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Keywords:

  • enthesis;
  • fibrocartilage;
  • force transmission;
  • insertion site;
  • type II collagen

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

The distribution of type II collagen in sagittal sections of the Achilles tendon has been used to reconstruct the three-dimensional (3D) shape and position of three fibrocartilages (sesamoid, periosteal and enthesis) associated with its insertion. The results showed that there is a close correspondence between the shape and position of the sesamoid and periosteal fibrocartilages – probably because of their functional interdependence. The former protects the tendon from compression during dorsiflexion of the foot, and the latter protects the superior tuberosity of the calcaneus. When the zone of calcified enthesis fibrocartilage and the subchondral bone are mapped in 3D, the reconstructions show that there is a complex pattern of interlocking between pieces of calcified fibrocartilage and bone at the insertion site. We suggest that this is of fundamental importance in anchoring the tendon to the bone, because the manner in which a tendon insertion develops makes it unlikely that many collagen fibres pass across the tissue boundary from tendon to bone. When force is transmitted to the bone from a loaded tendon, it is directed towards the plantar fascia by a series of highly orientated trabeculae that are clearly visible in 3D in thick resin sections.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

The Achilles tendon inserts on the posterior surface of the calcaneus, and immediately above its attachment, the space between the tendon and the bone is occupied by the retrocalcaneal bursa. The anterior wall of this bursa is formed by the calcaneus and the posterior by the tendon (Rufai et al. 1995). As the foot is dorsiflexed, the tendon bends near its attachment, the bursa flattens and its walls become opposed, i.e. the distal part of the tendon is pressed against the bone.

The term ‘enthesis organ’ has recently been coined to describe the tendon insertion site itself, together with the bursa and its walls (Benjamin & McGonagle, 2001). The word ‘enthesis’ collectively embraces the concept of a tendon, ligament or joint capsule attachment to bone – be it the origin or insertion of a tendon, or the equivalent attachments at the two ends of a ligament. An ‘enthesis organ’ is thus a collection of related tissues that act together to protect both the tendon and bone from wear and tear. The enthesis organ of the Achilles tendon includes three fibrocartilages (FC) – an enthesis FC at the tendon–bone junction, together with two fibrocartilages that form the bursal walls and protect them from compression (Rufai et al. 1995). These are a sesamoid FC in the deep surface of the tendon and a periosteal FC covering the superior tuberosity of the calcaneus (Rufai et al. 1995).

A detailed three-dimensional (3D) picture of the orientation of the fibrocartilages associated with the human Achilles tendon insertion is of clinical relevance, as modern MRI techniques have now developed to the point at which subtle signal abnormalities can be demonstrated in patients with chronic Achilles tendon pain, for example that associated with Haglund’s deformity, ectopic tendon calcification, calcaneal spurs or retrocalcaneal bursitis. Movin et al. (1998), for example, have correlated histopathological signs of increased glycosaminoglycan production and altered fibre structure in patients with achillodynia, with changing signal intensity. As MRI is essentially a 3D technique (albeit commonly presented in 2D), the imaging capability requires an anatomical knowledge of the 3D arrangement of the fibrocartilages in the Achilles tendon, if we are to improve our understanding of what is ‘normal’ or ‘abnormal’ in MRI. Furthermore, a knowledge of the nature and orientation of the fibrocartilages is relevant to surgeons who must choose between different tendon resection methods when treating posterior heel pain (Kolodziej et al. 1999).

Current views on the functional significance of the components of the enthesis organ are coloured by the 2D nature of the studies on which these have been based. Thus, Schneider (1956) has suggested that fibrocartilaginous entheses (i.e. those like the Achilles tendon) have a ‘two-tier protection mechanism’ reducing the risk of wear and tear. He argues that the irregularity of the interface between calcified enthesis FC and bone protects tendons against shear, while the presence of uncalcified FC enables them to withstand compression. However, 2D images cannot show whether the pieces of calcified FC and bone actually interlock with each other in 3D. If they do, this must be important in anchoring the tendon to the bone. Thus, the purpose of our study is to describe the 3D structure of the calcified enthesis FC–bone interface in the Achilles tendon insertion, and the three fibrocartilages associated with its enthesis organ.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

For immunohistochemistry, Achilles tendon insertions were removed from four fresh cadavers, within 48 h of death (three males, one female; age range 32–73 years) and fixed for 24 h in 90% methanol at 4 °C. Fine saw cuts were made in the sagittal plane, either side of the midline of the calcaneus, so that approximately 8-mm-wide strips of tissue were sampled from the insertion site. Each strip contained the distal part of the tendon and its insertion, together with the superior tuberosity of the calcaneus. Excess cancellous bone was removed from beneath the insertion site, and the specimens stored in methanol at –20 °C until required. The tissue was decalcified for 1–2 weeks in 5% EDTA (the exact end point was determined radiographically), rinsed in phosphate-buffered saline (PBS), infiltrated overnight with 5% sucrose in PBS and supported in Cryo-M-Bed embedding compound (Bright Instrument Company Ltd, UK) on cryostat chucks. The tendons were frozen with dry ice and sectioned at 12 µm in the sagittal plane. Preliminary sections taken were from the edge of the block and stained with toluidine blue in order to locate the exact position of the sesamoid and periosteal FC. This allowed the block face to be trimmed with a single-edged razor blade, so that sections of a size suitable for antibody application could then be collected at 1.5-mm intervals throughout the block. These sections were pretreated with a cocktail of hyaluronidase (1.5 units mL−1; Sigma) and chondroitinase ABC (0.25 units mL−1; Sigma) at 37 °C and labelled with a monoclonal antibody against type II collagen (CIICI from the Developmental Studies Hybridoma Bank, maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242, under contract NO1-HD-7-3263 from the NICHD). Endogenous peroxidase activity was blocked by pretreating the sections with 0.3% hydrogen peroxide in methanol for 30 min and non-specific binding of the secondary antibody was minimized by blocking sections with appropriate serum for 40 min. For control sections, the primary antibody was omitted or sections incubated with nonimmune mouse immunoglobulins (10 µg mL−1). Antibody binding was detected with a Vectastain ABC ‘Elite’ avidin/biotin/peroxidase kit. Sections were digitized at a magnification of ×5 and the outline of the three fibrocartilages (enthesis, sesamoid and periosteal) was traced interactively. Three-dimensional images of these datasets were generated using Surfdriver© software purchased from the University of Hawaii (http://www.surfdriver.com).

For routine histology, the Achilles tendon insertion was removed from four embalmed, dissecting room cadavers (three males; one female; ages 70–81 years). The cadavers had been perfusion-fixed in embalming fluid containing 4% formaldehyde and 25% alcohol for 72 h. Eight-mm-wide strips of tissue were taken either side of the midline of the tendon insertion, as described above, and the specimens were further fixed in 10% neutral buffered formal saline for 1 week. The material was decalcified in 2% nitric acid, dehydrated in graded alcohols and embedded in paraffin wax. Twelve serial sagittal sections (8 µm thick) were collected from the centre of each block and stained with toluidine blue. The junction between calcified enthesis FC and bone was reconstructed in 3D using the Surfdriver© software. Sections were digitized using a ×10 objective lens and the outlines of the bone and calcified FC were traced interactively at three different locations at the insertion (proximal, central and distal as shown in Figure 1c). Three-dimensional images of these datasets were generated as described above and the volume fraction of the calcified FC was calculated using the Surfdriver© software. Significant differences between the three regions of the insertion site were assessed by Student’s t-test.

image

Figure 1. (a) A posterior view of 3D reconstructions of the enthesis (red), periosteal (blue) and sesamoid (grey) fibrocartilages in the Achilles tendon. Column 1 shows the enthesis fibrocartilage by itself and columns 2 and 3 successively add reconstructions of the periosteal and sesamoid fibrocartilage. Column 4 shows all three fibrocartilages viewed against the background of the tendon itself. Note the close correspondence in both the position and the shape of the periosteal and sesamoid fibrocartilages. (b) Three-dimensional reconstructions of the zone of calcified enthesis fibrocartilage (red) and the underlying bone (grey) in the proximal (I), central (II) and distal (III) thirds of the Achilles tendon insertion as illustrated in (c). Regions where the two tissues overlap in 3D appear in shades of pink (asterisk) and these are most obvious in this specimen in region II. (c) A sagittal section of the posterior part of the calcaneus showing the Achilles tendon (T) enthesis organ. Note the relationships of the enthesis (EF), periosteal (PF) and sesamoid (SF) fibrocartilages in the distal part of the tendon. Zones I, II and III at the insertion site correspond to those used for the reconstructions illustrated in (b). Note also that the regularly aligned trabeculae in the inferior part of the calcaneus (arrows) are orientated along the lines of force transmission from the Achilles tendon. ST, superior tuberosity. Unstained resin section. Scale bar = 0.5 cm.

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Two additional hindfeet were fixed in formalin, frozen at –20 °C, cut into 5-mm-thick, sagittal sections and embedded in epoxy resin according to the method of Von Hagens et al. (1987) and Fritsch (1996). They were examined unstained under a dissecting microscope.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

The 2D relationships of the enthesis, sesamoid and periosteal FC associated with the Achilles tendon are shown in Figure 1(c). Each fibrocartilage has been defined by a separate region of immunohistochemical labelling for type II collagen (Figure 2a,b). Three-dimensional reconstructions of these fibrocartilages based on the distribution of the immunolabel are illustrated in Figure 1(a). In all specimens, the reconstructions showed that there was a close correspondence between the shape and position of the sesamoid and periosteal FC. There was also a consistent overlap between the sesamoid and the enthesis FC (Figure 1a). The sesamoid FC covered most of the periosteal FC, but showed a moderate overlap with the proximal part of the enthesis FC.

The enthesis FC itself has a larger zone of uncalcified tissue in which the cells are arranged in longitudinal rows between parallel collagen fibres, and a much smaller zone of calcified tissue in which the FC cells are less numerous (Figure 2c). The calcified and uncalcified FC are separated from each other by a basophilic line called the tidemark. This is relatively straight compared with the irregular interface between the calcified FC and the bone (Figure 2c). Three-dimensional reconstructions of this interface in three different locations with respect to the position of the bursa are illustrated in Figure 1(b). The figures show that there is a complex pattern of 3D interlocking between the zones of calcified FC and subchondral bone that was greatest in the central part of the insertion site. They also indicate that isolated pieces of calcified FC can be seen as ‘islands’ locked within the subchondral bone. The volume of the calcified FC was significantly higher (P < 0.05) near both the proximal and the central parts of the insertion site than in the distal part (Figure 3).

image

Figure 2. Immunohistochemical labelling for type II collagen in (a) the periosteal (PF) and sesamoid (SF) fibrocartilages and (b) the enthesis fibrocartilage (EF), in the Achilles tendon. The distribution of such labelling was used as the basis for defining the extent of the fibrocartilages in the reconstructions shown in (a). B, bone. Scale bars = 100 µm. (c) A toluidine blue section showing the irregular interface (arrows) between the zone of calcified enthesis fibrocartilage (CEF) and the underlying bone (B). Serial sections of this type were used to make the 3D reconstructions shown in (b). Note that the zones of calcified and uncalcified (UEF) fibrocartilage are separated from each other in this case by two tidemarks (TM). Wherever this occurred, reconstructions were made up to the tidemark furthest from the bone. Scale bar = 50 µm.

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image

Figure 3. A histogram showing the mean values (+ standard error) for the volume fraction of calcified enthesis fibrocartilage (CEF) in the proximal, central and distal thirds of the tendon insertion. There are significant differences (P < 0.05) between the distal part of the insertion and each of the other two regions.

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In the resin section illustrated in Figure 1(c), there is a conspicuous bundle of highly orientated trabeculae in the postero-inferior part of the calcaneus. These trabeculae are aligned along the direction of the fascicles in the Achilles tendon and are orientated towards the proximal attachment of the plantar fascia.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

This is the first demonstration of the 3D distribution of a molecule typical of cartilage in the fibrocartilages associated with a tendon insertion. We have used the pattern of distribution of type II collagen to create a picture of the 3D shape of the enthesis, sesamoid and periosteal FC. The 3D data suggest that there is a close correspondence between both the position and the size of the sesamoid and periosteal FC – probably because of their functional interdependence. The superior tuberosity of the calcaneus (on which the periosteal FC is located) acts as a pulley for the distal part of the Achilles tendon. A corresponding area of the tendon must thus be structurally modified to withstand the compression on the tendon that comes from its contact with that pulley – hence the sesamoid FC. The uncalcified enthesis FC serves a number of roles (see Benjamin & Ralphs, 1998, for further details), but in particular it dissipates the bending of tendon fibres away from the hard tissue interface. Even though it is an integral part of the tendon itself, it could thus be regarded as acting as a further pulley located distal to (i.e. ‘downstream’ from) the superior tuberosity (Figure 4). At neither pulley is there much, if any, longitudinal excursion of tendon fibres. Thus, we suggest that the presence of two microscopic pulleys in series with each other provides an efficient moment arm for the Achilles tendon. The wedge shape of the uncalcified enthesis FC in the Achilles tendon (i.e. its greater prominence in the superior than the inferior part of the attachment site) promotes its role as a pulley. Such a shape has been reported previously for other entheses (Benjamin et al. 1986; Woo et al. 1988; Evans et al. 1990; Frowen & Benjamin, 1995).

image

Figure 4. A diagrammatic representation of how the periosteal (PF) and enthesis (EF) fibrocartilages could be viewed as two pulleys (P1 and P2) in series with each other, to increase the moment arm of the Achilles tendon at its insertion. The periosteal fibrocartilage, covering the superior tuberosity of the calcaneus, acts as a pulley for the Achilles tendon above the level of the broken line (P1), altering the direction of its collagen fibres. Below the line, the enthesis fibrocartilage acts as a pulley at the insertion site itself (P2).

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Previous work has shown that there are differences between the quantity of calcified enthesis FC in the different attachments of the quadriceps tendon/patellar ligament complex (Evans et al. 1991). Our results show that there are also regional differences in the quantity of calcified FC in different parts of the same enthesis. There is more calcified tissue proximally than distally and this corresponds to the distribution of the uncalcified FC. The complexity of the interface between calcified enthesis FC and bone is well known (Woo et al. 1988; Gao & Messner, 1996; Benjamin & Ralphs, 1998), but no-one has considered the shape of these zones in 3D. Our results show that the two tissues interlock with each other in a complex manner. Indeed, such is the degree of interlocking that it could form an important part of the fundamental mechanism by which tendons (or indeed ligaments) anchor to bone. We envisage that pieces of calcified enthesis FC ‘fit’ into the bone like pieces of a jigsaw and contribute substantially to anchoring the tissues together. Intriguingly, the 2D studies of Gao & Messner (1996) suggest that the interface is more complex in ligaments that are subject to greater tensile load. When the tendon/ligament is under tension (i.e. at the time when maintaining the integrity of the enthesis is critical) it is conceivable that its zone of calcified enthesis FC is clamped even more tightly to the underlying bone. This is supported by Mente & Lewis’ (1994) suggestion that the calcified zone of articular cartilage is 10 times less stiff than the underlying bone – even though the degree of mineralization is about the same. It should be noted, however, that a contrary view is presented by the back-scattering image analysis studies of Boyde et al. (1995), Vajda & Bloebaum (1999) and Shea et al. (2001).

It is worth drawing attention to the parallels between the calcified enthesis FC–bone junction in a tendon and the junction between epidermis and dermis in thick skin. The epidermal/dermal interface is similarly corrugated and this contributes to holding the two tissues together – because dermal ‘pegs’ protrude into the deep surface of the epidermis. There are also similarities between a tendon enthesis and the calcified cartilage/bone interface that characterizes articular cartilage (Müller-Gerbl et al. 1987; O’Connor, 1997). This too is highly irregular, especially in heavy load-bearing joints (Teshima et al. 1999), and again the irregular interface contributes greatly to the mechanical adhesion of cartilage to bone (Oegema et al. 1997). However, whether there is any comparable interlocking of cartilage and bone in 3D is unclear, for there has been no comparable study of serial sections. As with a tendon enthesis, the cartilage/bone interface must enable the region to withstand shear. However, an important difference is that the interface in a tendon is subject to tension, whereas that in articular cartilage is not. Oegema et al. (1997) have summarized a number of further interesting suggestions on the function of the zone, i.e. that it limits diffusion from the underlying bone, plays a role in the gradual transition of force transmission from articular cartilage to bone, and acts as a growth plate.

While the calcified enthesis FC–bone interface is critical for allowing force to be transferred from tendon to bone, it is the orientation of trabeculae within the calcaneus that determines the direction in which these forces are subsequently transmitted. It is thus pertinent to note that the highly regular, 3D orientation of trabeculae seen in the thick resin section (Figure 1c) suggests that force is relayed in the direction of the plantar fascia. Force transmission along this fascia may help to maintain the medial longitudinal arch of the foot. It is as if the fascicles of the Achilles tendon ‘continue’ through the bone in the guise of the orientated spicules. Such an osseous line of force transmission would complement the soft tissue link between the Achilles tendon and plantar fascia which exists because these structures are continuous below the calcaneus itself (Snow et al. 1995; Williams et al. 1995). This group of trabeculae is clearly visible in the illustrations of Yettram & Camilleri (1993) and Snow et al. (1995), though neither study comments on it. The trabeculae are clearly relevant to the development of finite-element, stress-analysis models of the calcaneus, though the model developed by Yettram & Camilleri (1993) is insufficiently complex to take them into account.

For developmental reasons, it is hard to envisage how any significant number of collagen fibres in a cartilage bone such as the calcaneus could pass from tendon into bone and contribute to its anchorage. During the formation of a fibrocartilaginous enthesis, the bone erodes the tendon by vascular invasion followed by endochondral ossification (Gao et al. 1996). Thus, tendon fibres are eroded as bone grows into tendon, rather than simply becoming embedded in bone. However, we are not arguing that collagen fibre continuity between tendon (or ligament) and bone does not occur at all, for this has been demonstrated by Clark & Stechschulte’s (1998) scanning electron microscopy study. Interestingly, however, they show that collagen fibres from the tendon largely interdigitate between groups of bone lamellae rather than penetrate within them. We also cannot exclude the possibility that there is some sort of molecular ‘glue’ at the calcified enthesis FC–bone interface that contributes to maintaining its integrity. Such a role has been suggested for type X collagen (Niyibizi et al. 1996; Fukuta et al. 1998; Gao, 2000).

Several studies have recently focused on the problem of how tendons/ligaments can best be re-attached surgically to bone (Petersen & Laprell, 2000; Shaieb et al. 2000; Aoki et al. 2001; Oguma et al. 2001). Sharpey’s fibres are commonly reported to develop at the new attachment site. During normal development, Sharpey’s fibres are typically a feature of fibrous rather than fibrocartilaginous entheses. Unfortunately, such features are seldom illustrated in the reattachment studies, so it is difficult to assess the structures to which the authors refer. The works do suggest, however, that at least during the early stages of tendon repair, the insertion mechanism may be different from that which operates at a healthy fibrocartilaginous enthesis. Enthesis FC can certainly be reconstituted in time (Jones et al. 1987; Petersen & Laprell, 2000), but it may well be that the initial, early repair does not involve FC–bone interaction.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

The work of Dr Milz in this study was supported by the Friedrich Baur Stiftung Munich.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References
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