Femke Mathot

Chapter 7 112 (IACUC protocol A2464-00), a 10 mm segmental defect of the sciatic nerve of 80 male Lewis rats weighing 250-300 grams (Envigo, Madison, WI, USA) was repaired with a 10 mm (i) reversed autograft, (ii) decellularized allograft (iii) decellularized allograft seeded with undifferentiated MSCs, or (iv) decellularized allograft seeded with differentiated MSCs. The decellularized allografts originated from Sprague-Dawley rats and were specifically chosen for their histocompatibility mismatch to Lewis rats. 29, 30 This simulates the clinical setting where an allogenic processed nerve graft is seeded with autologous MSCs. After 12 and 16 weeks, functional, histological and immunofluorescence outcomes were evaluated. Nerve allograft collection, processing and seeding Sixty sciatic nerve segments from 30 Sprague-Dawley rats (Envigo, Madison, WI, USA) weighing 250-350 grams served as nerve allografts. After anesthesia with isoflurane, rats were euthanized, shaved and sterilely prepped. The sciatic nerve was exposed, removed under an operating microscope (Zeiss OpMi6, Carl Zeiss Surgical GmbH, Oberkochen, Germany) and processed according to a previously published protocol. 7 After sterilization with g -irradiation, nerves were stored at 4 ° C in Phosphate Buffered Saline (PBS) until surgery. Stem cell preparation and differentiation MSCs were derived from the inguinal fat pad of inbred Lewis rats according to protocol.16 Cells were previously characterized by plastic adherence, pluripotency towards mesodermal lineages, the expression of mesenchymal stem cell markers CD29 (88.2%) and CD90 (88.3%) and the absence of hematopoetic cell markers CD34 (91.1% absent) and CD45 (86.0% absent). 26-28 Both cell-types were cultured in an incubator at 37 ° C (5% CO2) and the growth medium was changed every 72 hours. Passage six MSCs were used in this experiment for both differentiated and undifferentiated MSCs. MSCs-culture The stromal cell pellet was re-suspended in normal growth medium consisting of a -MEM (Advanced MEM (1x); Life Technologies Corporation, NY, USA), 5% platelet lysate (PLTMax®; Mill Creek Life Sciences, MN, USA), 1% Penicillin/Streptomycin (Penicillin-Streptomycin (10.000 U/mL; Life Technologies Corporation), 1% GlutaMAX (GlutaMAX Supplement 100X; Life Technologies Corporation) and 0.2% Heparin (Heparin Sodium Injection, USP, 1.000 USP units per mL; Fresenius Kabi, IL, USA). MSC differentiation MSCs were differentiated into Schwann cell-like cells using a differentiation cocktail containing 0.14% Forskolin (Sigma-Aldrich corp., MO, USA), 0.01% basis fibroblast growth factor (bFGF; PeproTech, NJ, USA), 0.005% platelet-derived growth factor (PDGF-AA; PeproTech) and 0.02% Neuregulin-1 ß1 (NRG1-b1; R&D systems Inc, MN, USA). 16 Differentiation was assessed by immunocytochemistry for the expression of S100 (S100; ThermoFisher Scientific, MA, USA), Glial fibrillary acidic protein (GFAP, mouse anti-GFAP; ThermoFisher Scientific) and neurotrophin receptor p75 (p75 NTR, rabbit anti-p75 NTR; ThermoFisher Scientific). Goat anti-rabbit fluorescein isothiocyanate (FITC) and goat anti-mouse cyanine 3 (CY3, both