Simulation of the Influence of the Radial Graded Porosity Distribution on Elastic Modulus of γ/ß Phase Ti-Based Alloy Foams for Bone Implant.

This research aims to examine how a radial graded porosity distribution affects the elastic modulus by conducting simulations on Ti-based alloy foams with face-centered cubic and body-centered cubic crystal structures. Four types of foams were analyzed; commercially pure-Ti, Ti-13Ta-6Mn (TTM), Ti-13Ta-(TT) and Ti-13Ta-6Sn (TTS), (all in at.%). Four radial graded porosity distribution configurations were modeled and simulated using the finite element analysis (FEA). The radial graded porosity distribution configurations were generated using a Material Designer (Ansys) with a pore range of 200 to 600 µm. These radial graded porosity distributions had average porosity values of 0, 20, 30 and 40%. The consolidated samples that were obtained through a powder metallurgy technique in two step samples were synthesized using a powder metallurgy technique, with the elastic moduli values of the aforementioned Ti based alloys being measured by ultrasound using ~110, ~69, ~61 and ~65 GPa, respectively. The results showed that the modulus decreased as a function of porosity level in all simulated materials. The TTM, TT and TTS foams, with average porosities of 20, 30 and 40%, exhibited an modulus smaller than 30 GPa, which is a requirement to be used as a biomaterial in human bones. The TT foams showed the lowest modulus when compared to the other foams. Finally, certain theoretical models were used to obtain the modulus, the best being; the Gibson-Ashby model (α = 1 and n = 2.5) for the cp-Ti foams and Knudsen-Spriggs model (b = 3.06) for the TTM, TT and TTS foams.

Computational Study of the Influence of α/ß-Phase Ratio and Porosity on the Elastic Modulus of Ti-Based Alloy Foams.

This work aims to perform a computational analysis on the influence that microstructure and porosity have on the elastic modulus of Ti-6Al-4V foams used in biomedical applications with different α/ß-phase ratios. The work is divided into two analyses, first the influence that the α/ß-phase ratio has and second the effects that porosity and α/ß-phase ratio have on the elastic modulus. Two microstructures were analyzed: equiaxial α-phase grains + intergranular ß-phase (microstructure A) and equiaxial ß-phase grains + intergranular α-phase (microstructure B). The α/ß-phase ratio was variated from 10 to 90% and the porosity from 29 to 56%. The simulations of the elastic modulus were carried out using finite element analysis (FEA) using ANSYS software v19.3. The results were compared with experimental data reported by our group and those found in the literature. The ß-phase amount and porosity have a synergic effect on the elastic modulus, for example, when the foam has a porosity of 29 with 0% ß-phase, and it has an elastic modulus of ≈55 GPa, but when the ß-phase amount increases to 91%, the elastic modulus decreases as low as 38 GPa. The foams with 54% porosity have values smaller than 30 GPa for all the ß-phase amounts.

Fabrication and Arc Erosion Behavior of Ag-SnO2-ZnO Electrical Contact Materials.

This study investigated the synthesis of Ag-SnO2-ZnO by powder metallurgy methods and their subsequent electrical contact behavior. The pieces of Ag-SnO2-ZnO were prepared by ball milling and hot pressing. The arc erosion behavior of the material was evaluated using homemade equipment. The microstructure and phase evolution of the materials were investigated through X-ray diffraction, energy-dispersive spectroscopy and scanning electron microscopy. The results showed that, although the mass loss of the Ag-SnO2-ZnO composite (9.08 mg) during the electrical contact test was higher than that of the commercial Ag-CdO (1.42 mg), its electrical conductivity remained constant (26.9 ± 1.5% IACS). This fact would be related to the reaction of Zn2SnO4's formation on the material's surface via electric arc. This reaction would play an important role in controlling the surface segregation and subsequent loss of electrical conductivity of this type of composite, thus enabling the development of a new electrical contact material to replace the non-environmentally friendly Ag-CdO composite.

Display of Heterologous Proteins in Bacillus Subtilis Biofilms for Enteric Immunization.

One of the foremost goals in vaccine development is the design of effective, heat-stable vaccines that simplify the distribution and delivery while conferring high levels of protective immunity. Here, we describe a method for developing a live, oral vaccine that relies on the biofilm-forming properties of the spore-former bacterium Bacillus subtilis. The amyloid protein TasA is an abundant component of the extracellular matrix of the biofilms formed by B. subtilis that can be genetically fused to an antigen of interest. Spores of the recombinant strain are then prepared and applied via the oral route in an animal model. Due to the intrinsic resistance of the spores, they can bypass the stomach barrier, germinate, and subsequently colonize the gut, where they develop into biofilms, expressing the antigen of interest. We describe here the steps necessary to produce spores, immunization, and downstream analysis of the vaccine efficacy.

A Tribological and Ion Released Research of Ti-Materials for Medical Devices.

The increase in longevity worldwide has intensified the use of different types of prostheses for the human body, such as those used in dental work as well as in hip and knee replacements. Currently, Ti-6Al-4V is widely used as a joint implant due to its good mechanical properties and durability. However, studies have revealed that this alloy can release metal ions or particles harmful to human health. The mechanisms are not well understood yet and may involve wear and/or corrosion. Therefore, in this work, commercial pure titanium and a Ti-6Al-4V alloy were investigated before and after being exposed to a simulated biological fluid through tribological tests, surface analysis, and ionic dissolution characterization by ICP-AES. Before exposure, X-ray diffraction and optical microscopy revealed equiaxed α-Ti in both materials and ß-Ti in Ti-6Al-4V. Scratch tests exhibited a lower coefficient of friction for Ti-6Al-4V alloy than commercially pure titanium. After exposure, X-ray photoelectron spectroscopy and surface-enhanced Raman spectroscopy results showed an oxide film formed by TiO2, both in commercially pure titanium and in Ti-6Al-4V, and by TiO and Al2O3 associated with the presence of the alloys. Furthermore, inductively coupled plasma atomic emission spectroscopy revealed that aluminum was the main ion released for Ti-6Al-4V, giving negligible values for the other metal ions.

The effect of Staphylococcus aureus on the electrochemical behavior of porous Ti-6Al-4V alloy.

Ti-6Al-4V alloy has been widely investigated for biomedical applications due to its low density, high specific strength, and favorable corrosion resistance. However, some reported failures have imposed a challenge to improve bone regeneration and fixation, as well as antibacterial properties. A further opportunity for solving this problem is the introduction of porosity. However, this can induce metallic release and corrosion product formation. In this work, a Ti-6Al-4V alloy was exposed to Hank's solution, sterilized and inoculated with Staphylococcus aureus at 37 °C. Surface analysis was carried out by SEM-EDS and XPS. Electrochemical measurements were also performed using chronopotentiometry at open circuit potential, polarization curves, and electrochemical impedance spectroscopy. After exposure, FE-SEM showed some colonies of S. aureus on the sample with 22% porosity. However, XPS analysis revealed that the presence of bacterium influenced the composition of the oxide layer, even more drastically with the increase in added porosity. Moreover, the impedance analysis showed De Levie's behavior, revealing a reduction of pore resistance and modulus of the impedance in the low frequency range in inoculated medium, and polarization curves showed that the passivity potential range was decreased, whereas the passivity current increased in the presence of the S. aureus.

Tribological Performance of Porous Ti-Nb-Ta-Fe-Mn Alloy in Dry Condition.

The tribological properties of a novel porous Ti-Nb-Ta-Fe-Mn alloy with 0%, 30%, and 60% porosity were evaluated for biomedical applications. The tribotesting was performed using a ball-on-disc under dry conditions, using an alumina ball and 1 N of a load. The coefficient of friction at the early stage of the porous samples was lower than that of the bulk, 0.2 and 0.7, respectively, but the samples with 30% porosity shift toward the bulk value after a variable number of cycles, while the samples with 60% remained stable after 100,000 cycles. The wear rate of the specimen with 60% porosity was twice as low as that of the bulk. The results are explained by shift in wear mechanism associated with the modified bearing ratio of the porous surface and by the accumulation of wear debris inside the pores, which prevented the development of three-body abrasion.

Effect of added porosity on a novel porous Ti-Nb-Ta-Fe-Mn alloy exposed to simulated body fluid.

Porous titanium materials have gained interest as prosthesis materials due to their similar mechanical properties to the human bone, biocompatibility, and high corrosion resistance. The presence of pores in the metal matrix implies a decrease in the elastic modulus and an increase in the active area, perhaps improving the osseointegration. Corrosion resistance is a critical consideration as corrosion may lead not only to mechanical failure but also the release of ions and/or particles to the bloodstream. In this work, a novel Ti-Nb-Ta-Fe-Mn alloy with varying percentage of porosity (25, 31 and 37 v/v%) was exposed to simulated body fluid (SBF) at 37 °C and its corrosion resistance was investigated using electrochemical techniques and surface analysis as a function of exposure time. Open circuit potential and polarization curves revealed that the effect of porosity was mainly on the shift of the corrosion potential to more negative values with a slight increase in the anodic current. A passive range was also observed, which was not influenced either by increased exposure time or increased porosity. Therefore, a change in the surface specific area could have taken place during the exposure, which is not necessarily related to a corrosion process. Moreover, a typical porous electrode behavior was identified by electrochemical Impedance spectroscopy, without any significant change over time. No release of metal ions was detected by on line ICP-AES, either at the open circuit potential or upon polarizing the samples up to 2 V vs. SCE, whereas only traces elements (Fe and Mn 1 nmol/s cm2) were detected in the electrolyte accumulating all released ions during 30 days of exposure. Additionally, the surface analysis showed thickening of the oxide layer with exposure time. Therefore, the stability of the passive layer and low release of ions indicate that the porous alloys are suitable for further study as prosthesis materials.

Linear Versus Nonlinear Acoustic Probing of Plasticity in Metals: A Quantitative Assessment.

The relative dislocation density of aluminum and copper samples is quantitatively measured using linear Resonant Ultrasound Spectroscopy (RUS). For each metallic group, four samples were prepared with different thermomechanical treatments in order to induce changes in their dislocation densities. The RUS results are compared with Nonlinear Resonant Ultrasound Spectroscopy (NRUS) as well as Second Harmonic Generation (SHG) measurements. NRUS has a higher sensitivity by a factor of two to six and SHG by 14⁻62%. The latter technique is, however, faster and simpler. As a main result, we obtain a quantitative relation between the changes in the nonlinear parameters and the dislocation density variations, which in a first approximation is a linear relation between these differences. We also present a simple theoretical expression that explains the better sensitivity to dislocation content of the nonlinear parameters with respect to the linear ones. X-Ray diffraction measurements, although intrusive and less accurate, support the acoustics results.

Mouse intestinal microbiota reduction favors local intestinal immunity triggered by antigens displayed in Bacillus subtilis biofilm.

BACKGROUND: We previously engineered Bacillus subtilis to express an antigen of interest fused to TasA in a biofilm. B. subtilis has several properties such as sporulation, biofilm formation and probiotic ability that were used for the oral application of recombinant spores harboring Echinococcus granulosus paramyosin and tropomyosin immunogenic peptides that resulted in the elicitation of a specific humoral immune response in a dog model. RESULTS: In order to advance our understanding of the research in oral immunization practices using recombinant B. subtilis spores, we describe here an affordable animal model. In this study, we show clear evidence indicating that a niche is required for B. subtilis recombinant spores to colonize the densely populated mice intestinal microbiota. The reduction of intestinal microbiota with an antibiotic treatment resulted in a positive elicitation of local humoral immune response in BALB/c mice after oral application of recombinant B. subtilis spores harboring TasA fused to E. granulosus (102-207) EgTrp immunogenic peptide. Our results were supported by a lasting prevalence of spores in mice feces up to 50 days after immunization and by the presence of specific secretory IgA, isolated from feces, against E. granulosus tropomyosin. CONCLUSIONS: The reduction of mouse intestinal microbiota allowed the elicitation of a local humoral immune response in mice after oral application with spores of B. subtilis harboring immunogenic peptides against E. granulosus.

Oral Application of Recombinant Bacillus subtilis Spores to Dogs Results in a Humoral Response against Specific Echinococcus granulosus Paramyosin and Tropomyosin Antigens.

Bacillus subtilis is known as an endospore- and biofilm-forming bacterium with probiotic properties. We have recently developed a method for displaying heterologous proteins on the surface of B. subtilis biofilms by introducing the coding sequences of the protein of interest into the bacterial genome to generate a fusion protein linked to the C terminus of the biofilm matrix protein TasA. Although B. subtilis is a regular component of the gut microflora, we constructed a series of recombinant B. subtilis strains that were tested for their ability to be used to immunize dogs following oral application of the spores. Specifically, we tested recombinant spores of B. subtilis carrying either the fluorescent protein mCherry or else selected antigenic peptides (tropomyosin and paramyosin) from Echinococcus granulosus, a zoonotic intestinal tapeworm of dogs and other carnivores. The application of the recombinant B. subtilis spores led to the colonization of the gut with recombinant B. subtilis but did not cause any adverse effect on the health of the animals. As measured by enzyme-linked immunosorbent assay and immunoblotting, the dogs were able to develop a humoral immune response against mCherry as well as against E. granulosus antigenic peptides. Interestingly, the sera of dogs obtained after immunization with recombinant spores of E. granulosus peptides were able to recognize E. granulosus protoscoleces, which represent the infective form of the head of the tapeworms. These results represent an essential step toward the establishment of B. subtilis as an enteric vaccine agent.

Heterologous expression of antigenic peptides in Bacillus subtilis biofilms.

BACKGROUND: Numerous strategies have been developed for the display of heterologous proteins in the surface of live bacterial carriers, which can be used as vaccines, immune-modulators, cancer therapy or bioremediation. Bacterial biofilms have emerged as an interesting approach for the expression of proteins of interest. Bacillus subtilis is a well-described, endospore-forming organism that is able to form biofilms and also used as a probiotic, thus making it a suitable candidate for the display of heterologous proteins within the biofilm. Here, we describe the use of TasA, an important structural component of the biofilms formed by B. subtilis, as a genetic tool for the display of heterologous proteins. RESULTS: We first engineered the fusion protein TasA-mCherry and showed that was widely deployed within the B. subtilis biofilms. A significant enhancement of the expression of TasA-mCherry within the biofilm was obtained when depleting both tasA and sinR genes. We subsequently engineered fusion proteins of TasA to antigenic peptides of the E. granulosus parasite, paramyosin and tropomyosin. Our results show that the antigens were well expressed within the biofilm as denoted by macrostructure complementation and by the detection of the fusion protein in both immunoblot and immunohistochemistry. In addition, we show that the recombinant endospores of B. subtilis preserve their biophysical and morphological properties. CONCLUSIONS: In this work we provide strong evidence pointing that TasA is a suitable candidate for the display of heterologous peptides, such as antigens, cytokines, enzymes or antibodies, in the B. subtilis biofilms. Finally, our data portray that the recombinant endospores preserve their morphological and biophysical properties and could be an excellent tool to facilitate the transport and the administration.

Evidence for the widespread production of DSF family signal molecules by members of the genus Burkholderia by the aid of novel biosensors.

Many bacteria employ cis-2-unsaturated fatty acids, referred to as DSF (diffusible signal factor) family signals, to communicate with each other. Such systems have been shown to control biofilm formation, motility, production of hydrolytic enzymes and expression of virulence factors. We report the construction of novel biosensors on the basis of components of the Burkholderia-DSF (BDSF) dependent circuitry of Burkholderia cenocepacia H111 and evaluated their utility for detecting the production of DSF family signal molecules. We show that a luxAB-based biosensor responds to nM levels of synthetic BDSF and is suitable to detect a wide range of cis-2 fatty acid molecules. Using this biosensor we show that the production of DSF family molecules is widespread among members of the B. cepacia complex and demonstrate for the first time that DSF-based molecules are also produced by plant-associated Burkholderia species.

σ54-Dependent Response to Nitrogen Limitation and Virulence in Burkholderia cenocepacia Strain H111.

Members of the genus Burkholderia are versatile bacteria capable of colonizing highly diverse environmental niches. In this study, we investigated the global response of the opportunistic pathogen Burkholderia cenocepacia H111 to nitrogen limitation at the transcript and protein expression levels. In addition to a classical response to nitrogen starvation, including the activation of glutamine synthetase, PII proteins, and the two-component regulatory system NtrBC, B. cenocepacia H111 also upregulated polyhydroxybutyrate (PHB) accumulation and exopolysaccharide (EPS) production in response to nitrogen shortage. A search for consensus sequences in promoter regions of nitrogen-responsive genes identified a σ(54) consensus sequence. The mapping of the σ(54) regulon as well as the characterization of a σ(54) mutant suggests an important role of σ(54) not only in control of nitrogen metabolism but also in the virulence of this organism.

The IclR-family regulator BapR controls biofilm formation in B. cenocepacia H111.

In Burkholderia cenocepacia H111, the large surface protein BapA plays a crucial role in the formation of highly structured communities, known as biofilms. We have recently demonstrated that quorum sensing (QS) is necessary for the maximal expression of bapA. In this study we identify BapR, a protein from the IclR family of transcriptional regulators that, in conjunction with QS, controls biofilm formation by affecting the expression of bapA. We present evidence that, in addition to bapA, BapR influences the expression of extracellular proteases, swimming motility and has a profound impact in the incidence of persister cells, making this regulator an interesting target for persister cells and biofilm eradication.

Two quorum sensing systems control biofilm formation and virulence in members of the Burkholderia cepacia complex.

The Burkholderia cepacia complex (Bcc) consists of 17 closely related species that are problematic opportunistic bacterial pathogens for cystic fibrosis patients and immunocompromised individuals. These bacteria are capable of utilizing two different chemical languages: N-acyl homoserine lactones (AHLs) and cis-2-unsaturated fatty acids. Here we summarize the current knowledge of the underlying molecular architectures of these communication systems, showing how they are interlinked and discussing how they regulate overlapping as well as specific sets of genes. A particular focus is laid on the role of these signaling systems in the formation of biofilms, which are believed to be highly important for chronic infections. We review genes that have been implicated in the sessile lifestyle of this group of bacteria. The new emerging role of the intracellular second messenger cyclic dimeric guanosine monophosphate (c-di-GMP) as a downstream regulator of the fatty acid signaling cascade and as a key factor in biofilm formation is also discussed.

The AHL- and BDSF-dependent quorum sensing systems control specific and overlapping sets of genes in Burkholderia cenocepacia H111.

Quorum sensing in Burkholderia cenocepacia H111 involves two signalling systems that depend on different signal molecules, namely N-acyl homoserine lactones (AHLs) and the diffusible signal factor cis-2-dodecenoic acid (BDSF). Previous studies have shown that AHLs and BDSF control similar phenotypic traits, including biofilm formation, proteolytic activity and pathogenicity. In this study we mapped the BDSF stimulon by RNA-Seq and shotgun proteomics analysis. We demonstrate that a set of the identified BDSF-regulated genes or proteins are also controlled by AHLs, suggesting that the two regulons partially overlap. The detailed analysis of two mutually regulated operons, one encoding three lectins and the other one encoding the large surface protein BapA and its type I secretion machinery, revealed that both AHLs and BDSF are required for full expression, suggesting that the two signalling systems operate in parallel. In accordance with this, we show that both AHLs and BDSF are required for biofilm formation and protease production.

Identification of functions linking quorum sensing with biofilm formation in Burkholderia cenocepacia H111.

Burkholderia cenocepacia has emerged as an important pathogen for patients suffering from cystic fibrosis (CF). Previous work has shown that this organism employs the CepIR quorum-sensing (QS) system to control the expression of virulence factors as well as the formation of biofilms. To date, however, very little is known about the QS-regulated virulence factors and virtually nothing about the factors that link QS and biofilm formation. Here, we have employed a combined transcriptomic and proteomic approach to precisely define the QS regulon in our model strain B. cenocepacia H111, a CF isolate. Among the identified CepR-activated loci, three were analyzed in better detail for their roles in biofilm development: (i) a gene cluster coding for the BclACB lectins, (ii) the large surface protein BapA, and (iii) a type I pilus. The analysis of defined mutants revealed that BapA plays a major role in biofilm formation on abiotic surfaces while inactivation of the type I pilus showed little effect both in a static microtitre dish-based biofilm assay and in flow-through cells. Inactivation of the bclACB lectin genes resulted in biofilms containing hollow microcolonies, suggesting that the lectins are important for biofilm structural development.

Cis-2-dodecenoic acid receptor RpfR links quorum-sensing signal perception with regulation of virulence through cyclic dimeric guanosine monophosphate turnover.

Many bacterial pathogens produce diffusible signal factor (DSF)-type quorum sensing (QS) signals in modulation of virulence and biofilm formation. Previous work on Xanthomonas campestris showed that the RpfC/RpfG two-component system is involved in sensing and responding to DSF signals, but little is known in other microorganisms. Here we show that in Burkholderia cenocepacia the DSF-family signal cis-2-dodecenoic acid (BDSF) negatively controls the intracellular cyclic dimeric guanosine monophosphate (c-di-GMP) level through a receptor protein RpfR, which contains Per/Arnt/Sim (PAS)-GGDEF-EAL domains. RpfR regulates the same phenotypes as BDSF including swarming motility, biofilm formation, and virulence. In addition, the BDSF(-) mutant phenotypes could be rescued by in trans expression of RpfR, or its EAL domain that functions as a c-di-GMP phosphodiesterase. BDSF is shown to bind to the PAS domain of RpfR with high affinity and stimulates its phosphodiesterase activity through induction of allosteric conformational changes. Our work presents a unique and widely conserved DSF-family signal receptor that directly links the signal perception to c-di-GMP turnover in regulation of bacterial physiology.