Femke Mathot

Chapter 8 134 cells did not change over time. SEM images of longitudinally cut segments of the Avance® Nerve Graft did not show any MSCs in the inner ultrastructure of the graft after 6, 12 and 24 hours of seeding ( figure 6 , left side). MSCs were present throughout the inner surface of the NeuraGen® Nerve Guides after 6, 12 and 24 hours, although the coverage of MSCs was clearly less than on the outside of the Nerve Guide (Figure 6, right side). Additionally, the MSC did not migrate into the substrate of the NeuraGen® Nerve Guides. These findings were confirmed by Hoechst staining of cross-sectional images of both groups, that revealed no staining of nuclei in cells inside of the Avance® Nerve Grafts but detectable nuclear staining of cells within the NeuraGen® Nerve Guides ( figure 7 ). Figure 4. Hoechst fluorescence intensity of the Avance® Nerve Graft and the NeuraGen® Nerve Guide when seeding with MSCs according to increasing seeding durations (n=3 per group per time point). ANOVA analysis did not demonstrate a significant interaction between seeding duration and Hoechst fluorescence (p=0.001) when merging the groups, but within groups analysis did not demonstrate any significant increases between time points (p>0.221 for the Avance® Nerve Grafts and p>0.083 for the NeuraGen® Nerve Guides). Figure 5. Scanning electron microscopy images showing the cell-coverage of the Avance® Nerve Graft (A and C) and the NeuraGen® Nerve Guide (B and D) after being dynamically seeded with human MSCs for 12 hours. Images A and B display overview images with 150X magnification. Images C and D display the areas that are encircled in red in images A and B, 500X magnification. Shown is a uniform distribution of partly aggregating MSCs on the porous surface of both nerve substitutes. Examples of cell contours are displayed in red in C and D. Before seeding 6 hours 12 hours 24 hours 0 10000 20000 30000 40000 50000 Hoechst fluorescence Mean Hoechst fluorescence intensity Avance® Nerve graft + MSCs NeuraGen® Nerve Guide + MSCs Seeding duration