Femke Mathot

9 MSC gene expression on nerve substitutes 149 Statistical analysis The gene expression profiles of the seeded MSCs on both nerve substitutes were expressed as a ratio of the average gene expression of unseeded MSCs (= reference group). Gene expression ratios of MSCs seeded onto Avance® Nerve Grafts were compared to gene expression ratios of MSCs seeded onto NeuraGen® Nerve Guides over time and were analyzed using a 2-factor ANOVA with repeated measures of one factor, with post-hoc Bonferroni correction. This analysis provided insight in the effects of the type of nerve substitute and the time passed after seeding. All results are expressed as the mean +/- SEM. A a <0.05 was considered statistically significant. RESULTS Control experiments with nerve substitutes The presence of RNAwas examined in unseeded Avance®Nerve Grafts (n=5) andNeuraGen® Nerve Guides (n=5) and no RNA was detected. Neurotrophic gene expression (Figure 1) Expression of five neurotrophic factors (NGF, GDNF, PTN, GAP43 and BDNF) was measured by qPCR to assess whether nerve substitutes can induce neurotrophic factors in MSCs. Nerve Growth factor (NGF) NGF is endogenously produced by cells like neurons and Schwann cells and is crucial for neuroplasticity since it promotes neuronmaturation and can induce cell repair and apoptosis. 32, 33 The factor time solely had a significant effect on NGF expression (p<0.001), while the type of nerve substitute did not (p=0.228). Directly after seeding, MSCs on Avance® Nerve Grafts expressed significantly increased levels of NGF compared to MSCs on NeuraGen® Nerve Guides (5.304 ± 0.731 versus 2.530 ± 0.419, p<0.001), while MSCs on NeuraGen® Nerve Guides expressed significantly increased NGF expression ratio 1 day after seeding (1.226 ± 0.187 versus 3.868 ± 1.379, p=0.002). After 1 to 3 days after seeding, the NGF expression of MSCs in both groups was comparable to the expression in unseeded MSCs. Thus, NGF expression is transiently elevated in MSCs seeded onto nerve substitutes shortly after seeding and subsides over time. Glial cell line-derived neurotrophic factor (GDNF) GDNF is described to have a crucial role in neuronal migration, proliferation and synaptogenesis. 34, 35 EnhancedGDNFdeliveryhasdemonstrated to result inearlier regeneration after nerve crush injuries 36 and its expression decreases coincidentally with the ingrowth of regenerating axons. 35 The GDNF expression ratio of seeded MSCs was significantly affected by culture duration after seeding (p<0.001); the expression slowly diminished over time with the exception of GDNF expression after 21 days in MSCs seeded onto Avance® Nerve Grafts. No significant differences were found in GDNF expression between both groups. The GDNF expression curves of both groups approximated the unseeded MSC expression from 1 day onwards.