Femke Mathot

8 Stem cells on nerve graft substitutes 131 eliminated by measuring the viability of two extra groups (MSCs + Avance® Nerve Graft and MSCs + NeuraGen® Nerve Guide) at each time point. After 1, 2, 3 and 7 days of incubation at 37°C the metabolic activity of three samples of each group were measured with the Infinite® 200 Pro TECAN Reader (Tecan Trading AG, Switzerland) at an absorbance wavelength of 490nm. The metabolic activity of group I (pHEMA + MSCs + Avance® Nerve Graft) and group II (pHEMA + MSCs + NeuraGen® Nerve Guide) were expressed as a ratio of the metabolic activity of the control group (pHEMA + MSCs) and compared to each other. Experimental Design and Measurement of Cell distribution, Migration and Seeding Efficiency In total, 20 Avance® Nerve Grafts and 20 NeuraGen® Nerve Guides of 10mm in length were used in this experiment. 18 samples per group were dynamically seeded according to the dynamic seeding strategy described by Rbia and colleagues. 27 Prior to seeding, all nerve substitute segments were soaked in a-MEM for two hours as an equilibration step to restore the salt balance. Conical tubes containing the nerve samples and one million MSCs per nerve sample in growth medium were rotated in a bioreactor placed in an incubator (37°C) for 6, 12 and 24 hours (n=6 per group per seeding duration). The viability of adherent MSCs was evaluated by live/dead Cell Viability Assays (Invitrogen, Life Technologies Corporation, NY, USA) after each of the different seeding durations. Hoechst staining was performed according to standard protocols (Hoechst stain solution; Sigma-Aldrich Corp., MO, USA) on the surface of the nerve substitutes to show the distribution and migration of cells after dynamic seeding. Both Live/Dead and Hoechst stains were performed on three samples per group per seeding duration and were visualized directly after seeding with a confocal microscope (Zeiss LSM 780 confocal microscope). To study cell distribution, Scanning Electron Microscopy (SEM) (n=3 per group per seeding duration) was performed. Samples were fixed in 2% Trump’s fixative solution (37% formaldehyde and 25% glutaraldehyde) directly after seeding. After 24 hours, samples were washed with phosphate buffer, rinsed in water, processed through graded series of ethanol (final 100% ethanol), critical point dried with carbon and mounted on an aluminum stub. After sputter-coating for 60 seconds using gold-palladium, sample-images were taken with a Hitachi S-4700 cold field emission SEM (Hitachi High Technologies America, Inc., IL, USA) at 5kV accelerating voltage. After obtaining images of the graft surface, samples were cut longitudinally and imaged to reveal cell distribution on the inside of the nerve substitutes. To reinforce the SEM-findings, two extra 10mm samples per group were seeded with 1 million MSCs according to the estimated optimal seeding duration, fixed in 10% formalin and processed and embedded in paraffin. Three sections of 5µM of the proximal- and mid- nerve substitutes were taken and Hoechst stained to evaluate for cells that migrated inside the nerve substitute (i.e. within the nerve allograft or nerve conduit material itself). The cells were visualized with a confocal microscope (Zeiss LSM 780 confocal microscope; Zeiss, Germany).