Femke Mathot

6 Vascularization in stem cell seeded nerve allografts 103 differentiated into Schwann-like cells is the most likely mechanism of the superior degree of (neo)angiogenesis in processed nerve allografts seeded with differentiated MSCs. 19, 20, 22, 25-27, 32, 36, 46 The role of the growth factors in the differentiation medium that might become embedded in the extracellular matrix of the cells cannot be ruled out, but they are not specifically known to stimulate neoangiogenesis. 25 The vascularization in autograft nerves and normal non-operated nerves did not significantly differ from both stem-cell groups. Revascularization in nerves is hypothesized to occur via two mechanisms: centripetal neovascularization (vessels sprouting into the graft from the surrounding tissues) and inosculation (vessels sprouting into the graft from both stump ends into the existing vascular tree). 47 In autografts, there is still an existing vascular tree surrounded by endothelial cells which is likely to increase the vascularization speed and improve the alignment of vessels which was demonstrated in this study. 1 In processed allografts, all cellular debris has been removed, leaving no directions for ingrowing vessels. 28 In contrast to the longitudinal alignment of vessels in normal non-operated and autograft nerves, the vascularization of the stem cell-seeded nerve allografts consisted of an extensive network of micro-vessels distributed among the entire nerve that were not in line with the expected direction of axon regeneration ( figure 3 ). Combining the described differences in revascularization pattern, we hypothesize the predominant mechanism of revascularization in autografts is inosculation, leading to well-aligned and accelerated revascularization. Centripetal neovascularization was hypothetically the predominant mechanism of vascularization or at least had a greater share in the revascularization of allografts seeded with MSCs. Based on the timeline of angiogenic gene expression profiles in previous research 32 and the known limited survivability of MSCs in vivo, 29 both type of MSCs are expected to accelerated revascularization mainly in the first few days after seeding with a slowly diminishing effect up to 29 days. The purpose of this study was to determine the effect of type of MSCs on enhancing vascularization of processed nerve allografts and to compare degree and pattern of vascularization to autograft and allograft nerves. It is thus underpowered to perform functional or histological analysis. Despite the small group size however, we were able to clearly demonstrate the effect of MSCs on vascularity. We successfully evaluated the vasculature of nerves in an accurate manner and were able to objectively quantify the amount of (neo)angiogenesis. A future study should focus on functional outcomes. The effective pixel size of the used micro-CT may have caused a loss in the detection of the smallest vessels, particularly present in both stem cell groups. The CT-scans enabled us to fairly compare the volume of the bigger vessels in the operated nerve to that of the bigger vessels in the non-operated side, but it sub-optimally displayed the smaller vessels. This could partially mask the effect of stem cell seeding, as the MSCs seem to lead to the formation of small vessels in particular. For future research, it is advised to use a micro-CT scanner with a smaller effective pixel size. This is likely to increase the correlation between the vascular volume and the vascular surface area.