© 2000 by The Society for Integrative and Comparative Biology
The Nucleus of the Intervertebral Disc from Development to Degeneration1
1 Physiology Laboratory, Oxford University, Oxford OX1 3PT, UK
2 Centre for Spinal Studies, RJAH Orthopaedic Hospital, Oswestry, Shropshire SY10 7AG, UK
3 Connective Tissue Biology Laboratory, School of Molecular and Medical Biosciences, University of Wales Cardiff, P.O. Box 911, Cardiff CF1 3US, UK
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SYNOPSIS |
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 SYNOPSIS INTRODUCTIONTHE STRUCTURE AND COMPOSITION...DEVELOPMENT OF THE DISC...CELLS OF THE DISCCHANGES IN THE DISC...MACROMOLECULAR FUNCTIONEFFECT OF MECHANICAL STRESS...CONCLUSIONSReferences |
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The nucleus of the intervertebral disc in humans shows the most dramatic changes with age of any cartilaginous tissue. It originates from the notochord. In the foetus and infant, the nucleus contains actively dividing and biosynthetically active notochordal cells. The proteoglycans and other matrix components produced have a high osmotic pressure, imbibe water and maintain a hydrated structure which, though it has little mechanical strength, has a high swelling pressure which maintains disc turgor. In some species, the notochordal cells and the mucoid nucleus pulposus persist throughout adult life. However by about 4 yr of age in humans, the notochordal cells have disappeared to be replaced by those of chondrocytic appearance but of unknown origin. These cells continue to produce proteoglycans but also synthesize significant amounts of collagen. The nucleus becomes firmer and less hydrated and loses its transparent appearance. The cell density of the adult nucleus is very low with cells occupying less than 0.5% of tissue volume; each cell thus has to turn over and maintain a large domain of extracellular matrix. The density of living cells decreases with age, possibly because of problems with nutrient supply to this large avascular tissue. Proteoglycan concentration also falls, and nucleus hydration decreases markedly, the disc discolours and in many cases clefts and fissures form. In most adults, the disc nucleus degenerates eventually to a stage where it can no longer fulfil its mechanical role.
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INTRODUCTION |
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SYNOPSISINTRODUCTIONTHE STRUCTURE AND COMPOSITION...DEVELOPMENT OF THE DISC...CELLS OF THE DISCCHANGES IN THE DISC...MACROMOLECULAR FUNCTIONEFFECT OF MECHANICAL STRESS...CONCLUSIONSReferences |
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The role of the intervertebral discs is mechanical. They are the joints of the spine, enabling it to bend and twist in all directions. They support compressive loads arising from body weight and muscle tension and anchor one vertebral body to the next. How the discs perform these mechanical tasks however, depends on the organisation and composition of the major macromolecules, which make up this tissue, the collagens and proteoglycans. Disc composition changes significantly during development, growth, ageing and degeneration and this in turn alters how the discs respond to changes in mechanical stress. Since the discs occupy around one third of the length of the vertebral column, changes in the behaviour of the disc can affect other spinal structures such as ligaments and muscles. This review will concentrate on the human nucleus pulposus and discuss its major biochemical components in relation to their functional roles, showing how changes with development and ageing may affect the biomechanical behaviour of the nucleus and thus of the whole disc.
The macromolecules of the disc are made by the disc's cells and changes in composition ultimately arise from changes in cellular metabolism. We will thus also review briefly factors, such as nutrient supply and mechanical load, that are known to affect cellular activity and that may be involved in the etiology of disc degeneration.
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THE STRUCTURE AND COMPOSITION OF THE DISC |
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The intervertebral discs lie between the vertebral bodies, separated from them by a thin cartilaginous endplate and consisting of two main regions, an inner, soft and highly hydrated structure, the nucleus pulposus, and an outer, firm, collagenous annulus fibrosus consisting of concentric lamellae encircling the nucleus (Fig. 1). These consist of bundles of collagen fibres running obliquely from one vertebral body to the next and firmly anchoring these to the bone or the cartilaginous endplate. The angle of the collagen bundles alternates between successive lamellae thus forming a cross-woven and re-inforced structure (Hukins 1984)
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The three major constituents of the disc are water, fibrillar collagens and aggrecan, the large aggregating proteoglycan consisting of a protein core to which up to 100 highly sulphated glycosaminoglycan (GAG) chains, principally chondroitin and keratan sulphate, are covalently attached (Muir, 1995)
. The proportion and organisation of these components vary considerably with position across the disc with the nucleus having a higher concentration of aggrecan and water and a lower collagen content than other regions of the disc. The fibrillar collagens also change across the disc, with the nucleus containing only type II collagen while both types I and type II are found in the annulus (Eyre and Muir, 1977). Changes in composition down the spine have also been described, though these have been less well characterised. The proportions of aggrecan and water in the nucleus fall steeply with age, while the proportion of collagen rises; a similar change is seen in degenerate discs. These changes appear to arise from loss of aggrecan rather than an increase in the amount of collagen produced and laid down (Antoniou et al., 1996b).
The nucleus contains many other components apart from type II collagen and aggrecan. Several minor collagens have been found in specific locations around the cells (type IX, type VI, type III) suggesting a functional role (Roberts et al., 1991b; Nerlich et al., 1997). Small proteoglycans such as decorin, biglycan, lumican and fibromodulin have been found in the nucleus though in lower concentrations than in the annulus (Sztrolovics et al., 1999). Other proteins and glycoproteins such as elastin and fibronectin are also present but have not been well characterised (Melrose and Ghosh, 1988).
The disc also contains enzymes, which can degrade the macromolecular components (Melrose and Ghosh, 1988). Since these proteases cleave molecules such as collagen and aggrecan at specific sites, antibodies against these ‘neo-epitopes’ have been used as markers of degradation and turnover in the disc (Antoniou et al., 1996a; Sztrolovics et al., 1997). With age and disc degeneration the level of active proteases present in the disc increases, supporting their suggested role in disc matrix turnover and degradation (Crean et al., 1997). More detailed information on disc composition and biochemistry is given in recent reviews (Oegema, 1993; Urban and Roberts, 1996; Johnstone and Bayliss, 1995).
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DEVELOPMENT OF THE DISC NUCLEUS FROM THE NOTOCHORD |
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The intervertebral discs develop embryologically from both the mesenchyme (the annulus fibrosus) and the notochord (the nucleus pulposus) (Walmsley, 1953)
. The mesenchymal tissue surrounding the rod-like notochord begins to segment in the human foetus at around 5–6 wk gestation, with the regularly spaced condensations which express the Pax gene, HuP48, eventually forming the annulus fibrosus of the future intervertebral discs; the non-condensed regions form the future vertebral bodies (Theiler, 1988; Goto and Uhthoff, 1986). Soon after condensation, both regions begin to form a hyaline-like cartilage and, in parallel with the process of chondrification, the notochord enlarges between the vertebral bodies to form the nucleus pulposus of the future intervertebral discs and disappears in the areas where vertebrae are developing (Theiler, 1988; Walmsley, 1953).
The notochordal nucleus pulposus, while rich in typical cartilage and disc matrix components such as sulphated glycosaminoglycans (GAGs) and hyaluronan has been shown to produce different proteins from those of other cartilage or disc cells (Gotz et al., 1995). The notochordal cells express type IIA collagen, the differentially spliced form of type II collagen typical of pre-chondrocytes, rather than the alternate type IIB collagen which is expressed by mature chondrocytes; they also express mRNAs such as versican and decorin, more characteristic of fibroblasts (Sandell, 1994). Finally notochordal cells also produce a large chondroitin sulphate proteoglycan which though a product of the aggrecan gene, differs from aggrecan and contains no keratan sulphate, possibly as the result of posttranslational mechanisms (Domowicz et al., 1995).
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CELLS OF THE DISC |
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In some animals the notochordal cells persist in the disc nucleus throughout life (Butler, 1989)
. In these species (e.g., rodents, cats and non-chondrodystrophoid dogs) the discs remain translucent and semi-liquid with a high proportion of sulphated GAGs and a low collagen content. In other species however, the notochordal cells have virtually disappeared by the time of birth (e.g., horses) or, as in humans, vanish within the first few years of life (Butler, 1989; Walmsley, 1953). It is at present not known whether in these species, the notochordal cells develop into the chondrocyte-like nucleus pulposus cells seen in these species, or whether they die off and are replaced by cells migrating from the inner annulus or endplate (Trout et al., 1982; Walmsley, 1953).
The mature human intervertebral disc has a very low cell density in comparison to other tissues with the cells occupying approximately 0.25–0.5% of the tissue volume. In adult human or bovine discs, the cells of the nucleus and inner annulus are chondrocyte-like, being rounded and enclosed in a capsule (Roberts et al., 1991a); unlike chondrocytes however they contain cytoplasm-filled processes (Errington et al., 1998). In contrast, the cells of the outer annulus are thin and extended along the collagen fibrils, rather like tendon cells (Postacchini et al., 1984). In adults, a large proportion of cells in the nucleus appear necrotic (Trout et al., 1982).
Although the disc has so few cells, their role is vital to the health of the disc since they synthesise and maintain an appropriate macromolecular composition, producing proteoglycans and other molecules throughout life. They also produce proteases and their inhibitors. The disc remains healthy while the rate of macromolecular synthesis and breakdown are in balance. However, if the rate of breakdown increases over synthesis, the disc matrix will ultimately disintegrate and the disc will degenerate.
The activity of disc cells can be regulated by growth factors and cytokines (Thompson et al., 1991; Shinmei et al., 1988) and by physical factors such as mechanical stress as discussed below. Nutrient supply to the avascular disc also affects cellular metabolism significantly (Ohshima and Urban, 1992) and loss of nutrient supply because of structural changes to the cartilage endplate, is thought to be a major cause of disc degeneration (Nachemson et al., 1970b; Urban and Roberts, 1995).
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CHANGES IN THE DISC NUCLEUS WITH AGE |
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As the notochordal cell population disappears, the nucleus loses its gelatinous appearance and its translucency. The composition of the extracellular matrix of the nucleus becomes more cartilage-like and in the child and young adult it is white and opaque consists mainly of a high concentration of aggrecan embedded in a fine network of collagen II fibrils.
There are large changes in appearance of the adult disc nucleus with increasing age; the nucleus becomes less hydrated and more collagenous. It discolours, changing from white to yellow-brown in colour through the accumulation of products of non-enzymatic glycosylation; these can also form cross-links between polypeptide chains and may alter the tissue's mechanical properties, reducing flexibility (Hormel and Eyre, 1991). An antibody to stabilised and processed forms of these products (CML) has shown they are present even in young (13 yr) discs, but that they accumulate with age (Nerlich et al., 1997). With age the boundary between nucleus and annulus becomes increasingly blurred and the annular rings thicken and appear more disorganised (Coventry et al., 1945). Type X collagen is found in the matrix of elderly and of scoliotic discs and may be associated with abnormal calcification in these areas (Nerlich et al., 1997). Eventually cracks and fissures appear in the endplate, nucleus and annulus and the disc thins and distorts. In the final stages of degeneration or ageing the matrix almost vanishes to be replaced by disorganised scar or granulation tissue (Thompson et al., 1990; Vernon-Roberts, 1992). It is difficult to distinguish ageing from pathological changes and at present there are no clear markers, either morphological or biochemical, distinguishing these two processes.
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MACROMOLECULAR FUNCTION |
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The dramatic changes in disc composition during life, strongly influence its mechanical role as can be seen from a model of cartilaginous tissues (Broom and Marra, 1985)
consisting of a string network filled with balloons. The balloons inflate the network, putting the string under tension while the string holds the balloons in place. Neither component on its own is able to support a load; the string would collapse and the balloons would fly apart. Together, however, they can withstand tensile and compressive loads with the degree of deformation depending on the load, the architecture and knots (cross-links) of the string network, and the number and degree of inflation of the balloons. A network containing a few floppy balloons will deform more than one stuffed full of highly inflated ones. In the tissue, the collagen network assumes the role of the string with variations in its weave governing its mechanical behaviour (Hukins, 1984). The role of the balloons is fulfilled by water, drawn into the tissue by the high swelling pressure arising mainly from the osmotic properties of proteoglycans (Urban et al., 1979).
Proteoglycan swelling pressure and development
These mechanical functions of proteoglycans and collagen have an important role in development. The stiff collagenous notochordal sheath resists osmotic swelling of the notochord. The resulting rise in internal pressure permits the notochord to elongate and straighten without being buckled by the surrounding tissues (Adams et al., 1990). The development of a pressure within the notochord because of the restraints imposed by the surrounding cartilaginous sheath is also necessary for formation of the intervertebral discs; this mechanical pressure induces notochordal cells to migrate from the vertebral bodies to the presumptive intervertebral disc area and so form the nucleus pulposus (Theiler, 1988; Rufai et al., 1995). No intervertebral discs form in mice with abnormal collagens and thus weakened cartilage, which is unable to exert the necessary mechanical stimulus on the notochord (Aszodi et al., 1998).
Aggrecan and disc water content
The osmotic pressure of aggrecan arises mainly from the highly sulphated GAG chains which confer a high negative fixed charge on the matrix. The concentration of fixed negative charges determines the concentration of extracellular ions in the tissue through the Gibbs-Donnan equilibrium (Maroudas and Evans, 1974). Positively charged molecules such as small cations are thus attracted into the tissue to balance the negative charges of the GAGs, while negatively charged molecules are repelled to some extent. In the centre of a normal disc [Na+] is around 400 mm and [Cl-] is around 80–100 mm compared to a concentration of around 140 mm for both ions in serum. The concentration of cations and anions varies directly with local GAG concentration. The osmotic pressure arising from these ions is mainly responsible for the swelling pressure of the disc; the size of the GAG chains or the integrity of the aggrecan molecule has little effect on tissue osmotic pressure (Urban et al., 1979).
The water content of the disc depends both on aggrecan content and on the pressure applied to the disc by external loads. Pressure on the disc arises more from muscular activity than from body weight and thus varies with posture and movement. In human lumbar discs, pressure is lowest when lying prone (at around 0.1–0.2 MPa) and increases 5–8 fold when standing or sitting (Nachemson and Elfstrom, 1970). In order to maintain osmotic equilibrium, fluid is expressed as pressure increases, but because of the disc's size and low hydraulic permeability, water loss is slow and equilibration takes many hours; the disc thus rarely achieves osmotic equilibrium. In humans, around 20–25% of the disc's water is expressed due to high loads imposed by muscle tensions during the day's activity; this water is regained during the decrease in load under rest at night (Boos et al., 1993). This cyclical change in fluid content is thought to be responsible for the oscillating length of the spine which is 1–2 cm longer in the morning than in the evening (DePuky, 1935), while increase of disc hydration under weightlessness could also account for the 5cm height gain in space flight (Brown, 1977).
In discs containing notochordal nucleus cells, the collagen content of the nucleus is very low, the discs remain highly hydrated throughout life and thus act hydrostatically when loaded, distributing pressure evenly to the adjacent annulus and endplate; these discs rarely show signs of degeneration such as clefts in the annulus or endplate (Bray and Burbidge, 1998). In humans and some animals however, aggrecan content/dry weight of the nucleus decreases with age as does hydration (Fig. 2). The nucleus eventually no longer acts hydrostatically (McNally, 1995), thus the annulus and endplate are exposed to high point stresses which might lead to the cracks and fissures seen in degenerate discs. In addition, since hydraulic permeability increases with aggrecan loss, as well as having a lower fluid content, degenerate discs will lose that fluid more quickly under load. As water is the main component of the disc, loss of fluid leads to a fall in disc height and abnormal loading of other spinal structures such as the apophyseal joints (Fig. 3) (Adams et al., 1990).
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EFFECT OF MECHANICAL STRESS ON CELLULAR FUNCTION |
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The disc is subjected to varying mechanical forces at all times. Exposure to heavy mechanical stress over long periods is thought to be another factor leading to disc degeneration. It has been suggested that under normal conditions the disc is subjected to Wolff's law, i.e., applied stress affects cellular activity and the disc remodels to build a matrix which minimises the stress (Brickley-Parsons and Glimcher, 1984)
. Some studies have found that the matrix synthesis and production of proteinases by disc cells can be influenced by mechanical signals such as hydrostatic pressure (Fig. 4) (Handa et al., 1997; Ishihara et al., 1996), change in fluid content (Ohshima et al., 1995); (Ishihara et al., 1997) and stretch (Matsumoto et al., 1999). These signals lead to alterations in production of both proteoglycans and of proteases and thus could potentially influence turnover of the matrix. At present however, it is difficult to relate these results to the disc's responses to load in vivo. Animal studies have however shown that the disc responds to prolonged changes in loading (Stokes et al., 1996) and that high levels of static compression can induce disc degeneration (Lotz et al., 1998).
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CONCLUSIONS |
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The biomechanical responses of the disc depend on its macromolecular composition which is ultimately regulated by the activity of the disc's cells. The health of the disc thus depends on the ability of the cells to produce a matrix appropriate for the mechanical loads routinely encountered.
Degeneration can occur through loss of nutrient supply to the cells or because the cells cannot respond adequately to inappropriate mechanical or chemical signals. At present, factors which regulate the behaviour of disc cells are poorly understood and until this area is better understood, the problem of disc degeneration in man and other species will not be solved.
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ACKNOWLEDGMENTS |
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This work was supported by the ARC (U0507).
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FOOTNOTES |
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1 From the Symposium The Function and Evolution of the Vertebrate Axis presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 6–10 January 1999, at Denver, Colorado.
2 E-mail: jpgu{at}physiol.ox.ac.uk
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References |
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