Biological response of the intervertebral disc to dynamic loading

Disc degeneration is a chronic remodeling process that results in alterations of matrix composition and decreased cellularity. This study tested the hypothesis that dynamic mechanical forces are important regulators in vivo of disc cellularity and matrix synthesis. A murine model of dynamic loading was developed that used an external loading device to cyclically compress a single disc in the tail. Loads alternated at a 50% duty cycle between 0MPa and one of two peak stresses (0.9 or 1.3MPa) at one of two frequencies (0.1 or 0.01Hz) for 6h per day for 7 days. An additional group received static compression at 1.3MPa for 3h/day for 7 days. A control group wore the device with no loading. Sections of treated discs were analyzed for morphology, proteoglycan content, apoptosis, cell areal density, and aggrecan and collagen II gene expression. Dynamic loading induced differential effects that depended on frequency and stress. No significant changes to morphology, proteoglycan content or cell death were found after loading at 0.9MPa, 0.1Hz. Loading at lower frequency and/or higher stress increased proteoglycan content, matrix gene expression and cell death. The results have implications in the prevention of intervertebral disc degeneration, suggesting that loading conditions may be optimized to promote maintenance of normal structure and function.     

Degeneration of the intervertebral disc

The intervertebral disc is a cartilaginous structure that resembles articular cartilage in its biochemistry, but morphologically it is clearly different. It shows degenerative and ageing changes earlier than does any other connective tissue in the body. It is believed to be important clinically because there is an association of disc degeneration with back pain. Current treatments are predominantly conservative or, less commonly, surgical; in many cases there is no clear diagnosis and therapy is considered inadequate. New developments, such as genetic and biological approaches, may allow better diagnosis and treatments in the future.     

Pathophysiology of the human intervertebral disc

Intervertebral disc degeneration is a common invalidating disorder that can affect the musculoskeletal apparatus in both younger and older ages. The chief component of the intervertebral disc is the highly organized extracellular matrix; maintenance of its organization is essential for correct spinal mechanics. The matrix components, mainly proteoglycans and collagens, undergo a slow and continuous cell-mediated turnover process that enables disc cells to adapt their environment to external stimuli. Cellular senescence and a history of chronic abnormal loading can upset this balance, leading to progressive tissue failure that results in disc degeneration. Although biological treatment approaches to disc repair are still far to come, advances in our understanding of disc biochemistry and in defining the role of genetic inheritance have provided a starting point for developing new concepts in the diagnosis, therapy and prevention of disc degeneration.     

Thermal analysis of the human intervertebral disc

Intervertebral disc (IVD) degeneration is one of the most common musculuskeletal disorders affecting western society. Degeneration alters the morphology and the mechanical properties of the discs. According to previous reports, DSC proved to be a suitable method for the demonstration of thermal consequences of local as well as global conformational changes in the structure of the human intervertebral discs. In the present study, a wide spectrum of degenerated IVD was examined by DSC. The results suggest that definitive differences exist between the stages of disc degeneration in calorimetric measures.     

Neutral proteinases of the human intervertebral disc

Disc tissue consisting of pooled annuli fibrosus and nuclei pulposus from the cadaver of an adolescent aged 19 years was extracted with 4.0 M Gu-HCl. Proteins of low buoyant density (p less than or equal to 1.38 g/ml) containing the disc enzymes and inhibitors were separated from proteoglycans of high buoyant density (p greater than or equal to 1.50 g/ml) by density gradient ultracentrifugation. Sephadex G-75F gel chromatography followed by trypsin affinity chromatography was then used to resolve disc proteolytic and trypsin inhibitory activities. The results obtained were strongly suggestive of the presence of a high molecular weight zymogen which upon activation generated a population of smaller molecular weight proteinases. The disc proteinases obtained by this process showed similar properties in terms of: their pH optima (7.4-7.6); their inhibition patterns by class-specific proteinase inhibitors; their variation of activity as a function of NaCl and lysine concentrations; and the hydrodynamic size of their proteoglycan degradation products. The activated disc neutral proteinase demonstrated many characteristics in common with plasmin; however, unlike the latter, the disc proteinases also showed some calcium dependence.     

The proteoglycans of the canine intervertebral disc

The high-buoyant-density proteoglycans of the nucleus pulposus and annulus fibrosus of the beagle intervertebral disc have been isolated by CsCl density gradient ultracentrifugation. The sulphated proteoglycans were labelled in vivo with 35SO4, 24 h and 60 days prior to killing. The hydrodynamic size and aggregation of the 24 h, 60 day and resident (from hexuronic acid and hexosamine analysis) proteoglycan subunit populations were determined by Sepharose CL-2B chromatography in the presence or absence of excess hyaluronic acid. The hydrodynamic size of the keratan sulphate-proteoglycan core protein complexes were also determined by Sepharose CL-2B chromatography after chondroitinase ABC digestion of proteoglycans. When initially synthesised (24 h) or after 60 days, the percentage aggregation and hydrodynamic size of the proteoglycans derived from the annulus fibrosus were larger than those present in the nucleus pulposus. Hexosamine, hexuronic and protein determination of the high-buoyant-density fractions showed that the proteoglycans of the nucleus pulposus were richer in chondroitin sulphate than those in the annulus. However there was no difference in Mr of the chondroitin sulphate and keratan sulphate attached to the proteoglycans of the two disc regions, nor were differences detected by HPLC between the proportions of chondroitin 4-sulphate and chondroitin 6-sulphate present in these high-density fractions. In contrast, the low-buoyant-density (1.54 greater than p greater than 1.45) proteoglycan fractions and tissue residues remaining after 4 M GuHCl extraction were found to contain dermatan sulphate, suggesting the presence of a third proteoglycan species possibly associated with the collagen of the fibrocartilagenous matrix.     

Proteoglycans of the intervertebral disc. Absence of degradation during the isolation of proteoglycans from the intervertebral disc.

Proteoglycans extracted with 4M-guanidinium chloride from pig intervetebral discs, and purified by equilibrium density-gradient centrifugation in CsCl, were of smaller hydrodynamic size than those extracted and purified in the same way from the laryngeal cartilage of the same animal. Whether this difference in size arose from degradation during the extraction and purification of the proteoglycans of the disc was investigated. Purified proteoglycans labelled either in the chondroitin sulphate chains or in the core protein were obtained from laryngeal cartilage by short-term organ culture. These labelled proteoglycans were added at the beginning of the extraction of the disc proteoglycans, and labelled cartilage and unlabelled disc proteoglycans were isolated and purified together. There was no appreciable loss of radioactivity after density-gradient centrifugation nor decrease in hydrodynamic size of the labelled cartilage proteoglycans on chromatography on Sepharose 2B, when these were present during the extraction of disc proteoglycans. It is concluded that disc proteoglycans are intrinsically of smaller size than cartilage proteoglycans and this difference in size does not arise from degradation during the extraction.     

Mechanical response of a simple finite element model of the intervertebral disc under complex loading

A simple axisymmetric finite element model of a human spine segment containing two adjacent vertebrae and the intervening intervertebral disc was constructed. The bodies and disc were modeled by three substructures; one to represent each of the vertebral bodies, the annulus fibrosus, and the nucleus pulposus. A semi-analytic technique was used to maintain the computational economies of a two-dimensional analysis when non- axisymmetric loads were imposed on the model. The response of the model to compression, shear, torsion and bending loads applied to the superior vertebral body was examined to determine the effects of disc geometry and material properties on response. Comparisons of model responses with experimentally measured responses were made to estimate material property values for which model behaviors are in agreement with measured behaviors.     

A simple model for the function of proteoglycans and collagen in the response to compression of the intervertebral disc.

The nucleus pulposus of the intervertebral disc exerts a pressure which enables it to support axial compression when contained by the annulus fibrosus. The disc was modelled as a thick-walled cylindrical pressure vessel in which the nucleus was contained radially by the annulus. As a result, the stress in the annulus had radial (compressive) as well as tangential (tensile) components. The radial stress at a given point in the annulus was considered to be balanced by the internal pressure which is expected to arise from the attraction of water by proteoglycans. There was a reasonable agreement between the calculated radial stress distribution and published results on the distribution of water within the annulus. As the internal pressure is expected to be isotropic, the annulus was expected to contribute to the axial resistance to compression of the disc; this contribution would be equal, in magnitude, to the radial stress. Predictions of the pressure distribution within the annulus were similar to published experimental measurements made in the radial and axial directions. The tangential stress within the annulus was considered to arise from the restoring stress in its strained collagen fibrils.     

Aquaporin expression in the human intervertebral disc

The nucleus pulposus (NP) of the human intervertebral disc (IVD) is a hyperosmotic tissue that is subjected to daily dynamic compressive loads. In order to survive within this environment the resident chondrocyte-like cells must be able to control their cell volume, whilst also controlling the anabolism and catabolism of their extra-cellular matrix. Recent studies have demonstrated expression of a range of bi-directional, transmembrane water and solute transporters, named aquaporins (AQPs), within chondrocytes of articular cartilage. The aim of this study was to use immunohistochemsitry to investigate the expression of aquaporins 1, 2 and 3 within the human IVD. Results demonstrated expression of both AQP-1 and -3 by cells within the NP and inner annulus fibrosus (AF), while outer AF cells lacked expression of AQP-1 and showed very low numbers of AQP-3 immunopositive cells. Cells from all regions were negative for AQP-2. Therefore this study demonstrates similarities in the phenotype of NP cells and articular chondrocytes, which may be due to similarities in tissue osmolarity and mechanobiology. The decrease in expression of AQPs from the NP to the outer AF may signify changes in cellular phenotype in response to differences in mechanbiology, osmolarity and hydration between the gelatinous NP and the fibrous AF.     

Substructuring and poroelastic modelling of the intervertebral disc

We proposed a substructure technique to predict the time-dependant response of biological tissue within the framework of a finite element resolution. Theoretical considerations in poroelasticity preceded the calculation of the sub-structured poroelastic matrix. The transient response was obtained using an exponential fitting method. We computed the creep response of an MRI 3D reconstructed L₅–S₁ intervertebral disc of a scoliotic spine. The FE model was reduced from 10,000 degrees of freedom for the full 3D disc to only 40 degrees of freedom for the sub-structured model defined by 10 nodes attached to junction nodes located on both lower and upper surfaces of the disc. Comparisons of displacement fields were made between the full poroelastic FE model and the sub-structured model in three different loading conditions: compression, offset compression and torsion. Discrepancies in displacement were lower than 10% for the first time steps when time-dependant events were significant. The substructuring technique provided an exact solution in quasi-static behavior after pressure relaxation. Couplings between vertical and transversal displacements predicted by the reference FE model were well stored by the sub-structured model despite the drastic reduction of degrees of freedom. Finally, we demonstrated that substructuring was very efficient to reduce the size of numerical models while respecting the time-dependant behavior of the structure. This result highlighted the potential interest of substructure techniques in large-scale models of musculoskeletal structures.     

A mechanical model for the human intervertebral disc

Stress relaxation experiments were performed on specimens from a human intervertebral disc. Specimens were made from the nucleus pulposus and from the external lamellae of the anulus fibrosus in two different orientations. Tests were run with varying moisture content so as to develop a relaxation master curve. A model was developed based on the experimental data. It was found that the short term master curve for the lamellae of the anulus and nucleus are similar, whereas the long term rubbery plateau is different between the lamellae and the nucleus. It was also established that the master curves for different lamellae and the nucleus were shifted relative to each other in the time domain due to changes in water content. The average relaxation modulus of the whole disc was obtained by averaging the properties between the anulus and nucleus. This model was then used for studies of Schmorl's nodes, of degenerated discs and for circumstances in which hydration is considered to be important.     

Type III collagen in the intervertebral disc.

Several collagen types have now been isolated from the intervertebral disc, although type III collagen has previously only been extracted from human pathological disc. In this study, type III collagen has been isolated from normal human and bovine intervertebral disc and immunolocalized in sections of rat, sheep, bovine and 'normal' human intervertebral disc of various ages. Staining with antisera to type III collagen is localized primarily around the cells. Results indicate that cells of the disc sit in 'chondrons', similar to those seen in the deep and mid zones of articular cartilage. We suggest that type III collagen is present in the intervertebral disc and hypothesize that it may be involved in the organization of the pericellular environment, perhaps linking the chondron capsule to the interterritorial matrix.