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.     

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.     

Uniaxial tensile testing of canine annulus fibrosus tissue under changing salt concentrations

Recent modelling efforts in the field of mechanics of the intervertebral disc, demonstrate that the deformation properties of intervertebral disc tissue are intimately linked to compositional changes. This paper presents uniaxial tensile relaxation experiments of canine annulus fibrosus tissue under stepwise changes of external salt concentration.     

Intercellular communication via gap junctions affected by mechanical load in the bovine annulus fibrosus

Cells in the intervertebral disc, as in other connective tissues including tendon, ligament and bone, form interconnected cellular networks that are linked via functional gap junctions. These cellular networks may be necessary to affect a coordinated response to mechanical and environmental stimuli. Using confocal microscopy with fluorescence recovery after photobleaching methods, we explored the in situ strain environment of the outer annulus of an intact bovine disc and the effect of high-level flexion on gap junction signalling. The in situ strain environment in the extracellular matrix of the outer annulus under high flexion load was observed to be non-uniform with the extensive cellular processes remaining crimped sometimes at flexion angles greater than 25°. A significant transient disruption of intercellular communication via functional gap junctions was measured after 10 and 20 min under high flexion load. This study illustrates that in healthy annulus fibrosus tissue, high mechanical loads can impede the functioning of the gap junctions. Future studies will explore more complex loading conditions to determine whether losses in intercellular communication can be permanent and whether gap junctions in aged and degenerated tissues become more susceptible to load. The current research suggests that cellular structures such as gap junctions and intercellular networks, as well as other cell–cell and cell–matrix interconnections, need to be considered in computational models in order to fully understand how macroscale mechanical signals are transmitted across scales to the microscale and ultimately into a cellular biosynthetic response in collagenous tissues.     

Modeling interlamellar interactions in angle-ply biologic laminates for annulus fibrosus tissue engineering

Mechanical function of the annulus fibrosus of the intervertebral disc is dictated by the composition and microstructure of its highly ordered extracellular matrix. Recent work on engineered angle-ply laminates formed from mesenchymal stem cell (MSC)-seeded nanofibrous scaffolds indicates that the organization of collagen fibers into planes of alternating alignment may play an important role in annulus fibrosus tissue function. Specifically, these engineered tissues can resist tensile deformation through shearing of the interlamellar matrix as layers of collagen differentially reorient under load. In the present work, a hyperelastic constitutive model was developed to describe the role of interlamellar shearing in reinforcing the tensile response of biologic laminates, and was applied to experimental results from engineered annulus constructs formed from MSC-seeded nanofibrous scaffolds. By applying the constitutive model to uniaxial tensile stress–strain data for bilayers with three different fiber orientations, material parameters were generated that characterize the contributions of extrafibrillar matrix, fibers, and interlamellar shearing interactions. By 10 weeks of in vitro culture, interlamellar shearing accounted for nearly 50% of the total stress associated with uniaxial extension in the anatomic range of ply angle. The model successfully captured changes in function with extracellular matrix deposition through variations in the magnitude of model parameters with culture duration. This work illustrates the value of engineered tissues as tools to further our understanding of structure–function relations in native tissues and as a test-bed for the development of constitutive models to describe them.     

3D non-affine finite strains measured in isolated bovine annulus fibrosus tissue samples

Understanding of the mechanics of disc tissue calls for measurement of strains in physiological conditions. Because the intervertebral disc is gripped between two vertebrae, the swelling is constrained in vivo, resulting in a intradiscal pressure of 0.1–0.2?MPa in supine position. The excision of isolated disc tissue samples results often in non-physiological swelling. The purpose of the present study is to measure 3D finite strains in isolated bovine disc tissue specimens under physiological osmolarity and pressure, particularly around discontinuities of the collagen network. The collagen is stained by means of CNA35 probe, and the (dead) cells are stained by means of propidium iodide. The tissue is observed under confocal microscopy, under an externally applied pressure generated by a PEG solution. The 3D finite strains are obtained through correlation of the texture of the 3D images. The correlation technique yields principal strains in all areas except within collagen-free areas. The deformation is strongly non-affine. Especially around discontinuities, the strain field is non-homogeneous. Macroscopic strains as computed from finite element analysis of whole discs are insufficient to predict microstrains around clefts or cells. Because of the small number of specimens, the present results should be considered preliminary.     

A comparison of uniaxial and biaxial mechanical properties of the annulus fibrosus: a porcine model

The annulus fibrosus of the intervertebral disk experiences multidirectional tension in vivo, yet the majority of mechanical property testing has been uniaxial. Therefore, our understanding of how this complex multilayered tissue responds to loading may be deficient. This study aimed to determine the mechanical properties of porcine annular samples under uniaxial and biaxial tensile loading. Two-layer annulus samples were isolated from porcine disks from four locations: anterior superficial, anterior deep, posterior superficial, and posterior deep. These tissues were then subjected to three deformation conditions each to a maximal stretch ratio of 1.23: uniaxial, constrained uniaxial, and biaxial. Uniaxial deformation was applied in the circumferential direction, while biaxial deformation was applied simultaneously in the circumferential and compressive directions. Constrained uniaxial consisted of a stretch ratio of 1.23 in the circumferential direction while holding the tissue stationary in the axial direction. The maximal stress and stress-stretch ratio (S-S) moduli determined from the biaxial tests were significantly higher than those observed during both the uniaxial tests (maximal stress, 97.1% higher during biaxial; p=0.002; S-S moduli, 117.9% higher during biaxial; p=0.0004) and the constrained uniaxial tests (maximal stress, 46.8% higher during biaxial; S-S moduli, 82.9% higher during biaxial). These findings suggest that the annulus is subjected to higher stresses in vivo when under multidirectional tension.     

Strain transfer in the annulus fibrosus under applied flexion

A detailed understanding of the anatomical and mechanical environment in the intervertebral disc at the scale of the cell is necessary for the design of tissue engineering repair strategies and to elucidate the role of mechanical factors in pathology. The objective of this study was to measure and compare the macroscale to microscale strains in the outer annulus fibrosus in various cellular regions of intact discs over a range of applied flexion. Macroscale strains were measured on the annulus fibrosus surface, and contrasted to in situ microscale strains using novel confocal microscopy techniques for dual labeling of the cell and the extracellular matrix. Fiber oriented surface strains were significantly higher than in situ fiber strains, which implies a mechanism of load redistribution that minimizes strain along the fibers. Non-uniformity of the strains and matrix distortion occurred immediately and most interestingly varied little with increase in flexion (3–16°), suggesting that inter-fiber shear is important in the initial stages of strain redistribution. Fiber oriented intercellular strains were significantly larger and compressive compared to in situ strains in other regions of the extracellular matrix indicating that the mechanical environment in this region may be unique. Further examination of the structural morphology in this pericellular region is needed to fully understand the pathway of strain transfer from the tissue to the cell. This study provides new knowledge on the complex in situ micro-mechanical environment of the annulus fibrosus that is essential to understanding the mechanobiological behavior of this tissue.     

Moderate mechanical stimulation rescues degenerative annulus fibrosus by suppressing caveolin-1 mediated pro-inflammatory signaling pathway

Mechanical loading can induce or antagonize the extracellular matrix (ECM) synthesis, proliferation, migration, and inflammatory responses of annulus fibrosus cells (AFCs), depending on the loading mode and level. Caveolin-1 (Cav1), the core protein of caveolae, plays an important role in cellular mechanotransduction and inflammatory responses. In the present study, we presented that AFCs demonstrated different behaviors when subjected to cyclic tensile strain (CTS) for 24 h at a magnitude of 0%, 2%, 5% and 12%, respectively. It was found that 5% CTS had positive effects on cell proliferation, migration and anabolism, while 12% CTS had the opposite effects. Besides, cells exposed to interleukin-1β stimulus exhibited an increase expression in inflammatory genes, and the expression of these genes decreased after exposure to moderate mechanical loading with 5% CTS. In addition, 5% CTS decreased the level of Cav1 and integrin β1 and exhibited anti-inflammatory effects. Moreover, the expression of integrin β1 and p-p65 increased in AFCs transfected with Cav1 plasmids. In vivo results revealed that moderate mechanical stimulation could recover the water content and morphology of the discs. In conclusion, moderate mechanical stimulation restrained Cav1-mediated signaling pathway and exhibited anti-inflammatory effects on AFCs. Together with in vivo results, this study expounds the underlying molecular mechanisms on the effect of moderate mechanical stimulation on intervertebral discs (IVDs) and may provide a new therapeutic strategy for the treatment of IVD degeneration.     

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.

     

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...     

Extra-fibrillar matrix mechanics of annulus fibrosus in tension and compression

The annulus fibrosus (AF) of the disk is a highly nonlinear and anisotropic material that undergoes a complex combination of loads in multiple orientations. The tensile mechanical behavior of AF in the lamellar plane is dominated by collagen fibers and has been accurately modeled using exponential functions. On the other hand, AF mechanics perpendicular to the lamella, in the radial direction, depend on the properties of the ground matrix with little to no fiber contribution. The ground matrix is mainly composed of proteoglycans (PG), which are negatively charged macromolecules that maintain the tissue hydration via osmotic pressure. The mechanical response of the ground matrix can be divided in the contribution of osmotic pressure and an elastic solid part known as extra-fibrillar matrix (EFM). Mechanical properties of the ground matrix have been measured using tensile and confined compression tests. However, EFM mechanics have not been measured directly. The objective of this study was to measure AF nonlinear mechanics of the EFM in tension and compression. To accomplish this, a combination of osmotic swelling and confined compression in disk radial direction, perpendicular to the lamella, was used. For this type of analysis, it was necessary to define a stress-free reference configuration. Thus, a brief analysis on residual stress in the disk and a procedure to estimate the reference configuration are presented. The proposed method was able to predict similar swelling deformations when using different loading protocols and models for the EFM, demonstrating its robustness. The stress-stretch curve of the EFM was linear in the range 0.9 < λ? < 1.3 with an aggregate modulus of 10.18±3.32 kPa; however, a significant nonlinearity was observed for compression below 0.8. The contribution of the EFM to the total aggregate modulus of the AF decreased from 70 to 30% for an applied compression of 50% of the initial thickness. The properties obtained in this study are essential for constitutive and finite element models of the AF and disk and can be applied to differentiate between functional degeneration effects such as PG loss and stiffening due to cross-linking.     

Fibronectin content of the annulus fibrosus in diabetic and non-diabetic sand rats

Annulus fibrosus of intervertebral discs from diabetic and non-diabetic sand rats were examined by microspectrophotometry for fibronectin content. This was higher in the diabetic animals both in the dorsal and ventral parts and in the outer and inner lamellae of the annulus. It is suggested that diabetes-related changes in fibronectin are similar to changes in annular collagen observed in species other than sand rats.     

The mechanical behaviour of bony endplate and annulus in prolapsed disc configuration

The mechanical response of intervertebral joints is deeply influenced by disc degeneration. The phenomenon is expressed in terms of variations in the biomechanical properties of the material, whose compressibility characteristics change because of the liquid content loss in the tissue and, what is even more important, to prolapse. In this work, the problem is investigated by means of a computational mechanics approach; a coupled material and geometric non-linear model is developed, representing vertebra, annulus and nucleus submitted to an axial load. A transversely isotropic law is assumed for cortical bone in the vertebral body and an isotropic law for the cancellous portion; a hyperelastic formulation is assumed for the disc, allowing effective interpretation of the mechanical characteristics of degeneration. The results obtained are reported with regard to bony endplate and annulus behaviour; interaction phenomena between bony endplate and nucleus are emphasized.     

Degeneration affects the fiber reorientation of human annulus fibrosus under tensile load

The angled, lamellar structure of the annulus fibrosus is integral to its load-bearing function. Reorientation of this fiber structure with applied load may contribute to nonlinear mechanical behavior and to large increases in tensile modulus. Fiber reorientation has not yet been quantified for loaded non-degenerated and degenerated annulus fibrosus tissue. The objective of this study was to measure fiber reorientation and mechanical properties (toe- and linear-region modulus, transition strain, and Poisson's ratio) of loaded outer annulus fibrosus tissue using a new application of FFT image processing techniques. This method was validated for quantification of annulus fiber reorientation during loading in this study. We hypothesized that annulus fibrosus fibers would reorient under circumferential tensile load, and that fiber reorientation would be affine. Additionally, we hypothesized that degeneration would affect fiber reorientation, toe-region modulus and Poisson's ratio. Annulus fibrosus fibers were found to reorient toward the loading direction, and degeneration significantly decreased fiber reorientation (the fiber reorientation parameter, m(FFT)=-1.70 degrees /% strain for non-degenerated and -0.95 degrees /% strain for degenerated tissue). Toe-region modulus was significantly correlated with age (r=0.6). Paired t-tests showed no significant difference in the fiber reorientation parameter calculated experimentally with that calculated using an affine prediction. Thus, an affine prediction is a good approximation of fiber reorientation. The findings of this study add to the understanding of overall disc mechanical behavior and degeneration.     

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.     

Changes in the interfacial shear resistance of disc annulus fibrosus from genipin crosslinking

Crosslinking soft tissue has become more common in tissue engineering applications, and recent studies have demonstrated that soft tissue mechanical behavior can be directly altered through crosslinking. Using a recently reported test method that shears adjacent disc lamella, the effect of genipin crosslinking on interlamellar shear resistance was studied using in vitro bovine disc annulus.