Ageing does not result in a decline in cell synthetic activity in an injury prone tendon.

Advancing age is a well-known risk factor for tendon disease. Energy-storing tendons [e.g., human Achilles, equine superficial digital flexor tendon (SDFT)] are particularly vulnerable and it is thought that injury occurs following an accumulation of micro-damage in the extracellular matrix (ECM). Several authors suggest that age-related micro-damage accumulates due to a failure of the aging cell population to maintain the ECM or an imbalance between anabolic and catabolic pathways. We hypothesized that ageing results in a decreased ability of tendon cells to synthesize matrix components and matrix-degrading enzymes, resulting in a reduced turnover of the ECM and a decreased ability to repair micro-damage. The SDFT was collected from horses aged 3-30 years with no signs of tendon injury. Cell synthetic and degradative ability was assessed at the mRNA and protein levels. Telomere length was measured as an additional marker of cell ageing. There was no decrease in cellularity or relative telomere length with increasing age, and no decline in mRNA or protein levels for matrix proteins or degradative enzymes. The results suggest that the mechanism for age-related tendon deterioration is not due to reduced cellularity or a loss of synthetic functionality and that alternative mechanisms should be considered.

Tendon overload results in alterations in cell shape and increased markers of inflammation and matrix degradation.

Tendon injury is thought to involve both damage accumulation within the matrix and an accompanying cell response. While several studies have characterized cell and matrix response in chronically injured tendons, few have assessed the initial response of tendon to overload-induced damage. In this study, we assessed cell response to cyclic loading. Fascicle bundles from the equine superficial digital flexor tendon were exposed to cyclic loading in vitro, designed to mimic a bout of high-intensity exercise. Changes in cell morphology and protein-level alterations in markers of matrix inflammation and degradation were investigated. Loading resulted in matrix damage, which was accompanied by cells becoming rounder. The inflammatory markers cyclooxygenase-2 and interleukin-6 were increased in loaded samples, as were matrix metalloproteinase-13 and the collagen degradation marker C1,2C. These results indicate upregulation of inflammatory and degradative pathways in response to overload-induced in vitro, which may be initiated by alterations in cell strain environment because of localized matrix damage. This provides important information regarding the initiation of tendinopathy, suggesting that inflammation may play an important role in the initial cell response to tendon damage. Full understanding of the early tenocyte response to matrix damage is critical in order to develop effective treatments for tendinopathy.

Capacity for sliding between tendon fascicles decreases with ageing in injury prone equine tendons: a possible mechanism for age-related tendinopathy?

Age-related tendinopathy is common in both humans and horses; the initiation and progression of which is similar between species. The majority of tendon injuries occur to high-strain energy storing tendons, such as the human Achilles tendon and equine superficial digital flexor (SDFT). By contrast, the low-strain positional human anterior tibialis tendon and equine common digital extensor (CDET) are rarely injured. It has previously been established that greater extension occurs at the fascicular interface in the SDFT than in the CDET; this may facilitate the large strains experienced during locomotion in the SDFT without damage occurring to the fascicles. This study investigated the alterations in whole tendon, fascicle and interfascicular mechanical properties in the SDFT and CDET with increasing age. It was hypothesised that the amount of sliding at the fascicular interface in the SDFT would decrease with increasing horse age, whereas the properties of the interface in the CDET would remain unchanged with ageing. Data support the hypothesis; there were no alterations in the mechanical properties of the whole SDFT or its constituent fascicles with increasing age. However, there was significantly less sliding at the fascicular interface at physiological loads in samples from aged tendons. There was no relationship between fascicle sliding and age in the CDET. The increase in stiffness of the interfascicular matrix in aged SDFT may result in the fascicles being loaded at an earlier point in the stress strain curve, increasing the risk of damage. This may predispose aged tendons to tendinopathy.

A review of tendon injury: why is the equine superficial digital flexor tendon most at risk?

Tendon injury is one of the most common causes of wastage in the performance horse; the majority of tendon injuries occur to the superficial digital flexor tendon (SDFT) whereas few occur to the common digital extensor tendon. This review outlines the epidemiology and aetiology of equine tendon injury, reviews the different functions of the tendons in the equine forelimb and suggests possible reasons for the high rate of failure of the SDFT. An understanding of the mechanisms leading to matrix degeneration and subsequent tendon gross failure is the key to developing appropriate treatment and preventative measures.

Equine digital tendons show breed-specific differences in their mechanical properties that may relate to athletic ability and predisposition to injury.

BACKGROUND: Throughout the ages, human subjects have selected horse breeds for their locomotor capacities. Concurrently, tissue properties may have diversified because of specific requirements of different disciplines. OBJECTIVES: The aim of this study was to compare the biomechanical properties of tendons with different functions between equine breeds traditionally selected for racing or sport. STUDY DESIGN: This study used ex vivo tendons and compared the mechanical properties of the common digital extensor tendon (CDET) and superficial digital flexor tendon (SDFT) between racehorses (Thoroughbred [TB]) and sports horses (Friesian Horse [FH], Warmblood [WB]). METHODS: The SDFT and CDET of FH (n = 12), WBs (n = 12) and TBs (n = 8) aged 3-12 years were harvested. The cross sectional area (cm2 ), maximal load (N), ultimate strain (%), ultimate stress (MPa) and elastic modulus (MPa) were determined and tested for significant differences between the breeds (P<0.05). RESULTS: The SDFT from WB horses had a significantly lower elastic modulus than TB horses and failed at a higher strain and load than both FHs and TBs. The mechanical properties of the CDET did not differ between breeds. In agreement with previous studies, the CDET failed at a higher stress and had a higher elastic modulus than the SDFT and, for the WB group of horses only, failed at a significantly lower strain. Interestingly, the mode of failure differed between breeds, particularly with respect to the FHs. MAIN LIMITATIONS: The exercise history of horses used in this study was unknown and the age-range was relatively large; both these factors may have influenced the absolute properties reported in this study. CONCLUSIONS: This study shows for the first time that mechanical properties of the SDFT differ between breeds. These properties are likely to be related to selection for high-speed vs. an extravagant elastic gait and may be an important indicator of performance ability. The Summary is available in Spanish - see Supporting Information.

Can exercise modulate the maturation of functionally different immature tendons in the horse?

Tendons can be considered in two functional groups, those contributing to energetics of locomotion and those acting solely to position the limb. The energy-storing tendons in both human and equine athletes have a high frequency of injury with similar pathophysiology. In previous studies, high-intensity exercise appears to induce a disruption of the matrix rather than functional adaptation in adults. Here we explore the hypothesis that the introduction of controlled exercise during growth would result in an adaptive response without deleterious effects. Young horses were given a controlled exercise program similar to that previously shown to induce matrix changes in energy-storing tendons of skeletally mature animals. The tendons were assessed in relation to mechanical properties, molecular composition, and morphology. Results showed a significant increase in cartilage oligomeric matrix protein (COMP) in the positional tendon but not in the energy-storing tendon. Other matrix properties and mechanical properties were not significantly changed. While the imposition of high-strain-rate exercise in immature horses failed to augment the development of the energy-storing tendon over and above that induced by normal pasture exercise, it did not induce deleterious changes, supporting an earlier introduction of athletic training in horses.

Effect on the mechanical properties of type I collagen of intra-molecular lysine-arginine derived advanced glycation end-product cross-linking.

Non-enzymatic advanced glycation end product (AGE) cross-linking of collagen molecules has been hypothesised to result in significant changes to the mechanical properties of the connective tissues within the body, potentially resulting in a number of age related diseases. We have investigated the effect of two of these cross-links, glucosepane and DOGDIC, on the tensile and lateral moduli of the collagen molecule through the use of a steered molecular dynamics approach, using previously identified preferential formation sites for intra-molecular cross-links. Our results show that the presence of intra-molecular AGE cross-links increases the tensile and lateral Young's moduli in the low strain domain by between 3.0-8.5% and 2.9-60.3% respectively, with little effect exhibited at higher strains.

Subchondral bone thickness, hardness and remodelling are influenced by short-term exercise in a site-specific manner.

It was hypothesised that subchondral bone thickness, hardness and remodelling are influenced by exercise intensity, and by location within a joint. Dorsal carpal osteochondral injury is a major cause of lameness in horses undergoing high intensity training. This project aimed to determine the subchondral bone thickness, formation, resorption and hardness at sites with high and low incidence of pathology in 2 year-old horses undergoing 19 weeks high intensity treadmill training or low intensity exercise, and to compare these factors between exercise groups. Dorsal and palmar test sites were identified on radial, intermediate and third carpal articular surfaces after euthanasia. Adjacent osteochondral samples from each test site underwent histomorphometric analysis (for subchondral bone thickness, osteoid perimeter, osteoid seam width, eroded cavity area and eroded cement line surface length) and microhardness testing. Bone from horses undergoing high intensity training was thicker with a greater osteoid perimeter, and at individual sites had a smaller osteoid seam width and eroded cavity. Exercise-related differences were most marked at dorsal locations. Maximal differences in bone formation indices were observed at dorsal radial and medial third carpal locations. Overall subchondral bone from dorsal sites was thicker with a greater osteoid perimeter. Subchondral bone from dorsal sites was approximately 35% harder than bone from palmar sites. These results show topographical variations in subchondral bone structure, formation, resorption and material properties and a site-specific response to exercise. The maximal response to exercise was at high load sites with a clinical predisposition to injury. These findings indicate that the combined effect of exercise and local load variations within a joint may lead to maximal adaptive responses or overload of these responses at sites predisposed to injury.

Determination of sumatriptan succinate in plasma and urine by high-performance liquid chromatography with electrochemical detection.

Methods are described for the determination of sumatriptan succinate (1) in plasma and urine. Prior to chromatography, plasma is subjected to liquid/liquid extraction and urine is diluted in pH 7 buffer without further pretreatment. Both procedures use reversed-phase high-performance liquid chromatography with electrochemical detection. The analytical range for the plasma assay is 1-30 ng/mL and that for the urine assay is 0.2-12 micrograms/mL. The assays are linear over the analytical ranges and specific with respect to endogenous interference and the major metabolite (2) of sumatriptan. For the plasma assay, intra-assay data (n = 6) indicate a maximum coefficient of variation (CV) and bias across the calibration range of 6.0 and 3.0%, respectively. The interassay CV (n = 4) is approximately 15% at the bottom of the calibration range, falling to 4% or less at 8 ng/mL and above. Bias is approximately 12% at the bottom, reducing to < 2% at 8 ng/mL and above. The urine intra-assay data indicate a maximum CV and bias of 8.9 and 8.3%, respectively. The interassay is CV 15% for the lowest calibrant, reducing steadily across the calibration range to < 2% for the top calibration value, and bias is < 7% across the range.

Factors affecting the ultrasonic properties of equine digital flexor tendons.

The velocity, attenuation and apparent backscattering coefficient of 6-11-MHz ultrasound were measured in three orthogonal directions in equine deep digital flexor (DDF) and superficial digital flexor (SDF) tendons at 0 degree C. Ultrasonic measurements were examined for correlation with tendon water, collagen, DNA and glycosaminoglycans contents, determined by chemical analyses and with structure observed by scanning electron microscopy. The SDF tendon contained more water, more DNA (i.e., more cells), less collagen and less glycosaminoglycans and exhibited lower velocities and attenuations than the DDF tendon. Velocities were governed primarily by the adiabatic bulk modulus and density, perturbed by a highly direction-dependent rigidity. Ultrasound propagating across tendon generated frequency-independent backscattering which appeared to derive from the large interfaces between the fascicles, while along the fibres backscattering varied as f3.62 +/- 0.88 and appeared to derive from small structures such as collagen fibres. The mechanisms by which ultrasound is attenuated by tendon remain unknown.

Oxidative energy metabolism in equine tendon cells.

Hypoxia has been suggested as a possible cause of tissue degeneration and subsequent rupture in equine tendons. To determine whether low oxygen tension is likely to be detrimental to tendon cell function, experiments were designed to investigate oxidative energy metabolism in freshly isolated and cultured equine tendon cells. Freshly isolated tenocytes and cultured fibroblasts possessed activities of the mitochondrial enzyme citrate synthase similar to those of other mammalian cells, with well defined oxidative metabolism. D-[6(-14)C]-glucose oxidation was measurable in both freshly isolated and explant-derived cells. The content of adenosine triphosphate (ATP) in cultured cells was decreased by incubation with a mitochondrial respiratory uncoupler. These data demonstrate that tendon cells are capable of oxidative energy metabolism and rely upon it to maintain cellular ATP levels. Hypoxia must therefore be considered as a possible factor leading to tendon degeneration and subsequent injury.

Macroscopic 'degeneration' of equine superficial digital flexor tendon is accompanied by a change in extracellular matrix composition.

Injuries to the superficial digital flexor tendon are common in horses required to gallop and jump at speed. Partial rupture of this tendon usually occurs in the central core of the midmetacarpal region and may be preceded by localised degenerative changes. Post mortem examination of apparently normal equine flexor tendons has revealed an abnormal macroscopic appearance in the central core, characterised by a reddish discolouration. We have previously shown that there is also physical damage to the collagen fibres. In the present study we tested the hypothesis that the abnormal appearance is accompanied by changes in the composition of the extracellular matrix of the tendon. Biochemical analysis of the extracellular matrix demonstrated an increase in total sulphated glycosaminoglycan content, increase in the proportion of type III collagen and decrease in collagen linked fluorescence in the central core of 'degenerated' tendons relative to tissue from the peripheral region of the same tendon. Dry matter content and total collagen content were not significantly different between tendon zones or normal and 'degenerated' tendons. These changes suggest a change in cell metabolism and matrix turnover in the central core of the tendon and are likely to contribute to a decrease in mechanical properties in this part of the tendon, predisposing to the characteristic partial rupture of the tendon.

Age-related changes to the molecular and cellular components of equine flexor tendons.

Specific tendons show a high incidence of partial central core rupture which is preceded by degeneration. In the performance horse, the superficial digital flexor tendon (SDFT) is most often affected. We have described previously the molecular changes that are associated with degeneration in the central core region of the equine SDFT. The pathophysiological mechanism leading to change in synthetic activity of central zone cells in degenerated tendons is not known. In this study, we test the hypothesis that ageing results in matrix composition changes within the central zone of the SDFT. Extracellular matrix composition and cellularity were analysed in equine SDFTs collected from Thoroughbred horses and compared with a flexor tendon which rarely shows degenerative change and subsequent injury (deep digital flexor tendon, DDFT). Data were examined for age-related changes to central and peripheral zone tissue of the SDFT and DDFT. Ageing in both tendons (SDFT and DDFT) resulted in a significant increase in collagen-linked fluorescence and a decrease in cellularity in the DDFT but not the SDFT. The central zone tissue from the SDFT had a significantly higher proportion of type III collagen than the peripheral zone of the tendon. The highest level of type III collagen was found in the central zone tissue of the SDFT from the older group of horses and this may represent the early stages of a degenerative change. Collagen content did not differ between the 2 flexor tendons; however, there were differences in collagen type and organisation. The SDFT had a higher type III collagen content, higher levels of the mature trifunctional collagen crosslink hydroxylysylpyridinoline, lower total chondroitin sulphate equivalent glycosaminoglycan content, smaller diameter collagen fibrils and a higher cellularity than the DDFT. In conclusion, differences in macromolecular composition exist between the flexor tendons and ageing contributes to a tendon specific change in composition.

Differential scanning calorimetric studies of superficial digital flexor tendon degeneration in the horse.

Differential scanning calorimetry (DSC) of equine superficial digital flexor tendons revealed the presence of a small exothermic peak at 23 degrees C of unknown origin, and a large endothermic peak at 70 degrees C due to denaturation of cross-linked collagen fibres. In the central degenerated core of damaged tendons the denaturation temperature remained at 70 degrees C but the enthalpy decreased in relation to the extent of degeneration of the tendon. We suggest that this reduction in enthalpy is due to depolymerisation and denaturation of the collagen fibres. This contention is supported by the observed increased activity of the degradative enzyme cathepsin B secreted by the fibroblasts. DSC analysis of cultured porcine tendon fibroblasts revealed a multicomponent endotherm, denaturation beginning at 46 degrees C, a temperature capable of being achieved within the tendon during intensive exercise. DSC clearly has considerable potential in complementing morphological and biochemical studies to determine the aetiology and progress of equine tendon degeneration.

Galloping exercise induces regional changes in bone density within the third and radial carpal bones of Thoroughbred horses.

This study was performed to test the hypothesis that a localised bone hypertrophy could occur within the subchondral cancellous architecture of the third and radial carpal bones. Using 2 levels of controlled and defined exercise, it was observed that a high intensity treadmill exercise protocol resulted in functional adaptation of the carpal bones. The increase in trabecular thickening and density was seen to be localised to those regions underlying common sites of cartilage degradation, the interface of the thickened trabeculae with the normal architecture in the third carpal bone was coincident with a common site of clinical fractures. The bone changes were determined both qualitatively on examination of slab radiographs and quantified by dual energy x-ray absorptiometry. The findings from this study are relevant to mechanical factors involved in the pathophysiology of joint degeneration. The potential clinical implications of this study are in relation to changes in the type and duration of exercise regimens used in training of equine athletes. The rapid response of bone to mechanical stimulation has implications in the longer term for localised cartilage degradation. Imaging techniques could be developed to monitor these early bone changes in the specific areas identified in this study and thus allow appropriate changes in training intensity to minimise subsequent damage to the articular surface.

Are the material properties and matrix composition of equine flexor and extensor tendons determined by their functions?

REASONS FOR PERFORMING STUDY: Injury to the superficial digital flexor tendon (SDFT) is common in competition horses. The SDFT contributes to locomotory efficiency by storing energy; such tendons have low safety margins. Tendons which merely position the limb, including the opposing common digital extensor tendon (CDET), are rarely injured. The current failure of strategies to prevent or effectively treat injury to the SDFT indicates the importance of understanding how it differs from tendons which are not injury-prone. HYPOTHESIS: That the structural and material properties and matrix composition of the SDFT and CDET differ, reflecting their specific functional requirements in vivo. METHODS: Forelimb tendons were harvested from 26 mature horses and loaded to failure prior to matrix composition analysis of specimens. RESULTS: The SDFT had a significantly higher cross-sectional area, structural stiffness, failure load and failure strain and a lower elastic modulus than the CDET (P < 0.0001). CONCLUSIONS: The SDFT has conflicting requirements for strength and elasticity; although as a whole it is a stiffer structure than the CDET, differences in the matrix molecular composition including water and total sulphated glycosaminoglycan contents allow it to remain more elastic as a material. POTENTIAL RELEVANCE: Further information on how the two tendons attain these different properties may be of use in the development of prevention and treatment strategies for SDFT rupture.

Mechanical properties of the equine superficial digital flexor tendon relate to specific collagen cross-link levels.

REASONS FOR PERFORMING STUDY: Damage to the flexor tendons, particularly the superficial digital flexor tendon (SDFT), is one of the most common musculoskeletal injuries sustained by horses competing in all disciplines. Our previous work has shown that SDFTs from different individuals show a wide variation in mechanical strengths; this is important clinically as it may relate to predisposition to injury. The high mechanical strength of tendon relies on the correct orientation of collagen molecules within fibrils and stabilisation by the formation of chemical cross-links between collagen molecules. It is not known whether the variation in SDFT mechanical properties between individuals relates to differences in collagen cross-link levels. HYPOTHESIS: Enzyme-derived, intermolecular cross-linking of tendon collagen correlates with mechanical properties of the SDFT. METHODS: SDFTs were collected from 38 horses and mechanically tested to failure. Structural and material properties were calculated from the load/deformation plot and cross-sectional area for each tendon. Following mechanical testing, pyrrolic cross-link levels were measured using a spectrophotometric assay for Ehrlich's reactivity and pyridinoline levels were quantified by HPLC. Cross-link levels were correlated with mechanical properties and statistical significance tested using a Pearson's correlation test. RESULTS: Pyrrole cross-link levels showed a significant positive correlation with ultimate stress (P = 0.004), yield stress (P = 0.003) and elastic modulus (P = 0.018) of the tendons, despite being a minor cross-link in these tendons. There was no significant correlation of mechanical properties with either hydroxylysyl- or lysyl-pyridinoline levels. CONCLUSIONS: Given the low absolute levels of pyrrole, we suggest that the correlation with high mechanical strength is through an indirect mechanism. Understanding the nature of the relationships between pyrrole cross-links, other matrix characteristics and tendon material properties may allow development of strategies to identify horses at risk from tendon injury and be of value in informing training practices.

Structure, function and biosynthesis of the Mycobacterium tuberculosis cell wall: arabinogalactan and lipoarabinomannan assembly with a view to discovering new drug targets.

In spite of effective antibiotics to treat TB (tuberculosis) since the early 1960s, we enter the new millennium with TB, currently the leading cause of death from a single infectious agent, killing more than three million people worldwide each year. Thus an understanding of drug-resistance mechanisms, the immunobiology of cell wall components to elucidate host-pathogen interactions and the discovery of new drug targets are now required for the treatment of TB. Above the plasma membrane is a classical chemotype IV PG (peptidoglycan) to which is attached the macromolecular structure, mycolyl-arabinogalactan, via a unique diglycosylphosphoryl bridge. This review will discuss the assembly of the mAGP (mycolyl-arabinogalactan-peptidoglycan), its associated glycolipids and the site of action of EMB (ethambutol), bringing forward a new era in TB research and focus on new drugs to combat multidrug resistant TB.

The effect of exercise-induced localised hyperthermia on tendon cell survival.

Tendons that store energy during locomotion, such as the equine superficial digital flexor tendon (SDFT) and human Achilles tendon, suffer a high incidence of central core degeneration which is thought to precede tendon rupture. Although energy storage contributes to the efficiency of locomotion, tendons are not perfectly elastic and some energy is lost in the form of heat. Recent studies have shown that the central core of equine SDFT reaches temperatures as high as 45 degrees C during high-speed locomotion. In this study, we test the hypothesis that hyperthermia causes tendon cell death and results in tendon central core degeneration. Tendon fibroblasts cultured from the core of the equine SDFT were subjected to a temperature of 45 degrees C in an in vitro system for 0-180 min, and cell survival fraction was measured and compared with that for equine dermal fibroblasts and a commercial rat kidney fibroblast cell line (NRK 49F). Tendon fibroblasts were significantly more resistant to hyperthermia than NRK 49F cells after 30, 45 and 60 min of heating and significantly more resistant than dermal fibroblasts after 45 and 60 min of heating. After 10 min of heating at 45 degrees C, the tendon fibroblast cell survival fraction was 91 +/- 4%, whereas heating for 10 min at 48 degrees C resulted in a drop in the cell survival fraction to 22 +/- 4%. In conclusion, while temperatures experienced in the central core of the SDFT in vivo are unlikely to result in tendon cell death, repeated hyperthermic insults may compromise cell metabolism of matrix components, resulting in tendon central core degeneration.

The pathobiology and repair of tendon and ligament injury.

Injury of the collagenous structures comprising tendons and ligaments, either from acute trauma or from repetitive strain lesions, results in protracted periods of disability. The resolution of such injuries often fails to restore the normal morphologic and functional characteristics of the structure and, therefore, either compromises the future performance of the individual or predisposes to an increased risk of recurrent injury.