Study design A review of current knowledge and opinions concerning the biologic changes that take place during development, maturation and degeneration of the intervertebral disc. Objective To provide an overview of the changes that occur in structure and composition of the extracellular matrix of the intervertebral disc and to explain the origin of such changes and their functional consequences. Summary of background data The structure of the intervertebral disc, and, in particular, the composition of its extracellular matrix, changes throughout life, ultimately resulting in tissue degeneration in the adult. Methods A review of the published scientific literature. Results In the young disc, the outer anulus fibrosus and inner nucleus pulposus have clear physical and molecular properties, although these differences become less distinct in the adult. The age changes are due to variations in both the abundance and structure of the macromolecules, particularly aggrecan, and the structural variations may be due to changes in both synthesis and degradation. It is not clear how many of the changes are by design to adapt to the altered environment of the growing spine. However, it is commonly thought that the degradative changes are detrimental to disc function, a property that is exacerbated by the inability of the mature avascular disc to remove and replace accumulated degradation products. The rate at which these detrimental changes occur may vary between individuals because of genetic, biomechanical, and nutritional differences. Such changes are thought to form the basis of tissue loss associated with disc degeneration. Conclusion Changes in intervertebral disc structure throughout life ultimately result in tissue degeneration and the need for medical intervention. Current research is aimed at trying to restore the integrity of the degenerate disc matrix by biologic means, although at present it is not clear what the structure of the most appropriate repair tissue should be or how it can be achieved.

Biology of intervertebral disc aging and degeneration: involvement of the extracellular matrix.

The messenger RNAs and core proteins of the two small chondroitin/dermatan sulfate proteoglycans, biglycan and decorin, were localized in developing human bone and other tissues by both 35S-labeled RNA probes and antibodies directed against synthetic peptides corresponding to nonhomologous regions of the two core proteins. Biglycan and decorin expression and localization were substantially divergent and sometimes mutually exclusive. In developing bones, spatially restricted patterns of gene expression and/or matrix localization of the two proteoglycans were identified in articular regions, epiphyseal cartilage, vascular canals, subperichondral regions, and periosteum, and indicated the association of each molecule with specific developmental events at specific sites. Study of non-skeletal tissues revealed that decorin was associated with all major type I (and type II) collagen-rich connective tissues. Conversely, biglycan was expressed and localized in a range of specialized cell types, including connective tissue (skeletal myofibers, endothelial cells) and epithelial cells (differentiating keratinocytes, renal tubular epithelia). Biglycan core protein was localized at the cell surface of certain cell types (e.g., keratinocytes). Whereas the distribution of decorin was consistent with matrix-centered functions, possibly related to regulation of growth of collagen fibers, the distribution of biglycan pointed to other function(s), perhaps related to cell regulation.

Expression and localization of the two small proteoglycans biglycan and decorin in developing human skeletal and non-skeletal tissues.

The focus of this review is on conceptual and functional advances in our understanding of the small leucine-rich proteoglycans. These molecules belong to an expanding gene class whose distinctive feature is a structural motif, called the leucine-rich repeat, found in an increasing number of intracellular and extracellular proteins with diverse biological attributes. Three-dimensional modeling of their prototype protein core proposes a flexible, arch-shaped binding surface suitable for strong and distinctive interactions with ligand proteins. Changes in the properties of individual proteoglycans derive from amino acid substitutions in the less conserved surface residues, changes in the number and length of the leucine-rich repeats, and/or variation in glycosylation. These proteoglycans are tissue organizers, orienting and ordering collagen fibrils during ontogeny and in pathological processes such as wound healing, tissue repair, and tumor stroma formation. These properties are rooted in their bifunctional character: the protein moiety binding collagen fibrils at strategic loci, the microscopic gaps between staggered fibrils, and the highly charged glycosaminoglycans extending out to regulate interfibrillar distances and thereby establishing the exact topology of fibrillar collagens in tissues. These proteoglycans also interact with soluble growth factors, modulate their functional activity, and bind to cell surface receptors. The latter interaction affects cell cycle progression in a variety of cellular systems and could explain the purported changes in the expression of these gene products around the invasive neoplastic cells and in regenerating tissues.

The family of the small leucine-rich proteoglycans: key regulators of matrix assembly and cellular growth.

Leucine-rich repeat glycoproteins of the extracellular matrix

Fibromodulin-null mice have abnormal collagen fibrils, tissue organization, and altered lumican deposition in tendon.

Polyclonal anti-peptide antibodies were raised to the C-terminal regions of human biglycan and decorin. These antibodies were used in immunoblotting to study structural variations with age in the proteoglycan core proteins present in extracts of human articular cartilage and intervertebral disc. Three forms of the biglycan core protein were identified. The largest form was detected only after chondroitinase treatment and represents the proteoglycan form of the molecule from which the glycosaminoglycan chains have been removed. However, chondroitinase treatment did not alter the electrophoretic mobility of the two smaller proteins, which appear to represent non-proteoglycan forms of the molecule, resulting either from a failure to substitute the intact proteoglycan core protein with glycosaminoglycan chains during its synthesis or from proteolytic processing of the intact proteoglycan causing removal of the N-terminal region bearing the glycosaminoglycan chains. The non-proteoglycan forms constituted a minor proportion of biglycan in the newborn, but were the major components in the adult. A similar trend was seen in both articular cartilage and intervertebral disc. In comparison, decorin appears to exist predominantly as a proteoglycan at all ages, with two core protein sizes being present after chondroitinase treatment. Non-proteoglycan forms were detected in the adult, but they were always a minor constituent.

Non-proteoglycan forms of biglycan increase with age in human articular cartilage

Dermatan sulphate proteoglycans were purified from juvenile human articular cartilage, with a yield of about 2 mg/g wet wt. of cartilage. Both dermatan sulphate proteoglycan I (DS-PGI) and dermatan sulphate proteoglycan II (DS-PGII) were identified and the former was present in greater abundance. The two proteoglycans could not be resolved by agarose/polyacrylamide-gel electrophoresis, but could be resolved by SDS/polyacrylamide-gel electrophoresis, which indicated average Mr values of 200,000 and 98,000 for DS-PGI and DS-PGII respectively. After digestion with chondroitin ABC lyase the Mr values of the core proteins were 44,000 for DS-PGI and 43,000 and 47,000 for DS-PGII, with the smaller core protein being predominant in DS-PGII. Sequence analysis of the N-terminal 20 amino acid residues reveals the presence of a single site for the potential substitution of dermatan sulphate at residue 4 of DS-PGII and two such sites at residues 5 and 10 for DS-PGI.

Dermatan sulphate proteoglycans of human articular cartilage. The properties of dermatan sulphate proteoglycans I and II

The mechanisms of aggrecan degradation in adult human articular, adult bovine nasal and fetal bovine epiphyseal cartilage in response to either interleukin-1beta (IL-1beta) or retinoic acid were compared using an explant culture system. Bovine nasal cartilage cultured with either IL-1beta or retinoic acid exhibited significant release of glycosaminoglycan (GAG). For both factors, aggrecan proteolysis occurred predominantly at the 'aggrecanase' site, with no evidence for the action of matrix metalloproteinases, and resulted in the appearance of the corresponding G1 fragment in tissue extracts and in culture media. In human cartilage, little effect of IL-1beta was seen, but abundant release of GAG occurred in the presence of retinoic acid, with evidence of aggrecanase action. Treatment of fetal epiphyseal cartilage with retinoic acid resulted in significant GAG release, whereas treatment with IL-1beta did not. In the retinoic acid-treated tissue, however, no evidence for the cleavage of aggrecan in the interglobular region was apparent. Thus, in the fetal system, agents in addition to aggrecanase and matrix metalloproteinases appear to be active. Taken together, these data demonstrate that the pathways utilized for aggrecan catabolism may vary between different cartilages for a given stimulatory agent, and that, for a given tissue, different factors may elicit aggrecan release via different pathways.

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The mechanism of aggrecan release from cartilage differs with tissue origin and the agent used to stimulate catabolism.

Changes with age in the structure of fibromodulin in human articular cartilage.

The proteoglycans decorin and biglycan in extracts of human articular cartilage were analysed by SDS/PAGE and immunoblotting, using antisera raised to peptide sequences present in the pro-regions and the mature core proteins. In adult cartilage, both pro-forms and mature processed forms of the proteoglycan core protein were observed for both decorin and biglycan. In the case of biglycan, it was also shown that additional proteolytic processing takes place after removal of the propeptide and that this accounts for the presence of non-glycanated forms of the molecule. For both decorin and biglycan, the relative abundance of the pro-forms was much less in the juvenile than the adult. Different adult connective tissues, including meniscus, tendon and intervertebral disc were also examined for the presence of pro-forms of the proteoglycans. While the mature form of decorin was present at a similar level in extracts of all tissues examined, the pro-form was only detected in the articular cartilage. In the case of biglycan, the abundance of the mature form was more varied, with high levels in articular cartilage, intermediate levels in meniscus and the annulus fibrosus of the intervertebral disc, low levels in the nucleus pulposus of the intervertebral disc, and non-detectable levels in the patellar tendon. The pro-form of biglycan was detected in the disc tissue extracts, albeit at a lower level than in articular cartilage, but was not detected in the meniscus or tendon. The proportion of the pro-form relative to the mature form of biglycan was, however, higher in the nucleus pulposus of the intervertebral disc than in articular cartilage. Thus, the persistence of pro-forms of both decorin and biglycan is a feature of the extracellular matrix of some connective tissues, although their abundance is both tissue- and age-dependent, with adult articular cartilage being a particularly rich source.

Robert J. White

Papers

Non-proteoglycan forms of biglycan increase with age in human articular cartilage

Dermatan sulphate proteoglycans of human articular cartilage. The properties of dermatan sulphate proteoglycans I and II

The mechanism of aggrecan release from cartilage differs with tissue origin and the agent used to stimulate catabolism.

Changes with age in the structure of fibromodulin in human articular cartilage.

Presence of pro-forms of decorin and biglycan in human articular cartilage