LUMBAR INTERVERTEBRAL DISC (IVD).
EXTRACELLULAR MATRIX (ECM)
International Publicized Data
GMCD Instructional Course Lectures
Dr. med. Guy M.C. Declerck MD (GMCD)
Medical FRCS-, FRCS Ed Orth-, M Ch Orth-, PhD-studies
Spinal Surgical and Research Fellow, Perth, Western Australia
Spinal Orthopaedic Surgeon and Surgical Instructor
Consultant R&D Innovative & Restorative Spinal Technologies
President International Association of Andullation Technology (IAAT)
Copywriter / Translator: Filip Vanhaecke PhD
Illustrative expertise: Lennart Benoot, Mincko, Halle, Flanders, Belgium
Artistic illustrations: Alonso Ríos Vanegas, sculptor and writer, Medellín, Colombia
Page layout: Lennart Benoot, Mincko, Halle, Flanders, Belgium
Review scientific literature: Medical Consulting Advice, Ostend, Flanders, Belgium
Support: International Association of Andullation Therapy (IAAT)
Legal advice: Anthony De Zutter, kornukopia.be
Dedication to all my families. February 2015.
I know … each step in my life began with disasters. When I failed, a (long) period of desperation followed. But always stood up again and again. Nothing seemed to work the first time. Sorry for my stubbornness but I always believed in my capabilities. And you see, suddenly a possible solution came to my mind. Always felt I had ideas. But translation of my ideas was a minefield for all of you. Decided to be independent and active and not forced to adjust to other orders and your systems. If people now criticize me in a nonscientific way, I completely ignore them because it’s not an argument. If it could have been a scientific attack, I would have taken it seriously and responded accordingly. Strangely enough, you knew nothing about me. Indeed, the opposite of love is indifference. And to paraphrase Albert Camus : 'Ce n’est pas si facile de devenir ce qu’on est’.
Relying on the work of giants is the lifeblood of scientific research. Indeed, if I have seen further, it is by standing on the shoulders of giants. One might even say that I have always depended on the kindness of strangers in this regard (*).
The continuous support by professor BA Kakulas (Neuropathology), professor JR Taylor (Spinal Anatomy and Human Biology), and Sir George M Bedbrook (Spinal Orthopaedic and Rehabilitation Surgeon) made it possible to analyse 23,539 post-mortem human spines, normal and pathological, in the Department of Neuropathology, Royal Perth Hospital/University Western Australia, Perth.
Note: in order not to disturb easy reading of the underneath scientifically based chapters, only a few authors are mentioned in the text where dr. Guy considered it essential. Their names are placed between brackets. Further information on their individual research can be read in the last chapter ‘Literature Encyclopedia’.
(*) Mirsky Steve. Technology is making it harder for word thieves to earn outrageous fortunes. Scientific American, February 2014, p. 64
1. Extracellular matrix (ECM) in the avascular intervertebral disc (IVD)
The healthy but avascular IVD mainly consists of an abundant extracellular matrix (ECM) with a very small amount of cells that help to maintain this ECM*.
* ECM is the part of the IVD tissue that surrounds the disc cells
2. Synthesis and breakdown of ECM by IVD cells
The integrity of the IVD relies on a healthy balance between synthesis and degradation of the components of ECM by the disc cells. The cells in the IVD synthesize new extracellular matrix but break down existing ECM as well. Simply said, the ECM of the IVD is a dynamic tissue which is subject to continuous deterioration and remaking. Interactions between receptors at the cell surface and components in the ECM are the mechanisms to provide important feedback to the cells which thereby obtain signals to modulate any degradation and/or repair (see below: enzymes).
In the normal mature and non-degenerate IVD, there exists a normal equilibrium between synthesis and degradation of ECM. The important part of the IVD tissue consists of non-collagenous proteins, proteoglycans, highly organised networks of fibrillar collagens, and proteinases. In normal circumstances the ECM is an elaborate framework of macromolecules that attract and hold water within the ECM yet allowing diffusion of water, ions, and small molecules.
When this equilibrium between synthesis and degradation starts being disturbed - based on (a combination of) genetic, familial, traumatic, aging, and degenerative factors - higher concentrations of aberrant molecules are produced (more destructive proteinases, abnormal proteoglycans and collagens, and cytokines) causing an alteration of the structural and functional properties of the extracellular matrices. At that time painful mechanical malfunctioning of the IVD may and can occur.
3. Mechanobiologic importance of ECM
The ECM not only provides structural support to the cells. At all times the available and viable IVD cells in combination with their surrounding ECM maintain the composition, structure, functions and mechanical properties of the IVD tissue. When the composition of the ECM changes, the mechanical integrity of the IVD starts getting lost. This situation may lead to the development of degenerative lesions in the three important structures of the IVD: endplates, annuli and nuclei.
In depth knowledge of the normal and changing molecular structure of the ECM in the IVD remains essential for a detailed understanding of both the biological and mechanical functions of the IVD. In other words, it is essential to know the (altering) components of the ECM for understanding its functional mechanobiology!
4. Elaborate framework of ECM macromolecules
Both the nucleus pulposus (NP) and the annulus fibrosus (AF) of a normal and non-degenerate IVD contain an ECM with a small amount of cells but an abundant amount of molecules. The matrices consist of non-collagenous proteins, proteoglycans, collagens, and enzymes (Table 4).
type I (0 - 80 %)
type II (0 - 80 %)
. collagenases (MMPs 1, 8, 13)
type III (< 5 %)
. gelatinases (MMPs 2, 9)
type V (1 - 2 %)
. stromelysin-1 (MMP 3)
type VI (10 - 20 %)
type IX (1 - 2 %)
type X (1 - 2 %)
type XI (1 - 2 %)
type XII (< 1 %)
type XIV (< 1 %)
Table 4. Most common components in the extracellular matrices of the mature lumbar intervertebral disc
5. ECM of nucleus pulposus and endplates versus ECM of annulus fibrosus
The extracellular matrix in the IVD can be divided into the ECM of the nucleus pulposus (NP) and the cartilaginous endplate which contain chondrocyte-like cells, and the ECM of the annulus fibrosus (AF) containing fibroblast-like cells.
The IVDs of nonchondrodystrophoid dogs, in which the notochordal nucleus cells never disappear, have a very low collagen content in their NP but their aggrecan content is very high. As a result, these IVDs remain highly hydrated throughout life. These discs act hydrostatically when loaded. This means that the NP distributes the pressures experienced during daily activities evenly to the adjacent AF and endplates. These IVDs rarely show signs of degeneration such as clefts, fissures, and ruptures in the annulus or endplate (Bray and Burbidge).
In humans, the ECM contains mainly collagens and proteoglycans, which are present in different proportions in the nucleus and in the annulus. The notochordal cells in the NP are replaced by chondrocytes around the age of 10 years but the density of these chondrocytes decreases with age as well. Therefore, the proportion of the proteoglycans and collagens varies considerably with time as well as their position across the IVD. The nucleus has the greatest concentration of proteoglycan and water, but a lower collagen content than other regions in the disc.
The NP has a ECM that mainly consists of the aggregated form of proteoglycan (= aggrecan) and type II collagen in a ratio of 20:1. In human articular cartilage (hips, knees, shoulders, …) the ratio is 2/1. The greatest concentration of collagen (type I) exists in the outer annulus fibrosus.
The collagens provide form and tensile properties while the proteoglycans, through interactions with water and creating of a swelling pressure, give the tissues stiffness, viscoelasticity, and resistance to compression.
6. Proteins in ECM
By 2013, the non-collagenous proteins such as asporin, CILP, elastin, and fibronectin have not been well characterised and their function(s) remain elusive.
7. Structure of a proteoglycan molecule in ECM
A proteoglycan molecule is constructed of a core protein from which radiate clusters of two types of negatively charged and highly sulphated glycosaminoglycan (GAG) chains: long chondroitin sulfate (CS) chains and shorter keratan sulfate (KS) chains. (Figure 7.).
The GAGs in the fetal nucleus pulposus are of chondroitin sulphate chains only reflecting its synthesis by the notochordal cells. The gradual increasing appearance of keratan sulfate during juvenile growth reflects the growing disappearance of the notochordal cells and the appearance of the chondrocyte-like cells.
Fig. 7. Simplified structural depiction of the aggregated aggrecan molecule. The central core protein
(blue line) bounds clusters of up to 100 highly sulphated and negatively charged (-) glycosaminoglycan
(GAGs) side chains [principally chondroitin sulfate (CS1 and CS2) and keratan sulfate (KS)]. At one of its ends the central core protein is connected to hyaluronan (H1 and H2) by a link protein (red circle). In the foetus, the aggrecan molecules only contain chondroitin sulphate chains. The elderly IVD nearly only contains keratan sulfate chains.
8. Variety of proteoglycans in ECM
A variety of proteoglycan molecules form the major component within the ECM in the nucleus. The nucleus pulposus contains the greatest concentration of the largest proteoglycan named aggrecan. Aggrecans are critical to attract, retain and maintain the water content giving the nucleus its main function in responding to the mechanical compression loads. Loss of aggrecans results in a decrease of hydration and of fluid pressure within the nucleus resulting in major changes in the biomechanical functions of the IVD.
Small proteoglycans such as biglycan, decorin, fibromodulin, hyaluronan, lumican, persican, versican are present in the nucleus though in lower concentrations than in the annulus.
In a young adult the proteoglycan molecules constitute 70% of the nucleus content in comparison to the water content that can exceed +/- 90%. In older IVDs, the tissue hydration falls continuously. With increasing age, the annulus becomes stiffer and weaker as its initially lower proteoglycan content decreases as well (Figure 8).
Fig. 8. The nucleus pulposus in the neonates contains a very high concentration of proteoglycans molecules responsible for a high water content (A and B in an IVD of a 1/12 old individual X83-478). In an older IVD (C and D in an 55-year-old individual X89-910) the gel-like character of the nucleus decreases due to the degradation of the proteoglycans. More collagen type I forms in the center of the IVD resulting in a more fibrous tissue and ultimately leading to a dissolution of the distinction between the nucleus, the annulus and the endplates
Original slides (A and C) by Declerck / Kakulas, Neuropathology, Perth, Western Australia
Artistic illustration (B and D) by Colombian Sculptor Alonso Ríos, www.alonsoriosescultor.com
The biochemical breakdown and loss of proteoglycans already starts during childhood so that the concentrations of the proteoglycans decline progressively with increasing age resulting in a concomitant and progressing decrease in water content. The decline arises more rapidly when a traumatic spinal event occurs or when the degenerative processes start in the IVD. Thus, it is evident that losing proteoglycans finally can lead to IVD dysfunction and the potential creation of low back pain.
9. Aggrecan: most important proteoglycan in ECM
Aggrecan is the most important (and abundant) proteoglycan component and is especially present in the ECM of the nucleus of a mature disc. The chondrocyte-like cells synthesize aggrecan molecules, part of which are joined to hyaluronan by a specific link protein to form aggregating proteoglycans (Figure 7). These very large molecules are trapped within the IVD tissue by an extensive network of collagen type II fibers and together they structure the ECM.
Aggrecan is highly hydrophilic, imbibing water with such avidity that it generates a swelling pressure sufficient to force apart the vertebral bodies.
Because of the continuous proteolytic processing of aggrecan in the IVD, the majority of aggrecan molecules exists as non-aggregating proteoglycans (see below: enzymes). The higher and increasing concentration of non-aggregated aggrecans in the nucleus is viewed as a prelude to subsequent IVD aging because the interaction with hyaluronan is no longer possible.
10. Aggregated aggrecan molecules create osmotic environment
The key function of the aggregated proteoglycan molecules is to create an osmotic environment. To balance the fixed negative charges of the sulphated GAGs (= glycosaminoglycans) in the ECM, cations (+) are attracted and anions (-) are repelled. The osmotic pressure arises from all these ions. GAGs (-) attract and retain water molecules which have positive (+) charges on either end. As such the aggregated aggrecan molecules contribute to the swelling pressure in the IVD (Maroudas; Urban) and are responsible for resisting and responding to the compressive loads during all daily activities.
Note: the higher concentration of non-aggregated proteoglycan molecules in the nucleus is viewed as a prelude to subsequent disc aging as the interaction with hyaluronan is not possible.
11. Aggrecan content decreases with age
The aggrecan content (measured by its dry weight) of the nucleus decreases with age as does the hydration, but the collagen content increases (Figure 8). The nucleus eventually no longer acts hydrostatically (McNally). This means that the annulus and endplate are exposed to high point stresses which might lead to the cracks and fissures seen in degenerate discs. These high point stresses are the reasons for acute low back pain attacks (Mulholland).
Having a lower fluid content and because hydraulic permeability increases with aggrecan loss, aging and degenerate discs will lose that fluid more quickly under load. As water is the main component of the IVD, loss of fluid leads to a fall in disc height and abnormal loading of other spinal structures such as the apophyseal joints (Adams).
12. Water content in lumbar IVD
Aging is associated with fragmentation, destruction, and reduction of proteoglycans which then no longer can attract and bind sufficient water molecules. Water content in the nucleus of the IVD declines and the hydrostatic pressure gets lost. This explains why the IVD becomes more and more unable to dissipate spinal forces ultimately resulting in a progressive functional biomechanical failure.
But water content not only depends on the aggrecan concentration but on the external mechanical loads on the IVD as well. Pressure on the IVD arises more from muscular activity than from body weight and thus varies with posture and movement.
In human lumbar IVDs, pressure is lowest when lying prone (at around 0.1–0.2 MPa) and increases five-to-eightfold when standing or sitting (Nachemson). In order to maintain an osmotic equilibrium, fluid is forced out of the disc as pressure increases, but because of the disc's size and low hydraulic permeability, water loss is slow and equilibration takes many hours. The IVD thus rarely achieves osmotic equilibrium.
Around 20 to 25% of the IVDs water is squeezed out due to high loads imposed by muscle tensions during the day's activity. This water is regained during the decrease in load while resting at night (Boos). This cyclical change in fluid content is thought to be responsible for the oscillating length of the spine which is 1 to 2 cm longer in the morning than in the evening (De Puky). Increase of IVD hydration under weightlessness could also account for the 5 cm height gain in space flight (Brown).
13. Other proteoglycans in ECM
Other proteoglycans are present in all regions of the IVD and have structural and functional properties. Some can bind to growth factors and play a role in the development of the nucleus pulposus.
Biglycan, decorin, fibromodulin, and lumican are essential for interacting tightly with the different types of collagen, for assisting in cross-linking of collagen, for the organisation, integrity, and stability of the collagen network.
Biglycan is involved in the interaction with type VI collagen. The presence of increased amounts of biglycan in older and in degenerated discs is the expression of the cells’ inability to proper repair.
Decorin is associated with the surface of type I and type II collagen fibrils.
The degradation fragments of fibromodulin, especially abundant in the annulus, initiate and propagate an inflammatory response locally at the site of disc degeneration.
Versican is important in attaching the annular lamellae to one another. It binds to hyaluronan and will undergo extensive degradation with age.
14. Different collagens in ECM
The ECM of the IVD includes type I, II, III, V, VI, IX, X, X1, XII, and XIV collagen fibrils which form the multiple different and complex networks in the IVD (Table 4). The proteoglycans biglycan, decorin, fibromodulin, and lumican are important in regulating the cross-bridging of the different collagen fibrils and their assemblage into a complex network.
The functions of most of these collagens, which are present in very small amounts in the ECM of the nucleus (type III, V, IX, X, XI, XII, and XIV), remain elusive. However, some of these collagens (type III and IX) are found in very specific locations around the chondrocytes. Nobody knows why.
Type X collagen is found in the ECM of IVDs in the elderly and in scoliotic IVDs.
15. Type I and type II collagen networks
The collagenous networks type I in the annulus and type II in the nucleus account for approximately 80 % of the ECM collagen content.
16. Type I collagen networks of peripheral annulus fibrosus
The fine type I collagen fibers are important in the annulus. On progressing from the outer to the inner annulus, the type I collagen level declines and that of the type II increases.
The annulus fibrosus of a mature lumbar IVD may possess up to 25 lamellar sheets, which have abundant collagen type I fibrils arranged parallel to one another (see Topic ‘IVD – Structural Details’). Because of the exceptional well-oriented deflection of these fibrils in the adjacent lamellae, the annulus has an unique capability for resisting the tensile forces induced by bending and twisting of the lumbar spine.
Note: the layers of collagen networks in the lamellae also contain proteoglycans.
17. Type II collagen networks of central nucleus pulposus
In the mature nucleus pulposus, the coarse type II collagen fibrillar networks are dominant and comprise more than 80 % of all collagens. The fibrils adopt a random orientation. They function as a framework which is interspersed by a proteoglycan-rich interfibrillar ECM responsible for resistance of the IVD to compressive loads during all daily activities.
18. Collagens provide viscoelastic properties to IVD
The viscoelastic behavior of the IVD is especially related to the viscoelastic characteristics of its collagenous fibrillar networks. Decreased or increased cross-bridging of the collagen fibrils will lead to altered mechanical properties of the collagen networks. This then results in an impaired ability of the annulus to resist tensile forces delivered by compression of the nucleus.
19. Collagens provide mechanical stability of ECM
To provide an optimal mechanical stability of the ECM, the dominant collagen fibers type I (in the annulus) and type II (in the nucleus) are cross-bridged by the smaller amounts of type V, X, XI, XII an d XIV collagen fibrils to form a hybrid network.
20. Collagens provide ECM of the nucleus with more fluid consistency
In the nucleus pulposus the type IX collagens, that cross-link with the type II collagens fibers, create a site for interaction with other matrix components providing the more fluid consistency to the ECM of the central part of the IVD.
21. Special function of collagen type VI
Type VI collagen accounts for 10 % to 20 % of the total collagen in the ECM of the IVD. Collagen type VI interacts with aggrecan and other proteoglycans to organise a collagen network which function is to carry load and to reinforce the pericellular ECM.
Type VI collagen is closely associated with NG2-(neural-glial antigen 2)-transmembrane proteoglycan which binds to growth factors in the nucleus pulposus.
22. Collagen type IX
In the mature and intact IVD, collagen type IX is found in the endplate, nucleus, and inner portion of the annulus. Collagen type IX is copolymerised with collagen type II. Because collagen type-IX collagen is located on the surface of the type II fibrils, it is thought to play a role in regulating the fibril diameter and to mediate interactions between the collagen fibril network and noncollagenous tissue proteins (see further).
23. Aging processes affect collagens
During fetal and juvenile growth the synthesis of collagen fibers by the disc cells increases. With increase in age the collagen content increases continuously.
During the aging processes, in the annulus and the inner annulus there is a progressive replacement of the fine type II collagen fibers by an increased deposition of coarse fibers of collagen type I. The fibers become stiffer and stronger as the collagenous networks become excessively cross-linked. It strengthens the annular tissue to tensile loading but limits the swelling properties of the nucleus.
In the long term the collagen fibers become more vulnerable as degenerative proteolytic damage increases (see below: enzymes). This does not necessarily results in collagen loss. Because of the extensive and increasing cross-linking, the damaged collagen molecules remain enclosed in the abundant networks. However, the accumulation of damaged collagens eventually weakens the mechanical strength of the IVD tissue and ultimately will result in tissue loss (Figure 23).
The accumulation of damaged collagens with age is responsible as well for the changes in consistency from a translucent fluid to a soft amorphous tissue. The amassing of damaged collagens is responsible as well for the gradual change in colour from white to the characteristic yellow-brown that occurs in the later stages of IVD degeneration (Figure 23).
Fig. 23. As the IVD ages it becomes stiffer as a result of increases cross-linking of the disc collagens. The accumulation of damaged collagens with age is responsible as well for the changes in consistency from a translucent fluid to a soft amorphous tissue. The amassing of damaged collagens is responsible as well for the gradual change in colour from white to the characteristic yellow-brown that occurs in the later stages of IVD degeneration.
(Declerck / Kakulas, Neuropathology, Perth, Western Australia)
24. Non-collagenous proteins
The ECM of the IVD contains all non-collagenous proteins that are present in the fibrous and cartilaginous structures of the human body. Of particular interest are fibronectin and elastin.
Fibronectin helps to bind the cells to their ECM. Fibronectin and its proteolytic fragments increase in the progressively aging disc and are abundant in degenerated discs.
Elastin is responsible for the ability of a connective tissue to recoil after being stretched. The elastic fibers then run parallel to the collagen fibrils of the annular lamellae. In the nucleus pulposus they are oriented both radially and axially as they need to restore deformation encountered during loading.
Cartilage intermediate layer protein (CILP) binds growth factors in the nucleus.
Cartilage oligomeric matrix protein (COMP) binds heavily to collagen and helps in maintaining the stiffness of the normal disc.
Asporin is associated with the occurrence of degeneration of the IVD
25. Enzymes that build and destroy ECM in the IVD
What are enzymes?
Enzymes (E) catalyze biochemical reactions, speeding up the conversion from substrate (S) to product (P) molecules. The quantitative description of the enzymes is characterized by the Michaelis-Menten equation (E +S ← → ES ← → E + P). An anabolic enzyme can be compared with a cement mixer where cement, sand, and water are mixed to produce concrete (E + S → ES). A catabolic enzyme can be compared with a demolition hammer destroying concrete in various parts (ES → E + P).
Disc cells of both nucleus pulposus and annulus fibrosus not only synthesize the molecular components of their ECM but are able to produce and activate destructive proteolytic enzymes as well. These proteases cleave the non-collagenous proteins, proteoglycans, and collagens at specific sites. In a mature IVD, and under normal circumstances, an equilibrium between production and recycling the different proteins maintains a healthy ECM.
26. Two major classes of degrading enzymes in ECM
Matrix metalloproteinases (MMPs) and proteases of the ADAMs (‘a desintegrin and metalloproteinase’) family are the two major classes of degrading enzymes in the ECM of the IVD. Both are involved in the normal turnover of the ECM molecules. Their catabolic genes are strongly upregulated when the IVD starts aging and during the degenerative processes.
27. Tissue inhibitors of metalloproteinases (TIMPs)
It seems very logical that a potential destructive and catabolic pathway within the ECM of the IVD by the MMPs and ADAMS enzymes cannot proceed without inhibition.
Chondrocytes contain anti-catabolic genes producing tissue inhibitors of MMPs (TIMPs) to counteract the degradative properties of the MMPs and ADAMS (aggrecanases) to prevent loss of the ECM.
In the normal, healthy, and nondegenerated and IVDs, TIMPS and MMPs balance their activities to maintain the structural and functional characteristics of the extracellular matrix.
28. Aging processes affect content of enzymes
With age and IVD degeneration the concentration of the entire group of active degradative proteases increases (MMPs and ADAMs) and the anti-catabolic proteases (TIMPs) decrease. This results in interrupting the healthy balance in favor of the destruction of the ECM-proteins.
29. Decisive factors for more degradative activity
The presence of unfavourable genetic factors, familial inheritance, and traumatic incidents decide upon the time and speed by which the imbalance between constructive and destructive agents in the IVD will occur. Final upregulation of the genes encoding these catabolic enzymes is initiated when the supply of nutrients to the IVD becomes deranged and/or when the loading conditions disturb or exceed the mechanical capacity of the IVD.
None of all these destructive enzymes is introduced in the IVD by diffusion, infiltrating cells or blood vessels through annular tears.
More active degradative activity of the proteases may start at any age. At that moment, the biomechanical weight-bearing functions of the IVD and the capacity of its ECM to remodel following the repetitive normal daily loading patterns will decrease and the hydration of the IVD no longer will be maintained.
30. When normal enzymatic balance shifts …
When the balance shifts to a higher production of proteolytic degradative enzymes, more MMPs and more ADAMs become active. It results in a further diffusion derangement of nutrients, interference with normal disc cell metabolism, increased cell apoptosis, loss of hydration, deleterious changes in the biomechanical properties of the disc, and stimulation of the production of proinflammatory cytokines.
To make it even more complex, the catabolic activation and expression of these proteases remains strongly regulated by the presence of TIMPs (= tissue inhibitors of metalloproteinases), growth factors, and the amount of proinflammatory cytokines.
31. Function of increased levels of enzymes
Increased levels of matrix metalloproteinases (MMPs) and ‘a desintegrin and metalloproteinase’ (ADAMs) are associated with the pathogenesis of disc degeneration (and degenerative disc herniation as well).
The main members of the MMP family are collagenases (MMPs 1, 8, and 13), gelatinases (MMPs 2 and 9), and stromelysin-1 (MMP 3). Each of these different MMPs has its specific matrix substrate so that all extracellular components within the disc (proteoglycans, and collagens, and non-collagenous proteins) can be degraded contributing to the age-changes taking place in the IVD.
Stromelysin-1 (MMP 3) is found mainly in the nucleus pulposus. It is a key enzyme that breaks down the non-collagenous proteins, the core protein and the glycosaminoglycan (GAG) chains of proteoglycans leaving isolated hyaluronan binding sites, degraded proteoglycan aggregates, and glycosaminoglycan fragments as breakdown products.
Collagenases and gelatinases are more prevalent in the annulus and cooperate in the breakdown of collagen. The proteases of the ADAMs family, aggrecanases and versicanases, degrade aggrecan and versican.
32. Growth factors in ECM
The chondrocytes in the IVDs possess genes to produce growth factors. They are especially stimulated to increase their synthesis as a consequence of a spinal injury traumatically rupturing the IVD (cannot be seen on MRI!) and during the IVD degenerating processes.
Growth factors are able to (a) avoid apoptosis (= programmed cell death), (b) stimulate the chondrocytes to increase their proteoglycan (aggrecan) production, (c) inhibit the production of the degradative matrix metalloproteases (MMPs), (d) restore or prevent further destruction of ECM and, (e) cross-bridge collagen fibers into new networks.
Growth factors are bound by cartilage intermediate layer proteins (CILP) but are released if the matrix starts to be degrading.
The anabolic growth genes synthesize bone morphogenetic proteins (BMP-2, and -7), transforming growth factor-β1 (TGF-β1), and insulin-like growth factor-1 (IGF-1).
33. Cytokines in ECM
When the IVD starts degenerating, the chondrocytes not only produce degradative MMP-enzymes but inflammatory and catabolic cytokines as well. The levels of interleukin-1 (IL-1), interleukin-8 (IL-8), and tumor necrosis factor-alfa (TNF-α) increase when degenerative tears appear in the annulus. At the time macrophages start entering the IVD, the concentrations of these inflammatory proteins raise further.
The appearance of such molecules in the ECM is associated directly with the occurrence of all known degenerative phenomena in the IVD: (a) reduced levels of TIMPs, (b) promotion of the production of the degradative enzymes MMPs and ADAMTSs, (c) increased proteolytic breakdown of proteoglycans and collagen type II, (d) decreased synthesis of the proteoglycans, (e) increased synthesis of collagen type I, and (f) excessive apoptotic cell death.
Tumor necrosis factor-alfa (TNF-α) not only degrades the ECM, but induces ingrowth of vessels and sensory nerve fibers in the annulus fibrosus. Once in contact with spinal nerve roots, TNF-α induces serious nerve root damage (fibrosis and even atrophy) as can be seen during spinal surgery in ‘long-standing’ painful CLBP situations.
34. Aging processes weaken ECM
The rate of synthesis of glycosaminoglycans, proteoglycans, link proteins and hyaluronan decreases progressively with age as they undergo continuous proteolytic degradation (MMPs and ADAMs). At the same time, the production of collagen type I increases. The number of cells diminishes as well.
This results in a number of changes. Aggrecan content in the nucleus pulposus drops significantly, and with it the ability of the ECM to attract, bind, and maintain water. The nucleus becomes progressively more fibrous and opaque, and with increased pigmentation. As the collagen content increases and changes from type II to type I, demarcation between the nucleus and annulus becomes less distinct and separation of adjacent annular laminae occurs. This delamination leads to the development of concentric tears in the annulus.
As long as the circumferential annulus and the cartilaginous endplates of the vertebrae remain intact, there is no easy but only a slow route for the aggrecan degradation products to be removed from the IVD by diffusion. Because the non-aggregating proteoglycan fragments remain entrapped in the nucleus but fulfil their function in attracting water, the occurrence of degenerative changes in one or all three structures of the IVD (endplates, nucleus pulposus, annulus fibrosus) only progress very slowly over decades.
35. Degeneration of ECM
Healthy discs express a balance between anabolic (growth factors & TIMPs) and catabolic (MMPs and cytokines) factors.
As the disc ages and degeneration progresses, TIMPs decrease, degradative enzymes MMPs and ADAMS (aggrecanases) become upregulated, anti-inflammatory cytokines such as the growth factors are stimulated, and proinflammatory cytokines (interleukins and tumor necrosis factors) become more active. These important but drastic biochemical changes finally result in a continuously decreasing production of the proteoglycans and collagens in the ECM. The decline in the synthesis of aggrecan and the increase in the concentrations of small proteoglycans become responsible for the disc's lack of reparative capabilities (Figure 35).
The daily mechanical loading of the IVD itself becomes a precipitating catabolic factor in furthering the degenerative processes. Decreased cellularity and altered biochemical matrices (less aggrecans and more collagens type II) affect the mechanical properties of the IVD and result in structural defects leading to microfractures in the endplate. Inflammatory cytokines and degradation products now can diffuse from the IVD and incite a sclerotic reaction in the adjacent vertebral endplates.
Fig. 35. The integrity of the intervertebral disc relies on a healthy balance between synthesis and degradation of the components of the extracellular matrix by the disc cells (especially proteoglycans and collagens type II). While the degrading IVD processes progress, higher concentrations of aberrant molecules appear (destructive proteolytic enzymes such as matrix metalloproteinases, abnormal proteoglycans and collagens, more collagens type I and cytokines) which cause alterations in the structure and function of the matrix. The mechanical properties of this ECM further become impaired because the nucleus of the IVD starts losing more water under load and becomes more fibrous. Moreover, and once the endplates start to sclerose and the transport endplate capillaries obliterate, the healthy balance is adversely influenced by the diminishing nutrient transport (glucose, amino acids, oxygen) and the daily loading patterns (flexion, extension, rotation, etc …). When the viscoelastic characteristics of the collagen networks in the nucleus and the annulus start decreasing, compression and tension forces become difficult to resist. Constructive damage occurs in the nucleus, endplates and the annulus. The endplates rupture and in-to-out fissures appear in the annulus.
36. Painful and painless degenerative IVDs
The aging processes in the IVD are associated with loss of its major proteoglycan: aggrecan. Normal amount of IVD aggrecans is responsible for its neurovascular growth-inhibitory role. Loss of aggrecan implicates the potential of developing pain by an increased sensory nerve and capillary vessel ingrowth in the IVD as occurs during the degenerative processes.
By the age of 40 years, a higher degree of neurovascularisation is seen in the annulus than in the endplate because the more proteoglycan-rich cartilaginous endplate forms a greater barrier for neurovascular ingrowth.
Painful IVDs have increased density of pain-transmitting neurons (nociceptors) at the vertebral endplate and outer annulus because the granulation tissue that is formed to heal matrix damage is invaded by a nociceptive neurovascular bundle.
These nociceptors can be sensitized by cytokines and lactate. Indeed, lactate has a clear nociceptor-stimulatory role (Cavanaugh; Keshari; Naves; McMahon)
Cellular dysfunction of the IVD further triggered by a confluence of stressful environmental inputs, may induce pain (Lotz).
37. Potential innovative molecular therapeutic approach???
In the future, innovative interleukin (IL)-proteins and TNF-α blockers might be developed to stabilize the evolving IVD degeneration and leg pain.
Apoptosis and degradation of IVD tissue might be reduced and even repaired by blocking the catabolic cytokines. Indeed, inhibition of inflammatory cytokines induce the suppression of MMPs and suppression of MMPs results in an increase of type II collagen expression.
Because of the central role of the ECM proteoglycans for the function of the IVD, innovative research is going on to develop bioactive molecules to inhibit the degradative MMPs and cytokines and/or to stimulate the chondrocytic production of growth factors.
The processes of IVD degeneration and the already degraded biochemical matrix may be, one day, stabilized or altered by transferring innovative molecules (anti-catabolics; mitogens; intracellular regulators) directly into the IVD by innovative non-invasive biological treatments (safe gene therapy or direct injection).
Innovative molecules may be able to suppress the altered matrix biochemistry and even to stimulate the remaining capabilities of proteoglycan.
Adams MA, Dolan P, Hutton WC, Porter RW
Diurnal changes in spinal mechanics and their clinical significance
J Bone Joint Surg, 1990, 72B:266
The internal mechanical functioning of intervertebral discs and articular cartilage, and its relevance to matrix biology
Matrix Biol, 2009, 28:384
Adams MA, McNally DS, Dolan P
‘Stress’ distributions inside intervertebral discs. The effects of age and degeneration
J Bone Joint Surg, 1996, 78B:965
What is intervertebral disc degeneration, and what causes it?
Spine, 2006, 31:2151
Adams P, Eyre DR, Muir H
Biochemical aspects of development and ageing of human lumbar intervertebral discs
Rheumatol Rehab, 1977, 16:22
Adams P, Muir H
Qualitative changes with age of proteoglycans of human lumbar discs
Ann Rheum Dis, 1976, 35:289
mRNA expression of cytokines and chemokines in herniated lumbar intervertebral discs
Spine, 2002, 27:911
Variation with age in the pattern of type X collagen expression in normal and scoliotic human intervertebral discs
Calcif Tissue Int, 1998, 63:263
The expression of NG2 proteoglycan in the human intervertebral disc
Spine, 2007, 32:306
An HS, Masuda K
Relevance of in vitro and in vivo models for intervertebral disc degeneration
J Bone Joint Surg, 2006, 88A(Suppl 2):88
Intradiscal administration of osteogenic protein-1 increases intervertebral disc height and proteoglycan content in the nucleus pulposus in normal adolescent rabbits
Spine, 2005, 30:25
Anderson DJ, Izzo MW, Hall DJ, Vaccaro AR, Hilibrand A, Arnold W, Tuan RS, Albert TJ
Comparative gene expression profiling of normal and degenerative discs. Analysis of a rabbit annular laceration model
Spine, 2002, 27:1291
Anderson GD, Li X, Tannoury T, Beck G , Balian G
A fibronectin fragment stimulates intervertebral disc degeneration in vivo
Spine, 2003, 28:2338
Antoniou J, Steffen T, Nelson F, Winterbottom N, Hollander AP, Poole RA, Aebi M, Alini M
The human lumbar intervertebral disc. Evidence for changes in the biosynthesis and denaturation of the extracellular matrix
with growth, maturation, ageing, and degeneration
J Clin Invest, 1996, 98:996
The relationship between apoptosis of endplate chondrocytes and aging and degeneration of the intervertebral disc
Spine, 2001, 26:2414
Bayliss MT, Johnstone B, 0’Brien JP
Proteoglycan synthesis in the human intervertebral disc. Variation with age, region and pathology. 1988 Volvo Award in Basic Science
Spine, 1988, 13:972
Bibby SR, Urban JP
Effect of nutrient deprivation on the viability of intervertebral disc cells
Eur Spine J, 2004, 13:695
Blakey PR, Happey F, Naylor A, Turner RL
Protein in the nucleus pulposus of the intervertebral disk
Nature, 1962, 195:73
Boos N, Wallin A, Gbedegbegnon T, Aebi M, Boesch C
Quantitative MR imaging of lumbar intervertebral disks and vertebral bodies. Influence of diurnal water content variations
Radiology, 1993, 188:351
Classification of age-related changes in lumbar intervertebral discs. 2002 Volvo Award in basic science
Spine, 2002, 27:2631
Bray JP, Burbidge HM
The canine intervertebral disk. Part two: Degenerative changes. Nonchondrodystrophoid versus chondrodystrophoid disks
J Am Animal Hospital Assoc, 1998, 34:135
Crew height measurement. Apollo-Soyuz test project. Medical report
NASA SP411, 1977, 119 (ASTP002)
Brown MD, Tsaltas TT
Studies on the permeability of the intervertebral disc during skeletal maturation
Spine, 1976, 1:240
Brown MF, Hukkanen MV, McCarthy ID, Redfern DR, Batten JJ, Crock HV, Hughes SP, Polak JM
Sensory and sympathetic innervation of the vertebral endplate in patients with degenerative disc disease
J Bone Joint Surg, 1997, 79B:147-153
Aging and degeneration of the human intervertebral disc
Spine, 1995, 20:1307
Spine update. Aging and degeneration of the human intervertebral disc
Spine, 1995, 20:1307
Buckwalter JA, Cooper RR, Maynard JA
Elastic fibers in human intervertebral discs
J Bone Joint Surg, 1976, 58A:73
Buckwalter JA, Einhorn TA, Simon SR
Orthopedic basic science. Biology and biomechanics of the musculoskeletal system
Buckwalter JA, Einhorn TA, Simon SR (eds)
American Academy of Orthopaedic Surgeons, Rosemont, 2nd edn., 2000:548
Buckwalter JA, Pedini-Mille A, Pedrini V,Tudisco C
Proteoglycans of human infant intervertebral disc. Electron microscopic and biochemical studies
J Bone Joint Surg, 1985, 67A:284
Human nucleus pulposis can respond to a pro-inflammatory stimulus
Spine, 2003, 28:2685
Burke JG, Watson RW, McCormack D, Dowling FE, Walsh MG, Fitzpatrick JM
Intervertebral discs which cause low back pain secrete high levels of proinflammatory mediators
J Bone Joint Surg, 2002, 84B:196
Bushell GR, Ghosh P, Taylor TKF, Akeson WH
Proteoglycan chemistry of intervertebral disks
Clin Orthop Relat Res, 1977, 129:115
Biochemistry of intervertebral disc degeneration and the potential for gene therapy applications
Spine J, 2001, 1:205
Mechanisms of low back pain. A neurophysiologic and neuroanatomic study
Clin Orthop Relat Res, 1997, 335:166
Effects of growth differentiation factor-5 on the intervertebral disc. In vitro bovine study and in vivo rabbit disc degeneration model study
Spine, 2006, 31:2909
Variations of the proteoglycans of the canine intervertebral disc with ageing
Biochim Biophys Acta, 1986, 880:209
Crean JK, Roberts S, Jaffray DC, Eisenstein SM, Duance VC
Matrix metalloproteinases in the human intervertebral disc. Role in disc degeneration and scoliosis
Spine, 1997, 22:2877
Cs-Szabo G, Ragasa-San JD, Turumella V, Masuda K, Thonar EJ, An HS
Changes in mRNA and protein levels of proteoglycans of the anulus fibrosus and nucleus pulposus during intervertebral disc degeneration
Spine, 2002, 27:2212
Age-related decrease in susceptibility of human articular cartilage to matrix metalloproteinase-mediated degradation. The role of advanced glycation end products
Arthritis Rheum, 2001, 44:2562
De Puky P
The physiological oscillation of the length of the body
Acta Orthop Scand, 1935, 338
Deyo RA, Tsui-Wu YJ
Descriptive epidemiology of low-back pain and its related medical care in the United States
Spine, 1987, 12:264
Dickson IR, Happey F, Pearson CH, Naylor A, Turner RL
Variations in the protein components of human intervertebral disk with age
Nature, 1967, 215:52
Di Fabio JL, Pearce RH, Caterson B, Hughes H
The heterogeneity of the non-aggregating proteoglycans of the human intervertebral disc
Biochem J, 1987, 244:27
Doege KJ, Sasaki M, Kimura T, Yamada Y
Complete coding sequence and deduced primary structure of the human cartilage large aggregating proteoglycan, aggrecan. Human-specific repeats, and additional alternatively spliced forms
J Biol Chem, 1991, 266:894
Doita M, Kanatani T, Harada T, Mizuno K
Immunohistologic study of the ruptured intervertebral disc of the lumbar spine
Spine, 1996, 21:235
Influence of macrophage infiltration of herniated disc tissue on the production of matrix metalloproteinases leading to disc resorption
Spine, 2001, 26:1522
Donohue PJ, Jahnke MR, Blaha JD, Caterson B
Characterization of link protein(s) from human intervertebral-disc tissues
Biochem J, 1988, 251:739
Dou CL, Levine JM
Inhibition of neurite growth by the NG2 chondroitin sulfate proteoglycan
J Neurosci, 1994, 14:7616-7628
Changes in collagen cross-linking in degenerative disc disease and scoliosis
Spine, 1998, 23:2545
Duffy MJ, Lynn DJ, Lloyd AT, O’Shea CM
The ADAMs family of proteins. From basic studies to potential clinical applications
Thromb Haemost, 2003, 89:622
Tensile properties of nondegenerate human lumbar anulus fibrosus
Spine, 1996, 21:452
Eyre DR, Matsui Y, Wu JJ
Collagen polymorphisms of the intervertebral disc
Biochem Soc Trans, 2002, 30:844
Eyre DR, Muir H
Quantitative analysis of types I and II collagens in human intervertebral discs at various ages
Biochim Biophys Acta, 1977, 492:29
Eyre DR, Muir H
Types I and II collagen in intervertebral disc: interchanging radial distributions in annulus fibrosus
Biochem J, 1976, 157: 267
Recent developments in cartilage research. Matrix biology of the collagen II/IX/XI heterofibril network
Biochem Soc Trans, 2002, 30:893
The biochemistry and physiology of the intervertebral disk
Clin Orthop Relat Res, 1969, 67:16
Extracellular matrix in disc degeneration
J Bone Joint Surg, 2006, 88A(Suppl 2):25
Degeneration of intervertebral discs. Current understanding of cellular and molecular events, and implications for novel therapies
Expert Rev Mol Med, 2001, 3:1
Freemont AJ, Peacock TE, Goupille P, Hoyland JA, O'Brien J, Jayson MI
Nerve ingrowth into diseased intervertebral disc in chronic back pain
The Lancet, 1997, 350:178
Freemont AJ, Watkins A, Le Maitre C, Baird P, Jeziorska M, Knight MT, Ross ER, O'Brien JP, Hoyland JA
Nerve growth factor expression and innervation of the painful intervertebral disc
J Pathol, 2002, 197:286
Interaction between tissue inhibitor of metalloproteinases-2 and progelatinase A. Immunoreactivity analyses
Biochem J, 1996, 313:827
Fujita K, Nakawaga T, Hirabayashi K, Nagai K
Neutral proteinases in human intervertebral disc. Role in degeneration and probable origin
Spine, 1993, 18:1766
Herniation of cervical intervertebral disc. Immunohistochemical examination and measurement of nitric oxide
Spine, 2001, 26:1110
Ghosh P, Bushell GR, Taylor TF, Akeson WH
Collagens, elastin and noncollagenous protein of the intervertebral disk
Clin Orthop Relat Res, 1977, 129:124
Ghosh P, Melrose J, Cole TC, Taylor T
A comparison of the high buoyant density proteoglycans isolated from the intervertebral discs of chondrodystrophoid and non-chondrodystrophoid dogs
Matrix, 1992, 12:148
High-affinity binding of basic fibroblast growth factor and platelet-derived growth factor-AA to the core protein of the NG2 proteoglycan
J Biol Chem, 1999, 274:16831
Immunohistochemical localization of the small proteoglycans decorin and biglycan in human intervertebral discs
Cell Tissue Res, 1997, 289:185
Goupille P, Jayson MIV, Valat JP, Freemont AJ
Spine, 1998, 23: 1612
Gower WE, Pedrini V
Age-related variations in proteinpolysaccharides from human nucleus pulposus, annulus fibrosus, and costal cartilage
J Bone Joint Surg, 1969, 51A:1154
Gruber HE, Hanley EN Jr
Ultrastructure of the human intervertebral disc during aging and degeneration. Comparison of surgical and control specimens
Spine, 2002, 27:798
Gruber HE, Norton HJ, Hanley EN Jr
Anti-apoptotic effects of IGF-1 and PDGF on human intervertebral disc cells in vitro
Spine, 2000, 25:2153
Guiot BH, Fessler RG
Molecular biology of degenerative disc disease
Neurosurgery, 2000, 47:1034
Immunoglobulins and alpha-1-proteinase inhibitor in human intervertebral disc material
Biochem Soc Trans, 1995, 23:212S
Haefeli M, Kalberer F, Saegesser D, Nerlich AG, Boos N, Paesold G
The course of macroscopic degeneration in the human lumbar intervertebral disc
Spine, 2006, 31:1522
The collagen and ground substance of human intervertebral disc at different ages
Acta Chem Scand, 1962, 16:705
Effects of hydrostatic pressure on matrix synthesis and matrix metalloproteinase production in the human lumbar intervertebral disc
Spine, 1997, 22:1085
Hansson TH, Keller TS, Spengler DM
Mechanical behavior of the human lumbar spine. II. Fatigue strength during dynamic compressive loading
J Orthop Res, 1987, 5:479
Hascall VC Proteoglycans. The chondroitin sulfate/keratan sulfate proteoglycan of cartilage
ISI Atlas Sci Biochem, 1988, 1:189
Cleavage of fibromodulin in cartilage explants involves removal of the N-terminal tyrosine sulfate-rich region by proteolysis at a site that is sensitive to matrix metalloproteinase-13
J Biol Chem, 2004, 279:6286
Heinegård D, Aspberg A, Franzén A, Lorenzo P
Glycosylated matrix proteins.
In: Connective tissue and its heritable disorders. Molecular, genetic, and medical aspects
Royce PM, Steinmann B, (eds)
2nd ed, New York, Wiley-Liss, 2002:271
Hickey DS, Hukins DWL
Collagen fibril diameters and elastic fibres in the annulus fibrosus of human fetal intervertebral disc
J Anat, 1981, 133:351
Hildebrand A, Romaris M, Rasmussen LM, Heinegård D, Twardzik DR, Border WA, Ruoslahti E
Interaction of the small interstitial proteoglycans biglycan, decorin and fibromodulin with transforming growth factor beta
Biochem J, 1994, 302(Pt 2):527
Hirsch C, Paulson S, Sylven B, Snellman O
Biophysical and physiological investigations on cartilage and other mesenchymal tissues. VI. Characteristics of human nuclei pulposi during aging
Acta Orthop Scand, 1953, 22:175
Hollander AP, Heathfield TF, Liu JJ, Pidoux I, Roughley PJ, Mort JS, Poole AR
Enhanced denaturation of the a1(II) chains of type-II collagen in normal adult human intervertebral discs compared with femoral articular cartilage
J Orthop Res, 1996, 14:61
Hormel SE, Eyre DR
Collagen in the ageing intervertebral disc: an increase in covalently bound fluorophores and chromophores
Biochim Biophys Acta, 1991, 1078:243
Horner HA, Urban JP
Effect of nutrient supply on the viability of cells from the nucleus pulposus of the intervertebral disc. 2001 Volvo award winner in basic science studies
Spine, 2001, 26:2543
Hoyland JA, Le Maitre C, Freemont AJ
Investigation of the role of IL-1 and TNF in matrix degradation in the intervertebral disc
Rheumatology, 2008, 47:809
Hsieh AH, Lotz JC
Prolonged spinal loading induces matrix metalloproteinase-2 activation in intervertebral discs
Spine, 2003, 28:1781
Disc structure and function
In: The biology of the intervertebral disc. Ghosh P (ed)
CRC Press, Boca Raton, 1988:1
Exogenous tumor necrosis factor-alpha mimics nucleus pulposus-induced neuropathology. Molecular, histologic, and behavioral comparisons in rats. 2000 Volvo Award winner in basic science studies
Spine, 2000, 25:2975
Structure and composition of proteoglycans from human annulus fibrosus
Connect Tissue Res, 1991, 26:47
Localization and distribution of cartilage oligomeric matrix protein in the rat intervertebral disc
Spine, 2006, 31:1539
Histochemical and ultrastructural observations on brown degeneration of human intervertebral disc
J Orthop Res, 1991, 9:78
Effects of low oxygen concentrations and metabolic inhibitors on proteoglycan and protein synthesis rates in the intervertebral disc
J Orthop Res, 1999, 17:829
Degradation of interleukin 1beta by matrix metalloproteinases
J Biol Chem, 1996, 271:14657
Jahnke MR, McDevitt CA
Proteoglycans of the human intervertebral disc. Electrophoretic heterogeneity of the aggregating proteoglycans of the nucleus pulposus
Biochem J, 1988, 251:347
Johnson EF, Chetty K, Moore IM, Stewart A, Jones W
The distribution and arrangement of elastic fibers in the intervertebral disc of the adult human
J Anat, 1982, 135:301
Johnson WE, Caterson B, Eisenstein SM, Hynds DL, Snow DM, Roberts S
Human intervertebral disc aggrecan inhibits nerve growth in vitro
Arthritis Rheum, 2002, 46:2658
Johnson WE, Caterson B, Eisenstein SM, Roberts S
Human intervertebral disc aggrecan inhibits endothelial cell adhesion and cell migration in vitro
Spine, 2005, 30:1139
'Rumours of my death may have been greatly exaggerated'. A brief review of cell death in human intervertebral disc disease and implications for cell transplantation therapy
Biochem Soc Trans, 2007, 35:680
Johnson WEB, Stephan S, Roberts S
The influence of serum, glucose and oxygen on intervertebral disc cell growth in vitro. Implications for degenerative disc disease
Arthritis Res Ther, 2008, 10:R46
Johnstone B, Bayliss MT
The large proteoglycans of the human intervertebral disc. Changes in their biosynthesis and structure with age, topography, and pathology
Spine, 1995, 20:674
Johnstone B, Markopoulos M, Neame P, Caterson B
Identification and characterization of glycanated and non-glycanated forms of biglycan and decorin in the human intervertebral disc
Biochem J, 1993, 292(Pt3):661
Spine, 1995, 20:59
Kääpä E, Holm S, Han X, Takala T, Kovanen, Vanharanta H
Collagens in the injured porcine intervertebral disc
J Orthop Res, 1994, 12:93
Kairemo KJ, Lappalainen AK, Kääpä E, Laitinen OM, Hyytinen T, Karonen Sl, Grönblad M
In vivo detection of intervertebral disk injury using a radiolabeled monoclonal antibody against keratan sulfate
J Nucl Med, 2001, 42:476
Kanemoto M, Hukuda S, Komiya Y, Katsuura A, Nishioka J
Immunohistochemical study of matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1 in
human intervertebral discs.
Spine, 1996, 21:1
Herniated lumbar intervertebral discs spontaneously produce matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2
Spine, 1996, 21:271
Herniated cervical intervertebral discs spontaneously produce matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2
Spine, 1995, 20:2373
Kang JD, Stefanovic-Racic M, McIntyre LA, Georgescu HI, Evans CH
Toward a biochemical understanding of human intervertebral disc degeneration and herniation. Contributions
of nitric oxide, interleukins, prostaglandin E2 and matrix metalloproteinases
Spine, 1997, 22:1065
Post-traumatic findings of the spine after earlier vertebral fracture in young patients. Clinical and MRI study
Spine, 2000, 25:1104
Lactic acid and proteoglycans as metabolic markers for discogenic back pain
Spine, 2008, 33:312
Alterations in biochemical components of extracellular matrix in intervertebral disc herniation: role of MMP-2 and TIMP-2 in type II collagen loss
Cell Biochem Funct, 2006, 24:431
Localization of degradative enzymes and their inhibitors in the degenerate human intervertebral disc
J Pathol, 2004, 204:47
The role of interleukin 1 in the pathogenesis of human intervertebral disc degeneration
Arthritis Res Ther, 2005, 7:R732
Matrix synthesis and degradation in human intervertebral disc degeneration
Biochem Soc Trans, 2007, 35:652
Li X, Leo BM, Beck G, Balian G, Anderson GD
Collagen and proteoglycan abnormalities in the GDF-5-deficient mice and molecular changes when treating disk cells with recombinant growth factor
Spine, 2004, 29:2229
Metaplastic proliferative fibrocartilage as an alternative concept to herniated intervertebral disc
Spine, 1988, 13:1055
Lipson SJ, Muir H
Experimental intervertebral disc degeneration. Morphologic and proteoglycan changes over time
Arthritis Rheum, 1981, 24:12
Lipson SJ, Muir H
Proteoglycans in experimental intervertebral disc degeneration. 1980 Volvo award in basic science
Spine, 1981, 6:194
Identification of human intervertebral disc stromelysin and its involvement in matrix degradation
J Orthop Res, 1991, 9:568
Lotz JC Animal models of intervertebral disc degeneration. Lessons learned
Spine, 2004, 29:2742
Intervertebral disc cell death is dependent on the magnitude and duration of spinal loading
Spine, 2000, 25:1477
Lotz JC, Ulrich JA
Innervation, inflammation, and hypermobility may characterize pathologic disc degeneration: review of animal model data
J Bone Joint Surg, 2006, 88A(Suppl 2):76
Lyons G, Eisenstein SM, Sweet MB
Biochemical changes in intervertebral disc degeneration
Biochim Biophys Acta, 1981, 673:443
Changes with age in proteoglycan synthesis in cells cultured in vitro from the inner and outer rabbit annulus fibrosus. Responses to interleukin-1 and interleukin-1 receptor antagonist protein
Spine, 2000, 25:166
Maroudas A, Evans H
A study of ionic equilibria in cartilage
Conn Tiss Res, 1974, 23, 1:69
Masuda K, Oegema TR Jr, An HS
Growth factors and treatment of intervertebral disc degeneration
Spine, 2004, 29:2757
Metalloproteinases and their inhibitors in matrix remodeling
Trends Gent, 1990, 6:121
Matsui Y, Maeda M, Nakagami W, Iwata H
The involvement of matrix metalloproteinases and inflammation in lumbar disc herniation
Spine, 1998, 23:863
Proteoglycans of the intervertebral disc
In: The biology of the intervertebral disc, Ghosh P (ed)
CRC Press, Boca Raton, Florida, 1988:151
McMahon SB, Cafferty WB
Neurotrophic influences on neuropathic pain
Novartis Found Symp, 2004, 261:68
Biomechanics of the intervertebral disc. Disc pressure measurements and significance
In: Lumbar Spine disorders: Current concepts
Aspden RM, Porter RW (eds)
World Scientific Publishing Co., Singapore, 1995:42
Melrose J, Ghosh P
The noncollagenous proteins of the intervertebral disc
In: The biology of the intervertebral disc, Ghosh P (ed)
CRC Press, Boca Raton, Florida, 1988:189
Melrose J, Ghosh P, Taylor T
A comparative analysis of the differential spatial and temporal distributions of the large (aggrecan, versican)
and small (decorin, biglycan, fibromodulin) proteoglycans of the intervertebral disc
J Anat, 2001,198:3
Melrose J, Ghosh P, Taylor TK, Hall A, Osti OL, Vernon-Roberts B, Fraser RD
A longitudinal study of the matrix changes induced in the intervertebral disc by surgical damage to the annulus fibrosus
J Orthop Res, 1992, 10:665
Melrose J, Roberts S, Smith S, Menage J, Ghosh P
Increased nerve and blood vessel ingrowth associated with proteoglycan depletion in an ovine anular lesion model
experimental disc degeneration
Spine, 2002, 27:1278
Michaelis L, Menten ML
Die Kinetik der Invertinwirkung
Biochem Z, 1913, 49:333
Mikawa Y, Hamagami H, Shikata J, Yamamuro T
Elastin in the human intervertebral disk. A histological and biochemical study comparing it with elastin in the human yellow
Arch Orthop Trauma Surg, 1986, 105:343
Miller JA, Schmatz C, Schultz AB
Lumbar disc degeneration. Correlation with age, sex, and spine level in 600 autopsy specimens
Spine, 1988, 13:173
Mott JD, Werb Z
Regulation of matrix biology by matrix metalloproteinases
Curr Opin Cell Biol, 2004, 16:558
Muir H The chondrocyte, architect of cartilage. Biomechanics, structure, function and molecular biology of cartilage matrix
Bioessays, 1995, 17:1039
The myth of lumbar instability: the importance of abnormal loading as a cause of low back pain
Eur Spine J 2008, 17:619
Mwale F, Demers CN, Petit A, Roughley P, Poole AR, Steffen T, Aebi M, Antoniou J
A synthetic peptide of link protein stimulates the biosynthesis of collagens II, IX and proteoglycan by cells of the intervertebral disc
J Cell Biochem, 2003, 88:1202
Distinction between the extracellular matrix of the nucleus pulposus and hyaline cartilage. A requisite for tissue engineering of intervertebral disc
Eur Cell Mater, 2004, 8:58
Nachemson A, Elfström G
Intravital dynamic pressure measurements in lumbar discs. A study of common movements, maneuvers and exercises
Scand J Rehabil Med Suppl, 1970, 1:1
In vitro diffusion of dye through the end-plates and the annulus fibrosus of human lumbar inter-vertebral discs
Acta Orthop Scand, 1970, 41:589
Nagase H, Woessner JF Jr
J Biol Chem, 1999, 274:21491
Naves LA, McCleskey EW
An acid-sensing ion channel that detects ischemic pain
Braz J Med Biol Res, 2005, 38:1561
Intervertebral disc prolapse and degeneration. The biochemical and biophysical approach
Spine, 1976, 1:108
The biochemical changes in the human intervertebral disc in degeneration and nuclear prolapse
Orthop Clin North Amer, 1971, 2:343
The biophysical and biochemical aspects of intervertebral disc herniation and degeneration
Ann Roy Coll Surg Ed, 1962, 31:91
Naylor A, Happey F, MacRae T
The collagenous changes in the intervertebral disc with age and their effect on elasticity. An X-ray crystallographic study
Brit Med J, 1954, 2:570
Matrix metalloproteinase-3 production by human degenerated intervertebral disc
J Spinal Disord, 1997, 10:493
Expression of fibronectin and TGF-beta1 mRNA and protein suggest altered regulation of extracellular matrix in degenerated disc tissue
Eur Spine J, 2005, 14:17
Immunolocalization of major interstitial collagen types in human lumbar intervertebral discs of various ages
Virchows Arch, 1998, 432:67
Immunohistologic markers for age-related changes of human lumbar intervertebral discs. 1997 Volvo Award winner in basic science studies
Spine, 1997, 22:2781
Biochemistry of the intervertebral disc
Clin Sports Med, 1993, 12:419
Oegema TR Jr, Bradford DS, Cooper KM
Aggregated proteoglycan synthesis in organ cultures of human nucleus pulposus
J Biol Chem, 1979, 254:10579
Oegema TR Jr, Johnson SL, Aguiar DJ, Ogilvie JW
Fibronectin and its fragments increase with degeneration in the human intervertebral disc
Spine, 2000, 25:2742
Ohshima H, Urban JPG
The effect of lactate and pH on proteoglycan and protein synthesis rates in the intervertebral disc
Spine, 1992, 17:1079
Okuda S, Myoui A, Ariga K, Nakase T, Yonenobu K, Yoshikawa H
Mechanisms of age-related decline in insulin-like growth factor-I dependent proteoglycan synthesis in rat
intervertebral disc cells
Spine, 2001, 26:2421
Okuma M, Mochida J, Nishimura K, Sakabe K, Seiki K
Reinsertion of stimulated nucleus pulposus cells retards intervertebral disc degeneration. An in vitro and in vivo experimental study
J Orthop Res, 2000, 18:988
Age-related changes in proteoglycans of human intervertebral discs
Z Rheumatol, 1994, 53:19
Inflammatogenic properties of nucleus pulposus
Spine, 1995, 20:665
Olmarker K, Larsson K
Tumor necrosis factor alpha and nucleus-pulposus-induced nerve root injury
Spine, 1998, 23:2538
Önnerfjord P, Heathfield TF, Heinegård D
Identification of tyrosine sulfation in extracellular leucine-rich repeat proteins using mass spectrometry
J Biol Chem, 2004, 279:26
Autocrine/paracrine mechanism of insulin-like growth factor-1 secretion, and the effect of insulin-like growth factor-1 on proteoglycan synthesis in bovine intervertebral discs
J Orthop Res, 1996,14:690
Regulation of gelatinase-A (MMP-2) production by ovine intervertebral disc nucleus pulposus cells grown in alginate bead culture by Transforming Growth Factor-beta(1)and insulin like growth factor-I
Cell Biol Int, 2001, 25:679
Pearce RH, Grimmer BJ
Target tissue models. The proteoglycans and degeneration of the human intervertebral disc
J Rheumatol Suppl, 1983, 11:108
Pearce RH, Grimmer BJ, Adams ME
Degeneration and the chemical composition of the human lumbar intervertebral disc
J Orthop Res, 1987, 5:198
Pearce RH, Mathieson JM, Mort JS, Roughley PJ
Effect of age on the abundance and fragmentation of link protein of the human intervertebral disc
J Orthop Res, 1989, 7:861
Peng B, Hao J, Hou S, Wu W, Jiang D, Fu X, Yang Y
Possible pathogenesis of painful intervertebral disc degeneration
Spine, 2006, 31:560
Pokharna HK, Phillips FM
Collagen crosslinks in human lumbar intervertebral disc aging
Spine, 1998, 23:1645
Porter S, Clark IM, Kevorkian L, Edwards DR
The ADAMTS metalloproteinases
Biochem J, 2005, 386:15
Razaq S, Wilkins RJ, Urban JP
The effect of extracellular pH on matrix turnover by cells of the bovine nucleus pulposus
Eur Spine J, 2003, 12:341
Evolution of disc degeneration in lumbar spine: a comparative histological study between herniated and postmortem retrieved disc specimens
J Spinal Disord, 1998, 11:41
Risbud MV, Albert TJ, Guttapalli A, Vresilovic EJ, Hillibrand AS, Vaccaro AR, Shapiro IM
Differentiation of mesenchymal stem cells towards a nucleus pulposus-like phenotype in vitro. Implications for cell-based transplantation therapy
Spine, 2004, 29:2627
Risbud MV, Fertala J, Vresilovic EJ, Albert TJ, Shapiro IM
Nucleus pulposus cells upregulate PI3K/Akt and MEK/ERK signaling pathways under hypoxic conditions and resist apoptosis induced by serum withdrawal
Spine, 2005, 30:882
Roberts S, Ayad S, Menage PJ
Immunolocalisation of type VI collagen in the intervertebral disc
Ann Rheum Dis, 1991, 50:787
Matrix metalloproteinases and aggrecanase. Their role in disorders of the human intervertebral disc
Spine, 2000, 25:3005
Roberts S, Evans H, Trivedi J, Menage J
Histology and pathology of the human intervertebral disc
J Bone Jt Surg, 2006, 88A:10
1991 Volvo award in basic sciences. Collagen types around the cells of the intervertebral disc and cartilage end plate. An immunolocalization study
Spine, 1991, 16:1030
Roberts S, Urban JP, Evans H, Eisenstein SM
Transport properties of the human cartilage endplate in relation to its composition and calcification
Spine, 1996, 21:415
Disc morphology in health and disease
Biochem Soc Trans, 2002, 30:864
Biology of intervertebral disc aging and degeneration. Involvement of the extracellular matrix
Spine, 2004, 29:2691
Roughley PJ, White RJ, Magny MC, Liu J, Pearce RH, Mort JS
Non-proteoglycan forms of biglycan increase with age in human articular cartilage
Biochem J, 1993, 295(Pt2):421
Roughley PJ, Melching LI, Heathfield TF, Pearce RH, Mort JS
The structure and degradation of aggrecan in human intervertebral disc
Eur Spine J, 2006, 15(Suppl 3):S326
Salisbury JR, Watt FM
Lack of keratan sulphate in the human notochord
J Anat, 1988, 157:175
Glycosaminoglycan accumulation in primary culture of rabbit intervertebral disc cells
Spine, 2001, 26:2653
Observations on fiber-forming collagens in the anulus fibrosus
Spine, 2000, 25:2736
Scott JE Proteoglycan. Collagen interactions and subfibrillar structure in collagen fibrils. Implications in the development and ageing of connective tissues
J Anat, 1990, 169:23
Scott JE, Bosworth TR, Cribb AM, Taylor JR
The chemical morphology of age-related changes in human intervertebral disc glycosaminoglycans from cervical, thoracic and
lumbar nucleus pulposus and annulus fibrosus
J Anat, 1994, 184:73
Collagenolytic enzyme systems in human intervertebral disc. Their control, mechanism, and their possible role in the initiation of biomechanical failure
Spine, 1982, 7:213
Séguin CA, Bojarski M, Pilliar RM, Roughley PJ, Kandel RA
Differential regulation of matrix degrading enzymes in a TNFalpha-induced model of nucleus pulposus tissue degeneration
Matrix Biol, 2006, 25:409
TNF-alpha induces MMP2 gelatinase activity and MT1-MMP expression in an in vitro model of nucleus pulposus tissue degeneration
Spine, 2008, 33:356
Séguin CA, Pilliar RM, Roughley PJ, Kandel RA
Tumor necrosis factor-alpha modulates matrix production and catabolism in nucleus pulposus tissue
Spine, 2005, 30:1940
A functional SNP in CILP, encoding cartilage intermediate layer protein, is associated with susceptibility to lumbar disc disease
Nat Genet, 2005, 37:607
Interleukin 1, tumor necrosis factor, and interleukin 6 as mediators of cartilage destruction
Semin Arthritis Rheum, 1989, 18:27
Sjöberg A, Onnerfjord P, Mörgelin M, Heinegård D, Blom AM
The extracellular matrix and inflammation: fibromodulin activates the classical pathway of complement by directly binding C1q
J Biol Chem, 2005, 280:32301
Quantitative analysis of gene expression in a rabbit model of intervertebral disc degeneration by real-time polymerase chain reaction
Spine J, 2005, 5:14
Stevens RL, Dondi PG, Muir H
Proteoglycans of the intervertebral disc. Absence of degradation during the isolation of proteoglycans from the intervertebral disc
Biochem J, 1979, 179:573
Sylven B On the biology of nucleus pulposus
Acta Orthop Scand, 1951, 20:275
Sylvén B, Paulson S, Hirsch C, Snellman O
Biophysical and physiological investigations on cartilage and other mesenchymal tissues. II The Ultrastructure of Bovine and Human Nuclei
J Bone Joint Surg, 1951, 33A:333
Sztrolovics R, Alini M, Mort JS, Roughley PJ
Age-related changes in fibromodulin and lumican in human intervertebral discs
Spine, 1999, 24:1765
Sztrolovics R, Alini M, Roughley PJ, Mort JS
Aggrecan degradation in human intervertebral disc and articular cartilage
Biochem J, 1997, 326 ( Pt 1):235
The characterization of versican and its message in human articular cartilage and intervertebral disc
J Orthop Res, 2002, 20:257
Inflammatory cytokines in the herniated disc of the lumbar spine
Spine, 1996, 21:218
Osteogenic protein-1 enhances matrix replenishment by intervertebral disc cells previously exposed to interleukin-1
Spine, 2002, 27:1318
Tang BL ADAMTS. A novel family of extracellular matrix proteases
Int J Biochem Cell Biol, 2001, 33:33
Taylor JR, Scott JE, Cribb AM, Bosworth TR
Human intervertebral disc acid glycosaminoglycans
J Anat, 1992, 180:137
Taylor TKF, Little K
Intercellular matrix of the intervertebral disk in ageing and in prolapse
Nature, 1965, 208:384
Tengblad A, Pearce RH, Grimmer BJ
Demonstration of link protein in proteoglycan aggregate from human intervertebral disc
Biochem J, 1984, 222:85
Thompson JP, Oegema TR Jr, Bradford DS
Stimulation of mature canine intervertebral disc by growth factors
Purification and cloning of aggrecanase-1. A member of the ADAMTS family of proteins
Science, 1999, 284:1664
Tsuji T, Chiba K, Imabayashi H, Fujita Y, Hosogane N, Okada Y, Toyama Y
Age-related changes in expression of tissue inhibitor of metalloproteinases-3 associated with transition from the notochordal nucleus pulposus to the fibrocartilaginous nucleus pulposus in rabbit intervertebral disc
Spine, 2007, 32:849
Repeated disc injury causes persistent inflammation. ISSLS prize winner
Spine, 2007, 32:2812
Nutrition of the intervertebral disk. An in vivo study of solute transport
Clin Orthop Relat Res, 1977,129:101
Urban JPG, Maroudas A
The chemistry of the intervertebral disc in relation to its physiological function and requirements
Clin Rheumat Dis, 1980, 6:51
Urban JPG, Maroudas A
Swelling of the intervertebral disc in vitro
Connect Tissue Res, 1981, 9:1
Urban JP, Maroudas A, Bayliss MT, Dillon J
Swelling pressures of proteoglycans at the concentrations found in cartilaginous tissues
Biorheol, 1979, 16:447
Urban JPG, Roberts S
Degeneration of the intervertebral disc
Arthritis Res Ther, 2003, 5:120
Urban JPG, Roberts S
The intervertebral disc
In: Structure and function of the extracellular matrix, Comper W (ed)
Gordon and Breach, Reading, UK, 1997:203
Urban JPG, Roberts S, Ralphs JR
The nucleus of the intervertebral disc from development to degeneration
Am Zool, 2000, 40:53
Urban JPG, Smith S, Fairbank JC
Nutrition of the intervertebral disc
Spine, 2004, 29:2700
Verzijl N, DeGroot J, Oldehinkel E, Bank RA, Thorpe SR, Baynes JW, Bayloss MT, Bijlsma JW, Lafeber FP, Tekoppele JM
Age-related accumulation of Maillard reaction products in human articular cartilage collagen
Biochem J, 2000, 350:381
Verzijl N, DeGroot J, Ben ZC, Brau-Benjamin O, Maroudas A, Bank RA, Mizrahi J, Schalkwijk CG, Thorpe SR, Baynes JW, Bijlsma JW,
Lafeber FP, TeKoppele JM
Crosslinking by advanced glycation end products increases the stiffness of the collagen network in human articular cartilage: a possible mechanism through which age is a risk factor for osteoarthritis
Arthritis Rheum, 2002, 46:114
Visse R, Nagase H
Matrix metalloproteinases and tissue inhibitors of metalloproteinases. Structure, function, and biochemistry
Circ Res, 2003, 92:827
Walker MH, Anderson DG
Molecular basis of intervertebral disc degeneration
Spine J, 2004, 4(Suppl 6):158S
Gene therapy applications for intervertebral disc degeneration
Spine, 2003, 28(15 suppl):S93
Gene transfer of the catabolic inhibitor TIMP-1 increases measured proteoglycans in cells from degenerated human intervertebral discs
Spine, 2003, 28:2331
Expression and distribution of tumor necrosis factor alpha in human lumbar intervertebral discs. A study in surgical specimen and autopsy controls
Spine, 2005, 30:44
2002 SSE Award competition in basic science. Expression of major matrix metalloproteinases is associated with intervertebral disc degradation and resorption
Eur Spine J, 2002, 11:308
Wiberg C, Klatt AR, Wagener R, Paulsson M, Bateman JF, Heinegärd D, Mörgelin M
Complexes of matrilin-1 and biglycan or decorin connect collagen VI microfibrils to both collagen II and
J Biol Chem, 2003, 278:37698
Wu JJ, Eyre DR
Intervertebral disc collagen. Usage of the short form of the a1(IX) chain in bovine nucleus pulposus
J Biol Chem, 2003, 278:24521
Wu JJ, Lark MW, Chun LE, Ayre DR
Sites of stromelysin cleavage in collagen types II, IX, X, and XI of cartilage
J Biol Chem, 1991, 266:5625
Enzyme kinetics, past en present
Science, 2013, 342:1457
The histology of lumbar intervertebral disc herniation. The significance of small blood vessels in the extruded tissue
Spine, 1993, 18:1761
Yoon ST, Kim KS, Li J, Park JS, Tomoyuki A, William AE, Hutton WC
The effect of bone morphogenetic protein-2 on rat intervertebral disc cells in vitro
Spine, 2003, 28:1773
Yoon ST, Patel NM
Molecular therapy of the intervertebral disc
Eur Spine J, 2006, 15(Suppl 3):S379
Yu J Elastic tissues of the intervertebral disc
Biochem Soc Trans, 2002, 30:848
Yu J, Fairbank JCT, Roberts S, Urban JPG
The elastic fiber network of the anulus fibrosus of the normal and scoliotic human intervertebral disc
Spine, 2005, 30:1815
Elastic fibre organization in the intervertebral discs of the bovine tail
J Anat, 2002, 201:465