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The Journal of Experimental Biology 203, 2187–2193 (2000) Printed in Great Britain © The Company of Biologists Limited 2000 JEB2802
THE DEVELOPMENT OF FATIGUE QUALITY IN HIGH- AND LOW-STRESSED TENDONS OF SHEEP (OVIS ARIES) ANNA V. L. PIKE*, ROBERT F. KER AND R. MCNEILL ALEXANDER School of Biology, University of Leeds, Leeds LS2 9JT, UK *e-mail:
[email protected]
Accepted 18 April; published on WWW 22 June 2000 Summary The time taken to rupture in cyclic fatigue tests, to a significantly with this stress. This relationship was not seen, stress of 45 MPa, was used to compare the fatigue quality however, in low-stressed tendons, which are not subjected of tendons from sheep of varying ages. Muscle and tendon to a comparable range of stresses over time. It is possible cross-sectional areas were used to calculate the stress-inthat cells modify tendon fatigue quality in response to life of each tendon. For any given age, high-stressed tendon loading history. Whilst Young’s modulus was seen plantaris tendons were of a higher fatigue quality than lowto increase with age, no difference was detected between stressed extensor tendons. Both fatigue quality and stresshigh- and low-stressed tendons. in-life increased with age for each tendon type. Highstressed tendons are subjected to large increases in Key words: tendon, development, fatigue damage, fatigue quality, stress-in-life, time to rupture, Young’s modulus, sheep, Ovis aries. stress-in-life during growth, and fatigue quality increased
Introduction Tendons are cyclically loaded during locomotion. The fluctuating stresses can be expected to cause damage, which will accumulate, leading eventually to failure unless it is repaired. In vitro experiments (in which repair processes do not operate) have confirmed that damage accumulation and eventual failure occur in both prolonged cyclic and prolonged static tests, even at fairly low stresses (Wang and Ker, 1995; Wang et al., 1995; Schechtman and Bader, 1997; Ker et al., 2000). Some tendons have to withstand much higher peak stresses than others. This was apparent from a study in which measurements were made of the cross-sectional areas of various tendons from a diverse selection of mammals and of the physiological cross-sectional areas of their muscles (Ker et al., 1988). The stresses that would act in the tendons, when their muscles exerted their estimated maximum isometric stress of 0.3 MPa, were far higher for gastrocnemius and plantaris tendons of species in which these tendons are important in running, as energy-saving springs, than for other tendons. In most respects, these high-stressed tendons have mechanical properties very like those of low-stressed tendons: they have similar values of Young’s modulus and energy dissipation (Bennett et al., 1986). It is also thought that they are similar with respect to ultimate tensile stress, although problems associated with clamping make this measurement difficult to determine with confidence (see Ker et al., 2000). Ker et al. (2000), however, have shown that high-stressed tendons are exceptionally resistant to damage due to static fatigue. They demonstrate a greater ‘fatigue quality’ compared
with low-stressed tendons. It will be shown here that these tendons are also more resistant to damage due to cyclic fatigue than are low-stressed tendons. The main purpose of this paper is to determine whether the superior fatigue quality of highstressed tendons is present from birth or whether it develops as the animal matures. One possibility is that it may develop in response to the tendon’s loading history. Certainly, the physical properties of tendon are known to change over time. There is a general increase in the density of cross-links of collagen molecules in tendons with age (Sinex, 1968), restricting the ability of aggregates of collagen molecules to slip past one another (Gillis et al., 1997). However, it is uncertain whether these physical changes correlate with the stress to which a tendon is subjected in life. Parry et al. (1978a) suggest that there may be a relationship between fibril diameter distribution patterns and the stress a tendon experiences. In support of this, horse flexor tendons routinely subjected to long periods of high stress seem to have a bimodal distribution of fibril diameters at maturity, whereas lower-stressed extensor tendons do not (Parry et al., 1978b). However, the proposed relationship between fibril diameter and stress cannot explain the bimodal distribution of fibril diameter in mature rat tail tendon (Parry and Craig, 1977, 1978) or human digital extensor tendons (Dyer and Enna. 1976), which experience relatively low stresses in life (Ker et al., 1988). There is evidence in the literature that certain mechanical properties of tendons change during the maturation of an
2188 A. V. L. PIKE, R. F. KER AND R. MCN. ALEXANDER animal. Tendons from a variety of species show an increase in their Young’s modulus throughout maturation as well as an increase in their tensile strength (Torp et al., 1975a; Vogel, 1978; Shadwick, 1990; Nakagawa et al., 1996), whilst failure strain decreases (Torp et al., 1975a; Galeski et al., 1977; Shadwick, 1990). Information on the development of resistance to damage in tendons is limited. Torp et al. (1975b) studied the effect of age and mechanical deformation on the ultrastructure of tendon by cyclically straining rat tail tendons to a point where their elastic modulus had reduced to a predetermined level. They found fibrillar dissociation to be the primary failure mechanism associated with this reduction. The development of fissures within the tendon structure occurred as a secondary failure mechanism. Damage was cumulative and ultimately led to failure of the tendon. Fibrillar dissociation occurred at all ages, but the severity of the mechanical treatment required to induce the same amount of damage (as determined by reduction in the elastic modulus) increased with age. These observations are consistent with the fact that crosslink density increases with age (Sinex, 1968). Few studies have attempted to correlate changes in mechanical behaviour of tendons during growth with the stresses they experience in life. Shadwick (1990) reported that alterations during maturation in the elastic properties of highstressed swine digital flexor tendons occurred to a significantly greater degree than in the low-stressed extensor tendons. However, further work (Pollock and Shadwick, 1994) revealed no inter- or intra-specific differences in elastic properties between mature high- and low-stressed tendons. The present study aims to establish whether the resistance to damage mechanisms of fatigue seen in mammalian tendons develops over a period of time or whether it is an innate quality. It further aims to correlate any changes seen in such a resistance to damage with the stress experienced by tendons in life. Plantaris (high-stressed) and extensor digitorum lateralis (low-stressed) tendons from sheep of varying ages were subjected to fatigue tests to achieve these aims. Each tendon was subjected to the same stress regime to allow comparison of their material properties. Ker et al. (2000) used the term ‘fatigue quality’ to describe the resistance of a tendon to timedependent rupture. The same term will be used here. Cyclic loading was chosen as a realistic representation of locomotion because tendon stress is oscillatory in life. Materials and methods The plantaris and extensor digitorum lateralis muscles and their corresponding tendons were dissected from the hind limbs of sheep (Ovis aries) of various known ages from birth (0 days old) to mature adult. The animals had either died of natural causes or been culled, and were stored in plastic bags in the deep freeze at −20 °C prior to dissection. During dissection, the tendons and muscles were kept moist with 0.9 % saline. Tendons not tested immediately after the dissection process were wrapped in moist tissue (0.9 % saline) and stored at −20 °C in sealed plastic bags until required.
The stress-in-life (Ker et al., 2000) was calculated for each tendon by multiplying the muscle to tendon area ratio (physiological cross-sectional area of the muscle/crosssectional area of its tendon) by 0.3 MPa, which was assumed to be the maximum isometric stress of the muscle (Wells, 1965). There is no information as to whether this stress remains the same or changes with age. We have assumed that it remains constant. This does not affect the experimental data presented in this paper because our tendons were not tested to their stressin-life. If a muscle is stretched while it is active, then this maximum isometric stress can be exceeded. There is, however, some experimental evidence to suggest that this does not apply during locomotion (for running turkeys, see Roberts et al., 1997; for hopping wallabies, see Biewener et al., 1998). The physiological cross-sectional area of each muscle was calculated from fascicle length using the method described by Ker et al. (1988). However, since the fascicle length of a muscle depends on the position of the joint on which it is acting, the resulting measurements were corrected to lengths obtained using an optimal sarcomere length of 2.2 µm (Pike, 1998). This was deemed necessary to allow a true comparison of the muscles because the joint positions that occur at optimal sarcomere lengths in the living animal are unlikely to be preserved post-mortem unless fixation of the tissues in their correct positions occurs immediately after death. Fixation is unsuitable for experiments requiring fresh tissues such as that described here. Physical disturbance, either by moving the cadaver or by pulling the muscle during the dissection process (albeit unintentionally), may also cause fascicle length distortion. Tendon cross-sectional area measurements As well as needing tendon cross-sectional area measurements to calculate muscle to tendon area ratios, values are required to calculate the force needed to produce any given stress in a mechanical test. The main body of the extensor digitorum lateralis tendon is of almost uniform shape, which enabled its cross-sectional area to be estimated by gravimetric methods using values of wet mass of the measured length and density. Density was assumed to be 1120 kg m−3 (Ker, 1981) for all tendons. The plantaris tendon, however, is of a nonuniform shape, which makes accurate measurements of the cross-sectional area of the test region difficult. These tendons consist of three regions: (i) a short, uniform proximal region leading from the aponeurosis; (ii) a large, broad region where the tendon passes over the hock of the animal and (iii) a longer, distal region that bifurcates near the point of insertion onto the toes. The part of the distal region between the hock and the toe bifurcation provides a useful specimen on which to perform mechanical tests. The hock region and the toe bifurcation point provide areas for effective clamping in the testing machine if they are left attached to the test region. These broad areas, however, elevate the value of mass per unit length of the test region, resulting in over-estimation of cross-sectional areas if direct gravimetric methods are used. To overcome this problem, Ker et al. (2000) took suitable measurements from
Development of fatigue in tendon 2189 the opposite limb and applied them to the test specimen, the assumption being that there is symmetry between the two. We present a different method for obtaining reasonable estimates of cross-sectional area using the opposite limb. Rather than relying on the assumption that the left and right limbs of an animal mirror each other in their development, this method relies on the assumption that different regions of the same plantaris tendon develop at similar rates to one another during growth. The shape of the plantaris tendon as a whole lends itself to this method. The uniform portions of both the proximal and distal regions of each ‘non-test’ plantaris tendon were dissected and their cross-sectional areas calculated using the gravimetric method described above. The relationship between the cross-sectional areas of the two portions was determined by linear regression. This relationship was then used to estimate the cross-sectional area of the distal region of the test tendons, once the cross-sectional area of their proximal regions had been determined.
tendon visible between the clamps) measured with the small pre-load applied. It is conventional to express the fatigue behaviour of a material as a function of the number of cycles involved in the test. Time, however, can be used if the frequency of the oscillation is known and remains constant, as is the case here. Extension over time was recorded by a Servogor 102 chart recorder and used to calculate the time to rupture of the test. Tests in which the specimen ruptured in or immediately next to the clamps were discarded to avoid false, clamp-induced rupture times. In addition, tests in which the specimen ruptured during the initial loading cycle were also discarded to avoid the possibility that rupture was caused by the application of a stress greater than the tendon’s ultimate tensile stress. Tendons that underwent two or more cycles of loading were deemed to have failed as a result of fatigue, despite the very short rupture times experienced by some tendons.
Mechanical testing The ends of each test specimen were air-dried, whilst the middle region was kept moist (Wang and Ker, 1995; Ker et al., 2000). The dry ends of the tendon were clamped and held between two serrated metal plates attached to an Instron 8500 servo-hydraulic testing machine. The tendon and clamps were immersed in a liquid paraffin bath, which was pre-heated to 37 °C and saturated with 0.9 % saline with the aim of preventing desiccation of the specimen during the test. Liquid paraffin is an inert medium. Tendons immersed in pure liquid paraffin tend to dry out in a similar manner to, but much more slowly than, those left in air: over time, the tendon becomes stiffer against bending. This is not observed in tendons immersed in saline-saturated liquid paraffin. A small pre-load of between 3 and 10 N, depending on the thickness of the tendon, was applied. This pre-load was necessary to maintain the machine in load control throughout the test. A minimum stress of zero would have been ideal but is not possible to achieve under load control with a specimen, such as tendon, that buckles rather than developing compressive load. However, because of the exponential dependence of stress on time to rupture (Ker et al., 2000), the pre-load was too small for any significant damage to occur. Each tendon was then subjected to a fatigue test, cycled from the pre-load to a maximum stress of 45 MPa, at a frequency of 2.2 Hz. This frequency is within the range of stride frequencies for trotting mammals of the mass of adult sheep (Heglund et al., 1974). A maximum stress of 45 MPa was chosen to give a reasonably short test time for adult plantaris tendons. Previous experience has shown us that, when subjected to lower stresses, these tendons can last for many days before rupture. The initial changes in load and actuator position (a measure of tendon extension) during the first cycle of the test were plotted on an SE 790 x,y plotter. The tangent Young’s modulus, E, of each specimen was calculated from the linear region of this graph (above a stress of approximately 20 MPa). The initial length of the specimen was taken to be the daylight length (the length of
Results Fig. 1 shows a significant, positive relationship between the cross-sectional areas of the proximal and distal regions of the plantaris tendons (F1,20=429.11, P
