Mechanisms for mechanical damage in the intervertebral disc annulus fibrosus

Intervertebral disc degeneration results in disorganization of the laminate structure of the annulus that may arise from mechanical microfailure. Failure mechanisms in the annulus were investigated using composite lamination theory and other analyses to calculate stresses in annulus layers, interlaminar shear stress, and the region of stress concentration around a fiber break. Scanning electron microscopy (SEM) was used to evaluate failure patterns in the annulus and evaluate novel structural features of the disc tissue. Stress concentrations in the annulus due to an isolated fiber break were localized to approximately 5 microm away from the break, and only considered a likely cause of annulus fibrosus failure (i.e., radial tears in the annulus) under extreme loading conditions or when collagen damage occurs over a relatively large region. Interlaminar shear stresses were calculated to be relatively large, to increase with layer thickness (as reported with degeneration), and were considered to be associated with propagation of circumferential tears in the annulus. SEM analysis of intervertebral disc annulus fibrosus tissue demonstrated a clear laminate structure, delamination, matrix cracking, and fiber failure. Novel structural features noted with SEM also included the presence of small tubules that appear to run along the length of collagen fibers in the annulus and a distinct collagenous structure representative of a pericellular matrix in the nucleus region.     

Mechanical damage to the intervertebral disc annulus fibrosus subjected to tensile loading

Damage of the annulus fibrosus is implicated in common spinal pathologies. The objective of this study was to obtain a quantitative relationship between both the number of cycles and the magnitude of tensile strain resulting in damage to the annulus fibrosus. Four rectangular tensile specimens oriented in the circumferential direction were harvested from the outer annulus of 8 bovine caudal discs (n = 32) and subjected to one of four tensile testing protocols: (i) ultimate tensile strain (UTS) test; (ii) baseline cyclic test with 4 series of 400 cycles of baseline cyclic loading (peak strain = 20% UTS); (iii & iv) acute and fatigue damage cyclic tests consisting of 4 x 400 cycles of baseline cyclic loading with intermittent loading to 1 and 100 cycles, respectively, with peak tensile strain of 40%, 60%, and 80% UTS. Normalized peak stress for all mechanically loaded specimens was reduced from 0.89 to 0.11 of the baseline control levels, and depended on the magnitude of damaging strain and number of cycles at that damaging strain. Baseline, acute, and fatigue protocols resulted in permanent deformation of 3.5%, 6.7% and 9.6% elongation, respectively. Damage to the laminate structure of the annulus in the absence of biochemical activity in this study was assessed using histology, transmission electron microscopy, and biochemical measurements and was most likely a result of separation of annulus layers (i.e., delamination). Permanent elongation and stress reduction in the annulus may manifest in the motion segment as sub-catastrophic damage including increased neutral zone, disc bulging, and loss of nucleus pulposus pressure. The preparation of rectangular tensile strip specimens required cutting of collagen fibers and may influence absolute values of results, however, it is not expected to affect the comparisons between loading groups or dose-response reported.     

Ultrastructural observations on collagen and proteoglycans in the annulus fibrosus of the intervertebral disc

Replicas of freeze-fractured collagen fibrils of peripheral and central parts of the annulus fibrosus of bovine intervertebral discs show microfibrils which run either arranged in parallel or in a helix with an inclination-angle ranging from 4 degrees to 8 degrees. According to results of freeze-fracutred tissues, thin sections of specimens treated with 4M guanidinium chloride show collagen fibrils with a parallel or a slightly wavy microfibrillar packing. Thin sections of specimens treated with alcian blue diluted in MgCl2 critical electrolyte solutions reveal interfibrillar proteoglycan particles with a filamentous shape in the peripheral zones of the annulus fibrosus and a predominant leaf-like appearance in the inner ones. These observations are discussed with reference to previous data concerning the variation in composition from the peripheral to the inner parts of the annulus fibrosus.     

A homogenization model of the annulus fibrosus

The objective of this study was to use a homogenization model of the anisotropic mechanical behavior of annulus fibrosus (AF) to address some of the issues raised in structural finite element and fiber-reinforced strain energy models. Homogenization theory describes the effect of microstructure on macroscopic material properties by assuming the material is composed of repeating representative volume elements. We first developed the general homogenization model and then specifically prescribed the model to in-plane single lamella and multi-lamellae AF properties. We compared model predictions to experimentally measured AF properties and performed parametric studies. The predicted tensile moduli (E theta and E z) and their dependence on fiber volume fraction and fiber angle were consistent with measured values. However, the model prediction for shear modulus (G thetaz) was two orders of magnitude larger than directly measured values. The values of E theta and E z were strongly dependent on the model input for matrix modulus, much more so than the fiber modulus. These parametric analyses demonstrated the contribution of the matrix in AF load support, which may play a role when protoeglycans are decreased in disc degeneration, and will also be an important design factor in tissue engineering. We next compared the homogenization model to a 3-D structural finite element model and fiber-reinforced energy models. Similarities between the three model types provided confidence in the ability of these models to predict AF tissue mechanics. This study provides a direct comparison between the several types of AF models and will be useful for interpreting previous studies and elucidating AF structure-function relationships in disc degeneration and for functional tissue engineering.     

A chemo-mechanical model for osmo-inelastic effects in the annulus fibrosus

Biomechanics and Modeling in Mechanobiology - The annulus fibrosus exhibits complex osmotic and inelastic effects responsible for unusual transversal behavior with a Poisson’s ratio higher...     

Types I and II collagens in intervertebral disc. Interchanging radial distributions in annulus fibrosus.

Intervertebral disc is a highly specialized cartilaginous tissue, containing two genetic types of collagen (I and II). Analysis of peptides from a CNBr digest of collagen showed that the proportions of I and II varied gradually and inversely across pig annulus fibrosus, with exclusively type I at the extreme outer edge and exclusively type II in the nucleus pulposus.     

Effect of overload on changes in mechanical and structural properties of the annulus fibrosus of the intervertebral disc

The research focussed on analysing structural and mechanical properties in the intervertebral disc (IVD), caused by long-term cyclic loading. Spinal motion segments were divided into two groups: the control (C), and the group in which it was analysed the impact of posterior column in the load-bearing system of the spine—specimens with intact posterior column (IPC) and without posterior column (WPC). To evaluate the structural and mechanical changes, the specimens were tested with simulation of 100,000 compression-flexion load cycles after which it was performed macroscopic analysis. Mechanical properties of the annulus fibrosis (AF) from the anterior and posterior regions of the IVD were tested at the uniaxial tension test. The stiffness coefficient values were statistically 32% higher in the WPC group (110 N/mm) than in the IPC (79 N/mm). The dynamics of increase in this parameter does not correspond with the course of decrease in height loss. WPC segments revealed clear structural changes that mainly involve the posterior regions of the IVD (bulging and delamination with the effect of separation of collagen fibre bundles). Pathological changes also caused decreases in the value of stress in the AF. The greatest changes in the stress value about group C (7.43 ± 4.49 MPa) were observed in the front part of the fibrous ring, where this value was for IPC 4.49 ± 4.78 MPa and WPC 2.56 ± 1.01 MPa. The research indicates that the applied load model allows simulating damage that occurs in pathological IVD. And the posterior column’s presence affects this change’s dynamics, structural and mechanical properties of AF.

     

GAG content,fiber stiffness,and fiber angle affect swelling-based residual stress in the intact annulus fibrosus

Biomechanics and Modeling in Mechanobiology - Biological tissues with a high glycosaminoglycan (GAG) content have an excellent ability to swell by absorbing water molecules from the surrounding...     

Collagen fibre orientation in the annulus fibrosus of intervertebral disc during bending and torsion measured by x-ray diffraction

X-ray diffraction has been used to measure the orientation of the collagen fibres in the ventral annulus fibrosus of intact L1/2 rabbit intervertebral disc during in vitro bending and torsion. Fibres are tilted with respect to the axis of the spine. As predicted by theory, fibre tilt decreases in those regions of the annulus which are stretched by bending but increases in the slackened regions. Good agreement with the quantitative predictions of bending theory was obtained in three of the six series of experiments, the predicted trend being found in all six. Tilt direction alternates in successive lamellae of the annulus. When discs were subjected to both clockwise and anticlockwise torsion of 5°, the two families of titled fibres reoriented in the expected directions.     

Formation of lamellar cross bridges in the annulus fibrosus of the intervertebral disc is a consequence of vascular regression

Cross bridges are radial structures within the highly organized lamellar structure of the annulus fibrosus of the intervertebral disc that connect two or more non-consecutive lamellae. Their origin and function are unknown. During fetal development, blood vessels penetrate deep within the AF and recede during postnatal growth. We hypothesized that cross bridges are the pathways left by these receding blood vessels.Initially, the presence of cross bridges was confirmed in cadaveric human discs aged 25 and 53 years. Next, L1-L2 intervertebral discs (n = 4) from sheep ranging in age from 75 days fetal gestation to adult were processed for paraffin histology. Mid-sagittal sections were immunostained for endothelial cell marker PECAM-1. The anterior and posterior AF were imaged using differential interference contrast microscopy, and the following parameters were quantified: total number of distinct lamellae, total number of cross bridges, percentage of cross bridges staining positive for PECAM-1, cross bridge penetration depth (% total lamellae), and PECAM-1 positive cross bridge penetration depth.Cross bridges were first observed at 100 days fetal gestation. The overall number peaked in neonates then remained relatively unchanged. The percentage of PECAM-1 positive cross bridges declined progressively from almost 100% at 100 days gestation to less than 10% in adults. Cross bridge penetration depth peaked in neonates then remained unchanged at subsequent ages. Depth of PECAM-1 positive cross bridges decreased progressively after birth. Findings were similar for both the anterior and posterior.The AF lamellar architecture is established early in development. It later becomes disrupted as a consequence of vascularization. Blood vessels then recede, perhaps due to increasing mechanical stresses in the surrounding matrix. In this study we present evidence that the pathways left by receding blood vessels remain as lamellar cross bridges. It is unclear whether the presence of cross bridges in the aging and degenerating intervertebral disc would be advantageous or detrimental, and this question should be addressed by future studies.     

Effect of removing the nucleus pulposus on the deformation of the annulus fibrosus during compression of the intervertebral disc

Eighteen frozen ovine discs were bisected, in the mid-sagittal plane, to produce 36 specimens. The cut surfaces were marked at the inner and outer annulus boundaries of the annulus fibrosus, both anteriorly and posteriorly, with Alcian blue stain. The sections were sealed by a transparent plate, and thawed. A compression of 1mm at a rate of 0.2mms(-1) was applied. The displacements of the Alcian blue marks were measured from the video images, recorded during the tests, using interactive image analysis software. Before removal of the nucleus, the inner boundaries of the annulus moved outwards during compression (P<0.001, anterior; P=0.01, posterior). However, after removal of the nucleus, both inner boundaries moved inwards (P<0.001, anterior and posterior). The outer boundaries moved outwards both before and after removal of the nucleus (P<0.001). The results showed that total removal of the nucleus changes the response of the annulus to compression.     

Cyclic tensile stretch modulates proteoglycan production by intervertebral disc annulus fibrosus cells through production of nitrite oxide

Degeneration of the intervertebral disc is the main pathophysiological process implicated in low back pain and is a prerequisite to disc herniation. Clinically, mechanical forces are important modulators of the degeneration, but the underlying molecular mechanism is not known and needs investigation to identify the biological target. The aim of this work was to study, at the molecular level, the effects of cyclic tensile stretch (CTS) on the production of proteoglycan by intervertebral disc annulus fibrosus cells since proteoglycans seem to be implicated in the dynamic process of intervertebral disc degeneration. Such cells of rabbit were cultured at high density on plates with a flexible bottom. CTS was applied with use of a pressure-operated instrument to deform the plates. With CTS at 1% elongation (1 Hz frequency), the level of (35)S-labeled neosynthesized proteoglycans that accumulated in the cellular pool or were secreted in the culture medium did not change, but at 5% elongation, the level was significantly reduced after 8 h of stimulation (30 and 21%, respectively) and further reduced at 24 h (43 and 41%, respectively). Introducing the protein synthesis inhibitor cycloheximide had no effect on this result. Neither aggrecan and biglycan expression nor proteoglycan physical properties were modified. The level of nitrite oxide production significantly increased by 3.5 times after 8 h of 5% elongation. Introducing the nitric oxide synthase (NOS) inhibitors N(G)-methyl-l-arginine or N-omega nitro-l-arginine diminished the effects of CTS on the production of nitrite oxide and proteoglycans. By contrast, introducing N-iminoethyl-l-lysine (a more specific inhibitor of inductible NOS [iNOS]) had little or no effect. Taken together, these results suggest that cNOS activation seems to be more implicated in the 5% CTS modulation of proteoglycan production than iNOS activation. These results suggest that CTS can help regulate the intervertebral disc matrix by decreasing proteoglycan production through a post-translational regulation involving nitrite oxide. This result could be of interest in the development of local therapeutic strategies aimed at controlling intervertebral disc degeneration.     

A comparison between porcine,ovine, and bovine intervertebral disc anatomy and single lamella annulus fibrosus tensile properties

This project aimed to compare gross anatomical measures and biomechanical properties of single lamellae from the annulus fibrosus of ovine and porcine lumbar vertebrae, and bovine tail vertebrae. The morphology of the vertebrae of these species differ significantly both from each other and from human, yet how these differences alter biomechanical properties is unknown. Geometric parameters measured in this study included: 1) absolute and relative intervertebral (IVD) and vertebral body height and 2) absolute and relative intervertebral disc (IVD) anterior‐posterior (AP) and medial‐lateral (ML) widths. Single lamella tensile properties included toe‐region stress and stretch ratio, stiffness, and tensile strength. As expected, the bovine tail IVD revealed a more circular shape compared with both the ovine and porcine lumbar IVD. The bovine tail also had the largest IVD to vertebral body height ratio (due to having the highest absolute IVD height). Bovine tail lamellae were also found to be strongest and stiffest (in tension) while ovine lumbar lamellae were weakest and most compliant. Histological analysis revealed the greatest proportion of collagen in the bovine corroborating findings of increased strength and stiffness. The observed differences in anatomical shape, connective tissue composition, and tensile properties need to be considered when choosing an appropriate model for IVD research. J. Morphol. 277:244–251, 2016. © 2015 Wiley Periodicals, Inc.     

Hyperelastic anisotropic microplane constitutive model for annulus fibrosus

In a recent paper, Peng et al. (2006, "An Anisotropic Hyperelastic Constitutive Model With Fiber-Matrix Interaction for the Human Annulus Fibrosis," ASME J. Appl. Mech., 73(5), pp. 815-824) developed an anisotropic hyperelastic constitutive model for the human annulus fibrosus in which fiber-matrix interaction plays a crucial role in simulating experimental observations reported in the literature. Later, Guo et al. (2006, "A Composites-Based Hyperelastic Constitutive Model for Soft Tissue With Application to the Human Fibrosis," J. Mech. Phys. Solids, 54(9), pp. 1952-1971) used fiber reinforced continuum mechanics theory to formulate a model in which the fiber-matrix interaction was simulated using only composite effect. It was shown in these studies that the classical anisotropic hyperelastic constitutive models for soft tissue, which do not account for this shear interaction, cannot accurately simulate the test data on human annulus fibrosus. In this study, we show that the microplane model for soft tissue developed by Caner and Carol (2006, "Microplane Constitutive Model and Computational Framework for Blood Vessel Tissue," ASME J. Biomech. Eng., 128(3), pp. 419-427) can be adjusted for human annulus fibrosus and the resulting model can accurately simulate the experimental observations without explicit fiber-matrix interaction because, in microplane model, the shear interaction between the individual fibers distributed in the tissue provides the required additional rigidity to explain these experimental facts. The intensity of the shear interaction between the fibers can be adjusted by adjusting the spread in the distribution while keeping the total amount of the fiber constant. A comparison of results obtained from (i) a fiber-matrix parallel coupling model, which does not account for the fiber-matrix interaction, (ii) the same model but enriched with fiber-matrix interaction, and (iii) microplane model for soft tissue adapted to annulus fibrosus with two families of fiber distributions is presented. The conclusions are (i) that varying degrees of fiber-fiber and fiber-matrix shear interaction must be taking place in the human annulus fibrosus, (ii) that this shear interaction is essential to be able to explain the mechanical behavior of human annulus fibrosus, and (iii) that microplane model can be fortified with fiber-matrix interaction in a straightforward manner provided that there are new experimental data on distribution of fibers, which indicate a spread so small that it requires an explicit fiber-matrix interaction to be able to simulate the experimental data.     

Proteoglycan-collagen interactions in intervertebral disc. A chondroitin sulphate proteoglycan associates with collagen fibrils in rabbit annulus fibrosus at the d-e bands

Rabbit annulus fibrosus and nucleus pulposus were analysed for hydroxyproline, chondroitin sulphate, keratan sulphate and dermatan sulphate. Tissue proteoglycans were stained for electron microscopy with Cupromeronic blue, used in the critical electrolyte concentration mode, with and without prior digestion by chondroitinase AC or ABC, hyaluronidase or keratanase. Collagen bands, a-e were demonstrated with UO2++. A chondroitin sulphate proteoglycan was found orthogonally associated with loosely packed collagen fibrils in annulus fibrosus at the d and e bands. The close metabolic and structural analogies with the dermatan sulphate proteoglycans previously shown to be located at collagen d-e bands in tendon, skin, etc. (Scott and Haigh (1985) Biosci. Rep. 5:71-81), are discussed. Tightly packed annulus collagen fibrils were surrounded by axially oriented proteoglycan filaments, mostly without specific locations.     

Elastic fibers in the anulus fibrosus of the dog intervertebral disc

A light microscopic investigation of the anulus fibrosus in cervical intervertebral discs of the dog was conducted to ascertain the arrangement and distribution of elastic fibers. Elastic fibers were observed in all lamellae of the anulus fibrosus. However, collagenous fibers were the predominant type of connective tissue fiber, and elastic fibers were randomly dispersed among them. Intralamellar (collagenous and elastic) fibers were vertically and obliquely oriented in both superficial and deep lamellae of the anulus fibrosus. All intralamellar fibers were densely and regularly arranged in superficial lamellae, but they were more loosely organized in deep lamellae. A narrow border of interlamellar, elastic fibers was observed between broader, contiguous lamellae in the superficial zone of the anulus fibrosus. Interlamellar elastic fibers wer vertically and obliquely arranged in superficial lamellae; however, they were radially oriented in deep lamellae. The deepest lamella of the anulus fibrosus consisted of a loose, three-dimensional network of intermeshing collagenous and elastic fibers. These observations suggest that elastic fibers are integral components of the articular and shock absorption mechanisms of the anulus fibrosus, and the cervical intervertebral disc of the dog is a suitable model for experimental investigation of the role of elastic fibers in intervertebral disc herniation.     

Recent advances in biological therapies for disc degeneration: tissue engineering of the annulus fibrosus,nucleus pulposus and whole intervertebral discs

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