Tiam Mana Saffari

215 MICRO CT ANALYSIS AFTER STEM CELL DELIVERY AND ANGIOGENESIS 10 Nerve allograft harvest and processing Ten male Sprague-Dawley rats (Envigo, Madison, WI, USA), weighing 250-300 grams, served as donors for harvesting a 15-mm segment of the sciatic nerve bilaterally. Sprague-Dawley rats were used to obtain a major histocompatibility complex mismatch with the recipient Lewis rats 13,14 . After rats were anesthetized in an isof lurane inducted chamber and euthanized with an overdose of Pentobarbital Sodium (Fatal Plus, 390 mg/mL, Vortech, Dearborn, MI, USA), sciatic nerves were collected, cleaned from external debris and processed using a five-day decellularization protocol utilizing elastase and chondroitinase to become acellular allografts 15 . After processing, the nerves were sterilized using g-irradiation and stored in phosphate buffer saline at 4°C. All steps were carried out at room temperature with agitation under sterile conditions and in a laminar flow hood. Mesenchymal stem cell preparation and culture Rat MSCs from a previously characterized lineage were used for experiments 16 . These MSCs were derived from the inguinal fat pad of inbred Lewis rats 17 , characterized by plastic adherence, pluripotency towards mesodermal lineages, the expression of MSC surface markers CD29 and CD90, and absence of hematopoietic cell surface markers CD34 and CD45 18 . The stromal cell pellet was re-suspended in growth medium consisting of advanced Minimum Essential Medium (a-MEM (1x); Life Technologies Corporation (LTC)), 5% platelet lysate (PLTMax®; Mill Creek Life Sciences, MN, USA), 1% Penicillin/Streptomycin (Penicillin-Streptomycin (10.000 U/mL; LTC), 1% GlutaMAX (GlutaMAX Supplement 100X; LTC) and 0.2% Heparin (Heparin Sodium Injection, USP, 1.000 USP units per mL; Fresenius Kabi, IL, USA). Mesenchymal stem cell differentiation MSCs were differentiated into Schwann cell-like cells according to protocol 17 . This protocol has shown to morphologically change 81.5% of the MSCs exposed to the differentiation medium into a typical spindle-like shape 17 . Cells were treated with ß-mercaptoethanol (Sigma-Aldrich, MO, USA) and all-trans-retinoic acid (1:1000 of