Peptide gels of fully-defined composition and mechanics for probing cell-cell and cell-matrix interactions in vitro.

Current materials used for in vitro 3D cell culture are often limited by their poor similarity to human tissue, batch-to-batch variability and complexity of composition and manufacture. Here, we present a "blank slate" culture environment based on a self-assembling peptide gel free from matrix motifs. The gel can be customised by incorporating matrix components selected to match the target tissue, with independent control of mechanical properties. Therefore the matrix components are restricted to those specifically added, or those synthesised by encapsulated cells. The flexible 3D culture platform provides full control over biochemical and physical properties, allowing the impact of biochemical composition and tissue mechanics to be separately evaluated in vitro. Here, we demonstrate that the peptide gels support the growth of a range of cells including human induced pluripotent stem cells and human cancer cell lines. Furthermore, we present proof-of-concept that the peptide gels can be used to build disease-relevant models. Controlling the peptide gelator concentration allows peptide gel stiffness to be matched to normal breast (<1 kPa) or breast tumour tissue (>1 kPa), with higher stiffness favouring the viability of breast cancer cells over normal breast cells. In parallel, the peptide gels may be modified with matrix components relevant to human breast, such as collagen I and hyaluronan. The choice and concentration of these additions affect the size, shape and organisation of breast epithelial cell structures formed in co-culture with fibroblasts. This system therefore provides a means of unravelling the individual influences of matrix, mechanical properties and cell-cell interactions in cancer and other diseases.

Model-based image analysis of a tethered Brownian fibre for shear stress sensing.

The measurement of fluid dynamic shear stress acting on a biologically relevant surface is a challenging problem, particularly in the complex environment of, for example, the vasculature. While an experimental method for the direct detection of wall shear stress via the imaging of a synthetic biology nanorod has recently been developed, the data interpretation so far has been limited to phenomenological random walk modelling, small-angle approximation, and image analysis techniques which do not take into account the production of an image from a three-dimensional subject. In this report, we develop a mathematical and statistical framework to estimate shear stress from rapid imaging sequences based firstly on stochastic modelling of the dynamics of a tethered Brownian fibre in shear flow, and secondly on a novel model-based image analysis, which reconstructs fibre positions by solving the inverse problem of image formation. This framework is tested on experimental data, providing the first mechanistically rational analysis of the novel assay. What follows further develops the established theory for an untethered particle in a semi-dilute suspension, which is of relevance to, for example, the study of Brownian nanowires without flow, and presents new ideas in the field of multi-disciplinary image analysis.

Transformations of citrate and Tween coated silver nanoparticles reacted with Na2S.

Silver nanoparticles (Ag NPs) are susceptible to transformations in environmental and biological media such as aggregation, oxidation, dissolution, chlorination, sulfidation, formation/replacement of surface coatings following interaction with natural organic matter (NOM). This paper investigates the impact of surface coating and Suwannee River fulvic acid (SRFA) on the transformations and behavior of Ag NPs (citrate coated and Tween coated; cit-Ag NPs and Tween-Ag NPs, respectively), following reaction with different concentrations of Na2S solution (as a source of sulfide species, H2S and HS(-)). These transformations and the dominant mechanisms of transformations were investigated using UV-vis and scanning transmission electron microscopy coupled with electron energy loss spectroscopy. Here, we have shown that Ag NP surface coating impacts their dissolution following dilution in ultrahigh purity water, with higher extent of dissolution of Tween-Ag NPs compared with cit-Ag NPs. Tween-Ag NPs are susceptible to dissolution following their sulfidation at low S/Ag molar ratio. Suwannee River fulvic acid (SRFA) slows down the dissolution of Tween-Ag NPs at low sulfide concentrations and reduces the aggregation of cit-Ag NP in the presence of sodium sulfide. Sulfidation appears to occur by direct interaction of sulfide species with Ag NPs rather than by indirect reaction of sulfide with dissolved Ag species subsequent to dissolution. Furthermore, the sulfidation process results in the formation of partially sulfidized Ag NPs containing unreacted (metallic) subgrains at the edge of the NPs for Tween-Ag NPs in the presence of high sulfide concentration (2000nM Na2S), which occurred to less extent at lower Na2S concentration for Tween-Ag NPs and at all concentrations of Na2S for cit-Ag NPs. Thus, sulfidized Ag NPs may preserve some of the properties of the Ag NPs such as their potential to shed Ag(+) ions and their toxic potential of Ag NPs.

Similar endothelial glycocalyx structures in microvessels from a range of mammalian tissues: evidence for a common filtering mechanism?

The glycocalyx or endocapillary layer on the luminal surface of microvessels has a major role in the exclusion of macromolecules from the underlying endothelial cells. Current structural evidence in the capillaries of frog mesentery indicates a regularity in the structure of the glycocalyx, with a center-to-center fiber spacing of 20 nm and a fiber width of 12 nm, which might explain the observed macromolecular filtering properties. In this study, we used electron micrographs of tissues prepared using perfusion fixation and tannic acid treatment. The digitized images were analyzed using autocorrelation to find common spacings and to establish whether similar structures, hence mechanisms, are present in the microvessel glycocalyces of a variety of mammalian tissues. Continuous glycocalyx layers in mammalian microvessels of choroid, renal tubules, glomerulus, and psoas muscle all showed similar lateral spacings at ∼19.5 nm (possibly in a quasitetragonal lattice) and longer spacings above 100 nm. Individual glycocalyx tufts above fenestrations in the first three of these tissues and also in stomach fundus and jejunum showed evidence for similar short-range structural regularity, but with more disorder. The fiber diameter was estimated as 18.8 (± 0.2) nm, but we believe this is an overestimate because of the staining method used. The implications of these findings are discussed.

Cartilage collagen matrix reorientation and displacement in response to surface loading.

An investigation of collagen fiber reorientation, as well as fluid and matrix movement of equine articular cartilage and subchondral bone under compressive mechanical loads, was undertaken using small angle X-ray scattering measurements and optical microscopy. Small angle X-ray scattering measurements were made on healthy and diseased samples of equine articular cartilage and subchondral bone mounted in a mechanical testing apparatus on station ID18F of ESRF, Grenoble, together with fiber orientation analysis using polarized light and displacement measurements of the cartilage matrix and fluid using tracers. At surface pressures of up to approximately 1.5 MPa, there was reversible compression of the tangential surface fibers and immediately subjacent zone. As load increased, deformation in these zones reached a maximum and then reorientation propagated to the radial deep zone. Between surface pressures of 4.8 MPa and 6.0 MPa, fiber orientation above the tide mark rotated 10 deg from the radial direction, with an overall loss of alignment. With further increase in load, the fibers "crimped" as shown by the appearance of subsidiary peaks approximately +/-10 deg either side of the principal fiber orientation direction. Failure at higher loads was characterized by a radial split in the deep cartilage, which propagated along the tide mark while the surface zone remained intact. In lesions, the fiber organization was disrupted and the initial response to load was consistent with early rupture of fibers, but the matrix relaxed to an organization very similar to that of the unloaded tissue. Tracer measurements revealed anisotropic solid and fluid displacement, which depended strongly on depth within the tissue.

Modeling flow in collecting lymphatic vessels: one-dimensional flow through a series of contractile elements.

The lymphatic system comprises a series of elements, lymphangions, separated by valves and possessed of active, contractile walls to pump interstitial fluid from its collection in the terminal lymphatics back to the main circulation. Despite its importance, there is a dearth of information on the fluid dynamics of the lymphatic system. In this article, we describe linked experimental and computational work aimed at elucidating the biomechanical properties of the individual lymphangions. We measure the static and dynamic mechanical properties of excised bovine collecting lymphatics and develop a one-dimensional computational model of the coupled fluid flow/wall motion. The computational model is able to reproduce the pumping behavior of the real vessel using a simple contraction function producing fast contraction pulses traveling in the retrograde direction to the flow.

Solute transport in the deep and calcified zones of articular cartilage.

OBJECTIVES: (1) To establish whether the tidemark and calcified cartilage are permeable to low molecular weight solutes, thereby providing a potential pathway for nutrition of cells in the deep cartilage. (2) To investigate transport from the subchondral microcirculation into calcified cartilage in an intact perfused joint and the effects on transport of static loading. METHODS: The permeability of the tidemark and calcified cartilage was investigated in plugs of cartilage and subchondral bone which formed the membrane of a diffusion cell. Transport from the subchondral microcirculation and the effects of load were studied in an intact perfused joint. Both preparations used the metacarpophalangeal joints of mature horses and fluorescein and rhodamine (m.w. approximately 400 Da) were employed as tracers, assayed by quantitative fluorescence microscopy on histological sections. RESULTS: Calcified cartilage was permeable to both solutes, both from the superficial and the subchondral sides. The effective diffusivity of both solutes was of the order of 9 x 10(-9) cm(2) s(-1), fivefold less than in the uncalcified cartilage. The calcified zone was heterogeneous, with high uptake of both tracers in the vicinity of the tidemark. The distribution volume of rhodamine B was higher than for fluorescein, consistent with a significant anionic charge in the calcified matrix. Static loading of the intact joint did not affect the transport of rhodamine B but caused a significant decrease in concentration of fluorescein both in the surface and deep zones of the tissue. CONCLUSIONS: Calcified cartilage is permeable to small solutes and the subchondral circulation may make a significant contribution to the nutrition of deep cartilage in the mature horse. Static loading reduces the uptake of small anionic solutes in the intact joint.