Equine Superficial Digital Flexor Tendon Research Articles

Non invasive measurement of equine superficial digital flexor tendon loading at high speed trot on different track surfaces

Click to increase image sizeClick to decrease image sizeKeywords: tendon loadultrasoundhigh speedtrackhorse

Matrix metabolism rate differs in functionally distinct tendons

Tendon matrix integrity is vital to ensure adequate mechanical properties for efficient function. Although historically tendon was considered to be relatively inert, recent studies have shown that tendon matrix turnover is active. During normal physiological activities some tendons are subjected to stress and strains much closer to their failure properties than others. Tendons with low safety margins are those which function as energy stores such as the equine superficial digital flexor tendon (SDFT) and human Achilles tendon (AT). We postulate therefore that energy storing tendons suffer a higher degree of micro-damage and thus have a higher rate of matrix turnover than positional tendons. The hypothesis was tested using tissue from the equine SDFT and common digital extensor tendon (CDET). Matrix turnover was assessed indirectly by a combination of measurements for matrix age, markers of degradation, potential for degradation and protein expression. Results show that despite higher cellularity, the SDFT has lower relative levels of mRNA for collagen types I and III. Non-collagenous proteins, although expressed at different levels per cell, do not appear to differ between tendon types. Relative levels of mRNA for MMP1, MMP13 and both pro-MMP3 and MMP13 protein activity were significantly higher in the CDET. Correspondingly levels of cross-linked carboxyterminal telopeptide of type I collagen (ICTP) were higher in the CDET and tissue fluorescence lower suggesting more rapid turnover of the collagenous component. Reduced or inhibited collagen turnover in the SDFT may account for the high level of degeneration and subsequent injury compared to the CDET.

Graded Arrangement of Collagen Fibrils in the Equine Superficial Digital Flexor Tendon

By using ultramorphological and biochemical methods, we analyzed the regional differences between the three parts of the equine superficial digital flexor tendon (SDFT), namely, the myotendinous junction (MTJ), middle metacarpal (mM), and osteotendinous junction (OTJ). Cross-sectional images showed unique distributions of collagen fibrils of varying diameters in each region. Small collagen fibrils (diameter <100 nm) were distributed predominantly in the MTJ region, and the OTJ region was relatively rich in large collagen fibrils (diameter >200 nm). In the mM region, the collagen fibrils were intermediately distributed between the MTJ and OTJ. The results indicate a graded arrangement of collagen fibrils in the tendon. Type V collagen was detected preferentially in the MTJ region. Since type V collagen is believed to be one of the collagens regulating collagen fibril formation, its possible functionality in the MTJ region in terms of fibril formation and fibril arrangement in the tendon has been discussed here.

Acoustodynamométrie : Un procédé innovant de mesure non-invasive de la force qui s'exerce dans un tendon au cours du mouvement

A novel technique was developed to measure the tendon load in people and in horses in motion. This non-invasive technique is based on a relation, demonstrated experimentally in vitro on isolated tendons and limbs, between the velocity of ultrasound in the tendon and the load to which it is subjected. A prototype was created to examine the equine superficial digital flexor tendon. The reproducibility of these measurements assessed in five horses was very good in a given subject, but marked differences were observed between the animals, in terms of plot shape and ultrasound velocity. These differences appeared directly related to the horse’s digital standing conformation. The technique thus validated was then used to examine the effect of four types of orthopaedic shoes compared to a standard shoe, on hard as well as soft ground.

Characteristics of Collagen Fibrils in the Entire Equine Superficial Digital Flexor Tendon

The superficial digital flexor tendon (SDFT) is one of the longest tendons in the horse. In racehorses, disturbance of the locomotor functions of the SDFT occurs most frequently in the central area of the mid-metacarpal region. While many studies have investigated the equine SDFT, there are no reports to date of the morphological characteristics of collagen fibrils in the central and peripheral areas of each of the three regions that comprise the entire tendon: the myotendinous junction (MTJ), the mid-metacarpal region (mM) and the osteotendinous junction (OTJ). Mass average diameter (MAD), which provides functional information on the mean collagen fibril diameter and tensile strength of the tendon, was found to be smaller in the central area than in the peripheral area of all three regions. The MAD value was lowest in both the central and peripheral areas in the MTJ region, and tended to increase generally in a distal direction in the OTJ. The OTJ is important parts that unite with the bone. We conclude that morphological structure suggested that it corresponds to biomechanical function in some region of the equine SDFT.

Effects of platelet-derived growth factor-BB on the metabolic function and morphologic features of equine tendon in explant culture

To evaluate the effects of recombinant human platelet-derived growth factor-BB (rhPDGF-BB) on the metabolic function and morphologic features of equine superficial digital flexor tendon (SDFT) in explant culture. Animals-6 euthanized horses (2 to 5 years old). Forelimb SDFT explants were cultured for 6 days as untreated control specimens or treated with rhPDGF-BB (1, 10, 50, or 100 ng/mL of medium). Treatment effects on explant gene expression were evaluated via real-time PCR analysis of collagen type I, collagen type III, PDGF-A, and PDGF-B mRNA. Explants were assayed for total collagen, glycosaminoglycan, and DNA content; histologic changes were assessed via H and E staining and immunohistochemical localization of collagen types I and III. No morphologic or proliferative changes were detected in tendon explant sections. After high-dose rhPDGF-BB treatment, gene expression of collagen types I and III was increased and decreased, respectively. Expression of PDGF-A and PDGF-B mRNA was significantly increased at 24 hours, but later decreased to have few or negative autoinductive effects. Although PDGF gene expression waned after 48 hours of culture, collagen type I gene expression was significantly increased at 48 hours and reached peak value on day 6. Glycosaminoglycan and DNA content of explants were unchanged with rhPDGF-BB treatment. Results suggest that rhPDGF-BB use may be of benefit in the repair of equine tendon, particularly through induction of collagen type I mRNA. Positive autoinductive effects of PDGF-BB in equine SDFT explants were detected early following culture medium supplementation, but these diminished with time.

Effect of uphill exercise on equine superficial digital flexor tendon forces at trot and canter

One cause of overstrain injury to the superficial digital flexor tendon (SDFT) in horses is the force loaded on the SDFT during repeated running. Therefore, decreasing this force may reduce SDFT injury. It has been reported that strain on the SDFT decreases with a toe-wedge shoe. Uphill courses are used for training of racehorses, and the angle of hoof-sole to the horizon during uphill running is similar to that of the toe-wedge shoe. To determine the effects of uphill exercise on the force on the SDFT during trotting and cantering. Arthroscopically implantable force probes (AIFP) were implanted into the SDFT of the left or right forelimb of 7 Thoroughbred horses and AIFP output recorded during trotting and cantering on a treadmill inclined at slopes of 0, 3 or 8%, and then 0% again. Superficial digital flexor tendon force was calculated as a relative value, with the amplitude of AIFP output voltage at initial 0% slope equal to 100. Out of 14 sets of experiments, AIFP data were analysed successfully in 9 at the trot, in 3 at the canter in the trailing forelimb on a slope of 3 and 8%, and in 2 at the canter in the leading forelimb on a slope of 3%. Increasing the incline from 0-8% tended to decrease peak force in the SDFT at the trot, and in the trailing forelimb at the canter. However, force in the SDFT was unchanged in the leading forelimb at the canter on the 3% incline. The force in the SDFT trotting or cantering uphill is unchanged or lower than that loaded at the same speed on a flat surface. Because at similar speeds the workload for uphill exercise is greater than on the flat, uphill running increases exercise intensity without increasing force in the SDFT. Uphill exercise may reduce the risk of SDFT injury as both running speed and SDFT force are decreased on an incline as compared to the flat, even when exercise intensity is the same. Further study is needed to confirm these findings at canter in a larger population of horses.

Thrombospondin-4 and Cartilage Oligomeric Matrix Protein form Heterooligomers in Equine Tendon

Injuries of the equine superficial digital flexor tendon are common in racing horses. Knowledge of the tendon matrix composition is crucial to understand physiological and pathological processes in the tendon. The aim of this study was to analyze TSP-4 expressed in equine tendon. Equine tendons were extracted with 10 mM EDTA-containing buffer and TSP-4 purified with ion-exchange chromatography followed by heparin affinity chromatography. The purified TSP-4 was analyzed by one- and two-dimensional SDS-PAGE, immunoblotting, and MALDI-TOF mass spectrometry. Purified TSP-4 gave bands reacting with a TSP-4 specific antiserum, but also with an antiserum to COMP, when submitted to SDS-PAGE under nonreducing conditions. Two-dimensional SDS-PAGE (nonreducing followed by reducing conditions) and immunoprecipitation as well as MALDI-TOF mass spectrometry analysis showed that TSP-4 and COMP are both present in equine tendon and cannot be separated under nonreducing conditions despite significant differences in subunit size. This suggests that they are connected via disulfide bridges into heterooligomers.

Effect of heat on synthesis of gelatinases and pro-inflammatory cytokines in equine tendinocytes

The aim of this study was to clarify whether matrix metalloproteinases (MMP-2 and -9: gelatinases) and pro-inflammatory cytokines [tumor necrosis factor (TNF) alpha and interleukin (IL)-1beta] are induced by heat in tendon tissue in vitro and to test the hypothesis that heat exposure causes tendinocytes to synthesize pro-inflammatory cytokines and that synthesis of these cytokines, in turn, leads to up-regulation of synthesis of gelatinases. Isolated tendinocytes from equine superficial digital flexor tendons were cultured and all experiments were performed on cells passaged 3 or 4 times. In the cells exposed to heat (37 to 45 degrees C, 0 to 60 min), the survival rate decreased sharply in a temperature- and time-dependent manner, especially at 42 and 45 degrees C. Cells exposed at 40 degrees C, however, showed little change in survival rate and morphology. Gelatin zymograms revealed that proMMP-2 and -9 were the only two MMPs remaining in the supernatant of the cultured tendinocytes, including that of untreated cells. Addition of TNFalpha and IL-1beta to the culture medium of tendinocytes accelerated proMMP-9 synthesis considerably. Heating the tendinocytes (40 degrees C) led to a three-fold increase in proMMP-9 synthesis in a short time. Only TNFalpha was detected in tendinocytes after heat exposure for 30 and 60 min. In contrast, IL-1beta was under the detectable level in ELISA. Cooling of heat-exposed cells from 40 degrees C to 37 degrees C considerably down-regulated cellular proMMP-9 synthesis. Furthermore, proMMP-9 level was greatly reduced in cells treated at lower temperatures, 20 degrees C and 5 degrees C. These findings support our hypothesis that hyperthermia in the horse tendon induces tendinocytes to synthesize pro-inflammatory cytokines and that the synthesis of these cytokines results in the up-regulation of gelatinases.

Functional adaptation through changes in regional biochemical characteristics during maturation of equine superficial digital flexor tendons

To quantify and compare biochemical characteristics of the extracellular matrix (ECM) of specimens harvested from tensional and compressive regions of the superficial digital flexor tendon (SDFT) of horses in age classes that include neonates to mature horses. Tendon specimens were collected on postmortem examination from 40 juvenile horses (0, 5, 12, and 36 months old) without macroscopically visible signs of tendonitis. Central core specimens of the SDFT were obtained with a 4-mm-diameter biopsy punch from 2 loaded sites, the central part of the mid-metacarpal region and the central part of the mid-sesamoid region. Biochemical characteristics of the collagenous ECM content (ie, collagen, hydroxylysylpyridinoline crosslink, and pentosidine crosslink concentrations and percentage of degraded collagen) and noncollagenous ECM content (percentage of water and glycosaminoglycans, DNA, and hyaluronic acid concentrations) were measured. The biochemical composition of equine SDFT was not homogeneous at birth with respect to DNA, glycosaminoglycans, and pentosidine concentrations. For most biochemical variables, the amounts present at birth were dissimilar to those found in mature horses. Fast and substantial changes in all components of the matrix occurred in the period of growth and development after birth. Unlike cartilage, tendon tissue is not biochemically blank (ie, homogeneous) at birth. However, a process of functional adaptation occurs during maturation that changes the composition of equine SDFT from birth to maturity. Understanding of the maturation process of the juvenile equine SDFT may be useful in developing exercise programs that minimize tendon injuries later in life that result from overuse.

Extracellular Matrix Compositon of the Equine Superficial Digital Flexor Tendon: Relationship with Age and Anatomical Site

The objectives of the present study were to test the hypotheses that: (1) the composition of the extracellular matrix of the equine superficial digital flexor tendon (SDFT) shows great functional similarities with articular cartilage, i.e. that significant differences exist in biochemical composition of differently loaded areas (which in the case of tendons may be more apparent as tendon shows more obvious differences than cartilage); and (2) that, as in articular cartilage, no substantial alterations in biochemical composition take place during ageing once adulthood has been attained. Tendon samples were taken from 60 adult slaughter horses from a central area at cross-section in the mid-metacarpal region and at the height of the proximal sesamoid bones (sesamoid region) of the SDFT. Contents of collagenous and non-collagenous components were determined. None of the parameters were correlated with age in either region, except for a significant increase in pentosidine cross-links with age in the sesamoid region. Between the two anatomical regions, there were significant differences in all variables, except for hydroxylysylpyridinoline cross-links. It was concluded that in the equine SDFT, similar to articular cartilage, most molecular parameters are not influenced by age in mature horses, indicating a low remodelling rate. Tendon composition is clearly different between regions, apparently reflecting different specific modes of biomechanical loading at the points sampled.

Control of the Collagen Fibril Diameter in the Equine Superficial Digital Flexor Tendon in Horses by Decorin

The distribution pattern of collagen fibril diameter in the equine superficial digital flexor tendon (SDFT) is known to differ in central and peripheral areas of some regions. This study reports the essence of collagen fibril differences among different regions of the equine SDFT by transmission electron microscopic (TEM) and high-voltage electron microscopic observations and biochemical analysis. The distribution of large collagen fibrils increased but the density of collagen fibrils decreased from the proximal metacarpal region to the distal metacarpal region. Large collagen fibrils with an irregular cross-sectional profile were found more frequently in the middle metacarpal region than in other regions. Three-dimensional reconstruction of images of irregularly shaped collagen fibrils revealed that these fibrils are formed through fusion of small collagen fibrils with large ones. The amount of decorin, which reportedly inhibits the lateral fusion of collagen fibrils, decreased in the direction of the distal metacarpal region. On the other hand, the size of decorin gradually increased in the direction of the distal metacarpal region. These results suggest that regional differences in collagen fibril distribution and density of collagen fibrils in the SDFT are due, at least in part, to fusion of collagen fibrils and the concomitant regional differences in the amount and size of decorin.

Differences in Tumor Necrosis Factor (TNF).ALPHA. and TNF Receptor-1-Mediated Intracellular Signaling Factors in Normal, Inflamed and Scar-Formed Horse Tendons

Tumor necrosis factor (TNF) receptors (TNF-R)-mediated cell survival or apoptosis has been demonstrated in many cells, but little is known about survival or apoptotic signals via TNF-R1 in tendinocytes. In this study, we focused on four signaling factors, TNFalpha, TNF-R1, TNFR-associated factor2 (TRAF2) and caspase-3, in order to elucidate the signaling events in tendinocytes. Samples were obtained from normal, inflamed and scar-formed equine superficial digital flexor tendons. To detect these signaling factors, samples were subjected to immunohistochemistry and Western blot analysis, and some samples were also subjected to reverse transcription-polymerase chain reaction (RT-PCR), PCR-Southern blot analysis and in situ hybridization to detect the expression of TNFalpha mRNA. Distribution of the four factors differed depending on the tendon condition, normal, inflamed or scar-formed. In the normal tendon, large amounts of TRAF2 were found in tendinocytes, but the amounts of TNF-R1 were small. TNFalpha mRNA was expressed most highly in the inflamed tendon. TNF-R1, which was only faintly detected in the normal tendon, was detected at a high level in the inflamed tendon, and the amounts of TRAF2 and caspase-3 also increased. Activated caspase-3 was only detected in the inflamed tendon. TNFalpha mRNA was also expressed in the scar-formed tendon, though it showed weak signals, and the expression levels of TNF-R1, TRAF2 and caspase-3 proteins were very low. Two distinct intracellular signaling pathways of TNFalpha, which lead to cell survival and apoptosis, might be present in tendinocytes mediated through TNF-R1. These results, which reflect the dynamism of TNFalpha, provide important clues for means to prevent tendinopathy.

Functional ability of tendinocytes to take up Fe substances in an inflamed tendon

The fate of iron (Fe) after bleeding has been analyzed in various connective tissues, but there have been only a few inconclusive studies on Fe in the tendon. In this study, energy dispersive X-ray microanalysis and an iron staining method were used to determine the localization of Fe in cells of the equine superficial digital flexor tendon. In inflamed tendons, Fe was detected in tendinocytes as well as macrophages. In contrast, negative or weak reactions were observed in many cells in the normal tendon. Although the accepted theory states that the reticuloendothelial system (macrophages and reticuloendothelial cells) is mainly responsible for the uptake and decomposition of erythrocytes, and tendon cells under conditions of inflammation show a functional ability to take up Fe substances. It has been reported that tendinocytes have multiple functions, including synthesis and secretion of collagen, cytokines, and matrix metalloproteinases. Taking these functions into consideration, indicates that the tendinocyte is not only an active cell but also a multi-functional cell.

Correlations between mean echogenicity and material properties of normal and diseased equine superficial digital flexor tendons: an in vitro segmental approach

The objective of this study was to test the hypothesis that tendon echogenicity is associated with the material properties of the corresponding tendon site, especially in case of lesions, due to local changes in tendon matrix composition. Four normal and nine spontaneously injured equine superficial digital flexor tendons (SDFT) were isolated then ultrasonographically examined under tension, in a special device placed in a water bath. Ultrasonographic transversal images (7.5 MHz linear transducer) of five segments along each tendon were digitized, and analyzed in order to measure the mean cross-sectional area (MCSA) and mean echogenicity (ME) of each segment. The tendons were then tested in traction until rupture in a testing machine. For each segment, stress and strain were determined throughout the test, and the elastic modulus (EM) was evaluated. The tendon lesions were also documented by histology. No correlation was found between ME and the material properties of normal tendon segments. At the rupture sites of the nine diseased tendons, ME was positively correlated with maximal stress and EM, whereas no correlation was demonstrated with maximal strain. Besides, a positive correlation was demonstrated between ME and both MCSA and EM, when the three metacarpal segments of the diseased tendons were considered. Although ME gives only rough information about tendon matrix structure, it does show, under these in vitro conditions, significant correlations with material properties of pathological tendon segments, which may improve the functional significance and therefore the pronostic value of the ultrasonographic examination of tendon lesions.

A non-invasive method of tendon force measurement

The ability to measure the forces exerted in vivo on tendons and, consequently, the forces produced by muscles on tendons, offers a unique opportunity to investigate questions in disciplines as varied as physiology, biomechanics, orthopaedics and neuroscience. Until now, tendon loads could be assessed directly only by means of invasive sensors implanted within or attached to these collagenous structures. This study shows that the forces acting on tendons can be measured, in a non-invasive way, from the analysis of the propagation of an acoustic wave. Using the equine superficial digital flexor tendon as a model, it is demonstrated that the velocity of an ultrasonic wave propagating along the main axis of a tendon increases with the force applied to this tendon. Furthermore, we show that this velocity measurement can be performed even in the presence of skin overlying the tendon. To validate this measurement technique in vivo, the ultrasonic velocity plots obtained in the Achilles tendon at the walk were compared to the loads plots reported by other authors using invasive transducers.

Mechanical and functional properties of the equine superficial digital flexor tendon

The in vitro and in vivo mechanical properties of the superficial digital flexor tendon have been described. To date the focus has been on single load to failure testing, however refined in vivo methods may prove useful to evaluate the effects of treatment and exercise on tendons. During maximal exercise, the adult superficial digital flexor tendon operates close to its functional limits with a narrow biomechanical safety margin. This combined with exercise and age associated microdamage, and a limited adaptive ability may increase the risk of fatigue failure. Studies evaluating treatment regimens for tendonitis have focused on repair and regeneration and yielded varying results. It would appear that the superficial digital flexor tendon has a limited ability if any to adapt positively to exercise after maturity. In contrast, the foal’s superficial digital flexor tendon may have a greater adaptive ability and may respond to an appropriate exercise regimen to produce a more functionally adapted tendon. Recent studies have shown that foals allowed free pasture exercise develop a larger, stronger, more elastic tendon compared to foals that were confined or subjected to a training program. Effects on the non-collagenous matrix appear to be responsible for these differences. In contrast, training or excess exercise may have permanent detrimental effects on the biomechanical and functional properties of the superficial digital flexor tendon in the foal. The implication is that the determination of optimum exercise intensity and timing, and the role of the non-collagenous matrix in tendon physiology in the young horse may hold the key to developing tendons more capable of resisting injury.

Structural changes in loaded equine tendons can be monitored by a novel spectroscopic technique.

This study aimed to investigate the preferential collagen fibril alignment in unloaded and loaded tendons using elastic scattering spectroscopy. The device consisted of an optical probe, a pulsed light source (320-860 nm), a spectrometer and a PC. Two probes with either 2.75 mm or 300 microm source-detector separations were used to monitor deep and superficial layers, respectively. Equine superficial digital flexor tendons were subjected to ex vivo progressive tensional loading. Seven times more backscattered light was detected parallel rather than perpendicular to the tendon axis with the 2.75 mm separation probe in unloaded tendons. In contrast, using the 300 microm separation probe the plane of maximum backscatter (3-fold greater) was perpendicular to the tendon axis. There was no optical anisotropy in the cross-sectional plane of the tendon (i.e. the transversely cut tendon surface), with no structural anisotropy. During mechanical loading (9-14% strain) backscatter anisotropy increased 8.5- to 18.5-fold along the principal strain axis for 2.75 mm probe separation, but almost disappeared in the perpendicular plane (measured using the 300 microm probe separation). Optical (anisotropy) and mechanical (strain) measurements were highly correlated. We conclude that spatial anisotropy of backscattered light can be used for quantitative monitoring of collagen fibril alignment and tissue reorganization during loading, with the potential for minimally invasive real-time structural monitoring of fibrous tissues in normal, pathological or repairing tissues and in tissue engineering.

Morphological study of the Golgi tendon organ in equine superficial digital flexor tendon.

The Golgi tendon organ (GTO) is an encapsulated fusiform mechanoreceptor siding in the musculo-tendinous junction of many animal species. Inhibitory function of afferent nerve fibers distributed from nearby motor units, the organ responds to active tension exerted onto the muscle. The morphological features of the equine GTO have not yet been elucidated. Additionally, there is some controversy regarding to the existence of the GTO in the equine superficial digital flexor tendon (SDFT). Therefore, immunohistochemistry and immunoelectron microscopy using alcian blue (pH 2.5) staining and the silver-enhanced colloidal gold method were carried out to determine both the location and characteristics of the GTO at the musculo-tendinous junction of the SDFT. A GTO with a fusiform structure of approximately 3 mm in length was found in the tendinous part. The lumen of the GTO was divided into compartments by septal cells. Each compartment contained collagen fibrils, nerve fibers and Schwann cells. This is the first report of the equine GTO.

The effect of enrofloxacin on cell proliferation and proteoglycans in horse tendon cells.

Fluoroquinolone antibiotics have been used widely in humans and domestic animals, including horses, because of their broad-spectrum bactericidal activity, and relative safety. The use of fluoroquinolones, however, is not without risk. Tendonitis and spontaneous tendon rupture have been reported in people during or following therapy with fluoroquinolones. We have studied the effects of enrofloxacin, a fluoroquinolone antibiotic used commonly in domestic animals, on tendon cell cultures established from equine superficial digital flexor tendons. Effects on cell proliferation and morphology were studied using cell counting and scanning electron microscopy. Monosaccharide content and composition was determined by gas chromatography-mass spectrometry analysis. Western and Northern blot analyses were utilized to evaluate the synthesis and expression of two proteoglycans, biglycan and decorin. Our data demonstrate that enrofloxacin inhibits cell proliferation, induces morphological changes, decreases total monosacharide content and alters small proteoglycan synthesis at the glycosylation level in equine tendon cell cultures. These effects are more pronounced in juvenile tendon cells than in adult equine tendon cells. We hypothesize that morphological changes and inhibition of cell proliferation are a result of impaired production of biglycan and decorin, proteoglycans involved in fibrillogenesis of collagen, the most important structural component of the tendon of enrofloxacin-treated tendon cells. Our findings suggest that fluoroquinolones should be used with caution in horses, especially in foals.