and Key Terms: Abstract Despite the potential for biomechanical conditioning with prosthetic use, the soft tissues of residual limbs following lower -limb amputation are vulnerable to damage . Imaging studies revealing mor phological changes in the se soft tissue s have not distinguished between superficial an d intramuscular adipose distribution, despite the recognition that intramuscular fat levels indicate reduced tolerance to mechanical loading . Furthermore, it is unclear h ow these changes may alter tissue tone and stiffness. 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 reson ance imagin g in combination with tissue structural stiffness. The results revealed h igher 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 i ntramuscular 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 p atellar tendon site , which plays a key role in load transfer across the limb-prosthesis interface . The tissue changes following amputation can have relevance in the clinical understanding of prosthetic socket design variables and sof t tissue damage risk in this vulnerable group. Keywords: Transtibial amputation, magnetic resonance imaging, infiltrating adipose , remodelling, muscle atrophy Tissue composition and load response after amputation Introduction Following lower limb amputation, t he residual skin and soft tissues form a critical interface with the bespoke ‘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 [1]. The resulting t issue deformations may cause skin and soft tissue damage , with reported prevalence values between 36% and 66% [1]–[3]. Experimental and numerical models indicate that large deformation s over short periods of time represent the most important factor in the caus al pathway for Deep Tissue Injury (DTI), which initiat es in muscle tissues [4]–[12]. By contrast, superficial pressure ulcers (PUs) are generally caused by external pressures and shear forces . The tissue tolera nce to loading magnitude and duration varies between individuals [13], and is influenced by many intrinsic factors [14]. There has been relatively little research into skin damage in individuals with lower limb amputations , despite the specific risk factors and h igh prevalence in this group [15]. Indeed the r esidual limbs are exposed to challenging biomechanical conditions, impaired load tolerance due to comorbidities, considerable variab ility in anato my and surgical reconstruction, and the presence of scar tissue over vulnerable sites [16]. 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 [17]–[19]. The se characteristics change post -amputation due to oedema, muscle atrophy and tissue remodelling in biomechanical adaptation to prosthetic load bearing. The oedematous response to the trauma of am putation decreases gradually in the months following surgery [20]. Physiotherapy exercises are prescribed to promote range of motion in the residual lim b join ts, and reduce muscle atrophy and oedema [21]. Despite these interventions, residual muscle s atrophy due to denervation and disuse, with a subsequent inf iltration of adipose or fibrous tissue s [22], [23] . 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 938mmHg (0.5 to 125kPa) and 8 to 389mmHg (1 to 52kPa), respectively [24]–[26]. 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, w ith an increased epidermal thicknes s in transtibial residua compared to the contralateral limb, and higher microvascular function [27]. To date there is li mited evidence of how biomechanical loading affects the vulnerable residuum muscle and adipose tissues during the early rehabilitation phase . Volume imaging modalities have been used to observe res idual limb adaptation . These include Magnetic Resonance Imaging (MRI) to visualis e muscle morphology changes and differentiate between changes due to oedema and muscular atrophy [17], and computed tomography ( CT) to determine the proportion of muscle and fat mass in the residual limb compared to contralateral limbs [28]. MRI has been used to evaluate fatty infiltration in other tissues [29], [30] , but previous studies have not quantified adipose 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 [31]. Furthermore, it remains unclear how these changes may alter tissue tone and stiffness, which represent key biomechanical characteristics at the interface with the prosthetic socket . Indeed, these properties Tissue composition and load response after amputation have be en shown to change due to a range of factors relevant to the amputee population including ageing, stroke, exe rcise and post -exercise massage [32]–[35], as well as in the spinal cord injury population [36]. This study was designed to compare the morphology and biomechanical response of residual and intact limb tissues during representative prosthetic loading in individuals with and without transtibial amputation. This involved characterising the superficial adipose and a dipose infiltrating muscle tissue using MRI, in terms of the tissues’ composition and their deformation under mechanical loading in situ , 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 unilateral transtibial amputation . Inclusion criteria involved p articipants over 18 years o f age , in good healt h with n o active skin -related conditions at sites relevant to the study . Participants without amputation had additional exclusion criteria of ne urological and vascular pathologies. Local E thics Committee approval for th e test protocol was granted by the Un iversity of Southampton (ERGO IDs: 29696 and 41864) and participants provided informed consent in writing . Test Protocol Pressure was applied t o the right proximal calf of control participants without amputation, and both calves of participants with unila teral transtibial amputation using a n inflatable cuff (Ref 0124 Aneroid Sphygmomanometer, Bosch + Sohn GmbH, Germany) according to a previous publi cation by the authors [37]. To review briefly, a prosthetic liner ( 6mm ContexGel Liner, NMA21L200/XXL, RSL Steeper, UK) was posit ioned underneath the cuff to provide a representative material to interface with the skin. Three 50mm square sites were selected for measur ement on each limb representing load bearing regions of differing tissue composition , namely the patellar tendon, lateral calf, and posterior calf ( Figure 1A). Cylindrical polymer indenters of 17mm diameter and 15mm height were positioned undern eath the cuff at the three measurement sites , containing sunflower oil capsules to facilitate identification within the MR images (Figure 1B). Participants were scanned in the supine, feet-first position using a 3T MRI scanner (MAGNETOM Skyra, Siemens, Germany) , with their test -limb elevated and resting on foam supports. MR images were acquired using an 18 -channel body array coil placed on t op of the test limb, as well as the in -built s pine coil in the scanner cou ch underneath the limb. Images were ac quired at baseline and at a cuf f inflation pressure of 60mmHg (8kPa) to characterise direct tissue deformation and visualise morphology and tissue composition [37] (Figure 1C). Volumetric MRI data were acquired using a 3D T1 DIXON sequenc e [38] with an echo time (TE) of 6.15ms and a repetition time (TR) of 17.10ms , a 134mm field of view, an in-slice resolution of 0.6mm x 0.6mm and a corresponding slice thickness of 1.2mm . The acquisition time was 5 minutes 19 seconds. This sequence generates a set of 4 volumetric axial image datasets, each with a different contrast: in-phase, o pposed -phase, fat -only and fat -suppressed (water -only) images. Tissue composition and load response after amputation Figure 1 A: Measurement sites on the right lower limb, each of area 50 x 50mm ; 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 (m iddle) and MRI test set up prior to imaging (bottom), C: Timeline of the MRI test protocol . Figure 2: Image p rocessing steps applied to the axial MRI fat-only slice of the lower limb at the posterior calf measurement site, showin g 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 un inflated cuff baseline (solid line) and 8kPa inflated cuff (dashed line) conditions . The volume of both s uperficial - and muscle -infiltrating adipose tissue was quantified by processing the MR images in ImageJ 1.52p (Rasband, W. National Institute of Healt h, US) . Background noise was removed by subtracting a pixel intensity of 10 , Tissue composition and load response after amputation 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 (Figure 2). Gross compressive strain under each indenter was estimated by selecting single MR slices correspondi ng to the centre of the measurement sites, measuring the normal distance from the indenter surface to the nearest bony prominence and comparing these values at both unloaded baseline (0kPa) and inflated cuff test conditions (8kPa). Prior to imaging, interface pressure and soft tissue stiffness measurements were recorded for each participant 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 f oam supports. I ndenter -skin i nterface pressures were measured using a pneumatic pressure monitoring system (Mk II I, Talley Medical, Romsey, UK) with 28mm diameter measurement cells, which have a reported mean error of 12 ± 1% and a repeatability of ±0.53 m mHg [39]. The MyotonPro ( Myoton AS, Talinn, Estonia) was used to apply a 15ms, 0.4N mechanical impulse at each of the measurement sites in order to estimate the composite tissue stiffness . This device has been demonstrated to show reliable lower limb skeletal muscle structural stiffness measurements [40] which correlate with shear wave elastography [41]. Data Analysis Raw data from each of the measurement technique s were processed and analysed using MATLAB (Math Works, USA) and SPSS Statistics (IBM, USA). After testing for normality, MRI data are presented using non -parametric desc riptors (median, quartiles and range), w hereas i nterface pressure and tissue stiffness data are presented using parametric descriptors (mean and standard deviation ). Differences in tissue composition, deformation and strain between control, contralateral a nd residual limb groups were assessed for statistical significance using a Mann -Whitney -U test , and differences between structural stiffness were assessed using T -Tests. Relationships between percentage 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 were considered to be statistically significant at the 5% level (p<0.05). engrXiv Pre-Print Paper v0.1 , posted 16th April 2021 : Results Ten participants without am putation and 10 participants with unil ateral transtibial amputation were recruited for this study (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 ran ge of time since amputation , which ranged from 1 to 35 years . Table 1: Participant characteristics, reported as median (range). Controls Participan ts with Amputation Characteristic All (n=10) Male (n=6) Female (n=4) All (n=10) Male (n=8) Female (n=2) Age (yrs) 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/m2) 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 Circumferenc e (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 amput ation (yrs) - - - 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 1 0 Daily socket use (hrs) - - - 12.5 (6 -16) 11.5 (6 -616) 14.5 (14 -15) engrXiv Pre-Print Paper v0.1 , posted 16th April 2021 : Soft Tissue Composition Residual limbs were observed to have a smaller cross -sectional area and generally a less consistent ly round shape than intact limbs, although the residual limbs often revealed distorted shape artefacts resulting from the foam support (Figure 3). Figures in the Supplementary Data detail the percentage volumes of superficial adipose tissue, adipose infiltrating muscle and muscle tissue across the limb sections for all participants. Residual limbs displayed greater adipose tissue infiltrating muscle than intact limbs ( Figure 3), reaching significance (p<0.05) when compar ed to the residual and control limbs ( Figure 4). 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 the co ntrol (C) and residual (R) limbs for ten p articipants with transtibial amputation (right columns, #1A -#10A). Tissue composition and load response after amputation 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. Correla tion analysis was performed between the percentage volumes of adipose tissue , and three intrinsic factors, namely BMI, time since amputation and daily socket use (Figure 5, Table 2). A significant, positive correlation was observed between the levels of both adipose tissue types in the residual and contralateral limbs ( Figure 5 A&B). There was a negative correlation between adipose infiltrating muscle and estimated daily socket use in both contralateral and residual limbs, although this was only statistically significant for the former ( r=-0.88, p<0.0 1, Figure 5C). In contrast, no correlation was evident between the adipose infiltrating muscle values and the time since amputation (r= -0.06, p=0.88, Figure 5D). It was also interesting to note that the re was a positive trend between the adipose infiltrating muscle tissue of the control limb and the BMI in the non -amputated group , although th e correlation was not sta tistically significant (r=0.55) . With respec t to superficial adipose values, there were no significant correlations with any of the three intrinsic factors (Table 2). Tissue composition and load response after amputation Figure 5: Posit ive correlations were observed between residual limb and contralateral limb superficial adipose ( A) and infiltrating adipose ( B). Negative correlation was observed between p ercentage volume of infiltrating adipose tissue in contralateral limbs and estimate d daily socket use (C), though no correlation was seen between contralateral limb infiltrating adipose and time since amputation ( D). Number indicates participant ID. engrXiv Pre-Print Paper v0.1 , posted 16th April 2021 : Table 2: Correlation analysis for percentage volume of inf iltrating and superfic ial 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 represents significance at the 5% level . Correlation between Limb Correlation r significance P Percentage volume of infiltrating adipose and: BMI Control 0.55 0.10 Contralatera l 0.35 0.33 Residual 0.12 0.75 Time Since Amputation Contralatera l -0.06 0.88 Residual 0.45 0.19 Est. Daily Socket Use Contralatera l -0.88 <0.01 * Residual -0.38 0.28 Percentage volume of superficial adipose and: BMI Control 0.18 0.63 Contralatera l 0.08 0.83 Residual -0.19 0.60 Time Since Amputation Contralatera l 0.12 0.75 Residual 0.20 0.59 Est. Daily Socket Use Contralatera l 0.07 0.85 Residual -0.06 0.88 Table 3: Interface pressure at th ree 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 Mean (S.D.) Interface Pressure at Applied Cuff Pressure Study gro up Measurement Site 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) engrXiv Pre-Print Paper v0.1 , posted 16th April 2021 : Interface Pressure At a cuff inflation pressure of 60mmHg, the mean interface pressures ranged from 66 to 74mmHg, 70 to 75mmHg and 72 to 84mmHg 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 change s from baseline to a cuff pressure of 60mmHg (8kPa) , visualised from MR images ( Figures in the Supplementary Data ) were converted into two parameters, gross tissue deformation and strain. These data revealed that deformation was significantly higher (p<0.01) in control limbs than residual limbs at all three sites (Figure 6). Deformation was also higher in control limbs than the contralateral limbs , with statistical ly significant differences 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 site s (p<0.0 5). Strain results revealed similar trends to that of deformation . However, there were no significant differences betwe en groups at the posterior calf site , with all three sites observed to demonstrate similar strain magnitudes . The high deformation at the posterior calf site produced relatively low strains owing to its high soft tissue layer thickness. Few notable correla tions were revealed between either gross tissue deformation or compressive strain and percentage volume of superficial adipose or time since amputation (Supplementary Data Table S1). 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 Tissue composition and load response after amputation 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 ( Figure 7). The highest stiffness values (mean 739 ± 187N/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 f ew notable correlations were revealed between structural stiffness and percentage volume of superficial adipose or time since amputation (Supplementary Data Tabl e S2). Figure 7: Mean v alues 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. Tissue composition and load response after amputation Discussion This study was designed to investigate residual limb soft tissue composition and how loading affects tissue deformation . It presents, f or the first time , the combination of MRI data and structural stiffness measurements using a commercial device . Two cohorts were recruited, namely those with and without transtibial amputation who were imaged using MRI pr ior to and during the application of representative mechanical loads via a pressure cuff . The results revealed significant 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 show to demonstrate significantly incre ased stiffness in the residual limb compared 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 (Figure 3, Figure 4). Comparison between the control and amputee groups demonstrates how pathology resulted in a markedly increased variability in limb tissue composition and morpholog y. Residual limbs containe d approximately three times more infiltrating adipose tissue than control limbs of participants 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 lower limb muscle atrophy, and the other (#5A) had Type 1 diabetes which has been ass ociated with increased adipose infiltrating muscle [42]. These observations reflect the well -established changes in tissue composition associated with muscle atrophy post-amputation associated with denervation and disuse [17], [22], [23] . However, this study has proved novel in discriminating between super ficial and adipose infiltrating muscle tissues in residual limbs , thus providing insight in the potential for both to disease progression [29], [30], [43] and an enhanced risk for DTI [44]–[46]. Correlation analysis provided insight s into the relationship s between tissue composition in the contralateral and residual limbs and intrinsic factors associated with the individuals post-amputation (Table 2). Residual limb adipose was observed to correlate significantly with contralateral limb adipose for both superficial and infiltrating types ( Figure 5 top). However, a high percentage volume of superficial adipose tissue did not necess arily correspond with high infiltrating adipose indicating that various factors may be responsible for the infiltration . It is of note that s uperficial adipose did not correlate with BMI, time since amputation or estimated hours of prosthesis use. By contr ast, a significant negative correlatio n was revealed between infiltrating adipose in contralateral limbs and estimated daily prosthesis us age, which supports the suggestion that infiltrating adipose may represent a biomechanical adaptation to disuse atroph y. More active limbs present ed with more lean muscle mass , and th e lack of correlation for residual limbs may indicate the influence of other factors such as gait compensations where participants favour their intact limb [47]. Adipose tissues change in size and function in response to a number of factors including loading, exercise, temperature and nutrition, w ith hypertrophy observed under static tension [48], [49] . This may have influenced the structural stiffness values measured and is worthy of further explor ation. Tissue composition and load response after amputation The composition and status of soft tissues will affect how they respond to and tolerate mechanical loading. The pressure cuff was used to apply pres sure representative of PPAM aid use during rehabilitation [50]. Loading was applied through a 60mmHg cuff inflation, with some non -uniformity of inter face pressure s, with the patellar tendon with its relatively thin soft tissue coverage demonstrating the highest pressure s. Low, non - zero pressure was measured between the indenters and limb at baseline (0mmHg cu ff pressure) although these were within the reported error s of the pressure measurement system [39]. Using MRI to eval uate tissue deformation pre - and post -loading, and resulting gross compressive strain, revealed the l owest values at the residual limb patellar tendon site . This corresponded to the highest tissue stiffness measurements (Figure 7), and is the location at which many prosthetist s focus loading using specific socket designs (e.g. patella tendon bearing socket [51]), and could be attributed to local biomechanical adaptation in response to repetitive loading at this location . Residual limbs were al so generally smaller than contralateral limbs , resulting in higher compressive strains for equivalent deformations at residual limb sites (Figure 6). At the calf sites, the highest stiffness values were observed in the contralateral limbs, which could again indicate adaptation in response to compensatory gait patterns, prior to- or follow ing amputation [47]. During periods of loading application, the magnitude and duration of mechanical strain is considered to represent the most important factor in the causal pathway for damage of soft tissue [4]–[12]. The largest strains observed in this study were between 20 and 30%, applied over a 15 minute period. Th ese conditions represent a lower range than that observed in examining tissue damage in model systems [52] and in clinical situations . Limitations The small sample size and hetero geneity of the individuals with amputation limits the study’s generalisability . Nonetheless the study was designed to e valuate the heterogeneous nature of a cohort with lower limb amputations with a wide range of tissue responses, and thus could provide in sight into the factors that can affect tissue adaptation and load tolerance post -amputation. During the testing session s it was often difficult to support residual limbs in a consistent man ner, and participants with short residua l limbs and knee flexion contracture required support from below. These factors dictated the length of all the limbs which could be consistently i maged, at ≈60 mm. With respect to structural measurements , although limbs were kept in a consistent ly supported posit ion, relaxation of the muscles was not achieved objectively . Indeed, contracted muscles could have present ed with higher stiffness and elastic ity values [32]. Furthermore, the MyotonPRO measurement system is mainly designed for measuring the stiffness of superficial tissues, so any adapta tion of deeper muscular tissues may be less apparent in participants with higher superficial adipose tissue [34]. 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 demons trated less strain under external pressure than intact limbs. Understanding the changes in tissue composition can provide clinicians with new insights into how residual limb tissue adapt to representative prosthetic loading and could offer strategies to pr event skin and sub - dermal damage, w hich is common in this population. Tissue composition and load response after amputation The evidence of superficial tissue biomechanical adaptation in response to increased mechanical loads, notably at the patellar tendon, extend s evidence from case stud ies reporting incre ased soft tissue tolerance to ische mia under loading at this critical location which is exploited by prosthetists for residuum -prosthesis load transfer [37]. The results also show indicators of muscle atrophy, presenting as elevated adipose infiltrating muscle tissue in residual limbs. This is a well -established marker of pressure ulcer risk in individuals with spinal cord injury and accumulates over time [53], 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 [54]. This study demonstrates how residual limb soft tissues can change post -amputation in a small population with a r ange of amputation causes, and longitudinal studies could help to determine more predictive variables that affect tissue composition and tolerance to loading post -amputation. This insig ht will help to further understanding of how the soft tissues adapt to tolerate prosthetic loading, to help reduce the risk of tissue da mage during prosthetic use. 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 Acad emy 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. Disclosures: Each aut hor 1) made an important contribution to the conception and design, acquisition of data, or analysis and interpretation of data in the study; 2) drafte d or revised the manuscript critically for intellectual content; and 3) approved the final version of th e submitted manuscript. None of the authors has any conflict of interest to declare. Raw data will be made openly available from the University of Southampton re pository at upon peer reviewed pu blication. Tissue composition and load response after amputation