New findings confirm the viscoelastic behaviour of the inter-lamellar matrix of the disc annulus fibrosus in radial and circumferential directions of loading.

While few studies have improved our understanding of composition and organization of elastic fibres in the inter-lamellar matrix (ILM), its clinical relevance is not fully understood. Moreover, no studies have measured the direct tensile and shear failure and viscoelastic properties of the ILM. Therefore, the aim of this study was, for the first time, to measure the viscoelastic and failure properties of the ILM in both the tension and shear directions of loading. Using an ovine model, isolated ILM samples 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) and a ramp test to failure was performed at a strain rate of 10%s-1. The findings from this study identified that the stiffness of the ILM was significantly larger at faster strain rates, and energy absorption significantly smaller, compared to slower strain rates, and the viscoelastic and failure properties were not significantly different under tension and shear loading. We found a strain rate dependent response of the ILM during dynamic loading, particularly at the fastest rate. The ILM demonstrated a significantly higher capability for energy absorption at slow strain rates compared to medium and fast strain rates. A significant increase in modulus was found in both loading directions and all strain rates, having a trend of larger modulus in tension and at faster strain rates. The finding of no significant difference in failure properties in both loading directions, was consistent with our previous ultra-structural studies that revealed a well-organized (±45°) elastic fibre orientation in the ILM. The results from this study can be used to develop and validate finite element models of the AF at the tissue scale, as well as providing new strategies for fabricating tissue engineered scaffolds. STATEMENT OF SIGNIFICANCE: While few studies have improved our understanding of composition and organization of elastic fibres in the inter-lamellar matrix (ILM) of the annulus in the disc no studies have measured the direct mechanical failure and viscoelastic properties of the ILM. The findings from this study identified that the stiffness of the ILM was significantly larger at faster strain rates, and energy absorption significantly smaller, compared to slower strain rates. The failure properties of the ILM were not significantly different under tension and shear.

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

The relationship between elastic fibre disorders and disc degeneration, aging and progression of spine deformity have been discussed in a small number of studies. However, the clinical relevance of elastic fibres in the annulus fibrosus (AF) of the disc is poorly understood. Ultrastructural visualization of elastic fibres is an important step towards understanding their structure-function relationship. In our previous studies, a novel technique for visualization of elastic fibres across the AF was presented and their ultrastructural organization in intra- and inter-lamellar regions was compared. Using the same novel technique in the present study, the ultrastructural organization of elastic fibres in the partition boundaries (PBs), which are located between adjacent collagen bundles, is presented for the first time. Visualization of elastic fibres in the PBs in control and partially digested (digested) samples was compared, and their orientation in two different cutting planes (transverse and oblique) were discussed. The ultrastructural analysis revealed that elastic fibres in PBs were a well-organized dense and complex network having different size and shape. Adjacent collagen bundles in a cross section (CS) lamella appear to be connected to each other, where elastic fibres in the PBs were merged in parallel or penetrated into the collagen bundles. There was no significant difference in directional coherency coefficient of elastic fibres between the two different cutting planes (p = .35). The present study revealed that a continuous network of elastic fibres may provide disc integrity by connecting adjacent bundles of CS lamellae together. Compared to our previous studies, the density of the elastic fibre network in PBs was lower, and fibre orientation was similar to the intra-lamellar space and inter-lamellar matrix. STATEMENT OF SIGNIFICANCE: 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.

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

The inter-lamellar matrix (ILM)-located between adjacent lamellae of the annulus fibrosus-consists of a complex structure of elastic fibers, while elastic fibers of the intra-lamellar region are aligned predominantly parallel to the collagen fibers. The organization of elastic fibers under low magnification, in both inter- and intra-lamellar regions, was studied by light microscopic analysis of histologically prepared samples; however, little is known about their ultrastructure. An ultrastructural visualization of elastic fibers in the inter-lamellar matrix is crucial for describing their contribution to structural integrity, as well as mechanical properties of the annulus fibrosus. The aims of this study were twofold: first, to present an ultrastructural analysis of the elastic fiber network in the ILM and intra-lamellar region, including cross section (CS) and in-plane (IP) lamellae, of the AF using Scanning Electron Microscopy (SEM) and second, to -compare the elastic fiber orientation between the ILM and intra-lamellar region. Four samples (lumbar sheep discs) from adjacent sections (30µm thickness) of anterior annulus were partially digested by a developed NaOH-sonication method for visualization of elastic fibers by SEM. Elastic fiber orientation and distribution were quantified relative to the tangential to circumferential reference axis. Visualization of the ILM under high magnification revealed a dense network of elastic fibers that has not been previously described. Within the ILM, elastic fibers form a complex network, consisting of different size and shape fibers, which differed to those located in the intra-lamellar region. For both regions, the majority of fibers were oriented near 0° with respect to tangential to circumferential (TCD) direction and two minor symmetrical orientations of approximately±45°. Statistically, the orientation of elastic fibers between the ILM and intra-lamellar region was not different (p=0.171). 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. STATEMENT OF SIGNIFICANCE: 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.