Corrosion Rate and Mechanism of Degradation of Chitosan/TiO2 Coatings Deposited on MgZnCa Alloy in Hank's Solution.

Overly fast corrosion degradation of biodegradable magnesium alloys has been a major problem over the last several years. The development of protective coatings by using biocompatible, biodegradable, and non-toxic material such as chitosan ensures a reduction in the rate of corrosion of Mg alloys in simulated body fluids. In this study, chitosan/TiO2 nanocomposite coating was used for the first time to hinder the corrosion rate of Mg19Zn1Ca alloy in Hank's solution. The main goal of this research is to investigate and explain the corrosion degradation mechanism of Mg19Zn1Ca alloy coated by nanocomposite chitosan-based coating. The chemical composition, structural analyses, and corrosion tests were used to evaluate the protective properties of the chitosan/TiO2 coating deposited on the Mg19Zn1Ca substrate. The chitosan/TiO2 coating slows down the corrosion rate of the magnesium alloy by more than threefold (3.6 times). The interaction of TiO2 (NPs) with the hydroxy and amine groups present in the chitosan molecule cause their uniform distribution in the chitosan matrix. The chitosan/TiO2 coating limits the contact of the substrate with Hank's solution.

Morphological and Surface Potential Characterization of Protein Nanobiofilm Formation on Magnesium Alloy Oxide: Their Role in Biodegradation.

The formation of a protein nanobiofilm on the surface of degradable biomaterials such as magnesium (Mg) and its alloys influences metal ion release, cell adhesion/spreading, and biocompatibility. During the early stage of human body implantation, competition and interaction between inorganic species and protein molecules result in a complex film containing Mg oxide and a protein layer. This film affects the electrochemical properties of the metal surface, the protein conformational arrangement, and the electronic properties of the protein/Mg oxide interface. In this study, we discuss the impact of various simulated body fluids, including sodium chloride (NaCl), phosphate-buffered saline (PBS), and Hanks' solutions on protein adsorption, electrochemical interactions, and electrical surface potential (ESP) distribution at the adsorbed protein/Mg oxide interface. After 10 min of immersion in NaCl, atomic force microscopy (AFM) and scanning Kelvin probe force microscopy (SKPFM) showed a higher surface roughness related to enhanced degradation and lower ESP distribution on a Mg-based alloy than those in other solutions. Furthermore, adding bovine serum albumin (BSA) to all solutions caused a decline in the total surface roughness and ESP magnitude on the Mg alloy surface, particularly in the NaCl electrolyte. Using SKPFM surface analysis, we detected a protein nanobiofilm (∼10-20 nm) with an aggregated and/or fibrillary morphology only on the Mg surface exposed in Hanks' and PBS solutions; these surfaces had a lower ESP value than the oxide layer.

Mechanical stress affects methylation pattern of GNAS isoforms and osteogenic differentiation of hAT-MSCs.

Mechanical stress exerts a substantial role on skeletal-cell renewal systems, whereas accumulating evidence suggests that epigenetic mechanisms induce changes and differential gene expression. Although the underlying mechanisms remain to be fully elucidated, our study suggests that the influence of the long term mechanical stimulation elicits epigenetic modifications controlling osteogenic differentiation of human adipose tissue multipotential stromal cells (hAT-MSCs) and contributes to an accelerating in vitro osteogenesis. GNAS imprinting gene acts as a critical regulator of osteoblast differentiation and is implicated in human genetic disorders with pathological formation of ectopic-skeletal bone. Investigating a wide variety of stimuli, we showed that daily mechanical stretch on hAT-MSCs of 7th and 15th days' intervals induced a significant down-regulation in DNA methylation status of critical CpG sites of NESP and GNASXL isoforms, accompanied by up-regulation of the corresponding gene transcripts, and osteogenic differentiation earlier in culture. Importantly, methylation analysis of differentiating bone marrow-derived MSCs revealed similar methylation patterns. Bioinformatic analysis further showed that all CpG islands exhibiting significant methylation alterations encompassed transcriptional repressor CTCF binding sites. We hereby emphasize the need to investigate the epigenetic alterations on hAT-MSCs during environmental mechanical forces and to consider how the knowledge gained through these studies may foster new means of symptoms prevention and management of ectopic bone formation in the clinic.

Clinical Symptomatology of Anxiety and Family Function in Adolescents-The Self-Esteem Mediator.

BACKGROUND: Family, the child's first environment, shapes their psycho-emotional balance. The literature links adolescent anxiety to family relationships, interactions, and dynamics. The self-esteem of adolescents appears to protect their mental health. GOAL: This study examines whether family cohesion and adaptability affect adolescent anxiety symptoms. It also examines whether teen self-esteem mediates this relationship. METHOD: This cross-sectional, descriptive study included 166 Attica youth aged 12-18 from schools and educational units. The adolescents completed Olson's FACES-III cohesion and adaptability scale, Spielberger's STAI-C, Rosenberg's self-esteem scale, and a socio-demographic questionnaire. RESULTS: Family cohesion, but not adaptability, was negatively correlated with state (rho = -0.25, p = 0.001) and trait (rho = -0.46, p < 0.001) anxiety in the adolescents. Teenagers from extreme families with the lowest cohesion and adaptability had higher trait anxiety (x2(2) = 6.91, p = 0.032) than those from moderately balanced/balanced families. Self-esteem mediated the relationship between the family cohesion functioning and adolescent's state anxiety (p = 0.005) and trait anxiety (p = 0.011). CONCLUSIONS: The findings show that family dysfunction negatively impacts adolescent anxiety, as well as their self-esteem, which protects mental balance.

Albumin Protein Impact on Early-Stage In Vitro Biodegradation of Magnesium Alloy (WE43).

Mg and its alloys are promising biodegradable materials for orthopedic implants and cardiovascular stents. The first interactions of protein molecules with Mg alloy surfaces have a substantial impact on their biocompatibility and biodegradation. We investigate the early-stage electrochemical, chemical, morphological, and electrical surface potential changes of alloy WE43 in either 154 mM NaCl or Hanks' simulated physiological solutions in the absence or presence of bovine serum albumin (BSA) protein. WE43 had the lowest electrochemical current noise (ECN) fluctuations, the highest noise resistance (Zn = 1774 Ω·cm2), and the highest total impedance (|Z| = 332 Ω·cm2) when immersed for 30 min in Hanks' solution. The highest ECN, lowest Zn (1430 Ω·cm2), and |Z| (49 Ω·cm2) were observed in the NaCl solution. In the solutions containing BSA, a unique dual-mode biodegradation was observed. Adding BSA to a NaCl solution increased |Z| from 49 to 97 Ω·cm2 and decreased the ECN signal of the alloy, i.e., the BSA inhibited corrosion. On the other hand, the presence of BSA in Hanks' solution increased the rate of biodegradation by decreasing both Zn and |Z| while increasing ECN. Finally, using scanning Kelvin probe force microscopy (SKPFM), we observed an adsorbed nanolayer of BSA with aggregated and fibrillar morphology only in Hanks' solution, where the electrical surface potential was 52 mV lower than that of the Mg oxide layer.

Electronic properties and surface potential evaluations at the protein nano-biofilm/oxide interface: Impact on corrosion and biodegradation.

The formation of a protein nano-biofilm, which exhibits a special electronic behavior, on the surface of metals or oxide biomaterials considerably influences the crucial subsequent interactions, particularly the corrosion and biodegradation processes. This study discusses the impact of electrical surface potential (ESP) of a single or nano-biofilm of albumin protein on the electrochemical interactions and electronic property evolutions (e.g., charge carriers, space charge capacitance (SCC), and band bending) occurring on the surface oxide of CoCrMo implants. Scanning Kelvin probe force microscopy (SKPFM) results indicated that ESP or surface charge distribution on a single or nano-biofilm of the albumin protein is lower than that of a CoCrMo complex oxide layer, which hinders the charge transfer at the protein/electrolyte interface. Using a complementary approach, which involved performing Mott-Schottky analysis at the electrolyte/protein/oxide interface, it was revealed that the albumin protein significantly increases the SCC magnitude and number of n-type charge carrier owing to increased band bending at the SCC/protein interface; this facilitated the acceleration of metal ion release and metal-protein complex formation. The nanoscale SKPFM and electrochemical analyses performed in this study provide a better understanding of the role of protein molecules in corrosion/biodegradation of metallic biomaterials at the protein nano-biofilm/oxide interface.

On Growth and Morphology of TiO2 Nanotubes on CP-Ti by Anodic Oxidation in Ethylene Glycol Electrolyte: Influence of Electrolyte Aging and Anodization Parameters.

Anodic oxidation of CP-Ti, for production of TiO2 nanotubes, has been extensively described in terms of the electrochemical mechanism of tubular growth or the effect of the parameters on the final tube morphology. Recently, a kinetic growth model was proposed to describe the distinct morphologies of the anodic oxide layer as phases of the nanotubular development process, offering a new perspective for the tuning of nanotube production. In this work, the anodizing behavior of a CP-Ti alloy in an ethylene glycol electrolyte was investigated in light of this new model. The final morphology of the nanotubes was characterized by SEM, considering the effects of electrolyte aging, the microstructure, the applied potential difference and time on the morphological development of nanotubes. Electrolyte aging was shown to lead to a decreased dissolution effect on the oxide. The applied potential difference was shown to lead to an increased dissolution effect and more rapid nanotube growth kinetics, while time resulted in extended dissolution. Moreover, the obtained results were analyzed considering a previous study focused on the anodizing behavior of the α- and ß-phases of Ti6Al4V alloy. Overall, the tube morphology resembled that obtained for the Al-containing α-phase of the Ti6Al4V alloy, but the growth kinetics were considerably slower on CP-Ti.

On Growth and Morphology of TiO2 Nanotubes on Ti6Al4V by Anodic Oxidation in Ethylene Glycol Electrolyte: Influence of Microstructure and Anodization Parameters.

Different studies demonstrated the possibility to produce TiO2 nanotubes (TNTs) on Ti6Al4V alloy by electrochemical anodization. However, the anodizing behavior of α and ß-phases in organic electrolytes is not yet clarified. This study reports on the anodizing behavior of the two phases in an ethylene glycol electrolyte using different applied potentials and anodizing times. Atomic force and scanning electron microscopies were used to highlight the anodic oxides differences in morphology. It was demonstrated that the initial compact oxide grew faster over the ß-phase as the higher Al content of the α-phase caused its re-passivation, and the higher solubility of the V-rich oxide led to earlier pores formation over the ß-phase. The trend was inverted once the pores formed over the compact oxide of the α-phase. The growth rate of the α-phase TNTs was higher than that of the ß-phase ones, leading to the formation of long and well defined nanotubes with thin walls and a honeycomb tubular structure, while the ones grown over the ß-phase were individual, shorter, and with thicker walls.

Plastic electrode decorated with polyhedral anion tetrabutylammonium octamolybdate [N(C4H9)4]4 Mo8O26 for nM phosphate electrochemical detection.

Inorganic phosphorous (as phosphate (PO43-), is one of the essential nutrients for all living forms, either terrestrial or marine. In oligotrophic seawaters, this macronutrient is limited (10-9 M) and its ratio with other elements (nitrogen or carbon) is denoting the health state of the marine environment; a small variation of its concentration can produce eutrophication. The gold standard method used for PO43- detection is based on colorimetric detection of phosphomolybdate. The colored complex is obtained by mixing water-soluble molybdenum salts (Mo(VI)) and reducing agents in acid media, along with the sample containing PO43-. Moreover, the kinetic of complex formation is slow, about 1 h is generally required for color to develop, exposing the assay to the drawbacks of interferences as those from silica. The detection is preferably performed in a controlled environment (i.e. in a laboratory) because several chemicals and steps of preparations are required as well as the optical instrumentation is not intended for in-field use. Electrochemical sensors offer portability and simplicity making them a practical option for on-site detection applications. To gain an analytical alternative in measuring low quantities of PO43- (10-9 M), and overcome some of the drawbacks of the classical approaches, we optimised a new easy way to produce a plastic electrode decorated with an alkyl Mo-polyoxometalate (Mo8O264-), that is soluble in organic solvents. This tetra-butyl-ammonium octamolybdate powder, [N (C4H9)4]4 Mo8O26, purposely synthetized was identified with FTIR, Raman, MS methods, and the electroactivity and reactivity with PO43- was confirmed in solution with cyclic voltammetry (CV). When the Mo-decorated electrode was in contact with PO43-, an electroactive phosphomolybdate aggregate formed at the electrode surface that was electrochemically detectable with square wave voltammetry (SWV). A remarkably low detection limit of 6.1 nM, to PO43-, as well as good stability and selectivity were obtained also in real samples. In fact, PO43- was measured in saline simulated and real seawater samples at nM concentrations in less than 5 min. The present investigation provides a new alternative to the current standard colorimetric methods to detect low phosphate concentrations, showing the potential to be used for monitoring nutrients in oligotrophic seawater.

Membrane perturbation, altered morphology and killing of Staphylococcus epidermidis upon contact with a cytocompatible peptide-based antibacterial surface.

One possibility to prevent prosthetic infections is to produce biomaterials resistant to bacterial colonization by anchoring membrane active antimicrobial peptides (AMPs) onto the implant surface. In this perspective, a deeper understanding of the mode of action of the immobilized peptides should improve the development of AMP-inspired infection-resistant biomaterials. The aim of the present study was to characterize the bactericidal mechanism against Staphylococcus epidermidis of the AMP BMAP27(1-18), immobilized on titanium disks and on a model resin support, by applying viability counts, Field Emission Scanning Electron Microscopy (FE-SEM), and a fluorescence microplate assay with a membrane potential-sensitive dye. The cytocompatibility to osteoblast-like MG-63 cells was investigated in monoculture and in co-culture with bacteria. The impact of peptide orientation was explored by using N- and C- anchored analogues. On titanium, the ∼50 % drop in bacteria viability and dramatically affected morphology indicate a contact-killing action exerted by the N- and C-immobilized peptides to the same extent. As further shown by the fluorescence assay with the resin-anchored peptides, the bactericidal effect was mediated by rapid membrane perturbation, similar to free peptides. However, at peptide MBC resin equivalents the C-oriented analogue proved more effective with more than 99 % killing and maximum fluorescence increase, compared to half-maximum fluorescence with more than 90 % killing produced by the N-orientation. Confocal microscopy analyses revealed 4-5 times better MG-63 cell adhesion on peptide-functionalized titanium both in monoculture and in co-culture with bacteria, regardless of peptide orientation, thus stimulating further studies on the effects of the immobilized BMAP27(1-18) on osteoblast cells.

A Rapid Fluorescence-Based Microplate Assay to Investigate the Interaction of Membrane Active Antimicrobial Peptides with Whole Gram-Positive Bacteria.

BACKGROUND: Membrane-active antimicrobial peptides (AMPs) are interesting candidates for the development of novel antimicrobials. Although their effects were extensively investigated in model membrane systems, interactions of AMPs with living microbial membranes are less known due to their complexity. The aim of the present study was to develop a rapid fluorescence-based microplate assay to analyze the membrane effects of AMPs in whole Staphylococcus aureus and Staphylococcus epidermidis. METHODS: Bacteria were exposed to bactericidal and sub-inhibitory concentrations of two membrane-active AMPs in the presence of the potential-sensitive dye 3,3'-dipropylthiadicarbocyanine iodide (diSC3(5)) and the DNA staining dye propidium iodide (PI), to simultaneously monitor and possibly distinguish membrane depolarization and membrane permeabilization. RESULTS: The ion channel-forming gramicidin D induced a rapid increase of diSC3(5), but not PI fluorescence, with slower kinetics at descending peptide concentrations, confirming killing due to membrane depolarization. The pore-forming melittin, at sub-MIC and bactericidal concentrations, caused, respectively, an increase of PI fluorescence in one or both dyes simultaneously, suggesting membrane permeabilization as a key event. CONCLUSIONS: This assay allowed the distinction between specific membrane effects, and it could be applied in the mode of action studies as well as in the screening of novel membrane-active AMPs.