Femke Mathot

4 Interaction between MSCs and a nerve allograft 61 INTRODUCTION Although many efforts have been made to find a neural tissue substitute equivalent to nerve autografts, autografts currently remain the gold standard in the reconstruction of critical nerve defects. 1-5 Nerve allografts are a promising option, but decellularization is required to prevent an immune response in the recipient. While necessary for successful transplantation, decellularization removes all cellular components including Schwann cells from allograft nerve tissues. 6, 7 Schwann cells are fundamental for peripheral nerve formation and play a critical role in peripheral nerve regeneration by producing axonotrophic factors and providing remyelination. 8, 9 It has been postulated that direct or indirect addition of growth factors may enhance nerve regeneration in processed nerve allografts, replacing or mimicking the function of the absent Schwann cells. Yet, direct delivery of growth factors has not resulted in improved outcomes 10-12 , suggesting that cellular mechanisms are necessary for the growth factors to be functional. Indirect delivery of growth factors could be provided by mesenchymal stromal cells (MSCs). MSCs are biologically important for tissue repair and regeneration by influencing the immune system, enhancing angiogenesis and inhibiting scar formation. 13, 14 MSCs are mostly obtained from either bone marrow or adipose tissue. Adipose tissue can be easily harvested and contains large amounts of MSCs that proliferate rapidly in optimized cell culture medium. 15-18 When added to decellularized nerve allografts, adipose-derived MSCs produce growth factors that support tissue repair in response to interactions with the extracellular matrix (ECM) of the allograft nerve. 19-24 The interaction between the ECM of the decellularized nerve allograft and the MSCs influences the differentiation state of MSCs and their growth factor production. 25, 26 It has been proposed that inducing neural differentiation of MSCs prior to the addition to a decellularized allograft, may enhance nerve regeneration when compared to undifferentiated MSCs. Multiple studies have shown that it is possible to differentiate adipose derived MSCs into Schwann-like cells 18, 27-29 and in vitro studies demonstrated increased neurite outgrowth of motor neurons when exposed to differentiated MSCs compared to undifferentiated MSCs. 22, 30-36 While it is plausible that the biological properties of differentiated MSCs may be altered upon interaction with the ECM of nerve allografts, there is a paucity of molecular data that characterizes these putative differences. The purpose of this study was to evaluate temporal profiles of gene expression in undifferentiated and differentiated MSCs at multiple time points after dynamic seeding 37, 38 onto a decellularized nerve allograft. Determination of differences in gene expression may provide mechanistic insight into pathways that could be further leveraged to improve peripheral nerve repair. MATERIALS AND METHODS General design This study was approved by the IACUC institutional review committee and the Institutional Review Board (IACUC protocol A3053-16). The experimental design is shown in table 1 .