J Clin Pathol: first published as 10.1136/jcp.s3-12.1.7 on 1 January 1978. Downloaded from J. clin. Path., 31, Suppl. (Roy. Coll. Path.), 12, 7-13

Chondrocytes

R. A. STOCKWELL From the Department ofAnatomy, University ofEdinburgh

Cartilage is a skeietal tissue largely consisting of Ultrastructure extracellular matrix populated by a small number of cells-the . All hyalinecartilages consist Typical chondrocytes are ovoid cells ranging in of about 75% water while fibies and pro- maximum diameter from about 10 /tm in articular teoglycan molecules make up a large proportion of to about 30 ,tm in other hyaline the dry mass. Elastic cartilage contains elastic (Stockwell and Meachim, 1973). The pericellular tiss-e in addition to collagen fibres, while in fibro- matrix is usually of a finer texture than the coarsely cartilage the amounts and proportions of collagen fibrous tissue more remote from the cell (Fig. 1). and are such that the fibres are visible The junction is often sharply demarcated and con- to the naked eye and accessible to routine histo- stitutes the lacunar rim. The cell has a scalloped logical stains. The matrix is responsible for the surface which increases the surface area :volume rigidity and resilience of the tissue, the mechanical ratio. Projecting cell processes are occasionally properties reflecting the physicochemical organisa- several micrometres long and may reach beyond tion of the matrix macromolecules. The chondro- the . A monocilium is sometimes found in cytes produce the matrix during growth and, despite adult cartilage (Fig. 2) but more commonly in copyright. their small numbers, maintain the tissue during adult immature tissue. While it is difficult to identify a life. Since cartilage lacks blood vessels, lymphatics, glycocalyx in the presence of ground substance, and nerves-except in cartilage canals during growth chondrocytes enzymatically stripped of their matrix -the cells must be sustained by nutrient and gas carry major histocompatibility antigens (Elves, diffusion over large distances compared with vascular 1976). tissues. The cell nucleus in older chondrocytes is often

Cartilage is easily recognised histologically in irregular and lobated, a feature which once appeared http://jcp.bmj.com/ normal circumstances, with its scattered cells in to substantiate the old erroneous view that chondro- lacunae lying in basophilic matrix. The itself has many typical features but nevertheless the cell type is difficult to define in structural terms. Identification in practice often depends on recognis- ing its synthetic and secretory products. The histo- logist is prone to rely ultimately on the pericellular localisation of metachromatic material or, in electron on September 25, 2021 by guest. Protected micrographs, of banded collagen fibres in amor- phous electron-lucent ground substance. The bio- chemist can use more specific criteria to characterise the phenotype, such as the secretion of type II 'cartilage' collagen (Miller and Matukas, 1974) and of that can aggregate with hyaluronic acid (Muir, 1977). Recent immunofluorescent techni- R ques for type II collagen (Von der Mark et al., 1976) may offer a more precise means of histological identification. However, changes can occur in the types of collagen and proteoglycan secreted by chondrocytes in vitro, depending on the culture Fig. 1 Human femoral condylar cartilage, 56 years. conditions. Similar changes may occur in osteo- Part of chondrocyte with finely textured pericellular arthrosis (Muir, 1977). matrix (x 8000). R = lacunar rim. 7 J Clin Pathol: first published as 10.1136/jcp.s3-12.1.7 on 1 January 1978. Downloaded from 8 R. A. Stockwell cytes replicated by amitosis. The predominant' cytoplasmic organelles are the granular endoplasmic reticulum and the Golgi complex (Fig. 2), both- associated with the synthesis and secretion of col- lagen and proteoglycans (Revel and Hay, 1963; Horwitz and Dorfman, 1968; Ross, 1975). Golgi vacuoles are more numerous in the cells of growing cartilage. Lysosomes function in the removal of effete intracellular material but in chondrocytes they

for matrix synthesis. The large amounts of lipid 4 -A http://jcp.bmj.com/ in chondrocytes has never been explained satis- -' Fig. 4 Human femoral condylar cartilage, 56 years. Chondrocyte with several lipid globules. Note numerous dense bodies (extracellular lipid) (arrows) near distinct lacunar rim (x 4000).

..',§ '. ,factorily.e...... Fat is a normal inclusion (Fig. 4), but on September 25, 2021 by guest. Protected there is little evidence that it is used either by the cell itself or by the whole organism. However, as in the aorta (Buddecke et at., 1973), utilisation of NADH for fat synthesis might be an important factor in the reoxidation of the coenzyme, helping to stabilise the NAD/NADH ratio in the chondro- cyte. Fat globules increase in size during maturation - ->, ~~~~~~~~~andare smaller in articular than other hyaline carti- -. lages. Recent investigations of lipoarthrosis show that the superficial articular chondrocytes become engorged with lipid and that cell death and cartilage Fig. 2 Human femoral condylar cartilage, 21 years. fibrillation ensue (Ghadially et at., 1970; Sprinz and Chondrocyte with profuse endoplasmic reticulum and Stockwell, 1977). Seemingly the cells selectively in- Golgi membranes (x 7000). C = single cilium. corporate fatty acids (Fig. 5) rather than the glycerol J Clin Pathol: first published as 10.1136/jcp.s3-12.1.7 on 1 January 1978. Downloaded from

Chondrocytes 9

vascular organs (Bywaters, 1937). Species differ- ences in cartilage also reveal greater variation in cellular oxygen uptake than in glycolytic rate. There is an age-related diminution of chondrocyte res- piration though not of glycolysis; this is associated with loss of cytochromes (Rosenthal et al., 1941). However it is not known whether these age changes affect synthetic activity significantly. Manufacture of collagen and proteoglycans depends on an adequate supply of precursors such as amino-acids and sugars. Proteoglycan formation can be studied using 35S-sulphate and collagen by using 3H-proline. In collagen synthesis hydroxy- lation, important both for stability of the collagen Fig. 5 Rabbit femoral condylar cartilage, 3 months. molecule and for intermolecular cross-linkage, After intra-articular injection of (a) glyceryl-2-H3 trioleate, (b) glyceryl tri (oleate-9,10-H3). Frozen section requires factors such as atmospheric oxygen and autoradiographs, incident light ( x 280). vitamin C. In proteoglycan synthesis the production (from Sprinz and Stockwell (1976), by permission of the and interconversion of the nucleotide sugar donor Editor and publishers of the Journal of Anatomy) molecules are affected by the redox state of the cell, particularly the NAD/NADH ratio (Phelps and Stevens, 1975). Such requirements for synthesis moiety of intra-articular triglyceride (Sprinz and depend ultimately on adequate diffusion of nutrients, Stockwell, 1976). itself affected by the physicochemical state of the matrix (Maroudas, 1973). The availability of Metabolism proteoglycan core-protein and the intracellular levels of enzyme inhibitors-for example, UDP-xylose

Oxygen tension in the middle of small blocks of --also affect rates of synthesis. Secretion occurs copyright. cartilage is only one-third of that in vascular tissues via the Golgi complex, although steric effects may (Silver, 1975). The chondrocyte is remarkable for its hinder the extracellular passage of large proteo- ability to synthesise and secrete matrix components glycans through the matrix. Proteoglycans influence in an avascular environment. Nevertheless, while collagen fibrogenesis by electrostatic interactions. they tolerate very low oxygen tensions more suc- Endogenous mechansims for the degradation of cessfully than do fibroblasts, chondrocytes do not collagen in cartilage are ill-understood, although of thrive under such conditions. Synthetic activity is chondrocytes produce cathepsin B1 capable http://jcp.bmj.com/ also reduced by very high oxygen tensions (Marcus, cleaving collagen fibres at an acid pH (Dingle, 1975). 1973; Brighton et al., 1974). However, little or no turnover of collagen occurs in Since cartilage cellularity varies over a wide range adult cartilage. Proteoglycan degradation is achieved the glycolytic and respiratory rates (Table 1) of the most effectively by disrupting the protein core; a whole tissue tend to reflect cell density. This factor number of cathepsins (B1, D, and F) have been accounts for much of the greater activity of imma- implicated. Initial partial cleavage of macromole- ture compared with mature cartilage. While the cules occurs in the extracellular matrix by release of glycolytic rate per cell is comparable to that in other degradative enzymes. There are, however, bio- on September 25, 2021 by guest. Protected tissues, oxygen utilisation is much lower than in chemical (Dingle, 1975) and anatomical difficulties

Table 1 Glycolytic and respiratory rates of whole cartilage samples Oxygen uptake Anaerobic glycolysis Cell density Authors (ul/mg dry weight/h) (jg lactic acid produced lmg (cells x JO0/MM3) dry weight/h) Rat costal cartilage 0-22, 0-68 5-44, 7 40 25 Dickens and Weil- Malherbe (1936) Rabbit articular cartilage 0-15 2-56 12-8 Bywaters (1937); Hills (1940) Bovine articular cartilage 0-024 1-75 4-7 Rosenthal et al. (1941) Rabbit liver 7-7 13-6 62 Bywaters (1937) Rabbit kidney 10-8 20-8 110 Bywaters (1937) J Clin Pathol: first published as 10.1136/jcp.s3-12.1.7 on 1 January 1978. Downloaded from 10 R. A. Stockwell regarding the turnover of matrix, at least in regions produced by tumours (Brem and Folkman, 1975; remote from the cell. Eisenstein et al., 1975) and lymphocytes and may be Topographical differences in proteoglycan meta- a protease inhibitor. bolism in articular cartilage are well documented (Collins and McElligott, 1960; Maroudas, 1975). Cellularity The synthetic activity of the superficial cells is much less than in the deeper tissue. Such zonal variations Cartilage increases in size both by appositional and occur in other cartilages. There is some evidence interstitial growth. Chondrocyte mitotic rates in the also of local functional heterogeneity among epiphyseal growth plates are important factors in chondrocytes, superimposed on the zonal variation. the rate of increase in length of long (Kember Results indicate that some cells produce chondroitin and Sissons, 1976). Most other cartilages are thought sulphate-rich rather than keratan sulphate-rich to be dependent on appositional rather than inter- proteoglycans (Kincaid etal., 1972), that in immature stitial cell proliferation in the later stages of growth. cartilage some are more active in collagen than in Mitotic rates in articular cartilage are only 1/20th proteoglycan synthesis (Mazhuga and Cherkasov, of those in the growth plate during development and 1974), and that there is also heterogeneity in degrada- no thymidine-labelling can be detected in the normal tive enzyme release (Poole, 1975). Variability in adult (Mankin, 1964). Cell proliferation is resumed if activity could be either cyclical or permanent. It is tissue damage occurs, as in cell cluster formation in not known if the same cell produces at the same time fibrillated cartilage (Dustmann et al., 1974) or if cells both hyaluronic acid and the proteoglycan 'subunit' freed from their matrix are grown in culture molecules which bind together to produce the (Sokoloff, 1976). aggregates characteristic of cartilage matrix. Cellularity in human cartilage is reduced sevenfold Control of cartilage metabolism is not well under- during maturation from birth to adult life (Stockwell, stood. Low oxygen tension and mechanical com- 1967). A major factor is the secretion and inter- pression favour chondrocyte expression (Bassett and position of new matrix between cells, but the contri- Herrmann, 1961). Little is known about the mode bution made by cell death has never been measured.

of action of mechanical forces on chondrocyte Cell distribution is not uniform within the adult copyright. metabolism although calcium ions and cyclic AMP tissue. At a microscopically local level cells lie either may be involved (Norton et al., 1977). Proteoglycans singly or in isogeneous groups, while decreasing stimulate (Nevo and Dorfman, 1972) and hyaluronic gradients of cell density are observed from the acid inhibits (Wiebkin and Muir, 1975) proteoglycan tissue periphery to its centre. Such gradients also synthesis through interactions at or near the cell occurwherea is lacking-for example, surface. Local cation concentration also affects at articular surfaces-but the tissues near most

synthesis (Shulman and Opler, 1974). A number of osteochondral boundaries (though not in the inter- http://jcp.bmj.com/ hormones, particularly growth hormone (via soma- vertebral disc) show little change in cellularity. The tomedin) and thyroxin, stimulate chondrocyte maximum cell density found in the superficial zone metabolism. Corticosteroids depress synthesis and of articular cartilage must be attributed to proximity degradation of ground substance. to synovial fluid and its nutrient and other contents. Apart from their main products and associated Overall cell density of the tissue varies enormously enzymes, chondrocytes have an active role in carti- between different adult cartilages. This is easiest to lage calcification. Matrix vesicles 0-1-1-0 ,um investigate in joint cartilage where the articular diameter containing alkaline phosphatase (Ali et al., surface is not covered by a perichondrium. Femoral on September 25, 2021 by guest. Protected 1970) are deposited in the longitudinal septa of the condylar cartilage shows a 25-fold difference between growth plate by chondrocyte cell processes. They small species (mice) which have very cellular appear to act as vectors for calcium by virtue of their cartilage and large species such as man (Table 2). On membrane lipids (for example, phosphatidylserine) the other hand, a similar range of cellularity is found and other factors, thereby nucleating apatite crystals between the small and large joints of a single large (Wuthier, 1977). The chondrocytes also meciate the species. Similar observations apply to other non- removal of proteoglycan-hyaluronate complexes articular cartilages. Hence a scale effect is involved: (thought to be inhibitory to calcification) from the the overall cellularity of the tissue is inversely related hypertrophic zone by producing the enzyme lysozyme to the common factor, cartilage thickness (Fig. 6). which disaggregates them (Kuettner et al., 1975). It follows from such a relationship that the total Chondrocytes also have a positive role in the number of chondrocytes nourished by diffusion from exclusion of blood vessels from the tissue. A factor unit area of tissue boundary (for example, the which prevents endothelial proliferation is produced synovial fluid bathing the articular surface) is of the by cartilage. This antagonises the angiogenic factors same order in all cartilages and that the degree of J Clin Pathol: first published as 10.1136/jcp.s3-12.1.7 on 1 January 1978. Downloaded from

Chondrocytes 11 Table 2 Cellularity of femoral condylar cartilage 30-50% during adult life, but in normal humeral- (from Stockwell, 1971) head cartilage no such changes are observed Source of Car tilage Cell density No. of cells (x 104) (Meachim and Collins, 1962; Stockwell, 1967; cartilage thickness (cells x deep to 1 mm' Vignon et al., 1976). In the femoral head, though not (mm) 104/mm3) articular surface the condylar cartilage, the superficial zone losses are Man 2-26 1-4 31 large enough to cause a change in the overall Cow 1-68 2-0 3-3 cell density of the whole thickness of the tissue. Sheep 0-84 5-3 4-2 Dog 0-67 4-4 2-7 Probably in arthritis-prone joints such as the hip and Rabbit 0-21 18-8 3-7 knee the age-related cell loss of the superficial zone Cat 0 33 10-8 3-2 is associated with ultrastructural fissures and other Rat 0 07 26-5 1.9 Mouse 006 33-4 1 9 aberrations seen in surfaces judged intact by light microscopy. The lack of change in the shoulder suggests strongly that the cell loss in otherjoints is an 600 abnormal phenomenon rather than a physiological age change. Whether superficial cell loss is a primary event or secondary to other defects is not known, but it must reduce the capacity of the cartilage to I 500 I resist harmful agents. i References

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