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The nanostructure and nanomechanical properties of aggrecan monomers extracted and purified from human articular cartilage from donors of different ages (newborn, 29 and 38 year old) were directly visualized and quantified via atomic force microscopy (AFM)-based imaging and force spectroscopy. AFM imaging enabled direct comparison of full length monomers at different ages. The higher proportion of aggrecan fragments observed in adult versus newborn populations is consistent with the cumulative proteolysis of aggrecan known to occur in vivo. The decreased dimensions of adult full length aggrecan (including core protein and glycosaminoglycan (GAG) chain trace length, end-to-end distance and extension ratio) reflect altered aggrecan biosynthesis. The demonstrably shorter GAG chains observed in adult full length aggrecan monomers, compared to newborn monomers, also reflects markedly altered biosynthesis with age. Direct visualization of aggrecan subjected to chondroitinase and/or keratanase treatment revealed conformational properties of aggrecan monomers associated with chondroitin sulfate (CS) and keratan sulfate (KS) GAG chains. Furthermore, compressive stiffness of chemically end-attached layers of adult and newborn aggrecan was measured in various ionic strength aqueous solutions. Adult aggrecan was significantly weaker in compression than newborn aggrecan even at the same total GAG density and bath ionic strength, suggesting the importance of both electrostatic and non-electrostatic interactions in nanomechanical stiffness. These results provide molecular-level evidence of the effects of age on the conformational and nanomechanical properties of aggrecan, with direct implications for the effects of aggrecan nanostructure on the age-dependence of cartilage tissue biomechanical and osmotic properties., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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.

The ability of the nucleus pulposus of the intervertebral disc to resist compressive loads is due to its high content of the proteoglycan aggrecan. Degeneration of the intervertebral disc is preceded and accompanied by a loss of aggrecan due to proteolysis. Biological repair of intervertebral disc degeneration should strive to restore aggrecan content to its optimal functional level. One approach to such repair is to supplement the degenerate nucleus with cells that are capable of aggrecan synthesis. Such cells can be supported in a biomolecular scaffold, but it is essential that the scaffold is compatible with high aggrecan retention if a functional tissue is to be attained.

Study Design: An analysis of proteoglycans of the intervertebral disc using immunoblotting of tissue extracts., Objectives: To investigate the changes in structure and abundance of fibromodulin and lumican in human intervertebral discs during aging and degeneration., Summary of Background Data: Fibromodulin and lumican are keratan sulfate proteoglycan constituents of the disc's extracellular matrix, whose interaction with collagen fibrils may contribute to the mechanical properties of the tissue. Changes in their abundance and/or structure that occur with aging and degeneration therefore may have an impact on disc function., Methods: Lumbar intervertebral discs were obtained from individuals of different ages, and extracts of anulus fibrosus and nucleus pulposus were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting using antibodies specific for fibromodulin and lumican., Results: The major changes in abundance observed with age were a decrease in fibromodulin in the adult nucleus pulposus and an increase in lumican in anulus fibrosus during early juvenile development. In addition, fibromodulin in the anulus fibrosus exhibited a structural change with increasing age, characterized by a shift toward the predominance of its glycoprotein form lacking keratan sulfate. Fibromodulin was more abundant in the anulus fibrosus than in nucleus pulposus at all ages, whereas lumican was much more abundant in nucleus pulposus than in anulus fibrosus in the young juvenile; in the adult, however, lumican was present in comparable levels in both tissues. With increasing degrees of degeneration, fibromodulin exhibited an increase in abundance., Conclusions: Growth, aging, and degeneration of the intervertebral disc are associated with changes in the abundance and structure of fibromodulin and lumican, which presumably influence the functional properties of the tissue.

An anti-peptide antibody was raised in a rabbit against the carboxy terminal region of the human fibromodulin core protein. The antibody was purified from other components of the resulting antiserum by affinity chromatography using the immobilized peptide, and was used to study the structural heterogeneity of fibromodulin extracted from human articular cartilage of different ages by the use of immunoblotting following sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the extracted macromolecules. In the fetus and neonate, fibromodulin was visualized as a diffuse component with a relative molecular weight of 70-110 kDa, whereas in the mature adult a more discrete component of smaller size was apparent with a relative molecular weight of 67 kDa. The size of the fibromodulin from mature adult cartilage could not be altered by pretreatment of the samples with keratanase II or endo-beta-galactosidase before analysis. In contrast, the size of the fibromodulin from younger cartilage could be decreased with both glycosidases, with the endo-beta-galactosidase yielding a smaller product than the keratanase. The size of the product resulting from endo-beta-galactosidase treatment of the fibromodulin from young cartilage was the same as that of the intact fibromodulin from mature adult cartilage. Thus, fibromodulin is present in human articular cartilage at all ages, but the extracted molecules only appear to exist in a proteoglycan form possessing keratan sulfate chains in the juvenile and young adult, and the size of these chains decreases with age. In the mature adult the fibromodulin does not possess either keratan sulfate or non-sulfated polylactosamine chains, though it appears to possess the same number of N-linked oligosaccharides as its counterparts from the younger tissue, but they are not modified further. The majority of the fibromodulin extracted from arthritic cartilage is of the same size as that found in the normal mature adult, although there is evidence for proteolytic processing. The degree of such processing is greater for the fibromodulin obtained from the cartilage of rheumatoid arthritic joints than osteoarthritic joints.

Biochemical and biophysical studies have shown that the composition and sedimentation velocity of cartilage proteoglycans change with age, but these investigations cannot demonstrate the alterations in molecular structure responsible for these changes. Development of quantitative electron microscopic methods has made it possible to define the age-related structural changes in aggregating proteoglycans and to correlate the alterations in their structure with changes in tissue composition and morphology. Electron microscopic measurement of human and animal hyaline cartilage proteoglycans has shown that with increasing age the length of the chondroitin sulfate-rich region of aggregating proteoglycan monomers (aggrecan molecules) decreases, the variability in aggrecan length increases, the density of aggrecan keratan sulfate chains increases, the number of monomers per aggregate decreases, and the proportion of monomers that aggregate declines. Proteoglycans from the nucleus pulposus of the intervertebral disc show similar but more dramatic age-related alterations. At birth, nucleus pulposus aggrecan molecules are smaller and more variable in length than those found in articular cartilage. Within the first year of human life, the populations of aggregates and large aggrecan molecules analogous to those found in articular cartilage decline until few if any of these molecules remain in the central disc tissues of skeletally mature individuals. The mechanisms of the age-related changes in cartilage proteoglycans have not been fully explained, but measurement of proteoglycans synthesized by chondrocytes of different ages suggests that alterations in synthesis produce at least some of the age-related changes in aggrecan molecules. Degradation of aggrecan chondroitin sulfate-rich regions in the matrix probably also contributes to the structural changes seen by electron microscopy. Age-related changes in proteoglycan aggregation may be due to alterations in link protein function or inhibition of aggregation of newly synthesized aggrecan molecules by accumulation of degraded aggrecan molecules.

In chondrocytes isolated directly from human articular cartilage, without subsequent culture, biglycan mRNA levels decreased with the age of the donor, whereas those for decorin increased. In cultured chondrocytes in the presence of FCS, mRNA levels for biglycan remained similar to those in uncultured counterparts, while decorin transcription became depressed. The differential effect on decorin and biglycan mRNA expression was mimicked by the addition of TGF-beta. In the absence of exogenously added growth factors (TGF-beta or FCS) biglycan mRNA levels decreased, while those for decorin increased. In contrast, IGF-I showed no differential modulation of the relative abundance of the two messages. The opposite regulation of these two proteoglycans by TGF-beta was also reflected at the level of protein synthesis. It would appear that the need of articular cartilage for decorin is greatest in the adult, whereas the need for biglycan is greatest in the juvenile.

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.

Adult human articular cartilage contains a protein of Mr 55,000 which is deficient in newborn cartilage. In the adult the molecule represents one of the most abundant non-collagenous, non-proteoglycan molecules in 4 M guanidinium chloride extracts of the tissue. The molecular size of the protein on SDS-PAGE remains constant under reducing and non-reducing conditions, suggesting that it does not exist as a disulphide-bonded multimer, nor do intramolecular disulphide bonds greatly influence its conformation. The protein has the ability to interact with some immunoglobulin preparations making its detection possible by Western blotting with some non-specific antisera. Labeling with [3H]leucine in organ culture indicates that protein of this size is being made by the chondrocytes. However, during purification the newly synthesized molecules do not behave as the resident protein on ion-exchange chromatography, suggesting that the protein may accumulate with age rather than being a major synthetic product of the adult chondrocytes. Amino terminal protein sequence analysis indicates that the N-terminus of the protein is blocked. Sequences derived from peptides generated with cyanogen bromide do not show homology with previously characterized proteins. Molecules of a similar size and composition have been described in bovine cartilage.

Proteoglycan subunits from human articular cartilage were fractionated by caesium sulphate density gradient centrifugation. A single heterogeneous population of molecules was produced whose average density decreased with increasing age of the individual from which they were obtained. At no density did the carbohydrate composition of any adult fraction resemble that of any newborn fraction, although there was considerable overlap in density. However, there was a similarity in amino acid composition between the most dense proteoglycans from the adult and those of least density from the newborn. The carbohydrate content of a 2-year-old proteoglycan was intermediate in composition, with high density fractions resembling the newborn and low density fractions resembling the adult. In addition, the proteoglycans of lowest density in both the newborn and two year preparations showed additional bands on agarose/polyacrylamide gel electrophoresis resembling the adult material. These results indicate that while a core protein of adult composition may occur in the juvenile proteoglycan it need not necessarily be glycosylated in an adult manner, suggesting that glycosylation is to some extent independent of the origin of core protein heterogeneity.

After chromatography on Sepharose CL-2B under associative conditions, high-buoyant-density human articular-cartilage proteoglycans were analysed biochemically and by radioimmunoassay with monoclonal antibodies to a core-protein-related epitope and to keratan sulphate. An examination of proteoglycans from individuals of different ages revealed the presence at 1 year of mainly a single polydisperse population containing chondroitin sulphate (uronic acid) and keratan sulphate. From 4 years onwards a smaller keratan sulphate-rich and chondroitin sulphate-deficient population appears in increasing amounts until 15 years. At the same time the larger population shows a progressive decrease in size from 1 year onward. By 23 years and after the proportion of keratan sulphate in the larger chondroitin sulphate-rich proteoglycan increases. Both adult proteoglycan populations are shown immunologically to aggregate with hyaluronic acid, with the smaller showing a greater degree of interaction. The larger population is richer in serine and glycine, and the smaller population contains more glutamic acid/glutamine, alanine, phenylalanine, lysine and arginine; its protein content is also higher. Whether the larger post-natal population represents a different gene product from the single polydisperse population found in the human fetus, which has a different amino acid composition, remains to be established. The smaller population, which represents approximately one-third the mass of the larger population in the adult, may represent a degradation product of the larger population, in which the hyaluronic acid-binding region and keratan sulphate-rich region are conserved.

Human articular-cartilage link proteins are resolved into three components by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, indicative of three different structures. The action of the proteinase clostripain yields a single link-protein component with electrophoretic properties analogous to those of the smallest (most mobile) native link protein, suggesting that this link protein may be derived naturally from one or both of the larger molecules by proteolytic cleavage in situ. Upon chemical deglycosylation of native link protein two components are resolved, suggesting that two of the link proteins differ only in their degree and/or type of oligosaccharide substitution. This pattern is compatible with a proteolytic origin for the smallest link protein. During aging further proteolytic fragmentation occurs, though it is only apparent on reduction of disulphide bonds. This fragmentation occurs at identical sites in all three native link proteins, indicating the existence of a large region common to all the link proteins, which appears to consist predominantly of the C-terminal half of the molecules. These observations are compatible with the variation in oligosaccharide and proteolytic heterogeneity occurring at the N-terminus of the link proteins.

Rabbits were immunized with adult or foetal human articular cartilage proteoglycans and cell-mediated and antibody immunity assessed in vitro. Cellular immunity to proteoglycans of different ages was measured by the stimulation of 3H-thymidine incorporation into cultured spleen cells. Antibodies to proteoglycans in the rabbit sera were detected with an enzyme-linked immunosorbent assay. The ability of adult and foetal proteoglycans to preabsorb antibody activity in this assay was compared. The results from both the cellular and antibody studies support the same conclusion, that adult proteoglycans express all foetal antigenic determinants and have additional determinants not seen on foetal proteoglycans. The reactivity of rabbit spleen cells to proteoglycans of different ages indicated that these determinants appear during maturation when the major changes in proteoglycan structure occur. The results did not appear to be due to differences in chondroitin sulphate content of the preparations, since the same observations were made with chondroitinase ABC-digested proteoglycans. The observations might be explained by other differences in glycosylation between the preparations or by postulating the synthesis during maturation of an additional proteoglycan species with a different core-protein. Such changes in the antigenicity of articular cartilage proteoglycans with age could account for the development of autoimmunity to proteoglycans in inflammatory joint diseases and may play a role in their pathogenesis.

The effects of treatment of purified neonatal human articular-cartilage proteoglycan aggregate with H2O2 were studied. (1) Exposure of proteoglycan aggregate to H2O2 resulted in depolymerization of the aggregate and modification of the core protein of both the proteoglycan subunits and the link proteins. (2) Treatment of the proteoglycan aggregate with H2O2 rendered the proteoglycan subunits unable to interact with hyaluronic acid, with minimal change in their hydrodynamic size. (3) Specific cleavages of the neonatal link proteins occurred. The order in which the major products were generated and their electrophoretic mobilities resembled the pattern observed during human aging. (4) The proteolytic changes in the link proteins were inhibited in the presence of transition-metal-ion chelators, thiourea or tetramethylurea, suggesting that generation of hydroxyl radicals from H2O2 by trace transition-metal ions via a site-specific Fenton reaction may be responsible for the selective cleavages observed. (5) Cleavage of the link proteins in proteoglycan aggregates by H2O2 was shown to have a limited effect on the susceptibility of these proteins to cleavage by trypsin. (6) The relationship between these changes and those observed in cartilage during human aging suggests that some of the age-related changes in the structure of human cartilage proteoglycan aggregate may be the result of radical-mediated damage.

The link proteins of the human intervertebral disc were studied in tissue extracts by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE), followed by immunoblotting, using a specific monoclonal antibody. Three link proteins were detected, corresponding in electrophoretic mobility to those present in articular cartilage. As with articular cartilage, the largest link protein predominates in the young, whereas in the adult the smallest link protein is equally abundant and internal fragmentation of the link proteins occurs. Only in the newborn is the quantity of extractable link protein comparable to that from articular cartilage. In the adult, the disc contains much less link protein than is present in autologous articular cartilage. Neither the amount nor heterogeneity of the link protein differs among different levels within the lumbar spine, although the proportions of the three proteins can differ between the anulus fibrosus and nucleus pulposus. The anulus always contained more extractable link protein relative to tissue wet weight than the nucleus, and the nuclear link protein, at least in adolescents, contained a greater proportion of the smallest link protein. Such changes in the quantity and structure of the disc link proteins may affect the properties of the proteoglycan aggregates and, thus, could influence disc function.

Link proteins were identified immunologically in human articular-cartilage protein preparations from various individuals. Irrespective of age, all cartilages contained three link proteins of mol.wts. 48000, 44000 and 41000. However, with increasing age, multiple additional components of mol.wts. 26000-30000 were commonly observed under conditions where disulphide bonds were reduced.

The structure of the aggregating proteoglycans from human articular cartilage changes continuously from the early fetus to the mature adult, due to variation in both synthetic and degradative processes. The changes due to synthesis are most prominent prior to the end of growth, and are responsible for variations in the structure and abundance of the glycosaminoglycan chains. In contrast, the changes due to degradation occur throughout life, but predominate during aging of the adult. They result in proteolytic fragmentation of both the proteoglycan subunits and the link proteins, with those products possessing hyaluronic acid-binding capability accumulating in the tissue.

The proteoglycan subunits isolated from human articular cartilage of various ages (ranging from the newborn to the mature adult) were analyzed for their content of histidinoalanine. It was noted that while the young juveniles and adolescents contained about 1 nmole of histidinoalanine per mg protein, the skeletally mature adults contained about twice this amount. Unlike many age-related changes in proteoglycan structure that occur prior to the end of growth, this variation would appear to be more related to maturation of the adult.