Femke Mathot

6 Vascularization in stem cell seeded nerve allografts 97 Cell culture The obtained MSCs were cultured in growth medium that contained 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, NY, USA), 1% GlutaMAX (GlutaMAX Supplement 100X; Life Technologies Corporation, NY, USA) and 0.2% Heparin (Heparin Sodium Injection, USP, 1.000 USP units per mL; Fresenius Kabi, IL, USA). Differentiation of MSCs The differentiation of MSCs into Schwann cell-like cells was performed and verified according to a previously described protocol. This protocol has shown to morphologically change 81.5% of the MSCs exposed to the differentiation medium into a typical spindle-like shape and lets approximately 40-45% of the exposed MSCs express glial cell marker GFAP, Schwann cell marker S100 and neurotrophin receptor p75.36 After two preparatory steps with ß-mercaptoethanol (Sigma-Aldrich corp., MO, USA) and all-trans-retinoic acid (1:1000, dilution of stock; Sigma-Aldrich Corp., MO, USA), a differentiation cocktail was introduced to their growth medium. This differentiation cocktail consisted of Forskolin (Sigma-Aldrich corp., MO, USA), basic fibroblast growth factor (bFGF; PeproTech, NJ, USA), platelet derived growth factor (PDGF-AA; PeproTech, NJ, USA), and neuregulin-1 ß1 (NRG1-b1; R&D systems Inc, MN, USA). According to the protocol, MSCs differentiation was verified by immunocytochemistry for S100, GFAP and neurotrophin receptor p75 (Rabbit anti-S100, mouse anti-GFAP and rabbit anti-p75 NTR; all ThermoFisher Scientific, MA, USA). Secondary antibodies goat-anti rabbit FITC and goat anti-mouse Cyanine-3 (both ThermoFisher Scientific, MA, USA) were used to visualize the expression of the before-mentioned markers in differentiated MSCs. Undifferentiated MSC and Schwann cells were used as respectively negative and positive control. Stem cell seeding The decellularized allografts were dynamically seeded with 1x10^6 undifferentiated or differentiated MSCs for 12 hours at 37 ° C. Previous testing has shown that this is the most efficient seeding duration, leading to a seeding efficiency of 80% to 95% respectively. 30, 31 Surgical procedure Twenty male inbred Lewis rats (Envigo, Madison, WI, USA) weighing 250-300 gram, served as recipient rats. The surgical procedure was performed as previously described. 41 The 10 mm section of the sciatic nerve was reversed in the autograft group. The processed allografts, either unseeded, seeded with undifferentiated MSC or seeded with differentiated MSCs, were used to reconstruct a 10 mm sciatic nerve gap in the other three groups. No immunosuppression was administered postoperatively. Non-survival procedure – measurements During the non-survival procedures after 16 weeks, all rats (n=5) of each group were