Peter Winlove

The elastin network: its relationship with collagen and cells in articular cartilage as visualized by multiphoton microscopy

A combination of two‐photon fluorescence (TPF), second harmonic generation (SHG) and coherent anti‐Stokes Raman scattering (CARS) imaging has been used to investigate the elastin fibre network in healthy equine articular cartilage from the metacarpophalangeal joint. The elastin fibres were identified using their intrinsic two‐photon fluorescence and immuno‐staining was used to confirm the identity of these fibres. SHG was used to reveal the collagen matrix and the collagen fibre orientations were determined from their SHG polarization sensitivity, while CARS was used to clearly delineate the cell boundaries. Extensive elastin fibre networks were found in all the joint regions investigated. The elastin was found predominantly in the superficial zone (upper 50 μm) and was aligned parallel to the articular surface. Elastin was also detected in the pericellular matrix surrounding the superficial chondrocytes; however, individual fibres could not be resolved in this region. Variations in the density and organization of the fibres were observed in different regions on the joint surface.

Lamellar and fibre bundle mechanics of the annulus fibrosus in bovine intervertebral disc

UNLABELLED The intervertebral disc is a multicomposite structure, with an outer fibrous ring, the annulus fibrosus, retaining a gel-like core, the nucleus pulposus. The disc presents complex mechanical behaviour, and it is of high importance for spine biomechanics. Advances in multiscale modelling and disc repair raised a need for new quantitative data on the finest details of annulus fibrosus mechanics. In this work we explored inter-lamella and inter-bundle behaviour of the outer annulus using micromechanical testing and second harmonic generation microscopy. Twenty-one intervertebral discs were dissected from cow tails; the nucleus and inner annulus were excised to leave a ring of outer annulus, which was tested in circumferential loading while imaging the tissues collagen fibres network with sub-micron resolution. Custom software was developed to determine local tissue strains through image analysis. Inter-bundle linear and shear strains were 5.5 and 2.8 times higher than intra-bundle strains. Bundles tended to remain parallel while rotating under loading, with large slipping between them. Inter-lamella linear strain was almost 3 times the intra-lamella one, but no slipping was observed at the junction between lamellae. This study confirms that outer annulus straining is mainly due to bundles slipping and rotating. Further development of disc multiscale modelling and repair techniques should take into account this modular behaviour of the lamella, rather than considering it as a homogeneous fibre-reinforced matrix. STATEMENT OF SIGNIFICANCE The intervertebral disc is an organ tucked between each couple of vertebrae in the spine. It is composed by an outer fibrous layer retaining a gel-like core. This organ undergoes severe and repeated loading during everyday life activities, since it is the compliant component that gives the spine its flexibility. Its properties are affected by pathologies such as disc degeneration, a major cause of back pain. In this article we explored the micromechanical behaviour of the discs outer layer using second harmonic generation, a technique which allowed us to visualize, with unprecedented detail, how bundles of collagen fibres slide relative to each other when loaded. Our results will help further the development of new multiscale numerical models and repairing techniques.

The molecular structure and physical properties of elastin fibers as revealed by Raman microspectroscopy

Raman microspectroscopy has been used to investigate the structure of alpha-elastin and fibrous elastin from ligament and aorta, and to explore changes associated with mechanical strain and temperature. Although no vibrational modes associated with cross-linking of the fibers could be identified, the secondary structure of dehydrated fibrous elastin was significantly different from alpha-elastin. The former differed from previous experimental measurements, but was close to the theoretical predictions with 36% beta-structures, 46% unordered, and 18% alpha-helix. Alpha-elastin contained 29% beta-structures, 53% unordered, and 18% alpha-helix. In nuchal fibers the amide I mode was polarized, consistent with the peptide bond. Strains of up to 60% in ligament fiber bundles resulted in no significant shifts in peak position or in secondary structure. Polarization measurements revealed that the peptide bonds and several side chains re-orientated closer to the fiber axis. Heating nuchal fibers to 60 degrees C to increase the energetic component of the elasticity was associated with a 30% increase in the proportion of beta-structures in the amide I band, a 50% increase in the amide III band, and a 50% reduction in the signal from bound water.

Bovine and degenerated human annulus fibrosus: a microstructural and micromechanical comparison.

The complex structure of the annulus fibrosus is strongly related to its mechanical properties. Recent work showed that it is possible to observe the relative movement of fibre bundles in loaded cow tail annulus; the aim of this work was to describe and quantify annulus fibrosus micromechanics in degenerated human disc, and compare it with cow tail annulus, an animal model often used in the literature. Second harmonic generation was used to image the collagen matrix in twenty strips of annulus fibrosus harvested from intervertebral disc of seven patients undergoing surgery. Samples were loaded to 6% tensile strain in 1% steps. Elastic modulus was calculated from loading curves, and micromechanical strains were calculated from the images using custom software. The same protocol was applied to twenty strips of annulus harvested from cow tail discs. Significant morphological differences were found between human and cow tail samples, the most striking being the lack of collagen fibre crimp in the former. Fibres were also observed bending and running from one lamella to the other, forming a strong flexible interface. Interdigitation of fibre bundles was also present at this interface. Quantitative results show complex patterns of inter-bundle and inter-lamellar behaviour, with inter-bundle sliding being the main strain mechanism. Elastic modulus was similar between species, and it was not affected by the degree of degeneration. This work gives an insight into the complex structure and mechanical function of the annulus fibrosus, which should be accounted for in disc numerical modelling.

The effects of needle damage on annulus fibrosus micromechanics

Needle puncture of the intervertebral disc can initiate a mechanical and biochemical cascade leading to disc degeneration. Punctures mechanical effects have been shown near the puncture site, mechanical effects should be observed far, relative to needle size, from the puncture site, given the disc-wide damage induced by the stab. The aim of this work was to quantify these far-field effects, and to observe the local structural damage provoked by the needle. Strips of cow tail annulus fibrosus underwent two consecutive mechanical loadings to 5% tensile strain; fifteen samples were punctured in a radial direction with a randomly assigned needle between the two loadings (needle gauges between 19 and 23). Ten samples (control group) were not punctured. During loading, the tissue strains were imaged using second harmonic generation microscopy in a <600×800µm region about 4.4mm from the puncture site. After mechanical testing, the puncture site was imaged in 3D. Puncture had no significant effect on annulus elastic modulus. Imaging showed a modest change in the shearing between fibre bundles however, the linear strain between bundles, intra-bundle shear and linear strain were not significantly affected. At the puncture site, detached lumps of tissue were present. These results suggest that the mechanical effects observed in intact discs are due to the depressurization of the disc, rather than the local damage to the annulus. Needle profiles could be designed, aiming at separating fibre bundles rather than cutting through them, to avoid leaving dying tissue behind. STATEMENT OF SIGNIFICANCE Needle puncture of the intervertebral disc can initiate a mechanical and biochemical cascade leading to disc degeneration, but the link between the local damage of the puncture and the disc-wide effects is not well understood. This work aimed at determining the micro-mechanical effects of the puncture far from its site, and to observe the damage induced by the puncture with high resolution imaging. Results show that the puncture had modest effect far from the puncture, but lumps of tissue were left by the needle, detached from the disc; these could cause further damage through friction and inflammation of the surrounding tissues. This suggests that the cascade leading to degeneration is probably driven by a biochemical response rather than disc-wide mechanical effects.

Measuring blood flow dynamics using DOCT and Doppler amplitude optical coherence tomography (DAOCT)

Flow dynamics in blood vessels of a few hundred microns in diameter were investigated using Doppler optical coherence tomography (DOCT) and Doppler Amplitude optical coherence tomography (DAOCT), a novel extension of DOCT. The motivation behind this work is discussed, followed by a brief explanation of the theory underlying the motion of blood cells in small conduits and blood vessels. Preliminary results are presented and compared to the predictions expected from theory. All significant findings are analysed along with their importance to microvascular research.

Dual scale biomechanics of extracellular matrix proteins probed by Brillouin scattering and quasistatic tensile testing

The biomechanics of living tissues are critical to normal tissue function and disturbances in these properties are widely implicated in aging and disease. Protein fibres of the extracellular matrix (collagen and elastin) are the fundamental mechanical structures in connective tissues such as bone, cartilage and vasculature. We applied Brillouin light scattering (BLS) spectroscopy and quasistatic stress-strain testing to the study of the mechanics and structure of collagen and elastin fibres purified from connective tissues. BLS probes mechanical properties on a microscopic scale in biological tissues and thereby providing insights into structure-function relationships under normal and pathological conditions. The sensitivity of BLS measurements to fibre structure and hydration was investigated using samples mounted onto reflective substrates. We obtained a complete characterization of the mechanical tensor and elastic moduli which could be compared with complementary data from quasistatic stress-strain measurements at different hydration levels, hence giving the full description of fibre viscoelasticity.

PSOCT studies of intervertebral disk

Polarization-sensitive optical coherence tomography (PSOCT) is an emerging optical imaging technique that is sensitive to the birefringence properties of tissues. It thus has applications in studying the large-scale ordering of collagen fibers within connective tissues. This ordering not only provides useful insights into the relationship between structure and function for various anatomical structures but also is an indicator of pathology. Intervertebral disk is an elastic tissue of the spine and possesses a 3-D collagen structure well suited to study using PSOCT. Since the outer layer of the disk has a lamellar structure with collagen fibers oriented in a trellis-like arrangement between lamellae, the birefringence fast-axis shows pronounced variations with depth, on a spatial scale of about 100 μm. The lamellar thickness varies with age and possibly with disease. We have used a polarisation-sensitive optical coherence tomography system to measure the birefringence properties of freshly excised, hydrated bovine caudal intervertebral disk and compared this with equine flexor tendon. Our results clearly demonstrate the ability of PSOCT to detect the outer three lamellae, down to a depth of at least 700 μm, via discontinuities in the depth-resolved retardance. We have applied a simple semi-empirical model based on Jones calculus to quantify the variation in the fast-axis orientation with depth. Our data and modeling is in broad agreement with previous studies using x-ray diffraction and polarization microscopy applied to histological sections of dehydrated disk. Our results imply that PSOCT may prove a useful tool to study collagen organisation within intervertebral disk in vitro and possibly in vivo and its variation with age and disease.

Polarization-sensitive optical coherence tomography applied to intervertebral disk

Polarization-sensitive optical coherence tomography (PSOCT) is a powerful new optical imaging modality that is sensitive to the birefringence properties of tissues. It thus has potential applications in studying the large-scale ordering of collagen fibers within connective tisues and changes related to pathology. As a tissue for study by PSOCT, intervertebral disk respresents an interesting system as the collagen organization is believed to show pronounced variations with depth, on a spatial scale of about 100 μm. We have used a polarization-sensitive optical coherence tomography system to measure the birefringence properties of bovine caudal intervertebral disk and compared this with equine flexor tendon. The result for equine tendon, δ = (3.0 ± 0.5)x10-3 at 1.3 μm, is in broad agreement with values reported for bovine tendon, while bovine intervertebral disk displays a birefringence of about half this, δ = 1.2 x 10-3 at 1.3 μm. While tendon appears to show a uniform fast-axis over 0.8 mm depth, intervertebral disk shows image contrast at all orientations relative to a linearly polarized input beam, suggesting a variation in fast-axis orientation with depth. These initial results suggest that PSOCT could be a useful tool to study collagen organization within this tissue and its variation with applied load and disease.

The synovial lining: a paradigm for connective tissue research?

It is to be expected that the extensive researches of Professor Levick and his group on the exchange of water and solutes between synovial fluid and the microcirculation of the synovial lining will advance understanding of the nutrition and lubrication of the synovial joint and of the ways in which disturbances in these processes are involved in joint disease. What is less expected is the way in which this work has raised, and often definitively answered, questions that are pertinent to many other areas of physiology and biophysics.