Neil D. Broom

In Vitro and in Vivo Effects of Adiponectin on Bone

Fat mass impacts on both bone turnover and bone density and is a critical risk factor for osteoporotic fractures. Adipocyte-derived hormones may contribute to this relationship, and adiponectin is a principal circulating adipokine. However, its effects on bone remain unclear. We have, therefore, investigated the direct effects of adiponectin on primary cultures of osteoblastic and osteoclastic cells in vitro and determined its integrated effects in vivo by characterizing the bone phenotype of adiponectin-deficient mice. Adiponectin was dose-dependently mitogenic to primary rat and human osteoblasts ( approximately 50% increase at 10 microg/ml) and markedly inhibited osteoclastogenesis at concentrations of 1 microg/ml or greater. It had no effect on osteoclastogenesis in RAW-264.7 cells or on bone resorption in isolated mature osteoclasts. In adiponectin knockout (AdKO) male C57BL/6J mice, trabecular bone volume and trabecular number (assessed by microcomputed tomography) were increased at 14 wk of age by 30% (P = 0.02) and 38% (P = 0.0009), respectively. Similar, nonsignificant trends were observed at 8 and 22 wk of age. Biomechanical testing showed lower bone fragility and reduced cortical hardness at 14 wk. We conclude that adiponectin stimulates osteoblast growth but inhibits osteoclastogenesis, probably via an effect on stromal cells. However, the AdKO mouse has increased bone mass, suggesting that adiponectin also has indirect effects on bone, possibly through modulating growth factor action or insulin sensitivity. Because adiponectin does influence bone mass in vivo, it is likely to be a contributor to the fat-bone relationship.

Effect of loading rate and hydration on the mechanical properties of the disc.

STUDY DESIGN The mechanical response of bovine intervertebral discs to axial compression at different loading rates and hydration levels was quantified. OBJECTIVES To quantify the effects of hydration and loading rate on the mechanical response of the intervertebral disc to compressive axial load. SUMMARY OF BACKGROUND DATA The disc is known to be viscoelastic, but there are few experimental data showing the effect of loading rate and hydration on its response to compression. METHODS Hydration level reduced by creep-loading from a fully hydrated starting point. Four groups were tested: Group A: fully hydrated (n = 5), six loading rates, from 0.3 kPa/sec to 30 MPa/sec; Group B: after 30 minutes of creep (n = 4); and Group C: after 2 hours of creep (n = 4) under a static load of 1 MPa, loading rates 3 MPa/sec, 30 kPa/sec, and 0.3 kPa/sec; Group D: at 5-minute intervals, during an 8-hour period of creep (n = 3) under a static load of 1 MPa, loading rate 3 MPa/sec. Data normalized by disc area and height: nominal stress, strain, and modulus calculated. RESULTS Group A: Modulus increased with load and rate of loading, with significant differences among the lower three loading rates. The highest three loading rates were significantly different from the lower rates, but not from each other. Group B: At the two higher loading rates, modulus was greater than in group A. At the lowest loading rate the modulus was similar to that in Group A. Group C: At the highest loading rate, the modulus was less than that of Groups A and B. At the lower two loading rates, the modulus was similar to that in Group A. Group D: The modulus increased in the first 30 minutes and decreased in the interval from 60 to 480 minutes. CONCLUSIONS Intervertebral disc compressive mechanical properties are significantly dependent on loading rate and hydration.

A Study of the Structural Response of Wet Hyaline Cartilage to Various Loading Situations

A direct view has been obtained of the manner in which the fibrous components components and chondrocytes in hyaline cartilage respond to the application of uniaxial tensile loading and plane-strain compressive loading. A micro-mechanical testing device has been developed which inserts directly into the stage of a high-resolution optical microscope fitted with Nomarski interference contrast and this has permitted simultaneous morphological and mechanical observations to be conducted on articular cartilage maintained in its wet functional condition. Aligned and crimped fibrous arrays surround the deeper chondrocytes and can be observed to undergo well-defined geometric changes with applied stress. It is thought that these arrays may act as displacement or strain sensors transmitting mechanical information from the bulk matrix to their associated cells thus inducing a specific metabolic response. The process of tissue recovery following sustained high levels of compressive loading can also be observed with this experimental technique.

The stress/strain and fatigue behaviour of glutaraldehyde preserved heart-valve tissue.

Abstract Static tensile tests and accelerated fatigue tests were used to investigate the mechanical durability of glutaraldehyde preserved bovine and porcine mitral tissue with a view to gaining some understanding of the long-term functional behaviour of preserved heterografts. As well as there being a reduction in the tissue compliance following fixation, the effects of cyclic loading over a wide range of peak stresses were to reduce further the tissue compliance. Fatigue induced structural changes leading finally to gross disruption of the collagen fibre array were observed using optical and electron microscopy.

Intralamellar relationships within the collagenous architecture of the annulus fibrosus imaged in its fully hydrated state

The anisotropic, inhomogeneous, multiply collagenous architecture of the annulus reflects the complex pattern of mainly tensile stresses developed in this region of the disc during normal function. Structural and mechanical responses of fully hydrated in‐plane sections taken from within single lamellae of the outer annulus of healthy bovine caudal discs have been investigated using a micromechanical technique in combination with simultaneous high‐resolution differential interference contrast optical imaging. Responses both along and across (i.e. transverse to) the primary direction of the mono‐array of collagen fibres were studied. Stretching along the alignment direction revealed a biomechanical response consistent with the behaviour of an array whose overall strength is governed primarily by the strength of embedding of the fibres in the vertebral endplates, rather than from interfibre cohesion along their length. The mono‐aligned array, even when lacerated, is highly resistant to any further tearing across the alignment direction. Although not visible in the relaxed mono‐arrays, transverse stretching revealed a highly complex set of interconnecting structures embodying hierarchical relationships not previously revealed. It is suggested that these structures might play an important role in the containment under pressure of the nuclear contents. The dramatic differences in rupture behaviour observed along vs. across the primary fibre direction are consistent with the known clinical consequences arising from varying degrees of annular wall damage, and might also explain various types of disc herniation. The lamellar architecture of the healthy disc revealed by this investigation provides an important reference framework for exploring structural changes associated with disc trauma and degeneration.

The structural basis of interlamellar cohesion in the intervertebral disc wall

The purpose of this study was to investigate the structural mechanisms that create cohesion between the concentric lamellae comprising the disc annulus. Sections, 50–60 µm thick, were obtained using a carefully chosen cutting plane that incorporated the fibrous component in alternating lamellae as in‐plane and cross‐sectioned arrays. These sections were then subjected to microtensile stretching both across (radial) and along (tangential) the in‐plane fibre direction, in their fully hydrated state. Structural responses were studied by simultaneously viewing the sections using high‐resolution Nomarski interference contrast light microscopy. Additional bulk samples of annulus were fixed while held in a constant, radially stretched state in order to investigate the potential for interlamellar separation to occur in a state more representative of the intact disc wall. The study has provided a detailed picture of the structural architecture creating disc wall cohesion, revealing a complex hierarchy of interconnecting relationships within the disc wall, not previously described. Importantly, because our experimental approach offers a high‐resolution view of the response of the interlamellar junction to deformation in its fully hydrated condition, it is a potentially useful method for investigating subtle changes in junction cohesion resulting from both early degeneration and whole‐disc trauma.

Is classical consolidation theory applicable to articular cartilage deformation

In this paper, classical consolidation theory has been used to investigate the time-dependent response of articular cartilage to static loading. An experimental technique was developed to measure simultaneously the matrix internal pressure and creep strain under conditions of one-dimensional consolidation. This is the first measurement of the internal stress state of loaded cartilage. It is demonstrated that under static compression the applied load is shared by the components of the matrix (i.e. water, the proteoglycans, and the collagen fibrillar meshwork), during which time a maximum hydrostatic excess pore pressure is developed as initial water exudation occurs. This pressure decays as water is further exuded from the matrix and effective consolidation begins with a progressive transfer of the applied stress from water to the collagen fibrils and proteoglycan gel. Consolidation is completed when the hydrostatic excess pore pressure is reduced to zero and the solid components sustain in full the applied load.

Biomechanics of load-bearing of the intervertebral disc: an experimental and finite element model.

This paper presents an experimental and finite element study of the biomechanical response of the intervertebral disc to static-axial loading in which classical consolidation theory was used to analyse its time-dependent response. A newly developed experimental technique was employed to load the disc in compression and measure simultaneously the matrix internal pressure and creep strain for the full consolidation process. It is shown that, upon loading, the disc develops a maximum hydrostatic excess pore pressure which gradually decays as water is exuded from the matrix. During this decay process, the applied load is progressively transferred to the solid components of the matrix until the load is borne in full by the solid at the end of consolidation. Material properties for the tissue were then obtained from the experimental stress-strain data and used in the finite element study in the development of a finite element solution based on Biots theory of coupled solid-fluid interaction. An axisymmetric formulation was employed and the disc modelled as an anisotropic, non-linear poroelastic solid. A sensitivity analysis of the material properties for the structural components of the disc was carried out and the biomechanical response to compressive loading evaluated and compared to experimental data. The results show that the matrix permeability plays a significant role in determining the transient response of the tissue. Annular disruptions of the disc were shown to result in an increase in the nuclear principal stresses suggesting that disrupted regions of the annulus fibrosus play a reduced role in load bearing.

A degeneration‐based hypothesis for interpreting fibrillar changes in the osteoarthritic cartilage matrix

The collagen fibrillar architectures in the general matrix of cartilage slices removed from both normal and osteoarthritic femoral heads were examined by both differential interference light microscopy and scanning electron microscopy. Whereas the normal general matrix contained a finely differentiated pseudo‐random weave of fibrils developed from an interconnected array of radial elements, the osteoarthritic general matrix was characterised by the presence of structurally distinct regions consisting of strongly aligned radial bundles of fibrils and associated intense tangles or ‘knotted’ features. Simple structural models were developed to explore possible transformation structures based on two different types of interconnectivity in the three‐dimensional fibrillar network. These models support the hypothesis that the distinctive ultrastructural features of the osteoarthritic general matrix can develop as a consequence of largely passive degradative changes occurring in the fibrillar weave originally present in the normal matrix. This could, in principle, occur independently of any new structure that might develop as a consequence of any upregulation of collagen associated with the osteoarthritic process.

ISSLS Prize Winner: Microstructure and Mechanical Disruption of the Lumbar Disc Annulus Part I: A Microscopic Investigation of the Translamellar Bridging Network

Study Design. Microstructural investigation of interlamellar connectivity. Objective. To reveal the macro and micro structure of the translamellar bridging network in the lumbar annulus. Summary of Background Data. Contrary to the view that there is minimal interconnection between lamellar sheets, experimental data reveal a significant contribution to the material behavior of the annulus from interactions between fiber populations of alternating lamellae. Recent microstructural studies indicate a localized rather than a homogeneous or dispersed mode of interconnectivity between lamellae. Methods. Anterior segments of ovine lumbar discs in 2 age groups were sectioned along the oblique fiber angle. A 3-dimensional picture of the translamellar bridging network is developed using structural information obtained from fully hydrated unstained serial sections imaged by differential interference contrast optics. Results. A high level of connectivity between apparently disparate bridging elements was revealed. The extended form of the bridging network is that of occasional substantial radial connections spanning many lamellae with a subsidiary fine branching network. The fibrous bridging network is highly integrated with the lamellae architecture via a collagen-based system of interconnectivity. Conclusion. This study demonstrates a far greater complexity to the interlamellar architecture of the disc annulus than has previously been recognized. Our findings are clearly relevant to disc biomechanics. Significant degrading of the translamellar bridging network may result in annular weakening leading potentially to disc failure. Most importantly this work opens the way to a much clearer understanding of the microanatomy of the disc wall.