Femke Mathot

9 MSC gene expression on nerve substitutes 159 curves, we identified four general trends; a linear decline (NGF, GDNF, CD31) a linear increase (PTN, GAP43, BDNF), a stable curve (PMP22, MPZ, COL1A1, LAMB2, CD96) and a U-shaped curve (VEGF-a, COL3A1, FBLN1). We believe that those trends are more reliable and therefore a more important finding than the individual expression ratios at each of the time points. The in vitro setting of our study does not provide the required micro-environmental signals that are essential to mimic the described regeneration cascade. Studying the effects on gene expression that is solely caused by the interaction between MSCs and nerve substitutes, does demonstrate the potential of MSCs to interfere in the previously mentioned cascade steps when dynamically seeded on the outer surface of clinically available nerve graft substitutes. It is recognized that corroborating the mRNA expression changes to protein expression changes could have contributed to the described findings. However, measuring protein levels is a costly technique, vulnerable to flaws and the absence of environmental regenerative signals in vitro would have resulted in outcomes that cannot per definition be related to in vivo protein expression and would still need translation to an in vivo model. Therefore, this study is used to demonstrate that interaction between MSCs and the nerve substitutes occurs, effects a wide range of genes and that it lasts on the long term, while limiting the costs and still preventing unnecessary sacrifices of extra animals in the future. Although it was hypothesized that the biological composition of the Avance® Nerve Grafts (i.e. neural tissue) would lead to more expression of neurotrophic genes in MSCs, analysis demonstrated that the MSCs seeded onto NeuraGen® Nerve Guides expressed higher levels of neurotrophic (GDNF and PTN), angiogenic (CD31 and VEGF-a), ECM (COL3A1 and FBLN1) and immunoglobulin (CD96) genes. Higher seeding efficiency and a different composition of the guide may have resulted in improved sustainability of the graft/MSCs in vitro and better cell proliferation in the long-term, leading to the described enhanced gene expressions. Most neurotrophic factors mediate other processes that are not involved in nerve regeneration which could explain enhanced gene expression levels in the absence of any neural material in the NeuraGen® Nerve Guide group. 13 While our data suggest that nerve autograft substitutes could benefit from the addition of MSCs, future studies are necessary to determine gene expression patterns and the resulting trophic factor production of MSCs in the presence of injured nerve tissue. Furthermore, the in vivo effects on functional outcomes of the described interactions need to be correlated and compared to determine the clinical relevance of our findings. CONCLUSION When human MSCs are dynamically seeded onto the surfaces of Avance® Nerve Grafts and NeuraGen® Nerve Guides, their interaction with the ECM of these nerve substitutes results in a change and mostly an upregulation of the expression of numerous genes important for nerve regeneration over time. The in vitro interaction of MSCs with the NeuraGen® Nerve