C. Peter Winlove

The collagen structure of bovine intervertebral disc studied using polarization-sensitive optical coherence tomography

Polarization-sensitive optical coherence tomography (PS-OCT) is used to measure the birefringence properties of bovine intervertebral disc and equine flexor tendon. For equine tendon the birefringence delta n is (6.0 +/- 0.2) x 10(-3) at a wavelength of 1.3 microm. This is somewhat larger than the values reported for bovine tendon. The surface region of the annulus fibrosus of a freshly excised intact bovine intervertebral disc displays an identical value of birefringence, delta n = (6.0 +/- 0.6) x 10(-3) at 1.3 microm. The nucleus pulposus does not display birefringence, the measured apparent value of delta n = (0.39 +/- 0.01) x 10(-3) being indistinguishable from the effects of depolarization due to multiple scattering. A clear difference is found between the depth-resolved retardance of equine tendon and that of bovine intervertebral disc. This apparently relates to the lamellar structure of the latter tissue, in which the collagen fibre orientation alternates between successive lamellae. A semi-empirical model based on Jones calculus shows that the measurements are in reasonable agreement with previous optical and x-ray data. These results imply that PS-OCT could be a useful tool to study collagen organization within the intervertebral disc in vitro and possibly in vivo and its variation with applied load and disease.

Tissue oxygenation and perfusion in patients with systemic sepsis.

ObjectiveMultiple organ dysfunction is associated with systemic sepsis. To investigate whether this is attributable to peripheral tissue hypoperfusion and/or cellular hypoxia, simultaneous measurements of tissue perfusion and oxygenation were made in patients with severe sepsis and in controls. DesignProspective, observational study. SettingAdult intensive care unit, tertiary referral center. PatientsVolunteers (group C, n = 7), patients undergoing cardiopulmonary bypass (group B, n = 6), and patients with severe sepsis (group S, n = 6). InterventionsLimb ischemia and reperfusion. Measurements and Main Results Tissue oxygenation and microvascular flow were measured by using microelectrodes inserted into brachoradialis muscle and overlying subcutaneous tissue. Forearm cutaneous red cell flux and regional blood flow were measured simultaneously. Responses to 20 mins of limb ischemia and subsequent reperfusion were observed. Baseline muscle tissue oxygenation was greater in sepsis (1.7 ± 0.2, 1.5 ± 0.7, and 4.4 ± 0.6 kPa for groups C, B, and S, respectively, mean ± sem, p < .05), although baseline subcutaneous tissue oxygenation did not vary between groups. During ischemia tissue oxygenation, values decreased in muscle (to 1.3 ± 0.2, 1.0 ± 0.4, and 1.5 ± 0.4 kPa for groups C, B, and S, respectively) and subcutaneous tissue (to 2.0 ± 0.3, 1.7 ± 0.5, and 2.3 ± 0.2 kPa for groups C, B, and S, respectively). Decline in tissue oxygen tension was initially more rapid in septic muscle compared with controls (25% decrease, 68 ± 23 vs. 176 ± 38 for group S vs. group C, p < .05, and 50% decrease, 126 ± 34 vs. 398 ± 72 secs for group S vs. group C, p < .01). However, overall rate of tissue decline was similar (95% decrease, 444 ± 122 vs. 614 ± 96 for group S vs. group C, p > .05). After reperfusion, significant differences in muscle tissue oxygenation reappeared between groups (2.0 ± 0.3, 1.5 ± 0.7, and 4.0 ± 0.4 kPa for groups C, B, and S, respectively, p < .05). There were no differences in time to 25%, 50%, or 95% tissue oxygen recovery. Whole limb reperfusion was significantly less in patient groups compared with controls (10.6 ± 0.9, 4.5 ± 1.2, and 4.3 ± 1.6 mL·100 mL−1·min−1 for groups C, B, and S, respectively, p < .05). ConclusionsSignificant differences in tissue oxygenation distribution between muscle and subcutaneous tissues occur in patients with severe sepsis. High baseline muscle tissue oxygen levels are accompanied by rapid extraction of oxygen during stagnant ischemia.

Microfibrils, elastin fibres and collagen fibres in the human intervertebral disc and bovine tail disc

The distribution of microfibrils was studied immunohistochemically in intervertebral discs taken from young normal human surgical cases and from the bovine tail. Co‐localization of microfibrils and elastin fibres was examined by dual immunostaining of fibrillin‐1 and elastin. Collagen fibre network orientation was studied by using polarized filters. A similar microfibrillar network was seen in both bovine and human discs with network organization being completely different from region to region. In the outer annulus fibrosus (OAF), abundant microfibrils organized in bundles were mainly distributed in the interterritorial matrix. In addition, the microfibril bundles were orientated parallel to each other and co‐localized highly with elastin fibres. Within each lamella, co‐localized microfibrils and elastin fibres were aligned in the same direction as the collagen fibres. In the interlamellar space, a dense co‐localized network, staining for both microfibrils and elastin fibres, was apparent; immunostaining for both molecules was noticeably stronger than within lamellae. In the inner annulus fibrosus, the microfibrils were predominantly visible as a filamentous mesh network, both in the interterritorial matrix and also around the cells. The microfibrils in this region co‐localized with elastin fibres far less than in the OAF. In nucleus pulposus, filamentous microfibrils were organized mainly around the cells where elastin fibres were hardly detected. By contrast, sparse elastin fibres, with a few of microfibrils, were visible in the interterritorial matrix. The results of this study suggest the microfibrillar network of the annulus may play a mechanical role while that around the cells of the nucleus may be more involved in regulating cell function.

Collagen fiber arrangement in normal and diseased cartilage studied by polarization sensitive nonlinear microscopy

Second harmonic generation (SHG) and two-photon fluorescence (TPF) microscopy is used to image the intercellular and pericellular matrix in normal and degenerate equine articular cartilage. The polarization sensitivity of SHG can be used directly to determine fiber orientation in the superficial 10 to 20 microm of tissue, and images of the ratio of intensities taken with two orthogonal polarization states reveal small scale variations in the collagen fiber organization that have not previously been reported. The signal from greater depths is influenced by the birefringence and biattenuance of the overlying tissue. An assessment of these effects is developed, based on the analysis of changes in TPF polarization with depth, and the approach is validated in tendon where composition is independent of depth. The analysis places an upper bound on the biattenuance of tendon of 2.65 x 10(-4). Normal cartilage reveals a consistent pattern of variation in fibril orientation with depth. In lesions, the pattern is severely disrupted and there are changes in the pericellular matrix, even at the periphery where the tissue appears microscopically normal. Quantification of polarization sensitivity changes with depth in cartilage will require detailed numerical models, but in the meantime, multiphoton microscopy provides sensitive indications of matrix changes in cartilage degeneration.

Scanning electrochemical microscopy as a local probe of oxygen permeability in cartilage

The use of scanning electrochemical microscopy, a high-resolution chemical imaging technique, to probe the distribution and mobility of solutes in articular cartilage is described. In this application, a mobile ultramicroelectrode is positioned close ( approximately 1 microm) to the cartilage sample surface, which has been equilibrated in a bathing solution containing the solute of interest. The solute is electrolyzed at a diffusion-limited rate, and the current response measured as the ultramicroelectrode is scanned across the sample surface. The topography of the samples was determined using Ru(CN)(6)(4-), a solute to which the cartilage matrix was impermeable. This revealed a number of pit-like depressions corresponding to the distribution of chondrocytes, which were also observed by atomic force and light microscopy. Subsequent imaging of the same area of the cartilage sample for the diffusion-limited reduction of oxygen indicated enhanced, but heterogeneous, permeability of oxygen across the cartilage surface. In particular, areas of high permeability were observed in the cellular and pericellular regions. This is the first time that inhomogeneities in the permeability of cartilage toward simple solutes, such as oxygen, have been observed on a micrometer scale.

Structural Analysis of Glycosaminoglycans and Proteoglycans by Means of Raman Microspectrometry

Raman spectra have been determined for hyaluronan, chondroitin-4-sulfate, chondroitin-6-sulfate, aggrecan monomers and aggregates. The nature of the saccharides and the pattern of sulfation can be discerned. There were only small spectral changes with pH and ionic composition. Differences between hydroxyl vibrations, bulk water and solution conditions are shown. The spectrum of aggrecan is dominated by chondroitin sulfate contribution. The sulfation pattern and ratio of protein to glycosaminoglycan and the secondary structure of the core protein were determined.

Spectroscopy on the wing: Naturally inspired SERS substrates for biochemical analysis

We show that naturally occurring chitinous nanostructures found on the wings of the Graphium butterfly can be used as substrates for surface-enhanced Raman scattering when coated with a thin film of gold or silver. The substrates were found to exhibit excellent biocompatibility and sensitivity, making them ideal for protein assaying. An assay using avidin/biotin binding showed that the substrates could be used to quantify protein binding directly from changes in the surface-enhanced Raman scattering (SERS) spectra and were sensitive over a concentration range comparable with a typical enzyme-linked immunosorbent assays (ELISA) assay. A biomimetic version of the wing nanostructures produced using a highly reproducible, large-scale fabrication process, yielded comparable enhancement factors and biocompatibility. The excellent biocompatibility of the wings and biomimetic substrates is unparalleled by other lithographically produced substrates, and this could pave the way for widespread application of ultrasensitive SERS-based bioassays.

The collagen structure of equine articular cartilage, characterized using polarization-sensitive optical coherence tomography

Optical coherence tomography and polarization-sensitive optical coherence tomography images of equine articular cartilage are presented. Measurements were made on intact joint surfaces. Significant (e.g. × 2) variations in the intrinsic birefringence were found over spatial scales of a few millimetres, even on samples taken from young (18 month) animals that appeared visually homogeneous. A comparison of data obtained on a control tissue (equine flexor tendon) further suggests that significant variations in the orientation of the collagen fibres relative to the plane of the joint surface exist. Images of visually damaged cartilage tissue show characteristic features both in terms of the distribution of optical scatterers and of the birefringent components.

Measuring red blood cell flow dynamics in a glass capillary using Doppler optical coherence tomography and Doppler amplitude optical coherence tomography

Blood, being a suspension of deformable red cells suspended in plasma, displays flow dynamics considerably more complicated than those of an ideal Newtonian fluid. Flow dynamics in blood capillaries of a few hundred micrometers in diameter are investigated using Doppler optical coherence tomography (DOCT) and Doppler amplitude optical coherence tomography (DAOCT), a novel extension of DOCT. Velocity profiles and concentration distributions of normal and rigidified in vitro red blood cell suspensions are shown to vary as functions of mean flow velocity, cell concentration, and cell rigidity. Deviation from the parabolic velocity profile expected for Pouseille flow is observed for both rigid and normal cells at low flow rates. Axial red cell migration both toward and away from the tube axis is observed for both rigid and normal cells as a function of flow velocity. Good agreement is found between our measurements, and theoretical expectations.

Observation and characterisation of the glycocalyx of viable human endothelial cells using confocal laser scanning microscopy

This paper describes the use of confocal laser scanning microscopy (CLSM) to observe and characterise the fully hydrated glycocalyx of human umbilical vein endothelial cells (HUVECs). Viable HUVECs in primary culture were studied at room temperature in HEPES-buffered, phenol red- and serum-free CS-C cell culture medium. A fluorescein isothiocyanate-linked wheat germ agglutinin (WGA-FITC) (2 μg ml−1, 30 min) was used to detect N-acetylneuraminic (sialic) acid, which is a significant component of the endothelial glycocalyx. Single confocal sections, less than 1.3 μm thick, were collected at intervals of 0.5 μm, scanning through the entire z-axis of a series of cells. Cell-surface associated staining was observed, which enabled the glycocalyx thickness to be deduced as 2.5 ± 0.5 μm. This dimension is significantly greater than that measured by electron microscopy, for glutaraldehyde-fixed cells (0.10 ± 0.04 μm). The specificity of WGA-FITC staining was demonstrated by treatments with several enzymes, known to degrade glycocalyx (heparatinase, chondroitinase, hyaluronidase and neuraminidase), of which neuraminidase (1 U ml−1, 30–60 min) was the most effective, removing up to 78 ± 2% of WGA-FITC binding to HUVECs. Cell viability was assessed simultaneously with ethidium homodimer-1 staining and confirmed by standard colorimetric 3-[4,5]dimethylthiazol-2,5diphenyltetrazolium bromide (MTT) test. CLSM thus provides a useful approach for in situ visualisation and characterisation of the endothelial glycocalyx in viable preparations, revealing a thickness that is an order of magnitude greater than found in ex situ measurements on fixed cells.