Shear Wave Elastography Measures of the Achilles Tendon: Influence of Time of Day, Leg Dominance and the Impact of an Acute 30-Minute Bout of Running

The mechanical properties of human tendons are likely to be in uenced by factors known to affect elastic structures, including patterns of loading and unloading during the day. However, the exact scale and relevance of these variables to tendon stiffness remains unclear. The aim of this study was to (1) measure Achilles tendon (AT) stiffness over the course of the day, (2) examine AT stiffness between dominant and non-dominant standing leg tendons and (3) assess the impact of previous activity on AT stiffness. To assess the impact of time of day and leg dominance, 15 healthy participants (6 females, 9 males; mean age 28 4 year, mean VISA-A score 99.0 1.2) had shear wave elastography (SWE) measures taken at 08:00 h, 12:30 h and 17:00 h on both dominant and non-dominant legs. To assess the impact of exercise, 24 tendons were analysed (7 females, 5 males; mean age 27 4 year, mean VISA-A Score 99.1 1.1) with participants randomly assigned to either a control (CONT) group or a running (RUN) group. The RUN group performed a 30-min run at a subjective intensity of 13–15 on rating of perceived exertion (RPE) scale and had SWE measures taken before, immediately after, 6 h 24 h, 48 h and 72 h following the run. There were no signi cant differences in AT stiffness over the course of a day or between dominant and non-dominant leg. Signi cant increases in AT stiffness were noted pre-post run (0.27 m/s, 2.95%,p= 0.037). Leg dominance does not affect SWE values from asymptomatic ATs or change throughout a day, but a 30-min run signi cantly increases AT stiffness.

run. There were no signi cant differences in AT stiffness over the course of a day or between dominant and non-dominant leg. Signi cant increases in AT stiffness were noted pre-post run (0.27 m/s, 2.95%,p= 0.037). Leg dominance does not affect SWE values from asymptomatic ATs or change throughout a day, but a 30-min run signi cantly increases AT stiffness. Leg dominance and timing of clinical appointments are unlikely to affect SWE results, however a prior bout of physical activity may cause changes within the AT resulting in a signi cantly different SWE measure. Clinicians and researchers should be cautious of interpreting SWE results if weight bearing exercise has been performed beforehand. Keywords: Achilles tendon; shear wave elastography; ultrasound elastography; time of day; leg dominance; prior activity 1. Introduction The mechanical properties of human tendons are likely to be in uenced by factors known to affect elastic structures, including patterns of loading and unloading during the day. The scale and relevance of these variables to tendon structure and function remain relatively unclear [1] and there are few systematic reports on how the stiffness of a human tendon alters throughout the course of a day [2,3]. Periods of sleep, rest and activity throughout a day will cause altered loading and unloading at differing frequencies and intensities on tendons, which will impact their stiffness. The research available into the effect of time of day on tendon structuresin vivohas been conducted using the patella tendon, with tendon stiffness estimated using an isokinetic dynamometer to measure force Appl. Sci.2018,8, 1170; doi:10.3390/app8071170

Appl. Sci.2018,8, 1170 2 of 14 and B-mode ultrasound to measure length [2]. This research measured a decrease in tendon stiffness of 20.2 9.5% between the testing times of 08:00 h and 18:00 h [2]. No research has yet assessed the impact of time of day on tendon stiffness using shear wave elastography (SWE). The term mechanotransduction refers to the processes converting mechanical loading, such as exercise, into measurable cellular responses that may result in structural or functional change [4]. Exercise has been shown capable of altering both the structural and chemical makeup of human tendon by inducing increases in its cross-sectional area and increasing the concentration of metabolic enzymes to increase collagen turnover and prostaglandin production [5,6]. Mechanical stimulus is also postulated to initiate changes within the extracellular matrix of tendon that results in a more damage resistant tissue with optimal force transmission properties [7]. The early adaptive responses to mechanotransduction can initiate longer term alterations in the mechanical properties of a tendon [8], however the majority of alterations brought about by mechanotransduction are noted as the result of repeated exercise (loading) programmes. This could be because the adaptation in the mechanical properties of tendons takes a long time, or the impact of acute bouts of exercise have not yet been extensively studied. Regardless, the exact in uence of acute bouts of speci c forms of exercise on the mechanical properties of healthy human tendon remains relatively unclear [1,9]. Long term exercise training such as running may increase the mass, collagen content, ultimate tensile strength and load to failure of a tendon [5], but changes in relation to acute exercise bouts are less well understood. A systematic review of the immediate effect of exercise on AT properties was conducted in 2013 [10]. The conclusion of this paper was that acute bouts of exercise impact AT mechanical properties in a manner dependent on both mode and dose of exercise as the differences in tendon stress-strain characteristics, such as rate, duration and frequency, between different types of exercise will cause varying changes to the mechanical properties of tendons over time [10].

in 2013 [10]. The conclusion of this paper was that acute bouts of exercise impact AT mechanical properties in a manner dependent on both mode and dose of exercise as the differences in tendon stress-strain characteristics, such as rate, duration and frequency, between different types of exercise will cause varying changes to the mechanical properties of tendons over time [10]. Of the papers included in the review by Obst et al. (2013), 14 assessed AT stiffness with 12 of these using data obtained from ultrasonography and dynamometry [11–22]. Whilst the remaining two studies also utilised ultrasonography, they used either a force platform or a force transducer to obtain measures of force [23,24]. Six papers reported a decrease in AT stiffness following exercise, one reported a signi cant increase and the remaining seven reported no differences [10]. Of the articles that found a decrease in AT stiffness, four assessed the effect of stretching, whilst others assessed isometric or concentric muscle contractions and not speci cally running. One study found a signi cant increase in tendon stiffness post 10 minutes of static stretching with stiffness calculated using ultrasonography and isokinetic dynamometry [18]. Only two articles included in the review assessed tendon stiffness after running, one study found no difference in AT stiffness post running [23]. The other study found no signi cant difference in stiffness, however, it only looked at changes after a 6-min warm up jog and stretching [19]. Also noted in the Obst et al. (2013) paper was the lack of studies evaluating the mechanical properties of the `free' AT [10], with `free' AT referring to the part of the tendon without any other attachment to either bony or muscular structures. This current study therefore aims to focus on the `free' portion of the AT to address this gap in the literature. Although not directly assessing stiffness, research using ultrasound tissue characterisation (UTC) has assessed the structure of the humanin vivoAT before and after exercise. The results of studies using UTC suggest there were changes in tendon structure and integrity following a bout of exercise, including a decrease in aligned

`free' portion of the AT to address this gap in the literature. Although not directly assessing stiffness, research using ultrasound tissue characterisation (UTC) has assessed the structure of the humanin vivoAT before and after exercise. The results of studies using UTC suggest there were changes in tendon structure and integrity following a bout of exercise, including a decrease in aligned tendon brils and an increase in the separation and waviness of brils, both of which returned to baseline over the following 72 h [25]. A decrease in the alignment of the tendon bres may result in decreased tendon stiffness. Habitual running of long distances (>80 km/week for >3 years) results in a marked increase in the cross-sectional area of the AT of approximately 22% in comparison with a non-running control group [26]. This increase in cross-sectional area may be a compensatory mechanism as pathological tendons can compensate for signi cant areas of disorganised bres by increasing thickness to reduce stress and maintain structural homeostasis to ensure adequate load bearing [27,28]. Despite a large body of work surrounding the

Appl. Sci.2018,8, 1170 3 of 14 effects of habitual running, studies exploring the impact of a single acute bout of running on stiffness measures of normal, healthy human ATs in vivo remain scarce [10,19]. Most research into tendon stiffness utilise direct methods of calculating stiffness using ultrasound to measure change in tendon length and a dynamometer to measure force. This can produce accurate results, but is time consuming, requires complex procedures and a lot of equipment and space to run. Hence, a quick, easy and non-invasive measure of tendon stiffness is required. This study proposes to measure shear wave velocity (SWV), a surrogate measure of stiffness, in the ATin vivo using shear wave elastography (SWE). The relatively recent introduction of SWE offers a novel way to quantitatively assess tendon stiffness by measuring SWV through a tissue, providing information on tissue stiffness [29]. In recent years, there have been several studies published using SWE to assess tendon stiffness [30–34] and many reporting the reliability and validity of SWE [33,35,36]. Despite this, the reported information on the many variables that potentially in uence SWE measurements remain unclear [32]. It is necessary to understand normal variation in AT stiffness with relation to leg dominance [37], as ATs have been shown to be signi cantly different between dominant and non-dominant legs [38] having different mechanical properties attributed to different loading pro les of both legs during daily activity due to foot dominance [39]. No studies have examined differences in AT stiffness with leg dominance using SWE or the alterations in SWE measurements experienced within a healthy human AT in vivo, in response to the time of day or an acute bout of running. It is important to understand the normal variation in SWV measures within the ATin vivoand the in uence of external loads on these values. Without such information, abnormal or clinically relevant values cannot be decided upon and accurate interpretation is not possible. The aim of this study is three-fold. Firstly, it will measure the stiffness of the AT using SWE in the morning (08:00 h), afternoon (12:30 h) and evening (17:00

within the ATin vivoand the in uence of external loads on these values. Without such information, abnormal or clinically relevant values cannot be decided upon and accurate interpretation is not possible. The aim of this study is three-fold. Firstly, it will measure the stiffness of the AT using SWE in the morning (08:00 h), afternoon (12:30 h) and evening (17:00 h) to see whether any measurable differences are apparent dependent on time of day. Secondly, it will examine SWE measures obtained in ATsin vivobi-laterally to assess measurable differences between dominant and non-dominant standing leg tendons. Thirdly, this study will use SWE to assess measures of stiffness taken before, immediately after, 6 h, 24 h, 48 h and 72 h after an acute 30-min bout of running to trace the time course of SWV alterations in the ATin vivo after exercise. 2. Materials and Methods 2.1. Participants To assess the impact of time of day and leg dominance, 15 healthy participants were examined (6 females, 9 males; mean age 28 4 year, mean VISA-A score 99.0 1.2). To determine foot dominance, participants were asked to identify which foot they would kick a ball with [40–42]. To assess the impact of a 30-min bout of running, 24 tendons from 12 participants were analysed (7 females, 5 males; mean age 27 4 year, mean VISA-A Score 99.1 1.1). Inclusion criteria was set as both males and females over the age of 18 years old who achieved a minimum score of 96/100 on the VISA-A to rule out subjectively symptomatic tendons. Exclusion criteria included previous diagnosis of Achilles tendinopathy, history of pain in the AT area lasting for more than 24 h, pregnancy, previous medical or surgical intervention on the AT or abnormal features consistent with Achilles Tendinopathy on conventional 2D ultrasound. Participants were recruited by word of mouth from the University department where the testing took place. All participants provided written informed consent to participate in the study and all procedures performed involving human participants were commenced following ethical approval for the study being obtained from the University of Brighton ethics committee, in

consistent with Achilles Tendinopathy on conventional 2D ultrasound. Participants were recruited by word of mouth from the University department where the testing took place. All participants provided written informed consent to participate in the study and all procedures performed involving human participants were commenced following ethical approval for the study being obtained from the University of Brighton ethics committee, in line with the 1964 Helsinki declaration and its later amendments.

Appl. Sci.2018,8, 1170 4 of 14 2.2. Methods To assess the impact of time of day, all participants were scanned three times throughout the course of their normal working day. Firstly, between 08:00–08:30 h, secondly between 12:30–13:00 h and lastly between 17:00–17:30 h. To assess the impact of leg dominance, SWE measures were taken on both the dominant and non-dominant legs. Participants were asked to maintain any daily walking activity they deemed normal but refrain from any other exercise for 48 h before testing and during the testing period. To assess the impact of an acute bout of running, participants were randomly placed into the intervention group of running (RUN) or the control (CONT) group. If assigned to the CONT group, participants were asked to remain seated in the examination room for a period of at least 30 min, and no more than 40 min during their testing session. The participants were asked to remain seated keeping their legs still during this period and not move around, the experimenter was always present to ensure this occurred. Following assignment to each intervention group, the measures outlined below were taken from each participant immediately before and immediately after their sitting or running intervention. Subsequent follow up measures were also taken at 6 h, 24 h, 48 h and 72 h post intervention. In the RUN group, participants were given the possibility to warm up on the treadmill for no more than 5 min. Following the warm up, they were asked to keep the incline on the treadmill at 1% and increase the speed of the treadmill to a pace they felt comfortable with, and to run for a period of 30 min. A Rating of Perceived Exertion (RPE) scale [43] was placed in the direct eye line of the participants and they were asked to maintain an RPE of at least 13 (representing an intensity of somewhat hard), but below 15 (hard). The participants were asked every 5 min to rate their current RPE value and were encouraged by the experimenter to maintain an RPE of between 13 and 15 if it

in the direct eye line of the participants and they were asked to maintain an RPE of at least 13 (representing an intensity of somewhat hard), but below 15 (hard). The participants were asked every 5 min to rate their current RPE value and were encouraged by the experimenter to maintain an RPE of between 13 and 15 if it was outside of these values. The participants were able to alter their speed during their 30-min run to maintain RPE between these boundaries. Following 30 min of running, participants could complete a cool down at a walking speed of their choice for a period of no more than 5 min. 2.3. Scanning Techniques During all measures, participants lay prone with both feet hanging clear of an examination table and an amount of ultrasound gel suf cient to maintain good contact between the ultrasound probe and the skin was applied. All measures were taken with a Siemens ACUSON S3000™HELX EVOLUTION Ultrasound System (Siemens Medical Solutions, Mountain View, CA, USA). Measures obtained were shear wave velocity (SWV) which was used for analysis without converting to Young's modulus. 2.4. Conventional Ultrasound Technique During each measurement, extended eld of view `SieScape' images were taken on grey scale ultrasound by a single operator (CP), with three years imaging experience, using a 14L5SP probe to visualize the `free' AT length, between insertion of the AT the calcaneus to the lowest bres of soleus, following previous methodology [44]. Tendon mid-point was calculated as half AT length and used as the reference point for all subsequent measures to ensure all were taken at the tendon mid-point, relative to each participant. Measures of maximum anterior-posterior (max AP) diameter were calculated at the tendon mid-point relative to each participant using a 14L5 probe. A transverse image of the AT was captured from the tendon mid-point and the ultrasound software used to measure the maximum distance in millimetres (mm) from the anterior border of the tendon to the posterior border of the tendon. As used in previous research [45], three consecutive measures were taken, ensuring all measures fell within 5 mm

a 14L5 probe. A transverse image of the AT was captured from the tendon mid-point and the ultrasound software used to measure the maximum distance in millimetres (mm) from the anterior border of the tendon to the posterior border of the tendon. As used in previous research [45], three consecutive measures were taken, ensuring all measures fell within 5 mm of each other, with the mean of the three measurements taken to represent max AP diameter.

Appl. Sci.2018,8, 1170 5 of 14 2.5. Shear Wave Elastography Following conventional ultrasound, the system was placed into Virtual Touch IQ (VTIQ) mode, an acoustic radiation force-based method that produces both qualitative and quantitative maps of SWV ranging between 0.5 and 10.0 m/s [46,47]. Images were obtained by the same operator (CP) using a linear-array 9L4 transducer probe. Due to the saturation limit of the technology, a SWV value measured above 10 m/s was returned by the software as `High'. Only 4 of these were returned throughout the course of the study and when they did occur, they were discounted from the study. Image quality was closely monitored throughout examination, tissue compression was avoided. Quality maps were assessed to ensure images conformed to a high level of quality, speci cally checking for a quality map all green in colour, which indicates that a correct amount of pressure was used. All elastograms were taken in a longitudinal plane with the foot in a relaxed foot position, as this has been demonstrated to provide the most reproducible measures [44]. Ten set size and shape regions of interest (ROI) were placed manually in the same order on longitudinal elastograms, at a standardised depth of 0.5 cm, along the tendon length, starting proximally and working distally (See Figure).Appl. Sci. 2018, 8, x FOR PEER REVIEW 5 of 13 Image quality was closely monitored throughout examination, tissue compression was avoided. Quality maps were assessed to ensure images conformed to a high level of quality, specifically checking for a quality map all green in colour, which indicates that a correct amount of pressure was used. All elastograms were taken in a longitudinal plane with the foot in a relaxed foot position, as this has been demonstrated to provide the most reproducible measures [44]. Ten set size and shape regions of interest (ROI) were placed manually in the same order on longitudinal elastograms, at a standardised depth of 0.5 cm, along the tendon length, starting proximally and working distally (See Figure 1). Figure 1. Longitudinal shear wave elastogram of the right Achilles tendon taken from a 24‐year‐old

This study investigates factors affecting Achilles tendon stiffness using shear wave elastography.