Femke Mathot

4 Interaction between MSCs and a nerve allograft 63 signals reflection protein expression in differentiated MSCs was compared to expression in Schwann cells and undifferentiated MSCs. Nerve allograft decellularization 65 sciatic nerves were obtained from 33 Sprague-Dawley rats (Envigo, Madison, WI, USA) weighing 250-350 grams and were processed according to the protocol of Hundepool and colleagues. 7 Sprague-Dawley rats were specifically selected, because there is a known histocompatibility mismatch with Lewis rats. 39, 40 The nerves were sterilized using g -irradiation and stored at 4°C for a maximum of 24 hours. Dynamic MSC seeding The 65 processed nerve allografts of 10mm in length were dynamically seeded for 12 hours with either 1x106 undifferentiated or differentiated MSCs from Lewis rats. This method has been previously demonstrated to be successful for both cell types. 37, 38 Once the seeding was completed, the seeded nerve allografts were placed in normal growth medium in 6-well plates. The growth medium was changed every 72 hours. Real Time Reverse Transcriptase Quantitative Polymerase Chain Reaction (qPCR) analysis Gene expression of decelllularized allografts dynamically seeded with undifferentiated and differentiated MSCs was measured for 5 duplicates per group at 6 different time points. Cells were harvested directly after seeding (T0) and 1 (T1), 3 (T2), 7 (T3), 14 (T4) and 21 (T5) days after seeding. Baseline gene expression (Tc) of both cell groups was obtained from 5 samples of undifferentiated and differentiated MSCs, while 5 unseeded decellularized allograft samples served as negative controls (T-). At each time point, nerve tissues where frozen at -80 ° C in Qiazol. Each sample was minced with a sterile needle to enable ribonucleic acid (RNA) extraction according to the manufacturer’s protocol (Direct-zolTM RNA MiniPrep Kit; Zymo Research, Irvine, CA, USA). After measuring RNA concentration with the NanoDropTM 2000/2000c Spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA), complementary deoxyribonucleic acid (cDNA) was obtained in a concentration of 10ng/µL. Expression of mRNAs was determined by qPCR analysis for the neurotrophic markers nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), pleiotrophin (PTN), growth associated protein 43 (GAP43) and peripheral protein 22 (PMP22); for the angiogenic markers platelet endothelial cell adhesion molecule 1 (PECAM1/ CD31) and vascular endothelial cell growth factor alpha (VEGF1); for extracellular matrix (ECM) markers collagen type I (COL1A1), collagen type III (COL3A1), Fibulin 1 (FBLN1) and laminin subunit beta 2 (LAMB2); and for regulatory cell cycle markers caspase 3 (CASP3) and Cyclin B2 (CCBN2). Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) served as reference gene. Primers were manufactured by Sigma and are listed in table 2 . Function descriptions for the evaluated genes are provided in table 3 . After preparing the reactions containing primers, real time-qPCR master mix (Qiagen, MD, USA) and cDNA, the reactions were amplified by real time-qPCR using a thermocycler (Bio-rad Laboratories, Inc., CA, USA) with the following