Femke Mathot

7 Motor results of MSC-seeded nerve allografts 121 DISCUSSION Despite advances in decellularization techniques for allograft nerves, nerve autograft remain the gold standard for segmental defect reconstruction of critical motor or sensory nerves. 1, 6 To overcome the limitations of decellularized allograft nerves, MSCs have been hypothesized to improve outcomes of decellularized allograft nerves 7 by producing proteins and cytokines that establish a micro-environment favorable for neural regeneration. 8, 12-14, 37 Differentiated MSCs have been demonstrated to exert their neurotrophic effect immediately after implementation by expressing increased levels of neurotrophic genes, while undifferentiated MSCs require additional time to interact with the surrounding tissue prior to expressing neurotrophic genes. 14 The purpose of this study was to determine the effect of dynamically seeding undifferentiated and differentiated MSCs onto decellularized nerve allografts 7 with respect to functional and histologic outcomes in a rat sciatic defect model, in order to determine which cell-type has greatest clinical potential. In this study, MSCs were successfully differentiated into Schwann cell-like cells 16 and dynamically seeded onto decellularized nerve allografts. 27 Compared to unseeded allografts, undifferentiated MSCs led to significant improvement of both ITF and CMAP (p=0.017 and p=0.004) outcomes at 12 weeks, while differentiated MSCs only led to significant improved CMAP outcomes (p<0.001). These findings correspond to the study of Hou and colleagues whom observed that (differentiated) MSC-seeded grafts recovered earlier than acellular grafts when measuring electrophysiology, with significant results at 12 weeks. 38 Differences between groups normalized at 16 weeks which is consistent with the study of Tang and colleagues, that demonstrated normalizing ITF measurements at 16 weeks of follow-up. 39 Functional assessment did not result in any significant differences between both cell-types for all functional outcome measures at 12 and 16 weeks, which is in line with published studies of Orbay and Watanabe. 22, 23 The hypothesized consequences of different effective phases of both cell-types could not be confirmed in this study. At 16 weeks, no significant differences in functional outcomes between groups were found, except for muscle mass recovery that was significantly better in autografts than in allografts seeded with differentiated MSCs (p=0.002). Although muscle mass is easily obtainable, it is an indirect measurement of motor outcome as enlarged muscle fibers do not neccesarily feature improved contractility. 36 ITF has been described to objectively quantify contractility of muscle fibers and is easily reproducible. 36 The vulnerability of CMAP measurements, which is affected by the placement of all individual electrodes, may explain why the CMAP outcomes are greater than the ITF measures. 40 Histologically, the autografts had significantly better N-ratios in the peroneal nerves at both time points compared to all other groups. Although not examined, this could be explained by less formation of fibrosis in autografts. 41 Due to small groups and insufficient sensitivity of density measures, the histology outcomes could not be significantly confirmed by immunofluorescence outcomes, but unseeded nerve allografts subjectively seem to contain less Schwann cells and axons compared to all other groups.