Penetration Time Profiles for Two Class 3B Lasers in In Situ Human Achilles at Rest and Stretched

Abstract

Background and objective: The majority of studies investigating penetration of laser light are performed in vitro on skin flaps, with measures of immediate penetration depth and energy loss. The aim of this study was to investigate the penetration time profiles for two different lasers used in low-level laser therapy, during 150 sec of exposure both in stretched and relaxed human Achilles in situ. Materials and methods: Thirty-four Achilles tendons from 17 healthy volunteers were irradiated by an 810 nm, 200 mW, continuous- and a 904 nm, 60 mW, super-pulsed laser. Irradiation was performed with the Achilles tendons in relaxed and stretched condition. The energy penetrating skin–skin was measured every 30 sec using an optical power meter. Results: The 810 nm laser penetration ability did not differ significantly in relaxed and stretched condition with 0.17% [standard error of the mean (SEM) 0.02] of mean output power (MOP) and 0.02% (SEM 0.004) of MOP, respectively. The 904 nm laser demonstrated a statistical significant (p < 0.05) and almost linear increasing penetration ability both in relaxed and stretched Achilles from 0.25% (SEM 0.03) to 0.38% (SEM 0.04) of MOP and from 0.05% (SEM 0.01) to 0.13% (SEM 0.01) of MOP, respectively. The penetrated ability differed between lasers and tissue conditions at all measure points (p < 0.05). Conclusions: The 904 nm laser penetrates relatively more energy than the 810 nm laser in in situ human Achilles. Moreover, penetration from the super-pulsed 904 nm laser increased during exposure time, whereas penetration from the 810 nm laser was constant. In addition, stretching the Achilles causes a higher energy attenuation by the tissue.

Background

Low-level laser therapy (LLLT) is a form of photobiomodulation performed with class 3B lasers with red or infrared wavelengths.^1

–5 Empirical evidence from clinical trials suggests that lasers with infrared wavelengths 780–860 nm operating in continuous mode/wave (CW) need to deliver nearly double doses of energy, compared to super-pulsed wave (SPW) 904 nm lasers, to generate similar clinical effects in tendinopathies.^3,6 However, the guidelines for treating tendinopathies with LLLT originates from clinical trials and lack basic research verification. Thus, all wavelengths covered above act to positively modulate inflammation and tissue structures in tendinopathy when following recommended dosage guidelines,^3,7
–9 but the question is open why the apparent differences in energy occur.¹⁰

It is well known that longer wavelengths penetrate deeper than shorter.^2,11,12 To generate clinical effects in tendinopathies, the light energy first has to overcome the skin barrier. Dermal melanin, elastin, and collagen content attenuate laser light penetration through mechanisms of absorption and scattering even during the first millimeters of epidermis.^13

–16 These optical properties do not merely depend on dermal tissue content but also on dermal structure and tissue organization, exemplified by the bundle networks of elastin and collagen.¹² The differences in skin penetration ability between 780–860 and 900 nm theoretically is <20%,¹⁷ and thus inadequate to completely explain the abovementioned differences in energy needs between CW 780–860 nm and SPW 904 nm lasers.

However, Joensen et al.¹⁰ investigated the influence of laser types on penetration abilities in rat skin irradiating the skin flaps with an SPW 904 nm and a CW 810 nm laser during 150 sec. Both lasers had stable mean output power (MOP) during 150 sec of exposure, measured directly into an optical power meter (OPM). They found that the 904 nm laser almost linearly increased its skin energy penetration ability through rat skin from 38.7% to 58% of its MOP during the entire exposure time. The 810 nm laser presented a constant energy penetration around 20.4% of its MOP during exposure. According to the authors, these penetration time profiles may partly explain World Association for Laser Therapy's LLLT differentiated dosage recommendations for 904 and 810 nm lasers, respectively, as the 904 nm laser increases its penetration over time and thus potentially achieves the optimal dosage deliverance faster than the 810 nm laser.

The majority of studies investigating penetration of laser light are being performed in vitro or in vivo on human or animal skin flaps, eventually combined with other tissues, with measures of immediate penetration depth and energy loss.^{13
–15,18
–20} Thus, the CW and SPW laser penetration abilities over time have not yet been tested on humans (in situ).These properties may have important clinical implications as proposed by Joensen et al.¹⁰

In clinical practice, the tissue treated is usually in rested position during LLLT. However, positive results from LLLT have also been reported on stretched tendons.²¹ Further, several structural changes are known to occur in collagen formation during tendon elongation.^22

–28 It is an open question whether energy absorption in biological tissue differs between relaxed and stretched tendon positions. The same is true for the penetration abilities of 3B lasers²⁹ operating in different modes over time in humans. The aim of this study is to investigate the penetration time profiles for a CW 810 nm and an SPW 904 nm laser in both stretched and rested human Achilles in situ during 150 sec of exposure.

Materials and Methods

Samples

Seventeen healthy volunteers (mean = 27.1 years, standard deviation = 3.9) from Bergen University College were included as convenience sample. The sample consisted of 16 females and 1 male, with light skin color and no history of Achilles tendon pathology the last 3 months. Both right and left Achilles tendons of the subjects were subjected to irradiation (n = 34 tendons).

Instruments

Two commercially available therapeutic class 3B lasers were used for laser irradiation. (1) The 904 nm wavelength laser (Irradia, Sweden) set to super-pulsed mode: Peak power 20 W, super-pulsed width 100 ns (10⁻⁹ sec) with a pulse train frequency of 6 kHz; 60 mW MOP, spot size 0.0364 cm², and power density 1.67 W/cm² (manufacturer's specification); and (2) 810 nm wavelength laser (Thor-DD, United Kingdom) set to continuous mode with MOP of 200 mW, spot size 0.0314 cm², and power density of 6.37 W/cm² (manufacturer's specification). To measure the laser MOP, an optical power meter system (Thorlabs Instruments, United Kingdom) consisting of a PM100 Display unit with sample rate of 6 Hz and accuracy of ±1%, and an S121B silicon sensor were used. Manufacturer's specifications reported the S121B sensor's diameter Θ=9.5mm and an input optical power range of 500 nW–500 mW (accuracy ±5%).

Real-time ultrasonography (RTUS) images were captured with a Logiq-S8 (GE Healthcare, Minneapolis), consisting of a 19" LCD Screen, operating in B-mode with High Definition Speckle Reduction Imaging, CrossXBeam resolution, and Coded Harmonic imaging. The linear matrix transducer (ML6-15-D) was tuned to frequency, 12 MHz.

Time was measured with a stopwatch displaying minutes, seconds, and hundredths.

Experimental protocols

The study was based on a single-factor experimental design with repeated measurements. We performed two separate experiments.

Initial procedure

All subjects' right Achilles and left Achilles were pen marked with a thin pen line on the dorsal side of the tendon ∼2 cm proximal from the calcaneal bone. The ankle joints were in resting position.

Experiment 1: Laser procedure with Achilles tendon in rested position

Initial procedures

Subjects were prone lying with the ankles in neutral position. RTUS images were performed in the longitudinal and transversal plane in the Achilles area. These images covered the 2 cm marker point. Images were saved for later tissue thickness score.

The experiment

The subjects were positioned in a comfortable side-lying position with the actual ankle in resting position on a wedge (Fig. 1). The laser, stabilized in an adjustable tripod stand, was set in sufficient skin contact on the lateral side at the level of the 2 cm marker point. The OPM was tuned to match the operating laser's wavelength and thereby placed and held in skin contact with a stable pressure on the medial side, facing the laser beam output direction. Researcher 1 controlling the laser and OPM positions signaled to researcher 2 at the start of irradiation. Researcher 2 synchronized the timing of irradiation with a stopwatch and further noted the measured penetration values displayed on the OPM every 30 sec during 150 sec of total irradiation. The OPM display was hidden from researcher 1 and thus leaving him blinded to power measurements. The right Achilles and left Achilles were irradiated in a sequential manner with both lasers.

FIG. 1.

Setup experiment 1 (Achilles at rest): researcher 1 controls the laser being blinded for the OPM values and timing controlled by researcher 2. Achilles is rested on a wedge. OPM, optical power meter.

Experiment 2: Laser procedure with Achilles tendon in stretched position

Initial procedures

Subjects were standing on a step case (Fig. 2). RTUS images of both Achilles once again were performed longitudinally and transversally at the level of the 2 cm marker point with the foot placed at 20° of dorsiflexion on a wedge, without hyperextending the knee. Images were saved for later tissue thickness scoring.

FIG. 2.

Setup experiment 2 (Achilles at stretched). Researcher 1 controls the laser being blinded for the OPM values and timing controlled by researcher 2. The ankle is dorsiflexed on a wedge sloping 20°. Cornered: Laser and OPM setups: The tripod-supported laser and the handheld OPM were set opposite to each other in the frontal plane of Achilles in both experiments (with tissues at rest and at stretched), set at the level of the skin mark made 2 cm proximal to the calcaneal bone (the picture is from experiment 2, Achilles at stretched).

The experiment

The subjects sustained with the ankle wedged to 20° of dorsiflexion without hyperextension in the respective knee (Fig. 2). The experimental procedure was identical to that of experiment 1 with the laser in the tripod stand placed in skin contact on the lateral side and the OPM placed in skin contact on the medial side of Achilles (Fig. 2).

Laser MOP control procedures

The laser's MOP was measured directly into the OPM immediately after every irradiation procedure, with the laser still switched on.

Postexperiment procedures

After subjects had gone through the laser experiments, tissue thickness was measured on saved RTUS images. To measure tissue thickness as reliably as possible between the laser source and the OPM sensor, transversal images were selected. All measurements were performed at a fixed point. Prior tests indicated that the 4 mm level anterior from the dorsal skin surface was most likely the location where the laser beam passed on its way to the OPM sensor. In longitudinal images, tendon thickness was measured 2.5 cm proximal to the calcaneus.

Main outcome measures

The residual amount of optical energy was measured in mW, which is penetrating through in situ human Achilles from lateral to medial side (skin to skin) at every 30 sec during 150 sec of irradiation from an 810 nm CW laser and a super-pulsed 904 nm LLLT device.

Statistical analysis

Statistical descriptive analysis, Student's t-tests, 95% p-value statistical significant differences, and graphs were made using Microsoft Office Excel 2016. Statistical analysis of the penetration time profile was made by generalized estimating equations (GEE) in Statistical Package for the Social Sciences (SPSS, v.19).

Ethical approval

LLLT is considered to be nonhazardous when applied to healthy tissues.^30
–32 Due to the established harmlessness of LLLT and only participation of adult healthy individuals in this study, no special ethical approval was necessary. All subjects agreed to an informed consent before participation.

Results

Real-time ultrasonography

In the direction of irradiation (mediolateral) the Achilles thickness (tendon and skin to skin) did not change significantly from a rested to stretched position (p > 0.05). However, in the anterior–posterior plane (longitudinal images), tendon thickness was reduced significantly (p < 0.05) from rested to stretched position (Table 1).

Table 1.

Ultrasonography Measured Thickness of the Achilles Tendon and Surrounding Tissues (Skin–Skin)

RTUS measurement direction			Transversal
Value	Tissue condition	Long. tendon	Tendon	Skin–skin
Avg. (SEM)	Rested	0.50 (0.01)	1.20 (0.03)	2.17 (0.09)
Avg. (SEM)	Stretched	0.45 (0.01)	1.20 (0.03)	2.27 (0.09)
RTUS t-tests	p Value	0.000	0.993	0.128

Longitudinal measure at 2.5 cm Prox. from calcaneus. Trans. measure is 0.4 cm profund from the skin surface (n = 34).

RTUS, real-time ultrasonography; SEM, standard error of the mean.

Laser's MOP

The MOP direct (no tissue between) into OPM of each laser did not change significantly during the experiments or between measurements (95% p > 0.05) (Table 2).

Table 2.

Penetrated Laser Energy Achilles Skin–Skin In Situ: Measurements of Penetrated Energy

		Irradiation time (sec)
	n	1	30	60	90	120	150	MOP in mW (SEM)
810 nm
mW (SEM)
At rest	34	0.333 (0.038)	0.333 (0.038)	0.330 (0.038)	0.332 (0.039)	0.330 (0.038)	0.330 (0.038)	199.8 (0.5)
At stretched	34	0.047 (0.009)	0.044 (0.008)	0.042 (0.008)	0.042 (0.007)	0.041 (0.007)	0.041 (0.007)	199.0 (0.6)
p Value (t-test)		0.000	0.000	0.000	0.000	0.000	0.000
%MOP (SEM)
At rest	34	0.17 (0.02)	0.17 (0.02)	0.17 (0.02)	0.17 (0.02)	0.17 (0.02)	0.17 (0.02)
At stretched	34	0.02 (0.004)	0.02 (0.004)	0.02 (0.004)	0.02 (0.004)	0.02 (0.004)	0.02 (0.004)
p Value (t-test)	34	0.000	0.000	0.000	0.000	0.000	0.000	0.296
Energy loss mW/cm (SEM)
At rest	34	96.627 (3.486)	96.627 (3.48)	96.628 (3.486)	96.627 (3.486)	96.628 (3.486)	96.628 (3.486)
At stretched	34	92.194 (3.481)	92.196 (3.482)	92.197 (3.482)	92.197 (3.482)	92.197 (3.482)	92.198 (3.482)
p Value (t-test)		0.072	0.072	0.072	0.072	0.072	0.072
904 nm
mW (SEM)
At rest	34	0.143 (0.017)	0.156 (0.018)	0.176 (0.021)	0.192 (0.021)	0.204 (0.023)	0.218 (0.024)	58.1 (0.3)
At stretched	34	0.031 (0.005)	0.038 (0.005)	0.049 (0.006)	0.058 (0.007)	0.068 (0.007)	0.079 (0.008)	58.7 (0.3)
p Value (t-test)		0.000	0.000	0.000	0.000	0.000	0.000
%MOP (SEM)
At rest	34	0.25 (0.03)	0.27 (0.03)	0.31 (0.04)	0.33 (0.04)	0.34 (0.04)	0.38 (0.04)
At stretched	34	0.05 (0.01)	0.06 (0.01)	0.08 (0.01)	0.10 (0.01)	0.12 (0.01)	0.13 (0.01)
p Value (t-test)	34	0.000	0.000	0.000	0.000	0.000	0.000	0.130
Energy loss mW/cm (SEM)
At rest	34	28.052 (4.811)	28.046 (4.810)	28.036 (4.808)	28.029 (4.807)	28.023 (4.806)	28.017 (4.805)
At stretched	34	27.160 (1.022)	27.158 (1.022)	27.152 (1.021)	27.148 (1.021)	27.143 (1.021)	27.138 (1.021)
p Value (t-test)		0.25	0.25	0.26	0.26	0.26	0.26

t-tests %MOP between lasers: At rest: All time points: p < 0.05. At stretched: All time points: p < 0.05.

MOP, mean output power.

Experiment 1: Tissue condition Achilles at rest

The 810 nm laser showed a stable and statistically nonsignificant (95% p > 0.05) change in penetration ability with around 0.17% of MOP [standard error of the mean (SEM) 0.02] during 150 sec of irradiation (Table 2 and Fig. 3). There was a small change in energy penetration per 30-sec time interval of −0.0003 mW/cm (SEM = 0.0001). The penetration time profile, GEE estimated slope, of −0.001 mW per 30 sec was not a statistically significant change over time [p = 0.42; 95% confidence interval (CI): −0.004 to 0.002]. The 904 nm laser showed a statistically significant and almost linear increase in penetration ability, with residual energy rising from 0.25% of MOP to 0.38% of MOP (SEM 0.03 and 0.04, respectively) during 150 sec of irradiation (95% p < 0.001). Thus, the 904 nm laser's penetration ability was significantly higher than the 810 nm laser during the entire experiment (95% p < 0.05) (Table 2 and Fig. 3). The average increase in energy penetration per 30-sec interval for the 904 nm was 0.007 mW/cm of tissue (SEM = 0.0001). The penetration time profile, GEE estimated slope, of 0.030 mW per 30 sec was a statistically significant change over time (p < 0.05; 95% CI: 0.021–0.038).

FIG. 3.

Measured OPM values skin–skin as %MOP from two 3B lasers in the Achilles area over 150 sec in two tissue length conditions (rest and stretched). MOP, mean output power.

Experiment 2: Tissue condition Achilles stretched

The 810 nm laser presented a stable and statistically nonsignificant (95% p > 0.05) change in penetration ability, corresponding to around 0.020% of MOP (SEM 0.004) during 150 sec of irradiation (Table 2 and Fig. 3). The average change in energy penetration per 30-sec time interval was −0.001 mW/cm of tissue (SEM = 0.0001). The penetration time profile, GEE estimated slope, of −0.003 mW per 30 sec was a statistically significant change over time (p < 0.05; 95% CI: −0.005 to −0.001). The penetration ability of the 904 nm laser increased significantly and almost linearly with residual energy rising from 0.05% to 0.13% MOP (SEM 0.01 for both) during 150 sec of irradiation (95% p < 0.001). The average increase in energy penetration per 30-sec interval was 0.004 mW/cm of tissue (SEM = 0.0004). The penetration time profile, GEE estimated slope, of 0.020 mW per 30 sec was a statistically significant change over time (p < 0.05; 95% CI: 0.015–0.024). In addition, the 904 nm penetrated with a statistically significant higher proportion of its MOP compared to the 810 nm during the entire experiment (95% p < 0.05) (Table 2 and Fig. 3).

Discussion

In this study on LLLT penetration in in situ human tissue, we investigated the penetration time profiles from two commercial class 3B lasers. Healthy human Achilles, at rest and in stretched position, was irradiated for 150 sec.

We found that the 904 nm and the 810 nm lasers distinctly differ in their respective penetration time profiles during 150 sec of irradiation. In both tissue conditions, the 904 nm laser presented a statistically significant higher percent of MOP (%MOP) penetrating the tissues compared to the 810 nm laser at all measured time points and further almost linearly increased its penetration ability over time. Thus, proportionally, more energy is then available for photobiomodulation in any deeper structure during an irradiation session with the 904 nm laser. In contrast, energy penetration from the 810 nm laser appeared unchanged over time in the same experiments, reflecting a stable and constant penetration ability of this device.

This study is the first study to investigate the influence of laser parameters in laser energy penetration abilities in in situ human tissue. The results presented correlate well with the findings in an in vitro study by Joensen et al.¹⁰ on rat skin, which shared the discovery of a relatively higher and an almost linear increasing %MOP penetrating from the 904 nm laser compared to the relatively stable energy penetration presented by the 810 nm laser over time. In contrast, Enwemeka¹⁸ found a nonsignificant difference in %MOP penetrating rabbit skin + tendon from a continuous and pulsed He–Ne 632.8 nm laser, and an SPW 904 nm laser operating in both 292 and 2336 pps. However, distinctly different laser output powers were used, as the SPW laser MOPs were 6.5 and 0.8 mW versus MOPs at 60 and 200 mW in our study.

The difference in penetration abilities between the wavelengths 800 and 900 nm in the skin has previously been calculated to ∼10%¹² and ∼15%¹⁷ in theoretical models. Our two lasers shared a relatively similar probe spot size and presented stable MOPs during both experiments (Table 2). The current initial energy penetrations expressed as %MOP from the 904 nm laser nonetheless were initially 1.5 and 2.26 times higher than the parallel values from the 810 nm laser in experiment 1 and 2, respectively. For each of the time points at 30 sec and above, these differences in penetration abilities increased and exceeded what can reasonably be explained by wavelength alone. On the contrary, our residual energy values are notably very small due to a substantial energy attenuation in the tissues, and caution should be taken when interpreting these differences.

Mathematically, the abovementioned relationships between the first measured time point %MOP penetrating the tissues may to some degree relate to the methods of measurements. The laser beams needed to penetrate the skin twice to become sensed by the OPM—each skin layer absorbing and scattering the light due to melanin content, and the structures of collagen and elastin fibers.¹² In rat skin, penetration of one skin layer at the first measurement time point allowed 20% of the applied MOP to penetrate from the 810 nm laser and 38% of MOP for the 904 nm laser.¹⁰ Thus, adding another skin layer, residual energy would then become further reduced to the same extent in the second skin layer for both lasers. In the presence of similar patterns in human skin, the differences in penetrated energy between the 810 and 904 nm lasers therefore arise when doubling the number of skin layers.

Next, we speculate if the demonstrated superior penetration abilities of the 904 nm laser are further explained by either the photobleaching effect³³ or cellular destructions,³⁴ which propose the existence of protein rearrangements and formations of micropores, respectively, when irradiating with strong pulses. Moreover, alterations in lipid formations have been demonstrated after high-power laser irradiations.³⁵ Thus, the 904 nm laser light may penetrate better over time due to structural changes in the skin or underlying tissues during exposure from the 904 nm laser. However, we speculate around the relative impact of pulse frequency and peak power to provoke either of the abovementioned alterations in cellular structures using the 904 nm SPW laser.

Based on the presented penetration time profiles, we confirm that the 904 nm laser may need to deliver a smaller dose than the 810 nm lasers to achieve similar therapeutically efficient dosages in tissue beneath the skin. One study³⁶ compared the inflammatory modulation in rat knees when irradiating with CW and SPW 905 nm laser light (different peak powers) in similar irradiation durations. An inflammatory upregulation was seen from the SPW mode. The authors themselves hypothesized a photothermal effect from the SPW 905 nm laser. However, in the presupposition of a higher and increasing penetration ability over time from the SPW 904 nm laser, we propose that an irradiation overdose may also occur during the SPW laser exposure.

Traditionally, tendons treated with LLLT are relatively superficially located.³⁷ However, the demonstrated 904 nm laser penetration time profile may suggest the super-pulsed laser potentials for treating tissues, where a deeper penetration is needed. The 904 nm super-pulsed lasers have previously demonstrated better penetration in muscle tissues than lasers with shorter wavelength operating in the continuous mode,¹⁸ and long exposure time and high dosages from super-pulsed lasers have been successfully used to decrease pain and increase function in deeper conditions of the neck and lower back.^38
–40 However, our penetration time profiles cannot explain these treatment effects, but underpin the need for further double-blinded studies to investigate the super-pulsed and continuous operating lasers' ability to evoke clinical effects in deeper structures.

Second, we found significantly more residual energy penetrating the tissue in a relaxed manner compared to the stretched position. Previous penetration studies have examined tissue propagations in the rested position only, but dorsiflexion has successfully been used in the treatment of Achilles tendinopathies.⁴¹ The 20° of ankle dorsiflexion mainly involves tendinous elongation rather than dermal structural formation changes due to the tendon's proportional higher collagen content.^42
–44 The detected longitudinal reductions in tendon thickness in experiment 2 are small and relatively lesser than the extent of energy attenuation. Whether this increased energy loss has a clinical implication remains to be investigated.

An interesting question arises when one may ask what clinical relevance or consequences these demonstrated optical differences might have. In addition, in the perspective of a dose–response window in LLLT,^3,4,6 are there any benefits or disadvantages from faster energy delivery? However, penetration time profiles found in our study may suggest that irradiation time is an interesting and possibly double-sided parameter in LLLT studies.

Further research should focus on the extent and relevance of these penetration properties on specific tissue pathology. In addition, identifying internal relationships between laser parameters (peak power, wavelength, pulse frequency) for optimal treatment should be addressed in future research.

Also, tendon stretching yields some optical but unknown possible consequences for LLLT treatment. Further investigation should compare clinical tissue reactions from like-a-like dosages applied to various tissue conditions.

On the contrary, we suggest caution as the current results originate from a relatively homogenous sample consisting mainly of healthy young-adult light-skinned females. Tissue structure is less affected by gender,⁴⁵ but is shown to be influenced by age,^46

–49 disease,^{42,50

–54} and inactivity.⁵⁵ Also, light penetration is affected by pigmentation^11,12 and skin color.^10,56
–58 Thus, we should be careful in generalizing our results.

Conclusions and Summary

Our study demonstrates that the SPW 904 nm laser and the CW 810 nm laser yield distinctly different penetration time profiles during 150 sec of exposure in in situ healthy human Achilles. First, more energy from the SPW 904 nm laser penetrates Achilles than from the CW 810 nm laser. Second, the penetration of the SPW 904 nm laser increases significantly during the irradiation, whereas the CW 810 nm laser exhibits a stable penetration throughout an irradiation session. It is, however, important to remember that our findings do not address the clinical effectiveness of these two laser types in tendinopathy treatment, but merely that dosage must be differently titrated for them.

Irradiation time emerges as an important treatment parameter in LLLT. In addition, we have shown that proportionally less laser energy penetrates in tendon tissue in a stretched position than a rested position.

Clinical implications

The current study and its presented penetration time profiles intensify the need to differentiate the effective therapeutic dosages from 904 and 810 nm lasers in treatment protocols. However, all irradiated tissues in the Achilles area are sufficiently irradiated by both lasers, highlighting that all structures here could be sufficiently photobiomodulated. The 904 nm laser may be able to target deeper body structures than Achilles.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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