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Original Article
Changes in Tissue Composition and Load Response After Transtibial
Amputation Indicate Biomechanical Adaptation
J. L. BRAMLEY,
1
P. R. WORSLEY,
2
D. L. BADER,
2
C. EVERITT,
3
A. DAREKAR,
3
L. KING,
3
and A. S. DICKINSON
1
1
School of Engineering, Faculty of Engineering and Physical Sciences, University of Southampton, Mailpoint M7, University
Road, Southampton SO17 1BJ, UK;
2
School of Health Sciences, Faculty of Environmental and Life Sciences, University of
Southampton, Southampton, UK; and
3
University Hospital Southampton NHS Foundation Trust, Southampton, UK
(Received 20 April 2021; accepted 20 August 2021)
Associate Editor Stefan M Duma oversaw the review of the article.
Abstract—Despite the potential for biomechanical condi-
tioning with prosthetic use, the soft tissues of residual limbs
following lower-limb amputation are vulnerable to damage.
Imaging studies revealing morphological changes in these
soft tissues have not distinguished between superficial and
intramuscular adipose distribution, despite the recognition
that intramuscular fat levels indicate reduced tolerance to
mechanical loading. Furthermore, it is unclear how these
changes may alter tissue tone and stiffness, which are key
features in prosthetic socket design. This study was designed
to compare the morphology and biomechanical response of
limb tissues to mechanical loading in individuals with and
without transtibial amputation, using magnetic resonance
imaging in combination with tissue structural stiffness. The
results revealed higher adipose infiltrating muscle in residual
limbs than in intact limbs (residual: median 2.5% (range 0.2–
8.9%); contralateral: 1.7% (0.1–5.1%); control: 0.9% (0.4–
1.3%)), indicating muscle atrophy and adaptation post-
amputation. The intramuscular adipose content correlated
negatively with daily socket use, although there was no
association with time post-amputation. Residual limbs were
significantly stiffer than intact limbs at the patellar tendon
site, which plays a key role in load transfer across the limb-
prosthesis interface. The tissue changes following amputation
have relevance in the clinical understanding of prosthetic
socket design variables and soft tissue damage risk in this
vulnerable group.
Keywords—Transtibial amputation, Magnetic resonance
imaging, Infiltrating adipose, Remodelling, Muscle atrophy.
INTRODUCTION
Following lower limb amputation, the residual skin
and soft tissues form a critical interface with the be-
spoke ‘socket’ component of a prosthetic limb. These
tissues are vulnerable to damage, particularly during
the early rehabilitation phase, prior to adequate
biomechanical conditioning arising from mechanical
loading.
35
The resulting tissue deformations may cause
skin and soft tissue damage, with reported prevalence
between 36 and 66%.
12,35,36
Experimental and numerical models indicate that
large deformations over short periods of time represent
the most important factor in the causal pathway for
Deep Tissue Injury (DTI), which initiates in muscle
tissues.
8,15,31–33,39,40,53,54
By contrast, superficial pres-
sure ulcers (PUs) are generally caused by external
pressures and shear forces. The tissue tolerance to
loading magnitude and duration varies between indi-
viduals,
19
and is influenced by many intrinsic factors.
10
There has been relatively little research into skin
damage in individuals with lower limb amputations,
despite the specific risk factors and high prevalence in
this group.
18
Indeed the residual limbs are exposed to
challenging biomechanical conditions, impaired load
tolerance due to comorbidities, considerable variability
in anatomy and surgical reconstruction, and the pres-
ence of scar tissue over vulnerable sites.
41
Tissue loading at the residuum-prosthesis interface
is influenced by the socket design, with the prosthetist
considering both the morphology of the local tissues
and their load tolerance.
28,42,46
These characteristics
change post-amputation due to oedema, muscle atro-
Address correspondence to A. S. Dickinson, School of Engi-
neering, Faculty of Engineering and Physical Sciences, University of
Southampton, Mailpoint M7, University Road, Southampton SO17
1BJ, UK. Electronic mail: alex.dickinson@soton.ac.uk
Annals of Biomedical Engineering (2021)
https://doi.org/10.1007/s10439-021-02858-0
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2021 The Author(s)
phy and tissue remodelling in biomechanical adapta-
tion to prosthetic load bearing. The oedematous
response to the trauma of amputation decreases
gradually in the months following surgery.
29
Physio-
therapy exercises are prescribed to promote range of
motion in the residual limb joints, and reduce muscle
atrophy and oedema.
43
Despite these interventions,
residual muscles atrophy due to denervation and dis-
use, with a subsequent infiltration of adipose or fibrous
tissues.
51,58
In addition, the superficial tissues adapt in
response to increased repetitive loading, with pressures
and shear stresses at the limb-prosthesis interface
ranging widely, from 4 to 938 mmHg (0.5 to 125 kPa)
and 8 to 389 mmHg (1 to 52 kPa), respectively.
56,62,63
The skin and subdermal tissues may thicken, and callus
is formed to adapt their vascular function. Such
changes have been reported using optical coherence
tomography, with an increased epidermal thickness in
transtibial residua compared to the contralateral limb,
and higher microvascular function.
55
To date there is limited evidence of how biomechan-
ical loading affects the vulnerable residuum muscle and
adipose tissues during early rehabilitation. Volume
imaging modalities have been used to observe residual
limb adaptation. These include Magnetic Resonance
Imaging (MRI) to visualise muscle morphology changes
and differentiate between changes due to oedema and
muscular atrophy,
28
and computed tomography (CT) to
determine the proportion of muscle and fat mass in the
residual limb compared to contralateral limbs.
50
MRI
has been used to evaluate fatty infiltration in other tis-
sues,
20,21
but previous studies have not quantified adi-
pose tissue or distinguished between superficial and
intramuscular distribution in amputees. This is despite
the recognition that intramuscular fat levels represent an
indicator of the risk for severe pressure ulcers, as
observed in the gluteal region of individuals with spinal
cord injury prone to DTI.
59
Furthermore, it remains
unclear how these changes may alter tissue tone and
stiffness, which represent key biomechanical character-
istics at the interface with the prosthetic socket, likely to
influence residuum-socket load transfer patterns and in
turn the soft tissue damage risk. Indeed, these properties
have been shown to change due to a range of factors
relevant to the amputee population including ageing,
stroke, exercise and post-exercise massage,
2,9,14,25
as
well as in the spinal cord injury population.
48
Changes in both soft tissue morphology and
mechanical response to loading following amputation
have clinical relevance to the understanding of effective
prosthetic socket designs. Research has identified the
potential application of prosthesis-limb interface sen-
sors and/or numerical predictions to support the pre-
vention of soft tissue damage.
11,45
However, there is still
limited research which assesses the composition and
structural features which are critical in determining tis-
sue tolerance to mechanical loading. Accordingly, this
study was designed to characterise residual limb soft
tissue morphology, composition and mechanical
response to representative prosthetic loading. To assess
changes arising from amputation and prosthetic limb
use, the study compared residual and intact limbs of
individuals with unilateral transtibial amputation, and
intact controls. This involved characterising the pro-
portions of both superficial adipose and adipose infil-
trating muscle tissue using MRI, their deformation and
gross strain under in situ mechanical loading with
indenters, and measuring the structural stiffness of the
combined soft tissue layers.
METHODOLOGY
Study Design and Recruitment
An observational comparison study was conducted
with participants recruited from the local community
population, including those with and without unilat-
eral transtibial amputation. Inclusion criteria involved
participants over 18 years of age, in good health with
no active skin-related conditions at sites relevant to the
study. Participants without amputation had additional
exclusion criteria of neurological and vascular
pathologies. Local Ethics Committee approval for the
test protocol was granted by the University of
Southampton (ERGO IDs: 29696 and 41864) and
participants provided informed consent in writing.
Test Protocol
Pressure was applied to the right proximal calf of
control participants without amputation, and both
calves of participants with unilateral transtibial
amputation using an inflatable cuff (Ref 0124 Aneroid
Sphygmomanometer, Bosch + Sohn GmbH, Ger-
many) according to a previous publication.
6
A pros-
thetic liner (6mm ContexGel Liner, NMA21L200/
XXL, RSL Steeper, UK) was positioned underneath
the cuff to provide a representative material to inter-
face with the skin. Three 50 mm square sites were se-
lected for measurement on each limb representing load
bearing regions of differing tissue composition, namely
the patellar tendon, lateral calf, and posterior calf
(Fig. 1a). Cylindrical polymer indenters of 17 mm
diameter and 15 mm height were positioned under-
neath the cuff at the three measurement sites, con-
taining sunflower oil capsules to facilitate identification
within the MR images (Fig. 1b).
Participants were scanned supine, feet-first using a
3T MRI scanner (MAGNETOM Skyra, Siemens,
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BRAMLEY ET AL
Germany), with their test-limb elevated and resting on
foam supports. MR images were acquired using an 18-
channel body array coil placed on top of the test limb,
as well as the spine coil in the scanner couch under-
neath the limb. Images were acquired at baseline and
at a cuff inflation pressure of 60 mmHg (8 kPa) to
characterise direct tissue deformation and visualise
morphology and tissue composition
6
(Fig. 1c). Volu-
metric MRI data were acquired using a 3D T1 DIXON
sequence
34
with an echo time (TE) of 6.15 ms and a
repetition time (TR) of 17.10 ms, a 134 mm field of
view, an in-slice resolution of 0.6 mm 90.6 mm and a
corresponding slice thickness of 1.2 mm. The acquisi-
tion time was 5 min 19 s. This sequence generates a set
of 4 volumetric axial image datasets, each with a dif-
ferent contrast: in-phase, opposed-phase, fat-only and
fat-suppressed (water-only) images.
The volume of both superficial- and muscle-infil-
trating adipose tissue was quantified by processing the
MR images in ImageJ 1.52p (Rasband, W. National
Institute of Health, US). Background noise was
removed by subtracting a pixel intensity of 10, and
binary images created with the Auto Threshold Stack
tool. Masks were created representing the whole soft
tissue area, tibia, fibula and muscle, and Boolean
operations were applied to generate superficial- and
muscle-infiltrating adipose tissue masks whose areas
were calculated (Fig. 2). Gross deformation and com-
pressive strain under each indenter was estimated by
selecting single MR slices corresponding to the centre
of the measurement sites, measuring the normal dis-
tance from the indenter surface to the nearest bony
prominence and comparing these values at both un-
loaded baseline (0 kPa) and inflated cuff test condi-
tions (8 kPa). Deformation was calculated as
d¼d0d8
ðÞand strain as e¼d0d8
ðÞ=d0.
Prior to imaging, interface pressure and soft tissue
stiffness measurements were recorded for each partici-
pant in a seated position on a commercial hospital bed
with adjustable backrest (Enterprise, Arjo Huntleigh,
Bedforshire, UK), with their test-limb elevated and
resting on foam supports. Indenter-skin interface pres-
sures were measured using a pneumatic pressure moni-
toring system (Mk III, Talley Medical, Romsey, UK)
with 28 mm diameter measurement cells, which have a
reported mean error of 12 ±1% and a repeatability of
FIGURE 1. (a) Measurement sites on the right lower limb, each of area 50 350 mm; (b) 3D printed indenter positioned at each
measurement site via adhesive fixation ring, enclosed by a pressure cuff with the limb in the supported test position (middle) and
MRI test set up prior to imaging (bottom), (c) Timeline of the MRI test protocol.
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±0.53 mmHg.
4
The cuff and liner were then doffed, and
the MyotonPro (Myoton AS, Talinn, Estonia) device
was used to apply a 15 ms, 0.4 N mechanical impulse at
each of the measurement sites in order to estimate the
superficial tissue structural stiffness. This device esti-
mates dynamic stiffness using S¼amax mprobe =Dl,
where amax is the impulse probe’s maximum accelera-
tion, mprobe is its mass and Dlis its displacement at the
point of maximum acceleration. The MyotonPRO has
been demonstrated to show reliable lower limb skeletal
muscle structural stiffness measurements
3
which corre-
late with shear wave elastography.
23
Data Analysis
Raw data from each of the measurement techniques
were processed and analysed using MATLAB (Math-
Works, USA) and SPSS Statistics (IBM, USA). After
testing for normality, MRI data were analysed using non-
parametric descriptors (median, quartiles and range),
whereas interface pressure and tissue stiffness data were
analysed using parametric descriptors (mean and stan-
dard deviation). Differences in tissue composition,
deformation and strain between control, contralateral
and residual limb groups (non-parametric) were assessed
for statistical significance using a Mann-Whitney-U test,
and differences between structural stiffness (parametric)
were assessed using T-Tests. Relationships between per-
centage of infiltrating and superficial adipose tissue, the
time since amputation, socket use, structural stiffness and
deformation were evaluated using scatter plots and
Spearman’s correlation. Differences wereconsidered to be
statistically significant at the 5% level (p<0.05).
RESULTS
Ten participants without amputation and 10 par-
ticipants with unilateral transtibial amputation were
recruited (Table 1). The control group was younger
than the group with amputation and presented with a
lower median weight and BMI. There was a variety of
causes of amputation in the latter group, and a wide
range of time since amputation, from 1 to 35 years.
Soft Tissue Composition
Residual limbs were observed to have a smaller
cross-sectional area and a less consistently round shape
than intact limbs, although the residual limbs often
revealed distorted shape artefacts resulting from the
foam support (Fig. 3). Figures in the Supplementary
Data detail the percentage volumes of superficial adi-
pose tissue, adipose infiltrating muscle and muscle
tissue across the limb sections for all participants.
Residual limbs displayed greater adipose tissue infil-
trating muscle than intact limbs (Fig. 3), reaching
significance (p<0.05) when compared to the residual
and control limbs (Fig. 4).
Correlation analysis was performed between the per-
centage volumes of adipose tissue, and three intrinsic
factors, namely BMI, time since amputation and daily
socket use (Fig. 5, Table 2). A significant positive corre-
lation was observed between the levels of both adipose
tissue types in the residual and contralateral limbs
(Fig. 5a and b). There was a negative correlation between
adipose infiltrating muscleand estimated daily socket use
in both contralateral and residual limbs, although this
was only statistically significant for the former (r=
20.87, p<0.01, Fig. 5c). In contrast, no correlation was
evident between the adipose infiltrating muscle values
and the time since amputation (r=20.05, p=0.88,
Fig. 5d). It was also interesting to note that there was a
positive trend between the adipose infiltrating muscle
tissue and the BMI in the control, non-amputated group,
although the correlation was not statistically significant
(r=0.46, p=0.18). With respect to superficial adi-
pose values, there were no significant correlations with
any of the three intrinsic factors (Table 2).
FIGURE 2. Image processing steps applied to the axial MRI
fat-only slice of the lower limb at the posterior calf
measurement site, showing (a) original image, and (b) after
binarization and masking. (c) superficial adipose mask
(yellow) and muscle-infiltrating adipose mask (red)
superimposed over the corresponding opposed-phase
image at same slice, and (d) superimposed outlines of limb
under uninflated cuff baseline (solid line) and 8 kPa inflated
cuff (dashed line) conditions. Example measures are shown
for calculating the displacement and gross strain arising from
cuff inflation between the posterior calf indenter to the
nearest bony prominence, uninflated (d
0
) and at 8 kPa
inflation (d
8
).
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Interface Pressure
At a cuff inflation pressure of 60 mmHg, the mean
interface pressures ranged from 66 to 74 mmHg, 70 to
75 mmHg and 72 to 84 mmHg in control, residual and
contralateral limbs, respectively (Table 3). The highest
pressures and variability generally occurred at the
patellar tendon, which represented the measurement
site with the lowest soft tissue coverage over the
underlying bony anatomy.
Soft Tissue Deformation and Strain
The soft tissue shape changes from baseline to a cuff
pressure of 60 mmHg (8 kPa), visualised from MR
images (Figures in the Supplementary Data) were
converted into two parameters, gross tissue deforma-
tion and strain. These data revealed that deformation
was significantly higher (p<0.01) in control limbs
than residual limbs at all three sites (Fig. 6). Defor-
mation was also higher in control limbs than the
contralateral limbs, with statistically significant dif-
ferences at the patellar tendon (p<0.01) and the
posterior calf (p<0.05). Within the individuals with
amputation, deformation was significantly different
between their residual and contralateral limbs at the
lateral (p<0.01) and posterior calf sites (p<0.05).
Strain results revealed similar trends to that of defor-
mation. However, there were no significant differences
between groups at the posterior calf site, with all three
sites observed to demonstrate similar strain magni-
tudes. The high deformation at the posterior calf site
produced relatively low strains owing to its high soft
tissue layer thickness. Few notable correlations were
revealed between either gross tissue deformation or
compressive strain and percentage volume of superfi-
cial adipose or time since amputation (Supplementary
Data Table S1).
Soft Tissue Stiffness
Structural stiffness values were highest at the
patellar tendon site, which has the least soft tissue
coverage, adjacent to a bony prominence (Fig. 7). The
highest stiffness values (mean 740 ±190 N/m) were
observed in the residual limb group at the patella site,
which were significantly higher than those estimated
from the control group (p<0.05). No differences were
observed between groups in the lateral calf or posterior
calf, and few notable correlations were revealed
between structural stiffness and percentage volume of
superficial adipose or time since amputation (Supple-
mentary Data Table S2).
TABLE 1. Participant characteristics, reported as median (range).
Characteristic
Controls Participants with Amputation
All (n=10) Male (n=6) Female (n=4)
All (n=10)
Male (n=8) Female (n=2)
Age (years) 28 (23–36) 26 (23–34) 28 (27–36) 41 (25–62) 45 (25–62) 38 (30–46)
Height (m) 1.78 (1.60–1.92) 1.82 (1.75–1.92) 1.66 (1.60–1.76) 1.76 (1.63–1.88) 1.79 (1.65–1.88) 1.65 (1.63–1.68)
Mass (kg) 66 (56–90) 78 (66–90) 58 (56–64) 79 (51–127) 79 (73–127) 76 (51–100)
BMI (kg/m
2
) 22.1 (18.3–29.4) 23.6 (18.3–29.4) 21.5 (18.4–23.5) 27.3 (19.2–37.5) 27.3 (20.7–37.5) 27.4 (19.2–35.6)
Max Calf Circumference (mm)
Residual – – – 290 (250–450) 300 (260–450) 270 (250–290)
Contralateral 360 (320–410) 390 (350–410) 360 (320–360) 390 (340–530) 390 (350–530) 390 (340–440)
Residual limb length (mm) – – – 150 (100–300) 150 (100–300) 210 (140–270)
Time since amputation (years) – – – 7.5 (1–35) 5.0 (1–35) 18.5 (8–29)
Amputation cause
CRPD – – – 2 1 1
Congenital – – – 2 1 1
Trauma – – – 5 5 0
PVD –––1 10
Daily socket use (h) – – – 12.5 (6–16) 11.5 (6–616) 14.5 (14-15)
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FIGURE 3. Exemplar transverse MRI slices in the calf with superficial adipose (yellow) and adipose infiltrating muscle (red) tissue
overlays. Images represent the right control limb of ten participants without amputation (left columns #1-10), and both the control
(C) and residual (R) limbs for ten participants with transtibial amputation (right columns, #1A-#10A).
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DISCUSSION
This study was designed to investigate residual limb
soft tissue composition and how loading affects tissue
deformation. It presents, for the first time, the com-
bination of MRI data and structural stiffness mea-
surements using a commercial device. Two cohorts
were recruited, with and without transtibial amputa-
tion, and they were imaged using MRI prior to and
during the application of representative mechanical
loads via a pressure cuff. The results revealed signifi-
cant changes to soft tissue composition in the residual
limb, with a higher proportion of muscle-infiltrating
adipose tissue, which was associated with the amount
of daily socket use. A critical load bearing site, the
patellar tendon, was also shown to demonstrate sig-
nificantly increased stiffness in the residual limb com-
pared to intact control limbs.
Measurements and Analysis
MRI data enabled clear visualisation of the soft
tissues with the specific distinction of bone, muscle and
adipose tissues (Figs. 3,4). Comparison between the
control and amputee groups demonstrates how
pathology resulted in a markedly increased variability
in limb tissue composition and morphology. Residual
limbs contained approximately three times more infil-
trating adipose tissue than control limbs of partici-
pants without amputation. Adipose infiltrating muscle
was particularly apparent in more established residual
limbs (#4A, #6A and #9A), and in two people with
shorter time since amputation (#1A and #5A). One of
these participants (#1A) used a wheelchair for mobility
for several years prior to amputation which may have
caused additional atrophy in the lower limb muscles,
and the other (#5A) had Type 1 diabetes which has
been associated with increased adipose infiltrating
muscle.
5
These observations reflect the well-established
changes in tissue composition associated with muscle
atrophy post-amputation associated with denervation
and disuse.
28,51,58
However, this study has proved no-
vel in discriminating between superficial and adipose
infiltrating muscle tissues in residual limbs, thus pro-
viding insight into the potential for both disease pro-
gression
1,20,21
and an enhanced risk for DTI.
37,52,57
Correlation analysis provided insights into the
relationships between tissue composition in the con-
tralateral and residual limbs and intrinsic factors
associated with the individuals post-amputation (Ta-
ble 2). Residual limb adipose was observed to correlate
significantly with contralateral limb adipose for both
superficial and infiltrating types (Fig. 5top). However,
a high percentage volume of superficial adipose tissue
did not necessarily correspond with high infiltrating
adipose indicating that various factors may be
responsible for the infiltration. It is of note that
superficial adipose did not correlate with BMI, time
since amputation or estimated hours of prosthesis use.
By contrast, a significant negative correlation was re-
vealed between infiltrating adipose in contralateral
limbs and estimated daily prosthesis usage, which
supports the suggestion that infiltrating adipose may
represent a biomechanical adaptation, namely muscu-
lar atrophy due to disuse. More active limbs presented
with more lean muscle mass, and the lack of correla-
tion for residual limbs may indicate the influence of
other factors such as gait compensations where par-
ticipants favour their intact limb.
30
Adipose tissues
change in size and function in response to a number of
factors including loading, exercise, temperature and
nutrition, with hypertrophy observed under static
tension.
27,61
This may have influenced the structural
stiffness values measured and is worthy of further
exploration.
The composition and status of soft tissues will affect
how they respond to and tolerate mechanical loading.
The pressure cuff was used to apply pressure repre-
sentative of PPAM aid use during rehabilitation.
47
Loading was applied through a 60 mmHg cuff infla-
tion, with some non-uniformity of interface pressures.
The patellar tendon, with its relatively thin soft tissue
coverage, demonstrated the highest pressures. Low,
non-zero pressure was measured between the indenters
FIGURE 4. Median, interquartile range (IQR) and range in
percentage of tissue constituents of the overall limb, in a
60mm segment distal from the tibial plateau. + indicates
outliers; * indicates significance at p£0.05; ** indicates
significance at p£0.01.
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and limb at baseline (0mmHg cuff pressure) although
these were within the reported errors of the pressure
measurement system.
4
Using MRI to evaluate tissue
deformation pre- and post-loading, and resulting gross
compressive strain, revealed the lowest values at the
residual limb patellar tendon site. This corresponded
to the highest tissue stiffness measurements (Fig. 7),
and is the location at which many prosthetists focus
loading (i.e. using patella tendon bearing socket de-
signs
44
), and could be attributed to local biomechani-
cal adaptation in response to repetitive loading at this
location. Residual limbs were also generally smaller
than contralateral limbs, resulting in higher compres-
sive strains for equivalent deformations at residual
limb sites (Fig. 6). At the calf sites, the highest stiffness
values were observed in the contralateral limbs, which
could again indicate adaptation in response to com-
pensatory gait patterns, prior to- or following ampu-
tation.
30
Though the MyotonPRO assesses the
superficial tissues only, it produced structural stiffness
results consistent with gross mechanical indentation in
the transtibial amputated limb’s anterior aspect (ap-
proximately 400–500 N/m).
49
The recorded structural
stiffness values were in the same range as reported in
other myotonometry studies of skeletal muscles in the
lower limb. These studies reported mean values rang-
FIGURE 5. Positive correlations were observed between residual limb and contralateral limb superficial adipose (a) and
infiltrating adipose (b). Negative correlation was observed between percentage volume of infiltrating adipose tissue in contralateral
limbs and estimated daily socket use (c), though no correlation was seen between contralateral limb infiltrating adipose and time
since amputation (d). Number indicates participant ID.
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ing from approximately 275–450 N/m for the gas-
trocnemius,
13,22,24,26
and 350–400 N/m for the tibialis
anterior,
24,26
which correspond with the present
study’s posterior calf and lateral calf sites, respectively.
Literature studies reporting MyotonPro assessment of
the patella tendon have primarily recruited athletes, for
whom the stiffness values might be elevated. However,
neglecting elite athlete studies and considering control
groups, mean values have been reported ranging from
780 to 900 N/m,
7,60
consistent with the present study’s
results.
During periods of loading application, the magni-
tude and duration of mechanical strain is considered to
represent the most important factor in the causal
pathway for damage of soft tissue.
8,15,31–33,39,40,53,54
The largest strains observed in this study were between
20 and 30%, applied over a 15 minute period. These
conditions represent a lower range than that observed
in examining tissue damage in model systems
17
and in
clinical situations.
Limitations
The study’s generalisability is limited by its small
sample size and heterogeneity of the individuals with
amputation. The group has a substantial range in time
since amputation (1–35 years), although this variety is
largely representative of the community accessing
prosthetics services in our Southern UK area.
Accepting this limitation, the study was designed to
evaluate the heterogeneous nature of a cohort with
lower limb amputations with a wide range of individ-
ual demographics, reasons for amputation and asso-
ciated tissue morphologies and responses. Accordingly,
the study used correlation analysis and prioritised
comparison between the residual and contralateral
limbs. Thus, the present study has provided insight
TABLE 2. Correlation analysis for selected intrinsic factors (BMI, time since amputation and estimated daily socket use) and the
percentage volume of infiltrating and superficial adipose from the tibial plateau to 60mm distally, in the right control limbs of ten
participants without amputation and the contralateral and residual limbs of ten participants with unilateral transtibial amputation.
Bold text and ** represents significance at the 1% level.
Correlation between Limb Correlation rSignificance p
Percentage volume of infiltrating adipose and: BMI Control 0.46 0.18
Contralateral 0.35 0.33
Residual 0.12 0.75
Time Since Amputation Contralateral 20.05 0.88
Residual 0.45 0.19
Est. Daily Socket Use Contralateral 20.87 <0.001*
Residual 20.34 0.34
Percentage volume of superficial adipose and: BMI Control 0.12 0.75
Contralateral 0.08 0.83
Residual 20.19 0.60
Time Since Amputation Contralateral 0.12 0.75
Residual 0.20 0.59
Est. Daily Socket Use Contralateral 0.09 0.82
Residual 20.04 0.92
TABLE 3. Interface pressure at three measurement sites, at baseline and a cuff pressure of 60mmHg, applied to the right control
limb of 10 participants without amputation and both residual and contralateral limbs of 10 participants with unilateral transtibial
amputation
Measurement site
Mean (S.D.) interface pressure at applied cuff pressure
0 mmHg (Baseline) 60 mmHg
Control limbs Patella Tendon 13.1 (7.4) 73.7 (8.2)
Lateral Calf 4.7 (2.9) 72.6 (5.5)
Posterior Calf 0.5 (1.3) 66.2 (5.0)
Contralateral limbs Patella Tendon 17.7 (15.2) 83.6 (34.3)
Lateral Calf 7.7 (11.1) 75.1 (6.7)
Posterior Calf 2.8 (6.3) 72.0 (11.7)
Residual limbs Patella Tendon 13.6 (13.4) 73.1 (21.6)
Lateral Calf 13.9 (12.4) 75.1 (11.4)
Posterior Calf 11.9 (13.4) 69.9 (12.7)
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Tissue Composition and Load Response After Amputation
into the factors that can affect tissue adaptation and
load tolerance post-amputation, with further studies
needed to explore how these differences could affect
their tolerance to prosthetic loading. The two cohorts
were not matched by their demographics (age, weight
and BMI in particular) and this may substantially af-
fect the comparisons observed between the people with
amputations and the intact controls. However, the
intact control data are interesting to assess the soft
tissue characteristics of a young, healthy group of
people without amputations, whose lower limb soft
tissues might represent the start point of prosthetic
rehabilitation in cases of amputation due to trauma or
neoplasia. The sample size is a recurring issue for lower
limb prosthetics studies with a small population of
eligible participants, so the presented data do not cover
the full variance of physiological conditions experi-
enced in the wider population of people who use
prosthetics. The present findings provide focus for
further study on more homogeneous groups of par-
ticular interest or concern.
During the testing sessions it was often difficult to
support residual limbs in a consistent manner, and
participants with short residual limbs and knee flexion
contracture required support from below. These fac-
tors dictated the length of all the limbs which could be
consistently imaged, at 60 mm. With respect to
structural measurements, although limbs were kept in
a consistently supported position, relaxation of the
muscles was not achieved objectively. Indeed, con-
tracted muscles could have presented with higher
stiffness and elasticity values.
14
Furthermore, the
MyotonPRO measurement system is mainly designed
for measuring the stiffness of superficial tissues, so any
adaptation of deeper muscular tissues may be less
apparent in participants with higher superficial adipose
tissue.
9
Considering tissue strain measurement, the
reported values are equivalent to a measure of engi-
neering strain. Therefore, these estimates are not di-
rectly comparable to the principal and shear Green-
Lagrange strain components most commonly em-
ployed in imaging and finite element analysis (FEA)
estimation of tissue damage risk thresholds.
8,31,32,54
The paired, aligned MR images collected in this study
would enable Green-Lagrange strain prediction using
FEA but this was outside the present scope.
Summary and Clinical Implications
A higher proportion of muscle-infiltrating adipose
was observed in residual limbs compared to intact
limbs, indicating muscle atrophy post-amputation.
Residual limbs were also stiffer at the patellar tendon
site and demonstrated less strain under external pres-
sure than intact limbs. Understanding the changes in
tissue composition can provide clinicians with new
insights into how residual limb tissues adapt to repre-
sentative prosthetic loading and could offer strategies
FIGURE 6. Median, interquartile range (IQR) and range of
lower limb soft tissue deformation under 60mmHg pressure
cuff loading for all participant groups. * indicates significance
at p£0.05 and ** indicates significance at p£0.01.
FIGURE 7. Mean values of tissue structural stiffness at three
measurement sites on the right control limb of eight
participants without amputation and both contralateral and
residual limbs of ten participants with unilateral transtibial
amputation. * indicates p£0.05.
BIOMEDICAL
ENGINEERING
SOCIETY
BRAMLEY ET AL
to prevent skin and sub-dermal damage, which is
common in this population.
The evidence of superficial tissue biomechanical
adaptation in response to increased mechanical loads,
notably at the patellar tendon, extends evidence from case
studies reporting increased soft tissue tolerance to ische-
mia under loading at this critical location which is
exploited by prosthetists for residuum-prosthesis load
transfer.
6
The results also show indicators of muscle
atrophy, presenting as elevated adipose infiltrating mus-
cle tissue in residual limbs. This is a well-established
marker of pressure ulcer risk in individuals with spinal
cord injury, and accumulates over time,
16
so this evidence
contributes to our understanding of these individuals’
risk of deep tissue injury, as well as metabolic syndrome,
cardiovascular disease and related mortality.
38
This study demonstrates how residual limb soft
tissues can change post-amputation in a small popu-
lation with a range of amputation causes, and longi-
tudinal studies could help to determine more predictive
variables that affect tissue composition and tolerance
to loading post-amputation. This insight will help to
further understanding of how the soft tissues adapt to
tolerate prosthetic loading, to help reduce the risk of
tissue damage during prosthetic use.
AUTHOR CONTRIBUTIONS
JLB: Conceived and designed research; Per-
formed experiments; Analyzed data; Interpreted results
of experiments; Prepared figures; Drafted manuscript;
Edited and revised manuscript; Approved final version
of manuscript. ASD: Conceived and designed research;
Analyzed data; Interpreted results of experiments;
Prepared figures; Drafted manuscript; Edited and
revised manuscript; Approved final version of manu-
script. PRW, DLB: Conceived and designed research;
Analyzed data; Interpreted results of experiments;
Edited and revised manuscript; Approved final version
of manuscript. CE, AD, LK: Conceived and designed
research; Performed experiments; Approved final ver-
sion of manuscript.
CONFLICT OF INTEREST
Each author (1) made an important contribution to the
conception and design, acquisition of data, or analysis
and interpretation of data in the study; (2) drafted or
revised the manuscript critically for intellectual con-
tent; and (3) approved the final version of the sub-
mitted manuscript. None of the authors has any
conflict of interest to declare. Raw data are openly
available from the University of Southampton reposi-
tory at https://doi.org/10.5258/SOTON/D1941.
OPEN ACCESS
This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits
use, sharing, adaptation, distribution and reproduction
in any medium or format, as long as you give appro-
priate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and
indicate if changes were made. The images or other
third party material in this article are included in the
article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is
not included in the article’s Creative Commons licence
and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need
to obtain permission directly from the copyright
holder. To view a copy of this licence, visit http://crea
tivecommons.org/licenses/by/4.0/.
ACKNOWLEDGMENTS
The authors would like to thank the following for
their financial support: JLB: the University of
Southampton’s Institute for Life Sciences (IfLS), and
EPSRC Doctoral Training Program (ref EP/N509747/
1). PRW, DLB: the EPSRC-NIHR ‘‘Medical Device
and Vulnerable Skin Network’’ (ref EP/N02723X/1),
ASD: the Royal Academy of Engineering, UK, (ref
RF/130). Ethics Committee approval for this protocol
was granted by the University of Southampton
(ERGO ID: 29696 and 41864). We would like to thank
all of the individuals who participated in this study.
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