Femke Mathot

Chapter 11 188 extracellular matrix and cell cycle genes at multiple time points after seeding. The gene expression profile of both cell-types changed significantly upon interaction with processed nerve allografts. Undifferentiated MSCs demonstrated enhanced expressions of neurotrophic (NGF, GDNF, PMP22), extracellular matrix (FBLN1, LAMB2) and regulatory cell cycle genes (CCNB2) 7 days after seeding. DifferentiatedMSCs expressed enhanced levels of neurotrophic (NGF, GDNF, GAP43), angiogenic (VEGF1), extracellular matrix (FBLN1) and regulatory cell cycle genes (CASP3, CCNB2) in the first 72 hours after seeding. The differences in gene- profiles and effective phases suggests that both cell-types can affect nerve regeneration in different ways and at different time points, which should be confirmed in vivo. Vascularization has been postulated to be essential for nerve regeneration by guiding regenerating nerves and providing the supply of necessary trophic factors. As tools to visualize and quantify vascularization patterns in transplanted nerves are lacking, the study described in chapter 5 focusses on the development of a technique that provides a three- dimensional visualization and quantification of the peripheral neural vascularity in a rodent model. Vascularization in autografts (n=12) and untreated nerves (n=12) was quantified by the vascular surface area using conventional photography (2D) and the vascular volume was calculated using micro-computed tomography (3D). Combining both methods accurately reflects the degree of vascularization in rat nerves. This easily reproducible strategy allows objective assessment of the level and the organization of vascularization in nerves and could be extrapolated to any other desired organ ex vivo. In chapter 6 , the technique validated in chapter 5 is used to determine the beneficial effect on vascularization when seeding undifferentiated and differentiated MSCs onto a decellularized nerve allograft in a rat sciatic nerve defect model. The vascularization of normal nerves, autografts, allografts seeded with differentiated MSCs and allografts seeded with undifferentiated MSCs was quantified and compared at 16 weeks after surgery. Unseeded allografts had a significantly lower vascular surface area percentage than normal non- operated nerves and allografts seeded with differentiated MSCs. Although it was significantly correlated to the vascular surface area, no significant differences in vascular volume were obtained between groups. The vascularization pattern in MSC-seeded allografts consisted of an extensive non-aligned network of micro-vessels with a centripetal pattern, while the vessels in autografts and normal nerves were more aligned with longitudinal patterns. The more extensive network might facilitates better oxygen and nutrient supply throughout the graft, but can be detrimental for the alignment of sprouting axons considering the directional function of vasculature. The study in chapter 7 aimed to determine whether the previous elucidated rationale of MSC- seeding onto nerve allografts could be confirmed by in vivo evaluation of functional outcomes. Autografts (n=20), allografts (n=20), allografts seeded with undifferentiated MSCs (n=20) and allografts seeded with differentiated MSCs (n=20) were used to reconstruct a ten millimeter sciatic nerve defect in a rodent model. Cross sectional tibial muscle ultrasound measurements evaluated functional recovery over time. At 12 and 16 weeks after surgery (n=10 per group