LUMBAR INTERVERTEBRAL DISC (IVD).

EXTRACELLULAR MATRIX (ECM)

International Publicized Data

GMCD Instructional  Course Lectures

Author :  

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)

www.guy-declerck.com

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’.

J

Acknowledgements

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

Contents

1. The extracellular matrix (ECM) in the avascular IVD

2. Synthesis and breakdown of ECM by IVD cells

3. Mechanobiologic importance of ECM

4. Elaborate framework of ECM macromolecules

5. ECM of nucleus pulposus and endplates versus ECM of annulus fibrosus

6. The proteins in the ECM

7. Structure of a proteoglycan molecule in the ECM

8. A variety of proteoglycans in ECM

9. Aggrecan: most important proteoglycan in the ECM

10. Aggregated aggrecan molecules create an osmotic environment

11. Aggrecan content decreases with age

12. Water content in the lumbar IVD

13. The other proteoglycans in the ECM

14. Different collagens in the ECM

15.Type I and type II collagen networks

16. Type I collagen networks of the peripheral annulus fibrosus

17. Type II collagen networks of the central nucleus pulposus

18. Collagens provide viscoelastic properties to the IVD

19. Collagens provide mechanical stability of the ECM

20. Collagens provide the ECM of the nucleus a more fluid consistency

21. Special function of collagen type VI

22. Collagen type IX

23. Aging processes affect the collagens

24. Non-collagenous proteins

25. Enzymes that build and destroy the ECM in the IVD

26. Two major classes of degrading enzymes in the ECM

27. Tissue inhibitors of metalloproteinases (TIMPs)

28. Aging processes affect the content of enzymes

29. Decisive factors for more degradative activity

30. When the normal enzymatic balance shifts …

31. Function of increased levels of enzymes

32. Growth factors in the ECM

33. Cytokines in the ECM

34. Aging processes weaken the ECM

35. Degeneration of the ECM

36. Painful and painless degenerative IVDs

37. Potential for innovative molecular therapeutic approach

38. Literature Encyclopaedia

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).

Proteins

Proteoglycans

Collagens

Proteinases

asporin

aggrecan

type I (0 - 80 %)

metalloproteinases (MMPs)

CILP

biglycan

type II (0 - 80 %)

. collagenases (MMPs 1, 8, 13)

elastin

decorin

type III (< 5 %)

. gelatinases (MMPs 2, 9)

fibronectin

fibromodulin

type V (1 - 2 %)

. stromelysin-1 (MMP 3)

hyaluronan

type VI (10 - 20 %)

ADAMS (aggrecanases)

lumican

type IX (1 - 2 %)

perlecan

type X (1 - 2 %)

TIMPs

versican

type XI (1 - 2 %)

Growth factors

type XII (< 1 %)

type XIV (< 1 %)

Cytokines

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.

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