Femke Mathot

7 Motor results of MSC-seeded nerve allografts 113 ThermoFisher Scientific) were used as secondary antibodies. Cell nuclei were labeled with 4’,6-diamidino-2-phenylindole (DAPI). 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 undifferentiated MSCs or 1x10^6 differentiated MSCs in 10mL growth medium were 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% CO2). After the bioreactor had rotated for 12 consecutive hour, 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/kgBuprenorphine andonedoseof 30mg/kgdiluted 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). Nonsurvival procedure At 12 and 16 weeks, ten rats of each group underwent a non-survival procedure. Anesthesia was induced by isoflurane, followed by intraperitoneal injection of Ketamine (80mg/kg) and Xylazine (10mg/kg) and maintained by additional doses of Ketamine (40mg/kg). Compound Muscle Action Potentials (CMAP) - A miniature bipolar electrode was clamped around the sciatic nerve proximal to the nerve graft. One ground electrode was placed in surroundingmusculature and two recording electrodes were superficially placed in the anterior tibial muscle. The CMAP was measured using a VikingQuest portable electromyelogram (Nicolet Biomedical, Madison, WI). A non-recurrent single stimulation with a duration of