Regional variations in discrete collagen fibre mechanics within intact intervertebral disc resolved using synchrotron computed tomography and digital volume correlation.

Many soft tissues, such as the intervertebral disc (IVD), have a hierarchical fibrous composite structure which suffers from regional damage. We hypothesise that these tissue regions have distinct, inherent fibre structure and structural response upon loading. Here we used synchrotron computed tomography (sCT) to resolve collagen fibre bundles (∼5µm width) in 3D throughout an intact native rat lumbar IVD under increasing compressive load. Using intact samples meant that tissue boundaries (such as endplate-disc or nucleus-annulus) and residual strain were preserved; this is vital for characterising both the inherent structure and structural changes upon loading in tissue regions functioning in a near-native environment. Nano-scale displacement measurements along >10,000 individual fibres were tracked, and fibre orientation, curvature and strain changes were compared between the posterior-lateral region and the anterior region. These methods can be widely applied to other soft tissues, to identify fibre structures which cause tissue regions to be more susceptible to injury and degeneration. Our results demonstrate for the first time that highly-localised changes in fibre orientation, curvature and strain indicate differences in regional strain transfer and mechanical function (e.g. tissue compliance). This included decreased fibre reorientation at higher loads, specific tissue morphology which reduced capacity for flexibility and high strain at the disc-endplate boundary. STATEMENT OF SIGNIFICANCE: The analyses presented here are applicable to many collagenous soft tissues which suffer from regional damage. We aimed to investigate regional intervertebral disc (IVD) structural and functional differences by characterising collagen fibre architecture and linking specific fibre- and tissue-level deformation behaviours. Synchrotron CT provided the first demonstration of tracking discrete fibres in 3D within an intact IVD. Detailed analysis of regions was performed using over 200k points, spaced every 8 µm along 10k individual fibres. Such comprehensive structural characterisation is significant in informing future computational models. Morphological indicators of tissue compliance (change in fibre curvature and orientation) and fibre strain measurements revealed localised and regional differences in tissue behaviour.

Investigation of fibrillin microfibrils in the canine cruciate ligament in dogs with different predispositions to ligament rupture.

Cranial cruciate ligament disease (CCLD) is the most common cause of pelvic limb lameness in dogs but its precise aetiopathogenesis is uncertain. Fibrillin microfibrils (FM) are complex macro-molecular assemblies found in many tissues including ligaments, where they are thought to play an important mechanical role. We hypothesised that FM ultrastructural variation correlates with the differing predisposition of canine breeds to CCLD. Non-diseased cranial and caudal cruciate ligaments (CCLs and CaCLs) were obtained from Greyhound (GH) and Staffordshire Bull Terrier (SBT) cadavers. Fibrillin microfibrils were extracted from the ligaments by bacterial collagenase digestion, purified by size-exclusion chromatography and subsequently visualized by atomic force microscopy (AFM). With AFM, FMs have a characteristic beads-on-a-string appearance. For each FM, periodicity (bead-bead distance) and length (number of beads/FM) was measured. Fibrillin microfibril length was found to be similar for GH and SBT, with non-significant inter-breed and inter-ligament differences. Fibrillin microfibril periodicity varied when comparing GH and SBT for CCL (GH 60.2 ± 1.4 nm; SBT 56.2 ± 0.8 nm) and CaCL (GH 55.5 ± 1.6 nm; SBT 61.2 ± 1.2 nm). A significant difference was found in the periodicity distribution when comparing CCL for both breeds (P < 0.00001), further, intra-breed differences in CCL vs CaCL were statistically significant within both breeds (P < 0.00001). The breed at low risk of CCLD exhibited a periodicity profile which may be suggestive of a repair and remodelling within the CCL.

Synchrotron tomography of intervertebral disc deformation quantified by digital volume correlation reveals microstructural influence on strain patterns.

The intervertebral disc (IVD) has a complex and multiscale extracellular matrix structure which provides unique mechanical properties to withstand physiological loading. Low back pain has been linked to degeneration of the disc but reparative treatments are not currently available. Characterising the disc's 3D microstructure and its response in a physiologically relevant loading environment is required to improve understanding of degeneration and to develop new reparative treatments. In this study, techniques for imaging the native IVD, measuring internal deformation and mapping volumetric strain were applied to an in situ compressed ex vivo rat lumbar spine segment. Synchrotron X-ray micro-tomography (synchrotron CT) was used to resolve IVD structures at microscale resolution. These image data enabled 3D quantification of collagen bundle orientation and measurement of local displacement in the annulus fibrosus between sequential scans using digital volume correlation (DVC). The volumetric strain mapped from synchrotron CT provided a detailed insight into the micromechanics of native IVD tissue. The DVC findings showed that there was no slipping at lamella boundaries, and local strain patterns were of a similar distribution to the previously reported elastic network with some heterogeneous areas and maximum strain direction aligned with bundle orientation, suggesting bundle stretching and sliding. This method has the potential to bridge the gap between measures of macro-mechanical properties and the local 3D micro-mechanical environment experienced by cells. This is the first evaluation of strain at the micro scale level in the intact IVD and provides a quantitative framework for future IVD degeneration mechanics studies and testing of tissue engineered IVD replacements. STATEMENT OF SIGNIFICANCE: Synchrotron in-line phase contrast X-ray tomography provided the first visualisation of native intact intervertebral disc microstructural deformation in 3D. For two annulus fibrosus volumes of interest, collagen bundle orientation was quantified and local displacement mapped as strain. Direct evidence of microstructural influence on strain patterns could be seen such as no slipping at lamellae boundaries and maximum strain direction aligned with collagen bundle orientation. Although disc elastic structures were not directly observed, the strain patterns had a similar distribution to the previously reported elastic network. This study presents technical advances and is a basis for future X-ray microscopy, structural quantification and digital volume correlation strain analysis of soft tissue.

Mass spectrometry-based proteomics reveals the distinct nature of the skin proteomes of photoaged compared to intrinsically aged skin.

OBJECTIVE: With increasing age, skin is subject to alterations in its organization, which impact on its function as well as having clinical consequences. Proteomics is a useful tool for non-targeted, semi-quantitative simultaneous investigation of high numbers of proteins. In the current study, we utilize proteomics to characterize and contrast age-associated differences in photoexposed and photoprotected skin, with a focus on the epidermis, dermal-epidermal junction and papillary dermis. METHODS: Skin biopsies from buttock (photoprotected) and forearm (photoexposed) of healthy volunteers (aged 18-30 or ≥65 years) were transversely sectioned from the stratum corneum to a depth of 250 µm. Following SDS-PAGE, each sample lane was segmented prior to analysis by liquid chromatography-mass spectrometry/mass spectrometry. Pathway analysis was carried out using Ingenuity IPA. RESULTS: Comparison of skin proteomes at buttock and forearm sites revealed differences in relative protein abundance. Ageing in skin on the photoexposed forearm resulted in 80% of the altered proteins being increased with age, in contrast to the photoprotected buttock where 74% of altered proteins with age were reduced. Functionally, age-altered proteins in the photoexposed forearm were associated with conferring structure, energy and metabolism. In the photoprotected buttock, proteins associated with gene expression, free-radical scavenging, protein synthesis and protein degradation were most frequently altered. CONCLUSION: This study highlights the necessity of not considering photoageing as an accelerated intrinsic ageing, but as a distinct physiological process.

OBJECTIF: Avec l'âge, la peau est sujette à des altérations dans son organisation, et outre le fait d'avoir des conséquences cliniques cela a un impact sur sa fonction. La protéomique est un outil utile pour l'évaluation non ciblée, semi-quantitative, simultanée d'un nombre élevé de protéines. Dans cette étude, nous utilisons la protéomique pour caractériser et comparer les différences associées à l'âge entre une peau photoexposée et une peau photoprotégée, avec une attention particulière sur l'épiderme, la jonction dermo-épidermique et le derme papillaire. MÉTHODES: Des biopsies de peau de la fesse (photoprotégée) et de l'avant-bras (photoexposée) de volontaires sains (âgés de 18 à 30 ans ou de ≥ 65 ans) ont été sectionnées transversalement depuis la couche cornée jusqu'à une profondeur de 250 µm. Suite à une électrophorèse SDS-PAGE, chaque échantillon a été segmenté avant l'analyse par chromatographie en phase liquide couplée à la spectrométrie de masse/spectrométrie de masse. Une analyse des voies de signalisation a été réalisée à l'aide d'Ingenuity IPA. RÉSULTATS: La comparaison des protéomes de la peau des sites des fesses et de l'avant-bras a révélé des différences dans l'abondance relative de protéines. Le vieillissement de la peau de l'avant-bras photoexposée montre une augmentation de 80% des protéines altérées avec l'âge, contrairement à la peau des fesses photoprotégée où une réduction de 74 % des protéines altérées avec l'âge a été mesurée. Sur le plan de la fonction, les protéines altérées par l'âge dans la peau de l'avant-bras photoexposée étaient associées à une structure, une énergie et un métabolisme. Dans la peau des fesses photoprotégée, les protéines associées à l'expression génique, la neutralisation des radicaux-libres, la synthèse des protéines et la dégradation des protéines étaient le plus fréquemment altérés. CONCLUSION: Cette étude souligne la nécessité de ne pas considérer le photovieillissement comme un vieillissement accéléré intrinsèque, mais comme un processus physiologique distinct.

A review of techniques for visualising soft tissue microstructure deformation and quantifying strain Ex Vivo.

Many biological tissues have a complex hierarchical structure allowing them to function under demanding physiological loading conditions. Structural changes caused by ageing or disease can lead to loss of mechanical function. Therefore, it is necessary to characterise tissue structure to understand normal tissue function and the progression of disease. Ideally intact native tissues should be imaged in 3D and under physiological loading conditions. The current published in situ imaging methodologies demonstrate a compromise between imaging limitations and maintaining the samples native mechanical function. This review gives an overview of in situ imaging techniques used to visualise microstructural deformation of soft tissue, including three case studies of different tissues (tendon, intervertebral disc and artery). Some of the imaging techniques restricted analysis to observational mechanics or discrete strain measurement from invasive markers. Full-field local surface strain measurement has been achieved using digital image correlation. Volumetric strain fields have successfully been quantified from in situ X-ray microtomography (micro-CT) studies of bone using digital volume correlation but not in soft tissue due to low X-ray transmission contrast. With the latest developments in micro-CT showing in-line phase contrast capability to resolve native soft tissue microstructure, there is potential for future soft tissue mechanics research where 3D local strain can be quantified. These methods will provide information on the local 3D micromechanical environment experienced by cells in healthy, aged and diseased tissues. It is hoped that future applications of in situ imaging techniques will impact positively on the design and testing of potential tissue replacements or regenerative therapies. LAY DESCRIPTION: The soft tissues in our bodies, such as tendons, intervertebral discs and arteries, have evolved to have complicated structures which deform and bear load during normal function. Small changes in these structures can occur with age and disease which then leads to loss of function. Therefore, it is important to image tissue microstructure in 3D and under functional conditions. This paper gives an overview of imaging techniques used to record the deformation of soft tissue microstructures. Commonly there are compromises between obtaining the best imaging result and retaining the samples native structure and function. For example, invasive markers and dissecting samples damages the tissues natural structure, and staining or clearing (making the tissue more transparent) can distort tissue structure. Structural deformation has been quantified from 2D imaging techniques (digital image correlation) to create surface strain maps which help identify local tissue mechanics. When extended to 3D (digital volume correlation), deformation measurement has been limited to bone samples using X-ray micro-CT. Recently it has been possible to image the 3D structure of soft tissue using X-ray micro-CT meaning that there is potential for internal soft tissue mechanics to be mapped in 3D. Future application of micro-CT and digital volume correlation will be important for soft tissue mechanics studies particularly to understand normal function, progression of disease and in the design of tissue replacements.

Extracellular matrix fragmentation in young, healthy cartilaginous tissues.

Although the composition and structure of cartilaginous tissues is complex, collagen II fibrils and aggrecan are the most abundant assemblies in both articular cartilage (AC) and the nucleus pulposus (NP) of the intervertebral disc (IVD). Whilst structural heterogeneity of intact aggrecan ( containing three globular domains) is well characterised, the extent of aggrecan fragmentation in healthy tissues is poorly defined. Using young, yet skeletally mature (18-30 months), bovine AC and NP tissues, it was shown that, whilst the ultrastructure of intact aggrecan was tissue-dependent, most molecules (AC: 95 %; NP: 99.5 %) were fragmented (lacking one or more globular domains). Fragments were significantly smaller and more structurally heterogeneous in the NP compared with the AC (molecular area; AC: 8543 nm2; NP: 4625 nm2; p < 0.0001). In contrast, fibrillar collagen appeared structurally intact and tissue-invariant. Molecular fragmentation is considered indicative of a pathology; however, these young, skeletally mature tissues were histologically and mechanically (reduced modulus: AC: ≈ 500 kPa; NP: ≈ 80 kPa) comparable to healthy tissues and devoid of notable gelatinase activity (compared with rat dermis). As aggrecan fragmentation was prevalent in neonatal bovine AC (99.5 % fragmented, molecular area: 5137 nm2) as compared with mature AC (95.0 % fragmented, molecular area: 8667 nm2), it was hypothesised that targeted proteolysis might be an adaptive process that modified aggrecan packing (as simulated computationally) and, hence, tissue charge density, mechanical properties and porosity. These observations provided a baseline against which pathological and/or age-related fragmentation of aggrecan could be assessed and suggested that new strategies might be required to engineer constructs that mimic the mechanical properties of native cartilaginous tissues.

Visualising the 3D microstructure of stained and native intervertebral discs using X-ray microtomography.

Intervertebral disc degeneration (IVDD) is linked to low back pain. Microstructural changes during degeneration have previously been imaged using 2D sectioning techniques and 3D methods which are limited to small specimens and prone to inducing artefacts from sample preparation. This study explores micro computed X-ray tomography (microCT) methods with the aim of resolving IVD 3D microstructure whilst minimising sample preparation artefacts. Low X-ray absorption contrast in non-mineralised tissue can be enhanced using staining and phase contrast techniques. A step-wise approach, including comparing three stains, was used to develop microCT for bovine tail IVD using laboratory and synchrotron sources. Staining successfully contrasted collagenous structures; however not all regions were stained and the procedure induced macroscopic structural changes. Phase contrast microCT of chemically fixed yet unstained samples resolved the nucleus pulposus, annulus fibrosus and constituent lamellae, and finer structures including collagen bundles and cross-bridges. Using the same imaging methods native tissue scans were of slightly lower contrast but free from sample processing artefacts. In the future these methods may be used to characterise structural remodelling in soft (non-calcified) tissues and to conduct in situ studies of native loaded tissues and constructs to characterise their 3D mechanical properties.

A new in vitro assay to test UVR protection of dermal extracellular matrix components by a flat spectrum sunscreen.

The efficacy of topical sunscreens is currently assessed by crude, costly and time consuming in vivo assays. We have previously demonstrated that components of the dermal extracellular matrix (ECM), rich in UV-absorbing amino acids, are susceptible to damage by solar simulated radiation (SSR) in vitro. Here we developed an in vitro method to test the ability of sunscreens to protect fibrillin-rich microfibrils (FRM) and fibronectin, key components of the dermal ECM from UV-induced damage. Solutions of FRM or fibronectin were irradiated without protection, in the presence of a vehicle or a commercially-available flat-spectrum sunscreen. The effect of SSR on molecular structure was determined by atomic force microscopy (FRM) and SDS-PAGE (fibronectin). Following irradiation, FRM periodicity became bi-modally distributed (peaks: 40nm & 59nm) compared to the unimodal distribution in unexposed controls (peak: 50nm). Irradiation in the presence of flat-spectrum sunscreen protected against this change, maintaining the unimodal distribution. SSR induced significant aggregation of fibronectin (p=0.005), which was abrogated by sunscreen. These results demonstrate that this in vitro assay system is sufficiently sensitive to act as an initial/additional screen of sunscreen efficacy. We conclude that sunscreen can reduce UV-mediated damage of key dermal ECM in vitro and thereby prevent remodelling associated with photoageing.

Organization of the dermal matrix impacts the biomechanical properties of skin.

BACKGROUND: Human skin has the crucial roles of maintaining homeostasis and protecting against the external environment. Skin offers protection against mechanical trauma due to the reversible deformation of its structure; these biomechanical properties are amenable to dynamic testing using noninvasive devices. OBJECTIVES: To characterize the biomechanical properties of young, black African/African-Caribbean and white Northern European skin from different anatomical sites, and to relate underlying skin architecture to biomechanical function. METHODS: Using cutometry and ballistometry, the biomechanical properties of buttock and dorsal forearm skin were determined in black African/African-Caribbean (n = 18) and white Northern European (n = 20) individuals aged 18-30 years. Skin biopsies were obtained from a subset of the volunteers (black African/African-Caribbean, n = 5; white Northern European, n = 6) and processed for histological and immunohistochemical detection of the major elastic fibre components and fibrillar collagens. RESULTS: We have determined that healthy skin from young African and white Northern European individuals has similar biomechanical properties (F3): the skin is resilient (capable of returning to its original position following deformation, R1), exhibits minimal fatigue (R4) and is highly elastic (R2, R5 and R7). At the histological level, skin with these biomechanical properties is imbued with strong interdigitation of the rete ridges at the dermoepidermal junction (DEJ) and candelabra-like arrays of elastic fibres throughout the papillary dermis. Dramatic disruption to this highly organized arrangement of elastic fibres, effacement of the rete ridges and alterations to the alignment of the fibrillar collagens is apparent in the white Northern European forearm and coincides with a marked decline in biomechanical function. CONCLUSIONS: Maintenance of skin architecture - both epidermal morphology and elastic fibre arrangement - is essential for optimal skin biomechanical properties. Disruption to underlying skin architecture, as observed in the young white Northern European forearm, compromises biomechanical function.

Novel approaches to characterize age-related remodelling of the dermal-epidermal junction in 2D, 3D and in vivo.

BACKGROUND/PURPOSE: The dermal-epidermal junction (DEJ) forms epidermal protrusions down into the dermis (rete ridges) and dermal projections up into the epidermis (dermal papillae). Usually visualized in two-dimensions (2D), our knowledge of how the DEJ changes with ageing is limited. We aimed to characterize how this structure exists in 3D and changes with age. METHODS: Photoprotected and photoexposed skin were imaged using reflectance confocal microscopy (RCM) in young and aged individuals. Biopsies of the imaged areas were processed for histological sectioning and for imaging using micro-computed X-ray tomography (microCT). RESULTS: Images obtained from RCM and microCT were used to 3D reconstruct the DEJ. DEJ heights obtained from microCT images showed strong correlation with histology-measured heights. We proposed a novel definition of rete ridges (RRm ) and dermal papillae (DPm ), which allowed easier automated measurement of reduced DPm and RRm volumes in aged skin from microCT reconstructions. An algorithm to map DPm connectivity showed reduced lengths of DPm branches with age. CONCLUSION: Three-dimensional images illustrated the complex topography of the DEJ and highlighted the distinct morphology of dermal papillae compared with rete ridges, which is not evident when evaluating 2D sections. Ex vivo imaging was more successful in differentiating DEJ architecture with respect to age.

Frequency-modulated atomic force microscopy localises viscoelastic remodelling in the ageing sheep aorta.

Age-related aortic stiffening is associated with cardiovascular diseases such as heart failure. The mechanical functions of the main structural components of the aorta, such as collagen and elastin, are determined in part by their organisation at the micrometer length scale. With age and disease both components undergo aberrant remodelling, hence, there is a need for accurate characterisation of the biomechanical properties at this length scale. In this study we used a frequency-modulated atomic force microscopy (FM-AFM) technique on a model of ageing in female sheep aorta (young: ~18 months, old: >8 years) to measure the micromechanical properties of the medial layer of the ascending aorta. The novelty of our FM-AFM method, operated at 30kHz, is that it is non-contact and can be performed on a conventional AFM using the ×³cantilever tune' mode, with a spatial (areal) resolution of around 1.6µm(2). We found significant changes in the elastic and viscoelastic properties within the medial lamellar unit (elastic lamellae and adjacent inter-lamellar space) with age. In particular, there was an increase in elastic modulus (Young; geometric mean (geometric SD)=42.9 (2.26)kPa, Old=113.9 (2.57)kPa, P<0.0001), G' and G″ (storage and loss modulus respectively) (Young; G'=14.3 (2.26)kPa, Old G'=38.0 (2.57)kPa, P<0.0001; Young; G″=14.5 (2.56)kPa, Old G″=32.8 (2.52)kPa, P<0.0001). The trends observed in the elastic properties with FM-AFM matched those we have previously found using scanning acoustic microscopy (SAM). The utility of the FM-AFM method is that it does not require custom AFM hardware and can be used to simultaneously determine the elastic and viscoelastic behaviour of a biological sample.

Localized micro- and nano-scale remodelling in the diabetic aorta.

Diabetes is strongly associated with cardiovascular disease, but the mechanisms, structural and biomechanical consequences of aberrant blood vessel remodelling remain poorly defined. Using an experimental (streptozotocin, STZ) rat model of diabetes, we hypothesized that diabetes enhances extracellular protease activity in the aorta and induces morphological, compositional and localized micromechanical tissue remodelling. We found that the medial aortic layer underwent significant thickening in diabetic animals but without significant changes in collagen or elastin (abundance). Scanning acoustic microscopy demonstrated that such tissue remodelling was associated with a significant decrease in acoustic wave speed (an indicator of reduced material stiffness) in the inter-lamellar spaces of the vessel wall. This index of decreased stiffness was also linked to increased extracellular protease activity (assessed by semi-quantitative in situ gelatin zymography). Such a proteolytically active environment may affect the macromolecular structure of long-lived extracellular matrix molecules. To test this hypothesis, we also characterized the effects of diabetes on the ultrastructure of an important elastic fibre component: the fibrillin microfibril. Using size exclusion chromatography and atomic force microscopy, we isolated and imaged microfibrils from both healthy and diabetic aortas. Microfibrils derived from diabetic tissues were fragmented, morphologically disrupted and weakened (as assessed following molecular combing). These structural and functional abnormalities were not replicated by in vitro glycation. Our data suggest that proteolysis may be a key driver of localized mechanical change in the inter-lamellar space of diabetic rat aortas and that structural proteins (such as fibrillin microfbrils) may be biomarkers of diabetes induced damage.

Geographical ancestry is a key determinant of epidermal morphology and dermal composition.

BACKGROUND: Geographical ancestry plays a key role in determining the susceptibility of human skin to external insults and dermatological disease. Despite this, studies of skin from individuals of diverse geographical ancestry focus primarily on epidermal pigmentation. Few reports characterize the gross morphology and composition of the dermis and dermal-epidermal junction (DEJ). OBJECTIVES: To characterize epidermal morphology and dermal composition in skin from individuals of diverse geographical ancestry. METHODS: Immunohistochemical techniques were used to assess epidermal morphology and protein composition of the DEJ and dermal extracellular matrix in photoprotected skin from young African, Eurasian and Far East Asian individuals (n = 7 per group; age 18-30 years). RESULTS: The epidermis of African skin was thicker, with deeper rete ridges and a more convoluted DEJ than Eurasian and Far East Asian skin. Compared with Eurasians, protein composition of the DEJ was collagen VII poor in African and Far East Asian skin (P < 0·001 and P < 0·01, respectively); the dermis of African skin was enriched in fibrillar collagens (P < 0·05), but was relatively elastin poor (P < 0·05). African dermis was abundant in fibrillin-rich microfibrils and fibulin-5 (P < 0·001 and P < 0·001, respectively) compared with Eurasian and Far East Asian skin. CONCLUSIONS: We demonstrate that fundamental differences exist in skin structure and composition in individuals of diverse geographical ancestry. Disparate environmental pressures encountered by ancestral human populations living at different latitudes may have driven adaptations in skin structure and composition. Further research into the functional significance and clinical consequences of these differences is warranted.

A new wrinkle on old skin: the role of elastic fibres in skin ageing.

Cutaneous ageing is the result of two distinct, biological processes which may occur concurrently: (i) the passage of time, termed intrinsic ageing and (ii) environmental influences, termed extrinsic ageing. Intrinsic ageing of the skin is a slow process which causes changes in tissue structure and impairs function in the absence of additional biological, chemical and physical factors. The clinical features of intrinsically aged skin are not usually evident until old age when, although smooth and unblemished, the skin surface appears pale and is characterized by fine wrinkles with occasional exaggerated expression lines. Functionally, intrinsically aged skin is dry and less elastic than more youthful skin. In contrast, extrinsically aged skin is exemplified by deep, coarse wrinkles, mottled hyperpigmentation and a marked loss of elasticity and recoil. The two major environmental influences which induce extrinsic ageing are: (i) chronic exposure to solar ultraviolet (UV) irradiation (termed photoageing) and (ii) smoking. This review discusses the changes associated with the ageing process in the skin, with particular emphasis on the role played by the elastic fibre network in maintaining dermal function. The review concludes with a discussion of a short-term assay for independent assessment of the efficacy of anti-ageing cosmetic products using the elastic fibre component fibrillin-1 as a biomarker of extracellular matrix repair.

Nanoindentation of histological specimens: Mapping the elastic properties of soft tissues.

Although alterations in the gross mechanical properties of dynamic and compliant tissues have a major impact on human health and morbidity, there are no well-established techniques to characterize the micromechanical properties of tissues such as blood vessels and lungs. We have used nanoindentation to spatially map the micromechanical properties of 5-mum-thick sections of ferret aorta and vena cava and to relate these mechanical properties to the histological distribution of fluorescent elastic fibers. To decouple the effect of the glass substrate on our analysis of the nanoindentation data, we have used the extended Oliver and Pharr method. The elastic modulus of the aorta decreased progressively from 35 MPa in the adventitial (outermost) layer to 8 MPa at the intimal (innermost) layer. In contrast, the vena cava was relatively stiff, with an elastic modulus >30 MPa in both the extracellular matrix-rich adventitial and intimal regions of the vessel. The central, highly cellularized, medial layer of the vena cava, however, had an invariant elastic modulus of ~20 MPa. In extracellular matrix-rich regions of the tissue, the elastic modulus, as determined by nanoindentation, was inversely correlated with elastic fiber density. Thus, we show it is possible to distinguish and spatially resolve differences in the micromechanical properties of large arteries and veins, which are related to the tissue microstructure.

Fibrillin-rich microfibrils: elastic biopolymers of the extracellular matrix.

Fibrillin-rich microfibrils are evolutionarily ancient macromolecular assemblies of the extracellular matrix. They have unique extensible properties that endow vascular and other tissues with long-range elasticity. Microfibril extensibility supports the low pressure closed circulations of lower organisms such as crustaceans. In higher vertebrates, microfibrils act as a template for elastin deposition and are components of mature elastic fibres. In man, the importance of microfibrils is highlighted by the linkage of mutations in their principal structural component, fibrillin-1, to the heritable disease Marfan syndrome which is characterised by severe cardiovascular, skeletal and ocular defects. When isolated from tissues, fibrillin-rich microfibrils have a complex ultrastructural organisation with a characteristic 'beads-on-a-strong' appearance. X-ray fibre diffraction studies and biomechanical testing have shown that microfibrils are reversibly extensible at tissue extensions of 100%. Ultrastructural analysis and 3D reconstructions of isolated microfibrils using automated electron tomography have revealed new details of how fibrillin molecules are aligned within microfibrils in untensioned and extended states, and delineated the role of calcium in regulating microfibril beaded periodicity, rest length and molecular organisation. The molecular basis of how fibrillin molecules assemble into microfibrils, the central role of cells in regulating this process, and the identity of other molecules that may coassemble into microfibrils are now being elucidated. This information will enhance our understanding of the elastic mechanism of these unique extracellular matrix polymers, and may lead to new microfibril-based strategies for repairing elastic tissues in ageing and disease.

The supramolecular organization of fibrillin-rich microfibrils.

We propose a new model for the alignment of fibrillin molecules within fibrillin microfibrils. Automated electron tomography was used to generate three-dimensional microfibril reconstructions to 18.6-A resolution, which revealed many new organizational details of untensioned microfibrils, including heart-shaped beads from which two arms emerge, and interbead diameter variation. Antibody epitope mapping of untensioned microfibrils revealed the juxtaposition of epitopes at the COOH terminus and near the proline-rich region, and of two internal epitopes that would be 42-nm apart in unfolded molecules, which infers intramolecular folding. Colloidal gold binds microfibrils in the absence of antibody. Comparison of colloidal gold and antibody binding sites in untensioned microfibrils and those extended in vitro, and immunofluorescence studies of fibrillin deposition in cell layers, indicate conformation changes and intramolecular folding. Mass mapping shows that, in solution, microfibrils with periodicities of <70 and >140 nm are stable, but periodicities of approximately 100 nm are rare. Microfibrils comprise two in-register filaments with a longitudinal symmetry axis, with eight fibrillin molecules in cross section. We present a model of fibrillin alignment that fits all the data and indicates that microfibril extensibility follows conformation-dependent maturation from an initial head-to-tail alignment to a stable approximately one-third staggered arrangement.

Fibrillin-rich microfibrils of the extracellular matrix: ultrastructure and assembly.

Fibrillin-rich microfibrils are a unique class of extensible connective tissue macromolecules. Their critical contribution to the establishment and maintenance of diverse extracellular matrices was underlined by the linkage of their principal structural component fibrillin to Marfan syndrome, a heritable connective tissue disorder with pleiotropic manifestations. Microscopy and preparative techniques have contributed substantially to the understanding of microfibril structure and function. The supramolecular organisation of microfibrillar assemblies in tissues has been examined by tissue sectioning and X-ray diffraction methods. Published findings are discussed and new information reported on the organisation of microfibrils in the ciliary zonular fibrils by environmental scanning electron microscopy. This review summarises microscopy and X-ray diffraction studies that are informing current understanding of the ultrastructure of fibrillin-rich microfibrils.