. The mechanical properties of human adipose tissues and their relationships to the structure and composition of the extracellular matrix. Am J Physiol Endocrinol Metab 305: E1427-E1435, 2013. First published October 8, 2013 doi:10.1152/ajpendo.00111.2013.-Adipose tissue (AT) expansion in obesity is characterized by cellular growth and continuous extracellular matrix (ECM) remodeling with increased fibrillar collagen deposition. It is hypothesized that the matrix can inhibit cellular expansion and lipid storage. Therefore, it is important to fully characterize the ECM's biomechanical properties and its interactions with cells. In this study, we characterize and compare the mechanical properties of human subcutaneous and omental tissues, which have different physiological functions. AT was obtained from 44 subjects undergoing surgery. Force/extension and stress/relaxation data were obtained. The effects of osmotic challenge were measured to investigate the cellular contribution to tissue mechanics. Tissue structure and its response to tensile strain were determined using nonlinear microscopy. AT showed nonlinear stress/strain characteristics of up to a 30% strain. Comparing paired subcutaneous and omental samples (n ϭ 19), the moduli were lower in subcutaneous: initial 1.6 Ϯ 0.8 (means Ϯ SD) and 2.9 Ϯ 1.5 kPa (P ϭ 0.001), final 11.7 Ϯ 6.4 and 32 Ϯ 15.6 kPa (P Ͻ 0.001), respectively. The energy dissipation density was lower in subcutaneous AT (n ϭ 13): 0.1 Ϯ 0.1 and 0.3 Ϯ 0.2 kPa, respectively (P ϭ 0.006). Stress/relaxation followed a two-exponential time course. When the incubation medium was exchanged for deionized water in specimens held at 30% strain, force decreased by 31%, and the final modulus increased significantly. Nonlinear microscopy revealed collagen and elastin networks in close proximity to adipocytes and a larger-scale network of larger fiber bundles. There was considerable microscale heterogeneity in the response to strain in both cells and matrix fibers. These results suggest that subcutaneous AT has greater capacity for expansion and recovery from mechanical deformation than omental AT.

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.

Objectives:(1) To establish whether the tidemark and calcified cartilage are permeable to low molecular weight solutes, thereby providing a potential pathway for nutrition of cells in the deep cartilage. (2) To investigate transport from the subchondral microcirculation into calcified cartilage in an intact perfused joint and the effects on transport of static loading.Methods: The permeability of the tidemark and calcified cartilage was investigated in plugs of cartilage and subchondral bone which formed the membrane of a diffusion cell. Transport from the subchondral microcirculation and the effects of load were studied in an intact perfused joint. Both preparations used the metacarpophalangeal joints of mature horses and fluorescein and rhodamine (m.w. w 400 Da) were employed as tracers, assayed by quantitative fluorescence microscopy on histological sections.Results: Calcified cartilage was permeable to both solutes, both from the superficial and the subchondral sides. The effective diffusivity of both solutes was of the order of 9 Â 10 À9 cm 2 s À1 , fivefold less than in the uncalcified cartilage. The calcified zone was heterogeneous, with high uptake of both tracers in the vicinity of the tidemark. The distribution volume of rhodamine B was higher than for fluorescein, consistent with a significant anionic charge in the calcified matrix. Static loading of the intact joint did not affect the transport of rhodamine B but caused a significant decrease in concentration of fluorescein both in the surface and deep zones of the tissue.Conclusions: Calcified cartilage is permeable to small solutes and the subchondral circulation may make a significant contribution to the nutrition of deep cartilage in the mature horse. Static loading reduces the uptake of small anionic solutes in the intact joint.

Through its ability to make relatively noninvasive and repeatable measurements, MRI has a great deal to offer, not only to clinical diagnosis of intervertebral disc disorders but also as a tool for basic research into disc physiology and the etiology of disc degeneration. In this brief review we outline the structure of the disc, the composition and organization of its macromolecules, and the changes that occur during disc degeneration, attempting to summarize features that have been or could become targets of MRI characterization. It is important to recognize, however, the fundamental limitation that most of the changes so far observed in MRI are consequences of alterations in cellular metabolism that occurred months to years previously and provide little insight into the current functional status of the tissue. There is therefore a need to develop MR techniques that directly characterize cellular activity and factors such as nutrient delivery on which it is critically dependent. We therefore briefly review cellular energy metabolism and nutrient transport into the avascular disc and consider the ability of MRI to reveal information about such processes. As a corollary of this discussion we also consider the constraints that the unusual transport properties of the disc impose on the delivery of contrast agents to the disc, since an understanding of these limitations is central to interpretation of the resulting images. MOST MRI INVESTIGATIONS of the spine are currently undertaken in an effort to establish the causes of back pain. However, the links between back pain and degeneration of the intervertebral disc are still, like most conditions involving congenital or acquired abnormalities of the disc, surprisingly poorly understood. This unsatisfactory situation reflects, in large part, our poor understanding of the physiology of the normal disc, and in particular, the relationships between function, or malfunction, and structure at the cellular and molecular levels. The requisite information is difficult to acquire due, largely, to experimental difficulties of various sorts. The intervertebral disc is closely coupled structurally, physiologically, and biomechanically, to the vertebral body and surrounding ligaments and musculature. The value of many measurements on excised discs in vitro is therefore severely compromised because these relationships are inevitably disrupted. In vivo approaches are also limited. Because these measurements generally involve extensive interventions, they have generally been confined to animals, but there is great variability in disc structure between species and few animals show degenerative changes resembling those in the human. Through its ability to make relatively noninvasive and repeatable measurements, MRI may here have a great deal to offer, not only to clinical diagnosis, but also as a tool for basic research (1).In this brief review we initially summarize the structure of the disc, the composition and organization of its macromolecules, and the changes that occur during dis...

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Brillouin light scattering (BLS) spectroscopy is a technique that is able to detect thermally excited phonons within a material. The speed of propagation of these phonons can be determined from the magnitude of the Brillouin frequency shift between incident and scattered light, thereby providing a measure of the mechanical properties of the material in the gigahertz range. The mechanical properties of the extracellular matrices of biological tissues and their constituent biopolymers are important for normal tissue function and disturbances in these properties are widely implicated in disease. BLS offers the prospect of measuring mechanical properties on a microscopic scale in living tissues, thereby providing insights into structure-function relationships under normal and pathological conditions. In this study, we investigated BLS in collagen and elastin-the fibrous proteins of the extracellular matrix (ECM). Measurements were made on type I collagen in rat tail tendon, type II collagen in articular cartilage and nuchal ligament elastin. The dependence of the BLS spectrum on fibre orientation was investigated in a backscattering geometry using a reflective substrate. Two peaks, a bulk mode arising from phonon propagation along a quasi-radial direction to the fibre axis and a mode parallel to the surface, depending on sample orientation relative to the fibre axis, could be distinguished. The latter peak was fitted to a model of wave propagation through a hexagonally symmetric elastic solid, and the five components of the elasticity tensor were combined to give axial and transverse Young's, shear and bulk moduli of the fibres. These were 10.2, 8.3, 3.2 and 10.9 GPa, and 6.1, 5.3, 1.9 and 8 GPa for dehydrated type I collagen and elastin, respectively. The former values are close to those previously reported. A microfocused BLS approach was also applied providing selection of single fibres. The moduli of collagen and elastin are much higher than those measured at lower frequency using macroscopic strains, and the difference between them is much less. We therefore believe, like previous investigators, that molecular-scale viscoelastic effects are responsible for the frequency dependence of the fibre biomechanics. Combining BLS with larger-scale mechanical testing methods therefore should, in the future, provide a means of following the evolution of mechanical properties in the formation of the complex structures found in the ECM.

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.