Femke Mathot

10 General discussion and future perspectives 173 differentiated MSCs to a better extent than in vitro outcomes, resulting in the assumption that differentiated MSCs enhance angiogenesis in nerve allografts to a greater extent than undifferentiated MSC. ANGIOGENESIS & FUNCTIONAL OUTCOMES The used techniques to measure angiogenesis validated in chapter 5 , are unique and innovative in describing 2D and 3D angiogenesis. Disadvantages of these techniques are the dependency on the effective pixel size of the used micro CT and adequate injection of the radiopaque compound. However, Microfil® has demonstrated before to reach even the smallest vessels, allowing analysis of the entire vascular network and adequate compound injection can be easily checked at the nailbeds of the rats. 54 Even if no micro CT is available, an impression of the vascular network and analysis of the vascular surface area can be readily obtained with the 2D technique. Although the 2D and 3D techniques cannot be combined with histological analysis of vessels since they require different processing techniques, histological evaluation falls short in quantifying and describing vascularity volumes and vascularization patterns and forms therefore no loss to the evaluation of angiogenesis. Vascular distribution and vessel sizes analyses on micro CT samples could add valuable information about neoangiogenesis in future studies in addition to vascular volume and surface area. Compared to unseeded, decellularized nerve allografts, both MSC-groups enhanced functional outcomes, which could be allocated to their improved angiogenesis described in chapter 6 . Since angiogenesis is granted a crucial role in axon guidance and nerve regeneration, it was expected that particularly differentiated MSCs would improve the functional outcomes of nerve allografts. There were no significant differences between the functional outcomes of undifferentiated and differentiated MSCs. However, compared to unseeded allografts, undifferentiated MSCs led to significantly improved CMAP and ITF outcomes, while differentiated MSCs only led to significantly enhanced CMAP outcomes. The small study groups and small animal model can be put forward as explanations that other significant differences were absent. Although most motor outcomes of both MSC-groups equaled the motor outcomes of nerve autografts, autografts histologically outperformed MSC-seeded allografts despite the comparable or even enhanced level of neoangiogenesis in the MSC-groups. The alignment of the new vasculature can be a clarification to this subtle discrepancy between the level of angiogenesis, the histological outcomes and the functional outcomes, as measured by CMAP, ITF and muscle mass. The vasculature was significantly enhanced in nerve grafts seeded with differentiated MSC in particular, but it led to an extensive, non-aligned network of small vessels. Considering their guiding role, enhanced angiogenesis should lead to improved regeneration, but might be counterproductive if it extents a particular level or becomes less aligned. When subjectively comparing the angiogenesis samples of the MSC- groups and the autograft group, the mal-alignment of vasculature seems to be of greater