Femke Mathot

8 Stem cells on nerve graft substitutes 137 DISCUSSION We examined the clinical potential of dynamic seeding of MSCs onto commercially available nerve graft substitutes by testing whether (I) the NeuraGen® Nerve Guide and the Avance® Nerve Graft affect the viability of human MSCs, (II) human MSCs can be dynamically seeded and distribute uniformly onto these nerve substitutes, and (III) dynamic seeding and optimized timing improves the efficiency of MSC-seeding. MTS assays demonstrated that both the Avance® Nerve Graft and the NeuraGen® Nerve Guide did not affect the metabolic activity or survivability of human adipose derived MSCs. The human MSCs were able to adhere in a uniform manner of the surfaces of both nerve substitutes, with an optimal dynamic seeding duration of 12 hours. The significantly better seeding efficiency of NeuraGen® Nerve Guides after 12 hours of seeding is most likely due to their hollow conduit configuration, enabling MSCs to adhere on the outer and inner surface of the conduit. The MSCs did not migrate inside the Avance® Nerve Graft or into the substrate of the NeuraGen® Nerve Guides. It is hypothesized that the decrease in seeding efficiency after 12 hours of dynamic seeding is related to cell damage due to the rotational forces of the seeding process, leading to a decreased ability of cells to adhere to the surfaces of the nerve substitutes. The concept that MSCs inside a nerve substitute support the regeneration of axons is relevant for the applicability of the dynamic seeding strategy. MSCs used in peripheral nerve repair potentially differentiate into Schwann cells in vivo, and may have a structural function by replacing injured tissue. However, studies supporting that MSCs need to be delivered inside nerve substitutes are limited and most papers show that only a fraction of the added MSCs are differentiated into actual Schwann cells and survive on the long term. 33, 34 Other studies reported trophic effects of MSCs, producing proteins and molecules that stimulate tissue regeneration, form extracellular matrix components, enhance angiogenesis, inhibit scar formation, and attenuate inflammation without the MSCs actually being physically integrated into the regenerating tissue. 35-39 Robust expression of secreted proteins including growth factors and morphogens by MSCs when introduced to injured tissues supports the concept that MSCs may be more effective as tissue repair catalysts rather than architectural participants. 40, 41 The trophic concept forms the basis of the proposed delivery method of MSCs. Considering the shown mismatch in size between the axon fascicules and the MSCs ( figure 6 ), delivery of MSCs inside a graft may block the ingrowth of regenerating axons. As MSCs do not need to be delivered inside the nerve graft to produce growth factors and cytokines that support nerve regeneration, it is preferred that MSCs are added in a simple, efficient and systematic manner that is non-traumatic and avoids damage to the nerve substitute. In contrast to microinjection and soaking methods, the dynamic seeding strategy of Rbia and colleagues meets these requirements. 22, 26-28 This strategy has demonstrated to enhance the neuroregenerative gene expression in the MSCs 19, 20 and revealed an in vivo survival of the MSCs up to 29 days. 42 The absence of MSCs on the inner surface of the Avance® Nerve Graft therefore does not