Tendon biomechanics

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Tendon biomechanics

M . K J á R , Bispebjerg Hospital and University of Copenhagen, Denmark, S . P . M A G N U S S O N , University of Copenhagen, Denmark and A . M A C K E Y , Bispebjerg Hospital, Denmark

Abstract: Tendon connective tissue adapts to mechanical loading with increased synthesis and turnover of matrix proteins. Collagen formation and degradation increase with acute loading of tendon and skeletal muscle, and this is associated with local and systemic release of growth factors (e.g. IGF1, TGF-beta). Chronic loading of tissue, such as with physical training, leads to increased collagen turnover and a net collagen synthesis together with a modification of the mechanical properties of the tendon, including a reduction in tendon stress. The adaptation time to chronic loading is longer in tendon tissue than in contractile elements of skeletal muscle or heart, and only very prolonged loading will significantly change gross dimensions of the tendon. Mechanical signalling in the tissue leads to biochemical changes in the matrix that can be converted into adaptations in morphology, structure and biomechanical properties of the tendon. Key words: tendon connective tissue, chronic loading, collagen synthesis.

15.1

Introduction

Tendons, ligaments, bone and intramuscular connective tissue have been studied for decades, and the biomechanical properties of these tissues are well appreciated. However, at least for tendon and ligaments the structures have been considered to be relatively inert (Kjñr 2004). The methods available to study these collagen-rich tissues in vivo have in the past been somewhat limited, and often findings were based solely on studies of cadaver tissue properties, or relied on cell culture work. With the present methodological developments and renewed interest for metabolic, circulatory and tissue protein turnover in collagen tissue such as tendon ± in conjunction with a simultaneous determination of morphology and biomechanical tissue properties ± a greater insight into the way that mechanical loading influences both the cells and the matrix of tendon has been achieved. As an example, it has been shown that in response to mechanical loading the human tendon increases its blood flow, metabolic activity and substrate uptake (e.g. glucose) by 3±4-fold, acutely associated with exercise (for references see Kjñr 2004).

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Biochemical adaptation of tendon to loading

Matrix adaptation to loading involves several proteins, but the most mechanically important load-bearing structure in tendon tissue is collagen type I. Several indirect methods have been used so far to indicate changes of collagen turnover in connective tissue. Determination of increased pro-collagen mRNA in tissue indicates an upregulation of transcriptional activity, but does not guarantee for any mature collagen formation. Furthermore, activity in the intracellular biosynthesis pathway for collagen can be estimated by determination of enzymes such as prolyl-4-hydroxylase (P-4-H), galactosylhydroxy-lysylglucotransferase (GGT) or lysyl hydroxylase (LHy) and increased collagen synthesis has been estimated from such methods in animal experiments (Kovanen 1989). Collagen formation in the tendon is shown to rise with acute and chronic loading, as determined by the use of the microdialysis technique. With this technique, catheters are put into the region of interest (e.g. around or through a tendon) and the interstitial concentration of the pro-collagen propeptides that are cleaved off in the maturation from procollagen to collagen (PICP or PINP) are determined of both previously loaded and unloaded tendon (Langberg et al. 1999, 2001). Using this technique locally around a tendon provides the possibility to determine local changes of PICP or PINP in regions that otherwise would only contribute marginally to any change in these parameters globally (i.e. changes in concentrations of PINP or PICP in the circulating blood stream) (Langberg et al. 2000).

15.2.1 In vivo collagen turnover in tendon: stable isotopes In collagen-containing tissues such as tendon and bone, other methods such as the use of radioactive isotopes for incorporation of representative tracers into tissue protein have been used in animal experiments. More recently the use of non-radioactive stable isotopes also provided the use in human experiments. Given that representative samples of the tissue can be obtained, direct protein synthesis can be determined as incorporation of the tracer into the connective tissue (Babraj et al. 2005; Miller et al. 2006b). Tendon tissue sampling can be performed in humans by percutanous tendon biopsies, and has been used in protocols where protein turnover, mRNA transcription and collagen fibril diameter have been determined in both young and elderly, patients and healthy subjects (Miller et al. 2006b). The use of stable isotope techniques to study incorporation of labelled amino acids into tissue in order to study the kinetics of collagen has been tried in tendon, ligament and bone (Babraj et al. 2005). Briefly, the principle of the direct incorporation technique using the precursor± product approach applicable on tendon tissue is to label the amino acid, proline, e.g. L-13C-proline or L-15N-proline. Proline is abundant in collagen, and is incorporated directly into new collagen proteins. Newly synthesised pro-

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collagens are post-translationally hydroxylated at the proline residues (forming hydroxyproline) before assembling into the triplehelical structure. Thus, collagen-specific hydroxyproline will be labelled. By measuring the enrichment of hydroxyproline from a tendon sample, this provides a very specific synthesis measure of collagen protein in the tendon tissue (Babraj et al. 2005). Using this method, acute exercise has been shown to increase the fractional synthesis rate of collagen in the patella tendon from approximately 0.05%/hour to around 0.10%/hour within 24 hours after exercise, showing a significant rise 6 hours post-exercise (Miller et al. 2005). This corresponds to a collagen synthesis that on a 24 hour level increases from around 1% at rest to 2±3% after exercise. The collagen synthesis rate remains elevated for at least 2±3 days after acute exercise (Heinemeier et al. 2003). Similar to collagen synthesis, there is indication that protein degradation is activated after exercise, in that local levels of matrix metalloproteinases in tendon and muscle tissue are increased after acute exercise (Koskinen et al. 2004). Although no good determination of collagen degradation has been provided so far, it indirectly points towards an increased collagen turnover with exercise. It is difficult to state how much of this increased turnover is in fact turned into assembled load-bearing collagen, but there is reason to believe that at least a certain percentage of the newly formed collagen will end up as insoluble collagen type I in the final tendon structure (Laurent 1987). Within skeletal muscle, the collagen synthesis increases with acute exercise by 2±3-fold and over a time that resembles that of myofibrillar protein synthesis, suggesting a coordinated response with regards to structural proteins both in contractile elements and in the extracellular matrix of human skeletal muscle. Prolonged, repeated mechanical loading chronically elevates the collagen synthesis in tendon almost 3±4-fold (Langberg et al. 2001), and more recently it has been shown that inactivity for only 10 days resulted in a decrease in protein synthesis of both collagen and myofibrillar protein of around 20±25% (DeBoer et al. 2007). Interestingly, neither of these studies resulted in any dramatic morphological changes of the tendon when determined by ultrasound. Clearly, in none of the studies an accurate determination of protein degradation could be performed, and perhaps more importantly, it has been shown that determination of collagen synthesis per se does not always correlate with true incorporation of new load-bearing mature collagen into existing structures (Laurent 1987). Finally, it cannot clearly be stated from cross-measurements of a tendon diameter whether or not the total collagen content of the tendon has been changed.

15.2.2 Regulation of collagen adaptation to mechanical loading An important question in relation to increased collagen turnover is how tendon senses the external loading during muscular contraction and, more specifically,

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what factors are involved in this regulation. The first example of factors involved in collagen synthesis regulation comes from the observation that apparently a gender difference exists, in that females respond less than males with regards to the increase in collagen formation after exercise (Miller et al. 2006b). The expected increase in collagen synthesis to exercise was less pronounced in women than in men, and furthermore the basal collagen synthesis rate was also lower in women compared with men (Miller et al. 2006b). Further experiments in women who have varying levels of sex hormones (e.g. oral contraceptives) suggest that oestradiol may contribute to a diminished collagen synthesis response in women (Hansen et al. 2008). Interestingly, this finding of a lower collagen synthesis rate both at rest and in response to exercise in women vs men, is correlated to a demonstration of lower stress tolerance in patella tendons of women compared with men (Haraldsson et al. 2005). Overall this may contribute to our understanding with regards to the fact that women experience a larger number of soft tissue injuries in, for example, cruciate ligaments than men, and may also be important in understanding of how rapid humans of both sexes not only adapt to physical training but also how well they may resist prolonged periods of reduced activity. What exact mechanism lies behind this gender-specific response is not definitively answered, but the finding of a correlation between increased oestrogen levels and a reduction in collagen synthesis in humans, is supported by in vitro studies where it has been shown that oestradiol receptors are present in ligaments (Sciore et al. 1998) and the finding that oestradiol per se can exert an collagen synthesis inhibiting effect in tendons and ligaments (Liu et al. 1996; Yu et al. 2001). In addition to a suggested direct inhibiting effect of oestradiol upon collagen synthesis, it may also be that oestradiol levels exert an indirect effect by influencing other hormonal components of the human endocrine system. As an example, lately high levels of circulating oestradiol in women have been shown to be associated with low levels of circulating insulin-like growth factor (IGF-I), a substance that may be directly coupled to the degree of collagen synthesis rise with exercise. Thus the gender differences may not be an effect of estradiol directly, but rather via an influence of estradiol on IGF-I. An important regulating factor in collagen synthesis is the growth hormone (GH) ± insulin-like growth factor 1 (IGF-1) axis, where in vitro data have shown a role for collagen formation (Dùssing and Kjñr 2005). As an example, it has been shown that IGF-I (and IGF-II) administration in rabbits will accelerate the protein synthesis in tendons (Abrahamsson 1997), and likewise that the recovery after tendon injury was accelerated when IGF-I was administered (Kurtz et al. 1999). Although GH in skeletal muscle has been shown to exert an effect upon muscle growth in GH-deficient individuals, the effect of GH supplementation upon muscle protein synthesis is absent in both young and elderly humans (Lange et al. 2002). Despite this, it seems that GH/IGF-I influences connective tissue, and administration of GH over 3 weeks has been shown to elevate

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circulation blood levels of procollagen propeptides (Longobardi et al. 2000). Thus it may be tempting to speculate that GH/IGF-I has a stimulating effect upon tendon tissue. In dwarf rats, GH administration has been shown to increase the expression of both collagen type I and III in intramuscular fibroblasts (Wilson et al. 1995). Recently it has been shown that IGF-1 is present in human Achilles tendon linked directly to fibroblasts, and furthermore a detectable interstitial concentration has been demonstrated in human tendon (Olesen et al. 2006). Although this supports the possibility that IGF-I may play a role in human tendon, the question, however, remains: to what extent is IGF-1 upregulated with exercise? By analysing RNA extracted from whole rodent tendons, it has recently been shown that functional overloading of a single muscle attached to the tendon±muscle (by ablation of other muscle synergists) leads to increased expression of IGF-I within the tendon, and, furthermore, that short-term strength training induces the expression of both TGF- -1 and IGF-I in rat Achilles tendon (Heinemeier et al. 2006). Quantification of specific mRNA species has also been used as a tool for studying human tendon pathology, and by analysing RNA extracted from surgical tendon specimens several changes in expression of matrix proteins and enzymes have been identified in overused/damaged tendons compared with healthy tendons (Riley et al. 2002). Furthermore, increased expression in tendon of an IGF-1 isoform called MGF (mechano growth factor) was also found (Heinemeier et al. 2007). This is notable since until recently this isoform was believed to exist only in skeletal muscle cells. In the rodent overload study mentioned above (where synergist muscles were ablated), also an up-regulation of transforming growth factor beta 1 (TGF- 1) was found already after 2 days of stimulation, and it preceded the rise in collagen expression. As an indication that TGF- 1 could play a role in collagen synthesis (Heinemeier et al. 2007), the mRNA for connective tissue growth factor (CTGF) was increased similar to TGF- 1. CTGF is thought to mediate many of the effects of IGF-1 in relation to matrix proteins. A number of in vitro studies have demonstrated a coupling between mechanical loading of tissue and TGF-beta expression (Skutek et al. 2001), and furthermore it has been shown that mechanically induced type I collagen synthesis can be ablated by inhibiting TGF- activity (Lindahl et al. 2002). In human tendon, the presence of TGF- 1 has been demonstrated in association with fibroblasts, and in human skeletal muscle TGF- 1 has been demonstrated to be located mainly in the perimysium in relation to fibroblasts and endothelium (Heinemeier et al. 2007). Furthermore, after exercise of human skeletal muscle TGF- 1 was up-regulated by 2.5 hours after exercise followed by increased collagen synthesis 6 hours post-exercise. Using the microdialysis technique around the Achilles tendon in humans, it was found that both local and circulating levels of TGF- increased in response to running, and furthermore, the time relation between TGF- and indicators of local collagen type I synthesis supported a role of TGF- in regulation of local

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collagen type I synthesis of tendon-related connective tissue (Heinemeier et al. 2003). In a recent study, different types of muscular contraction were used in order to study responses of collagen synthesis. Both concentric and eccentric contractions were performed in two different experimental groups. When performing eccentric contractions, the loading of both muscle and tendon was higher than with concentric contractions, whereas strain was not assumed to be different to a major degree in the two situations (Heinemeier et al. 2006). The expression of IGF-1 and TGF- 1 in muscle increased the most during eccentric exercise, whereas the response in tendon was independent of type of contraction (Heinemeier et al. 2007). Likewise, the procollagen expression was similar in concentric and eccentric exercise, indicating that stress was not a major determiner in the magnitude of collagen synthesis response in tendon, and the findings suggests a more prominent role for strain in signalling a growth factor release and subsequent increase in collagen formation. Taken together, the data supports the view that TGF- plays a role in collagen synthesis of tendon with mechanical loading of collagen-rich tissue. In addition, it is important to note that whereas acute TGF- responses to mechanical loading most likely represents an important physiological response, a more prolonged and chronic elevation of TGF- in association with a variety of pathological situations more likely will result in an uncontrolled formation of fibrotic tissue (Leask et al. 2002).

15.2.3 Matrix and contracting skeletal muscle: playmates? Developing skeletal muscle clearly demonstrates a close coupling between myogenesis and development of the intramuscular extracellular matrix components. It is more unclear to what extent such an interplay exists in mature skeletal muscle, but given the important role that intramuscular collagen plays in force transmission, it would be relevant to assume some kind of coordinated response to loading. Experiments have shown that the myogenic stem cell in skeletal muscle, the satellite cell, is activated not only in pathological situations associated with skeletal muscle injury, but also in physiological situations associated with muscle hypertrophy development (Kadi et al. 2005), but to what extent the connective tissue within the skeletal muscle is involved in this activation of satellite cells is not known. When human muscle is subjected to eccentric loading, it will result in activation of satellite cells in the absence of major damage of the muscle cell itself (Crameri et al. 2004a). In fact, the activation of satellite cells was accompanied by increased staining intensity for the procollagen propeptide PINP and for tenacin-C, a marker for connective tissue and known to reinforce lateral adhesion of the myofibre to the surrounding endomysium (Crameri et al. 2004b). These observations suggest that activation of collagen synthesis in the intramuscular connective tissue is associated with

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satellite cell activation. Although this suggests a potential coupling between signalling to the myofibre that involves activation of the collagen-rich tissue within the muscle, it has to be stated that performing even more heavy eccentric exercise in association with electrical stimulation can result in a marked degree of myofibre injury and this will cause an even more pronounced activation of the satellite cells (Crameri et al. 2007). This illustrates that even if a coupling between connective tissue and the muscle cell exists in mature muscle, it certainly does not exclude a direct link between muscle cell injury and satellite cell activation. A further dissociation between muscle cells and connective tissue arises from a recent study that investigated protein synthesis in response to heavy resistance training and more prolonged low-resistance exercise, respectively. Whereas the exercise-induced rise in protein synthesis for myofibrilar proteins was dependent upon the exercise intensity, the rise in protein synthesis for collagen of the muscle was similar in the two situations where intensity was varied but the total work output was the same (Holm et al., unpublished). This may illustrate that muscle cells are sensing the exercise intensity, whereas the collagen-rich tissue does not have a similar detailed sensing of mechanical loading.

15.2.4 Stem cells and their importance for tendon adaptation The ability to harvest mesenchymal cells from the bone marrow, grow them in culture and re-introduce them into an injured tendon has been widely exploited in racehorses, despite a lack of controlled studies endorsing this treatment (Smith and Webbon 2005). In a recent controlled study, however, mesenchymal cells, extracted and treated in a similar way, were applied to severed rabbit tendons, where some positive findings support the use of these stem cells in this way, at least in the early stages of tendon healing (Chong et al. 2007). The most limiting factor in stem cell research in tendon tissue, as in all tissues, is the availability of suitable markers. Many of the traditional markers, used in cellsorting methods, stain endothelial cells, pericytes and inflammatory cells as well as stem cells, are rendered unsuitable for use in situ where these different cell types are present. In vitro studies do not suffer from this complication and recently cells derived from human hamstring tendons were observed to behave similarly to bone marrow-derived cells in vitro, suggesting the presence of a population of cells with differentiation potential in human tendon (de Mos et al. 2007). The temporal appearance of tendon-produced and circulation-derived cells during tendon healing was elegantly demonstrated with the aid of green fluorescent protein (GFP) chimeric rats (Kajikawa et al. 2007). There, circulation-derived GFP cells were observed in the tendon wound 24 hours after injury, whereas tendon-derived GFP cells were not evident until 3 days after the injury, but appeared to take over from the circulation-derived cells by day 7. The labelling and tracing method employed in this study opens up a new

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way of investigating tendon remodelling in response to injury. In vivo cell tracking using magnetic resonance imaging (MRI) of magnetically labelled cells is the latest non-invasive method for following the movement of cells around the body. The success of this method in tracking the migration of adult stem cells in the central nervous system has recently been reviewed (Sykowa and Jendelova 2007), and one can surmise that it is merely a matter of time before this method will be applied to the area of tendon research, which, together with the other methods mentioned here, will help enrich our understanding of the involvement of stem cells in adaptation of this tissue.

15.3

Biomechanics of human tendon

Human lower extremity tendons are subjected to appreciable loads during human locomotion. In fact, the Achilles and patellar tendons can be subjected to several times the body weight during running. Unfortunately, tendons are unable to adapt to certain loading conditions and therefore end up with pathology, including so-called overuse injuries and complete tendon ruptures (Kannus and Jozsa 1991; Maffulli et al. 1999). However, whether over-use injuries and ruptures are associated with predisposing morphological changes, or whether the tendon inadvertently exceeds a narrow safety margin remains unknown. Nevertheless, the mechanical properties of the tendon are generally thought to play a pivotal role. Because the aetiology of these injuries, and exactly how tendons adapt to physical activity or lack thereof, are at best incompletely understood, the ability of clinicians to provide optimal treatment and prevent injury remains circumscribed.

15.3.1 Does tendon adapt to loading by hypertrophy? Tendons subjected to considerable stress, including the Achilles tendon, are believed to operate as springs by storing and releasing elastic strain energy during locomotion (Alexander and Bennet-Clark 1977; Biewener and Roberts 2000). From this standpoint it is advantageous to have a thin (and long) tendon. On the other hand, a thicker tendon, which would yield less strain energy, would reduce the average stress (force/area) across the tendon and thereby provide a greater safety margin. For example, if the tendon were to increase in size (hypertrophy) as a function of physical activity/exercise, the average stress would be reduced for a given applied force (e.g. some multiple of the body weight). However, the time course and to what extent human tendon adapts by changing its material and/or undergo hypertrophy in response to exercise remain an enigma. Animal data show that tendon may undergo both qualitative (Buchanan and Marsh 2001), hypertrophic changes (Birch et al. 1999; Woo et al. 1982), or both (Woo et al. 1982) in response to endurance-type exercise and do therefore not provide a coherent picture.

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In humans cross-sectional data suggest that habitual long-distance running (over a time span of 5 years) is associated with a markedly greater crosssectional area (CSA; 22%) of the Achilles tendon compared with that of nonrunners (Rosager et al. 2002). However, a total training stimulus of ~9 months of running in previously untrained young subjects did not result in tendon hypertrophy of the Achilles tendon (Hansen et al. 2003). At the same time it has been shown that resistance training for 3 months induced remarkable changes in the material properties of human tendon in the absence of any tendon hypertrophy in older people (Reeves et al. 2003), while the opposite was demonstrated in young male subjects (Kongsgaard et al. 2007). It is possible that these apparent discrepancies may relate to the training mode (endurance versus resistance training), age and tendon type (Achilles vs patellar tendon). It is also possible that gender (hormonal milieu) may play a role (Miller et al. 2006a): recent cross-sectional data indirectly suggest that the ability of the patellar tendon to adapt in response to habitual loading such as running is attenuated in women as evidenced by the similar tendon CSA, stiffness and modulus in habitual runners and non-runners (Westh et al. 2008). Therefore, although it has been elegantly and repeatedly shown in a human model that there is an increased metabolic activity of human tendon tissue in response to an acute bout of loading (Bojsen-Moller et al. 2006; Langberg et al. 1999; Miller et al. 2005), it is not entirely understood to what extent this results in a larger structure or a different material. Finally, it may be that adaptation of tendon morphology to exercise just requires extremely long time, a notion that is recently supported by the finding that in athletes that load their two legs (and their patella tendons) differently, a significant difference could be demonstrated, in that the patella tendon that was subjected to repetitive high and eccentric muscle loading was thicker than the contralateral tendon (Couppe et al. 2008).

15.3.2 Tendon regional differences in the morphology Tendon stress is calculated commonly based on a single or average value of the tendon CSA. However, it appears that there is a large variation in the tendon CSA along the length of both the human Achilles and patellar tendon (Kongsgaard et al. 2005; Magnusson and Kjñr 2003; Rosager et al. 2002; Westh et al. 2008). In fact, both the Achilles and patellar tendon can have a CSA that differs by more than 50% along its length such that the most proximal segment is considerably smaller than the most distal tendon segment. Stress along the length of the tendon may consequently differ considerably. It is interesting to note that clinical conditions such as patellar and Achilles tendinopathy occur in the region with the greatest average stress for a given applied force, and the aetiology may therefore be somehow related to the stress in the region. Furthermore, if in fact there is tendon hypertrophy in response to increased

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loading (see above), there is some evidence that it may occur in a region-specific manner (Kongsgaard et al. 2007; Magnusson and Kjñr 2003).

15.3.3 Methods to study tendon in vivo: the ultrasonography method Much of the current knowledge regarding mechanical behaviour is based on data from experimental animal or cadaver tissue, while some more recent studies have studied mechanics on a microscopic level. Currently, the most widely used technique to investigate, in vivo, human tendon displacement during muscle contractions is B-mode ultrasonography, which was developed in the mid-1990s for measurements in the whole tendon±aponeurosis complex (Fukashiro et al. 1995) and late 1990s for measurements in isolated tendon (Maganaris and Paul 1999). The non-invasive approach of studying human tendons in vivo is attractive, and many limitations have been successfully addressed, but several details have to be considered. The method accounts for only two dimensions of structural deformation (i.e. in the sagittal plane). The technique was originally intended for the displacement of intramuscular fascicular structures, and therefore the resulting deformation does not represent that of the tendon per se, but rather the total deformation of the combined tendon and aponeurosis distal to the measurement site. This can, in some muscle± tendon complexes, but not all, be circumvented by identifying the very junction between the aponeurosis and free tendon (the myotendinous junction), or alternatively by introducing a visible landmark such as a needle (Magnusson et al. 2003). For the patellar tendon the problem can be overcome by having two bony landmarks (tibia and patella) (Hansen et al. 2006). The technique is most commonly employed during `isometric' conditions in which small amounts of joint rotation or body movement may take place that can affect the displacement measurements, which need to be accounted for (Magnusson et al. 2003). A true resting length of the tendon (i.e. 0% strain) may be difficult to obtain in vivo, but may be defined as that corresponding to zero net joint moment. The current time resolution of ultrasonography (frequency of sampling) and of video recording apparatus is a limiting factor that precludes analysis of faster contraction velocities. However, the technique still lacks an associated accurate measurement of tendon force. Notwithstanding these methodological difficulties, the technique has provided recent insights into human, in vivo, tendon behaviour.

15.3.4 Aponeurosis shear in human muscle±tendon complex The facts that the CSA of tendon differs along its length and that the stress seen by the tendon therefore also differs considerably for a given applied force prompt questions as to how force is transmitted in general throughout the tendon. In this regard the human Achilles tendon is of interest because of its

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unique anatomy and because it is often implicated clinically. The human triceps surae muscle±tendon structure is composed of three separate muscle compartments that merge via their aponeuroses into one common tendon to ultimately insert on the calcaneus. To what extent activation of the individual muscles of the triceps surae complex influences aponeurosis and tendon strain is uncertain. An in vitro study demonstrated that differences in medial and lateral forces in the Achilles tendon can be observed when single muscles of the triceps surae were subjected to force (Arndt et al. 1999). Non-uniform tendon force would theoretically result in intratendinous shear strain and cause sliding between planes of tissue layers parallel to the acting forces. Because the triceps surae includes the gastrocnemii muscles that cross both the ankle and knee joints, and the soleus muscle that crosses the ankle joint alone, the relative contribution of these muscles to the tendon force will be influenced by the degree of knee flexion (Cresswell et al. 1995). It has been shown that during maximal isometric contractions with the plantarflexor muscles, there is a differential displacement between the soleus and gastrocnemius aponeuroses proximal to the junction with the Achilles tendon (Bojsen-Moller et al. 2004). When the knee joint was extended, displacement of the medial gastrocnemius aponeurosis exceeded that of the soleus aponeurosis, whereas the converse occurred when the knee joint was flexed. These differences in aponeurosis displacement created a `shear' effect with a direction that was governed by the knee joint position. Since the collagen fibres of the aponeurosis fuse distally to become free tendon structures, any difference in displacement at the level of the aponeuroses may be manifested as shear strain at the level of the tendon, although this has yet to be confirmed. The difference in displacement between separate aponeuroses within the triceps surae amounted to more than 30% of the maximal observed displacement, indicating that a considerable `shear potential' exists during movement tasks. The observed differential aponeurosis displacement was likely caused by differences in force output of the medial gastrocnemius and soleus muscles.

15.3.5 Mechanical properties of individual human tendon fascicles Patellar tendinopathy chiefly concerns the proximal and posterior portion of the patellar tendon (Johnson et al. 1996), and this poses the question as to whether the mechanical properties differ by region within a whole tendon. Data on strain properties of the anterior and posterior portion of the human patellar tendon is sparse and conflicting. In cadaver knees of older persons it has been shown that during quadriceps loading tensile strain was uniform in the anterior and posterior region with the knee in full extension, but tensile strain increased on the anterior side and decreased on the posterior with knee flexion (Almekinders et al. 2002). It has also been shown, however, that quadriceps loading in flexion caused a

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greater strain on the posterior compared to the anterior side of the patellar tendon (Basso et al. 2002). An alternative way to examine if the mechanical properties differ by region in the human patellar tendon is to test the isolated portions of the tendon. It was recently possible to obtain human tissue from healthy young men in association with elective anterior cruciate ligament reconstruction (Haraldsson et al. 2005). Thin collagen bundles that were ~35 mm in length and ~3.5 mm in diameter were obtained from the anterior and posterior portion of the harvested tendon. From these collagen bundles, individual strands of fascicles ( ~ 300 m) were dissected and tested for mechanical properties. The data showed that tendon fascicles from the anterior portion of the human patellar tendon in young men displayed substantially greater peak and yield stress and tangent modulus compared with the posterior portion of the tendon. This observation suggests that regions of the patellar tendon may have markedly different mechanical properties. It is not surprising since there may be regional differences when it comes to the structural components of the tendon. In the horse there may be region-specific differences with respect to collagen fibril diameter (PattersonKane et al. 1997), and it has also been shown that there may be a site-specific loss of larger size collagen fibrils in the core of ruptured human Achilles tendons (Magnusson et al. 2002). It remains to be established if the above noted differences in mechanical properties of the patellar tendon can be attributed to factors such as fibril size and density (Parry et al. 1978), cross-links (Thompson and Czernuszka 1995), fibril length (Redaelli et al. 2003) and components of the extracellular matrix (Kjñr 2004). It is also unknown to what extent training regimes with varying degrees of knee flexion may influence the region-specific material properties of the tendon, and if such exercises can reduce the sitespecific patellar tendinopathy. There is some understanding of force transmission in skeletal muscle, but relatively little is known about force transmission in tendon. Skeletal muscle fibres do not always extend from one tendon plate to the other, which requires that contractile force can be transmitted laterally to adjacent fibres to initiate movement, which was deomonstrated elegantly by Street (1983). Force is transmitted from the muscle fibres to the aponeurosis via the myotendinous junction, and can also be transmitted to parallel adjacent structures via the aponeurosis (Huijing and Jaspers 2005). The aponeurosis, which has different mechanical properties than free tendon (Magnusson et al. 2003), may be loaded heterogeneously during skeletal muscle contraction (Finni et al. 2003), although how this affects tendon force transmission and to what extent lateral force transmission exists in human free tendon remains unknown. The tendon is a hierarchical structure (Kastelic et al. 1978), and to understand how force is transmitted within the whole tendon it is necessary to examine the mechanical properties at the various levels. A disparity in magnitude of strain at different hierarchical levels has been observed, and while the exact mechanism

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for such a hierarchy of strain distribution within the tendon structure remains unknown, it suggests that `gliding' processes may be involved (Mosler et al. 1985; Puxkandl et al. 2002). Studies on force pathways in tendons have largely focused on molecular strain and intermolecular force transmission (Mosler et al. 1985; Puxkandl et al. 2002; Sasaki and Odajima 1996), fibril±fibroblast mechanotransduction (Arnoczky et al. 2002) and contributing factors to interfibrillar force transmission (Scott 2003; Vesentini et al. 2005). The tendon fascicle, however, is a distinct entity within the tendon hierarchy. The ability of the fascicle to transmit force to the adjacent parallel fascicle was recently examined in human patellar and Achilles tendon tissue obtained during surgery. From each of the tendon specimens, two adjoining strands of collagen fascicles enclosed in an intact fascicular membrane were carefully dissected and special care was taken to ensure that the fascicular membrane between adjoining fascicles was left intact. The specimens were subjected to a series of load±displacement cycles to a given displacement (~3% strain). The first load±displacement cycle was performed with both fascicles and fascicular membrane intact. The second load±displacement cycle was performed with one fascicle transversally cut while the other fascicle and the fascicular membrane remained intact. The third load±displacement cycle was performed with both fascicles transversally cut on opposite ends while the fascicular membrane was kept intact. Theoretically, if there is no or inconsequential lateral transfer of force, a reduction in the CSA of a structure by 50% (one of two fascicles of similar size) should reduce the stiffness of the structure by half. Severing one fascicle from the patellar tendon reduced the material stiffness by ~40% while the same procedure reduced the material stiffness by more than 50% in the Achilles tendon, indicating that lateral force transmission between adjacent human tendon fascicles was marginal. Cycle 3 represented two adjacent fascicles that could potentially transmit force only in parallel rather than in series, and these data confirm the findings of the comparison between cycle 1 and 2 by demonstrating that the lateral force transmission between fascicles was relatively small or negligible. Of course, these data do not show to what extent fascicles are functionally `independent' in whole tendon in vivo. It appears that the magnitude of strain at the various hierarchical levels differ such that strain of the whole tendon exceeds that of the strain at the molecule and fibril level (Mosler et al. 1985; Puxkandl et al. 2002; Sasaki and Odajima 1996). Specifically, it has been suggested that during loading the triple helix itself may elongate, that the gap region between longitudinally adjoining molecules increases, that there is relative slippage between laterally adjacent molecules, and that fibrils themselves may slide relative to one another. At the level of the molecule, strain will be taken up by cross-links (Puxkandl et al. 2002), while the strain between fibrils may be prevented by components of the extracellular matrix (Scott, 2003). Intuitively, this means that some of the force is `transferred' to the adjacent structure by

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means of lateral shear forces. The fascicle cutting experiment adds to the existing knowledge of tendon mechanical properties by demonstrating that gliding processes can take place freely between adjacent fascicles. However, the present data also suggests that the aforementioned mechanisms of lateral shear force are largely limited to within the fascicle.

15.4

References and further reading

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