., Samitier, J., Rovira, C., Fernàndez-Busquets, X., (2010). Modulation of A beta(42) fibrillogenesis by glycosaminoglycan structure FASEB Journal , 24, (11), 4250-4261

The role of amyloid beta (A beta) peptide in the onset and progression of Alzheimer's disease is linked to the presence of soluble A beta species. Sulfated glycosaminoglycans (GAGs) promote A beta fibrillogenesis and reduce the toxicity of the peptide in neuronal cell cultures, but a satisfactory rationale to explain these effects at the molecular level has not been provided yet. We have used circular dichroism, Fourier transform infrared spectroscopy, fluorescence microscopy and spectroscopy, protease digestion, atomic force microscopy (AFM), and molecular dynamics simulations to characterize the association of the 42-residue fragment A beta(42) with sulfated GAGs, hyaluronan, chitosan, and poly(vinyl sulfate) (PVS). Our results indicate that the formation of stable A beta(42) fibrils is promoted by polymeric GAGs with negative charges placed in-frame with the 4.8-angstrom separating A beta(42) monomers within protofibrillar beta-sheets. Incubation of A beta(42) with excess sulfated GAGs and hyaluronan increased amyloid fibril content and resistance to proteolysis 2- to 5-fold, whereas in the presence of the cationic polysaccharide chitosan, A beta(42) fibrillar species were reduced by 25% and sensitivity to protease degradation increased similar to 3-fold. Fibrils of intermediate stability were obtained in the presence of PVS, an anionic polymer with more tightly packed charges than GAGs. Important structural differences between A beta(42) fibrils induced by PVS and A beta(42) fibrils obtained in the presence of GAGs and hyaluronan were observed by AFM, whereas mainly precursor protofibrillar forms were detected after incubation with chitosan. Computed binding energies per peptide from -11.2 to -13.5 kcal/mol were calculated for GAGs and PVS, whereas a significantly lower value of -7.4 kcal/mol was obtained for chitosan. Taken together, our data suggest a simple and straightforward mechanism to explain the role of GAGs as enhancers of the formation of insoluble A beta(42) fibrils trapping soluble toxic forms.

Keywords: Alzheimer's disease, Amyloid fibril structure, Fibrillogenesis enhancers and inhibitors, Polysaccharides

Fumagalli, L., Gramse, G., Esteban-Ferrer, D., Edwards, M. A., Gomila, G., (2010). Quantifying the dielectric constant of thick insulators using electrostatic force microscopy Applied Physics Letters , 96, (18), 183107

Quantitative measurement of the low-frequency dielectric constants of thick insulators at the nanoscale is demonstrated utilizing ac electrostatic force microscopy combined with finite-element calculations based on a truncated cone with hemispherical apex probe geometry. The method is validated on muscovite mica, borosilicate glass, poly(ethylene naphthalate), and poly(methyl methacrylate). The dielectric constants obtained are essentially given by a nanometric volume located at the dielectric-air interface below the tip, independently of the substrate thickness, provided this is on the hundred micrometer-length scale, or larger.

Keywords: Borosilicate glasses, Finite element analysis, Insulating thin films, Mica, Nanostructured materials, Permittivity, Polymers, Scanning probe microscopy

Johansson, R., Torrents, E., Lundin, D., Sprenger, J., Sahlin, M., Sjöberg, B. M., Logan, D. T., (2010). High-resolution crystal structures of the flavoprotein NrdI in oxidized and reduced states – an unusual flavodoxin FEBS Journal , 277, (20), 4265-4277

The small flavoprotein NrdI is an essential component of the class Ib ribonucleotide reductase system in many bacteria. NrdI interacts with the class Ib radical generating protein NrdF. It is suggested to be involved in the rescue of inactivated diferric centres or generation of active dimanganese centres in NrdF. Although NrdI bears a superficial resemblance to flavodoxin, its redox properties have been demonstrated to be strikingly different. In particular, NrdI is capable of two-electron reduction, whereas flavodoxins are exclusively one-electron reductants. This has been suggested to depend on a lesser destabilization of the negatively-charged hydroquinone state than in flavodoxins. We have determined the crystal structures of NrdI from Bacillus anthracis, the causative agent of anthrax, in the oxidized and semiquinone forms, at resolutions of 0.96 and 1.4 Å, respectively. These structures, coupled with analysis of all curated NrdI sequences, suggest that NrdI defines a new structural family within the flavodoxin superfamily. The conformational behaviour of NrdI in response to FMN reduction is very similar to that of flavodoxins, involving a peptide flip in a loop near the N5 atom of the flavin ring. However, NrdI is much less negatively charged than flavodoxins, which is expected to affect its redox properties significantly. Indeed, sequence analysis shows a remarkable spread in the predicted isoelectric points of NrdIs, from approximately pH 4–10. The implications of these observations for class Ib ribonucleotide reductase function are discussed.

Keywords: Crystal structure, Flavin mononucleotide, Flavodoxin, NrdI, Ribonucleotide reductase

Garde, A., Sörnmo, L., Jané, R., Giraldo, B. F., (2010). Correntropy-based nonlinearity test applied to patients with chronic heart failure Engineering in Medicine and Biology Society (EMBC) 32nd Annual International Conference of the IEEE , IEEE (Buenos Aires, Argentina) , 2399-2402

In this study we propose the correntropy function as a discriminative measure for detecting nonlinearities in the respiratory pattern of chronic heart failure (CHF) patients with periodic or nonperiodic breathing pattern (PB or nPB, respectively). The complexity seems to be reduced in CHF patients with higher risk level. Correntropy reflects information on both, statistical distribution and temporal structure of the underlying dataset. It is a suitable measure due to its capability to preserve nonlinear information. The null hypothesis considered is that the analyzed data is generated by a Gaussian linear stochastic process. Correntropy is used in a statistical test to reject the null hypothesis through surrogate data methods. Various parameters, derived from the correntropy and correntropy spectral density (CSD) to characterize the respiratory pattern, presented no significant differences when extracted from the iteratively refined amplitude adjusted Fourier transform (IAAFT) surrogate data. The ratio between the powers in the modulation and respiratory frequency bands R was significantly different in nPB patients, but not in PB patients, which reflects a higher presence of nonlinearities in nPB patients than in PB patients.

Keywords: Practical, Theoretical or Mathematical, Experimental/cardiology diseases, Fourier transforms, Medical signal processing, Pattern classification, Pneumodynamics, Spectral analysis, Statistical analysis, Stochastic processes/ correntropy based nonlinearity test, Chronic heart failure, Correntropy function, Respiratory pattern nonlinearities, CHF patients, Nonperiodic breathing pattern, Dataset statistical distribution, Dataset temporal structure, Nonlinear information, Null hypothesis, Gaussian linear stochastic process, Statistical test, Correntropy spectral density, Iteratively refined amplitude adjusted Fourier transform, Surrogate data, Periodic breathing pattern

Fernandez, Javier G., Mills, C. A., Samitier, J., (2009). Complex microstructured 3D surfaces using chitosan biopolymer Small 5, (5), 614-620

A technique for producing micrometer-scale structures over large, nonplanar chitosan surfaces is described. The technique makes use of the rheological characteristics (deformability) of the chitosan to create freestanding, three-dimensional scaffolds with controlled shapes, incorporating defined microtopography. The results of an investigation into the technical limits of molding different combinations of shapes and microtopographies are presented, highlighting the versatility of the technique when used irrespectively with inorganic or delicate organic moulds. The final, replicated scaffolds presented here are patterned with arrays of one-micrometer-tall microstructures over large areas. Structural integrity is characterized by the measurement of structural degradation. Human umbilical vein endothelial cells cultured on a tubular scaffold show that early cell growth is conditioned by the microtopography and indicate possible uses for the structures in biomedical applications. For those applications requiring improved chemical and mechanical resistance, the structures can be replicated in poly(dimethyl siloxane).

Keywords: Biocompatible Materials/ chemistry, Cell Adhesion, Cell Culture Techniques/ methods, Cell Proliferation, Cells, Cultured, Chitosan/ chemistry, Crystallization/methods, Endothelial Cells/ cytology/ physiology, Humans, Materials Testing, Nanostructures/ chemistry/ ultrastructure, Nanotechnology/methods, Particle Size, Surface Properties, Tissue Engineering/methods

Ruiz, A., Mills, C. A., Valsesia, A., Martinez, E., Ceccone, G., Samitier, J., Colpo, P., Rossi, F., (2009). Large-area, nanoimprint-assisted microcontact stripping for the fabrication of microarrays of fouling/nonfouling nanostructures Small 5, (10), 1133-1137

Methods for the accurate positioning of nanometric beads on a substrate have been developed over a number of years, and range from serial atomic force microscopy (AFM)techniques for single-bead positioning to parallel techniques for the positioning of large populations of beads in monolayer or multilayer architectures, typically from a liquid suspension. For example, topographic cues have been used for bead-based protein array production, although in this case, there is a random distribution of beads within the topography. Bead patterning has also been achieved in capillaries using a micromolding in capillaries (MIMIC) technique. Line patterns with micrometer widths are possible with this technique, achieving good multilayer organization. For monolayer bead patterning at micrometer dimensions, electrostatic forces and similar electrostatic assemblies using nanoxerography, as well as patterning by selective chemical functionalization, by transfer of particles from a liquid–liquid interface, and by subtracting top–down processes, are possible.

Keywords: Microcontact stripping, Nanostructures, Poly(acrylic acid), Polystyrene, Surface patterning

Martinez, E., Lagunas, A., Mills, C. A., Rodriguez-Segui, S., Estevez, M., Oberhansl, S., Comelles, J., Samitier, J., (2009). Stem cell differentiation by functionalized micro- and nanostructured surfaces Nanomedicine 4, (1), 65-82

New fabrication technologies and, in particular, new nanotechnologies have provided biomaterial and biomedical scientists with enormous possibilities when designing customized supports and scaffolds with controlled nanoscale topography and chemistry. The main issue now is how to effectively design these components and choose the appropriate combination of structure and chemistry to tailor towards applications as challenging and complex as stem cell differentiation. Occasionally, an incomplete knowledge of the fundamentals of biological differentiation process has hampered this issue. However, the recent technological advances in creating controlled cellular microenvironments can be seen as a powerful tool for furthering fundamental biology studies. This article reviews the main strategies followed to achieve solutions to this challenge, particularly emphasizing the working hypothesis followed by the authors to elucidate the mechanisms behind the observed effects of structured surfaces on cell behavior.

Keywords: Cell pattering, Differentiation, Microcontact printing, Micropatterning, Microstructure, Nanoimprinting, Nanostructure, Stem cells

Caballero-Briones, F., Artes, J. M., Diez-Perez, I., Gorostiza, P., Sanz, F., (2009). Direct observation of the valence band edge by in situ ECSTM-ECTS in p-type Cu2O layers prepared by copper anodization Journal of Physical Chemistry C 113, (3), 1028-1036

Polycrystalline Cu2O layers have been selectively grown by electrochemical anodization of polycrystalline Cu electrodes in an alkaline medium (pH 12.85). Uniform layers with thicknesses around 100 nm have been obtained. Using electrochemical impedance spectroscopy, it was concluded that the Cu2O films behave as a p-type semiconductor. The Mott-Schottky plot gives a value for the flat band potential of U-FB = -255 mV vs silver/silver chloride electrode (SSC), an estimated carrier density N-A = 6.1 x 10(17) cm(-3), and the space charge layer width was calculated to be W-SCL = 9 nm at a band bending of 120 mV. The electronic structure of the Cu vertical bar Cu2O vertical bar electrolyte interface was for the first time probed by in situ electrochemical tunneling spectroscopy. The use of in situ electrochemical scanning tunneling microscopy allows us to directly observed the valence band edge and determine its position against the absolute energy scale to be E-VB = -4.9 eV. Finally, we constructed a quantitative electronic diagram of the Cu vertical bar Cu2O vertical bar electrolyte interface, where the positions of the valence and conduction band edges are depicted, as well as the edge of the previously reported electronic subband.

Keywords: 0.1 m NaOH, Electrochemical tunneling spectroscopy, Cuprous-oxide films, Anodic-oxidation, Electronic-structure, Alkaline-solution, Aqueous-solution, Initial-stages, Passive film, Thin-films

Mir, M., Cameron, P. J., Zhong, X., Azzaroni, O., Alvarez, M., Knoll, W., (2009). Anti-fouling characteristics of surface-confined oligonucleotide strands bioconjugated on streptavidin platforms in the presence of nanomaterials Talanta 78, (3), 1102-6

This work describes our studies on the molecular design of interfacial architectures suitable for DNA sensing which could resist non-specific binding of nanomaterials commonly used as labels for amplifying biorecognition events. We observed that the non-specific binding of bio-nanomaterials to surface-confined oligonucleotide strands is highly dependent on the characteristics of the interfacial architecture. Thiolated double stranded oligonucleotide arrays assembled on Au surfaces evidence significant fouling in the presence of nanoparticles (NPs) at the nanomolar level. The non-specific interaction between the oligonucleotide strands and the nanomaterials can be sensitively minimized by introducing streptavidin (SAv) as an underlayer conjugated to the DNA arrays. The role of the SAv layer was attributed to the significant hydrophilic repulsion between the SAv-modified surface and the nanomaterials in close proximity to the interface, thus conferring outstanding anti-fouling characteristics to the interfacial architecture. These results provide a simple and straightforward strategy to overcome the limitations introduced by the non-specific binding of labels to achieve reliable detection of DNA-based biorecognition events.

Keywords: DNA/ analysis, Gold, Nanostructures/ chemistry, Oligonucleotide Array Sequence Analysis/ instrumentation, Oligonucleotides/ chemistry, Streptavidin/ chemistry, Sulfhydryl Compounds

Martinez, E., Engel, E., Planell, J. A., Samitier, J., (2009). Effects of artificial micro- and nano-structured surfaces on cell behaviour Annals of Anatomy-Anatomischer Anzeiger , 191, (1), 126-135

Substrate topography, independently of substrate chemistry, has been reported to have significant effects on cell behaviour. Based on the use of fabrication techniques developed by the silicon microtechnology industry, numerous studies can now be found in the literature analyzing cell behaviour as to various micro- and nanofeatures such as lines, wells, holes and more. Most of these works have been found to relate the micro- and nano-sized topographical features with cell. orientation, migration, morphology and proliferation. In recent papers, even the influence of substrate nanotopography on cell gene expression and differentiation has been pointed out. However, despite the large number of papers published on this topic, significant general trends in cell behaviour are difficult to establish due to differences in cell type, substrate material, feature aspect-ratio, feature geometry and parameters measured. This paper intends to compile and review the relevant existing information on the behaviour of cells on micro- and nano-structured artificial substrates and analyze possible general behavioural trends.

Keywords: Microstructure, Topography, Cell behaviour, Cell morphology, Cell orientation

Engel, E., Martinez, E., Mills, C. A., Funes, M., Planell, J. A., Samitier, J., (2009). Mesenchymal stem cell differentiation on microstructured poly (methyl methacrylate) substrates Annals of Anatomy-Anatomischer Anzeiger , 191, (1), 136-144

Recent studies on 2D substrates have revealed the importance of surface properties in affecting cell behaviour. In particular, surface topography appears to influence and direct cell migration. The development of new technologies of hot embossing and micro-imprinting has made it possible to study cell interactions with controlled micro features and to determine how these features can affect cell behaviour. Several studies have been carried out on the effect of microstructures on cell adhesion, cell guidance and cell proliferation. However, there is still a lack of knowledge on how these features affect mesenchymal stem cell differentiation. This study was designed to evaluate whether highly controlled microstructures on PMMA could induce rMSC differentiation into an osteogenic lineage. Structured PMMA was seeded with rMSC and cell number; cell morphology and cell differentiation were evaluated. Results confirm that microstructures not only affect cell proliferation and alignment but also have a synergistic effect with osteogenic medium on rMSC differentiation into mature osteoblasts.

Keywords: Mesenchymal stem cells, Osteoblasts, Topography, Microstructures

Zazoua, A., Kherrat, R., Caballero, D., Errachid, A., Jaffrezic-Renault, N., Bessueille, F., Leonard, D., (2009). Characterisation of a Cr(VI) sensitive polysiloxane membrane by x-ray photoelectron spectrometry and atomic force microscopy Sensor Letters 6th Maghreb-Europe Meeting on Materials and Their Applications for Devices and Physical, Chemical and Biological Sensors , AMER SCIENTIFIC PUBLISHERS (Rabat, Morocco) 7, (5), 995-1000

Cr(VI) sensitive polysiloxane membranes containing tributylphosphate (TBP) or trioctylphosphine oxide (TOPO) were characterized in this study. TBP and TOPO as carriers, have a high selectivity for Cr(VI). The Potentiometric response of EMIS (Electrolyte/Membrane/Insulator/Semiconductor) sensors presents a quasi-nernstian response for Cr2O2-7 exchange. The ion exchange is shown by X-ray photoelectron spectrometry (XPS), the binding energy of the Cr 2p1/2 peak corresponding to Cr(VI) and the atomic composition after exposure to Cr(VI) shows a factor 1.7 higher for silopreneTBP membrane. The conformational topography of both polymeric membranes was characterized by Atomic Force Microscopy (AFM), the exchange of Cr(VI) leading to a heterogeneous topographic state. Adhesion force measurements are also performed to study the properties of adhesion of both selective membranes with a non-functionalized Si AFM tip and with an OTS functionalized one to study the interactions between the tip and the membrane, in liquid before and after the exposure of the membrane to ion chromium. The presence of the ionophores does not practically change the adhesion force compared to pure polysiloxane, showing a good solubility of the ionophore and the orientation of the alkyl chains towards the polysiloxane surface. After the exchange with Cr(VI), the adhesion force decreases drastically due to the hydrophilic character of the surface, complex of Cr(VI) with the P-O groups of both ionophore being oriented towards the surface.

Keywords: AFM, Electrolyte/membrane/insulator/semiconductor structures, Polysiloxane membrane, Xps

Lacroix, D., (2009). Biomechanical aspects of bone repair Bone repair biomaterials (ed. Planell, J. A., Lacroix, D., Best, S., Merolli, A.), Woodhead (Cambridge, UK)

A fundamental aspect of the rapidly expanding medical care sector, bone repair continues to benefit from emerging technological developments. This text provides researchers and students with a comprehensive review of the materials science and engineering principles behind these developments. The first part reviews the fundamentals of bone repair and regeneration. Further chapters discuss the science and properties of biomaterials used in bone repair, including both metals and biocomposites. Final chapters analyze device considerations such as implant lifetime and failure, and discuss potential applications, as well as the ethical issues that continually confront researchers and clinicians.

Keywords: Bone composition and structure, Biomechanical properties of bone, Bone damage and repair

Morales, R., Riss, M., Wang, L., Gavin, R., Del Rio, J. A., Alcubilla, R., Claverol-Tinture, E., (2008). Integrating multi-unit electrophysiology and plastic culture dishes for network neuroscience Lab on a Chip 8, (11), 1896-1905

The electrophysiological characterisation of cultured neurons is of paramount importance for drug discovery, safety pharmacology and basic research in the neurosciences. Technologies offering low cost, low technical complexity and potential for scalability towards high-throughput electrophysiology on in vitro neurons would be advantageous, in particular for screening purposes. Here we describe a plastic culture substrate supporting low-complexity multi-unit loose-patch recording and stimulation of developing networks while retaining manufacturability compatible with low-cost and large-scale production. Our hybrid polydimethylsilane (PDMS)-on-polystyrene structures include chambers (6 mm in diameter) and microchannels (25 mu m x 3.7 mu m 1 mm) serving as substrate-embedded recording pipettes. Somas are plated and retained in the chambers due to geometrical constraints and their processes grow along the microchannels, effectively establishing a loose-patch configuration without human intervention. We demonstrate that off-the-shelf voltage-clamp, current-clamp and extracellular amplifiers can be used to record and stimulate multi-unit activity with the aid of our dishes. Spikes up to 50 pA in voltage-clamp and 300 mu V in current-clamp modes are recorded in sparse and bursting activity patterns characteristic of 1 week-old hippocampal cultures. Moreover, spike sorting employing principal component analysis (PCA) confirms that single microchannels support the recording of multiple neurons. Overall, this work suggests a strategy to endow conventional culture plasticware with added functionality to enable cost-efficient network electrophysiology.

Keywords: Electrophysiological characterisation, Cultured neurons, Polydimethylsilane (PDMS)-on-polystyrene structures

Engel, E., Del Valle, S., Aparicio, C., Altankov, G., Asin, L., Planell, J. A., Ginebra, M. P., (2008). Discerning the role of topography and ion exchange in cell response of bioactive tissue engineering scaffolds Tissue Engineering Part A , 14, (8), 1341-1351

Surface topography is known to have an influence on osteoblast activity. However, in the case of bioactive materials, topographical changes can affect also ion exchange properties. This makes the problem more complex, since it is often difficult to separate the strictly topographical effects from the effects of ionic fluctuations in the medium. The scope of this paper is to analyze the simultaneous effect of topography and topography-mediated ion exchange on the initial cellular behavior of osteoblastic-like cells cultured on bioactive tissue engineering substrates. Two apatitic substrates with identical chemical composition but different micro/nanostructural features were obtained by low-temperature setting of a calcium phosphate cement. MG63 osteoblastic-like cells were cultured either in direct contact with the substrates or with their extracts. A strong and permanent decrease of calcium concentration in the culture medium, dependent on substrate topography, was detected. A major effect of the substrate microstructure on cell proliferation was observed, explained in part by the topography-mediated ion exchange, but not specifically by the ionic Ca(2+) fluctuations. Cell differentiation was strongly enhanced when cells were cultured on the finer substrate. This effect was not explained by the chemical modification of the medium, but rather suggested a strictly topographical effect.

Keywords: Alkaline Phosphatase/metabolism, Bone Cements/pharmacology, Calcium/metabolism, Calcium Phosphates/pharmacology, Cell Adhesion/drug effects, Cell Differentiation/drug effects, Cell Proliferation/drug effects, Cell Shape/drug effects, Cells, Cultured, Culture Media, Durapatite/pharmacology, Humans, Interferometry, Ion Exchange, Materials Testing, Osteoblasts/ cytology/drug effects/enzymology/ultrastructure, Phosphorus/metabolism, Powders, Tissue Engineering, Tissue Scaffolds

Oncins, G., Torrent-Burgues, J., Sanz, F., (2008). Nanomechanical properties of arachidic acid Langmuir-Blodgett films Journal of Physical Chemistry C 112, (6), 1967-1974

The nanomechanical properties of Langmuir-Blodgett monolayers of arachidic acid extracted at surface pressures of 1, 15, and 35 mN/m and deposited on mica were investigated by atomic force microscopy, force spectroscopy, and lateral force microscopy. It was experimentally demonstrated that the arachidic acid molecular orientation depends on the extraction pressure. According to this, tilting angles of 50, 34, and 22 degrees with respect to the surface perpendicular were detected and identified as conformations that maximize van der Waals interactions between the arachidic acid alkyl chains. The vertical force needed to puncture the monolayers with the AFM tip strongly depends on the molecular tilting angles attained at different monolayer extraction surface pressures, obtaining values that range from 13.07 +/- 3.24 nN for 50 degrees to 22.94 +/- 5.49 nN for 22 degrees tilting angles. The different molecular interactions involved in the monolayer cohesion are discussed and quantitatively related to the experimental monolayer breakthrough forces. The friction measurements performed from low vertical forces up to monolayer disruption reveal the existence of three well-defined regimes: first, a low friction response due to the elastic deformation of the monolayer, which is followed by a sharp increase in the friction force due to the onset of a sudden plastic deformation. The last regime corresponds to the monolayer rupture and the contact between tip and substrate. The friction coefficient of the substrate is seen to depend on the monolayer extraction pressure, a fact that is discussed in terms of the relationship between the sample compactness and its rupture mechanism.

Keywords: AFM, SAM, Reflection-absortion spectroscopy, Lipid-bilayers, Frictional-properies, Molecular-structure, Thermal behavior, Nanometer-scale, Chain-length, LB films

Fernandez, Javier G., Mills, C. A., Martinez, E., Lopez-Bosque, M. J., Sisquella, X., Errachid, A., Samitier, J., (2008). Micro- and nanostructuring of freestanding, biodegradable, thin sheets of chitosan via soft lithography Journal of Biomedical Materials Research - Part A , 85A, (1), 242-247

A technique for imparting micro- and nano-structured topography into the surface of freestanding thin sheets of chitosan is described. Both micro- and nanometric surface structures have been produced using soft lithography. The soft lithography method, based on solvent evaporation, has allowed structures similar to 60 nm tall and similar to 500 X 500 nm(2) to be produced on freestanding similar to 0.5 mm thick sheets of the polymer when cured at 293 K, and structures similar to 400 nm tall and 5 X 5 mu m(2) to be produced when cured at 283 K. Nonstructured chitosan thin sheets (similar to 200 mu m thick) show excellent optical transmission properties in the visible portion of the electromagnetic spectrum. The structured sheets can be used for applications where optical microscopic analysis is required, such as cell interaction experiments and tissue engineering.

Keywords: Chitin/chitosan, Microstructure, Nanotopography, Polymerization, Soft lithography

Pla, M., Fernandez, Javier G., Mills, C. A., Martinez, E., Samitier, J., (2007). Micro/nanopatterning of proteins via contact printing using high aspect ratio PMMA stamps and NanoImprint apparatus Langmuir 23, (16), 8614-8618

Micro- and nanoscale protein patterns have been produced via a new contact printing method using a nanoimprint lithography apparatus. The main novelty of the technique is the use of poly(methyl methacrylate) (PMMA) instead of the commonly used poly(dimethylsiloxane) (PDMS) stamps. This avoids printing problems due to roof collapse, which limits the usable aspect ratio in microcontact printing to 10:1. The rigidity of the PMMA allows protein patterning using stamps with very high aspect ratios, up to 300 in this case. Conformal contact between the stamp and the substrate is achieved because of the homogeneous pressure applied via the nanoimprint lithography instrument, and it has allowed us to print lines of protein similar to 150 nm wide, at a 400 nm period. This technique, therefore, provides an excellent method for the direct printing of high-density sub-micrometer scale patterns, or, alternatively, micro-/nanopatterns spaced at large distances. The controlled production of these protein patterns is a key factor in biomedical applications such as cell-surface interaction experiments and tissue engineering.

Keywords: Soft lithography, Cell-adhesion, Microstructures, Fabrication, Stability, Patterns

Mills, C. A., Pla, M., Martin, C., Lee, M., Kuphal, M., Sisquella, X., Martinez, E., Errachid, A., Samitier, J., (2007). Structured thin organic active layers and their use in electrochemical biosensors Measurement & Control , 40, (3), 88-91

Díez-Pérez, Ismael, Vericat, Carolina, Gorostiza, Pau, Sanz, Fausto, (2006). The iron passive film breakdown in chloride media may be mediated by transient chloride-induced surface states located within the band gap Electrochemistry Communications , 8, (4), 627-632

Despite its tremendous scientific and economic impact, the mechanism that triggers metal passive film breakdown in the presence of aggressive ions remains under discussion. We have studied the iron passive film in chloride media using X-ray photoelectron spectroscopy (XPS), electrochemical impedance spectroscopy and electrochemical tunneling spectroscopy (ECTS). Ex situ XPS reveal that the film consists exclusively of an Fe(III) oxide without chloride content. In situ ECTS has been used to build up conductance maps of the Fe electrode during its electrochemical oxidation in a borate buffer solution and its breakdown when the film is grown in the presence of chloride. This conductograms provide direct and in situ experimental evidence of chloride-induced surface states within the band gap of the oxide film (~3.3eV). These states enable new charge exchange pathways that allow hole capture at the surface of the n-type Fe(III) oxide. The blocking of VB processes that occurs in the iron passive film is no longer present in chloride media, and electrode corrosion can proceed through these new states. We propose a simple 3-step mechanism for the process, in which chloride anions form an oxidizing Fe(II) surface intermediate but do not participate directly in the reaction.

Keywords: Electrochemical tunneling spectroscopy, Electronic band structure, Fe passive film, Aqueous chloride corrosion, Semiconductor decomposition, Interface states