HENRY J. necessary to describe changes that occur in both these periods, since the. showing an abrupt change at the time of skeletal maturation.

Transcription

1 545 THE EFFECT OF AGING ON ARTICULAR CARTILAGE* HENRY J. MANKIN Director of Orthopaedics, Hospital for Joint Diseases Professor of Orthopaedics, The Mount Sinai School of Medicine, New York, N. Y. B ECAUSE of its unique appearance and staining characteristics, articular cartilage attracted the attention of early anatomists and histologists. Its microscopic features were described many years ago. Its unusual structural properties, however, sharply limited exploration of its biochemical and metabolic characteristics, and it was not until the last few decades that these areas were even tentatively explored. In recent years a large mass of data has accumulated regarding the structural, metabolic, and biochemical changes that occur in the articular surfaces with aging. The purpose of this report is to review and summarize these changes and to speculate on their significance. Before describing changes observed in the process of aging, it is essential to define the natural course of the process of maturation of this tissue. Articular cartilage, like bone and epiphyseal plate, is involved in the complex process of net synthesis for longitudinal growth, and certain characteristics are present in an immature cartilaginous mass; these change slowly over the period of skeletal maturation to the "adult" pattern. Subsequent alterations that occur in this mature cartilage may be considered to reflect senescence and might be more logically called the true pattern of "aging." In discussing the subject, however, it is necessary to describe changes that occur in both these periods, since the biochemical structure and metabolic activities vary gradually instead of showing an abrupt change at the time of skeletal maturation. The discussion will be divided into three areas: cell population, biochemical composition, and metabolism. CELL POPULATION If one compares the histologic pattern of immature and adult articular cartilage, there are noticeable general differences that are present *Based on a paper presented at a meeting of the New York Rheumatism Association, held at The New York Academy of Medicine, April This investigation was supported in part by Public Health Service Research Grant AM from the National Institute of Arthritis and Metabolic Diseases, Bethesda, Md. Vol. 44, No. 5, May 1968

2 H. H. J. MANKIN EFFECT OF AGING ON CELL COUNT PER UNIT WET WEIGHT OF RABBIT ARTICULAR CARTILAGE 2.25 to -J 2. E,,, 1.75 a X~~~~~~~ J w C.) 1. 6 Mo AGE IN YEARS Fig. 1. Effect of aging on cell count per unit wet weight of rabbit articular cartilage. Direct cell counts of rabbit articular surfaces were performed microscopically on animals of four ages to provide cells per unit volume. Volumes were converted to weight by determinations of specific density.' It should be noted that there is a progressive dedine in cell count with advancing years. regardless of species or joint studied. Immature cartilage cells are rounded or ovoid, and their nuclei are large and moderately basophilic. Adult cells are flattened and more basophilic, and the nuclear size considerably is diminished."2 What is perhaps more striking is the change in cell density (the number of cells per unit volume or unit mass), which has been shown in both rabbit 4 and bovine3 articular surfaces to decrease steadily with advancing age (Figure i). Immature cartilage cells are known to undergo mitotic division, and the DNA replications are not uniformly spaced throughout the cartilage mass.5'6 In the immature rabbit dividing cells (as defined by tritiated thymidine studies and mitotic counts) are located in two zones (Figure 2): one subjacent to the gliding surface and probably representing a growth zone for the articular surface; and the second deep to it, in proximity to the epiphyseal nucleus and probably representing the proliferative zone for a microepiphyseal plate for endochondral ossification of the bony nucleus.5 7 As the animal ages, the mitotic potential in these two areas appears to decline and the superficial zone Bull. N. Y. Acad. Med.

3 AGING AND ARTICULAR CARTILAGE 5 47 A B C (2 mo.) (6 mo.) (18 mo.) o Ooo O' 1.9~25 ~5226xig19 ;Cell Count cells /mm.x elscellis/mm.3 1 M itotic Index 32/,, 66 Fig. 2. Effect of aging on localization of mitotic figures, height of cartilage surface, cell and mitotic counts in rabbit articular cartilage. This composite drawing shows the changes that occur in the articular surface with maturation. In the immature animal (A) the cartilage surface is thicker and more cellular, and mitotic activity occurs in essentially two zones: Zone A, presumably for growth of the articular surface; and Zone B, which probably serves as a proliferative zone for endochondral ossification of the underlying epiphyseal nucleus. As the animal ages (B), the height diminishes, and the cell and mitotic counts decrease. Cell proliferation is found only in Zone B. With maturity, the thickness is further decreased, and no mitotic activity is present. See text. disappears (Figure 2). With the approach of maturity and the development of a well-defined zone of calcified cartilage in the basal area of the cartilaginous mass, mitotic activity ceases.8 In normal, unstressed articular cartilage from mature animals, mitotic figures are never seen, and autoradiographic studies with tritiated thymidine8 and cytophotometric determinations of DNA conteht9 have failed to show evidence for any form of cell division-mitotic or amitotic (Figure 2). The pattern described above suggests that the cellular replications occur only during net synthesis and that, in contrast to epithelial tissue such as skin, they are not a factor in compensation for normal attrition. Vol. 44, No. 5, May 1968

4 5485 H. J. MANKIN Al EFFECT OF AGING ON RATE OF INCORPORATION OF GLYCINE-H3 PER UNIT DNA OF RABBIT Z ARTICULAR CARTILAGE ole t Z 8 r 6 cn4 WJZ o 2 < _ amp 66Mo >1; AGE IN YEARS Fig. 3. Effect of aging on rate of incorporation of glycine-h8 per unit DNA of rabbit articular cartilage. Glycine-H8 has been shown to be an excellent indicator of protein synthesis in articular cartilage.2 21 When the rates of incorporation were measured and compared on the basis of quantity of DNA (which is equivalent to the number of cells), there is a rapid decline in rate up until the time of maturity (one year). After this the synthetic activity appears to be constant throughout the animal's life span. This would also suggest that the cells of mature articular surfaces are extraordinarily long-lived, since the rate of decline in the population of a cell is quite slow despite the inability of the cells to renew themselves. BIOCHEMICAL COMPOSITION The changes in biochemical composition that occur with aging of articular surfaces are surprisingly small, at least to the extent that they have been studied. Water content, electrolyte concentrations, and organic solid composition remain surprisingly constant throughout the maturation and postmaturation phases.' 11 Collagen accounts for about So per cent of the dry weight of cartilages 11 and probably, like most body collagen, turns over very slowly after net synthesis ceases This may serve to stabilize the chemical structure, but it also could make detection of minor changes in other organic components more difficult. The constituent protein polysaccharides are primarily chondroitin-4 and chondroitin-6-sulfates,14 and their ratio remains essentially constant Bull. N. Y. Acad. Med.

5 AGING AND ARTICULAR CARTILAGE 5 49 ( D -e 4 w D I U. z U It DEGRADATION RATE OF INCORPORATED GLYCINE-H3 AND S35 INTO ADULT ARTICULAR CARTILAGE 1 GLYCINE - H I I I I I I 1 s35o; 9 8 I7.6 5 _ 4-3 _.i I I I I DAYS Fig. 4. Rate of disappearance of incorporated S3O4= and glycine-h8 in adult articular cartilage. The articular cartilage of adult rabbits was "flash" labeled in vivo with the isotopes, the animals serially killed, and the cartilage surfaces assayed for incorporated substrates. From these data it is evident that the turnover of both the protein moieties (as represented by the glycine-h3) and the polysaccharides (SwO,=) are rapid with a half-life of approximately eight days. The variation for the two curves may be accounted for by the heterogeneity of the protein products. with advancing age in articular cartilage. Unlike costal cartilage,15 keratosulfate is present in only small quantities, and the concentration does not increase with age.'6 METABOLISM Little is known of the metabolism of this peculiar tissue. Its avascular state suggests that anaerobic glycolysis is well developed,9 and that lactate levels are high throughout the animal's life-span.17 Determinations of the utilization of carbohydrate and of the consumption of oxygen are hampered by the relative acellularity of the tissue,3 but proteinpolysaccharide and collagen synthesis have been explored reasonably well by the use of isotopically labeled substrates.'82' Cartilage cells incorporate large amounts of glycine both in vivo and in vitro, and several studies have demonstrated the validity of this technique as an Vol. 44, No. 5, May 1968

6 5 5 H. J. MANKIN DEGRADATION RATES FOR INCORPORATED GLYCINE-HH AND S35 INTO ARTICULAR CARTILAGE OF RABBITS gx I I I IT I I I I I o o 35 3O1 - GLYCINE-H3 S.I_ 1 HALF LIFE to DAYS HALF LIFE - IS DAYS -J9 9 O 7 7T 4 UA. S o 6-6 w - Fu 4-4 U Ia lo TIME (DAYS) Fig. 5. Rate of disappearance of incorporated SI*O,= and glycine-h31 in immature articuilar cartilage. This experiment was performed in a similar fashion to that described in Figure 4. Note the essential similarity between the curves obtained at the two age groups. The glycine-h3 curve is somewhat different presumably because it represents collagen synthesis as well as protein of protein-polysaccharide. These values have less validity than those shown in Figure 4, since there has been no correction for net synthesis. indicator of synthesis of protein of protein-polysaccharide (and collagen-although in a mature animal where collagen is stable, the amount incorporated is presumed minimal). 21 When glycine-h3 incorporation per microgram of DNA of the cartilage mass was determined and compared for various ages of rabbits, it is evident that the highest rates were present during the phase of net synthesis' (Figure 3). With maturity, however, the rate maintains a fairly constant level throughout senescence. This would seem to indicate that despite the inability of the mature cell to replicate its DNA, it is capable of maintaining a steady rate of protein synthesis that is independent of age. It would seem likely, therefore, that the mechanism of compensation for attritional loss is one of matrix production rather than of cell reproduction. When this concept is scrutinized, however, and other data are added, it is apparent that the synthetic rate defined in this and other studies is too rapid to be purely a compensation for attritional loss. This sug- Bull. N. Y. Acad. Med.

7 AGING AND ARTICULAR CARTILAGE 5 5 I gests an internal remodeling system; indeed, such a system has been described.22 A cathepsin found both within lysosomes and throughout the cytoplasm that is believed to be specific for the protein of proteinpolysaccharide is considered to exert a catabolic action on the matrix; presumably it provides a feedback stimulus to synthesis. When degradative rates are determined for the articular surfaces of the rabbit it becomes apparent that the turnover is not only very rapid, but virtually unaffected by the age of the animal. Half lives for both glycine-h3 and S354= are under eight days for the adult animal (Figure 4) and the similarity of the curves obtained reinforces the concept that little collagen is being synthesized (or degraded). Half lives for the same isotopes in the immature animal (Figure 5) are slightly longer but these values have less validity since net synthesis is occurring and the protein product that includes collagen is more heterogeneous. The data shown above leave little doubt, however, that the turnover rates for both immature and adult cartilage are not only of the same order of magnitude but quite similar. In summary, the data presented above suggest that considerable change occurs in the articular surfaces in the period of maturation but that little variation is noted with senescence. The characteristics of the early phase are a reflection of net synthesis and DNA replication, and increased rates of protein polysaccharide (and collagen) synthesis are presumably related not only to growth of the articular surface but to endochondral ossification of the underlying nucleus of the epiphysis. With maturity, however, a steady state is reached in which the measurable parameters vary little with senescence. No further synthesis of DNA occurs, but the rate of synthesis and the chemical composition of the matrix remain constant. Furthermore, the internal remodeling system, presumably necessary for maintenance of elasticity and lubrication, functions at approximately the same rate in both immature and adult animals. At this time the crudity of the methods available and the structural peculiarities of the tissue make further exploration difficult, and it is possible and even likely that some other changes are present during senescence but cannot be appreciated. Furthermore, the relation (or lack of relation) of aging to osteoarthritis is an unsettled problem. It is hoped that technical advances and continued interest will provide answers to some of the enigmas of this intriguing tissue. Vol. 44, No. 5, May 1968

8 5 5 2 H. J. MANKIN REFERENCES 1. Mankin, H. J. and Baron, P. A. The effect of aging on protein synthesis in articular cartilage of rabbits. Lab. Invest. 14:658, Silberberg, M. and Silberberg, R. Aging changes in cartilage and bone. In: Bourne, G. H., ed., Structural Aspects of Aging. New York, Hafner, 1961, p Rosenthal, O., Bowie, M. A. and Waguer, G. Studies in the metabolism of articular cartilage. J. Cell. Comp. Physiol. 17:221, Barnett, C. H., Cochrane, W. and Palfrey, A. J. Age changes in articular cartilage of rabbits. Amer. Rheum. Dis. 22:389, Mankin, H. J. Localization of tritiated thymidine in articular cartilage of rabbits: I. Growth in immature cartilage. J. Bone Joint Surg. 44:682, Mankin, H. J. Mitosis in articular cartilage of immature rabbits. CGin. Orthop. 34:17, Mankin, H. J. The calcified zone (basal layer) of articular cartilage of rabbits. Anat. Rec. 145:73, Mankin, H. J. Localization of tritiated thymidine in articular cartilage of rabbits: III. Mature articular cartilage. J. Bone Joint Surg. 45:529, Mankin, H. J. Unpublished data. 1. Miles, J. and Eichelberger, L. Biochemical studies of human cartilage during the aging process. J. Amer. Geriat. Soc. 12:1, Anderson, C. E., Ludowieg, J., Harper, H. A. and Engleman, E. P. The composition of the organic component of human articular cartilage. Relationship to age and degenerative joint disease. J. Bone Joint Surg. 46:1, 176, Neuberger, A. and Slack, H. G. B. The metabolism of collagen from liver, bone, skin and tendon in the normal rat. Biochem. J. 53:47, Robertson, W. van B. In: Connective Tissues: Intercellular Macromolecules. Boston, Little, Brown, 1964, p Muir, H. In: International Review of Connective Tissue, D. A. Hall, ed. New York, Academic Press, 1964, vol. 2, p Kaplan, D. and Meyer, K. Aging of human cartilage. Nature (London) 183: 1267, Kuhn, R. and Lepelmann, H. J. Galaktosamin und Glucosamin im Knorpel im Abhangigkeit vom Lebensalter. Ann. Chem. (Liebig's) 67:22, Lutwak-Mann, C. Enzyme systems in articular cartilage, Biochem. J. 34:517, Bostrom, H. On the metabolism of the sulfate group of chondroitin sulfuric acid. J. Biol. Chem. 196:477, Dziewiatkowski, D. D. Effect of age on some aspects of sulfate metabolism in the rat. J. Exp. Med. 99:283, Mankin, H. J. and Orlic, P. A. A method of estimating the "health" of rabbit articular cartilage by assays of RNA and protein synthesis, Lab. Invest. 13: 465, Mankin, H. J. The Metabolism of the Matrix of Articular Cartilage. In press, Fessel, J. M. and Chrisman,. D. Enzymatic degradation of chondromucoprotein by cell-free extracts of human cartilage. Arthritis Rheum. 7:398, Bull. N. Y. Acad. Med.