Microstructural characterization of annulus fibrosus by ultrasonography: a feasibility study with an in vivo and in vitro approach.

The main function of the intervertebral disc is biomechanical function, since it must resist repetitive high loadings, while giving the spine its flexibility and protecting the spinal cord from over-straining. It partially owes its mechanical characteristics to the lamellar architecture of its outer layer, the annulus fibrosus. Today, no non-invasive means exist to characterize annulus lamellar structure in vivo. The aim of this work was to test the feasibility of imaging annulus fibrosus microstructure in vivo with ultrasonography. Twenty-nine healthy adolescents were included. Ultrasonographies of L3-L4 disc were acquired with a frontal approach. Annulus fibrosus was segmented in the images to measure the thickness of the lamellae. To validate lamellar appearance in ultrasonographies, multimodality images of two cow tail discs were compared: ultrasonography, magnetic resonance and optical microscopy. In vivo average lamellar thickness was 229.7 ± 91.5 µm, and it correlated with patient body mass index and age. Lamellar appearance in the three imaging modalities in vitro was consistent. Lamellar measurement uncertainty was 7%, with good agreement between two operators. Feasibility of ultrasonography for the analysis of lumbar annulus fibrosus structure was confirmed. Further work should aim at validating measurement reliability, and to assess the relevance of the method to characterize annulus alterations, for instance in disc degeneration or scoliosis.

The effects of needle damage on annulus fibrosus micromechanics.

Needle puncture of the intervertebral disc can initiate a mechanical and biochemical cascade leading to disc degeneration. Puncture's mechanical effects have been shown near the puncture site, mechanical effects should be observed far, relative to needle size, from the puncture site, given the disc-wide damage induced by the stab. The aim of this work was to quantify these far-field effects, and to observe the local structural damage provoked by the needle. Strips of cow tail annulus fibrosus underwent two consecutive mechanical loadings to 5% tensile strain; fifteen samples were punctured in a radial direction with a randomly assigned needle between the two loadings (needle gauges between 19 and 23). Ten samples (control group) were not punctured. During loading, the tissue strains were imaged using second harmonic generation microscopy in a <600×800µm region about 4.4mm from the puncture site. After mechanical testing, the puncture site was imaged in 3D. Puncture had no significant effect on annulus elastic modulus. Imaging showed a modest change in the shearing between fibre bundles however, the linear strain between bundles, intra-bundle shear and linear strain were not significantly affected. At the puncture site, detached lumps of tissue were present. These results suggest that the mechanical effects observed in intact discs are due to the depressurization of the disc, rather than the local damage to the annulus. Needle profiles could be designed, aiming at separating fibre bundles rather than cutting through them, to avoid leaving dying tissue behind. STATEMENT OF SIGNIFICANCE: Needle puncture of the intervertebral disc can initiate a mechanical and biochemical cascade leading to disc degeneration, but the link between the local damage of the puncture and the disc-wide effects is not well understood. This work aimed at determining the micro-mechanical effects of the puncture far from its site, and to observe the damage induced by the puncture with high resolution imaging. Results show that the puncture had modest effect far from the puncture, but lumps of tissue were left by the needle, detached from the disc; these could cause further damage through friction and inflammation of the surrounding tissues. This suggests that the cascade leading to degeneration is probably driven by a biochemical response rather than disc-wide mechanical effects.

Bovine and degenerated human annulus fibrosus: a microstructural and micromechanical comparison.

The complex structure of the annulus fibrosus is strongly related to its mechanical properties. Recent work showed that it is possible to observe the relative movement of fibre bundles in loaded cow tail annulus; the aim of this work was to describe and quantify annulus fibrosus micromechanics in degenerated human disc, and compare it with cow tail annulus, an animal model often used in the literature. Second harmonic generation was used to image the collagen matrix in twenty strips of annulus fibrosus harvested from intervertebral disc of seven patients undergoing surgery. Samples were loaded to 6% tensile strain in 1% steps. Elastic modulus was calculated from loading curves, and micromechanical strains were calculated from the images using custom software. The same protocol was applied to twenty strips of annulus harvested from cow tail discs. Significant morphological differences were found between human and cow tail samples, the most striking being the lack of collagen fibre crimp in the former. Fibres were also observed bending and running from one lamella to the other, forming a strong flexible interface. Interdigitation of fibre bundles was also present at this interface. Quantitative results show complex patterns of inter-bundle and inter-lamellar behaviour, with inter-bundle sliding being the main strain mechanism. Elastic modulus was similar between species, and it was not affected by the degree of degeneration. This work gives an insight into the complex structure and mechanical function of the annulus fibrosus, which should be accounted for in disc numerical modelling.

Lamellar and fibre bundle mechanics of the annulus fibrosus in bovine intervertebral disc.

UNLABELLED: The intervertebral disc is a multicomposite structure, with an outer fibrous ring, the annulus fibrosus, retaining a gel-like core, the nucleus pulposus. The disc presents complex mechanical behaviour, and it is of high importance for spine biomechanics. Advances in multiscale modelling and disc repair raised a need for new quantitative data on the finest details of annulus fibrosus mechanics. In this work we explored inter-lamella and inter-bundle behaviour of the outer annulus using micromechanical testing and second harmonic generation microscopy. Twenty-one intervertebral discs were dissected from cow tails; the nucleus and inner annulus were excised to leave a ring of outer annulus, which was tested in circumferential loading while imaging the tissue's collagen fibres network with sub-micron resolution. Custom software was developed to determine local tissue strains through image analysis. Inter-bundle linear and shear strains were 5.5 and 2.8 times higher than intra-bundle strains. Bundles tended to remain parallel while rotating under loading, with large slipping between them. Inter-lamella linear strain was almost 3 times the intra-lamella one, but no slipping was observed at the junction between lamellae. This study confirms that outer annulus straining is mainly due to bundles slipping and rotating. Further development of disc multiscale modelling and repair techniques should take into account this modular behaviour of the lamella, rather than considering it as a homogeneous fibre-reinforced matrix. STATEMENT OF SIGNIFICANCE: The intervertebral disc is an organ tucked between each couple of vertebrae in the spine. It is composed by an outer fibrous layer retaining a gel-like core. This organ undergoes severe and repeated loading during everyday life activities, since it is the compliant component that gives the spine its flexibility. Its properties are affected by pathologies such as disc degeneration, a major cause of back pain. In this article we explored the micromechanical behaviour of the disc's outer layer using second harmonic generation, a technique which allowed us to visualize, with unprecedented detail, how bundles of collagen fibres slide relative to each other when loaded. Our results will help further the development of new multiscale numerical models and repairing techniques.

Elastic fibre organization in the intervertebral discs of the bovine tail.

Elastic fibres have been revealed by both elastin immunostaining and conventional histological orcein-staining in the intervertebral discs of the bovine tail. These fibres are distributed in all regions of the disc but their organization varies from region to region. In the centre of the nucleus, long (> 150 microm) elastic fibres are orientated radially. In the transitional region between nucleus and annulus, the orientation of the elastic fibres changes, producing a criss-cross pattern. In the annulus itself, elastic fibres appear densely distributed in the region between the lamellae and also in 'bridges' across the lamellae, particularly in the adult. Elastic fibres are apparent within the lamellae, orientated parallel to the collagen fibres of each lamella, particularly in the young (12-day-old) discs. In the region between the disc and the cartilaginous endplate, elastic fibres appear to anchor into the plate and terminate there. The results of this study suggest that elastic fibres contribute to the mechanical functioning of the intervertebral disc. The varying organization of the elastic fibres in the different regions of the disc is likely to relate to the different regional loading patterns.

Interface properties of simplified tear-like fluids in relation to lipid and aqueous layers composition.

The Langmuir trough approach to the study of the physical properties of an in vitro system representing the natural tear lipid-aqueous interface gives useful information on the effect produced by changes of composition of both phases. We found that variations of the composition of the lipid mixture affect more strongly the characteristics of the film rather than changes in the aqueous phase composition. Therefore, future investigations should consider the possibility of searching for and optimising additions of lipid mixtures to the natural tear film. These novel mixtures should stabilise the lipid layer, and thus treat the evaporative Dry Eye Syndrome. More critical and worthy of further investigation is the effect of surface-active water-soluble components that can reduce and ultimately destroy the lipid film integrity and effectiveness. The beneficial effect of water-soluble components, either naturally occurring or artificially added, may be more precisely compared with possible side effects on the stability of the lipid tear layer. In any case, these side effects may be largely compensated by the presence of stability enhanced lipid composition. The present work may be considered as an introductory investigation that takes the physical-chemical approach into the realm of debated data about the tear film structure and properties. Refining the model we have adopted in the present work is certainly necessary. For instance, introducing into the model tear composition other components such as ceramides and cerebrosides will provide insight to their contribution to the packing structure. We may also couple different techniques such as measuring elastic and rheological properties of the films that may be relevant to its physiological behaviour.