Substantial Achilles adaptation following strength training has no impact on tendon function during walking.

Tendons are responsive to mechanical loading and their properties are often the target of intervention programs. The tendon's mechanical properties, particularly stiffness, also govern its function, therefore changes to these properties could have substantial influence on energy-saving mechanisms during activities utilizing the stretch-shortening cycle. We investigated Achilles tendon (AT) function in vivo during walking with respect to a training intervention that elicited significant increases in AT stiffness. 14 men and women completed 12-weeks of isometric plantarflexor strength training that increased AT stiffness, measured during isometric MVC, by ~31%. Before and after the intervention, participants walked shod at their preferred velocity on a fully-instrumented treadmill. Movement kinematics, kinetics and displacement of the gastrocnemius medialis muscle-tendon junction were captured synchronously using 3D motion capture and ultrasound imaging, respectively. A MANOVA test was used to examine changes in AT force, stress, strain, stiffness, Young's modulus, hysteresis and strain energy, measured during walking, before and following strength training. All were non-significant for a main effect of time, therefore no follow-up statistical tests were conducted. Changes in joint kinematics, tendon strain, velocity, work and power and muscle activity during the stance phase were assessed with 1D statistical parametric mapping, all of which also demonstrated a lack of change in response to the intervention. This in vivo examination of tendon function in walking provides an important foundation for investigating the functional consequences of training adaptations. We found substantial increases in AT stiffness did not impact on tendon function during walking. AT stiffness measured during walking, however, was unchanged with training, which suggests that increases in stiffness may not be evident across the whole force-elongation relation, a finding which may help explain previously mixed intervention results and guide future investigations in the functional implications of tendon adaptation.

Impact of rest duration on Achilles tendon structure and function following isometric training.

Intervention programs are often sought to strengthen the Achilles tendon (AT) due to its high injury rate. Long rest periods between loading cycles have been found to increase collagen synthesis by tenocytes, suggesting rest duration may be important for tendon adaptation in vivo; however, exercise programs comparing long and short rest duration have not been directly compared. Fourteen adults completed a 12-week progressive training intervention; training sessions consisted of 5×10 isometric plantarflexion contractions each of 3-s duration performed at 90% of MVC three times weekly. Each leg was randomly allocated to long (LRT, 10-s rest) or short rest training (SRT, 3-s rest). We hypothesized that the leg allocated to LRT would demonstrate superior AT collagen organization compared to the leg receiving SRT, which would be related to improved biomechanical function. AT collagen organization and morphology were measured using ultrasound tissue characterization. AT properties were assessed before and after the intervention using a combination of dynamometry, ultrasound imaging, EMG, and motion capture. Contrary to our hypothesis, collagen organization did not improve following either training protocol; conversely, an unexpected decrease in echotype I proportion was seen after SRT (P<.001) but not LRT (P=.58), indicating an apparent protective effect of rest on collagen organization during isometric training. In contrast, AT adaptation was not appreciably enhanced by increasing intercycle rest duration; both protocols were equally effective at inducing significant strength gains and AT mechanical and material adaptation (P≤.001). Further research is necessary to identify optimal loading characteristics for injury prevention and rehabilitation.

In vivo biological response to extracorporeal shockwave therapy in human tendinopathy.

Extracorporeal shock wave therapy (ESWT) is a non-invasive treatment for chronic tendinopathies, however little is known about the in-vivo biological mechanisms of ESWT. Using microdialysis, we examined the real-time biological response of healthy and pathological tendons to ESWT. A single session of ESWT was administered to the mid-portion of the Achilles tendon in thirteen healthy individuals (aged 25.7 ± 7.0 years) and patellar or Achilles tendon of six patients with tendinopathies (aged 39.0 ± 14.9 years). Dialysate samples from the surrounding peri-tendon were collected before and immediately after ESWT. Interleukins (IL)-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-17A, vascular endothelial growth factor and interferon-γ were quantified using a cytometric bead array while gelatinase activity (MMP-2 and -9) was examined using zymography. There were no statistical differences between the biological tissue response to ESWT in healthy and pathological tendons. IL-1ß, IL-2, IL-6 and IL-8 were the cytokines predominantly detected in the tendon dialysate. IL-1ß and IL-2 did not change significantly with ESWT. IL-6 and IL-8 concentrations were elevated immediately after ESWT and remained significantly elevated for four hours post-ESWT (p < 0.001). Pro-forms of MMP-2 and -9 also increased after ESWT (p < 0.003), whereas there were no significant changes in active MMP forms. In addition, the biological response to ESWT treatment could be differentiated between possible responders and non-responders based on a minimum 5-fold increase in any inflammatory marker or MMP from pre- to post-ESWT. Our findings provide novel evidence of the biological mechanisms underpinning ESWT in humans in vivo. They suggest that the mechanical stimulus provided by ESWT might aid tendon remodelling in tendinopathy by promoting the inflammatory and catabolic processes that are associated with removing damaged matrix constituents. The non-response of some individuals may help to explain why ESWT does not improve symptoms in all patients and provides a potential focus for future research.

Effects of resistance training on tendon mechanical properties and rapid force production in prepubertal children.

Children develop lower levels of muscle force, and at slower rates, than adults. Although strength training in children is expected to reduce this differential, a synchronous adaptation in the tendon must be achieved to ensure forces continue to be transmitted to the skeleton with efficiency while minimizing the risk of strain-related tendon injury. We hypothesized that resistance training (RT) would alter tendon mechanical properties in children concomitantly with changes in force production characteristics. Twenty prepubertal children (age 8.9 ± 0.3 yr) were equally divided into control (nontraining) and experimental (training) groups. The training group completed a 10-wk RT intervention consisting of 2-3 sets of 8-15 plantar flexion contractions performed twice weekly on a recumbent calf-raise machine. Achilles tendon properties (cross-sectional area, elongation, stress, strain, stiffness, and Young's modulus), electromechanical delay (EMD; time between the onset of muscle activity and force), rate of force development (RFD; slope of the force-time curve), and rate of electromyographic (EMG) increase (REI; slope of the EMG time curve) were measured before and after RT. Tendon stiffness and Young's modulus increased significantly after RT in the experimental group only (∼29% and ∼25%, respectively); all other tendon properties were not significantly altered, although there were mean decreases in both peak tendon strain and strain at a given force level (14% and 24%, respectively; not significant) which may have implications for tendon injury risk and muscle fiber mechanics. A decrease of ∼13% in EMD was found after RT for the experimental group, which paralleled the increase in tendon stiffness (r = -0.59); however, RFD and REI were unchanged. The present data show that the Achilles tendon adapts to RT in prepubertal children and is paralleled by a change in EMD, although the magnitude of this change did not appear to be sufficient to influence RFD. These findings are of importance within the context of the efficiency and execution of movement.

Range of motion, neuromechanical, and architectural adaptations to plantar flexor stretch training in humans.

The neuromuscular adaptations in response to muscle stretch training have not been clearly described. In the present study, changes in muscle (at fascicular and whole muscle levels) and tendon mechanics, muscle activity, and spinal motoneuron excitability were examined during standardized plantar flexor stretches after 3 wk of twice daily stretch training (4 × 30 s). No changes were observed in a nonexercising control group (n = 9), however stretch training elicited a 19.9% increase in dorsiflexion range of motion (ROM) and a 28% increase in passive joint moment at end ROM (n = 12). Only a trend toward a decrease in passive plantar flexor moment during stretch (-9.9%; P = 0.15) was observed, and no changes in electromyographic amplitudes during ROM or at end ROM were detected. Decreases in H(max):M(max) (tibial nerve stimulation) were observed at plantar flexed (gastrocnemius medialis and soleus) and neutral (soleus only) joint angles, but not with the ankle dorsiflexed. Muscle and fascicle strain increased (12 vs. 23%) along with a decrease in muscle stiffness (-18%) during stretch to a constant target joint angle. Muscle length at end ROM increased (13%) without a change in fascicle length, fascicle rotation, tendon elongation, or tendon stiffness following training. A lack of change in maximum voluntary contraction moment and rate of force development at any joint angle was taken to indicate a lack of change in series compliance of the muscle-tendon unit. Thus, increases in end ROM were underpinned by increases in maximum tolerable passive joint moment (stretch tolerance) and both muscle and fascicle elongation rather than changes in volitional muscle activation or motoneuron pool excitability.

Neuromuscular factors influencing the maximum stretch limit of the human plantar flexors.

Maximum joint range of motion is an important parameter influencing functional performance and musculoskeletal injury risk. Nonetheless, a complete description of the muscle architectural and tendon changes that occur during stretch and the factors influencing maximum range of motion is lacking. We measured muscle-tendon elongation and fascicle lengthening and rotation sonographically during maximal plantar flexor stretches in 21 healthy men. Electromyogram (EMG) recordings were obtained synchronously with ultrasound and joint moment data, and H-reflex measurements were made with the ankle at neutral (0°) and dorsiflexed (50% maximal passive joint moment) positions; the maximum H amplitude (normalized to maximum M-wave amplitude; M(max)) and H-amplitude elicited at a stimulation intensity that evoked 10% M(max) were obtained. Maximal stretch was accomplished through significant muscle (14.9%; 30 mm) and tendon lengthening (8.4%; 22 mm). There were similar relative changes in fascicle length and angle, but planimetric modeling indicated that the contribution of fascicle rotation to muscle lengthening was small (<4 mm). Subjects with a greater range of motion showed less resistance to stretch and a greater passive joint moment at stretch termination than less flexible subjects (i.e., greater stretch tolerance). Also, greater fascicle rotation accompanied muscle elongation (9.7 vs. 5.9%) and there was a greater tendon length at stretch termination in more flexible subjects. Finally, a moderate correlation between the angle of EMG onset and maximum range of motion was obtained (r = 0.60, P < 0.05), despite there being no difference in H-reflex magnitudes between the groups. Thus clear differences in the neuromuscular responses to stretch were observed between "flexible" and "inflexible" subjects.

Age-related changes in mechanical properties of the Achilles tendon.

The stiffness of a tendon, which influences muscular force transfer to the skeleton and increases during childhood, is dependent on its material properties and dimensions, both of which are influenced by chronic loading. The aims of this study were to: (i) determine the independent contributions of body mass, force production capabilities and tendon dimensions to tendon stiffness during childhood; and (ii) descriptively document age-related changes in tendon mechanical properties and dimensions. Achilles tendon mechanical and material properties were determined in 52 children (5-12 years) and 19 adults. Tendon stiffness and Young's modulus (YM) were calculated as the slopes of the force-elongation and stress-strain curves, respectively. Relationships between stiffness vs. age, mass and force, and between YM vs. age, mass and stress were determined by means of polynomial fits and multiple regression analyses. Mass was found to be the best predictor of stiffness, whilst stress was best related to YM (< 75 and 51% explained variance, respectively). Combined, mass and force accounted for up to 78% of stiffness variation. Up to 61% of YM variability could be explained using a combination of mass, stress and age. These results demonstrate that age-related increases in tendon stiffness are largely attributable to increased tendon loading from weight-bearing tasks and increased plantarflexor force production, as well as tendon growth. Moreover, our results suggest that chronic increases in tendon loading during childhood result in microstructural changes which increase the tendon's YM. Regarding the second aim, peak stress increased from childhood to adulthood due to greater increases in strength than tendon cross-sectional area. Peak strain remained constant as a result of parallel increases in tendon length and peak elongation. The differences in Achilles tendon properties found between adults and children are likely to influence force production, and ultimately movement characteristics, which should be explicitly examined in future research.

Can Achilles tendon moment arm be predicted from anthropometric measures in pre-pubescent children?

Muscle-tendon moment arm magnitudes are essential variables for accurately calculating muscle forces from joint moments. Their measurement requires specialist knowledge and expensive resources. Research has shown that the patellar tendon moment arm length is related to leg anthropometry in children. Here, we asked whether the Achilles tendon moment arm (MA(AT)) can be accurately predicted in pre-pubescent children from surface anthropometry. Age, standing height, mass, foot length, inter-malleolar ankle width, antero-posterior ankle depth, tibial length, lower leg circumference, and distances from the calcaneus to the distal head of the 1st metatarsal and medial malleolus were determined in 49 pre-pubescent children. MA(AT) was calculated at three different ankle positions (neutral, 10° plantarflexion, and 10° dorsiflexion) by differentiating tendon excursion, measured via ultrasonography, with respect to ankle angle change using seven different differentiation techniques. Backwards stepwise regression analyses were performed to identify predictors of MA(AT.) When all variables were included, the regression analysis accounted for a maximum of 49% of MA(AT) variance at the neutral ankle angle when a third-order polynomial was used to differentiate tendon excursion with respect to ankle angle. For this condition, foot length and the distance between calcaneus and 1st metatarsal were the only significant predictors, accounting for 47% of the variance (p<0.05). The absolute error associated with this regression model was 3.8±4.4 mm, which would result in significant error (mean=14.5%) when estimating muscle forces from joint moments. We conclude that MA(AT) cannot be accurately predicted from anthropometric measures in children.

ORP-3, a human oxysterol-binding protein gene differentially expressed in hematopoietic cells.

Using differential display polymerase chain reaction, a gene was identified in CD34(+)-enriched populations that had with low or absent expression in CD34(-) populations. The full coding sequence of this transcript was obtained, and the predicted protein has a high degree of homology to oxysterol-binding protein. This gene has been designated OSBP-related protein 3 (ORP-3). Expression of ORP-3 was found to be 3- to 4-fold higher in CD34(+) cells than in CD34(-) cells. Additionally, expression of this gene was 2-fold higher in the more primitive subfraction of hematopoietic cells defined by the CD34(+)38(-) phenotype and was down-regulated with the proliferation and differentiation of CD34(+) cells. The ORP-3 predicted protein contains an oxysterol-binding domain. Well-characterized proteins expressing this domain bind oxysterols in a dose-dependent fashion. Biologic activities of oxysterols include inhibition of cholesterol biosynthesis and cell proliferation in a variety of cell types, among them hematopoietic cells. Characterization and differential expression of ORP-3 implicates a possible role in the mediation of oxysterol effects on hematopoiesis.

Flow cytometric analysis of intercellular adhesion between B-cell precursor acute lymphoblastic leukemic cells and bone marrow stromal cells.

The growth of B-cell precursor acute lymphoblastic leukemic (BCP ALL) cells in vitro is dependent on interactions with bone marrow (BM) stromal cells. We have recently demonstrated that the rate of cell division of BCP ALL cells increases when cultured in direct contact with BM stromal cells. In this study we describe a new method for examining the direct binding of BM stromal cells and BCP ALL cells at a cellular level. For this binding assay, BCP ALL cells from six patient samples were first stained with the lipophilic fluorescent probe PKH 26 GL and mixed with BM stromal cells in suspension. In all cases, aggregates between BCP ALL and BM stromal cells were identified by flow cytometry and isolated. Using this assay we have examined some of the mechanisms involved in this binding process. The pattern of aggregate formation at various leukemic/stromal cell ratios showed that the aggregate formation increased by increasing the number of either cell type and that the binding could not be saturated. This suggests that the interaction between these cells is an equilibrium reaction. Functional studies showed that the majority of BCP ALL-BM stromal cell binding is dependent on the presence of divalent cations and requires active cellular metabolism. Finally, by use of inhibitory monoclonal antibodies (moAbs) directed against cell adhesion molecules including anti-CD29, VCAM and CD18, we have demonstrated that the involvement of these molecules in the direct cellular interactions could be detected by this method. However, the maximum inhibition observed was 36% which suggests either that the avidity is low or that other adhesion molecules are involved. The data show that the use of flow cytometric analysis of aggregate formation (rather than cell binding to intact cell layers) allows the study of cell interactions at the individual cell level which can reveal additional cellular adhesion mechanisms.