Femke Mathot

9 MSC gene expression on nerve substitutes 157 As described NGF, GDNF, PTN, GAP43 and BDNF play a part in the stimulation of axonal outgrowth and the proliferation of neurons and Schwann cells (step 4 and 9, figure 6). 69-71 Previous in vivo research in a rat model demonstrated that particularly NGF is expressed in a significant higher manner in nerve autografts than in decellularized allografts. 72 In our study, NGF and GDNF expression was enhanced in the first 0-24 hours after seeding and the GDNF expression after 21 days seemed to increase again in the NeuraGen® Nerve Guide group. BDNF expression was enhanced in both groups among the entire follow-up period (up to 21 days), but PTN and GAP43 expressions did not increase in comparison to unseeded MSCs (ratio <1.0). The long-term low expression of PTN and GAP43 and the enhanced expression of BDNF after seeding was correspondingly described in in vitro research using human nerve allografts that were processed with elastase and stored at 4°C. In the study of Rbia and colleagues enhanced BDNF expression also led to enhanced levels of BDNF growth factor production. In contradiction to our findings, NGF and GDNF were not enhanced in that particular study, which might be due to differences in the ECM as a result of the different decellularization process. 73 Our results suggests that the interaction between MSCs and the ECM of nerve substitutes stimulates neural proliferation or may enhance neural outgrowth, particularly by upregulation of NGF, GDNF and BDNF. MPZ and PMP22 are mainly expressed in Schwann cells, which initiate axon myelination, occurring approximately 2 weeks after injury (step 7, figure 6). 19 The short-term (first 24 hours) enhanced expression of PMP22 and MPZ demonstrated in this study corresponds to previous in vitro research using the same seeding strategy on different nerve allografts. 73 Since transdifferentiation into Schwann-like cells is unlikely to have occurred in the described time-span, the elevated level of PMP22 might be subscribed to its role in the development of intercellular junctions. 74 The PMP22 and MPZ expression was not significantly altered on the long term (from 7 days onwards) by the interaction with the nerve substitutes in the current study; this could be due to the absence of Schwann cells in this in vitro setting. Previously, rat autograft nerves did not express significantly different levels of PMP22 and MPZ in vivo than unseeded processed allografts, which could insinuate that these genes are not pivotal for improving nerve regeneration in processed nerve allografts to a level equal to autografts. 72 VEGF-a functions particularly in axon regeneration and guidance (step 6 and 9, figure 6) by stimulating formation of blood vessels and enhancing Schwann cell and neuron survival. 71, 75 In vivo, rat autograft nerves previously demonstrated to express significantly higher levels of VEGF-a than unseeded processed nerve allografts. 72 The demonstrated upregulation of VEGF-a expression in this study in the first 24 hours and from two weeks onwards after seeding is in accordance with the described nerve regeneration cascade and with previous in vivo research, supporting the role that MSCs can play in revascularization. 72 CD31 is a platelet endothelial cell adhesion molecule (Pecam1) that is required for the motility and organization of endothelial cells, essential for angiogenesis (step 9, figure 6). 76 Autografts do not express significantly different levels of CD31 in vivo than processed nerve allografts. 72 Our data describes enhanced CD31 expression directly after seeding that diminishes after 1 to 3 days after seeding.