Femke Mathot

6 Vascularization in stem cell seeded nerve allografts 95 INTRODUCTION Compared to decellularized allografts or bioabsorbable synthetic conduits, autograft nerves continue to result in superior functional outcomes after peripheral nerve repair. One hypothesis for autografts superiority is the ability of autografts to revascularize. 1-3 Revascularization of injured tissue is an essential process in tissue regeneration as it relieves injury-induced hypoxia at the regeneration site while facilitating the delivery of nutrients and cells essential for the regeneration process. 4 Revascularization occurs as early as 2 days after nerve injury and precedes the axonal regeneration. 5, 6 Hypoxia in peripheral nerves causes macrophages to secrete vascular endothelial growth factor (VEGF), which induces (neo)angiogenesis and facilitates trophic factors to arrive at the regeneration site. 7 The endothelial cells of newly formed blood vessels have a directional function by guiding Schwann cells across the nerve- gap, which direct the regenerating axons in the correct direction. 6, 8 A sufficient volume of well-organized newly formed vessels in both nerve stumps is requisite for a functional outcome after peripheral nerve repair and has been confirmed in several animal-models. 3, 9, 10 Improved revascularization and diminished duration of avascularity has been suggested to prevent central fibrosis or necrosis in autografts compared to decellularized allografts, especially in large nerve gaps. 11-14 Improved vascularization could therefore lead to improved nerve regeneration in decellularized allografts. In order to improve vascularization, numerous studies have evaluated the addition of VEGF to nerve reconstructions. Although some studies demonstrated improved vascularization by the addition of VEGF, they failed to prove any benefit of VEGF with respect to functional outcomes demonstrating that the addition of a single growth factor was insufficient to replicate the complex angiogenesis cascade. 15-19 Adipose derived Mesenchymal Stem Cells (MSCs), when seeded onto decellularized allograft nerves, result in the production of neurotrophic and angiogenic factors, one of which is VEGF. 20 While the exact role of MSCs (structural vs immunomodulatory) continues to be defined, it has been demonstrated that MSCs have a finite lifespan, stimulate tissue repair via release of trophic factors and produce proteins and cytokines that stimulate tissue regeneration with immunomodulatory effects. 20-25 MSCs differentiated into Schwann-like cells (differentiated MSCs) in vitro lead to increased gene expressions of angiogenic (VEGF) and neurotrophic genes when compared to undifferentiated MSCs 20, 22, 26, 27 and are considered nerve regeneration catalysts. 19 The disadvantages of differentiating MSC include the extra preparation time, expense and extra handling of the cells. The effect of differentiation thus needs to be carefully investigated. In order to equal the results of nerve autografts, efforts have been recently made to optimize the quality of decellularized allografts. 28 To improve their outcomes a non-traumatic dynamic seeding strategy has been developed to seed undifferentiated and differentiated MSCs to the allograft surfaces, which survive up to a month in vivo. 29-31 Furthermore, differences in gene expression levels (and thus growth factors produced) have been elucidated between differentiate and undifferentiated MSCs, and demonstrated that differentiated MSCs in