Femke Mathot

Chapter 6 96 particular showed enhanced expressions of VEGF after interacting with the outer surface of the decellularized allografts. 25, 32 The purpose of this study was to either substantiate or invalidate the hypothesis whether the previously demonstrated different gene expression profiles of differentiated and undifferentiated MSCs would lead to different levels and patterns of vascularization when dynamically seeded onto processed/decellularized nerve allografts, in comparison to unseeded allografts and autografts. METHODS This study was approved by the IACUC institutional review committee and our Institutional Review Board (IACUC protocol A2464-00). A 10 mm segment of the right sciatic nerve of 20 Lewis rats (Envigo, USA) was excised and was replaced with either (i) a reversed autograft, (ii) a processed/decellularized nerve allograft, (iii) a processed/decellularized nerve allograft seeded with undifferentiated MSCs or (iv) a processed/decellularized nerve allograft seeded with differentiated MSCs. All four groups were sacrificed after 16 weeks to determine and compare degree and patterns of revascularization of the nerves. The 16 week survival period was chosen based on previous research indicating 16 weeks is the time period in which nerve regeneration matures. 33 Nerve allograft harvest and processing Ten Sprague-Dawley rats (Envigo, Madison, WI, USA) weighing 250-350 grams served as donors of 20 sciatic nerve segments of approximately 15 mm each. Sprague-Dawley rats were used to obtain a major histocompatibility complex mismatch with the recipient Lewis rats, in order to mimic a clinical setting in which allogenous donor nerves will be used. 34, 35 The rats were anesthetized in an isoflurane induction chamber and euthanized with an overdose of pentobarbital once asleep. Both sciatic nerves were carefully dissected with sharp dissecting scissors under an operating microscope (Zeiss OpMi6, Carl Zeiss Surgical GmbH, Oberkochen, Germany). Directly after their harvest, the obtained sciatic nerve segments were processed/ decellularized according to a previously published five-day protocol that includes multiple washing steps and emerging in elastase. 28 Sterilization of the nerve segments was obtained with g -irradiation. Stem cell preparation and differentiation In accordance with a future clinical trial in which the MSCs will be harvested from a patient, MSCs were harvested from the inguinal fat pad of an inbred Lewis rat and processed per Kingham and colleagues protocol. 36 The obtained MSCs were cultured in previously described cell culture conditions. 37-39 The MSCs complied with the criteria for MSCs, defined by the International Society for Cellular Therapy previously: they were plastic adherent and multi- potent and expressed canonical MSC-markers like CD44 (88.2%) and CD90 (88.3%) while markers such as CD34 and CD45 were absent. 30, 40