Femke Mathot

Chapter 3 54 soaking in cell-solutions are used as delivery methods of MSCs with the rationale that they have a structural function as Schwann cells that need to be delivered within the nerve allograft itself. Injection may be traumatic to both MSCs and the ultrastructure of the nerve allograft and results in an unequal distribution of the delivered cells. 27, 38-40 The average diameter of MSCs exceeds the calibers of myelinated axon fibers, suggesting that MSCs can block axon ingrowth when delivered inside the nerve graft. 41-43 Soaking techniques deliver lower number of cells in a nonuniform distribution. 28 It has been recently reported that growth factors and cytokines produced by MSCs may enhance nerve regeneration, while not necessitating intraneural placement of the MSCs. 7, 44 The straightforward dynamic seeding strategy of Rbia and colleagues successfully attaches large numbers of undifferentiated MSCs to the surface of a processed nerve allograft without harming the inner ultra-structure of the allograft. 29 The same technique resulted in the same optimal seeding duration for undifferentiated MSCs in this study, indicating a high reproducibility of the Rbia method. The seeding of differentiated MSCs is less well established and differentiation of cells may decrease their potential to attach to processed nerve allografts, possibly due to their changes in cellular morphology (e.g., spindle-like shape). 10 This study demonstrated there was no decrease in attachment efficiency when MSCs are differentiated. Based on our experience, dynamic seeding of differentiated MSCs onto a processed nerve allograft is possible and results in a uniform distribution of large amounts of differentiated MSCs (and undifferentiated MSCs) on the surface of the nerve allograft which has not been accomplished by other previously described methods. Both cell types have previously shown to produce neurotrophic and angiogenic factors and have been allocated an immunomodulatory role. 7, 18, 19, 24, 25, 44 The porous epineurium of the processed nerve allografts (demonstrated in figure 3 ) allows for these factors to both regulate the immune response and angiogenesis in the surroundings of the regenerating nerve, and stimulate nerve regeneration inside the nerve allograft, while the cells remain on the outer surface of the graft. The seeded MSCs form an addition to the circulating stem cells normally attracted to the regenerating nerve, also functioning from outside the epineurium. A limitation of this study is the in vitro setting, whichmay not translate into results expected for an in vivo setting. In vitro studies permit testing of the seeding potential of undifferentiated MSCs and differentiated MSCs without having to sacrifice extra animals. With the described strategy, it would approximately take up two to five weeks after nerve injury to obtain a processed allograft seeded with patient’s own (undifferentiated or differentiated) MSCs. Although peripheral nerve injuries are ideally repaired as soon as possible after injury, a two- or five-week delay that eventually leads to the desirable improved nerve regeneration would be clinically applicable. Furthermore, the utility of methods to deliver undifferentiated MSCs and differentiated MSCs to the surface of a nerve graft relies on the idea that MSCs at least have a partly trophic function and that growth factors and cytokines produced by them will migrate through the epineurium and enhance nerve regeneration. This hypothesis needs to be confirmed both in vitro and in vivo. The current study is the essential first step in testing this hypothesis.