Anat Embryol (1992) 186: 611-618
Anatomyand Efiabry01ogy 9 Springer-Verlag 1992
Development and ageing of phenotypically distinct fibrocartilages associated with the rat Achilles tendon A. Rufai, M. Benjamin, and J.R. Ralphs Department of Anatomy, University of Wales College of Cardiff, PO Box 900, Cardiff CFI 3YF, Wales, U.K. Accepted September 7, 1992
Summary. We describe by routine histology and by immunohistochemistry three phenotypically and developmentally distinct fibrocartilages associated with the Achilles tendon of the rat. All the fibrocartilages develop after birth and show significant age-related changes in the composition of their extracellular matrix. Attachment-zone fibrocartilage occurs at the insertion of the tendon on the calcaneus. It derives from the cartilage rudiment of the calcaneus and f r o m the region where the tendon merges with the perichondrium. The extracellular matrix contain type II collagen and chondroitin sulphate. Compressive tendon fibrocartilage occurs in the deep part of the tendon where it presses against the calcaneus, and is derived by metaplasia of tendon cells. The cells label strongly for the intermediate filament vimentin, and the extracellular matrix contains chondroitin and keratan sulphates, but type II collagen only in very old animals ( > 2 years). Calcaneal fibrocartilage covered the posterior surface of the calcaneus where it was in contact with the Achilles tendon. It labelled intensely for type II collagen and contained chondroitin and keratan sulphates. The cells were rich in vimentin. This fibrocartilage was derived from the calcaneal perichondrium. Key words: Tendon fibrocartilage - Development - Ageing - Extracellular matrix - Vimentin
Introduction Fibrocartilage is found at the attachment of tendons and ligaments to bone and provides a gradual transition in mechanical properties between them (Woo et al. 1988). It also occurs where tendons pass around bony pulleys, resisting compression and providing smooth gliding surfaces (Vogel and K o o b 1989). The correspondCorrespondence to: J.R. Ralphs
ing bones are themselves protected witl~ fibrocartilage (Stilwell and G r a y 1954). In previous work, we have shown that functionally distinct fibrocartilages within the rat quadriceps tendon are phenotypically and developmentally different (Ralphs et al. 1991, 1992). In the present study, we have examined three further fibrocartilages, associated with the rat Achilles tendon, to assess further the range of tissues that can be called fibrocartilage, and to gain insight into the differentiated status of fibrocartilage cells. The fibrocartilages occur at the attachment of the tendon to the calcaneus, within the tendon itself, and on the corresponding bony surface of the calcaneus. We show that they are developmentally and structurally different, and have distinctive extracellular matrices that vary with age.
Materials and methods Animals. White Wistar rats were used throughout the study. The youngest examined were 21-day fetuses, and the following ages were examined after birth: neonates; 1, 2, 4, 6 and 8 weeks; and 3, 6, 12, 14, 18, 24 and 28 months. A minimum of four animals from each age group was examined, two histologically, and two by immunohistochemistry. Histology. Animals were killed by cervical dislocation after anaesthetising with ether. Ankles were fixed in 10% neutral buffered formol saline for 1 week, decalcified in 2% nitric acid, dehydrated in graded alcohols and embedded in paraffin wax. Serial 8-gm sections were cut sagitally and stained with Masson's trichrome or toluidine blue for metachromasia. Immunohistochemistry. Tissue was obtained as above, fixed in 95%
alcohol at 4~ C for 2 h and washed in 0.1 M phosphate-buffered saline (PBS). The specimens were decalcified in 5% EDTA in PBS for 1 week at 4~ C, washed in PBS and infiltrated overnight with PBS containing 5% sucrose. They were mounted on cryostat chucks, frozen with dry ice, and cryosections were cut at 10 gin. Sections were immunolabelled by standard procedures for immunofluorescence, using a panel of monoclonal antibodies to extracellular matrix (ECM) and cytoskeletal components. Antibody specificities and procedures have been described in detail previously
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Fig, 1 a-e. Histological appearance of the three fibrocartilages associated with the Achilles tendon of the adult rat. Stained with toluidine blue for metachromasia a Low-power view of attachment-zone fibrocartilage (AZ), compressive tendon fibrocartilage (CTF) and calcaneal fibrocartilage (CF). C, calcaneus; arrow, synovial fold. Note the overlying long flexor tendon of the toes. • 96. b Highpower micrograph of attachment-zone fibrocartilage. The cells are
rounded, arranged in rows and have intensely metachromatic pericellular matrix. B, bone. x 190. e High power micrograph of calcaneal fibrocartilage. The cells are rounded and arranged in a manner similar to that of articular cartilage. However, the cells are smaller and the ECM is more fibrous. Arrow, surface articulating with the Achilles tendon; B, bone. x 104
(Ralphs et al. 1991). Briefly, the primary antibodies were: CIICI to type II collagen (Holmdahl et al. 1986; from the Developmental Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Md., and the Department of Biology, University of Iowa, Iowa City, Iowa, under contract NO1-HD6-2915 from the NICHD); MZ15 to keratan sulphate (Zanetti et al. 1985; Mehmet et al. 1986; from the Kennedy Institute, London); CS56 to chondroitin sulphate (Avnur and Geiger 1984; Sigma Chemical Company, Poole, Dorset, UK); and Vim 13.2 to vimentin (Sigma Chemical Company). Controls were incubated with nonimmune mouse immunoglobulins (Sigma Chemical Company). Sections to be labelled with CIICI and MZI5 were pretreated with
a cocktail of hyaluronidase (1.5 IU/ml; type I-S, Sigma Chemical Company) and chondroitinase ABC (0.25 IU/ml; Sigma Chemical Company). Primary antibody binding was detected using FITCconjugated rabbit anti-mouse immunoglobulins (Dako, High Wycombe, Bucks., UK).
Results
Position and structure o f fibrocartilages in 3-month rats There were three fibrocartilages associated with the Achilles t e n d o n o f these sexually m a t u r e rats. T h e y were
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Fig. 2 a-g. Immunohistochemical localisation of ECM and cytoskeletal components in adult rats. a Distribution of type II collagen in attachment-zone fibrocartilage. Strong labelling is present at the attachment of the Achilles tendon (AT) to the calcaneus (C). Label is continuous with the calcaneal fibrocartitage (CF) and superficially with that at the attachment of the long plantar ligament (LPL) and on the deep surface of the attachment zone with spicules of calcified cartilage in the underlying bone (arrows). EP, epiphyseal plate, x 38. b Distribution of keratan sulphate in compressive tendon fibrocartilage. Strong label is present pericellularly and weaker label in the interterritorial matrix. Labelling for chondroitin
sulphate is similar, x 240. e Strong immunolabel for vimentin in rounded cells from the deep portion of the long flexor tendon where it presses against the Achilles tendon, x 240. d Strong immunolabel for vimentin in cells of compressive tendon fibrocartilage. The cells retain the organisation in rows typical of tendon. x 380. e-g Distribution of ECM components in calcaneal fibrocartilage. Arrow, surface articulating with the Achilles tendon, e Type II collagen ( x 240), f chondroitin sulphate ( x 240) and g keratan sulphate ( x 380). Note the uniform distribution of type II collagen, and the largely pericellular distribution of chondroitin and keratan sulphate
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attachment-zonefibrocartilage, where the tendon was inserted into the bone; compressive tendon fibrocartilage, where the tendon pressed against the calcaneus; and calcanealfibroeartilage, covering the corresponding surface of the calcaneus (Fig. 1 a). A bursa was present between the Achilles tendon and the calcaneus, and a fatty synovial fold protruded into it. Attachment-zone fibrocartilage contained rows of large rounded cells with metachromatic pericellular matrix, lying between parallel arrays of tendon fibres (Fig. i b). Superficial fibres merged with those of the opposing long plantar ligament, central fibres were inserted into calcaneal bone, and deep fibres were continuous with the calcaneal fibrocartilage. The attachment zone was strongly labelled for type II collagen and chondroitin sulphate, the latter being mainly pericellular in distribution. Type II collagen labelling was continuous with the calcaneal fibrocartilage, with the attachment of the long plantar ligament, and with spicules of calcified cartilage extending into the bone (Fig. 2 a). There was little labelling for keratan sulphate within the attachment zone itself, although pericellular label was present around adjacent tendon cells. Compressive tendon fibrocartilage occupied the deeper part of the tendon, proximal to the attachment zone, as demonstrated by metachromasia (Fig. l a). Staining progressively increased towards the deep surface. In the superficial part of the fibrocartilage the arrangement of cells and fibres resembled that of normal tendon, although the cells were larger and enclosed by a metachromatic pericellular matrix. Deeper in the fibrocartilage, metachromasia was more general and the organisation of cells and fibres less regular. The ECM contained chondroitin and keratan sulphate, with labelling being especially intense pericellularly (Fig. 2b), but type II collagen was absent. The fibrocartilage cells were strongly labelled for vimentin (Fig. 2d). Notably, chondroitin sulphate and vimentin were also present in the superficial part of the Achilles tendon and the deep part of the overlying long flexor tendon where the two tendons were pressed together (Fig. 2c). However, there was no histological evidence of fibrocartilage at these sites. Calcaneal fibrocartilage covered the posterior surface of the calcaneus. At low magnification the tissue resembled articular hyaline cartilage, but it was more highly cellular, and had smaller cells and strongly fibrous ECM (Fig. 1 c). The cells, particularly in the more superficial part of the fibrocartilage, were rich in vimentin. The ECM was intensely labelled for type II collagen, and also contained keratan and chondroitin sulphate, usually in pericellular distribution (Fig. 2 e-g).
ing cartilage prior to the formation of a secondary centre of ossification. This centre was well established by 4 weeks, and the greater part of the cartilage in the calcaneus had been eroded with the onset of endochondral ossification, leaving spicules of calcified cartilage embedded in the newly deposited bone, continuous with the deep surface of the attachment zone. Cells in the attachment zone itself were rounded but arranged in rows typical of tendon organisation (Fig. 3c). Up to 2 weeks, labelling for type II collagen corresponded to the distribution of cartilage (Fig. 4a). It progressively disappeared as the cartilage was eroded, with the calcified spicules remaining strongly positive (Fig. 4b; see also Fig. 2a). However, not all of the type II collagen in the attachment zone was derived from the cartilage. In neonates, labelling did not extend into the region where the tendon blended with perichondrium (Fig. 4a; see also Fig. 3b), but by 6 weeks it continued a short distance into the tendon itself (Fig. 4b). In animals older than 12 months, type II collagen spread progressively further with age from the attachment zone into the tendon (Fig. 4c). Chondroitin sulphate was present in all animals, but there was little keratan sulphate.
Compressive tendon fibrocartilage. This fibrocartilage was absent at birth. In fetuses and neonates the tendon was highly cellular, having small cells aligned in rows, separated by small amounts of ECM (Fig. 3b). By 2 weeks the tendon had thickened, with more ECM deposited between the cell rows. The fibrocartilaginous region could now be recognised, with enlarged ceils and metachromatic matrix. By 4 weeks the tendon was thicker and metachromasia of the fibrocartilage increased, particularly towards the deep surface (Fig. 3d). These changes became more pronounced as the animals matured. In aged rats, the fibrocartilage was strongly metachromatic. The cells of the deep region were larger and more rounded than in young animals. The composition of the ECM changed during development and ageing. Chondroitin sulphate was present at all ages (e.g. Fig. 4d), whereas keratan sulphate first appeared pericellularly at 1 week. Subsequently, the intensity of keratan sulphate labelling increased with age. Type II collagen was only present in animals older than 2 years. Label was weak and patchy in the interterritorial matrix, but strong pericellularly (Fig. 4 e). Fibrocartilage cells labelled brightly for vimentin at all ages where the fibrocartilage was recognisable histologically.
Fig. 3a-h. Histological appearance of the fibrocartilages in devel-
Development and ageing of fibrocartilages Attachment-zone fibrocartilage. At birth, the Achilles tendon was attached to the cartilage model of the calcaneus, where it blended with the perichondrium (Fig. 3 a, b). Superficially, it was continuous with the long plantar ligament via the perichondrium, as in adults. By 2 weeks, hypertrophic chondrocytes were present in the underly-
opment and ageing, a Low-power micrograph to show the posterior part of the ankle in a neonatal rat. A T, Achilles tendon; C, calcaneus; arrow, bursa. Masson's trichrome, x 45 b Higher-power view of the neonatal Achilles tendon (A T). The tendon is highly cellular, with little ECM between the rows of cells. It inserts into the perichondrium of the calcaneus (C). The position of the calcaneal fibrocartilage (CF) is represented by perichondrium containing flattened cells which lie parallel to the surface of the cartilage. The perichondrium is covered by the synovial lining of the bursa (arrow). Masson's trichrome, x 130. e Attachment of the Achilles tendon to
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the calcaneus in a 4-week-old rat. Most of the cartilage of the calcaneus has been eroded in endochondral ossification. Spicules of calcified cartilage (arrows) are continuous with the attachment zone (AZ). Toluidine blue, x 64. d Calcaneal (CF) and compressive tendon (CTF) fibrocartilage in a 6-week-old rat. Note typical tendon organisation (7) away from the articular surface, with less regular fibre arrangement and increased metachromasia towards that surface. The calcaneal fibrocartilage has remnants of the calcaneal cartilage beneath it (arrows). Toluidine blue, x 104. e--h Development and ageing of calcaneal fibrocartilage. Toluidine blue.
e In a 2-week-old rat, the calcaneal fibrocartilage (CF) is fibrous but not metachromatic. It covers the surface of the calcaneal cartilage rudiment. The underlying chondrocytes are hypertrophic. x 321. f In a 4-week-old rat, the calcaneal fibrocartilage has become metachromatic, and its cells more prominent. Much of the underlying cartilage has been eroded. • 321. g In a 6-week-old rat, the calcaneal fibrocartilage has thickened and become intensely metachromatic, x 321. h In a 2-year-old rat, the calcaneal fibrocartilage remains intensely metachromatic, and, like 4-month animals (Fig. lc) is less cellular than at 2, 4 or 6 weeks, x 321
Fig, 4a-g. Immunohistochemical localisation of ECM molecules in developing and ageing fibrocartilages, a Type [I collagen in the calcaneus of a neonatal rat. Label is present in the cartilage rudiment, but absent from the tendon (7) and the presumptive calcaneal fibrocartilage (arrow). x 96. b Type II collagen in the calcaneus of a 6-week-old rat. Replacement of cartilage by bone leaves residual type II collagen in calcified cartilage spicules (S) continuous with the deep part of the attachment zone, between the bone (B) and tendon (T). Away from the bone, type II collagen surrounds cells arranged in rows typical of tendon (arrows). x 96. c Type II collagen in the attachment zone of a 14-month-old rat. Note the spreading of immunolabel from the attachment zone (AZ) along the tendon (T). C, calcaneus, x 96. d Chondroitin sulphate
distribution in all three fibrocartilages in an 8-week-old rat. Strong labelling is present in the attachment zone (AZ), compressive tendon (CTF) and calcaneal (CF) fibrocartilages. Label is also present in the deep part of the overlying long flexor tendon (arrow). x 96. e Type II collagen in the compressive tendon fibrocartilage of a 28-month-old rat. Moderate pericellular and weak, patchy interterritorial matrix labelling is present. The calcaneal fibrocartilage is strongly labelled (CF). • 240. f-g Type II collagen in developing calcaneal fibrocartilage; f 2-week and g 6-week-old animals. Labelling of calcaneal fibrocartilage does not extend to the surface (arrow) at 2 weeks, but extends throughout the fibrocartilage at 6 weeks. Compare with Fig. 3 e-g. x 240
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Calcaneal fibrocartilage. In neonatal rats, the position of this fibrocartilage was occupied by the perichondrium and overlain by the synovial lining of the bursa (Fig. 3 a, b). Tissue having the histological appearance of fibrocartilage was present at this site by 2 weeks (Fig. 3 e). At this stage, the cells were smaller than the underlying chondrocytes and the ECM was not metachromatic. By 4 weeks (Fig. 3 f), the cartilage of the developing calcaneus had largely been eroded, leaving a small number of chondrocytes embedded in metachromatic matrix at the deep surface of the fibrocartilage. Metachromasia increased in the fibrocartilage, but was not as intense as that of the cartilage remnants. By 6 weeks, the fibrocartilage had thickened considerably and become intensely metachromatic (Fig. 3g). In aged animals (18-24 months) the resemblance between the fibrocartilage and hyaline cartilage was stronger than in 3- to 4-month-old animals (Fig. 3 h). However, the fibrocartilage remained more fibrous and cellular than the nearby tarsal articular cartilages. The fibrocartilage cells were strongly labelled for vimentin at all ages. There were changes in the composition of the ECM during development and ageing. Type II collagen was restricted to the deep portion of the fibrocartilage in 2-week rats, but subsequently the fibrocartilage was strongly positive throughout (Fig. 4f, g). As for compressive tendon fibrocartilage, calcaneal fibrocartilage was labelled for chondroitin sulphate at all ages (e.g. Fig. 4d). Pericellular labelling for keratan sulphate was first seen at 1 week and the intensity of labelling increased subsequently. The tissue remained strongly metachromatic, and the cells became larger and more rounded with age. Discussion
This study shows that the three functionally distinct fibroeartilages associated with the rat Achilles tendon are developmentally and phenotypically different. Attachment-zone fibrocartilage is similar in some respects to that of the rat quadriceps tendon (Ralphs et al. 1991, 1992), but the others are unlike those described previously. Thus, there are four phenotypically distinct fibrocartilages associated with just two rat tendons: attachmentzone fibrocartilage, the suprapatella of the quadriceps tendon, compressive tendon fibrocartilage of the Achilles tendon, and calcaneal fibrocartilage. Attachment-zone fibrocartilage is initially derived from the cartilage rudiment of the calcaneus, and later from the region where the tendon merges with the perichondrium. This region is continuous with the calcaneal fibrocartilage, which has a similar origin (see later). The origin of attachment-zone fibrocartilage from hyaline cartilage was seen in the quadriceps tendon (Ralphs et al. 1992), although at this site we did not observe subsequent differentiation of a perichondrial region. As the patella develops as a sesamoid bone within the differentiating quadriceps tendon there may be no comparable perichondrium; the potential for contribution of peripheral cells would therefore be limited. The typical arrangement of attachment-zone fibrocartilage cells in rows
seems to be due to spreading of type II collagen label a short distance into the tendon, followed by erosion of most of the cartilage-derived fibrocartilage beneath it in endochondral ossification. With age the spreading continues although at a much lower rate. This has been reported previously in the quadriceps tendon (Benjamin et al. 1991). Clinically, such age-related changes could predispose to bony spur formation, by providing a basis for endochondral ossification. Compressive tendon fibrocartilage develops within the tendon itself, and the maintenance of normal tendon architecture suggests that it forms by metaplasia of tendon cells. This development is similar to that of fibrocartilage experimentally induced in tendons in the foot (Ploetz 1937; Gillard et al. 1979; Vogel and Koob 1989 review). All of these fibrocartilages have the common feature that they accumulate glycosaminoglycans more usually associated with cartilage. These presumably serve a similar function in fibrocartilage, contributing to the pressure resistance of the tissue by attracting water into the ECM. The presence of keratan and chondroitin sulphate in compressive tendon fibrocartilage of all animals, but the restriction of type II collagen to very old animals, suggests that the glycosaminoglycan content of tendons can be modified relatively easily, but that it takes a long period (> 2 years in rats) to change the collagen type. Type II collagen has been detected in another ageing compressive fibrocartilage, the suprapatella of the quadriceps tendon. Here it appeared earlier, in animals older than 6 months (Benjamin et al. 1991 ; Ralphs et al. 1991). This difference could be due to the distinct origins of the fibrocartilages. Suprapatellar fibrocartilage develops from a cell population at the tendon periphery (Ralphs et al. 1992) that even in adults is not fully differentiated to the tendon phenotype. This is shown by the rapid cell division that occurs at this site following tendon injury (Gelberman et al. 1988). It may be easier, therefore, for such cells to express components not normally found in tendon, such as type II collagen. Alternatively, matrix differences could be due to the relative pressures exerted on the matrix at the two sites - the suprapatella could be under greater compressive load than the compressive tendon fibrocartilage of the Achilles tendon. It might, therefore, accumulate cartilage-like matrix components more rapidly. Whatever the reason, with age both fibrocartilages synthesise a collagen characteristic of cartilage. It may be that this forms a more efficient network with the glycosaminoglycans for pressure resistance. The accumulation of vimentin in cells of compressive tendon and calcaneal fibrocartilages is an early event in their differentiation, along with chondroitin sulphate deposition in the ECM. This is also the case in development of the suprapatella of the quadriceps tendon, where vimentin can be used as a differentiation marker. Accumulation of intermediate filaments may be important in the ability of the cells to resist compression (Ralphs et al. 1992). Calcaneal fibrocartilage forms mainly from the perichondrium of the calcaneus, with the deepest part de-
618 rived from the cartilage of the early calcaneus as for attachment-zone fibrocartilage. It is possible that superficially some cells could have come from the synovium of the overlying bursa, although the thinness of this layer would suggest that this was not a significant component. The early absence and rapid accumulation of type II collagen by this fibrocartilage is its most notable feature, and it shares this with the perichondrially-derived region of the attachment zone (see above). The rapid transformation of the E C M could again be related to the origin of the tissue. In early development, perichondrial cells are derived f r o m the outer part of the chondrogenic condensation (Rooney et al. 1984). It m a y be that, at least in young animals, the cells retain their ability to synthesise type II collagen in response to appropriate stimuli. Alternatively, mechanical influences m a y be important. This fibrocartilage could be the most compressed of the fibrocartilages examined, as it is positioned on an unyielding b o n y surface, and must withstand the entire force exerted by the overlying tendon. The fibrocartilage has a full complement of cartilage components (in the context of this study) suggesting that it is the most efficient at resisting compressive forces, and thus is particularly well adapted to its site. The stimulus for development of both compressive tendon and calcaneal fibrocartilages is likely to be the onset of locomotion in young animals, as neither is present in neonates. It is interesting that there is another region within the Achilles tendon that could be regarded as a form of fibrocartilage. Immunohistochemically, the superficial part of the tendon is like fibrocartilage, but histologically it is indistinguishable from tendon, even with toluidine blue staining. Functionally, this region probably represents a simple f o r m of compressive tendon fibrocartilage, as it is pressed by the overlying deep flexor tendon of the toes. It could be that the pressure is relatively weak, and vimentin and chondroitin sulphate synthesis represent low level responses to compression, with histological change appearing along with keratan sulphate and type II collagen in more highly stressed tissue. We would expect to find a continuous range of metaplastic tendon fibrocartilages according to mechanical conditions and age.
Acknowledgements. This work was funded in part by the Nuffield Foundation. Aminu Rufai is supported by the Nigerian Government.
References Avnur Z, Geiger B (1984) Immunocytochemical localisation of native chondroitin-sulfate in tissues and cultured cells using specific monoclonal antibody. Cell 38 : 811-822 Benjamin M, Tyers RNS, Ralphs JR (1991) Age-related changes in tendon fibrocartilage. J Anat 179:127 136 Gelberman R, Goldberg V, An K-N, Banes A (1988) Tendon. In: Woo SL-Y, Buckwalter JA (eds) Injury and repair of the musculoskeletal soft tissues. American Academy of Orthopaedic Surgeons, Illinois, pp 5-40 Gillard GC, Reilly HC, Bell-Booth PG, Flint MH (1979) The influence of mechanical forces on the glycosaminoglycan content of the rabbit flexor digitorum profundus tendon. Connect Tissue Res 7:37-46 Holmdahl R, Rubin K, Klareskog L, Larsson E, Wigzell H (1986) Characterisation of the antibody response in mice with type II collagen-induced arthritis, using monoclonal anti-type II collagen antibodies. Arthritis Rheum 29: 400-410 Mehmet H, Scudder P, Tang PW, Hounsell EF, Caterson B, Feizi T (1986) The antigenic determinants recognized by three monoclonal antibodies to keratan sulphate involve sulphated heptaor larger oligosaccharides of the poly (N-acetyllactosamine) series. Eur J Biochem 157 : 385-391 Ploetz E (1937) Funktioneller Bau und funktionelle Anpassung der Gleitsehne. Z Orthop 67:212-234 Ralphs JR, Benjamin M, Thornett A (1991) Ceil and matrix biology of the suprapatella in the rat: a structural and immunocytochemical study of fibrocartilage in a tendon subject to compression. Anat Rec 231:167-177 Ralphs JR, Tyers RNS, Benjamin M (1992) Development of fnnctionally distinct fibrocartilages at two sites in the quadriceps tendon of the rat: the suprapatella and the attachment to the patella. Anat Embryol 185 : 181-187 Rooney P, Archer CW, Wolpert L (1984) Morphogenesis of cartilaginous long bone rudiments. In: Trelstad RL (ed) The role of extracellular matrix in development. Liss, New York, pp 305-322 Stilwell DL, Gray DJ (1954) The structure of bony surfaces in contact with tendons. Anat Rec 118:358-359 Vogel KG, Koob TJ (1989) Structural specialization in tendons under compression. Int Rev Cytol 115 : 267-293 Woo S, Maynard J, Butler D, Lyon R, Torzilli P, Akeson W, Cooper R, Oakes B (1988) Ligament, tendon, and joint capsule insertions to bone. In: Woo SL-Y, Buckwalter JA (eds) Injury and repair of the musculoskeletal soft tissues. American Academy of Orthopaedic Surgeons, Illinois, pp 133-166 Zanetti M, Ratcliffe A, Watt FM (1985) Two subpopulations of differentiated chondrocytes identified with a monoclonai antibody to keratan sulfate. J Cell Biol 101 : 53-59
Anatomyand Efiabry01ogy 9 Springer-Verlag 1992
Development and ageing of phenotypically distinct fibrocartilages associated with the rat Achilles tendon A. Rufai, M. Benjamin, and J.R. Ralphs Department of Anatomy, University of Wales College of Cardiff, PO Box 900, Cardiff CFI 3YF, Wales, U.K. Accepted September 7, 1992
Summary. We describe by routine histology and by immunohistochemistry three phenotypically and developmentally distinct fibrocartilages associated with the Achilles tendon of the rat. All the fibrocartilages develop after birth and show significant age-related changes in the composition of their extracellular matrix. Attachment-zone fibrocartilage occurs at the insertion of the tendon on the calcaneus. It derives from the cartilage rudiment of the calcaneus and f r o m the region where the tendon merges with the perichondrium. The extracellular matrix contain type II collagen and chondroitin sulphate. Compressive tendon fibrocartilage occurs in the deep part of the tendon where it presses against the calcaneus, and is derived by metaplasia of tendon cells. The cells label strongly for the intermediate filament vimentin, and the extracellular matrix contains chondroitin and keratan sulphates, but type II collagen only in very old animals ( > 2 years). Calcaneal fibrocartilage covered the posterior surface of the calcaneus where it was in contact with the Achilles tendon. It labelled intensely for type II collagen and contained chondroitin and keratan sulphates. The cells were rich in vimentin. This fibrocartilage was derived from the calcaneal perichondrium. Key words: Tendon fibrocartilage - Development - Ageing - Extracellular matrix - Vimentin
Introduction Fibrocartilage is found at the attachment of tendons and ligaments to bone and provides a gradual transition in mechanical properties between them (Woo et al. 1988). It also occurs where tendons pass around bony pulleys, resisting compression and providing smooth gliding surfaces (Vogel and K o o b 1989). The correspondCorrespondence to: J.R. Ralphs
ing bones are themselves protected witl~ fibrocartilage (Stilwell and G r a y 1954). In previous work, we have shown that functionally distinct fibrocartilages within the rat quadriceps tendon are phenotypically and developmentally different (Ralphs et al. 1991, 1992). In the present study, we have examined three further fibrocartilages, associated with the rat Achilles tendon, to assess further the range of tissues that can be called fibrocartilage, and to gain insight into the differentiated status of fibrocartilage cells. The fibrocartilages occur at the attachment of the tendon to the calcaneus, within the tendon itself, and on the corresponding bony surface of the calcaneus. We show that they are developmentally and structurally different, and have distinctive extracellular matrices that vary with age.
Materials and methods Animals. White Wistar rats were used throughout the study. The youngest examined were 21-day fetuses, and the following ages were examined after birth: neonates; 1, 2, 4, 6 and 8 weeks; and 3, 6, 12, 14, 18, 24 and 28 months. A minimum of four animals from each age group was examined, two histologically, and two by immunohistochemistry. Histology. Animals were killed by cervical dislocation after anaesthetising with ether. Ankles were fixed in 10% neutral buffered formol saline for 1 week, decalcified in 2% nitric acid, dehydrated in graded alcohols and embedded in paraffin wax. Serial 8-gm sections were cut sagitally and stained with Masson's trichrome or toluidine blue for metachromasia. Immunohistochemistry. Tissue was obtained as above, fixed in 95%
alcohol at 4~ C for 2 h and washed in 0.1 M phosphate-buffered saline (PBS). The specimens were decalcified in 5% EDTA in PBS for 1 week at 4~ C, washed in PBS and infiltrated overnight with PBS containing 5% sucrose. They were mounted on cryostat chucks, frozen with dry ice, and cryosections were cut at 10 gin. Sections were immunolabelled by standard procedures for immunofluorescence, using a panel of monoclonal antibodies to extracellular matrix (ECM) and cytoskeletal components. Antibody specificities and procedures have been described in detail previously
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Fig, 1 a-e. Histological appearance of the three fibrocartilages associated with the Achilles tendon of the adult rat. Stained with toluidine blue for metachromasia a Low-power view of attachment-zone fibrocartilage (AZ), compressive tendon fibrocartilage (CTF) and calcaneal fibrocartilage (CF). C, calcaneus; arrow, synovial fold. Note the overlying long flexor tendon of the toes. • 96. b Highpower micrograph of attachment-zone fibrocartilage. The cells are
rounded, arranged in rows and have intensely metachromatic pericellular matrix. B, bone. x 190. e High power micrograph of calcaneal fibrocartilage. The cells are rounded and arranged in a manner similar to that of articular cartilage. However, the cells are smaller and the ECM is more fibrous. Arrow, surface articulating with the Achilles tendon; B, bone. x 104
(Ralphs et al. 1991). Briefly, the primary antibodies were: CIICI to type II collagen (Holmdahl et al. 1986; from the Developmental Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Md., and the Department of Biology, University of Iowa, Iowa City, Iowa, under contract NO1-HD6-2915 from the NICHD); MZ15 to keratan sulphate (Zanetti et al. 1985; Mehmet et al. 1986; from the Kennedy Institute, London); CS56 to chondroitin sulphate (Avnur and Geiger 1984; Sigma Chemical Company, Poole, Dorset, UK); and Vim 13.2 to vimentin (Sigma Chemical Company). Controls were incubated with nonimmune mouse immunoglobulins (Sigma Chemical Company). Sections to be labelled with CIICI and MZI5 were pretreated with
a cocktail of hyaluronidase (1.5 IU/ml; type I-S, Sigma Chemical Company) and chondroitinase ABC (0.25 IU/ml; Sigma Chemical Company). Primary antibody binding was detected using FITCconjugated rabbit anti-mouse immunoglobulins (Dako, High Wycombe, Bucks., UK).
Results
Position and structure o f fibrocartilages in 3-month rats There were three fibrocartilages associated with the Achilles t e n d o n o f these sexually m a t u r e rats. T h e y were
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Fig. 2 a-g. Immunohistochemical localisation of ECM and cytoskeletal components in adult rats. a Distribution of type II collagen in attachment-zone fibrocartilage. Strong labelling is present at the attachment of the Achilles tendon (AT) to the calcaneus (C). Label is continuous with the calcaneal fibrocartitage (CF) and superficially with that at the attachment of the long plantar ligament (LPL) and on the deep surface of the attachment zone with spicules of calcified cartilage in the underlying bone (arrows). EP, epiphyseal plate, x 38. b Distribution of keratan sulphate in compressive tendon fibrocartilage. Strong label is present pericellularly and weaker label in the interterritorial matrix. Labelling for chondroitin
sulphate is similar, x 240. e Strong immunolabel for vimentin in rounded cells from the deep portion of the long flexor tendon where it presses against the Achilles tendon, x 240. d Strong immunolabel for vimentin in cells of compressive tendon fibrocartilage. The cells retain the organisation in rows typical of tendon. x 380. e-g Distribution of ECM components in calcaneal fibrocartilage. Arrow, surface articulating with the Achilles tendon, e Type II collagen ( x 240), f chondroitin sulphate ( x 240) and g keratan sulphate ( x 380). Note the uniform distribution of type II collagen, and the largely pericellular distribution of chondroitin and keratan sulphate
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attachment-zonefibrocartilage, where the tendon was inserted into the bone; compressive tendon fibrocartilage, where the tendon pressed against the calcaneus; and calcanealfibroeartilage, covering the corresponding surface of the calcaneus (Fig. 1 a). A bursa was present between the Achilles tendon and the calcaneus, and a fatty synovial fold protruded into it. Attachment-zone fibrocartilage contained rows of large rounded cells with metachromatic pericellular matrix, lying between parallel arrays of tendon fibres (Fig. i b). Superficial fibres merged with those of the opposing long plantar ligament, central fibres were inserted into calcaneal bone, and deep fibres were continuous with the calcaneal fibrocartilage. The attachment zone was strongly labelled for type II collagen and chondroitin sulphate, the latter being mainly pericellular in distribution. Type II collagen labelling was continuous with the calcaneal fibrocartilage, with the attachment of the long plantar ligament, and with spicules of calcified cartilage extending into the bone (Fig. 2 a). There was little labelling for keratan sulphate within the attachment zone itself, although pericellular label was present around adjacent tendon cells. Compressive tendon fibrocartilage occupied the deeper part of the tendon, proximal to the attachment zone, as demonstrated by metachromasia (Fig. l a). Staining progressively increased towards the deep surface. In the superficial part of the fibrocartilage the arrangement of cells and fibres resembled that of normal tendon, although the cells were larger and enclosed by a metachromatic pericellular matrix. Deeper in the fibrocartilage, metachromasia was more general and the organisation of cells and fibres less regular. The ECM contained chondroitin and keratan sulphate, with labelling being especially intense pericellularly (Fig. 2b), but type II collagen was absent. The fibrocartilage cells were strongly labelled for vimentin (Fig. 2d). Notably, chondroitin sulphate and vimentin were also present in the superficial part of the Achilles tendon and the deep part of the overlying long flexor tendon where the two tendons were pressed together (Fig. 2c). However, there was no histological evidence of fibrocartilage at these sites. Calcaneal fibrocartilage covered the posterior surface of the calcaneus. At low magnification the tissue resembled articular hyaline cartilage, but it was more highly cellular, and had smaller cells and strongly fibrous ECM (Fig. 1 c). The cells, particularly in the more superficial part of the fibrocartilage, were rich in vimentin. The ECM was intensely labelled for type II collagen, and also contained keratan and chondroitin sulphate, usually in pericellular distribution (Fig. 2 e-g).
ing cartilage prior to the formation of a secondary centre of ossification. This centre was well established by 4 weeks, and the greater part of the cartilage in the calcaneus had been eroded with the onset of endochondral ossification, leaving spicules of calcified cartilage embedded in the newly deposited bone, continuous with the deep surface of the attachment zone. Cells in the attachment zone itself were rounded but arranged in rows typical of tendon organisation (Fig. 3c). Up to 2 weeks, labelling for type II collagen corresponded to the distribution of cartilage (Fig. 4a). It progressively disappeared as the cartilage was eroded, with the calcified spicules remaining strongly positive (Fig. 4b; see also Fig. 2a). However, not all of the type II collagen in the attachment zone was derived from the cartilage. In neonates, labelling did not extend into the region where the tendon blended with perichondrium (Fig. 4a; see also Fig. 3b), but by 6 weeks it continued a short distance into the tendon itself (Fig. 4b). In animals older than 12 months, type II collagen spread progressively further with age from the attachment zone into the tendon (Fig. 4c). Chondroitin sulphate was present in all animals, but there was little keratan sulphate.
Compressive tendon fibrocartilage. This fibrocartilage was absent at birth. In fetuses and neonates the tendon was highly cellular, having small cells aligned in rows, separated by small amounts of ECM (Fig. 3b). By 2 weeks the tendon had thickened, with more ECM deposited between the cell rows. The fibrocartilaginous region could now be recognised, with enlarged ceils and metachromatic matrix. By 4 weeks the tendon was thicker and metachromasia of the fibrocartilage increased, particularly towards the deep surface (Fig. 3d). These changes became more pronounced as the animals matured. In aged rats, the fibrocartilage was strongly metachromatic. The cells of the deep region were larger and more rounded than in young animals. The composition of the ECM changed during development and ageing. Chondroitin sulphate was present at all ages (e.g. Fig. 4d), whereas keratan sulphate first appeared pericellularly at 1 week. Subsequently, the intensity of keratan sulphate labelling increased with age. Type II collagen was only present in animals older than 2 years. Label was weak and patchy in the interterritorial matrix, but strong pericellularly (Fig. 4 e). Fibrocartilage cells labelled brightly for vimentin at all ages where the fibrocartilage was recognisable histologically.
Fig. 3a-h. Histological appearance of the fibrocartilages in devel-
Development and ageing of fibrocartilages Attachment-zone fibrocartilage. At birth, the Achilles tendon was attached to the cartilage model of the calcaneus, where it blended with the perichondrium (Fig. 3 a, b). Superficially, it was continuous with the long plantar ligament via the perichondrium, as in adults. By 2 weeks, hypertrophic chondrocytes were present in the underly-
opment and ageing, a Low-power micrograph to show the posterior part of the ankle in a neonatal rat. A T, Achilles tendon; C, calcaneus; arrow, bursa. Masson's trichrome, x 45 b Higher-power view of the neonatal Achilles tendon (A T). The tendon is highly cellular, with little ECM between the rows of cells. It inserts into the perichondrium of the calcaneus (C). The position of the calcaneal fibrocartilage (CF) is represented by perichondrium containing flattened cells which lie parallel to the surface of the cartilage. The perichondrium is covered by the synovial lining of the bursa (arrow). Masson's trichrome, x 130. e Attachment of the Achilles tendon to
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the calcaneus in a 4-week-old rat. Most of the cartilage of the calcaneus has been eroded in endochondral ossification. Spicules of calcified cartilage (arrows) are continuous with the attachment zone (AZ). Toluidine blue, x 64. d Calcaneal (CF) and compressive tendon (CTF) fibrocartilage in a 6-week-old rat. Note typical tendon organisation (7) away from the articular surface, with less regular fibre arrangement and increased metachromasia towards that surface. The calcaneal fibrocartilage has remnants of the calcaneal cartilage beneath it (arrows). Toluidine blue, x 104. e--h Development and ageing of calcaneal fibrocartilage. Toluidine blue.
e In a 2-week-old rat, the calcaneal fibrocartilage (CF) is fibrous but not metachromatic. It covers the surface of the calcaneal cartilage rudiment. The underlying chondrocytes are hypertrophic. x 321. f In a 4-week-old rat, the calcaneal fibrocartilage has become metachromatic, and its cells more prominent. Much of the underlying cartilage has been eroded. • 321. g In a 6-week-old rat, the calcaneal fibrocartilage has thickened and become intensely metachromatic, x 321. h In a 2-year-old rat, the calcaneal fibrocartilage remains intensely metachromatic, and, like 4-month animals (Fig. lc) is less cellular than at 2, 4 or 6 weeks, x 321
Fig, 4a-g. Immunohistochemical localisation of ECM molecules in developing and ageing fibrocartilages, a Type [I collagen in the calcaneus of a neonatal rat. Label is present in the cartilage rudiment, but absent from the tendon (7) and the presumptive calcaneal fibrocartilage (arrow). x 96. b Type II collagen in the calcaneus of a 6-week-old rat. Replacement of cartilage by bone leaves residual type II collagen in calcified cartilage spicules (S) continuous with the deep part of the attachment zone, between the bone (B) and tendon (T). Away from the bone, type II collagen surrounds cells arranged in rows typical of tendon (arrows). x 96. c Type II collagen in the attachment zone of a 14-month-old rat. Note the spreading of immunolabel from the attachment zone (AZ) along the tendon (T). C, calcaneus, x 96. d Chondroitin sulphate
distribution in all three fibrocartilages in an 8-week-old rat. Strong labelling is present in the attachment zone (AZ), compressive tendon (CTF) and calcaneal (CF) fibrocartilages. Label is also present in the deep part of the overlying long flexor tendon (arrow). x 96. e Type II collagen in the compressive tendon fibrocartilage of a 28-month-old rat. Moderate pericellular and weak, patchy interterritorial matrix labelling is present. The calcaneal fibrocartilage is strongly labelled (CF). • 240. f-g Type II collagen in developing calcaneal fibrocartilage; f 2-week and g 6-week-old animals. Labelling of calcaneal fibrocartilage does not extend to the surface (arrow) at 2 weeks, but extends throughout the fibrocartilage at 6 weeks. Compare with Fig. 3 e-g. x 240
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Calcaneal fibrocartilage. In neonatal rats, the position of this fibrocartilage was occupied by the perichondrium and overlain by the synovial lining of the bursa (Fig. 3 a, b). Tissue having the histological appearance of fibrocartilage was present at this site by 2 weeks (Fig. 3 e). At this stage, the cells were smaller than the underlying chondrocytes and the ECM was not metachromatic. By 4 weeks (Fig. 3 f), the cartilage of the developing calcaneus had largely been eroded, leaving a small number of chondrocytes embedded in metachromatic matrix at the deep surface of the fibrocartilage. Metachromasia increased in the fibrocartilage, but was not as intense as that of the cartilage remnants. By 6 weeks, the fibrocartilage had thickened considerably and become intensely metachromatic (Fig. 3g). In aged animals (18-24 months) the resemblance between the fibrocartilage and hyaline cartilage was stronger than in 3- to 4-month-old animals (Fig. 3 h). However, the fibrocartilage remained more fibrous and cellular than the nearby tarsal articular cartilages. The fibrocartilage cells were strongly labelled for vimentin at all ages. There were changes in the composition of the ECM during development and ageing. Type II collagen was restricted to the deep portion of the fibrocartilage in 2-week rats, but subsequently the fibrocartilage was strongly positive throughout (Fig. 4f, g). As for compressive tendon fibrocartilage, calcaneal fibrocartilage was labelled for chondroitin sulphate at all ages (e.g. Fig. 4d). Pericellular labelling for keratan sulphate was first seen at 1 week and the intensity of labelling increased subsequently. The tissue remained strongly metachromatic, and the cells became larger and more rounded with age. Discussion
This study shows that the three functionally distinct fibroeartilages associated with the rat Achilles tendon are developmentally and phenotypically different. Attachment-zone fibrocartilage is similar in some respects to that of the rat quadriceps tendon (Ralphs et al. 1991, 1992), but the others are unlike those described previously. Thus, there are four phenotypically distinct fibrocartilages associated with just two rat tendons: attachmentzone fibrocartilage, the suprapatella of the quadriceps tendon, compressive tendon fibrocartilage of the Achilles tendon, and calcaneal fibrocartilage. Attachment-zone fibrocartilage is initially derived from the cartilage rudiment of the calcaneus, and later from the region where the tendon merges with the perichondrium. This region is continuous with the calcaneal fibrocartilage, which has a similar origin (see later). The origin of attachment-zone fibrocartilage from hyaline cartilage was seen in the quadriceps tendon (Ralphs et al. 1992), although at this site we did not observe subsequent differentiation of a perichondrial region. As the patella develops as a sesamoid bone within the differentiating quadriceps tendon there may be no comparable perichondrium; the potential for contribution of peripheral cells would therefore be limited. The typical arrangement of attachment-zone fibrocartilage cells in rows
seems to be due to spreading of type II collagen label a short distance into the tendon, followed by erosion of most of the cartilage-derived fibrocartilage beneath it in endochondral ossification. With age the spreading continues although at a much lower rate. This has been reported previously in the quadriceps tendon (Benjamin et al. 1991). Clinically, such age-related changes could predispose to bony spur formation, by providing a basis for endochondral ossification. Compressive tendon fibrocartilage develops within the tendon itself, and the maintenance of normal tendon architecture suggests that it forms by metaplasia of tendon cells. This development is similar to that of fibrocartilage experimentally induced in tendons in the foot (Ploetz 1937; Gillard et al. 1979; Vogel and Koob 1989 review). All of these fibrocartilages have the common feature that they accumulate glycosaminoglycans more usually associated with cartilage. These presumably serve a similar function in fibrocartilage, contributing to the pressure resistance of the tissue by attracting water into the ECM. The presence of keratan and chondroitin sulphate in compressive tendon fibrocartilage of all animals, but the restriction of type II collagen to very old animals, suggests that the glycosaminoglycan content of tendons can be modified relatively easily, but that it takes a long period (> 2 years in rats) to change the collagen type. Type II collagen has been detected in another ageing compressive fibrocartilage, the suprapatella of the quadriceps tendon. Here it appeared earlier, in animals older than 6 months (Benjamin et al. 1991 ; Ralphs et al. 1991). This difference could be due to the distinct origins of the fibrocartilages. Suprapatellar fibrocartilage develops from a cell population at the tendon periphery (Ralphs et al. 1992) that even in adults is not fully differentiated to the tendon phenotype. This is shown by the rapid cell division that occurs at this site following tendon injury (Gelberman et al. 1988). It may be easier, therefore, for such cells to express components not normally found in tendon, such as type II collagen. Alternatively, matrix differences could be due to the relative pressures exerted on the matrix at the two sites - the suprapatella could be under greater compressive load than the compressive tendon fibrocartilage of the Achilles tendon. It might, therefore, accumulate cartilage-like matrix components more rapidly. Whatever the reason, with age both fibrocartilages synthesise a collagen characteristic of cartilage. It may be that this forms a more efficient network with the glycosaminoglycans for pressure resistance. The accumulation of vimentin in cells of compressive tendon and calcaneal fibrocartilages is an early event in their differentiation, along with chondroitin sulphate deposition in the ECM. This is also the case in development of the suprapatella of the quadriceps tendon, where vimentin can be used as a differentiation marker. Accumulation of intermediate filaments may be important in the ability of the cells to resist compression (Ralphs et al. 1992). Calcaneal fibrocartilage forms mainly from the perichondrium of the calcaneus, with the deepest part de-
618 rived from the cartilage of the early calcaneus as for attachment-zone fibrocartilage. It is possible that superficially some cells could have come from the synovium of the overlying bursa, although the thinness of this layer would suggest that this was not a significant component. The early absence and rapid accumulation of type II collagen by this fibrocartilage is its most notable feature, and it shares this with the perichondrially-derived region of the attachment zone (see above). The rapid transformation of the E C M could again be related to the origin of the tissue. In early development, perichondrial cells are derived f r o m the outer part of the chondrogenic condensation (Rooney et al. 1984). It m a y be that, at least in young animals, the cells retain their ability to synthesise type II collagen in response to appropriate stimuli. Alternatively, mechanical influences m a y be important. This fibrocartilage could be the most compressed of the fibrocartilages examined, as it is positioned on an unyielding b o n y surface, and must withstand the entire force exerted by the overlying tendon. The fibrocartilage has a full complement of cartilage components (in the context of this study) suggesting that it is the most efficient at resisting compressive forces, and thus is particularly well adapted to its site. The stimulus for development of both compressive tendon and calcaneal fibrocartilages is likely to be the onset of locomotion in young animals, as neither is present in neonates. It is interesting that there is another region within the Achilles tendon that could be regarded as a form of fibrocartilage. Immunohistochemically, the superficial part of the tendon is like fibrocartilage, but histologically it is indistinguishable from tendon, even with toluidine blue staining. Functionally, this region probably represents a simple f o r m of compressive tendon fibrocartilage, as it is pressed by the overlying deep flexor tendon of the toes. It could be that the pressure is relatively weak, and vimentin and chondroitin sulphate synthesis represent low level responses to compression, with histological change appearing along with keratan sulphate and type II collagen in more highly stressed tissue. We would expect to find a continuous range of metaplastic tendon fibrocartilages according to mechanical conditions and age.
Acknowledgements. This work was funded in part by the Nuffield Foundation. Aminu Rufai is supported by the Nigerian Government.
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