Mammalian Tendon Research Articles

An interferometric-based tensile tester to resolve damage events within reconstituted multi-filaments collagen bundles

Understanding how mechanical damage propagates in load-bearing tissues such as skin, tendons and ligaments, is key to developing regenerative medicine solutions for when these tissues fail. For collagenous tissues in particular, damage is typically assessed after mechanical testing using a broad range of microscopy techniques because standard tensile testing systems do not have the time and force sensitivity to resolve mechanical damage events. Here we introduce an interferometric detection scheme to measure the displacement of a cantilever with a resolution of 0.03% of full scale at a sampling rate of 5000 samples/s. The system is validated using collagen fibers engineered to mimic mammalian tendons. The system can detect sudden decrease in force due to slippage between collagen filaments, one to five microns in diameter, within a fiber in air. It can also detect yield events associated with local collagen unfolding or sliding within collagen fibrils within a fiber in liquid. This is opening the road to the sub-failure study of damage propagation within a broad range of hierarchical biomaterials.

Endogenous tenocyte activation underlies the regenerative capacity of the adult zebrafish tendon

Tendons are essential, frequently injured connective tissues that transmit forces from muscle to bone. Their unique highly ordered, matrix-rich structure is critical for proper function. While adult mammalian tendons heal after acute injuries, endogenous tendon cells, or tenocytes, fail to respond appropriately, resulting in the formation of disorganized fibrovascular scar tissue with impaired function and increased propensity for re-injury. Here, we show that, unlike mammals, adult zebrafish tenocytes activate upon injury and fully regenerate the tendon. Using a full tear injury model in the adult zebrafish craniofacial tendon, we defined the hallmark stages and cellular basis of tendon regeneration through multiphoton imaging, lineage tracing, and transmission electron microscopy approaches. Remarkably, we observe that zebrafish tendons regenerate and restore normal collagen matrix ultrastructure by 6 months post-injury (mpi). Tendon regeneration progresses in three main phases: inflammation within 24 h post-injury (hpi), cellular proliferation and formation of a cellular bridge between the severed tendon ends at 3–5 days post-injury (dpi), and re-differentiation and matrix remodeling beginning from 5 dpi to 6 mpi. Importantly, we demonstrate that pre-existing tenocytes are the main cellular source of regeneration, proliferating and migrating upon injury to ultimately bridge the tendon ends. Finally, we show that TGF-β signaling is required for tenocyte recruitment and bridge formation. Collectively, our work debuts and aptly positions the adult zebrafish tendon as an invaluable comparative system to elucidate regenerative mechanisms that may inspire new therapeutic strategies.

An in-House System for the Precise Measurement of Electrical Potentials and Mechanical Properties of Soft Tissues: Design and Validation Using Adult Mammalian Tendon Fascicle Bundles.

Tissues, such as skin, bones, and tendons, exhibit a piezoelectric effect, which may be an important phenomenon in terms of tissue renewal and regeneration as well as the possibility of modifying their mechanical behavior. In this article, we present the design and development of an in-house system for the precise measurement of electrical potentials and mechanical properties of tendons. The system was validated using tendon fascicle bundles derived from positional as well as energy-storing tendons from various adult mammals (porcine, bovine, and deer samples). The presented system is able to capture changes in elastic and viscoelastic properties of tissue as well as its time–voltage response and, thus, may be used in a broad spectrum of future studies to uncover factors influencing piezoelectric phenomena in tendons. This, in turn, will help to optimize current methods used in physiotherapy and postoperative treatment for effective tendon recovery.

Bringing tendon biology to heel: Leveraging mechanisms of tendon development, healing, and regeneration to advance therapeutic strategies.

Tendons are specialized matrix-rich connective tissues that transmit forces from muscle to bone and are essential for movement. As tissues that frequently transfer large mechanical loads, tendons are commonly injured in patients of all ages. Following injury, mammalian tendons heal poorly through a slow process that forms disorganized fibrotic scar tissue with inferior biomechanical function. Current treatments are limited and patients can be left with a weaker tendon that is likely to rerupture and an increased chance of developing degenerative conditions. More effective, alternative treatments are needed. However, our current understanding of tendon biology remains limited. Here, we emphasize why expanding our knowledge of tendon development, healing, and regeneration is imperative for advancing tendon regenerative medicine. We provide a comprehensive review of the current mechanisms governing tendon development and healing and further highlight recent work in regenerative tendon models including the neonatal mouse and zebrafish. Importantly, we discuss how present and future discoveries can be applied to both augment current treatments and design novel strategies to treat tendon injuries.

Advanced glycation end-product cross-linking inhibits biomechanical plasticity and characteristic failure morphology of native tendon.

Advanced glycation end-products (AGEs) are formed in vivo from the nonenzymatic reaction between sugars and proteins. AGEs accumulate in long-lived tissues like tendons, cross-linking neighboring collagen molecules, and are in part complicit in connective tissue pathologies experienced in aging and with diabetes. We have previously described discrete plasticity: a characteristic form of nanoscale collagen fibril damage consisting of serial fibril kinking and collagen denaturation that occurs in some mechanically overloaded tendons. We suspect that this failure mechanism may be an adaptive trait of collagen fibrils and have published evidence that inflammatory cells may be able to recognize and digest the denatured collagen produced by overload. In this study, we treated bovine tail tendons with ribose to simulate long-term AGE cross-linking in vitro. We hypothesized that a high degree of cross-linking would inhibit the intermolecular sliding thought to be necessary for discrete plasticity to occur. Tendons were mechanically overloaded, and properties were investigated by differential scanning calorimetry and scanning election microscopy. Ribose cross-linking treatment altered the mechanical response of tendons after the yield point, significantly decreasing postyield extensibility and strain energy capacity before rupture. Coincident with altered mechanics, ribose cross-linking completely inhibited the discrete plasticity failure mechanism of tendon. Our results suggest that discrete plasticity, which may be an important physiological mechanism, becomes pathologically disabled by the formation of AGE cross-links in aging and diabetes. NEW & NOTEWORTHY We have previously shown that mechanically overloaded collagen fibrils in mammalian tendons accrue nanoscaled damage. This includes development of a characteristic kinking morphology within a shell of denatured collagen: discrete plasticity. Here, using a ribose-incubation model, we show that advanced glycation end-product cross-linking associated with aging and diabetes completely inhibits this mechanism. Since discrete plasticity appears to cue cellular remodeling, this result has important implications for diabetic tendinopathy.

Tendon developmental plasticity and functional regeneration

Tendons enable movement by transferring force from muscle to bone. They are comprised of a highly organized collagen‐rich extracellular matrix. Disruption to the development and maintenance of tendons can lead to birth defects and result in injury in adults. We currently have an incomplete understanding of how tendons interact with their neighboring tissues to regulate the development and morphogenesis of the musculoskeletal system. In addition, once mature mammalian tendon tissue is formed, it has limited regenerative potential after injury. Central to this challenge is to uncover new mechanisms underlying tendon cell regulation during musculoskeletal development and regeneration. To accomplish this, we have turned to the zebrafish system because it is highly regenerative and amenable to genetic manipulations and live imaging. We have previously demonstrated that zebrafish tendons have molecular, developmental, morphological and biomechanical similarities to mammalian tendons, thereby making them an excellent vertebrate model to study tendon biology. However, no study has investigated their capacity to regenerate their tendons. Using the zebrafish, we developed methods to perform genetic tendon cell ablation, lineage tracing, live imaging, and mechanical testing to determine if developing and adult zebrafish can regenerate their tendon tissues. Following tendon cell ablation in developing zebrafish embryos, we observe defects in tendon gene expression and ECM composition. Cartilage and muscle tissue morphology was also disrupted following tendon cell loss. Tendon cells and the collagen organization returned after 1–2 weeks and 1–2 months, respectively. Interestingly, the pattern of the attachment sites was also restored, indicating the presence of regulatory programs that recruit and/or promote tendon cell proliferation and differentiation at the previous attachment sites. We are currently focused on identifying the developmental origins of the regenerating tendon cells and the pathways restoring the attachment site pattern. In addition to embryonic tendon regeneration, we also found that adult zebrafish appear to regenerate their collagen organization and mechanical properties after an acute physical injury. Together, our work demonstrates robust tendon regeneration at embryonic and adult stages in the zebrafish. These studies also provide a system to examine the morphogenetic and molecular events underlying tendon regeneration and musculoskeletal tissue‐tissue interactions, which has significance for regenerative biology approaches to musculoskeletal injuries.Support or Funding InformationNICHD HD06953, NIDCR DE024771, Charles H. Hood Foundation, HSCI Seed GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Using the zebrafish to understand tendon development and repair.

Tendons are important components of our musculoskeletal system. Injuries to these tissues are very common, resulting from occupational-related injuries, sports-related trauma, and age-related degeneration. Unfortunately, there are few treatment options, and current therapies rarely restore injured tendons to their original function. An improved understanding of the pathways regulating their development and repair would have significant impact in stimulating the formulation of regenerative-based approaches for tendon injury. The zebrafish provides an ideal system in which to perform genetic and chemical screens to identify new pathways involved in tendon biology. Until recently, there had been few descriptions of tendons and ligaments in the zebrafish and their similarity to mammalian tendon tissues. In this chapter, we describe the development of the zebrafish tendon and ligament tissues in the context of their gene expression, structure, and interactions with neighboring musculoskeletal tissues. We highlight the similarities with tendon development in higher vertebrates, showing that the craniofacial tendons and ligaments in zebrafish morphologically, molecularly, and structurally resemble mammalian tendons and ligaments from embryonic to adult stages. We detail methods for fluorescent in situ hybridization and immunohistochemistry as an assay to examine morphological changes in the zebrafish musculoskeleton. Staining assays such as these could provide the foundation for screen-based approaches to identify new regulators of tendon development, morphogenesis, and repair. These discoveries would provide new targets and pathways to study in the context of regenerative medicine-based approaches to improve tendon healing.

Method to extract minimally damaged collagen fibrils from tendon.

A new method is presented to extract collagen fibrils from mammalian tendon tissue. Mammalian tendons are treated with a trypsin-based extraction medium and gently separated with tweezers in an aqueous solution. Collagen fibrils released in the solution are imaged using both dark-field light microscopy and scanning electron microscopy. The method successfully extracts isolated fibrils from rat tail and patellar tendons. To examine whether the method is likely to damage fibrils during extraction, sea cucumber dermis fibril lengths are compared against those obtained using only distilled water. The two methods produce fibrils of similar lengths. This is contrasted with fibrils being shortened when extracted using a tissue homogenizer. Scanning electron microscopy shows the new method preserves D-banding features on fibril surfaces and that fibril diameter does not vary substantially compared with water extracted fibrils.

Tension tests on mammalian collagen fibrils.

A brief overview of isolated collagen fibril mechanics testing is followed by presentation of the first results testing fibrils isolated from load-bearing mammalian tendons using a microelectromechanical systems platform. The in vitro modulus (326 ± 112 MPa) and fracture stress (71 ± 23 MPa) are shown to be lower than previously measured on fibrils extracted from sea cucumber dermis and tested with the same technique. Scanning electron microscope images show the fibrils can fail with a mechanism that involves circumferential rupture, whereas the core of the fibril stays at least partially intact.

Mechanical properties of the compass depressors of the sea-urchin Paracentrotus lividus (Echinodermata, Echinoidea) and the effects of enzymes, neurotransmitters and synthetic tensilin-like protein.

The compass depressors (CDs) of the sea-urchin lantern are ligaments consisting mainly of discontinuous collagen fibrils associated with a small population of myocytes. They are mutable collagenous structures, which can change their mechanical properties rapidly and reversibly under nervous control. The aims of this investigation were to characterise the baseline (i.e. unmanipulated) static mechanical properties of the CDs of Paracentrotus lividus by means of creep tests and incremental force-extension tests, and to determine the effects on their mechanical behaviour of a range of agents. Under constant load the CDs exhibited a three-phase creep curve, the mean coefficient of viscosity being 561±365 MPa.s. The stress-strain curve showed toe, linear and yield regions; the mean strain at the toe-linear inflection was 0.86±0.61; the mean Young’s modulus was 18.62±10.30 MPa; and the mean tensile strength was 8.14±5.73 MPa. Hyaluronidase from Streptomyces hyalurolyticus had no effect on creep behaviour, whilst chondroitinase ABC prolonged primary creep but had no effect on secondary creep or on any force-extension parameters; it thus appears that neither hyaluronic acid nor sulphated glycosaminoglycans have an interfibrillar load transfer function in the CD. Acetylcholine, the muscarinic agonists arecoline and methacholine, and the nicotinic agonists nicotine and 1-[1-(3,4-dimethyl-phenyl)-ethyl]-piperazine produced an abrupt increase in CD viscosity; the CDs were not differentially sensitive to muscarinic or nicotinic agonists. CDs showed either no, or no consistent, response to adrenaline, L-glutamic acid, 5-hydroxytryptamine and γ-aminobutyric acid. Synthetic echinoid tensilin-like protein had a weak and inconsistent stiffening effect, indicating that, in contrast to holothurian tensilins, the echinoid molecule may not be involved in the regulation of collagenous tissue tensility. We compare in detail the mechanical behaviour of the CD with that of mammalian tendon and highlight its potential as a model system for investigating poorly understood aspects of the ontogeny and phylogeny of vertebrate collagenous tissues.

Tensile properties of craniofacial tendons in the mature and aged zebrafish.

The zebrafish Danio rerio is a powerful model for the study of development, regenerative biology, and human disease. However, the analysis of load-bearing tissues such as tendons and ligaments has been limited in this system. This is largely due to technical limitations that preclude accurate measurement of their mechanical properties. Here, we present a custom tensile testing system that applies nano-Newton scale forces to zebrafish tendons as small as 1 mm in length. Tendon properties were remarkably similar to mammalian tendons, including stress-strain nonlinearity and a linear modulus (515 ± 152 MPa) that aligned closely with mammalian data. Additionally, a simple exponential constitutive law used to describe tendon mechanics was successfully fit to zebrafish tendons; the associated material constants agreed with literature values for mammalian tendons. Finally, mature and aged zebrafish comparisons revealed a significant decline in mechanical function with age. Based on the exponential constitutive model, age-related changes were primarily caused by a reduction in nonlinearity (e.g., changes in collagen crimp or fiber recruitment). These findings demonstrate the utility of zebrafish as a model to study tendon biomechanics in health and disease. Moreover, these findings suggest that tendon mechanical behavior is highly conserved across vertebrates.

The development of zebrafish tendon and ligament progenitors.

Despite the importance of tendons and ligaments for transmitting movement and providing stability to the musculoskeletal system, their development is considerably less well understood than that of the tissues they serve to connect. Zebrafish have been widely used to address questions in muscle and skeletal development, yet few studies describe their tendon and ligament tissues. We have analyzed in zebrafish the expression of several genes known to be enriched in mammalian tendons and ligaments, including scleraxis (scx), collagen 1a2 (col1a2) and tenomodulin (tnmd), or in the tendon-like myosepta of the zebrafish (xirp2a). Co-expression studies with muscle and cartilage markers demonstrate the presence of scxa, col1a2 and tnmd at sites between the developing muscle and cartilage, and xirp2a at the myotendinous junctions. We determined that the zebrafish craniofacial tendon and ligament progenitors are neural crest derived, as in mammals. Cranial and fin tendon progenitors can be induced in the absence of differentiated muscle or cartilage, although neighboring muscle and cartilage are required for tendon cell maintenance and organization, respectively. By contrast, myoseptal scxa expression requires muscle for its initiation. Together, these data suggest a conserved role for muscle in tendon development. Based on the similarities in gene expression, morphology, collagen ultrastructural arrangement and developmental regulation with that of mammalian tendons, we conclude that the zebrafish tendon populations are homologous to their force-transmitting counterparts in higher vertebrates. Within this context, the zebrafish model can be used to provide new avenues for studying tendon biology in a vertebrate genetic system.

Musculoskeletal regeneration and its implications for the treatment of tendinopathy

Tendinopathies are common muskoloskeletal injuries that lead to pain and disability. Development and pathogenesis of tendinopathy is attributed to progressive pathological changes to the structure, function, and biology of tendon. The nature of this disease state, whether acquired by acute or chronic injury, is being actively investigated. Scarring, disorganized tissue, and loss of function characterize adult tendon healing. Recent work from animal models has begun to reveal the potential for adult mammalian tendon regeneration, the replacement of diseased with innate tissue. This review discusses what is known about musculoskeletal regeneration from a molecular perspective and how these findings can be applied to tendinopathy. Non-mammalian and mammalian models are discussed with emphasis on the potential of Murphy Roths Large mice to serve as a model of adult tendon regeneration. Comparison of regeneration in non-mammals, foetal mammals and adult mammals emphasizes distinctly different contributing factors to effective regeneration.

Production of a novel silk-like protein from sea anemone and fabrication of wet-spun and electrospun marine-derived silk fibers

Silk-based biomaterials, which for centuries were primarily used as the raw materials for textile fabrics, are now being used in the medical, food and industrial fields because of their extraordinary properties. Their advantageous characteristics include high strength, extensibility, biocompatibility, biodegradability and minimal production of inflammatory reactions. To date, only silkworm silk has been applied in mass-produced technological applications, but spider silk proteins have been extensively studied because of their superior mechanical stability. Here we report the discovery of a novel silk-like protein (named aneroin) from the sea anemone Nematostella vectensis and the successful fabrication of wet-spun and electrospun silk fibers from purified recombinant aneroins. Aneroin fibers have promising mechanical properties, similar to those of recombinant spider silks with molecular weights of ∼100 kDa and those of natural mammalian tendon collagen. The results of this study demonstrate that aneroin, a new repertoire of silk-like protein found in sea anemones, has potential for use as a novel fibrous biomaterial. Its use would expand the applications of silk in the development of multifunctional and bio-inspired materials. A highly sought-after fabric for thousands of years, silk has also proved valuable to the industrial, food and medical fields — with applications in wound dressings or tissue engineering scaffolds, for example. The varied uses of this biomaterial arise from its attractive set of properties: it is strong, extensible, biocompatible and biodegradable. Silk is composed of proteins that can be processed into fibres; those used for large-scale applications are typically derived from the silkworm Bombyx mori or, to some extent, spiders. A Korean-based team led by Hyung Joon Cha from the Pohang University of Science and Technology has now discovered a silk-like protein — aneroin — in a marine organism, the sea anemone Nematostella vectensis. The researchers used the bacterium Escherichia coli to produce a recombinant aneroin protein, which was subsequently processed through wet- and electro-spinning. The resulting fibres exhibited excellent mechanical properties — superior to those of spider silk with a similar molecular weight. A novel marine silk fiber (named aneroin) from sea anemone has been discovered, and wet-spun and electrospun silk fibers from purified recombinant aneroins have been successfully fabricated. Aneroin fibers have promising mechanical properties, suggesting that this protein has potential for use as a novel fibrous biomaterial. Its use would expand the applications of silk in the development of multifunctional and bio-inspired materials.

Constitutive modeling of crimped collagen fibrils in soft tissues

A microstructurally oriented constitutive formulation for the hyperelastic response of crimped collagen fibrils existing in soft connective tissues is proposed. The model is based on observations that collagen fibrils embedded in a soft matrix crimp into a smooth three-dimensional pattern when unloaded. Following ideas presented by Beskos and Jenkins [Beskos, D., Jenkins, J., 1975. A mechanical model for mammalian tendon. ASME Journal of Applied Mechanics 42, 755–758] and Freed and Doehring [Freed, A., Doehring, T., 2005. Elastic model for crimped collagen fibrils. Journal of Biomechanical Engineering 127, 587–593] the collagen fibril crimp is approximated by a cylindrical helix to represent the constitutive behavior of the hierarchical organized substructure of biological tissues at the fibrillar level. The model is derived from the nonlinear axial force–stretch relationship of an extensible helical spring, including the full extension of the spring as a limit case. The geometrically nonlinear solution of the extensible helical spring is carried out by an iterative procedure. The model only requires one material parameter and two geometrical parameters to be determined from experiments. The ability of the proposed model to reproduce the biomechanical response of fibrous tissues is demonstrated for fascicles from rat tail tendons, for porcine cornea strips, and for bovine Achilles tendons.

Evidence that collagen and tendon have monolayer water coverage in the native state

This paper investigates an alternative explanation for widely reported paradoxical intracellular water properties. The most frequent biological explanation assumes water structure extending multiple layers from surfaces of compactly folded macromolecules to explain large amounts of perturbed water. Long range water structuring, however, contradicts molecular models widely accepted by the scientific majority. This study questions whether the paradoxical cell water could result from larger than expected amounts of first layer interfacial water on internal protein surfaces rather than structured multilayers. Native mammalian tendon is selected for the study because (1) the organ consists of highly compact structures of a single macromolecular protein--collagen, (2) molecular structure and geometry of collagen is well characterized by X-ray diffraction, (3) molecular structure extends to the macroscopic tendon level and (4) perturbed water behavior similar to cellular water is reported on tendon. Native tendon holds 1.6 g water/g dry mass. The 62% native water content simulates the water content of many cell types. MicroCT studies of tendon dilatometry as a function of hydration are measured and correlated to X-ray diffraction measurements of interaxial separation. Correlations show that native tendon has sufficient water for only a single monolayer of interfacial water. Thus the paradoxical properties of water in native tendon are first-layer interfacial water properties. Similar water behavior on globular proteins suggests that paradoxical cell water behavior could be caused by larger than expected amounts of first layer interfacial water on internal and external macromolecular surfaces of cell components.

Interactions between muscle fibers and segment boundaries in zebrafish

The most obvious segmental structures in the vertebrate embryo are somites: transient structures that give rise to vertebrae and much of the musculature. In zebrafish, most somitic cells give rise to long muscle fibers that are anchored to intersegmental boundaries. Therefore, this boundary is analogous to the mammalian tendon in that it transduces muscle-generated force to the skeletal system. We have investigated interactions between somite boundaries and muscle fibers. We define three stages of segment boundary formation. The first stage is the formation of the initial epithelial somite boundary. The second “transition” stage involves both the elongation of initially round muscle precursor cells and somite boundary maturation. The third stage is myotome boundary formation, where the boundary becomes rich in extracellular matrix and all muscle precursor cells have elongated to form long muscle fibers. It is known that formation of the initial epithelial somite boundary requires Notch signaling; vertebrate Notch pathway mutants show severe defects in somitogenesis. However, many zebrafish Notch pathway mutants are homozygous viable suggesting that segmentation of their larval and adult body plans at least partially recovers. We show that epithelial somite boundary formation and slow-twitch muscle morphogenesis are initially disrupted in after eight ( aei) mutant embryos (which lack function of the Notch ligand, DeltaD); however, myotome boundaries form later (“recover”) in a Hedgehog-dependent fashion. Inhibition of Hedgehog-induced slow muscle induction in aei/deltaD and deadly seven ( des) /notch1a mutant embryos suggests that slow muscle is necessary for myotome boundary recovery in the absence of initial epithelial somite boundary formation. Because we have previously demonstrated that slow muscle migration triggers fast muscle cell elongation in zebrafish, we hypothesize that migrating slow muscle facilitates myotome boundary formation in aei/deltaD mutant embryos by patterning coordinated fast muscle cell elongation. In addition, we utilized genetic mosaic analysis to show that somite boundaries also function to limit the extent to which fast muscle cells can elongate. Combined, our results indicate that multiple interactions between somite boundaries and muscle fibers mediate zebrafish segmentation.

Zebrafish periostin is required for the adhesion of muscle fiber bundles to the myoseptum and for the differentiation of muscle fibers

The myoseptum of fishes, composed of dense collagen, is a connective tissue layer that forms in the embryo, dividing somites from the trunk, and its structure and function are similar to those of the mammalian tendon. Both the myoseptum and tendon serve as the transmitter of muscular contractility to bones and adjoining muscles, and their structure is indispensable for movement of vertebrate animals. We cloned the zebrafish periostin gene and examined its expression and function in the myoseptum. The expression in embryos started in the rostral part of each segmented somite in the early segmentation stage; and consequently, metameric stripes were observed. At the end of segmentation, the expression region shifted to the transverse myoseptum and the myotome–epidermis boundary, and each myotome was surrounded by periostin. Using a polyclonal antibody, we found that the periostin protein was localized to the transverse myoseptum. Consistently, periostin morpholino antisense oligonucleotide led to defects in myoseptum formation, a delay in the differentiation of myofibers, and disorder of connection between myofibrils and myoseptum. We demonstrated here that periostin is the first molecule involved in myoseptum formation and propose that periostin secretion on the surface of the myoseptum is required for the adhesion of muscle fiber bundles to the myoseptum and the differentiation of muscle fibers.

A novel fibrocartilaginous tendon from an elasmobranch fish ( Rhinoptera bonasus).

Tendons of the jaw adductor muscles of a hard prey crushing stingray exhibit similar adaptations to compressive and shear loads as those seen in mammalian tendons. Ventral intermandibular tendon from the cownose ray, Rhinoptera bonasus, has a prominent fibrocartilaginous pad that lies between a fibrous region of the tendon and the mineralized tissue of the jaw. Histologically the pad is similar to the fibrocartilaginous meniscus of mammals, and these tissues also share some biochemical traits. Proteoglycan (PG) content in the fibrocartilaginous pad is nearly four times higher than in the linearly arrayed tendinous tissue. The predominant PGs appear to be an aggrecan-like molecule and a decorin-like molecule. The decorin-like molecule is quite small when compared to mammalian decorin (20-80 kDa vs. 100-200 kDa). This study is the first to document adaptations to compressive/shear loading in tendon from a cartilaginous fish, and the similarities to the mammalian condition argue for the early evolution of this reactive ability of tendinous tissue.

Fatigue quality of mammalian tendons.

When excised tendons are subjected to a prolonged load, whether constant or oscillatory, fatigue damage accumulates, leading eventually to rupture. 'Fatigue quality', assessed by the time-to-rupture under a given stress, was found to vary hugely among the tendons of a wallaby hind limb. This material property correlates with the varied stresses to which tendons from different anatomical sites are exposed in life. The correlation was demonstrated by subjecting each excised tendon to a load equal to the maximum isometric force that its muscle could have developed. The time-to-rupture was then approximately the same for each tendon, on average 4.2 h. A model is introduced in which damage is proposed as the trigger for adaptation of fatigue quality. The model aims, in particular, to explain why low-stressed tendons are not made of a 'better' material, although this clearly exists since it is used in high-stressed tendons. The principle of design to a minimum quality is viable in biology because of the availability of self-repair to balance routine damage. Clinical symptoms, to be included under the general heading of 'overuse injuries', will only arise when this balance fails.