Overview of study design

A cohort study examining functional and histomorphologic outcomes following VML repair with acellular biologic scaffolds was conducted with informed subject consent and approvals from the Institutional Review Board of the University of Pittsburgh and the US Department of Defense Human Research Protection Office (ClinicalTrails.gov, identifier NCT01292876). Subjects were screened for established exclusion criteria.14 A total of 13 subjects were enroled and subjected to a custom designed physical therapy regimen both before and following implantation of one of three different xenogenic scaffold materials, all of which were composed of porcine ECM (Table 1). Patients were enroled in pre-operative physical therapy and required to reach a functional plateau before the surgical procedure so that post-operative improvements in function could not be attributed to therapy alone. Force production, functional task improvement, EMG analysis, CT or MRI imaging, and histology were used to evaluate return of strength, function and bioscaffold remodelling characteristics at 6–8 weeks, 10–12 weeks and 24–28 weeks post implantation.

Subject selection and screening

Participants ranging from 18 to 70 years of age with a minimum 20% structural volume deficit as determined by MRI or CT, and/or 25% functional deficit of the muscle group mass when compared with the contralateral limb were eligible for inclusion in the study. All study subjects acquired VML at least 6 months prior to study inclusion. Exclusion criteria included poor nutrition, chronic disease, active infection, neoplasia, denervation or other medical comorbidities with the potential to impair wound healing.

Prior to inclusion in the trial, all subjects were screened by a licensed physical therapist to establish strength and functional deficits related to the anatomic location of interest, with respect to the contralateral limb. A detailed subject history was taken and the subject’s goals for participation in the study were recorded. Active and passive range-of-motion measurements were obtained at the joints both proximal and distal to the affected area using a goniometer. Isometric strength of the affected muscles was quantified using a hand-held dynamometer. Specific functional outcome variables were selected and evaluated for each subject based on their functional deficits and the objective measurements of strength and joint range-of-motion. Patient-reported outcomes, including the Disabilities of the Arm Shoulder and Hand (DASH)29 scale and Lower Extremity Functional Scale (LEFS)30 were administered, as appropriate. Subjects were also asked to provide a self-report of functional status at each of the tested time points. Outcome variables were established a priori for each subject through a study team consensus based on findings from the clinical examination specific to each subject and their observed strength and functional deficits. When possible, outcome variables were selected that were previously established as valid, reliable and aligned with the subject’s goals for the trial. Video recordings were performed during the evaluations when possible so as to ensure consistency in the testing positions across time points.

Surgical procedure

All procedures were performed in a tertiary care medical centre under general anaesthesia, and tourniquet control of the extremity used. The injured muscle compartment was accessed, scar tissue was debrided and selective tenolysis performed. One of the following three ECM bioscaffolds was implanted at the site of missing muscle: MatriStem (ACell, Columbia, MD, USA); BioDesign (Cook Medical, Bloomington, IN, USA) or XenMatrix (C.R. Bard, Warwick, RI, USA) which were derived from porcine urinary bladder (UBM), SIS or dermis, respectively. All three scaffold materials were decellularized to meet established minimum criteria for DNA removal.31 MatriStem was used in the first six subjects, and the remaining seven subjects received either BioDesign or XenMatrix, randomly assigned. The ECM bioscaffold was cut to defect size-matched appropriate length and width, and implanted within the injury site with contact to adjacent native healthy tissue, and secured under tension with monofilament absorbable sutures. Care was taken to prevent folding or wrinkling and to ensure adequate soft tissue coverage. All empty space was closed before closure of the surgical site to ensure maximum scaffold-host tissue interaction, and a closed suction drain was placed.

Physical therapy

Pre-surgical

Subjects were required to participate in rigorous pre-operative physical therapy for 4–16 weeks prior to surgery. The goal of the pre-operative physical therapy programme was to maximize performance with respect to the strength and functional outcome deficits identified during the screening examination. Due to the unique clinical presentation of each subject, physical therapy programs were customised for each subject to address the specific strength and functional deficits identified during the screening visit. Subjects were evaluated weekly on their progress by the treating physical therapist. Subjects were cleared to proceed to surgery after they reached a plateau in performance on their involved side, defined as functional gains of <2–3% over the course of any 2-week period, as determined by the treating physical therapist. The treating physical therapist was not a member of the investigative team. Outcome variables were tested by the same evaluating physical therapist who was a member of the investigative team at each time point.

Post-surgical

Post-surgical physical therapy was initiated between 24 and 48 h following surgery. No limitations were placed on the exercises or functional movements within the limits of tolerable pain. As early as the first post-operative day, targeted exercises were performed with the goal of stimulating muscle contraction and load bearing across the scaffold implantation site. Pain level, range-of-motion, strength and functional capacity were evaluated at each visit, and exercises were continued as tolerated. The post-operative physical therapy phase lasted 24 weeks.

Isometric strength measurement

Isometric strength testing of the affected limb was measured 1–2 days prior to ECM implantation, and again at 6–8 weeks, 10–12 weeks and 24–28 weeks post-operatively. Measurements were taken using a hand-held dynamometer and standard manual muscle testing positions.32 Each measurement was repeated three times, and the average value of the three trials was calculated to represent as an indication of the isometric strength of the affected muscle.

Range-of-motion and functional task analysis

Range-of-motion and functional task analysis was conducted pre-operatively and at 6–8 weeks, 10–12 weeks and 24–28 weeks post-operatively. All tasks were performed on both the affected and contralateral limb. Each task was repeated three times, and the average of the three trials was calculated as representative of performance on the task.

Pre- and post-surgical imaging

Initial pre-operative CT imaging was performed on a 64-slice CT scanner (LightSpeed VCT, GE Healthcare, Chicago, IL, USA) at a slice thickness of 1.25 and 2.5 mm and a pitch of 1.375 in both bone and soft tissue algorithms. MRI protocols included a variety of sequences in sagittal, coronal and axial planes using T1-weighted spin echo, T2-weighted fast spin echo with or without fat suppression and STIR sequences. The kVp and mA were optimised with respect to the subject habitus and site imaged. Coronal and sagittal reformations were obtained. Three-dimensional volumetric reformatted imaging was also performed using Vitrea (Vital Images, Minnetonka, MN, USA) with surface rendering, as well as emphasis on the underlying musculature and osseous structures. Pre-operative imaging was reviewed by a musculoskeletal-trained radiologist (4 years’ experience). Initial CT imaging was assessed primarily for the presence of volumetric loss of bulk and/or fatty infiltration in the affected musculature. The overall percentage loss of muscle volume and severity of fatty infiltration was graded, where appropriate. Imaging was also evaluated for concomitant soft tissue (e.g., tendinous) and osseous injury. Post-operative imaging was performed at an ~7-month interval with similar imaging parameters. Post-operative imaging included characterisation of the location and appearance of the ECM scaffold, as well as a change in volume or appearance of the surrounding musculature. Overall percentage change in affected muscle volume was measured.

Ultrasound-guided core biopsy of ECM

Ultrasound-guided biopsy of the surgically-placed ECM was performed ~6 weeks and 26 weeks post-operatively. Pre-procedural grayscale and colour/Power Doppler ultrasound of the operative site was performed to identify and characterise the surgically-placed ECM. After an appropriate needle trajectory was selected, the area was prepped and draped in sterile fashion. Local anaesthesia with skin infiltration and deeper injection was achieved with 1% lidocaine. Under ultrasound guidance, biopsy samples of the ECM bioscaffold and surrounding soft tissue were obtained using an 18-gauge spring-loaded biopsy needle (Temno, CareFusion, McGaw Park, IL, USA). A total of eight core samples were obtained at two separate biopsy sites. Biopsies spanned the proximal to distal length and medial to lateral width of the implantation site. Specimens were snap-frozen in liquid nitrogen and stored at −80 °C.

Electrodiagnostic studies

As previously reported, nerve conduction and electromyography studies were performed for 8 of the 13 subjects using a Synergy EMG machine (Cardinal Health, Dublin, OH, USA).33 The specific nerve conduction studies completed and the specific muscles tested with needle examination were determined by location of the VML. Needle EMG analysis used concentric needle electrodes placed in the standard muscle belly and was performed at the proximal and distal site of the injured muscle if the standard muscle belly showed no evidence of electrical activity. Improvement in nerve conduction was defined as a ⩾20% increase in motor nerve conduction amplitude. For EMG studies, improvement was defined as either evidence of increased firing in volitional recruitment of muscles or a decrease in abnormal spontaneous activity compared with pre-operative results. Differences in amplitudes of CMAP were compared between pre- and post-ECM bioscaffold implant.

Histology and immunolabelling

Frozen tissue sections were fixed in an ice cold 50:50 solution of methanol/acetone for 5 min and washed in phosphate-buffered saline (PBS). Tissue sections were incubated in blocking buffer to prevent non-specific antibody binding composed of 1% (w/v) bovine serum albumin (BSA), 2% (v/v) normal horse serum, 0·05% (v/v) Tween-20, 0·05% (v/v) Triton X-100 in PBS for 1 h at room temperature. Tissue sections were then incubated with primary antibodies diluted in blocking buffer as follows: mouse monoclonal CD146 (Abcam, Cambridge, MA, USA) at 1:350 and rabbit polycloncal Neurogenin-2 (NG2, Millipore, Billerica, MA, USA) at 1:200 as a perivascular stem cell markers, monocloncal anti-desmin (Abcam) at 1:200 for a muscle cell marker and (4) β-III tubulin at 1:200, for a neurogenic marker. After 16 h of incubation at 4 °C, tissue sections were washed with PBS and incubated with fluorophore-conjugated secondary antibodies (Alexa Fluor donkey anti-mouse 488 or 594 or donkey anti-rabbit 488, Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. After secondary incubation, nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and slides were coated with anti-fade mounting media (Dako, Carpinteria, CA, USA). Tissue sections were imaged using a Zeiss Axio-observer Z1 microscope using a ×20, 0.4 numerical aperture objective with a ×1.6 optovar magnification changer (Carl Zeiss, Oberkochen, Germany). Three fields of view were taken from each biopsy sample.