Femke Mathot

Chapter 10 170 is supported by in vitro studies demonstrating improved neurite outgrowth and enhanced levels of trophic factors in culture media in the presence of MSCs. 18-20 Furthermore, studies reported positive effects of MSCs in vivo without confirmed in vivo differentiation or long term survival. 18, 21 This trophic concept led to the insight that MSCs could influence nerve regeneration without being delivered inside nerve grafts. Trophic factors can theoretically penetrate the epineurium by diffusion, just like they do from surrounding vasculature. To avoid injection, dynamic seeding on nerve substitutes was proposed and led to a uniform distribution of MSCs on the outer surface of nerve grafts. 4 Drawbacks of this strategy are the minimal 12 hours needed to let the MSCS adhere to the nerve grafts and the potential vulnerability of the MSCs as they are on the outer surface of the graft during surgery. However, MSCs survived up to 29 days in vivo, implying that they can withstand the mechanical impacts during surgery. 22 Due to its practicability and efficient cell delivery, dynamic seeding is labeled as an effective strategy to non-traumatically adhere MSCs to nerve grafts, after which they ideally exert their trophic function. IN VITRO RESEARCH After nerve injury, Schwann cells change to a proliferating state 23, 24 , producing trophic factors that contribute to formation of the growth cone, angiogenesis, bands of Büngner and axon regeneration. 25-32 Although undifferentiated MSCs possess trophic characteristics, it is uncertain if they meet up to the trophic levels of Schwann cells normally present at the side of nerve regeneration. Since Schwann cells are essential for nerve regeneration but require autologous nerve tissue, differentiating MSCs into Schwann cell-like cells has been suggested as required addition to processed nerve grafts to hopefully have even greater effects on nerve regeneration than undifferentiated MSCs. 20 Compared to undifferentiated MSCs, MSCs differentiated into Schwann cell-like cells demonstrated to express enhanced levels of neurotrophic and angiogenic factors in vitro and led to comparable nerve regeneration in vivo in previous studies. 18, 20, 33-36 However, delivery strategies have never been tested on differentiated MSCs in specific. As this is fundamental to fairly compare effects of undifferentiated and differentiated MSCs in vitro and in vivo, both their capabilities to adhere to the surface of decellularized nerve allografts were analyzed in chapter 3 . This revealed a seeding efficiency of 80% for undifferentiated and 95% for differentiated MSCs. Although not significant, the difference cannot be subscribed to laminin levels, previously described to improve cell attachment, since both cell types expressed comparable levels over time ( chapter 4 ). 37 The difference might subjectively be attributed to the tendency of differentiated MSCs to cluster together. The fact that undifferentiated and differentiated MSCs can be equally seeded onto decellularized nerve grafts is essential preliminary knowledge to further fairly compare both cell-types in their capabilities to enhance angiogenesis and nerve regeneration. In chapter 4 the genes expressed by the different cell-types at multiple time points after