Femke Mathot

3 MSC-seeding on processed nerve allografts 49 Table 2. Experimental design experiment 2. MSCs: Mesenchymal Stem Cells, SEM: Scanning Electron Microscopy Statistical analysis Seeding efficiencies are displayed ± Standard Error of the Mean. Significance of the interaction between cell type, time and outcome were analyzed with two-way ANOVA. If the interaction was significant, the within and between group comparisons were analyzed with the Kruskal-Wallis test, followed by pairwise comparisons using Wilcoxon rank-sum tests with Bonferroni correction. A value of p < 0.05 was considered statistically significant. RESULTS Mesenchymal stem cell collection Flow cytometric analysis showed that the cultured rat MSCs were positive for mesenchymal stem cell markers CD29 (88.2%) and CD90 (88.3%) and negative for the hematopoetic cell markers CD34 (91.1%) and CD45 (86.0%), demonstrating that MSCs were definitively the cell lineage utilized in this study. Mesenchymal stem cell differentiation into Schwann-like cells The morphology of differentiated MSCs changed in vitro in a more spindle-like shape, typical for Schwann cells. In contrast to undifferentiated MSCs, Schwann cells and differentiated MSCs showed positive immunofluorescence for Schwann cell surface markers S100 Calcium Binding Protein B (S100B), Glial Fibrillary Acidic Protein (GFAP) and Nerve Growth Factor Receptor (NGFR/P75NTR), which verified successful differentiation into Schwann-like cells ( figure 1 ). 18 Cell viability The cell viability of undifferentiated and differentiated MSCs were equal and remained constant during the first three time points, after which the viability of both cell types slightly decreased. No decrease in cell viability was observed upon co-culture of MSCs with the processed nerve allograft. The viability of differentiated MSCs in the vicinity of an allograft approaches the viability of differentiated MSCs alone over time (p=0.270), while the viability of undifferentiated MSCs with the allograft increased over time compared to undifferentiated MSCs alone; the increased viability ratio between 2 and 3 days of culture was statistically significant (p=0.025). Group Description Time points (seeding duration) Number of samples Outcome measurements I Undifferentiated MSCs + nerve allograft T1 = 6 hours T2 = 12 hours T3 = 24 hours 6 samples per group per time point • Cell counts (n=6) • Live/dead + Hoechst stains (n=3) • SEM (n=3) II Differentiated MSCs + nerve allograft