Femke Mathot

Chapter 8 132 To quantify seeding efficiency, cell counts of the cell supernatant after the rotation duration was completed, provided the number of MSCs that remained free floating in the media. This indirectly led to the number and percentages of MSCs that were attached to the nerve sample, in this manuscript expressed as seeding efficiency. 32 In addition to cell counts in the supernatant, seeding efficiency of MSCs after the various seeding durations was further evaluated by Hoechst fluorescence staining of the seeded nerve substitutes using the Infinite® 200 Pro TECAN Reader (Tecan Trading AG, Switzerland) at an absorbance wavelength of 340/458nm. Statistical analysis Cell counts were performed in triplicate per sample and are expressed as themean percentage ± standard deviation (SD). The different measurements were analyzed using a two-way ANOVA. When ANOVA indicated a significant interaction, both within and between group comparisons were analyzed with the Kruskal-Wallis test, followed by pairwise comparisons using Wilcoxon rank-sum tests with Bonferroni correction. Significance was set at a> 0.05. RESULTS Cell Viability upon interaction of MSCs with Nerve Graft Substitute: Analysis revealed no significant effect on cell viability of the pHEMA coating that was used in this experiment. The viability of MSCs when in presence with the Avance® Nerve Graft or the NeuraGen® Nerve Guide, expressed as a ratio of the viability of MSCs without either of the nerve substitutes in figure 1 , was not affected by the presence of both nerve substitutes indicating that there was no detrimental interaction of the manufacturing process to MSCs. There were no significant differences in cell-viability between and within the two groups over time as well (p=0.450). Cell distribution, migration and seeding efficiency of MSCs on Nerve Graft Substitute Live/dead staining and nuclear staining using Hoechst dye revealed a uniform distribution of viable MSCs over the entire surface of both nerve substitutes after all seeding durations. Figure 2 and 3 demonstrate example images after 12 hours of seeding of live/dead staining and Hoechst staining respectively. Hoechst fluorescent measurements demonstrated increased fluorescence as seeding duration time increases ( figure 4 ) (p=0.001), but there were no significant differences in Hoechst fluorescence between groups. Despite the different composition of the surfaces of the Avance® Nerve Graft and the NeuraGen® Nerve Guide, SEM images revealed a similar distribution of cells among their surface ( figure 5 ). Manual quantification could not be carried out reliably due to cell aggregation, but assessment of the samples showed a marked increase in cell coverage of both nerve substitutes between 6 and 12 hours of seeding. Between 12 and 24 hours of dynamic seeding, there were no appreciable differences in cell coverage. The morphology of