Elastic energy storage in unmineralized and mineralized extracellular matrices (ECMs): a comparison between molecular modeling and experimental measurements

In order to facilitate locomotion and limb movement many animals store energy elastically in their tendons. In the turkey, much of the force generated by the gastrocnemius muscle is stored as elastic energy during tendon deformation and not within the muscle. As limbs move, the tendons are strained causing the collagen fibers in the extracellular matrices to be strained. During growth, avian tendons mineralize in the portions distal to the muscle and show increased tensile strength, modulus, and energy stored per unit strain as a result. In this study the energy stored in unmineralized and mineralized collagen fibers was measured and compared to the amount of energy stored in molecular models. Elastic energy storage values calculated using the molecular model were slightly higher than those obtained from collagen fibers, but display the same increases in slope as the fiber data. We hypothesize that these increases in slope are due to a change from the stretching of flexible regions of the collagen molecule to the stretching of less flexible regions. The elastic modulus obtained from the unmineralized molecular model correlates well with elastic moduli of unmineralized collagen from other studies. This study demonstrates the potential importance of molecular modeling in the design of new biomaterials.     

The role of tenascin-C in adaptation of tendons to compressive loading

Although most tendon regions are subjected primarily to high tensile loads, selected regions, primarily those that directly contact bones that change the direction of the tendon, must withstand high compressive loads as well. Compressed tendon regions differ from regions subjected to primarily tensile loads: they have a fibrocartilaginous structure with spherical cells surrounded by a matrix containing aggrecan and collagen types I and II, in contrast regions not exposed to compression have a fibrous structure with spindle shaped fibroblasts surrounded by a matrix of dense, longitudinally oriented type I collagen fibrils. The spherical shape of cells in fibrocartilagenous regions indicates these cells are more loosely attached to the matrix than their spindle-shaped counterparts in fibrous regions, a feature that may help to minimize cell deformation during tendon compression. We hypothesized that expression of tenascin-C, an anti-adhesive protein, is part of the adaptation of tendon cells to compression that helps establish and maintain fibrocartilaginous regions. To test this hypothesis we compared tenascin-C content and expression in compressed (distal) versus uncompressed (proximal) segments of bovine flexor tendons. Immunohistochemistry and immunoblot analyses showed that tenascin-C content was increased in the distal tendon where it co-distributed with type II collagen and aggrecan. Tendon cells from the distal segments expressed more tenascin-C than did cells from the proximal segments for up to four days in cell culture, indicating that increased tenascin-C expression is a relatively stable feature of the distal cells. These observations support the hypothesis that tenascin-C expression is a cellular adaptation to compression that helps establish and maintain fibrocartilagenous regions of tendons.     

The mechanics of jumping by a dog (Canis familiaris)

A force platform has been used to obtain records of the forces exerted on the ground by an Alsatian dog, during take-off for running long jumps and standing scale jumps. The records have been analysed in conjunction with cinematograph film, taken simultaneously, and anatomical data. Stresses in the principal muscles of the hind limb, and in the triceps, have been calculated and the values obtained are compared with the stresses found by other investigators in isometric experiments with excised mammal muscles. Stresses in certain tendons and bones have been calculated, and the values obtained are compared with published values for the strength of tendon and bone. Evidence is presented that the gastrocnemius and plantaris muscles behave, in take-off for a jump, essentially as passive elastic bodies. Most of the elastic energy is probably stored in their tendons. A tendency for distal limb muscles to be pinnate, with much shorter fibres than proximal limb muscles, is noted and discussed.     

Morphometric properties and innervation of muscle compartments in rat medial gastrocnemius

The rat medial gastrocnemius (MG) muscle is composed of the proximal and distal compartments. In this study, morphometric properties of the compartments and their muscle fibres at five levels of the muscle length and the innervation pattern of these compartments from lumbar segments were investigated. The size and number of muscle fibres in the compartments were different. The proximal compartment at the largest cross section (25% of the muscle length) had 34% smaller cross-sectional area but contained a slightly higher number of muscle fibres (max. 5521 vs. 5360) in comparison to data for the distal compartment which had the largest cross-sectional area at 40% of the muscle length. The muscle fibre diameters revealed a clear tendency within both compartments to increase along the muscle (from the knee to the Achilles tendon) up to 46.9?μm in the proximal compartment and 58.4?μm in the distal one. The maximal tetanic and single twitch force evoked by stimulation of L4, L5, and L6 ventral roots in whole muscle and compartments were measured. The MG was innervated from L4 and L5, only L5, or L5 and L6 segments. The proximal compartment was innervated by axons from L5 or L5 and L4, and the distal one from L5, L5 and L6, or L5 and L4 segments. The forces produced by the compartments summed non-linearly. The tetanic forces of the proximal and distal compartments amounted to 2.24 and 4.86?N, respectively, and their algebraic sums were 11% higher than the whole muscle force (6.37?N).     

Collagen fibril size and crimp morphology in ruptured and intact Achilles tendons.

The present study examined the hypothesis that collagen fibril diameter and crimp angle in ruptured human Achilles tendons differed from that of intact ones. Tissue samples were obtained from the central core (distal core) and the posterior periphery (distal superficial) at the rupture site, and the proximally intact (proximal superficial) part of the tendon in 10 subjects (38+/-8 years) with a complete tendon rupture. For comparisons corresponding tissue samples were procured from age (38+/-7 years) and gender matched intact Achilles tendons during routine forensic autopsy. The cross-sectional area density and diameter distribution of fibrils were analyzed using stereological techniques of digitized electron microscopy biopsy cross-sections, while crimp angle was measured by the changing banding pattern of collagen fibers when rotated between crossed polars. Nine of 10 persons with tendon ruptures reported that the injury did not occur during exceedingly large forces, and none experienced any symptoms in the days or months prior to the injury. Fibril diameter distribution showed no region-specific differences in either the ruptured or intact tendons for either group. However, in the distal core there were fewer fibrils in the ruptured compared to the intact tendons in 60-150 nm range, P<0.01. Similarly, in the distal superficial portion there were fewer fibrils in the ruptured compared to the intact tendons in the 90-120 nm range, 2P<0.05, while there were no differences in the proximal superficial tendons. Crimp angle did not display any region-specific differences, or any difference between the rupture and intact tendons. In conclusion, these data suggest that although crimp morphology is unchanged there appears to be a site-specific loss of larger fibrils in the core and periphery of the Achilles tendon rupture site. Moreover, the lack of symptoms prior to the rupture suggests that clinical tendinopathy is not an etiological factor in complete tendon ruptures.     

Regional adaptations in three rat tendons

Although detailed histological and immunocytochemical studies have been published for the rat calcanear tendon (CT), little is known of the structure, composition and biomechanics of the deep (DFT) and superficial (SFT) flexor tendons. In this study, we examined the structural specialization of these three tendons in 90-day-old rats by applying histochemical and biochemical assays to different tendon regions (proximal, intermediate and distal regions of the DFT and SFT, and proximal and distal regions of the CT). There were regional differences in tissue structure, glycosaminoglycan type and content, swelling properties and in the amount and distribution of elastic fibers. Dermatan sulfate occurred in all regions, but chondroitin sulfate predominated in the intermediate region of the DFT and in the distal region of the CT. These two chondroitin sulfate-bearing regions showed swelling in water, while all other regions lost fluid in water. Fibrocartilaginous sites were observed on the CT, one at the insertion to the bone and another distally at the innermost area of the tendon. The intermediate region of the DFT showed round cells disposed in lacunae, while the proximal and distal regions were typically fibrous. The intermediate region of the SFT showed a wavy array of collagen bundles but neither toluidine blue staining in the matrix nor round cells. Elastic fibers were present in each region of the three tendons, but were more prominent in the intermediate zone of the SFT. These results demonstrate regional variation in the three tendons. Tendon differentiation may occur by an increase in the number of elastic fibers and by variations in the arrangement of collagen fibers, without fibrocartilage formation.     

Membrane-bound intracellular collagen fibrils in fibroblasts and myofibroblasts of regenerating rabbit calcaneal tendons.

The ultrastructures of 33 rabbit calcaneal tendons were studied to determine (1) whether vacuolar fibrils are present in three regions of tendons undergoing normal healing after tenotomy and repair, and (2) to stimulate collagen synthesis via functional loading, and hence determine the effect of loading on the presence of vacuolar fibrils in healing tendons. In all the loaded tendons, electron microscopy revealed membrane-bound collagen fibril equivalents in sections of neotendon obtained from the site of tenotomy, and in sections of tendon segments proximal and distal to the site of surgery. Similar vacuolar fibrils were visualized in sections of the proximal and distal segments of the non-loaded regenerating tendons, and also in sections of neotendons formed at the site of tenotomy after 12 and 15 days of healing without functional loading. No such fibrils were visualized in the non-tenotomized normal control tendons. These findings indicate that chemical agents and disease are not necessary to induce the appearance of intracytoplasmic fibrils in vivo and that functional loading augments the presence of fibril-bearing vacuoles in regenerating tendons.     

Collagen self-assembly and the development of tendon mechanical properties

The development of the musculoskeleton and the ability to locomote requires controlled cell division as well as spatial control over deposition of extracellular matrix. Self-assembly of procollagen and its final processing into collagen fibrils occurs extracellularly. The formation of crosslinked collagen fibers results in the conversion of weak liquid-like embryonic tissues to tough elastic solids that can store energy and do work. Collagen fibers in the form of fascicles are the major structural units found in tendon. The purpose of this paper is to review the literature on collagen self-assembly and tendon development and to relate this information to the development of elastic energy storage in non-mineralizing and mineralizing tendons. Of particular interest is the mechanism by which energy is stored in tendons during locomotion. In vivo, collagen self-assembly occurs by the deposition of thin fibrils in recesses within the cell membrane. These thin fibrils later grow in length and width by lateral fusion of intermediates. In vitro, collagen self-assembly occurs by both linear and lateral growth steps with parallel events seen in vivo; however, in the absence of cellular control and enzymatic cleavage of the propeptides, the growth mechanism is altered, and the fibrils are irregular in cross section. Results of mechanical studies suggest that prior to locomotion the mechanical response of tendon to loading is dominated by the viscous sliding of collagen fibrils. In contrast, after birth when locomotion begins, the mechanical response is dominated by elastic stretching of crosslinked collagen molecules.     

Elastic mechanisms in primate locomotion

Tendons that stretch elastically and recoil, as the forces on them rise and fall, can save energy in running by enabling the animal to make do with shorter or slower muscle fascicles, that can generate force more economically. Non-human primates have rather long fascicles and thick tendons in their distal leg muscles and so seem poorly adapted to save energy in this way. Additional savings are made possible by the elastic compliance of ligaments in the foot. Though tendon and ligament compliance tend to save energy, the compliance of branches tends to increase the energy cost of arboreal locomotion.     

Elastic properties of human Achilles tendon are correlated to muscle strength.

The purpose of this study was to investigate whether the mechanical properties of the Achilles tendon were correlated to muscle strength in the triceps surae in humans. Twenty-four men and twelve women exerted maximal voluntary isometric plantar flexion (MVIP) torque. The elongation (DeltaX) and strain of the Achilles tendon (epsilon), the proximal part of which is the composite of the gastrocnemius tendon and the soleus aponeurosis, at MVIP were determined from the displacement of the distal myotendinous junction of the medial gastrocnemius using ultrasonography. The Achilles tendon force at MVIP (F) was calculated from the MVIP torque and the Achilles tendon moment arm. There were no significant differences in either the F-DeltaX or F-epsilon relationships between men and women. DeltaX and epsilon were 9.8 +/- 2.6 mm and 5.3 +/- 1.6%, respectively, and were positively correlated to F (r = 0.39, P < 0.05; r = 0.39, P < 0.05), which meant that subjects with greater muscle strength could store more elastic energy in the tendon. The regression y-intercepts for the F-DeltaX (P < 0.01) and F-epsilon (P < 0.05) relationship were significantly positive. These results might indicate that the Achilles tendon was stiffer in subjects with greater muscle strength, which may play a role in reducing the probability of tendon strain injuries. It was suggested that the Achilles tendon of subjects with greater muscle strength did not impair the potential for storing elastic energy in tendons and may be able to deliver the greater force supplied from a stronger muscle more efficiently. Furthermore, the difference in the Achilles tendon mechanical properties between men and women seemed to be correlated to the difference in muscle strength rather than gender.     

Analysis of cumulative strain in tendons and tendon sheaths

Twenty-five fresh frozen flexor digitorum profundus tendons stratified by sex were subjected to uniaxial step stress and cyclic loads in twelve intact human cadaver hands. By attaching specially designed clip strain gage transducers on tendons just proximal and distal to an undisrupted carpal tunnel, the interactions of the tendons, tendon sheath and retinacula were measured. The elastic and viscous response of the tendon composites to step stresses were found to fit fractional power functions of stress and time respectively. A significant and quantifiable decrease in strain from the proximal to the distal tendon segment was found to be a function of wrist deviation. The results indicate that an accumulation of strain does occur in tendinous tissues during physiologic loading.     

The myotendinous junction of the smooth feather muscles (mm. pennati)

Summary Smooth feather muscles (mm. pennati) consist of bundles of smooth muscle cells which are attached to the feather follicles by short elastic tendons. In addition, some muscle bundles are interrupted by elastic tendons. The elastic tendon is composed of longitudinally arranged elastic fibers which branch and wavy bundles of collagen fibrils. Smooth muscle cells of the muscle bundles are attached to each other by desmosome-like junctions and by fusion of the basal laminae. The cytoplasm of the muscle cells is characterized by conspicuous thick filaments and abundant thin and intermediate filaments. These are attached to band-like dense patches (dense bands) at the plasma membrane which are particularly broad at the tapering end of the muscle cell. The contact surface between smooth muscle cells and their elastic tendon is considerably increased (i) by deep finger-like invaginations and indentations located at the tapering muscle end, and (ii) by branching of the coarse elastic fibers into slender processes, which are attached to the richly folded surface of the muscle cell endings by peripheral microfibrils. This intimate interlocking closely resembles the myotendinous junctions in skeletal muscle. In addition to fibroblasts and fibrocytes, the myotendinous junction of the young growing chicks contains numerous so-called myofibroblasts, which are suggested to represent smooth muscle cells differentiating into fibroblasts of the developing tendon.Dedicated to Professor Dr. Helmut Leonhardt on the occasion of his 60th birthdaySupported by a grant from the Deutsche Forschungsgemeinschaft (Dr. 91/1)     

Tendon crimps and peritendinous tissues responding to tensional forces

Tendons transmit forces generated from muscle to bone making joint movements possible. Tendon collagen has a complex supramolecular structure forming many hierarchical levels of association; its main functional unit is the collagen fibril forming fibers and fascicles. Since tendons are enclosed by loose connective sheaths in continuity with muscle sheaths, it is likely that tendon sheaths could play a role in absorbing/transmitting the forces created by muscle contraction. In this study rat Achilles tendons were passively stretched in vivo to be observed at polarized light microscope (PLM), scanning electron microscope (SEM) and transmission electron microscope (TEM). At PLM tendon collagen fibers in relaxed rat Achilles tendons ran straight and parallel, showing a periodic crimp pattern. Similarly tendon sheaths showed apparent crimps. At higher magnification SEM and TEM revealed that in each tendon crimp large and heterogeneous collagen fibrils running straight and parallel suddenly changed their direction undergoing localized and variable modifications. These fibril modifications were named fibrillar crimps. Tendon sheaths displayed small and uniform fibrils running parallel with a wavy course without any ultrastructural aspects of crimp. Since in passively stretched Achilles tendons fibrillar crimps were still observed, it is likely that during the tendon stretching, and presumably during the tendon elongation in muscle contraction, the fibrillar crimp may be the real structural component of the tendon crimp acting as shock absorber. The peritendinous sheath can be stretched as tendon, but is not actively involved in the mechanism of shock absorber as the fibrillar crimp. The different functional behaviour of tendons and sheaths may be due to the different structural and molecular arrangement of their fibrils.     

Comparative anatomy of rabbit and human achilles tendons with magnetic resonance and ultrasound imaging

We sought to describe the comparative anatomy of the Achilles tendon in rabbits and humans by using macroscopic observation, magnetic resonance imaging, and ultrasonography. The calcaneus-Achilles tendon-gastrocnemius-soleus complexes from 18 New Zealand white rabbits underwent ultrasound (US) and magnetic resonance (MR) imaging and gross anatomic sectioning; these results were compared with those from a cadaveric gastrocnemius-soleus-Achilles tendon-calcaneus specimen from a 68-y-old human male. The medial and lateral gastrocnemius muscle tendons merged 5.2 +/- 0.6 mm proximal to the calcaneal insertion macroscopically, at 93% of their course, different from the gastrocnemius human tendons, which merged at 23% of their overall course. The rabbit flexor digitorum superficialis tendon, corresponding to the flexor digitorum longus tendon in human and comparable in size with the gastrocnemius tendons, was located medial and anterior to the medial gastrocnemius tendon proximally and rotated dorsally and laterally to run posterior to the Achilles tendon-calcaneus insertion. In humans, the flexor digitorum longus tendon tracks posteriorly to the medial malleolus. The soleus muscle and tendon are negligible in the rabbit; these particular comparative anatomic features in the rabbit were confirmed on the MR images. Therefore the rabbit Achilles tendon shows distinctive gross anatomical and MR imaging features that must be considered when using the rabbit as a research model, especially for mechanical testing, or when generalizing results from rabbits to humans.     

The role of tendon elasticity in the locomotion of the camel (Camelus dromedarius)

Several ofthe distal leg muscles ofcamels have very short or even rudimentary muscle fibres. This makes it possible to calculate the elastic extensions of tendons that occur in running, from the leg positions observed in films. A series of experiments have been performed for this purpose on the dissected legs of a camel. The initial conclusions derived from them are modified in the light of estimates of the forces that act on the tendons, and of measurements of the elastic properties of one tendon. Evidence for movement at the intertarsal and tarso-metatarsal joints. and the corresponding joints of the fore leg, is examined. The importance of the various tendons as elastic energy stores in running is assessed.     

Myofascial force transmission: muscle relative position and length determine agonist and synergist muscle force.

Equal proximal and distal lengthening of rat extensor digitorum longus (EDL) were studied. Tibialis anterior, extensor hallucis longus, and EDL were active maximally. The connective tissues around these muscle bellies were left intact. Proximal EDL forces differed from distal forces, indicating myofascial force transmission to structures other than the tendons. Higher EDL distal force was exerted (ratio approximately 118%) after distal than after equal proximal lengthening. For proximal force, the reverse occurred (ratio approximately 157%). Passive EDL force exerted at the lengthened end was 7-10 times the force exerted at the nonlengthened end. While kept at constant length, synergists (tibialis anterior + extensor hallucis longus: active muscle force difference approximately -10%) significantly decreased in force by distal EDL lengthening, but not by proximal EDL lengthening. We conclude that force exerted at the tendon at the lengthened end of a muscle is higher because of the extra load imposed by myofascial force transmission on parts of the muscle belly. This is mediated by changes of the relative position of most parts of the lengthened muscle with respect to neighboring muscles and to compartment connective tissues. As a consequence, muscle relative position is a major codeterminant of muscle force for muscle with connectivity of its belly close to in vivo conditions.     

Structure and function of tuna tail tendons

The caudal tendons in tunas and other scombrid fish link myotomal muscle directly to the caudal fin rays, and thus serve to transfer muscle power to the hydrofoil-like tail during swimming. These robust collagenous tendons have structural and mechanical similarity to tendons found in other vertebrates, notably the leg tendons of terrestrial mammals. Biochemical studies indicate that tuna tendon collagen is composed of the (alpha1)(2),alpha2 heterotrimer that is typical of vertebrate Type I collagen, while tuna skin collagen has the unusual alpha1,alpha2,alpha3 trimer previously described in the skin of some other teleost species. Tuna collagen, like that of other fish, has high solubility due to the presence of an acid-labile intermolecular cross-link. Unlike collagen in mammalian tendons, no differences related to cross-link maturation were detected among tendons in tuna ranging from 0.05 to 72 kg (approx. 0.25-6 years). Tendons excised post-mortem were subjected to load cycling to determine the modulus of elasticity and resilience (mean of 1.3 GPa and 90%, respectively). These material properties compare closely to those of leg tendons from adult mammals that can function as effective biological springs in terrestrial locomotion, but the breaking strength is substantially lower. Peak tendon forces recorded during steady swimming appear to impose strains of much less than 1% of tendon length, and no more than 1.5% during bursts. Thus, the caudal tendons in tunas do not appear to function as elastic storage elements, even at maximal swimming effort.     

Macroscopic and microscopic analyses in flexor tendons of the tarsometatarso‐phalangeal joint of ostrich (Struthio camelus) foot with energy storage and shock absorption

Flexor tendons function as energy storage and shock absorption structures in the tarsometatarso‐phalangeal joint (TMTPJ) of ostrich feet during high‐speed and heavy‐load locomotion. In this study, mechanisms underlying the energy storage and shock absorption of three flexor tendons of the third toe were studied using histology and scanning electron microscopy (SEM). Macroscopic and microscopic structures of the flexor tendons in different positions of TMTPJ were analyzed. Histological slices showed collagen fiber bundles of all flexor tendons in the middle TMTPJ were arranged in a linear‐type, but in the proximal and distal TMTPJ, a wavy‐type arrangement was found in the tendon of the M. flexor digitorum longus and tendon of the M. flexor perforans et perforatus digiti III, while no regular‐type was found in the tendon of the M. flexor perforatus digiti III. SEM showed that the collagen fiber bundles of flexor tendons were arranged in a hierarchically staggered way (horizontally linear‐type and vertically linear‐type). Linear‐type and wavy‐type both existed in the proximal TMTPJ for the collagen fiber bundles of the tendon of the M. flexor perforatus digiti III, but only the linear‐type was found in the distal TMTPJ. A number of fibrils were distributed among the collagen fiber bundles, which were likely effective in connection, force transmission and other functions. The morphology and arrangement of collagen fiber bundles were closely related to the tendon functions. We present interpretations of the biological functions in different positions and types of the tendons in the TMTPJ of the ostrich feet.     

Mechanical properties of rat tail tendon in relation to proximal-distal sampling position and age

The mechanical properties of 3, 15 and 25 month-old rat tail tendons were investigated in relation to proximal-distal sampling location along the fibre length. For the 15 and 25 month-old tendons maximum load as well as collagen content per mm fibre length (unit collagen) increased markedly from the proximal to the distal location. A linear regression analysis of the collagen content and mechanical parameters (maximum load, maximum slope of the load-strain curve and energy absorption) showed that these parameters were linearly correlated to the collagen content. However, normalization of the mechanical parameters with regard to the collagen content did not cancel the dependency of the parameters on proximal-distal sampling location. Normalized load and energy values for the 3 month-old tendons and normalized slope values for the 15 and 25 month-old tendons were found to decrease from proximal to distal location. These findings showed that tail tendons are heterogeneous along their length in respect to mechanical strength. The regression analysis also indicated the existence of an inverse relationship between unit collagen and mechanical quality of the collagen. Alternatively, the mechanical properties of tendon fibres might be influenced by other components than collagen.     

Local strain measurement reveals a varied regional dependence of tensile tendon mechanics on glycosaminoglycan content

Proteoglycans (PG) and their associated glycosaminoglycan (GAG) side chains are known to play a key role in the bearing of compressive loads in cartilage and other skeletal connective tissues. In tendons and connective tissues that are primarily loaded in tension, the influence of proteoglycans on mechanical behavior is debated due to conflicting experimental evidence that alternately supports or controverts a functional role of proteoglycans in bearing tensile load. In this study we sought to better reconcile these conflicting data by investigating the possibility that GAG content is differentially related to tensile tendon mechanics depending upon the anatomical subregion one considers. To test this hypothesis, we quantified the mechanical consequences of proteoglycan disruption within specific tendon anatomical subregions using an optical–mechanical measurement approach.Achilles tendons from adult mice were treated with chondroitinase ABC to obtain two groups consisting of native tendons and GAG-depleted tendons. All the tendons were mechanically tested and imaged with high-resolution digital video in order to optically quantify tendon strains. Tendon surface strains were locally analyzed in three main subregions: the central midsubstance, and the proximal and distal midsubstance near the muscle and bone insertions, respectively. Upon GAG digestion, the tendon midsubstance softened appreciably near the bone insertion, while elastic modulus in the central and proximal thirds was unchanged. Thus the contribution of PGs to tensile tendon mechanics is not straightforward and points to a heterogeneous and complex structure–function relationship in tendon. This study further highlights the importance of performing local strain analysis with regard to tensile tendon mechanics.