Tiam Mana Saffari

216 CHAPTER 10 stock dilution, 35 mg/mL diluted in dimethyl sulfoxide (DMSO); Sigma-Aldrich), prior to introducing a differentiation solution to the growth medium containing 0.14% Forskolin (10 mM dissolved in DMSO, Sigma-Aldrich), 0.01% basis fibroblast growth factor (bFGF; 100 mg/mL dissolved in 5 mM Tris Hydrochloride buffer, PeproTech, NJ, USA), 0.005% platelet-derived growth factor (PDGF-AA; 100 mg/mL dissolved in 10 mM acetic acid, PeproTech) and 0.02% Neuregulin-1 ß1 (NRG1-b1; 100 µg/ mL reconstituted in phosphate buffered saline (PBS), R&D systems Inc, MN, USA). Successful differentiation was previously assessed by immunohistochemistry 18 . All MSC cultures were maintained at subconfluent levels in a 37 °C incubator with 5% CO2 and passaged with TrypLE (Invitrogen, UK). Growth mediumwas changed every 72 hours. In this experiment, passage five cells were used to seed allografts. Seeding allograft nerves with mesenchymal stem cells For group IV and V, ten allografts were dynamically seeded for 12-24 hours with 1x10^6 undifferentiated MSCs (N=5) and 1x10^6 differentiated MSCs (N=5), respectively 16 . Briefly, decellularized nerve allografts were sterilely combined with 1x10^6 cells in a TubeSpin® Bioreactor tube (Midwest Scientific, MO, USA) containing cell culture medium (Figure 1A). Tubes were placed on a bioreactor with a fixed speed of 18 RPM in the incubator for 12-24 hours until surgery. Seeding efficiency using this dynamic seeding strategy has been previously validated 16,18 . Surgical procedure Rats were anesthetized in an isoflurane chamber, shaved, prepped and positioned in the nosecone to maintain anesthesia throughout the procedure. Preoperatively, the following were administered subcutaneously: 5 mL of NaCl 0.9% solution (to prevent dehydration), Enrofloxacin (Baytril, Bayer, Germany, 10mg/kg, providing infection prophylaxis) and Buprenorphine SR (Buprenorphine SR-LAB, ZooPharm pharmacy, 0.6mg/kg, pain control). Body temperature during surgery was maintained at 37°C with a heating pad.