Femke Mathot

9 MSC gene expression on nerve substitutes 147 each group were used to study the interaction with MSCs over time. MSC seeding To seed MSCs on nerve substitutes in a non-traumatic manner, a previously described seeding strategy was applied. 25, 26 Previous studies have demonstrated a 66% and 94% seeding efficiency for the Avance® Nerve Grafts and the NeuraGen® Nerve Guides, respectively. 27 Prior to seeding, the Avance® Nerve Grafts and the NeuraGen® Nerve Guides were soaked in a-MEM to restore the salt balance and to remove any harmful detergents. The nerve substitutes were placed in conical tubes containing 1 million MSCs in 10mL growth medium. The conical tubes were rotated for 12 consecutive hours on a bioreactor placed in a 37°C incubator. Quantitative PCR analysis Quantitative PCR analysis was performed on 5 duplicates per group before seeding (n=5 per group, baseline gene expression) and at 6 time points after seeding: directly after seeding, 1 day, 3 days, 7 days, 14 days and 21 days (n=5 per group per time point). Between seeding and qPCR analysis, the samples were placed in wells containing growth media. To determine the baseline gene expression of the MSCs, qPCR analysis was performed on 5 samples of MSCs only. At each time point, the seeded nerve substitutes were removed from the wells, placed in Qiazol and frozen at -80°C. The seeded nerve substitutes were minced with a sterile needle to ensure the DNA of the MSCs was dissolved in the fluid. Ribonucleic acid (RNA) extraction took place according to the manufacturer’s protocol (Direct-zolTM RNA MiniPrep Kit; Zymo Research, Irvine, CA, USA). RNA concentration was measured with the NanoDropTM 2000/2000c Spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). Subsequently, complementary deoxyribonucleic acid (cDNA) was obtained (SuperScript III, Invitrogen, Carlsbad, CA; 3 minutes at 65°C, 90 minutes at 37°C and 5 minutes at 95°C) and diluted to establish a concentration of 10ng/µL. The obtained cDNA was combined with the selected primers (all manufactured by Sigma, table 1) and real time- qPCR master mix (Qiagen, MD, USA) and amplified by real time-qPCR using a thermocycler (Bio-rad Laboratories, Inc., CA, USA) under the following parameters: 15 minutes at 95°C, followed by 50 cycles of 95°C for 20s, 60°C for 35s and 72°C for 35s. This provided the mRNA expression levels of the investigated genes. Analysis of mRNA biomarkers In relation to the described Wallerian degeneration and axon regeneration process, the neurotrophic effects of the interaction between MSCs and nerve substitutes were measured by assessing a panel of mRNA biomarkers, including nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), pleiotrophin (PTN), growth associated protein 43 (GAP43) and brain-derived neurotrophic factor (BDNF). The expression of myelination marker genes peripheral protein 22 (PMP22) and myelin protein zero (MPZ) were measured. The angiogenic potential of the seeded MSCs was measured by the gene