Study Design.

The study was conducted at the Massachusetts Institute of Technology (MIT) Biomechatronics Group in Cambridge, MA, between March 2017 and March 2020 under approval of NCT03374319 (https://www.clinicaltrials.gov/) and MIT’s Committee on the Use of Humans as Experimental Subjects (protocol 1801183130). Twenty-two individuals with unilateral transtibial amputations were consented. Fifteen subjects had undergone a unilateral below-knee amputation incorporating AMIs (AB cohort) at the Brigham and Women’s Hospital. Seven subjects with traditional below-knee amputation served as controls (TB cohort). All subjects had undergone standard physical therapy directed toward achieving a normal gait pattern.

Participants.

Participants were eligible if they were between 18 and 65 y of age, had a fully healed residuum, and were active users of a standard prosthesis with an ability for ambulation with variable cadence. Exclusion criteria included cardiopulmonary instability manifest as coronary artery disease and/or chronic obstructive pulmonary disease, extensive peripheral neuropathy, and extensive microvascular compromise.

The study design allowed for the control group to represent a range of below-knee amputation (BKA) techniques, together representing the “standard amputation.” As such, subjects had undergone standard amputations by vascular and orthopedic surgeons, the traditional services performing amputations. One TB subject had undergone targeted muscle reinnervation (38), and another had undergone an osteomyoplastic “Ertl” amputation (39, 40), techniques not specifically aimed at restoring musculotendinous proprioceptive feedback. Since our outcome measures were largely targeted on aspects influenced by musculotendinous proprioception, we grouped these subjects in the TB category.

Randomization and Blinding.

Participant allocation was nonrandomized, and the study was not blinded as the AB cohort was drawn from a pool of patients who had previously undergone the AMI BKA procedure at the Brigham and Women’s Hospital (under NCT03374319) by Matthew Carty. Willingness to enroll in complementary functional outcomes testing at MIT was included as an eligibility criterion for enrollment in the Brigham and Women’s Hospital trial. Each subject in the TB cohort was prospectively matched to a subject within the AMI group to the degree possible based on age and time since amputation.

Interventions.

Participants in the AB cohort had received two surgically constructed AMIs in their residuum during their amputation according to the method described in ref. 12 (Fig. 1A). Briefly, AMIs were constructed for the ankle and subtalar joints; the tibialis anterior (TA) and lateral gastrocnemius (LG) were coapted through a synovial tunnel anchored on the medial flat of the tibia to form the ankle AMI. The peroneus longus (PL) and tibialis posterior (TP) were utilized similarly for the subtalar joint. The synovial canals as well as embedded tendons were harvested from the lateral or medial malleoli in the discarded ankle. Two tantalum bead markers (Halifax Biomedical Inc.; Halifax Bead Set, 1 mm) were inserted into each muscle and one in each synovial tunnel to enable noninvasive monitoring of the muscle dynamics postoperatively. RPNIs were created for any transected sensory, cutaneous, and motor nerves encountered during surgery.

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Fig. 1.

(A) Illustration of the AMI constructs in the residuum of a BKA. The TA and LG form the ankle AMI through coaptation to the tendon in the tarsal (synovial) tunnel anchored on the tibia. The TP and PL form the subtalar AMI and pass through a more distal tarsal tunnel. These AMIs are positioned superficially to enable strong and independent EMG control signals for a myoelectric prosthesis. RPNIs are created for transected cutaneous, sensory, and motor nerves for neuroma prophylaxis. Positioning of these elements is representative and not to scale. (B) Phantom sensation and pain scores are listed for each subject. Phantom sensation for each part of the leg is represented colorimetrically (dark blue, very vivid; light blue, less perceptible). Phantom sensations in the AB cohort (n = 15) are significantly greater than those in the TB cohort (n = 7) (P < 0.05, Student’s t test). Pain scores are considerably lower in the AB cohort compared to the TB cohort (n = 7, P = 0.08, Student’s t test).

Each participant within the TB group received a traditional BKA without surgically reconstructed AMIs.

Outcomes.

Testing in the AB cohort was categorized into “short term” (6 to 12 mo postoperatively) and “long term” (12+ mo) time frames to capture changes that occurred as neuromuscular constructs underwent remodeling. All AB subjects were tested once within the short-term frame and six AB subjects underwent additional testing in the long-term frame. Two subjects (AB 30 and TB 22) were unable to complete the EMG- and ultrasound-based testing due to logistical challenges.

Pain Score.

The Patient-Reported Outcomes Measurement Information System (PROMIS) Sleep Disturbance 6a tool and Pain Interference 6a survey (41) were administered to all AB subjects at the following time points: preoperatively and 3, 6, 9, 12, and 24 mo postoperatively. During their testing session at the Biomechatronics laboratory, both TB and AB subjects were administered the same survey orally.

Phantom Limb Sensation.

Using open-ended questions, subjects were asked to describe their phantom limb percepts during walking, as well as during rest with the prosthesis worn and with the prosthesis not worn. Subjects were then specifically asked to relate the level of perception of their lateral and medial malleoli, heel pad, arch of the foot, balls of the footpad, and toes. The more thorough and tangible the phantom limb felt to subjects, the greater the phantom sensation scored. Responses were independently graded on a 1 to 3 scale and compared between cohorts using a Student’s t test, as the data satisfied requirements for normality and variance in each cohort.

Characterization of Muscle Activation Using EMG.

Bony anatomical landmarks and target muscles were identified by palpation and their superficial projections were marked using a skin marker and reflective balls (SI Appendix, Fig. S1 A–C). Surface EMG electrodes were placed on the affected and unaffected limbs (SI Appendix, Fig. S1 D and E), focusing on the muscle regions. Initially, the EMG data were collected using eight wireless bipolar electrodes (Trigno; Delsys) overlying the LG, TA, TP, and PL muscles sampled at 1,200 Hz. Starting in June 2019 we utilized up to 128 H124SG Ag/AgCl electrodes (Covidien), sampled at 2,048 Hz (Twente Medical Systems International [TMSi] Refa136; Type Refa_Ext-128e4b4a; REF code 95-0121-6446-1). Transparent plastic strips, with electrodes positioned at ∼2.5-cm intervals, aided in the placement and alignment of electrodes in a grid-like fashion (SI Appendix, Fig. S1 D and E). On the unaffected limb, electrodes were positioned over the native anatomical locations of the muscles incorporated in the AMI. Raw EMG was high-pass filtered at 70 Hz, full-wave rectified, and integrated using a 100-ms moving average window to obtain the muscle activation (12). The optimal electrode placement for each muscle was selected using a semiautomated process, thresholding for the highest signal-to-noise ratios during composite movements.

Synchronous Measurement of Intended Phantom Movements.

Subjects were asked to recline in a supine position with their intact limb positioned on a trapezoidal foam riser pillow to allow free movement of the ankle while minimizing motion of the electrodes and wiring (SI Appendix, Fig. S2). A 2 degrees-of-freedom wireless goniometer (Bio-Metrics Ltd.; WS200 DataLITE 2 Goniometer system with W110-Ankle and W150-Knee, and DataLITE Management Software 51025-00) was secured to the posterior aspect of the unaffected leg (SI Appendix, Fig. S2A) and used to synchronously measure ankle–foot kinematics for the unaffected limb. For all tasks, subjects were asked to mirror phantom movements onto the contralateral foot. Time-synchronized EMG and goniometry data were simultaneously recorded for all tasks to provide a one-to-one temporospatial indicator of the efferent signaling and intended joint movement of the phantom limb.

Study of Muscle Coupling and Excursion, Fascicle Strain, and AMI Construct Morphology.

Muscle dynamics, tissue remodeling, scarring, and tantalum bead movement were assessed using a portable high-definition ultrasound scanner at 60 fps (LS128; Telemed) during volitional activation of the AMI muscles (SI Appendix, Fig. S3). First, subjects were instructed to plantarflex (PF) and dorsiflex (DF) the phantom ankle joint while recording ultrasound from the tibialis anterior. Then, ultrasound of the tibialis posterior was recorded during inversion (IN) and eversion (EV) of the phantom subtalar joint. EMG and ultrasound measurements were analyzed to characterize muscle coupling. Imaging of the synovial tunnels was performed to quantify movement of the AMI construct through the synovial tunnel and surveil for dehiscence or scarring. Ultrasonographic planes capturing tantalum bead markers or their artifacts were prioritized to allow for quantitative measurements of fascicle strain. In cases where the beads were not visualized in AB subjects and for all TB subjects, fascicle lengths were identified, tracked, and measured using the UltraTrack v2 MATLAB (R_2018a) package (42). In control subjects, the most closely related and palpable muscle mass associated with each movement was used as a proxy if the original target muscle was unavailable (12).

ROM Percepts.

Subjects were asked to demonstrate and mirror the ROM possible of the affected or phantom joints on their unaffected limb, pre- and postoperatively at least three times. The angles of PF and DF were measured using the Biometrics 2-axis goniometer attached to the ankle region and averaged across trials. Data were processed through custom scripts in MATLAB (Mathworks) to identify the maximum amplitude for each movement and normalized as a percentage of the amplitude of the contralateral limb.

Motor Control Performance Tasks.

Subjects were then asked to perform PF, DF, IN, and EV of the ankle and subtalar joints, respectively, for 20 repetitions each at three different speeds. Subjects were then asked to rotate the foot and ankle in clockwise and counterclockwise circles. Commands were presented through an audio recording to ensure consistency across trials and subjects. Prior to these trials, subjects were educated on the isolation of each movement and were encouraged to focus on moving the joint to a given position, rather than activating the muscles. Tasks were performed without visual feedback to force subjects to use proprioception to close the loop on their internal efferent–afferent neuromuscular physiology.

A positional differentiation task was created to determine the ability of subjects to produce phantom movements with precision in position and muscle activation level. Subjects were instructed to move their limb to 0, 25, 50, 75, and 100% of their full ROM for PF and DF. Subjects were instructed to move the subtalar joint to 50 and 100% of their ROM for inversion and eversion. Only two levels were assessed for inversion and eversion as the subtalar joint range of motion was relatively limited, compared to the ankle, constraining goniometer-based classification of activation level. Commands for the ankle and subtalar movements were presented 40 and 30 times, respectively, and in random order to each subject through a custom MATLAB script displayed on a screen (SI Appendix, Fig. S2). This task assayed the proprioceptive feedback received and incorporated into each subject’s motor control; subjects with greater proprioception would be able to better determine the position of the joint and thus more precisely execute the task at each gradation. The muscle activation level was quantified by the EMG signal amplitude at each commanded movement, as identified through time-synched EMG and commands. The control of movements and distinctness of the efferent signals were compared between cohorts using a heteroscedastic two-tailed test on the means between adjacent activation levels at P < 0.05. The variance of performance was compared using a repeated-measures F test at P < 0.05, Bonferroni corrected for multiple comparisons at a statistical significance alpha of P < 0.008.

Statistical Analysis.

MATLAB (Mathworks) was used for all data and statistical analyses. Data distribution was assumed to be normal, but was not formally tested. For temporal correlations, data were normalized to the maximal amplitude of each subject for EMG, fascicle strain, and joint angle to allow for uniform comparisons across subjects and groups. All data are reported as mean ± SD and unless otherwise noted, heteroscedastic two-tailed Student’s t tests were used to compare differences between groups with an alpha of P < 0.05.