Regeneration of Large‐gap Peripheral Nerve Injuries Using Acellular Nerve Allografts plus Amniotic Fluid Derived Stem Cells (AFS)

Abstract

IntroductionSurgical reconstruction of peripheral nerve lesions in the extremities is challenging and often results in impaired functional recovery. The “gold standard” for successful nerve repair is a primary tensionless epineural repair which often is not possible. Nerve guidance tubes as well as acellular nerve allografts (ANA) have been developed to provide repairs comparable to those obtained with autografts. In order to promote nerve regeneration across large nerve gaps, regenerating axons are capable of extending the gap distance for nerve recovery when in the presence of Schwann cells. Tissue engineering strategies have attempted to mimic this cell environment by adding other supportive types of cells such as stem cells to the nerve allograft.HypothesisWe hypothesized that acellular nerve allografts (ANA) can be seeded with amniotic fluid‐derived stem cells (AFS) to promote and accelerate nerve regeneration. The presence of the AFS provides support for the regenerating axons without the requirement of becoming Schwann cells.MethodsIn vitro study: 1×106 “Off the shelf” AFS cells were injected underneath the epineurium of the ANAs using a 26 G syringe. Seeded grafts were placed vertically at the bottom of a small centrifuge tube covered with DMEM containing 20% FBS for overnight then transferred to a 48 well plate for additional 48 hours. In vivo study: ANA with AFS cells for long gap nerve repairs were studied using Lewis Rats. A large gap nerve injury (1.5 cm) was created in the sciatic nerve, and the gap was repaired immediately with an ANA construct alone (Group 1), an ANA construct with AFS cells (Group 2), or with an autograft (Group 3). Outcome assessments include walking track analysis (DigiGait Imaging system, Figure 1) to document the return of motor control at 1 month and 2 months post‐injury.ResultsIn vitro AFS cells seeding to ANA: DAPI staining on longitudinal and cross sections of ANAs showed cells spread evenly through the nerve fibers (Figure 2.) In vivo gait analysis of 23 parameters of the autograft, ANA and ANA plus AFS cells groups at 2 months post‐injury indicated that there were no significant differences in stride, stance/swing ratio, paw area at peak stance, stance factor, midline distance, % swing/stride, % brake/stride, % propel/stride, % stance/stride, %brake/stance, % propel/stance, % hind limb shared stance, step angle degree, stride length, MAX dA/dT and MIN dA/dT among groups. The autograft group showed greater stance width, overlap distance, axis distance, paw angle and paw drag compared to the ANA and ANA plus AFS cell groups.( p<0.01 in all indices, Figure 3) ANA plus AFS cell group showed reduced swing time, %swing/stride at the end of 2 months compared with 1 month time point (1 month vs. 2 months: 0.17±0.01s vs.0.14±0.02s; 37.76±3.97% vs. 33.37±4.78%; p<0.01, p<0.05) In addition, ANA plus AFS cell group demonstrated a more robust motor function recovery compared to ANA alone group (paw angle and paw drag value are close to autograft group), indicating AFS cells facilitated the nerve regeneration 2 months following injury. We will keep tracking the motor function recovery as well as the electromyographic and histological outcomes till the end of 4 months following injury.SignificanceThe findings of the study may have a direct impact on the future of stem cell therapies to facilitate nerve regeneration in patients who sustain peripheral nerve injuries.Support or Funding InformationThe study is funded by Department of Defense USAMRAA (W81XWH‐13‐1‐0310)