Intervertebral disc degeneration : interplay of ageing, environmental and genetic factors (EURODISC)

As disc is an avascular tissue maintenance and repair processes depend on the secretion of autocrine and paracrine growth factors. In addition, an important question for understanding the role of mechanical forces in disc homeostasis is the ability of the latter to induce growth factor secretion. To this end, we have studied the role of exogenous and autocrine growth factors on disc cell proliferation and the signalling cascades involved. Known growth factors, whose expression was identified in discs by immunocytochemical studies, such as PDGF, basic FGF, IGF-I and TGF-β, were found to regulate cell proliferation; their effect seems to be determined by the presence of extracellular matrix components. Intracellular signaling pathways mediating these actions, e.g. MAPK, PI-3-K/Akt or Smads, have been also studied. Preliminary data inducate the central role of the PI-3K/Akt pathway in the proliferative response of disc cells. Furthermore, we showed that extreme stressful conditions of pH, osmolarity or oxygen tension strongly inhibit disc cell proliferation.

Fluid balance in the intervertebral disc under applied load is determined primarily by its swelling pressure, i.e. the external pressure at which it neither loses nor gains water. This depends on the composition of the tissue, in particular on its proteoglycan concentration. Proteoglycans develop a high osmotic pressure due to their fixed negatively charged groups. Because of their size, proteoglycans are excluded from the collagen's intrafibrillar volume; hence their osmotic activity is determined only by the extrafibrillar water. Here we show that in order to evaluate correctly the swelling pressure in the annuli fibrosi of human intervertebral disc, it is essential to evaluate its proportion of intrafibrillar water. We used low-angle X-ray scattering and osmotic stress techniques to determine the lateral packing of the collagen molecules in the fibrils of the annuli fibrosi (ages: 25-77). It was found that the lateral packing and hence the intrafibrillar water content depends on age, external osmotic pressure, and location in the tissue. Subtracting intrafibrillar water from total hydration yields the amount of extrafibrillar water, from which the true fixed charge density of the tissue could be estimated. From a force balance, it would appear that collagen tension plays only a minor role in the equilibrium of the human intervertebral disc under load, in contrast to articular cartilage, where collagen tension is important for load-bearing. These data, i.e. swelling pressure as well as the total hydration are especially relevant for implementation of artificial discs, where both mechanical as well as chemical properties ought to remain within physiological range. These finding are sent for publication (not confidential).

Purpose of the project was to study influences of mechanical loads on intervertebral disc cells. For the application of high hydrostatic pressure within a wide loading range (up to 3 MPa or even higher) we have developed a compression cylinder. During the mechanical loading experiments, cell-seeded scaffolds that are sealed into sterile infusion bags filled with culture medium are transferred into the cylindric tube of the device. Before closing the system, the cylinder is completely filled with fluid and any residual air has to be removed from the closed device before loading. The closed cylinder is placed into the hydraulic loading frame of an Instron machine that is calibrated for the application of defined loading conditions. Intermittent hydrostatic pressure is applied with a computer-based loading program and monitored by a pressure sensor situated inside the chamber. With this method disc cell loaded scaffolds - as done in this project - but also complete disc samples can be loaded by well-defined hydrostatic pressure by variation of the pressure magnitude, the duration of the applied loading cycle, the number of repetitions of loading cycles etc. Disc cell gene expression, cell viability and cell signalling molecules in conditioned media was analysed compared to control cultures without any load application.

In preliminary analyses, among the 28 candidate genes studied in EURODISC project and using both visual and digital (created by SpEx© software) parameters, we have identified 24 genes associated with different degenerative findings (3 times more than what had been identified earlier). In addition, we also identified four genes associated with back pain history.

Histology of the intervertebral disc shows it to be a highly organized matrix, laid down by relatively few cells in a very specific manner. The central gelatinous nucleus pulposus is contained within the more collagenous anulus fibrosus laterally and the cartilage endplates inferiorly and superiorly. The anulus consists of concentric rings or lamellae, with fibers in the outer ones continuing into the longitudinal ligaments and vertebral bodies. This arrangement allows the discs to facilitate movement and flexibility within what would be an otherwise rigid spine. The human disc begins life with some vascular supply, both within the cartilage endplates and the anulus fibrosus. These vessels recede soon after birth, leaving the disc with very little direct blood supply in the healthy adult. With increasing age, changes occur to both the cellular and matrix components of the disc. Water is lost from the matrix, and proteoglycan content also changes and diminishes. Morphologically the disc particularly the nucleus becomes less gelatinous and more fibrous; eventually cracks and fissures form. More blood vessels begin to grow into the disc via the outer anulus. Cellular changes include not only an increase in cell proliferation and the formation of cell clusters, but also increased cell death. The cartilage endplate also undergoes changes with age, including thinning, altered cell density, formation of fissures, and sclerosis of the subchondral bone. These changes are similar to those seen in degenerative disc disease, resulting in much discussion as to whether aging and degeneration are two separate processes, or the same process occurring over a different timescale. Additional disorders involving the intervertebral disc can demonstrate other changes in morphology. Discs from patients with spinal deformities, e.g., scoliosis, have ectopic calcification in the cartilage endplate and sometimes in the disc itself. Cells in these discs and also those from patients with spondylolisthesis have been demonstrated to have some very long cell processes. Disc cells in herniated discs show some further differences; for example, they appear to have a higher degree of cellular senescence than nonherniated discs and produce a greater abundance of matrix metalloproteinases. The role that abnormalities such as these play in the etiopathogenesis of the different disorders is not always clear. They may be due to a genetic predisposition or a response of the tissue to some insult or to an altered mechanical environment. Whatever the initial cause, if the morphology of the tissue changes, it is likely to alter the physiologic and mechanical functioning of the tissue.

Intervertebral discs demonstrate degenerative changes relatively early in life. Disc degeneration, in turn, is associated with back pain and disc herniation, both of which cause considerable clinical problems in the western world. Cell senescence has been linked to degenerative diseases of other connective tissues such as osteoarthritis. Thus we investigated the degree of cell senescence in different regions of discs from patients with different disc disorders. Discs were obtained from 25 patients with disc herniations, from 27 patients undergoing anterior surgery for either back pain due to degenerative disc disease (n=25) or spondylolisthesis (n=2) and from 6 patients with scoliosis. In addition, 4 discs were obtained post-mortem. Samples were classified as annulus fibrosus or nucleus pulposus and tissue sections were assessed for the degree of cell senescence (using the marker senescence- associated-â-galactosidase (SA-â-Gal)) and the number of cells present in clusters. There were significantly more SA-â-Gal positive cells in herniated discs (8.5% of cells) than those with degenerative disc disease, spondylolisthesis, scoliosis or cadaveric discs (0.5% of cells; p<0.001). There was more senescence of cells of the nucleus pulposus compared to those of the annulus fibrosus and in herniated discs a higher proportion of cells in cell clusters (defined as groups of 3 or more cells) were SA-â-Gal positive (25.5%) compared to cells not in clusters (4.2%, p<0.0001). This study demonstrates an increased degree of cell senescence in herniated discs, particularly in the nucleus where cell clusters occur. These clusters have been shown previously to form via cell proliferation, which is likely to explain the increased senescence. These findings could have 2 important clinical implications: firstly, that since senescent cells are known to behave abnormally in other locations they may lead to deleterious effects on the disc matrix and so contribute to the pathogenesis and secondly, cells from such tissue may not be ideal for cell therapy and repair via tissue engineering.

Study Design: An immunohistological study of pathological human intervertebral disc tissue. Objective: To examine the presence of pleiotrophin in diseased intervertebral disc tissue and the relationship between its presence and the extent of tissue vascularisation and innervation. Summary of Background Data: Increased levels of pleiotrophin, a growth / differentiation factor that is active in various pathophysiological processes, including angiogenesis, has been associated with osteoarthritic changes of human articular cartilage. The relationship between pleiotrophin expression and pathological conditions of the human intervertebral disc is unknown. Methods: Specimens of human lumbar intervertebral discs, obtained following surgical discectomy, were divided into 3 groups: non-degenerated discs (n=7), degenerated discs (n=6) and prolapsed discs (n=11). Serial tissue sections of each specimen were immunostained to determine the presence of pleiotrophin, blood vessels (CD34-positive endothelial cells) and nerves (neurofilament 200kD (NF200)-positive nerve fibres). Results: Pleiotrophin immunoreactivity was seen in disc cells, endothelial cells and in the extracellular matrix in most specimens of intervertebral disc, but was most prevalent in vascularised tissue in prolapsed tissue. There was a significant correlation between the presence of pleiotrophin-positive disc cells and that of CD34-positive blood vessels. NF200-positive nerves were seen in vascularised areas of more degenerated discs, but nerves did not appear to co-distribute with blood vessels or pleiotrophin-positivity in prolapsed discs. Discussion: Pleiotrophin is present in pathological human intervertebral discs and its relative prevalence and distribution suggests that it may play a role in neovascularisation of diseased or damaged tissue.

Intervertebral disc is an avascular tissue. Accordingly, factor secreted by the discs cells are crucial for the maintenance of its homeostasis. Intervertebral disc cell cultures originating either from nucleus pulposus or annulus fibrosus of bovine tail discs have been developed. The cells have been subjected to hydrostatic pressure or physical stresses and the corresponding serum-free conditioned media (CMs) have been collected. These CMs were tested for their ability to stimulate or inhibit the proliferation of cultures of the same (autocrine activity) or of the neighbouring (paracrine activity) cell type. We have shown that media conditioned by cells undergoing a hydrostatic pressure stress, as well as, low glucose, low pH, low oxygen tension or high osmolarity stresses do not exhibit any significant autocrine or paracrine activity.

Biological repair of degenerate discs involves regeneration of the disc matrix through stimulation of resident disc cells or by implanting new cells. However in many degenerate discs, the nutrient supply may be inadequate to support the requirements of cells involved in such treatments. Here we describe a method for assessing nutrient supply rapidly during routine surgical procedures. The method is based on measurement of diffusion of a low molecular weight tracer into the disc. Nitrous oxide (N2O), routinely used as an anaesthetic gas, is the tracer of choice as its concentration in the disc can be measured electrochemically in seconds. Silver needle electrodes and Ag/AgCl references electrodes were made in house and calibrated against known concentrations of nitrous oxide using a custom-built potentiostat adapted to fulfil requirements for medical use. The method for assessing transport into the disc was validated by inserting needle microelectrodes into an intact bovine disc in vitro and measuring N2O diffusion into the discs in an in-vitro, perfusion system. Measured profiles were in agreement with those calculated from diffusion theory, thus validating the reliability of the electrochemical measurements. The equipment was then used in vivo on patients undergoing surgery for treatment of back pain. Measurements of intra-discal N2O concentrations were made prior to discectomy in discs of 25 consenting patients (31-70 yrs) operated on for disc prolapse. The needle microelectrode was inserted into the disc to a calibrated distance during surgery after the disc was exposed but before excision. Measurements in patients found that N2O transport efficiency was low (below 40%) in 22/25 patients. All discs examined were degenerate (Grades 3 - 5); transport efficiency showed no correlation with grade assessed by MRI or with age showing that these parameters are not predictive of sufficiency of nutrient supply. Results show that transport efficiency into the disc can be measured rapidly during surgical procedures. The equipment is relatively inexpensive but requires development for routine use. It is of interest not only for the disc but also for other tissues where nutrient transport could be restricted and has proved of interest in investigation of rotator cuff tears or femoral heads. Methods could be adapted for outpatient procedures.

Study Design. The effect of human intervertebral disc aggrecan on endothelial cell growth was examined using cell culture assays. Objectives. To determine the response of endothelial cells to human intervertebral disc aggrecan and whether the amount and type of aggrecan present in the intervertebral disc may be implicated in disc vascularisation. Summary of Background Data. Intervertebral disc degeneration has been associated with a loss of proteoglycan and the ingrowth of blood vessels and nerves. Neovascularisation is a common feature also of disc herniation. Intervertebral disc aggrecan is inhibitory to sensory nerve growth, but the effects of disc aggrecan on endothelial cell growth are not known. Methods. Aggrecan monomers were isolated separately from the anulus fibrosi (AF) and nucleus pulposi (NP) of human lumbar intervertebral discs and characterised to determine the amount and type of glycosaminoglycan (GAG) sidechains present. The effects of these aggrecan isolates on the cellular adhesion and migration of the human endothelial cell lines, HMEC-1 and EAhy-926, were examined in vitro. Results. Homogenous substrata of disc aggrecan inhibited endothelial cell adhesion and cell spreading in a concentration-dependent manner. In substrata choice assays, endothelial cells seeded onto collagen type I migrated over the collagen until they encountered substrata of disc aggrecan, where they either stopped migrating, retreated onto the collagen, or, more commonly, changed direction to align along the collagen: aggrecan border. The inhibitory effect of aggrecan on endothelial cell migration was concentration-dependent and reduced by enzymic deglycosylation of the aggrecan monomers. AF aggrecan was more inhibitory to endothelial cell adhesion than NP aggrecan. This difference did not relate, however, to the extent to which the different aggrecan isolates were glycosylated or to marked differences in the relative levels of the GAG sidechains, chondroitin sulphate and keratan sulphate. Conclusions. Human intervertebral disc aggrecan is inhibitory to endothelial cell migration and this inhibitory effect appears to depend, in part, on the presence of GAG sidechains on the aggrecan monomer.

Study Design. Co-culture assays of the migration and interaction of human intervertebral disc (IVD) cells and chick sensory nerves on alternate substrata of collagen and aggrecan. Objectives. To examine the effects of aggrecan on disc cell migration, how disc cells and sensory nerves interact, and whether previously reported inhibitory effects of aggrecan on sensory nerve growth are affected by disc cells. Summary of Background Data. Human IVD aggrecan is inhibitory to sensory nerve growth in vitro, suggesting that a loss of aggrecan from the disc may have a role to play in the increased innervation seen in disc degeneration. Endothelial cells that appear to co-migrate with nerves into degenerated IVD express neurotrophic factors, but the effects of disc cells on nerve growth are not known. Methods. Human disc cells were seeded onto tissue culture plates that had been coated with type I collagen and human IVD aggrecan. Explants of chick dorsal root ganglia (DRG) were subsequently added to the plates and sensory neurite outgrowth stimulated by the addition of nerve growth factor (NGF). The migration and interaction of the disc cells and sensory neurites, in the context of the different matrix substrata, were examined by time-lapse video and fluorescence microscopy. The effects of disc cell conditioned medium on nerve growth were also examined. Results. Disc cells spread and migrated on collagen until they encountered the aggrecan substrata, where some cells but not all were repelled. In co-culture, DRG neurites extended onto the collagen / disc cells until they encountered the aggrecan where, like the disc cells, many were repelled. However, in the presence of disc cells, some neurites were able to cross onto this normally inhibitory substratum. The number of neurite crossings onto aggrecan correlated significantly with the number of disc cells present on the aggrecan. In control experiments using DRG alone, all extending neurites were repelled at the collagen / aggrecan border. Conditioned medium from disc cell cultures stimulated DRG neurite outgrowth on collagen, but did not increase neurite crossing onto aggrecan substrata. Conclusions. Human disc cells migrate across aggrecan substrata that are repellent to sensory DRG neurites. Disc cells synthesise neurotrophic factors in vitro that promote neurite outgrowth. Furthermore, the presence of disc cells in co-culture with DRG partially abrogates the inhibitory effects of aggrecan on nerve growth. These findings have important implications for the regulation of nerve growth into the intervertebral disc, but whether disc cells promote nerve growth in vivo remains to be determined.

The most commonly used estimates of disc degeneration, both in research and clinical work, are based on visual 3-7 point scores (normal, slight, moderate and severe) but the applying accurate digital measures of disc degeneration has been extremely limited. Based on current understanding, disc degeneration and spinal disorders have multifactorial and multigenetic aetiology, but each known risk factor and gene has a very small role explaining only few percents at most of degeneration and related symptoms. However, in etiologic research of degenerative disc findings and their associations with back pain, continues digital parameters would significantly enhance identification of the numerous weak associations. It is therefore obvious that precise measures of degeneration would be crucial for a breakthrough in genetic epidemiology of common spinal disorders. Results of the gene-environment interaction studies will provide rational preventive intervention strategies in prevention of common spinal disorders, which has so far fully failed. In addition, the software will significantly enhance our tools in follow-up studies to identify changes, and particular pain producing pathology.

Intervertebral discs have a primarily mechanical role in transmitting loads through the spine. The disc is subjected to a combination of elastic, viscous and osmotic forces; previous models have typically neglected osmotic forces. The fibril-reinforced poroviscoelastic swelling model of Wilson, which has recently been developed in our group, is used to compute the interplay of osmotic, viscous and elastic forces in an intervertebral disc under axial compressive load. The unloaded 3D finite element mesh equilibrates in physiological solution, exhibiting an intradiscal pressure of about 0.2 MPa. Before and after axial loading the simulated hydrostatic pressure compares well with the experimental ranges measured. Loading the disc decreases the height of the disc and results in an outward bulging of the outer annulus. Fiber stresses are highest on the most outward bulging on the posterior-lateral side. The osmotic forces resulted in tensile hoop stresses, which are higher than typical values in a non-osmotic disc. The computed axial stress profiles reproduced the main features of the stress profiles, in particular the characteristic posterior and anterior stress peaks, which were observed experimentally by McNally et al.

We have used the racemization of aspartic acid as marker for "molecular age" of collagen and aggrecan component of the human intervertebral disc matrix i.e. aggregating and non-aggregating proteoglycan as well as different buoyant density fractions of the aggrecan. By measuring the D/LAsp ratio of the different aggrecan species as a function of age, and using the values of the racemization constant, ki, found earlier for aggrecan in articular cartilage, we were able to establish directly the relative residence time of these molecules in human intervertebral disc matrix. For collagen, the half life value is around 125 years for both the nucleus pulposus and the annulus fibrosus. For the large monomer, the half life is 5.5 +/- 1.5 years - similar to the 3.4 years previously found on human articular cartilage, whereas for the binding region (A1D6), this value is 21.5 +/- 0.6 years - similar to 25 years in articular cartilage. For A1 preparations taken from normal tissue, half-life of 12.2.0 and 11.23.1.86 years were found for nucleus pulposus and annulus fibrosus, respectively; respective values from degenerate tissue are 8.77.2.17 and 8.41 +/- 2.83 years, suggesting increased synthesis of intact monomer (A1D1) in the degenerate tissue and/or increased rate of removal of small aggrecan fragments. A1-preparations from nucleus pulposus contain a lower proportion of aggregating proteoglycans compared to annulus fibrosus, suggesting increased proteolytic modification in the nucleus pulposus. In view of the high half life values of both collagen and aggrecan (especially collagen), questions arise as to the real possibilities of regenerating and repairing tissue. These finding are sent for publication (not confidential).

Disc cells depend on diffusion from blood vessels at the disc margins for supply of nutrients. Loss of supply is thought to lead to disc degeneration, but how loss of supply affects nutrient concentrations in the disc is not known; nutrient concentrations within discs can normally only be calculated, since concentration measurements are invasive. However, realistic predictions cannot be made until there is data from measurements of metabolic rates at conditions found in the disc in vivo, i.e. at low levels of oxygen, glucose and pH. Here in vitro measurements of metabolic rates of isolated bovine nucleus pulposus cells were made at varying levels of oxygen, glucose and pH to understand conditions centre of the disc. The objective was to obtain quantitative information on the interactions between oxygen and glucose concentrations and pH, and the rates of oxygen and glucose consumption and lactic acid production for disc nucleus cells. A metabolism chamber was designed to allow simultaneous recording of oxygen and glucose concentrations and of pH. These concentrations were measured electrochemically with custom-built glucose and oxygen sensors; lactic acid was measured biochemically. Bovine nucleus pulposus cells were isolated and inserted into the chamber, and simultaneous rates of oxygen and glucose consumption and of lactic acid production were measured over a range of glucose, oxygen and pH levels. There were strong interactions between rates of oxygen consumption and lactic acid production and pH. At atmospheric oxygen levels, oxygen consumption rate at pH6.2 was 32% of that at pH7.4. The rate fell by 60% as oxygen concentration was decreased from 21% to 5% at pH7.4, but only by 20% at pH6.2. Similar interactions were seen for lactic acid production and glucose consumption rates; we found that glycolysis rates fell at low oxygen and glucose concentrations and low pH. Equations were derived which satisfactorily predict the effect of nutrient and metabolite concentrations on rates of lactic acid production rate and oxygen consumption. Disc cell metabolism in air and at pH7.4 differs markedly from that found in the disc nucleus in vivo, where low levels of oxygen, glucose and pH all co-exist. Modelling and experimental work needs to consider interactions on metabolism; measurements in vitro under standard laboratory conditions will not predict behaviour in vivo.

Classically, intervertebral disc cells have been described as fibrocytic in the anulus fibrosus and chondrocytic in the nucleus pulposus. Recent animal studies, however, have suggested that disc cell morphology may be more complex than previously considered. Here, by utilizing labelling of components of the cytoskeleton in combination with confocal microscopy, we have examined the detailed morphology of human intervertebral disc cells in pathological and non-pathological tissue. Filamentous-actin- and vimentin-positive cells that appeared either fibrocytic or chondrocytic were observed in all intervertebral discs. However, in localized areas of the disc, stellate cells that extended multiple, branching cytoplasmic processes into their surrounding matrix were also seen. This stellate appearance formed a marked feature of disc cells regionally in certain pathologies, i.e., in cells of the outer anulus fibrosus in scoliotic discs and in inner anulus/nucleus pulposus cells in one spondylolisthetic disc. We conclude that the phenotypic variation of human intervertebral disc cells should be extended to include cells with a stellate appearance, which may be more prevalent in tissue that has been subjected to abnormal load or tension.

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