Biophotonic tools for probing extracellular matrix mechanics.

The complex, hierarchical and heterogeneous biomechanics of the extracellular matrix (ECM) are central to the health of multicellular organisms. Characterising the distribution, dynamics and above all else origins of ECM biomechanics are challenges that have captivated researchers for decades. Recently, a suite of biophotonics techniques have emerged as powerful new tools to investigate ECM biomechanics. In this mini-review, we discuss how the non-destructive, sub-micron resolution imaging capabilities of Raman spectroscopy and nonlinear microscopy are being used to interrogate the biomechanics of thick, living tissues. These high speed, label-free techniques are implemented during mechanical testing, providing unprecedented insight into the compositional and structural response of the ECM to changes in the mechanical environment.

Micro-mechanical damage of needle puncture on bovine annulus fibrosus fibrils studied using polarization-resolved Second Harmonic Generation(P-SHG) microscopy.

Needle injection has been widely used in spinal therapeutic or diagnostic processes, such as discography. The use of needles has been suspected in causing mild disc degeneration which can lead to long-term back pain. However, the localised microscopic damage caused by needles has not been well studied. The local progressive damage on a microscopic level caused by needle punctures on the surface of bovine annulus fibrosus was investigated. Four different sizes of needle were used for the puncture and twenty-nine bovine intervertebral discs were studied. Polarization-resolved second harmonic generation and fluorescent microscopy were used to study the local microscopic structural changes in collagen and cell nuclei due to needle damage. Repeated 70 cyclic loadings at ±5% of axial strain were applied after the needle puncture in order to assess progressive damage caused by the needle. Puncture damage on annulus fibrosus were observed either collagen fibre bundles being pushed aside, being cut through or combination of both with part being lift or pushed in. The progressive damage was found less relevant to the needle size and more progressive damage was only observed using the larger needle. Two distinct populations of collagen, in which one was relatively more organised than the other population, were observed especially after the puncture from skewed distribution of polarization-SHG analysis. Cell shape was found rounder near the puncture site where collagen fibres were damaged.

The hierarchical response of human corneal collagen to load.

Fibrillar collagen in the human cornea is integral to its function as a transparent lens of precise curvature, and its arrangement is now well-characterised in the literature. While there has been considerable effort to incorporate fibrillar architecture into mechanical models of the cornea, the mechanical response of corneal collagen to small applied loads is not well understood. In this study the fibrillar and molecular response to tensile load was quantified using small and wide angle X-ray scattering (SAXS/WAXS), and digital image correlation (DIC) photography was used to calculate the local strain field that gave rise to the hierarchical changes. A molecular scattering model was used to calculate the tropocollagen tilt relative to the fibril axis and changes associated with applied strain. Changes were measured in the D-period, molecular tilt and the orientation and spacing of the fibrillar and molecular networks. These measurements were summarised into hierarchical deformation mechanisms, which were found to contribute at varying strains. The change in molecular tilt is indicative of a sub-fibrillar "spring-like" deformation mechanism, which was found to account for most of the applied strain under physiological and near-physiological loads. This deformation mechanism may play an important functional role in tissues rich in fibrils of high helical tilt, such as skin and cartilage. STATEMENT OF SIGNIFICANCE: Collagen is the primary mediator of soft tissue biomechanics, and variations in its hierarchical structure convey the varying amounts of structural support necessary for organs to function normally. Here we have examined the structural response of corneal collagen to tensile load using X-rays to probe hierarchies ranging from molecular to fibrillar. We found a previously unreported deformation mechanism whereby molecules, which are helically arranged relative to the axis of their fibril, change in tilt akin to the manner in which a spring stretches. This "spring-like" mechanism accounts for a significant portion of the applied deformation at low strains (<3%). These findings will inform the future design of collagen-based artificial corneas being developed to address world-wide shortages of corneal donor tissue.

Microstructure and mechanics of human resistance arteries.

Vascular diseases such as diabetes and hypertension cause changes to the vasculature that can lead to vessel stiffening and the loss of vasoactivity. The microstructural bases of these changes are not presently fully understood. We present a new methodology for stain-free visualization, at a microscopic scale, of the morphology of the main passive components of the walls of unfixed resistance arteries and their response to changes in transmural pressure. Human resistance arteries were dissected from subcutaneous fat biopsies, mounted on a perfusion myograph, and imaged at varying transmural pressures using a multimodal nonlinear microscope. High-resolution three-dimensional images of elastic fibers, collagen, and cell nuclei were constructed. The honeycomb structure of the elastic fibers comprising the internal elastic layer became visible at a transmural pressure of 30 mmHg. The adventitia, comprising wavy collagen fibers punctuated by straight elastic fibers, thinned under pressure as the collagen network straightened and pulled taut. Quantitative measurements of fiber orientation were made as a function of pressure. A multilayer analytical model was used to calculate the stiffness and stress in each layer. The adventitia was calculated to be up to 10 times as stiff as the media and experienced up to 8 times the stress, depending on lumen diameter. This work reveals that pressure-induced reorganization of fibrous proteins gives rise to very high local strain fields and highlights the unique mechanical roles of both fibrous networks. It thereby provides a basis for understanding the micromechanical significance of structural changes that occur with age and disease.

Clostridium perfringensα-toxin interaction with red cells and model membranes.

The effects of Clostridium perfringensα-toxin on host cells have previously been studied extensively but the biophysical processes associated with toxicity are poorly understood. The work reported here shows that the initial interaction between the toxin and lipid membrane leads to measurable changes in the physical properties and morphology of the membrane. A Langmuir monolayer technique was used to assess the response of different lipid species to toxin. Sphingomyelin and unsaturated phosphatidylcholine showed the highest susceptibility to toxin lypolitic action, with a two stage response to the toxin (an initial, rapid hydrolysis stage followed by the insertion and/or reorganisation of material in the monolayer). Fluorescence confocal microscopy on unsaturated phosphatidylcholine vesicles shows that the toxin initially aggregates at discrete sites followed by the formation of localised "droplets" accumulating the hydrolysis products. This process is accompanied by local increases in the membrane dipole potential by about 50 (±42) mV. In contrast, red blood cells incubated with the toxin suffered a decrease of the membrane dipole potential by 50 (±40) mV in areas of high toxin activity (equivalent to a change in electric field strength of 10(7) V m(-1)) which is sufficient to affect the functioning of the cell membrane. Changes in erythrocyte morphology caused by the toxin are presented, and the early stages of interaction between toxin and membrane are characterised using thermal shape fluctuation analysis of red cells which revealed two distinct regimes of membrane-toxin interaction.

The micromechanics of the superficial zone of articular cartilage.

OBJECTIVE: To investigate the relationships between the unique mechanical and structural properties of the superficial zone of articular cartilage on the microscopic scale. DESIGN: Fresh unstained equine metacarpophalangeal cartilage samples were mounted on tensile and compressive loading rigs on the stage of a multiphoton microscope. Sequential image stacks were acquired under incremental loads together with simultaneous measurements of the applied stress and strain. Second harmonic generation was used to visualise the collagen fibre network, while two photon fluorescence was used to visualise elastin fibres and cells. The changes visualised by each modality were tracked between successive loads. RESULTS: The deformation of the cartilage matrix was heterogeneous on the microscopic length scale. This was evident from local strain maps, which showed shearing between different regions of collagen under tensile strain, corrugations in the articular surface at higher tensile strains and a non-uniform distribution of compressive strain in the axial direction. Chondrocytes elongated and rotated under tensile strain and were compressed in the axial direction under compressive load. The magnitude of deformation varied between cells, indicating differences in either load transmission through the matrix or the mechanical properties of individual cells. Under tensile loading the reorganisation of the elastin network differed from a homogeneous elastic response, indicating that it forms a functional structure. CONCLUSIONS: This study highlights the complexity of superficial zone mechanics and demonstrates that the response of the collagen matrix, elastin fibres and chondrocytes are all heterogeneous on the microscopic scale.

Micromechanical response of articular cartilage to tensile load measured using nonlinear microscopy.

Articular cartilage (AC) is a highly anisotropic biomaterial, and its complex mechanical properties have been a topic of intense investigation for over 60 years. Recent advances in the field of nonlinear optics allow the individual constituents of AC to be imaged in living tissue without the need for exogenous contrast agents. Combining mechanical testing with nonlinear microscopy provides a wealth of information about microscopic responses to load. This work investigates the inhomogeneous distribution of strain in loaded AC by tracking the movement and morphological changes of individual chondrocytes using point pattern matching and Bayesian modeling. This information can be used to inform models of mechanotransduction and pathogenesis, and is readily extendable to various other connective tissues.

The effect of oxidative stress on the membrane dipole potential of human red blood cells.

The membrane dipole potential (ψ(d)) is an important biophysical determinant of membrane function and a sensitive indicator of lipid organisation. In this study we have used the environmentally sensitive probe di-8-anepps to explore the effects of oxidative stress on the membrane dipole potential of human erythrocytes. Cells suspended in 0.15mM phosphate buffered saline containing 0.1mg/ml albumin maintained a mean value for ψ(d) of 270 (±20) mV over the course of 1hour. In the presence of 0.4mM cumene hydroperoxide there was an increase in ψ(d) of 14 (±7)%, accompanied by a decrease in cell diameter of ~14 (±2)%. Exposure of the cells to 0.4mM hydrogen peroxide caused ψ(d) to decrease by 13 (±8)% at the centre of the cell and 8 (±5)% at the edge whilst the diameter remained constant. In both cases the changes were equivalent to a change in transmembrane electric field of a magnitude of ~10MVm(-1), sufficient to influence membrane function. Raman microspectrometry supported the conclusion that cumene exerts its effect primarily on membrane lipids whilst hydrogen peroxide causes the formation of spectrin-haemoglobin complexes which stiffen the membrane.

Motion and mixing for multiple ferromagnetic microswimmers.

This paper concerns the interaction of several ferromagnetic microswimmers, their motion and the resulting fluid mixing. Each swimmer consists of two ferromagnetic beads joined by an elastic link, and is driven by an external, time-dependent magnetic field. The external field provides a torque on a swimmer and, together with the varying attraction between the magnetic beads, generates a time-irreversible motion leading to persistent swimming in a low Reynolds number environment. The aim of the present paper is to consider the interactions between several swimmers. A regime is considered in which identical swimmers move in the same overall direction, and their motion is synchronised because of driving by the external field. It is found that two swimmers tend to encircle one another while three undergo more complicated motion that may involve the braiding of swimmer trajectories. By means of approximations it is established that the interaction between pairs of swimmers gives circulatory motion which falls off with an inverse square law and is linked to their overall speed of motion through the fluid. As groups of two or more swimmers move through the fluid they process fluid, leaving behind a trail of fluid that has undergone mixing: this is investigated by following streak lines numerically.

Modeling the steady-state deformation of the solid phase of articular cartilage.

The transient response of articular cartilage (AC) to compressive loads has been described by complex multicomponent models. However, the steady-state behaviour is determined by the collagen network which is heterogeneous through the depth of the tissue, a characteristic which is omitted from most theoretical models. Experimental data are now available on the local responses of the network to compressive loads and the aim of this study was to develop minimal models capable of simulating this behaviour. A series of finite element models (FEMs) of AC under load were developed of increasing complexity, assuming the AC was i) completely homogeneous, ii) layered and isotropic and iii) layered and anisotropic. The geometry of the layered cartilage model was based on the recent experimental data. It is shown that a layered transversely isotropic elastic model is required to accurately recreate the experimental data. Stress distributions within the models are analysed, and the relevance of this work to transient modeling of AC is discussed. The work presented is a fundamental step forward in the understanding of the distribution of local physiological stresses and strains in AC, and has applications in modeling chondrocyte mechanotransduction as well as the effects of pathogenesis.

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.

Spectrin maintains the lateral order in phosphatidylserine monolayers.

We investigate the effect of the skeletal protein spectrin on the lateral order in dipalmitoyl phosphatidylserine monolayers spread on aqueous surfaces using grazing incidence X-ray diffraction. Without spectrin, the condensed lipid monolayer exhibits two-dimensional hexagonal packing, characterized by monotonic decrease in the d-spacing and increase in the degree of order with increasing surface pressure between 17 and 36 mN/m. Addition of spectrin to the aqueous subphase at high pressures preserves the monolayers structural parameters unchanged from 36 to 25 mN/m. These results demonstrate for the first time that spectrin could participate in sustaining the two-dimensional order in lipid domains through a direct interaction with phosphatidylserine species.

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.

Two-dimensional order in mammalian pre-ocular tear film.

We report a grazing incidence x-ray diffraction (GIXD) investigation of the surface lipid layer of the pre-ocular tear film. For the first time we demonstrate the existence of 2D order over a wide range of surface pressures in this system, with typical spicing of 3.75A and 4.16A independent of the monolayer surface pressure. Analogous lipid ordering is also found in an artificial lipid mixture of the major lipid components of the tear film, suggesting that the 2D ordering is set by generic lipid-lipid interactions. Fluorescence microscopy of the natural and artificial tear film mixture reveals the co-existence of a dilute and a much more condensed phase in the amphiphilic lipid matrix over the pressure range of 15-45mN/m investigated by GIXD, plus an additional structure due to the much more hydrophobic part of the mixture. This evidence supports the previous hypothesis that tear film has a layered structure.

Feasibility study using surface-enhanced Raman spectroscopy for the quantitative detection of tyrosine and serine phosphorylation.

We investigate the feasibility of colloid-based surface enhanced Raman scattering (SERS) as a highly sensitive technique for detecting peptide phosphorylation at serine and tyrosine residues. Using the recently reported drop-coating deposition Raman method we validate our SERS spectra against normal Raman spectra that would otherwise be unobtainable at such low concentrations. Compared with existing techniques for quantifying peptide phosphorylation, such as high-performance liquid chromatography (HPLC), the short scanning and processing time associated with SERS makes it an attractive alternative for near-real-time measurement at sub micro-molar concentrations. Following pre-processing by Savistky-Golay second derivative (SGSD), the degree of phosphorylation of synthetic peptides is determined using multivariate spectral classification, interval partial least squares (iPLS). Furthermore, our results show that the technique is robust to interference from complex proteins and other phosphorylated compounds present at concentrations typically found in a screening assay.

Regional variations of collagen orientation in normal and diseased articular cartilage and subchondral bone determined using small angle X-ray scattering (SAXS).

OBJECTIVE: To determine regional differences in the orientation of collagen in the articular cartilage of the equine metacarpophalangeal joint as well as describing cartilage orientation in lesions using small angle X-ray scattering (SAXS). DESIGN: SAXS diffraction patterns were taken at the European Synchrotron Radiation Facility (ESRF), with increasing depth into cartilage and bone cross sections. Results for healthy samples were taken at different regions along the joint which receive different loads and differences in collagen orientation were determined. Results were also taken from diseased samples and the collagen orientation changes from that of healthy samples observed. RESULTS: Regions subject to low loads show a lower degree of orientation and regions exposed to the highest loads possess oriented collagen fibres especially in the radial layer. In early lesions the orientations of the collagen fibres are disrupted. Subchondral bone fibres are twisted in regions where the joint receives shear forces. Changes in fibre orientation are also observed in the calcified cartilage even in regions where the cartilage is intact. In more advanced lesions where there is loss of cartilage the fibres in the calcified layer are realigned tangential to the surface. CONCLUSIONS: Regional variations in collagen arrangement show that the highly ordered layers of the articular cartilage are the most important elements in supporting high variable loads. In lesions changes occur in the deep tissue whilst the overlying cartilage appeared normal. We therefore suggest that the interface region is a key element in the early stages of the disease.

Viscoelastic properties of insoluble amphiphiles at the air/water interface.

The effects of the presence of a molecular monolayer on the dilatational properties of the air/water interface have been investigated. Two water insoluble amphiphiles, dipalmitoyl phosphatidyl choline and quercetin 3-O-palmitate, were spread onto a pendant drop and the dynamic surface pressure was measured by means of drop shape analysis. The surface dilatational elasticity and viscosity of the spread monolayers were also determined by the oscillating drop technique. Constraints on the range of measuring conditions were investigated and we demonstrated that the pressure-area isotherms derived from oscillatory dynamic measurements display phase behaviour similar to that found in equilibrium measurements, albeit at reduced resolution. Both the amphiphiles formed purely elastic films that were characterised by a dilatational modulus that depended on the surface concentration and obeyed a power scaling law. The exponent of the relationship could be related to the thermodynamic conditions prevailing at the interface. The phospholipid monolayer scaling exponent was 2.8 in a temperature range of 20-26 degrees C indicates a favourable solvency of molecules in the bidimensional matrix. A very high scaling exponent (11.8 at 7 degrees C) for quercetin palmitate was interpreted assuming that molecules self-organise in fibre-like structures. This interface structure and the phase behaviour was found consistent with observations of the surface film obtained by Brewster angle microscopy. The structured quercetin 3-O-palmitate monolayers are disrupted by temperature increase or by adding a 0.2 molar fraction of the immiscible dipalmitoyl phosphatidyl choline.

Oxygen partial pressure in outer layers of skin of human finger nail folds.

To gain insight into oxygen transport by the cutaneous microcirculation, we have developed oxygen-sensitive microelectrodes (tip diameter approximately 5 micro m) to measure the distribution of PO2 in dermal papillae of the finger nail folds of healthy human subjects. Oxygen entry into the tissue was minimised by covering the skin with a layer of paraffin oil. The finger was held under a dissecting microscope and microelectrodes were guided into position. PO2 varied from 5-25 % of its atmospheric value, Pair (approximately 160 mmHg), depending on the location within the papilla. Along the axis of a papillary loop, PO2 decreased from 40.0 +/- 4.8 mmHg (mean +/- S.E.M., n = 6) at the base to 30.4 +/- 5.2 mmHg (n = 6) at the tip. The lowest values of PO2, in the range of 5 % of Pair, were measured in the epidermis where the metabolism of cells was highest and the steepest PO2 gradients were recorded in the vicinity of the epidermal-dermal boundary. When the local circulation was abruptly reduced or stopped, PO2 fell exponentially with time, with a time constant of 8.4 +/- 1.5 s (n = 7). When flow was reinstated, PO2 rose exponentially to a new value with a time constant of 4.8 +/- 0.8 s (n = 6). The steady state PO2 following reperfusion was approximately 23 % higher than the pre-occlusion value (P < 0.05, ANOVA and two-tailed Student's t test) indicating localised reactive hyperaemia.

Effects of perfusion rate on permeability of frog and rat mesenteric microvessels to sodium fluorescein.

The permeability, P(S), to sodium fluorescein (Stokes-Einstein radius = 0.45 nm) has been measured in single mesenteric capillaries of pithed frogs and anaesthetised rats as perfusion velocity, U, was varied over a range from 400 up to 2000-10,000 microm s(-1). P(S) increased linearly with U. In 20 frog capillaries, mean (+/- S.E.M.) P(S) (in microm s(-1)) = 9.35 (+/- 1.55)U x 10(-5) + 0.244 (+/- 0.0291). Similarly, in nine rat venules, mean P(S) = 1.62 (+/- 0.385)U x 10(-4) + 0.375 (+/- 0.025). The flow-dependent component of permeability could be reversibly abolished in frog capillaries by superfusing with 100 microM noradrenaline and by superfusing rat venules with the nitric oxide synthase inhibitor, N(G)-nitro-L-arginine (20 microM). It was shown that changes in microvascular pressure accompanying changes in U during free perfusion could account for only 15 % of the changes in P(S), i.e. 85 % of the changes in P(S) were changes in the permeability coefficient itself. A comparison between the changes in P(S) with U and the previously described changes in microvascular permeability to K(+) with U, suggest that if the flow-dependent component of permeability is modelled as a population of pores of constant size, these have radii of 0.8 nm. Such a pathway would limit flow-dependent permeability to small hydrophilic molecules and have minimal effect on net fluid exchange.