Elastic Fiber Network Research Articles

New insights into the viscoelastic and failure mechanical properties of the elastic fiber network of the inter-lamellar matrix in the annulus fibrosus of the disc

The mechanical role of elastic fibers in the inter-lamellar matrix (ILM) is unknown; however, it has been suggested that they play a role in providing structural integrity to the annulus fibrosus (AF). Therefore, the aim of this study was to measure the viscoelastic and failure properties of the elastic fiber network in the ILM of ovine discs under both tension and shear directions of loading. Utilizing a technique, isolated elastic fibers within the ILM from ovine discs were stretched to 40% of their initial length at three strain rates of 0.1% s−1 (slow), 1% s−1 (medium) and 10% s−1 (fast), followed by a ramp test to failure at 10% s−1. A significant strain-rate dependent response was found, particularly at the fastest rate for phase angle and normalized stiffness (p < 0.001). The elastic fibers in the ILM demonstrated a significantly higher capability for energy absorption at slow compared to medium and fast strain rates (p < 0.001). These finding suggests that the elastic fiber network of the ILM exhibits nonlinear elastic behavior. When tested to failure, a significantly higher normalized failure force was found in tension compared to shear loading (p = 0.011), which is consistent with the orthotropic structure of elastic fibers in the ILM. The results of this study confirmed the mechanical contribution of the elastic fiber network to the ILM and the structural integrity of the AF. This research serves as a foundation for future studies to investigate the relationship between degeneration and ILM mechanical properties. Statement of SignificanceThe mechanical role of elastic fibres in the inter-lamellar matrix (ILM) of the disc is unknown. The viscoelastic and failure properties of the elastic fibre network in the ILM in both tension and shear directions of loading was measured for the first time. We found a strain-rate dependent response for the elastic fibres in the ILM. The elastic fibres in the ILM demonstrated a significantly higher capability for energy absorption at slow compared to medium and fast strain rates. When tested to failure, a significantly higher normalized failure force was found in tension compared to shear loading, which is consistent with the orthotropic structure of elastic fibres in the ILM.

Extracellular Matrix Regulation and Dysregulation in Exfoliation Syndrome.

Several lines of evidence, such as ultrastructural signs for activated fibrillogenesis and excessive production of elastic microfibrillar components in the anterior segment of the eye and throughout the body, indicate that exfoliation syndrome (XFS) is an elastic microfibrillopathy, leading to the accumulation of an abnormal extracellular fibrillar material (XFM). The upregulation of elastic microfibrillar components is paralleled by the selective upregulation of the cross-linking enzyme lysyl oxidase-like 1 (LOXL1) in the early phase of the disease, suggesting that LOXL1 participates in the stabilization of newly synthesized elastic proteins and finally in the stable accumulation of XFM. Whereas the excessive production of elastic proteins rises from early to late XFS stages, possibly mediated by increasing fibrogenic stimuli in the aqueous humor, LOXL1 is downregulated in late stages of the disease by as yet unknown mechanisms, possibly leading to the well-documented elastotic matrix alterations characteristic of eyes with late stage XFS. Several studies indicate complex changes of the proteolytic balance in the anterior segment of XFS eyes, characterized primarily by decreased matrix metalloproteinase-2 activity, the major aqueous matrix metalloproteinase, potentially leading to a shift from appropriate matrix turnover to progressive matrix accumulation. In contrast, in nearly all tissues of the posterior segment, XFM seems to be absent and differential gene expression is confined to the lamina cribrosa, characterized by a stage-independent, primary, and XFS-specific downregulation of LOXL1 and elastic components in XFS eyes. Concomitant with this deficiency, the laminar elastic fiber network displays prominent ultrastructural alterations, which may lead to increased vulnerability to glaucomatous damage. Various growth factors, stress conditions, or dietary factors have been supposed to potentially influence the manifestation of the disease. However, in spite of increasing knowledge, the pathogenetic factors initiating the abnormal matrix process still remain elusive. Such information would be critical for our understanding of disease progression and would disclose new options for pharmaceutical intervention at the onset of the disease.

Differential reorganisation of cutaneous elastic fibres: a comparison of the in vivo effects of broadband ultraviolet B versus solar simulated radiation

Long-term exposure of human skin to ultraviolet radiation (UVR) in sunlight negatively impacts its appearance and function with photoaged skin having a characteristic leathery, rough appearance, with deep wrinkles. These clinical features of photodamage are thought to result from UVR-induced remodelling of the dermal extracellular matrix, particularly the elastic fibre system. There are few in vivo human data on the impact of acute UVR exposure on this fibre system and particularly solar-simulated radiation (SSR)-mediated effects. We examined the differential effect of broadband UVB and SSR on the human dermal elastic fibre system, and specifically the microfibrillar components fibrillin-1, fibulin-2 and fibulin-5. Healthy white Caucasian adults (skin type II–III) were recruited and irradiated with 3× their minimal erythema dose of broadband UVB (n = 6) or SSR (n = 6) on photoprotected buttock skin. Punch biopsies were taken 24 h after irradiation and from unirradiated control skin. Overall, histological assessment of elastic fibres revealed significantly less elastic fibre staining in broadband UVB (P = 0.004) or SSR (P = 0.04) irradiated skin compared to unirradiated control skin. Significantly less staining of fibrillin-1-positive microfibrils was also observed in the papillary dermis of UVB irradiated skin (P = 0.02) but not skin exposed to SSR. Conversely, immunohistochemistry for fibulin-5-positive microfibrils revealed significantly less expression in skin exposed to SSR (P = 0.04) but not to broadband UVB. There was no significant change in fibulin-2-positive microfibrils following either broadband UVB or SSR irradiation. Thus, broadband UVB and SSR mediate differential effects on individual components of the dermal elastic fibre network in human skin. Further human studies are required to explore the mechanisms underlying these findings and the impact of potential photoprotective agents.

Studies on sporadic non-syndromic thoracic aortic aneurysms: II. Alterations of extra-cellular matrix components and focal adhesion proteins.

Background Sporadic non-syndromic thoracic aortic aneurysms (SNSTAAs) are less well understood than familial non-syndromic or syndromic ones. Here, we focused on morphologic and molecular changes of the extracellular matrix of the tunica media of SNSTAAs. Design Single centre design. Methods Surgical media samples from seven SNSTAAs and seven controls underwent quantitative polymerase chain reaction, proteomics-bioinformatics, immunoblotting, histology and immunohistochemistry analysis. Results A down-regulation of Decorin mRNA with unchanged protein levels associated with a remarkable increase of collagen fibres. A reduced and distorted network of elastic fibres partnered with an attenuated expression of microfibril-associated glycoprotein1 despite the rise of MFAP2 gene-encoded mRNA levels. An increasingly proteolysed paxillin (55 kDa PXN), a focal adhesion protein, combined with an upregulated 62 kDa PXN holoprotein, without changes in amount and phosphorylation of focal adhesion kinase (pp125FAK). The upregulation of SPOCK2-encoded Testican2 proteoglycan and of ectodysplasin (EDA) protein was coupled with a down-regulation of EDA2 receptor (EDA2R). Conclusions Several tunica media extracellular matrix-related changes favour SNSTAA development. A steady level of decorin and a microfibril-associated glycoprotein1 protein shortage cause the assembly of structurally defective collagen and elastic fibres. Up-regulation of PXN holoproteins perturbs PXN/pp125FAK interaction and focal adhesion functioning. Testican2 up-regulation suppresses the membrane-type matrix metalloproteinase inhibiting activities of other SPOCK family members thus enhancing extracellular matrix proteolysis. Finally, the altered EDA•EDA2R signalling would impact on the remodelling of SNSTAA tunica media. Altogether, our results pave the way to a deeper molecular understanding of SNSTAAs necessary to identify their early diagnostic biochemical markers.

Neuropilin-1 and platelet-derived growth factor receptors cooperatively regulate intermediate filaments and mesenchymal cell migration during alveolar septation.

Generation of secondary alveolar septa occurs primarily after birth in humans and is complete in mice postnatally, when mechanical stresses vary as air space pressure oscillates. Alveolar mesenchymal cells deposit elastic fibers, which limit cell strain; although when the elastic fiber network is incomplete, this function is also served by the intracellular cytoskeleton. Intermediate filament proteins support deformation during cell division and migration, which occur during septal elongation. Because platelet-derived growth factor receptor-α (PDGFRα) signaling is essential for alveolar septation, we hypothesized that neuropilin-1 (NRP1) may link PDGFRα to cytoskeletal deformation. During cell migration, NRP1 links receptor tyrosine kinase signaling to cytoskeletal and focal adhesion remodeling. Therefore, we examined the consequences of nrp1 gene deletion in alveolar mesenchymal cells (myofibroblasts and pericytes). NRP1 depletion reduced the proportion of mesenchymal cells that contain nestin and desmin within the subpopulation that lacked PDGFRα but contained PDGFRβ. Desmin was reduced at alveolar entry rings, air spaces were enlarged, and surface area was reduced after NRP1 depletion. PDGFRα and NRP1 colocalized to membrane lipid rafts, which are known to contain Src kinase. NRP1 depletion reduced alveolar mesenchymal cell migration and PDGF-A-mediated activation of Src kinase, which may limit accumulation of desmin at septal tips (alveolar entry rings). Cooperation between NRP1 and PDGF signaling is required for secondary septation, and manipulation of NRP1 could promote alveolar regeneration without producing fibrosis.

Topology determines force distributions in one-dimensional random spring networks.

Networks of elastic fibers are ubiquitous in biological systems and often provide mechanical stability to cells and tissues. Fiber-reinforced materials are also common in technology. An important characteristic of such materials is their resistance to failure under load. Rupture occurs when fibers break under excessive force and when that failure propagates. Therefore, it is crucial to understand force distributions. Force distributions within such networks are typically highly inhomogeneous and are not well understood. Here we construct a simple one-dimensional model system with periodic boundary conditions by randomly placing linear springs on a circle. We consider ensembles of such networks that consist of N nodes and have an average degree of connectivity z but vary in topology. Using a graph-theoretical approach that accounts for the full topology of each network in the ensemble, we show that, surprisingly, the force distributions can be fully characterized in terms of the parameters (N,z). Despite the universal properties of such (N,z) ensembles, our analysis further reveals that a classical mean-field approach fails to capture force distributions correctly. We demonstrate that network topology is a crucial determinant of force distributions in elastic spring networks.

Elastic Fibers and Large Artery Mechanics in Animal Models of Development and Disease.

Development of a closed circulatory system requires that large arteries adapt to the mechanical demands of high, pulsatile pressure. Elastin and collagen uniquely address these design criteria in the low and high stress regimes, resulting in a nonlinear mechanical response. Elastin is the core component of elastic fibers, which provide the artery wall with energy storage and recoil. The integrity of the elastic fiber network is affected by component insufficiency or disorganization, leading to an array of vascular pathologies and compromised mechanical behavior. In this review, we discuss how elastic fibers are formed and how they adapt in development and disease. We discuss elastic fiber contributions to arterial mechanical behavior and remodeling. We primarily present data from mouse models with elastic fiber deficiencies, but suggest that alternate small animal models may have unique experimental advantages and the potential to provide new insights. Advanced ultrastructural and biomechanical data are constantly being used to update computational models of arterial mechanics. We discuss the progression from early phenomenological models to microstructurally motivated strain energy functions for both collagen and elastic fiber networks. Although many current models individually account for arterial adaptation, complex geometries, and fluid-solid interactions (FSIs), future models will need to include an even greater number of factors and interactions in the complex system. Among these factors, we identify the need to revisit the role of time dependence and axial growth and remodeling in large artery mechanics, especially in cardiovascular diseases that affect the mechanical integrity of the elastic fibers.

Ultrastructural organization of elastic fibres in the partition boundaries of the annulus fibrosus within the intervertebral disc.

A detailed ultrastructural study in the partition boundaries of the annulus fibrosus within the disc revealed a well-organized elastic fibre network with a complex ultrastructure. The continuous network of elastic fibres may provide disc integrity by connecting adjacent bundles of cross section lamellae together. The density of the elastic fibre network in PBs was lower, and fibre orientation was similar to the intra-lamellar space and the inter-lamellar matrix.

Degradation of tropoelastin and skin elastin by neprilysin

Neprilysin is also known as skin fibroblast-derived elastase, and its up-regulation during aging is associated with impairments of the elastic fiber network, loss of skin elasticity and wrinkle formation. However, information on its elastase activity is still limited. The aim of this study was to investigate the degradation of fibrillar skin elastin by neprilysin and the influence of the donor's age on the degradation process using mass spectrometry and bioinformatics approaches. The results showed that cleavage by neprilysin is dependent on previous damage of elastin. While neprilysin does not cleave young and intact skin elastin well, it degrades elastin fibers from older donors, which may further promote aging processes. With regards to the cleavage behavior of neprilysin, a strong preference for Gly at P1 was found, while Gly, Ala and Val were well accepted at P1′ upon cleavage of tropoelastin and skin elastin. The results of the study indicate that the progressive release of bioactive elastin peptides by neprilysin upon skin aging may enhance local tissue damage and accelerate extracellular matrix aging processes.

Role of LTBP4 in alveolarization, angiogenesis, and fibrosis in lungs.

Deficiency of the extracellular matrix protein latent transforming growth factor-β (TGF-β)-binding protein-4 (LTBP4) results in lack of intact elastic fibers, which leads to disturbed pulmonary development and lack of normal alveolarization in humans and mice. Formation of alveoli and alveolar septation in pulmonary development requires the concerted interaction of extracellular matrix proteins, growth factors such as TGF-β, fibroblasts, and myofibroblasts to promote elastogenesis as well as vascular formation in the alveolar septae. To investigate the role of LTBP4 in this context, lungs of LTBP4-deficient (Ltbp4-/-) mice were analyzed in close detail. We elucidate the role of LTBP4 in pulmonary alveolarization and show that three different, interacting mechanisms might contribute to alveolar septation defects in Ltbp4-/- lungs: 1) absence of an intact elastic fiber network, 2) reduced angiogenesis, and 3) upregulation of TGF-β activity resulting in profibrotic processes in the lung.

The ultra-structural organization of the elastic network in the intra- and inter-lamellar matrix of the intervertebral disc.

Visualization of the intra-lamellar matrix under high magnification revealed a dense network of elastic fibers that has not been previously described. The present study used extracellular matrix partial digestion to address significant gaps in understanding of disc microstructure and will contribute to multidisciplinary ultrastructure-function studies.

749 Impact of chronic sun exposure on the elastic fiber network of African-American and Caucasian skin

749 Impact of chronic sun exposure on the elastic fiber network of African-American and Caucasian skin

Development of a rapid matrix digestion technique for ultrastructural analysis of elastic fibers in the intervertebral disc

Collagen and elastic fibers are two major fibrous constituents of the annulus fibrosus (AF) in the disc that contribute to its mechanical and viscoelastic properties. It was thought that elastic fibers play no substantial role in the function and properties of the disc as these fibers were irregularly distributed. Studies that have revealed highly organized elastic fibers with different regional orientation and distribution, while being strongly crosslinked with matrix, suggesting their contribution to disc structure-function properties. These studies that were performed by light microscopic analysis of histologically prepared samples, have not been able to reveal the fine-scale architectural details of the elastic fiber network. Since elastic fibers are intermingled with other fibrous components of the disc and mostly obscured by the extracellular matrix, it is difficult to demonstrate their ultra-structural organization using scanning electron microscopy (SEM).Therefore the aim of this study was to develop a rapid matrix digestion technique for ultrastructural analysis of the disc elastic fibers. This study provides a new method for fundamental visualization of elastic fibers and their architecture in the disc. Through the ultra-structural analysis, the relationship between structure and function, as well as the role of elastic fibers on AF mechanical properties can be studied. This method may be used to develop a three-dimensional map of elastic fibers distribution within the disc, which would provide valuable information for designing tissue engineered scaffolds for AF repair and replacement.

Elastic microfibril distribution in the cornea: Differences between normal and keratoconic stroma

The optical and biomechanical properties of the cornea are largely governed by the collagen-rich stroma, a layer that represents approximately 90% of the total thickness. Within the stroma, the specific arrangement of superimposed lamellae provides the tissue with tensile strength, whilst the spatial arrangement of individual collagen fibrils within the lamellae confers transparency. In keratoconus, this precise stromal arrangement is lost, resulting in ectasia and visual impairment. In the normal cornea, we previously characterised the three-dimensional arrangement of an elastic fiber network spanning the posterior stroma from limbus-to-limbus. In the peripheral cornea/limbus there are elastin-containing sheets or broad fibers, most of which become microfibril bundles (MBs) with little or no elastin component when reaching the central cornea. The purpose of the current study was to compare this network with the elastic fiber distribution in post-surgical keratoconic corneal buttons, using serial block face scanning electron microscopy and transmission electron microscopy.We have demonstrated that the MB distribution is very different in keratoconus. MBs are absent from a region of stroma anterior to Descemet's membrane, an area that is densely populated in normal cornea, whilst being concentrated below the epithelium, an area in which they are absent in normal cornea. We contend that these latter microfibrils are produced as a biomechanical response to provide additional strength to the anterior stroma in order to prevent tissue rupture at the apex of the cone. A lack of MBs anterior to Descemet's membrane in keratoconus would alter the biomechanical properties of the tissue, potentially contributing to the pathogenesis of the disease.

Histological and Electron Microscope Staining for the Identification of Elastic Fiber Networks.

Elastic fibers are a major component of the extracellular matrix and are present in many tissues. Routine histology and standard electron microscopy procedures often do not allow for clear identification of elastic fibers making their organization and ultrastructure difficult to study. In this paper, we describe staining methods and procedures to enhance the contrast of elastin at both the light and electron microscope levels.

Raman microspectroscopy as a diagnostic tool for the non-invasive analysis of fibrillin-1 deficiency in the skin and in the in vitro skin models.

Mutations in building blocks of the fibrillin microfibril/ elastic fiber network manifest in disease conditions such as aneurysms, emphysema or lax skin. Understanding how structural changes induced by fibrillin-1 mutation impact the architecture of fibrillin microfibrils, which then translates into an altered activation state of targeted growth factors, represents a huge challenge in elucidating the genotype-phenotype correlations in connective tissue disorders such as Marfan syndrome. This study shows that Raman microspectroscopy is able to reveal structural changes in fibrillin-1 microfibrils and elastic fiber networks and to discriminate between normal and diseased networks in vivo and in vitro. Therefore Raman microspectroscopy may be utilized as a non-invasive and sensitive diagnostic tool to identify connective tissue disorders and monitor their disease progression.

Novel perspectives on the role of the human microbiota in regenerative medicine and surgery.

Plastic surgery is transitioning from a fine craftsmanship to a regenerative science. In wound healing, the role of microorganisms is no longer considered to be just counteracting, but also promoting. Furthermore, host-microbe interactions are essential for numerous aspects of normal mammalian physiology, from metabolic activity to immune homeostasis. Each area of the human body hosts a unique microbial community, and the composition of microbiota is dependent on the host, age and the anatomical area, and it changes according to the characteristics of the microenvironment. Every squared centimeter of skin contains ~1 billion bacteria. The majority of microorganisms of the skin are commensal or temporary passing members. Skin flora mechanisms interacting or influencing the human physical skin barrier are not well defined. Resident skin bacteria provide the first line of defence against potentially dangerous pathogens and produce small molecules that influence their microbial neighbours. Furthermore, the microbiota activates and assists innate immunity and influences adaptive immunity. Various types of immune and non-immune cells contribute to wound healing. The proliferative phase of wound healing is inversely proportional to the extent of the post-traumatic inflammatory reaction. Topical bacterial lipopolysaccharide application markedly affects wound healing by accelerating the resolution of inflammation, increasing macrophage infiltration, enhancing collagen synthesis and altering the secretion of mediators involved in skin regeneration. Various studies have investigated the biological contents of thermal spring waters, and their anti-inflammatory and immune protective roles. In addition, the regenerative properties of thermal spring waters were analysed in an experimental animal wound model. The areas treated with thermal water healed faster than the areas treated with conventional dressings, and exhibited a collagen and elastic fiber network comparable with the normal skin. Thus, the microbial environment may be considered as a potential tool in regenerative medicine and surgery.

Rat Bile Duct Decellularization

The repair and reconstruction of bile ducts damaged by disease or trauma remains a vexing medical problem. In particular, surgeons have few options when it comes to a long segment reconstruction of the bile duct. Tissue-engineered substitutes, with properties similar to the native tissue, might represent a solution for the functional reconstruction of bile ducts. In particular, decellularized tissues and organs represent a suitable option for tissue engineering when specific scaffolds are needed. However, the optimal conditions to completely remove all the cellular components and minimally affect the structural and residual biochemical properties of the extracellular matrix are still to be found. This paper presents the characterization of rat bile ducts after implementing an established detergent-enzymatic decellularization method. One cycle was enough to generate a complete decellularized bile duct matrix, histologically and structurally similar to the native one. The network of collagen, reticular and elastic fibers, found in the native bile duct matrix was well preserved. Moreover, the decellularization approach did not affect the elastin content.

082 Effect of blue light overexposure on normal human keratinocytes and ex vivo skin, in vivo objectivation

In the visible spectrum, blue light (400-495 nm) represents the High Energy Visible (HEV) light associated with high photon energy. The majority of blue light comes from the sun, but it appears that we are more and more exposed to artificial light sources, including light-emitting electronic devices that emit significant amounts of blue light. Recently, opsin receptor expression has been reported in human epidermal cells, with three of them being sensitive to blue light. The role of blue light overexposure on the physiological skin condition has been poorly described. The aim of the present study was to evaluate the effect of blue light exposure on Normal Human Keratinocytes (NHK) using LED irradiation. Exposure conditions generating Reactive Oxygen Species (ROS) accumulation were first determined in NHKs. In these conditions, the levels of opsins OPN1 SW, OPN2 and OPN3 were observed to have decreased at specific wavelengths of blue LED. We also treated NHKs with a peptidic extract of Cocoa porcelana and observed a partial preservation of keratinocyte photoreceptors after blue LED exposure. Moreover, ex vivo studies on skin biopsies showed the effects of blue LED overexposure on the elastic fiber network and its preservation associated with the application of the extract. To evaluate the positive effect of a peptidic extract of Cocoa porcelana on the visible signs of aging linked to prolonged exposure to blue light, an in vivo study was performed on the faces of 20 volunteers. At the end of the treatment, wrinkle parameters were observed to have decreased significantly on the treated side compared to the placebo side. Taken together, our results showed that the expression of opsin receptors could be disrupted by blue LED overexposure in NHKs, associated with an oxidative stress response. Moreover, the in vivo study showed an association with an observed decrease in wrinkle appearance. These studies represent a new approach regarding skin sensitivity to blue light through opsin receptors.

Scanning Electron Microscopy of the Presbylarynx.

To describe the findings on the presbylarynx under scanning electron microscopy. Cadaver study. Universidade Estadual Paulista (Botucatu, São Paulo, Brazil). Sixteen vocal folds were removed during necropsies and distributed into 2 age groups: control (n = 8; aged 30-50 years) and elderly (n = 8; aged 75-92 years). The right vocal fold was dissected, fixed in glutaraldehyde 2.5%, and prepared for scanning electron microscopy. The thickness of the epithelium was measured using a scandium morphometric digital program. In the control group, the epithelium had 5 to 7 overlapped cell layers, rare desquamation cells, and little undulation with protruding intercellular junctions. The lamina propria showed a uniform network of collagen and elastic fibers in the superficial layer. A dense network of collagen was identified in the deeper layer. In the elderly group, the epithelium was atrophic (2-3 cells), with more desquamation cells and intercellular junctions delimited by deep sulci. The epithelial thickness was lower in elderly than in controls (mean [SD], 221.64 [145.90] µm vs 41.79 [21.40] µm, respectively). The lamina propria had a dense and irregular distribution of collagen and elastic fibers in the superficial layer. In the deep layers, the collagen fibers formed a true fibrotic and rigid skeleton. Scanning electron microscopy identified several changes in the elderly larynx, differentiating it from the controls. These alterations are probably related to the aging process of the vocal folds. However, the exact interpretation of these findings requires additional studies, even to the molecular level, having the fibroblasts as targets.