A Salmonella Typhimurium-Typhi genomic chimera: a model to study Vi polysaccharide capsule function in vivo.

The Vi capsular polysaccharide is a virulence-associated factor expressed by Salmonella enterica serotype Typhi but absent from virtually all other Salmonella serotypes. In order to study this determinant in vivo, we characterised a Vi-positive S. Typhimurium (C5.507 Vi(+)), harbouring the Salmonella pathogenicity island (SPI)-7, which encodes the Vi locus. S. Typhimurium C5.507 Vi(+) colonised and persisted in mice at similar levels compared to the parent strain, S. Typhimurium C5. However, the innate immune response to infection with C5.507 Vi(+) and SGB1, an isogenic derivative not expressing Vi, differed markedly. Infection with C5.507 Vi(+) resulted in a significant reduction in cellular trafficking of innate immune cells, including PMN and NK cells, compared to SGB1 Vi(-) infected animals. C5.507 Vi(+) infection stimulated reduced numbers of TNF-α, MIP-2 and perforin producing cells compared to SGB1 Vi(-). The modulating effect associated with Vi was not observed in MyD88(-/-) and was reduced in TLR4(-/-) mice. The presence of the Vi capsule also correlated with induction of the anti-inflammatory cytokine IL-10 in vivo, a factor that impacted on chemotaxis and the activation of immune cells in vitro.

Composite hydrogels for nucleus pulposus tissue engineering

Tissue engineering offers a paradigm shift in the treatment of back pain. Engineered intervertebral discs could replace degenerated tissue and overcome the limitations of current treatments, which substantially alter the biomechanical properties of the spine. The centre of the disc, the nucleus pulposus, is an amorphous gel with a large bound water content and it can resist substantial compressive loads. Due to similarities in their compositions, hydrogels have frequently been considered as substitutes for the nucleus pulposus. However, there has been limited work characterising the time-dependent mechanical behaviour of hydrogel scaffolds for nucleus pulposus tissue engineering. Poroelastic behaviour, which plays a key role in nutrient transport, is of particular importance. Here, we investigate the time-dependent mechanical properties of gelatin and agar hydrogels and of gelatin-agar composites. The time-dependent properties of these hydrogels are explored using viscoelastic and poroelastic frameworks. Several gel formulations demonstrate comparable equilibrium elastic behaviour to the nucleus pulposus under unconfined compression, but permeability values that are much greater than those of the native tissue. A range of time-dependent responses are observed in the composite gels examined, presenting the opportunity for targeted design of custom hydrogels with combinations of mechanical properties optimized for tissue engineering applications. © 2011 Elsevier Ltd.

A family of simple benzene 1,3,5-tricarboxamide (BTA) aromatic carboxylic acid hydrogels.

We present the characterisation of a hydrogel forming family of benzene 1,3,5-tricarboxamide (BTA) aromatic carboxylic acid derivatives. The simple, easy to synthesise compounds presented here exhibit consistent gel formation at low concentrations through the use of a pH trigger.

Separating poroviscoelastic deformation mechanisms in hydrogels

Hydrogels have applications in drug delivery, mechanical actuation, and regenerative medicine. When hydrogels are deformed, load-relaxation arising from fluid flow - poroelasticity - and from rearrangement of the polymer network - viscoelasticity - is observed. The physical mechanisms are different in that poroelastic relaxation varies with experimental length-scale while viscoelastic does not. Here, we show that poroviscoelastic load-relaxation is the product of the two individual responses. The difference in length-scale dependence of the two mechanisms can be exploited to uniquely determine poroviscoelastic properties from simultaneous analysis of multi-scale indentation experiments, providing insight into hydrogel physical behavior. © 2013 American Institute of Physics.

Mechanical behaviour of electrospun fibre-reinforced hydrogels.

Mechanically robust and biomimicking scaffolds are needed for structural engineering of tissues such as the intervertebral disc, which are prone to failure and incapable of natural healing. Here, the formation of thick, randomly aligned polycaprolactone electrospun fibre structures infiltrated with alginate is reported. The composites are characterised using both indentation and tensile testing and demonstrate substantially different tensile and compressive moduli. The composites are mechanically robust and exhibit large strains-to-failure, exhibiting toughening mechanisms observed in other composite material systems. The method presented here provides a way to create large-scale biomimetic scaffolds that more closely mimic the composite structure of natural tissue, with tuneable tensile and compressive properties via the fibre and matrix phases, respectively.

Mechanical behaviour of electrospun fibre-reinforced hydrogels

Mechanically robust and biomimicking scaffolds are needed for structural engineering of tissues such as the intervertebral disc, which are prone to failure and incapable of natural healing. Here, the formation of thick, randomly aligned polycaprolactone electrospun fibre structures infiltrated with alginate is reported. The composites are characterised using both indentation and tensile testing and demonstrate substantially different tensile and compressive moduli. The composites are mechanically robust and exhibit large strains-to-failure, exhibiting toughening mechanisms observed in other composite material systems. The method presented here provides a way to create large-scale biomimetic scaffolds that more closely mimic the composite structure of natural tissue, with tuneable tensile and compressive properties via the fibre and matrix phases, respectively. © 2014 Springer Science+Business Media New York.

Viscoelastic analysis of single-component and composite PEG and alginate hydrogels

Hydrogels, three-dimensional hydrophilic polymer networks, are appealing candidate materials for studying the cellular microenvironment as their substantial water content helps to better mimic soft tissue. However, hydrogels can lack mechanical stiffness, strength, and toughness. Composite hydrogel systems have been shown to improve upon mechanical properties compared to their singlecomponent counterparts. Poly (ethylene glycol) dimethacrylate (PEGDMA) and alginate are polymers that have been used to form hydrogels for biological applications. Singlecomponent and composite PEGDMA and alginate systems were fabricated with a range of total polymer concentrations. Bulk gels were mechanically characterized using spherical indentation testing and a viscoelastic analysis framework. An increase in shear modulus with increasing polymer concentration was demonstrated for all systems. Alginate hydrogels were shown to have a smaller viscoelastic ratio than the PEGDMA gels, indicating more extensive relaxation over time. Composite alginate and PEGDMA hydrogels exhibited a combination of the mechanical properties of the constituents, as well as a qualitative increase in toughness. Additionally, multiple hydrogel systems were produced that had similar shear moduli, but different viscoelastic behaviors. Accurate measurement of the mechanical properties of hydrogels is necessary in order to determine what parameters are key in modeling the cellular microenvironment. © 2014 The Chinese Society of Theoretical and Applied Mechanics; Institute of Mechanics, Chinese Academy of Sciences and Springer-Verlag Berlin Heidelberg.

Absence of branches from xylan in Arabidopsis gux mutants reveals potential for simplification of lignocellulosic biomass.

As one of the most abundant polysaccharides on Earth, xylan will provide more than a third of the sugars for lignocellulosic biofuel production when using grass or hardwood feedstocks. Xylan is characterized by a linear β(1,4)-linked backbone of xylosyl residues substituted by glucuronic acid, 4-O-methylglucuronic acid or arabinose, depending on plant species and cell types. The biological role of these decorations is unclear, but they have a major influence on the properties of the polysaccharide. Despite the recent isolation of several mutants with reduced backbone, the mechanisms of xylan synthesis and substitution are unclear. We identified two Golgi-localized putative glycosyltransferases, GlucUronic acid substitution of Xylan (GUX)-1 and GUX2 that are required for the addition of both glucuronic acid and 4-O-methylglucuronic acid branches to xylan in Arabidopsis stem cell walls. The gux1 gux2 double mutants show loss of xylan glucuronyltransferase activity and lack almost all detectable xylan substitution. Unexpectedly, they show no change in xylan backbone quantity, indicating that backbone synthesis and substitution can be uncoupled. Although the stems are weakened, the xylem vessels are not collapsed, and the plants grow to normal size. The xylan in these plants shows improved extractability from the cell wall, is composed of a single monosaccharide, and requires fewer enzymes for complete hydrolysis. These findings have implications for our understanding of the synthesis and function of xylan in plants. The results also demonstrate the potential for manipulating and simplifying the structure of xylan to improve the properties of lignocellulose for bioenergy and other uses.

Phase-variable surface structures are required for infection of Campylobacter jejuni by bacteriophages.

This study characterizes the interaction between Campylobacter jejuni and the 16 phages used in the United Kingdom typing scheme by screening spontaneous mutants of the phage-type strains and transposon mutants of the sequenced strain NCTC 11168. We show that the 16 typing phages fall into four groups based on their patterns of activity against spontaneous mutants. Screens of transposon and defined mutants indicate that the phage-bacterium interaction for one of these groups appears to involve the capsular polysaccharide (CPS), while two of the other three groups consist of flagellatropic phages. The expression of CPS and flagella is potentially phase variable in C. jejuni, and the implications of these findings for typing and intervention strategies are discussed.

In vivo regulation of the Vi antigen in Salmonella and induction of immune responses with an in vivo-inducible promoter.

Salmonella enterica serovar Typhi, the agent of typhoid fever in humans, expresses the surface Vi polysaccharide antigen that contributes to virulence. However, Vi expression can also be detrimental to some key steps of S. Typhi infectivity, for example, invasion, and Vi is the target of protective immune responses. We used a strain of S. Typhimurium carrying the whole Salmonella pathogenicity island 7 (SPI-7) to monitor in vivo Vi expression within phagocytic cells of mice at different times after systemic infection. We also tested whether it is possible to modulate Vi expression via the use of in vivo-inducible promoters and whether this would trigger anti-Vi antibodies through the use of Vi-expressing live bacteria. Our results show that Vi expression in the liver and spleen is downregulated with the progression of infection and that the Vi-negative population of bacteria becomes prevalent by day 4 postinfection. Furthermore, we showed that replacing the natural tviA promoter with the promoter of the SPI-2 gene ssaG resulted in sustained Vi expression in the tissues. Intravenous or oral infection of mice with a strain of S. Typhimurium expressing Vi under the control of the ssaG promoter triggered detectable levels of all IgG subclasses specific for Vi. Our work highlights that Vi is downregulated in vivo and provides proof of principle that it is possible to generate a live attenuated vaccine that induces Vi-specific antibodies after single oral administration.

Poroviscoelastic characterization of particle-reinforced gelatin gels using indentation and homogenization.

Hydrogels are promising materials for bioengineering applications, and are good model materials for the study of hydrated biological tissues. As these materials often have a structural function, the measurement of their mechanical properties is of fundamental importance. In the present study gelatin gels reinforced with ceramic microspheres are produced and their poroviscoelastic response in spherical indentation is studied. The constitutive responses of unreinforced gels are determined using inverse finite element modeling in combination with analytical estimates of material parameters. The behavior of composite gels is assessed by both analytical and numerical homogenization. The results of the identification of the constitutive parameters of unreinforced gels show that it is possible to obtain representative poroviscoelastic parameters by spherical indentation without the need for additional mechanical tests. The agreement between experimental results on composite gelatin and the predictions from homogenization modeling show that the adopted modeling tools are capable of providing estimates of the poroviscoelastic response of particle-reinforced hydrogels.

Wet-spinning of amyloid protein nanofibers into multifunctional high-performance biofibers.

Amyloid nanofibers derived from hen egg white lysozyme were processed into macroscopic fibers in a wet-spinning process based on interfacial polyion complexation using a polyanionic polysaccharide as cross-linker. As a result of their amyloid nanostructure, the hierarchically self-assembled protein fibers have a stiffness of up to 14 GPa and a tensile strength of up to 326 MPa. Fine-tuning of the polyelectrolytic interactions via pH allows to trigger the release of small molecules, as demonstrated with riboflavin-5'-phophate. The amyloid fibrils, highly oriented within the gellan gum matrix, were mineralized with calcium phosphate, mimicking the fibrolamellar structure of bone. The formed mineral crystals are highly oriented along the nanofibers, thus resulting in a 9-fold increase in fiber stiffness.

Nanoindentation of biological and biomimetic materials

Nanoindentation techniques have recently been adapted for the study of biological materials. This feature will consider the experimental adaptations required for such studies. Following a brief review of the structure and constitutive behavior of biological materials, we examine the experimental aspects in detail, including working with hydrated samples, time-dependent mechanical behavior and extremely compliant materials. The analysis of experimental data, consistent with the constitutive response of the material, will then be treated. Examples of nanoindentation data collected using commercially-available instruments are shown, including nanoindentation creep curves of biological materials and relaxation responses of biomimetic hydrogels. Finally, we conclude by examining the current state and future needs of the biological nanoindentation community. © 2011, Society for Experimental Mechanics.

Composite electrospun gelatin fiber-alginate gel scaffolds for mechanically robust tissue engineered cornea.

A severe shortage of good quality donor cornea is now an international crisis in public health. Alternatives for donor tissue need to be urgently developed to meet the increasing demand for corneal transplantation. Hydrogels have been widely used as scaffolds for corneal tissue regeneration due to their large water content, similar to that of native tissue. However, these hydrogel scaffolds lack the fibrous structure that functions as a load-bearing component in the native tissue, resulting in poor mechanical performance. This work shows that mechanical properties of compliant hydrogels can be substantially enhanced with electrospun nanofiber reinforcement. Electrospun gelatin nanofibers were infiltrated with alginate hydrogels, yielding transparent fiber-reinforced hydrogels. Without prior crosslinking, electrospun gelatin nanofibers improved the tensile elastic modulus of the hydrogels from 78±19 kPa to 450±100 kPa. Stiffer hydrogels, with elastic modulus of 820±210 kPa, were obtained by crosslinking the gelatin fibers with carbodiimide hydrochloride in ethanol before the infiltration process, but at the expense of transparency. The developed fiber-reinforced hydrogels show great promise as mechanically robust scaffolds for corneal tissue engineering applications.