Tiam Mana Saffari

192 CHAPTER 9 Seeding protocol for allograft nerves To attach the undifferentiated and differentiated MSCs to the decellularized nerve allografts they were dynamically seeded according to a previously described protocol 28 . Either 1x10^6 undifferentiatedMSCs or 1x10^6 differentiatedMSCs in 10mL growth mediumwere placed in a conical tube containing a decellularized nerve allograft. The conical tube was then placed on a bioreactor that was positioned in an incubator at 37°C (5% CO 2 ). After the bioreactor had rotated for 12 hours, the nerve grafts with the attached MSCs were taken out of the tubes and directly implemented in the Lewis rats. The dynamic seeding strategy previously resulted in 80% and 95% adherence of cells on the surface of the processed allografts for undifferentiated and differentiated MSCs, respectively 27,28 . Surgical procedure of the recipient animals Under isoflurane anesthesia the right sciatic nerve of the Lewis rat was exposed. A 10 mm segment of the sciatic nerve was excised and reconstructed with a 10 mm graft under an operating microscope (Zeiss OpMi6, Carl Zeiss Surgical GmbH, Oberkochen, Germany). The epineurium was sutured with six 10-0 sutures (10-0 Ethilon, Ethicon Inc., USA), the muscle was approximated (6-0 Vicryl Rapide, Ethicon Inc.) and the skin was closed with a continuous subcutaneous suture (5-0 Vicryl Rapide, Ethicon Inc.). All rats received 5mL of 0.9% saline solution, 0.6mg/kg Buprenorphine and one dose of 30mg/kg diluted trimethoprim/sulfadiazine subcutaneously (Tribissen, Five Star Compounding Pharmacy, Clive, IA). Postoperatively, the rats were individually housed and provided with food and water ad libitum with a 12-hour light-dark cycle. Ultrasound measurements The cross-sectional tibial muscle area of six randomly selected rats per group was evaluated with ultrasound measurements of both sides at baseline and at two, four, eight, twelve and sixteen weeks after surgery as previously described using a GE Vivid 7 Ultrasound system (General Electric, Fairfield, CT, USA) 31,32 . Cross-sectional area was calculated with Adobe Photoshop CC 2018 (Adobe Systems Incorporated, San Jose, CA, USA).