The Journal of Bone & Joint Surgery

The Journal of Bone & Joint Surgery, Volume 88, Issue suppl 2

Workshop Articles | April 01, 2006

Extracellular Matrix in Disc Degeneration

Haoyu Feng, MD; Mikael Danfelter, MSc; Björn Strömqvist, MD, PhD; Dick Heinegård, MD, PhD

The authors did not receive grants or outside funding in support of their research for or preparation of this manuscript. They did not receive payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, educational institution, or other charitable or nonprofit organization with which the authors are affiliated or associated.

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J Bone Joint Surg Am, 2006 Apr 01;88(suppl 2):25-29. doi: 10.2106/JBJS.E.01341

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Abstract

The extracellular matrix of the intervertebral disc structures contains many molecules also found in cartilage. The extremely polyanionic proteoglycans play a central role, particularly in the nucleus, by creating an osmotic environment leading to retention of water and ensuing resistance to deformation—important for the resilience of the tissue.

Another major structural entity particularly important in the anulus is the network of collagen fibers; fibril-forming collagen 1 is a major constituent. The collagen fibrils in the anulus are largely oriented in sheets around the nucleus. A number of molecules present in the matrix regulate and direct the collagen fibril assembly by interacting with the collagen molecule and also the formed fibril. Several of these molecules bind by one domain to the collagen fiber and present another functional domain to interact either with other fibers or with other matrix constituents. In this manner the collagen fibers are cross-linked into a network that provides tensile strength and distributes load over large parts of the anulus. Diminished function in these cross-bridging molecules will lead to loss of mechanical properties of the collagen network and result in an impaired ability of the anulus to resist forces delivered by compression of the disc and particularly the nucleus.

A different network abundant in the disc and in other load-bearing tissues is based on the beaded filaments of collagen 6. The basic building block is a tetramer of two pairs of antiparallel collagen-6 molecules arranged such that two N-terminal ends of collagen 6 are exposed at either end of the unit. Further assembly occurs both by end-to-end and side-to-side associations. This process is catalyzed by both biglycan and decorin, where the combined effect of direct binding of the core protein to the collagen-6 N-terminal globular domain and the presence of the glycosaminoglycan side chain is essential. These ligands are bound at the same site in complexes extracted from the tissue and then also have one bound molecule of matrilin-1, 2, or 3, in turn bound to a collagen fiber, a procollagen molecule, or an aggrecan.

Interactions at the cell surface provide signals to the cells with regard to the conditions of the matrix. Such interactions include binding by matrix components to various receptors at the cell surface.

Remodeling of the matrix takes place in response to various factors. An early event in disease is degradation of aggrecan by the members of the ADAMTS (a disintegrin-like and metalloprotease with thrombospondin motifs) family and degradation of molecules important in maintaining the collagen network.

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Although the extracellular matrix of the intervertebral disc structures contains many molecules also found in cartilage, distinct differences exist as well. An example is the presence of collagen type I in the anulus fibrosus. The arrangement of the collagen in the anulus is quite distinct from that in cartilage. It is laid down in the form of sheets parallel to the circumference of the disc. This arrangement requires a distinctly different regulation of collagen fiber formation and organization. A number of molecules have roles in regulating collagen fibril assembly, notably a number of matrix proteins/proteoglycans of the leucine-rich repeat (LRR) protein family. Their functions extend to adding stability to the collagen fibrils and their organization into networks.

Molecular Constituents of the Extracellular Matrix

The major constituents in the tissues of the intervertebral disc are the proteoglycan aggrecan, which provides the swelling pressure important for resistance to compression and water flow, and the highly organized networks of fibrillar collagen that provide the tensile properties. The networks of collagen are based on the fibril-forming collagens type I and II on the one hand and collagen type VI on the other¹.

Some other components of the intervertebral disc structures are shown in Figure 1, representing two-dimensional separations of extracts of noncollagenous proteins from the tissues and identification of proteins in spots by mass spectrometry. It is apparent that some major proteins present are actually derived from blood, but in a rather selective manner. Others are typically found in most cartilaginous tissues. Because most studies on the functions and properties of these molecules have been performed using cartilage-derived as well as recombinant proteins, the following description primarily relates to findings associated with the molecules from these sources. A schematic illustration of the proteins and their associations into and with larger structures is shown in Figure 2.

Aggrecan

The proteoglycan aggrecan is particularly enriched in the nucleus pulposus. It is comprised of approximately 100 chondroitin sulfate chains, each containing polysaccharide chains with some 100 negatively charged groups. These polysaccharides are built from some fifty repeat disaccharides that contain one carboxyl and one sulfate negatively charged group. The additional approximately thirty keratan sulfate chains are primarily clustered within a distinct region next to that carrying the chondroitin sulfate chains². We have now demonstrated that these keratan sulfate chains are longer in the intervertebral disc than in cartilage, although considerably shorter than in chondroitin sulfate. The polysaccharide chains are covalently bound via their reducing terminus to the large protein core, and they are grouped in clusters². The core protein has a globular domain in its N-terminal that binds specifically to hyaluronan. Thus, hundreds of aggrecan molecules are aggregated via their binding to hyaluronan. In addition, the C-terminal contains a globular domain with a lectin homology with the capacity to bind to fibulins and tenascins²; thus, the aggrecan aggregates can network to other assemblies in the matrix by way of ligands at the C-terminal.

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Fig. 1: Patterns of proteins extracted from normal human tissues after two-dimensional gel-electrophoretic separation (isoelectric focusing and SDS-PAGE). Numbers identify those spots that could be identified after trypsin digestion and mass spectrometry.

Compared with cartilage, the nucleus pulposus contains a larger amount of aggrecan; however, the molecules appear more degraded and a higher proportion is not bound to the hyaluronan³.

One function of the network of proteoglycan aggregates is to provide a high level of fixed charge density, which creates an osmotic environment important for retaining water and restricting water flux. The concentration of aggrecan will influence how the tissue reacts to loads delivered at different rates; a higher rate of loading will result in a stiffer tissue.

Major Collagen Fiber Network

The other major structural entity that is particularly important in the anulus is the network of type-I collagen fibers. The collagen fibrils in the anulus are largely oriented in sheets around the nucleus. Their key functions are retaining the nucleus and taking up and distributing the load exerted by this tissue during various types of exercise. In order to provide optimal mechanical stability, the collagen fibers are cross-bridged by a number of molecules bound at their surface. These include collagen type IX, which is largely covalently cross-linked to collagen type-II fibers in cartilage, for example, and in the nucleus and the inner anulus⁴. This collagen contains three collagenous, triple helical domains interrupted and capped by noncollagenous domains. The arrangement is such that the noncollagenous domains interact with the surrounding structures.

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Fig. 2: Schematic illustration of assemblies of matrix proteins into structures in the intervertebral disc. COMP = cartilage oligomeric matrix protein, CS = chondroitin sulfate, HA = hyaluronan, HS-PG = heparan sulfate proteoglycan, KS = keratan sulfate, MAT = matrilin, PRELP = proline arginine-rich end leucine-rich repeat protein.

Leucine-Rich Repeat (LRR) Proteins and Proteoglycans

Other molecules that tightly interact at the collagen surface include fibromodulin, decorin, and lumican². All of these molecules have a central portion consisting of ten or eleven repeats of each (on the average) twenty-five amino acids and have leucine residues at conserved locations. This core region is capped by two disulfide loop structures. These three proteoglycans contain an N-terminal extension, which, in the case of fibromodulin and lumican, carries a number of tyrosine residues that have been posttranslationally modified by sulfation, providing for an anionic domain with special character and function⁵. In most species, these LRR proteins also contain one or two keratan sulfate chains bound to the repeat domain. In the case of decorin, the N-terminal extension contains one very long chain of chondroitin-dermatan sulfate. This polysaccharide can directly interact with collagen and has been suggested to interact with another chain in an antiparallel fashion⁶. Thus, by combining binding to collagen via its protein core with binding either to another collagen fiber directly via the glycosaminoglycan chains or via self-interactions, decorin is able to cross-bridge and cross-link collagen fibers².

Another function of these LRR proteins is the regulation of collagen-fiber assembly. Although the exact role in vivo of the individual regulator is not known, many of the LRR proteins inhibit collagen fibrillogenesis in vitro. Furthermore, mice in which these proteins have been inactivated often contain larger and more irregular collagen fibers². However, in the case of the fibromodulin-null mouse, the collagen fibers are thinner than in its wild-type counterpart². At the same time, the closely related lumican is present in larger amounts, apparently due to a lack of replacement of this molecule during tissue development by fibromodulin that binds to the same collagen site². This can be interpreted to indicate coordinated roles of the two proteins at different stages of collagen fibrillogenesis.

Other interesting properties of these LRR proteins include binding of growth factors, particularly transforming growth factor-ß⁷, that are important in cartilage development.

A distinctly different LRR protein is chondroadherin, a protein without carbohydrate substituents and without an N-terminal extension². This protein, like most of the other LRR proteins, binds tightly to collagen at two sites². It has other interactions, most notably to the a₂ß₁ integrin². This interaction leads to the production of matrix constituents but not to cell migration or division. Chondroadherin may thus have a role in maintaining the chondrocyte phenotype.

Cartilage Oligomeric Matrix Protein

Cartilage oligomeric matrix protein (COMP) is a protein that is abundant in cartilage. It contains five identical subunits held together by a coiled-coil domain in the N-terminal portion and exposing unique C-terminal globular domains with roles such as collagen binding. This binding is of very high affinity (K_D 10^-9); there are four sites with identical affinity spaced along the collagen triple helical molecule such that there is one at each end and two evenly spaced in between². However, a given COMP molecule can only bind to one of these sites because it cannot be stretched to span the distance between two such binding areas. Therefore, the other four binding domains are available for interactions with other molecules. In studies of early collagen fibrillogenesis in vitro, COMP molecules were shown to almost instantaneously organize the collagen molecules closely spaced in parallel. This results in a much more rapid and efficient collagen fibril assembly, with the end result being a more well-defined fiber compared with when fibrillogenesis takes place without COMP present⁸. Thus, COMP plays a role in regulating collagen fibril assembly, consistent with its location close to the cells in the growing individual. The molecule also functions to maintain collagen network stability by binding to each of the globular domains of collagen type IX (Fig. 2)².

Collagen-6 Beaded Filament Network

A different fibrillar network is made up of beaded filaments of collagen type VI. This collagen is particularly abundant in the intervertebral disc, but also enriched in other tissues carrying load¹. The molecule is a trimer of three different a-chains, and has a central triple helical domain capped with a small C-terminal globular domain and a considerably larger N-terminal domain, both containing von Willebrand factor-A repeats. Within the cell, four such molecules are arranged pairwise in an antiparallel fashion such that the filamentous complex is capped by two of the N-terminal domains on each end. On secretion, these end domains will provide the basis for both end-to-end and side-to-side interactions, leading to the formation of the filamentous, beaded collagen type-VI network². Studies in vitro demonstrate that the assembly process is efficiently guided, most prominently by biglycan but also by decorin. These LRR proteoglycans bind via their protein core to only the N-terminal domain of the collagen type-VI molecule. However, the presence of the chondroitin-dermatan sulfate side chains on the proteoglycan is essential for fibril assembly; when these are removed, formation of a proper network does not occur².

In collagen-6 networks isolated from cartilaginous tissue, the biglycan/decorin molecules were still found at the N-terminal domains of the collagen type-VI molecule. They show an additional interaction in that they were also bound to a molecule of matrilin-1, 2, or 3⁹. This complex of the LRR proteoglycans and a matrilin actually was shown to represent a linker module, connecting the collagen type-VI network to other matrix constituents, such as aggrecan, collagen fibrils, and procollagen molecules⁹. Thus, it is possible that collagen type VI has a role in organizing the matrix closest to the cells (i.e., the territorial matrix).

Other Matrix Macromolecules

One protein present in the disc is cartilage intermediate layer protein (CILP), originally identified in articular cartilage and upregulated in early osteoarthritis¹⁰. This protein has been linked to an increased incidence of lumbar disc disease¹¹. Although the protein has been shown to contain a heparin-binding motif, its function is unclear. The protein is made as a larger precursor for two distinct proteins, where the N-terminal forms the CILP itself while the C-terminal portion shows a homology with another protein, NTPPHase².

Interactions at the cell surface between matrix constituents and receptors are likely to provide important feedback to the cells, which thereby obtain signals to modulate any response to degradation and/or repair. One such cell-surface receptor, referred to as CD 44, has the ability to bind hyaluronan. Implicated in cartilage turnover is the discoidin domain receptor (DDR) family, particularly discoidin domain receptor 2 (DDR2)¹². Many integrins are found among the cell-surface proteins that bind to matrix constituents, where the integrins differ in their specificity for distinct matrix macromolecules. Another family is represented by the heparan sulfate proteoglycans in the form of syndecans, which provide signals to the inside of the cell.

The classical cell-binding molecule fibronectin indeed has both binding sites for integrins, including the classical a₅ß₁ integrin with specificity for the Arg-Gly-Asp sequence, and for the heparan sulfate chains of syndecan in the form of two heparin-binding domains¹³. Interestingly, a fragment containing either of the heparin-binding domains will stimulate chondrocytes as well as fibroblasts to matrix breakdown via proteolytic cleavage¹⁴. This is one mechanism through which fragments of matrix constituents may play a role in propagating a tissue-destruction process.

Matrix Components in Pathology

Fibromodulin is implicated in several roles in disease development. On interleukin-1 stimulation of cartilage in explant culture, a specific cleavage in the N-terminal extension is induced, leading to loss of the tyrosine sulfate-rich domain¹⁵. This process separates this anionic functional unit that is involved particularly in ionic interactions from the other functional unit in the form of the collagen-binding domain, with the possible consequence that the integrity of the collagen network is at risk and its mechanical stability will decrease². This event, coupled with cleavage of the other molecules at the collagen fiber surface, precludes the cross-linking that is essential for the collagen network properties. It will likely lead to a loss of function, particularly in the anulus where an outcome could be herniation when pressure is exerted by the nucleus pulposus.

One consequence of fragmentation and release of fibromodulin is a potential role in an inflammatory response. Sjöberg et al.¹⁶ recently demonstrated that fibromodulin bound at a surface will tightly bind C1q via its head domains. The bound C1q will then bind and activate C4 and C3 such that deposition of C4b and C3b is observed¹⁶. This represents activation of the classical pathway of complement. Although the precise domain of fibromodulin interacting with the C1q has not been defined, it is possible that fragments may actually initiate and propagate an inflammatory response locally at the site of disc degeneration. This may be a factor in local pain and irritation at nerve roots.

It is indeed possible that proteolytic fragmentation of key elements that serve to maintain matrix integrity will result in a release of fragments with a capacity to bind to cell surface receptors such as integrins with an ability to modulate activities of the cell (such as production of proteases, accomplishing further degradation as well as repair responses). Furthermore, fragments may be created that induce or inhibit the inflammatory response. Inflammation, if present, will lead to release of cytokines, which can induce cells in the intervertebral disc to further degrade the matrix and impair any repair response. Thus, the components of a vicious circle are potentially at hand. Intervention could be aimed at inhibiting active proteases or blocking the effects of cytokines.

Conclusions

The identification and characterization of matrix constituents in the intervertebral disc structures as well as cartilage have been expanded to also include identification of specific functions. Studies are under way to describe matrix constituents as targets in matrix breakdown. Studies of the mechanisms of this breakdown will provide information on the proteases involved in the process, creating targets for therapeutic intervention. Recent studies indicate that released fragments may promote inflammation and thereby further enhance the destructive process.

Further studies of constituents of the intervertebral disc and their alterations in disease are likely to provide important new information with both diagnostic and therapeutic applications. ?

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Topics

drug interactions ; extracellular matrix ; cartilage ; collagen ; intervertebral disc ; leucine ; proteoglycan ; collagen fiber ; degenerative disc disease ; aggrecan

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Haoyu Feng, Mikael Danfelter, Björn Strömqvist, Dick Heinegård; Extracellular Matrix in Disc Degeneration. The Journal of Bone & Joint Surgery. 2006 Apr;88(suppl_2):25-29.

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